BVDV-IBRV bivalent mRNA vaccine and use thereof

Abstract

Provided are a BVDV-IBRV bivalent mRNA vaccine and the use thereof. The mRNA vaccine of the present application contains a polynucleotide sequence of any one of SEQ ID NOs: 42-77, and can be used for treating or preventing bovine viral diarrhea virus infections and / or infectious bovine rhinotracheitis virus infections, or treating or preventing bovine viral diarrhea and / or bovine rhinotracheitis.

Description

BVDV-IBRV bivalent mRNA vaccine and its application

[0001] priority

[0002] This application claims priority to Chinese invention patent application filed on August 2, 2024, application number: 202411061951.X, entitled "BVDV-IBRV bivalent mRNA vaccine and its application". Technical Field

[0003] This invention belongs to the field of vaccine technology, and in particular relates to an mRNA vaccine for the prevention of BVDV-IBRV and its preparation method. Background Technology

[0004] Bovine viral diarrhea virus (BVDV)

[0005] Bovine Viral Diarrhea Virus (BVDV) is the pathogen of bovine viral diarrhea-mucosal disease (BVD-MD). BVDV was first discovered in a herd of cattle in the United States suffering from acute gastroenteritis with a high mortality rate. Since then, BVDV has gradually become one of the important viral pathogens for prevention and control in many regions worldwide. BVDV is an enveloped, single-stranded, positive-sense RNA virus belonging to the genus *Phosphovirus* of the family Flaviviridae. The BVDV genome contains a large open reading frame (ORF) in the middle, which encodes a large polyprotein precursor. The polyprotein is degraded into structural and non-structural proteins by its own proteolytic enzymes. The structural proteins are mainly located at the front of the genome, including C, Erns, E1, and E2 proteins. The C protein is an important component of the viral capsid protein and promotes viral replication, but it is not essential for viral assembly. The other three structural proteins constitute the viral envelope. The Erns protein, also known as the E0 protein, is highly conserved and can induce the body to produce neutralizing antibodies. Therefore, the Erns protein can be used in subunit vaccines and as a diagnostic antigen. The E1 protein can form the E1-E2 heterodimer with the E2 protein, which is an essential component for viral entry into host cells. Furthermore, the E1 protein can assist in the localization of the E0 protein but cannot induce an immune response. The E2 protein is a protective antigenic protein containing the virus's major antigenic determinants and is the main site for antibody binding, host binding adsorption, and mediating the production of neutralizing antibodies. The E2 protein is prone to mutation; gene mutations make the virus more survivable, which is a major cause of persistent bovine infection and vaccine failure.

[0006] Persistent infection in animals plays a crucial role in controlling the spread of BVDV. Currently, the primary target of BVDV vaccination is the fetus, aiming to prevent clinical disease and viral-induced immune dysregulation. BVDV vaccination programs involve various types of live attenuated vaccines, inactivated vaccines, and subunit vaccines.

[0007] Live attenuated vaccines have the potential for virulence reversion and can infect the fetus through the placenta, leading to persistent infection in animals. Live attenuated vaccines can also cause immunosuppression and other side effects in vaccinated animals. Inactivated vaccines are safer than live attenuated vaccines and can be administered at any age and stage of pregnancy. However, inactivated vaccines have lower immunogenicity, require multiple injections, and develop immunity more slowly. Inactivated vaccines do not offer cross-protection against different strains, and vaccination with inactivated vaccines only provides partial protection for the fetus. Subunit vaccines can induce high levels of antibodies against the same virus. However, subunit vaccines have lower immunogenicity, requiring methods such as increasing dosage and multiple immunizations to improve their effectiveness. Furthermore, subunit vaccines cannot simultaneously induce humoral and cellular immune responses.

[0008] Infectious bovine rhinobronchitis virus (IBRV)

[0009] IBRV, also known as Bovine herpesvirus 1 (BHV-1), belongs to the genus Varicellavirus within the subfamily Alphaherpesvirinae of the family Herpesviridae. IBRV is a double-stranded DNA virus, and its genome encodes 30-40 structural proteins. Among them, gB, gC, gD, and gE are four major glycoproteins present on the surface of BHV-1 viral particles. The gB protein is encoded by the UL27 gene. The gB protein is distributed on the surface of the viral particle and, during viral invasion of host cells, can effectively adsorb cell surface polysaccharides, thereby promoting viral recognition of host surface receptors. gB is genetically stable and not easily altered in BHV-1 and is the most conserved protein among herpesviruses. gB is also a major immunogen that triggers the body's cellular immune response. The gD protein is encoded by the US6 gene. gD mainly mediates the recognition and binding of the virus to host cell receptors; the receptors that bind to gD are mainly HveA, HveB, and HveC. Therefore, the gD protein is the main target protein for inducing the body to produce neutralizing antibodies, and its ability to induce cellular immunity is stronger than that of the gB protein. Thus, gB and gD are the main target proteins for BHV-1 vaccine development.

[0010] Once infected with IBRV, cattle will carry the virus for life and continuously shed it. Currently, in China, IBRV infection is mainly prevented using inactivated IBRV vaccines. However, existing inactivated vaccines have limitations such as a short immune cycle, requiring multiple immunizations. Inactivated vaccines also have limitations such as poor immunogenicity and inability to stimulate cellular immune responses. Therefore, inactivated vaccines cannot completely eliminate IBRV from the bodies of infected cattle.

[0011] Inactivated IBRV vaccines have drawbacks such as requiring multiple immunizations, poor immunogenicity, and inability to elicit cellular immune responses. Live attenuated IBRV vaccines can recombine with wild-type virus after immunization, leading to typical clinical symptoms in infected cattle, indicating lower safety. Furthermore, attenuated vaccines have limitations such as virus shedding and inability to effectively distinguish between wild-type virus infection and vaccine immunization. IBRV subunit vaccines, on the other hand, have limitations including large vaccination doses, high production costs, and inability to elicit cellular immune responses.

[0012] There is still a need in this field for vaccines against BVDV and / or IBRV. Summary of the Invention

[0013] Compared with traditional vaccines, mRNA vaccines have many advantages, including high efficacy, safety, short production cycle, and low cost. The approval of two mRNA vaccines, BNT162b2 and mRNA-1273, in 2020 effectively reduced the risk of human infection with COVID-19, demonstrating the advantages of mRNA technology in the development of vaccines for infectious diseases.

[0014] The inventors discovered that, compared to the reversion of virulence and effects on the fetus of BVDV live attenuated vaccines, the BVDV mRNA vaccine of this invention has higher safety. Furthermore, compared to inactivated vaccines and subunit vaccines that only elicit humoral immune responses, the BVDV mRNA vaccine of this invention can simultaneously elicit high levels of both humoral and cellular immune responses.

[0015] The inventors also discovered that the IBRV mRNA vaccine of this invention has high safety. Compared to inactivated vaccines, which can only elicit humoral immune responses, the IBRV mRNA vaccine of this invention can simultaneously elicit high levels of both humoral and cellular immune responses.

[0016] The inventors prepared BVDV mRNA vaccine and IBRV mRNA vaccine, and selected the most effective single vaccine to prepare BVDV-IBRV bivalent mRNA vaccine. This bivalent mRNA vaccine can stimulate cattle to produce high levels of BVDV and IBRV neutralizing antibodies, and the neutralizing antibody level is significantly higher than that of commercially available BVDV-IBRV bivalent inactivated vaccine.

[0017] In one aspect, a fusion protein is provided, comprising, from the N-terminus to the C-terminus, a signal peptide, an immunogenic protein, and optionally a molecular chaperone motif, wherein the immunogenic protein is included:

[0018] The signal peptide is bovine IL2 signal peptide, tPA signal peptide, bovine IL6 signal peptide, or bovine IgG heavy chain signal peptide.

[0019] The immunogenic protein comprises one or more of BVDV-1 E2 protein or its extracellular domain, BVDV-2 E2 protein or its extracellular domain, and BVDV-3 E2 protein or its extracellular domain, or...

[0020] The immunogenic protein comprises one or more of IBRV gB protein or its transmembrane demembrane region and IBRV gD protein or its extracellular domain;

[0021] The molecular chaperone portion is bovine IgG Fc protein, T4 Folden protein, or Ft protein.

[0022] In one embodiment, the immunogenic protein comprises two or three of the following: BVDV-1 E2 protein or its extracellular domain, BVDV-2 E2 protein or its extracellular domain, and BVDV-3 E2 protein or its extracellular domain.

[0023] In one embodiment, the immunogenic protein comprises two of the following: IBRV gB protein or its transmembrane demembrane region and IBRV gD protein or its extracellular domain.

[0024] In one embodiment, the bovine IL2 signal peptide comprises SEQ ID NO: 39 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39. In one embodiment, the tPA signal peptide comprises SEQ ID NO: 40 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40. In one embodiment, the bovine IgG heavy chain signal peptide comprises SEQ ID NO: 41 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41. In one embodiment, the bovine IL6 signal peptide comprises SEQ ID NO: 98 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 98.

[0025] In one embodiment, the BVDV-1 E2 protein comprises a sequence of SEQ ID NO: 110 or having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 110. In one embodiment, the extracellular domain of the BVDV-1E2 protein comprises a sequence of SEQ ID NO: 109 or having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 109. In one embodiment, the BVDV-2 E2 protein comprises SEQ ID NO: 112 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 112. In another embodiment, the extracellular domain of the BVDV-2 E2 protein comprises SEQ ID NO: 111 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 111. In one embodiment, the BVDV-3 E2 protein comprises a sequence of SEQ ID NO: 114 or having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 114. In one embodiment, the extracellular domain of the BVDV-3 E2 protein comprises a sequence of SEQ ID NO: 113 or having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 113. In one embodiment, the IBRV gB protein comprises SEQ ID NO: 117 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 117. In one embodiment, the transmembrane demembrane region of the IBRV gB protein comprises SEQ ID NO: 118 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 118.In one embodiment, the IBRV gD protein comprises SEQ ID NO: 115 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 115. In one embodiment, the extracellular region of the IBRV gD protein comprises SEQ ID NO: 116 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 116.

[0026] In one embodiment, the bovine IgG Fc protein comprises SEQ ID NO: 107 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 107. In one embodiment, the T4Folden protein comprises SEQ ID NO: 105 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 105. In one embodiment, the Ft protein comprises SEQ ID NO: 108 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 108.

[0027] In one implementation, the fusion protein has any of the following structures:

[0028] Signal peptide - [immunogenic protein] x ;

[0029] Signal peptide - [immunogenic protein - optional linker - molecular chaperone] x ;or

[0030] Signal peptide - [immunogenic protein] x -Optional connector-molecular chaperone;

[0031] The immunogenic protein is one of BVDV-1 E2 protein or its extracellular domain, BVDV-2 E2 protein or its extracellular domain, and BVDV-3 E2 protein or its extracellular domain.

[0032] Where x is 1, 2 or 3.

[0033] In one embodiment, when x is 2 or 3, the units within [...] are connected by a connector. In one embodiment, when x is 2 or 3, the types of immunogenic proteins can be the same or different. For example, signal peptide-[immunogenic protein]2 can refer to signal peptide-[immunogenic protein]-[immunogenic protein], and the two immunogenic proteins can each be independently selected from one of BVDV-1 E2 protein or its extracellular domain, BVDV-2 E2 protein or its extracellular domain, and BVDV-3 E2 protein or its extracellular domain, for example, they can include signal peptide-BVDV-1 E2 protein-BVDV-1 E2 protein, signal peptide-BVDV-1 E2 protein-BVDV-2 E2 protein, signal peptide-BVDV-1 E2 protein-BVDV-3 E2 protein, etc.

[0034] In one implementation, the connector is (GGGGS). n n = 1-6, preferably 2, 3 or 4, more preferably 3. In one embodiment, the linker is a self-cleaving peptide, preferably a P2A, T2A, E2A or F2A self-cleaving peptide.

[0035] In one embodiment, the fusion protein comprises any one of the following structures:

[0036] IL2sp-BVDV-1 E2 extracellular domain;

[0037] IL2sp-BVDV-1 E2 extracellular domain-adaptor-bovine IgG Fc;

[0038] IL2sp-BVDV-1 E2 extracellular domain-adaptor-Ft protein;

[0039] IL2sp-BVDV-1 E2 protein;

[0040] IL2sp-BVDV-1 E2 protein-linker-bovine IgG Fc;

[0041] IL2sp-BVDV-1 E2 protein-adaptor-Ft protein;

[0042] IL2sp-BVDV-2 E2 extracellular domain;

[0043] IL2sp-BVDV-2 E2 extracellular domain-adaptor-bovine IgG Fc;

[0044] IL2sp-BVDV-2 E2 extracellular domain-connector-Ft;

[0045] IL2sp-BVDV-2 E2 protein;

[0046] IL2sp-BVDV-2 E2 protein-linker-bovine IgG Fc;

[0047] IL2sp-BVDV-2 E2 protein-adaptor-Ft protein;

[0048] IL2sp-BVDV-3 E2 extracellular domain;

[0049] IL2sp-BVDV-3 E2 extracellular domain-connector-bovine IgG Fc;

[0050] IL2sp-BVDV-3 E2 extracellular domain-adaptor-Ft protein;

[0051] IL2sp-BVDV-3 E2 protein;

[0052] IL2sp-BVDV-3 E2 protein-linker-bovine IgG Fc;

[0053] IL2sp-BVDV-3 E2 protein-adaptor-Ft protein;

[0054] IL2sp-BVDV-1 E2 extracellular domain-adaptor-BVDV-2 E2 extracellular domain-adaptor-Ft protein;

[0055] IL2sp-BVDV-2 E2 extracellular domain-adaptor-BVDV-1 E2 extracellular domain-adaptor-Ft protein;

[0056] IL2sp-BVDV-1 E2 extracellular domain-adaptor-BVDV-2 E2 extracellular domain-adaptor-BVDV-3 E2 extracellular domain-adaptor-Ft protein;

[0057] IL2sp-BVDV-1 E2 extracellular domain-adaptor-Ft-P2A-BVDV-2 E2 extracellular domain-adaptor-Ft protein;

[0058] IL2sp-BVDV-2 E2 extracellular domain-adaptor-Ft-P2A-BVDV-1 E2 extracellular domain-adaptor-Ft protein;

[0059] IL2sp-BVDV-1 E2 extracellular domain-adaptor-Ft-P2A-BVDV-2 E2 extracellular domain-adaptor-Ft-P2A-BVDV-3 E2 extracellular domain-adaptor-Ft protein.

[0060] In one implementation, the fusion protein has any of the following structures:

[0061] Signal peptides are immunogenic proteins.

[0062] Signal peptide - immunogenic protein - optional adapter - immunogenic protein;

[0063] The immunogenic protein is one or more of IBRV gB protein or its transmembrane demembrane region and IBRV gD protein or its extracellular domain.

[0064] In one implementation, the connector is (GGGGS). n n = 1-6, preferably 2, 3 or 4, more preferably 3; or the linker is a self-cleaving peptide, preferably P2A, T2A, E2A or F2A self-cleaving peptide.

[0065] In one implementation, the fusion protein has any of the following structures:

[0066] IL2sp-IBRV gB demembrane region;

[0067] IL2sp-IBRV gB protein;

[0068] IL2sp-IBRV gD extracellular domain;

[0069] IL2sp-IBRV gD protein;

[0070] IL2sp-IBRV gD extracellular domain-P2A-IBRV gB transmembrane demembrane region;

[0071] IL2sp-IBRV gD protein-P2A-IBRV gB protein;

[0072] IL2sp-IBRV gB demembrane region-P2A-IBRV gD extracellular domain;

[0073] IL2sp-IBRV gB protein-P2A-IBRV gD protein;

[0074] IL2sp-IBRV gD extracellular domain-connector-IBRV gB transmembrane demembrane region;

[0075] IL2sp-IBRV gB demembrane region-connector-IBRV gD extracellular domain;

[0076] IL2sp-IBRV gD protein-linker-IBRV gB protein;

[0077] IL2sp-IBRV gB protein-connector-IBRV gD protein.

[0078] In one embodiment, the fusion protein comprises the amino acid sequence of any one of SEQ ID NO: 1-36 and 135-136 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 1-36 and 135-136.

[0079] In another aspect, an isolated nucleic acid is provided that encodes a fusion protein as described herein.

[0080] In one implementation, the nucleic acid is a single-stranded or double-stranded DNA molecule or RNA molecule.

[0081] In one implementation, the RNA molecule is an mRNA molecule.

[0082] In one implementation, the mRNA includes one or more of the following elements: a 5' cap, a 5' UTR, a 3' URT, and a Poly-A tail.

[0083] In one embodiment, the 5' cap comprises a compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer, or isotopic variant thereof:

[0084] in,

[0085] --- indicates a single key or none.

[0086] X1 is selected from O, S, CH2, CH2CH2, CH=CH, CH=CHO, CH2O, OCH2, CH2CH2O, OCH2CH2, and tricyclic alkyl groups.

[0087] R1, R2, R3, and R4 are independently halogenated, OH-, unsubstituted, or OC-substituted, respectively. 1-3 Alkyl-substituted OC 1-3 Alkyl, unsubstituted or OC 1-3 Alkyl-substituted OC 1-3 alkyl,

[0088] B1 and B2 are each independently selected from natural, modified, or non-natural nucleoside bases.

[0089] More preferably, the compound of formula (I) is

[0090] In one embodiment, the 5'UTR comprises a polynucleotide sequence selected from any one of SEQ ID NO: 83, 85, 87, 89, 91 or 93, or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a polynucleotide sequence of any one of SEQ ID NO: 83, 85, 87, 89, 91 or 93.

[0091] In one embodiment, 3'URT comprises a polynucleotide sequence selected from any one of SEQ ID NO: 84, 86, 88, 90, 92 or 94, or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a polynucleotide sequence of any one of SEQ ID NO: 84, 86, 88, 90, 92 or 94.

[0092] In one embodiment, the nucleic acid comprises a polynucleotide sequence or a degenerate sequence variant thereof of any one of SEQ ID NO: 42-77, 121-130 and 132-133.

[0093] In one embodiment, the nucleic acid is an mRNA molecule, wherein the uracil, cytosine, adenine, or guanine nucleotide of the mRNA molecule contains a modifying group, the modifying group including at least one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 5-methylcytosine, 5-methoxycytosine, N1-methylcytosine, 2-thiouridine, 5-methoxyuridine, or N1-methyladenosine, N1-methylguanine, N1-methylguanine, and isoguanine.

[0094] In one embodiment, the mRNA molecule is a modified mRNA, the modification including the conversion of uridine nucleoside to pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, or 5-methoxyuridine; and / or, the conversion of cytosine nucleoside to 5-methylcytosine, 5-methoxycytosine, or N1-methylcytosine; and / or, the conversion of adenine nucleoside to N1-methyladenosine; and / or, the conversion of adenine nucleoside to N1-methylguanine, N1-methylguanine, or isoguanine.

[0095] In another aspect, a vector containing the nucleic acid described herein is provided.

[0096] In another aspect, cells are provided that contain the fusion proteins, nucleic acids, or vectors described herein.

[0097] In another aspect, compositions are provided that comprise a fusion protein, nucleic acid, or vector. In one embodiment, the composition comprises a first fusion protein and a second fusion protein, the first fusion protein being the fusion protein described above for BVDV and the second fusion protein being the fusion protein described above for IBRV.

[0098] In another aspect, vaccine compositions are provided that comprise one or more mRNA molecules as described herein.

[0099] In one embodiment, the vaccine composition is a BVDV-IBRV bivalent mRNA vaccine composition.

[0100] In one embodiment, the vaccine composition comprises an mRNA molecule against BVDV and an mRNA molecule against IBRV, wherein the mRNA molecule against BVDV comprises one or more of SEQ ID NO.42-SEQ ID NO.65 and 135, and the mRNA molecule against IBRV comprises one or more of SEQ ID NO.66-SEQ ID NO.77 and 136.

[0101] In one embodiment, the vaccine composition comprises mRNA molecules against BVDV and mRNA molecules against IBRV in a ratio of 1:100 to 100:1.

[0102] In one embodiment, the vaccine composition comprises lipid nanoparticles carrying mRNA molecules.

[0103] In one embodiment, the vaccine composition comprises an adjuvant.

[0104] In one embodiment, the adjuvant is a molecular adjuvant or a nucleic acid encoding the molecular adjuvant, preferably mRNA.

[0105] In one embodiment, the molecular adjuvant is IL-2, GM-CSF, CD40L, or a combination or fusion thereof.

[0106] In one embodiment, IL-2 comprises the amino acid sequence of SEQ ID NO: 95 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 95.

[0107] In one embodiment, GM-CSF comprises the amino acid sequence of SEQ ID NO: 96 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 96.

[0108] In one embodiment, CD40L comprises the amino acid sequence of SEQ ID NO: 97 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 97.

[0109] In one embodiment, the fusion is a fusion of two or three of IL-2, GM-CSF and CD40L linked by a linker. Preferably, the linker is a self-cleaving peptide, preferably a P2A, T2A, E2A or F2A self-cleaving peptide.

[0110] In one embodiment, the adjuvant is a molecular adjuvant comprising an amino acid sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 37, 38, 95-97, or 137, or a nucleic acid, preferably mRNA, encoding the molecular adjuvant.

[0111] In another aspect, the use of the fusion proteins, nucleic acids, vectors, compositions or vaccine compositions of this article in the preparation of medicaments for the treatment or prevention of bovine viral diarrhea virus infection and / or infectious bovine rhinobronchitis virus infection, or for the treatment or prevention of bovine viral diarrhea-mucosal disease and / or bovine rhinobronchitis.

[0112] In another aspect, methods are provided for treating or preventing bovine viral diarrhea virus infection and / or infectious bovine rhinobronchitis virus infection, or for treating or preventing bovine viral diarrhea-mucosal disease and / or bovine rhinobronchitis, comprising administering the nucleic acid or vaccine composition described herein to a mammal. In one embodiment, the mammal is a ruminant, such as a cow.

[0113] In another aspect, methods for stimulating an immune response in a subject are provided, comprising administering the nucleic acid or vaccine composition described herein to the subject. The immune response includes cellular immune responses and / or humoral immune responses. The subject may be a mammal, preferably a ruminant, such as a cow.

[0114] The advantages of this invention include:

[0115] 1. Compared to the reversion of virulence and the impact on the fetus of BVDV live attenuated vaccines, the BVDV mRNA vaccine of this invention has higher safety. Furthermore, compared to inactivated vaccines and subunit vaccines that only elicit humoral immune responses, the BVDV mRNA vaccine of this invention can simultaneously elicit high levels of both humoral and cellular immune responses.

[0116] 2. Compared to inactivated vaccines, which can only elicit humoral immune responses, the IBRV mRNA vaccine of the present invention can simultaneously elicit high levels of humoral and cellular immune responses.

[0117] 3. The bivalent mRNA vaccine of the present invention can stimulate cattle to produce high levels of neutralizing antibodies against BVDV and IBRV, and the level of neutralizing antibodies is significantly higher than that of commercially available BVDV-IBRV bivalent inactivated vaccines.

[0118] 4. This invention discovers that Ft protein can be applied in the field of mRNA vaccines. Ft protein also plays a significant role in cellular immunity, with effects superior to other molecular chaperones.

[0119] 5. This invention has verified that IBRV mRNA vaccines (e.g., IBRV-3 or IBRV-4) with the structural scheme of "gD protein-P2A-gB protein" have the best immunogenicity. Detailed Implementation

[0120] The following definitions are provided to enable those skilled in the art to understand the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the invention, preferred materials and methods are described herein. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0121] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, terms used herein include those in Janeway CA Jr, Travers P, Walport M, et al., *Immunobiology*, 5th edition, New York: Garland Science (2001), and “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, HGW, Nagel, B. and The definition is given in H. (ed., 1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0122] The term "fusion protein" refers to a protein formed by covalently linking two protein motifs that do not exist together under natural conditions. In this document, a fusion protein may comprise one or more selected from BVDV-1 E2 protein or its extracellular domain, BVDV-2 E2 protein or its extracellular domain, and BVDV-3 E2 protein or its extracellular domain. Alternatively, a fusion protein may comprise one or more selected from IBRV gB protein or its transmembrane demembrane region and IBRV gD protein or its extracellular domain. These proteins are directly linked or linked via suitable linkers. The choice of linker is conventional to those skilled in the art. For example, a linker may comprise, but is not limited to, (GGGGS)n or (G)n, where n is greater than or equal to 1, for example, 2-10 (e.g., 3, 4, 5, 6, 7, 8, 9). The linker may be a self-cleaving peptide, preferably a P2A, T2A, E2A, or F2A self-cleaving peptide.

[0123] The expression "optional" means that the component may or may not be present. For example, "optional connector" can mean that a connector may or may not be present.

[0124] The term "immunogenic protein" refers to proteins that can induce an immune response in the immune system. These proteins can be recognized by the immune system as foreign antigens, thereby activating immune cells and producing antibodies. Immunogenic proteins play an important role in vaccine development and immunotherapy. In some embodiments, immunogenic proteins include one or more of BVDV-1 E2 protein or its extracellular domain, BVDV-2 E2 protein or its extracellular domain, and BVDV-3 E2 protein or its extracellular domain. In some embodiments, immunogenic proteins include one or more of IBRV gB protein or its transmembrane demembrane region and IBRV gD protein or its extracellular domain.

[0125] The term "secretion signal peptide" or "signal peptide" refers to a peptide used to guide the translocation of a synthesized fusion protein into the secretory pathway. Signal peptides are generally essential for transmembrane translocation in the secretory pathway and thus universally control the entry of most proteins into the secretory pathway in eukaryotes and prokaryotes. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) guides ribosomes to the rough endoplasmic reticulum (ER) membrane and triggers the transport of growing peptide chains across this membrane for processing. ER processing produces mature proteins, in which the signal peptide is typically cleaved from the precursor protein by ER-resident signal peptidases of the host cell, or remains unclew and acts as a membrane anchor. Signal peptides can also promote protein targeting to the cell membrane. Secretion signal peptides can be located at the N-terminus of the fusion protein. The type of secretion signal peptide is not particularly limited, as long as it can guide the secretion of the synthesized fusion protein. Secretion signal peptides can include, but are not limited to, bovine IL2 signal peptide, tPA signal peptide, bovine IL6 signal peptide, or bovine IgG heavy chain signal peptide.

[0126] The terms “nucleic acid,” “polynucleotide,” and “polynucleotide sequence” are used interchangeably to refer to oligomers and polymers of any length that are essentially composed of nucleotides (such as deoxyribonucleotides and / or ribonucleotides). Nucleic acids can contain purine and / or pyrimidine bases and / or other natural (e.g., xanthine, inosine, hypoxanthine), chemically or biochemically modified (e.g., methylated), non-natural, or derived nucleotide bases. The backbone of a nucleic acid can contain sugar and phosphate groups normally present in RNA or DNA, and / or one or more modified or substituted sugars and / or one or more modified or substituted phosphate groups. Modifications to phosphate groups or sugars can be introduced to improve stability, resistance to enzymatic degradation, or some other useful properties. A “nucleic acid” can be, for example, double-stranded, partially double-stranded, or single-stranded. When single-stranded, a nucleic acid can be a sense strand or an antisense strand. A “nucleic acid” can be circular or linear. As used herein, the term “nucleic acid” encompasses DNA and RNA, including genomes, pre-mRNA, mRNA, cDNA, and recombinant or synthetic nucleic acids containing vectors. For the purposes described herein, it should be understood that polynucleotides can be modified by any method available in the art.

[0127] The term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same positions when aligned. Such calculations are typically performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984). Furthermore, in determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called "conserved" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, having little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art.

[0128] The term “identity” when used in conjunction with nucleic acids or fragments thereof means that, when an optimized alignment is performed with other nucleic acids (or their complementary strands), at least 50%, 60%, 70%, 80%, 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases have nucleotide sequence identity, as determined by any sequence identity algorithm well known in the art (such as FASTA, BLAST, or GAP) discussed below.

[0129] A read frame (ORF) is a continuous extension of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). ORFs typically encode proteins. In this paper, a read frame may encode a fusion protein.

[0130] The term "5' untranslated region" (UTR) refers to an mRNA region located directly upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript translated by the ribosome) and that does not encode a protein or peptide. When an RNA transcript is generated, the 5' UTR may contain promoter sequences. These promoter sequences are known in the art. It should be understood that these promoter sequences will not be present in the mRNA described herein.

[0131] The term "3' untranslated region" (UTR) refers to an mRNA region located directly downstream (i.e., 3') of a stop codon (i.e., the codon that transmits the translation termination signal in the mRNA transcript) and that does not encode a protein or peptide.

[0132] The term "Poly-A tail" refers to an mRNA region containing multiple consecutive adenosine monophosphates (ATPs) located downstream of the 3' UR. For example, a Poly-A tail can be located directly downstream of the 3' UR (i.e., at the 3'). A Poly-A tail can contain 10 to 300 ATPs, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ATPs. A Poly-A tail can protect mRNA, for example, from enzymatic degradation in the cytoplasm, and facilitate transcription termination and / or the export of mRNA from the nucleus for translation.

[0133] The term "ionizable lipid" refers to an amphiphilic molecule (e.g., lipid or lipidoid, such as synthetic lipid or lipidoid) containing a group (e.g., a head group) that can ionize, for example, dissociate under given conditions (e.g., pH) to produce one or more charged substances.

[0134] The term "pharmaceutically acceptable" means a molecule or composition that, when administered to a recipient, is harmless to the recipient or provides a benefit to the recipient that outweighs any adverse effects. Regarding carriers or excipients used to formulate compositions as disclosed herein, a pharmaceutically acceptable carrier or excipient must be compatible with the other components of the composition and be harmless to the recipient, or provide a benefit to the recipient that outweighs any adverse effects. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate, or zinc or stearic acid), or solvent encapsulating material, which participates in carrying or transporting a pharmaceutical agent from one part of the body to another (e.g., from one organ to another). Each carrier must be "acceptable" in the sense that it is compatible with the other components of the formulation and is not harmful to the patient.

[0135] Fusion protein

[0136] In this document, the fusion protein can be a fusion protein involving BVDV protein or a fusion protein involving IBRV protein. A fusion protein involving BVDV protein may comprise one or more selected from BVDV-1 E2 protein or its extracellular domain, BVDV-2 E2 protein or its extracellular domain, and BVDV-3 E2 protein or its extracellular domain. A fusion protein involving IBRV protein may comprise one or more selected from IBRV gB protein or its transmembrane demembrane region and IBRV gD protein or its extracellular domain. For the constituent proteins of the fusion protein, their amino acid sequences may contain mutations (e.g., deletions, additions, substitutions, or insertions), as long as the immunogenicity of the constituent proteins is preserved. The BVDV-1 E2 protein may comprise SEQ ID NO: 110 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 110. The extracellular domain of the BVDV-1 E2 protein may contain a sequence of SEQ ID NO: 109 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 109. The BVDV-2 E2 protein may contain a sequence of SEQ ID NO: 112 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 112. The extracellular domain of the BVDV-2 E2 protein may contain a sequence of SEQ ID NO: 111 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 111. The BVDV-3 E2 protein may contain a sequence of SEQ ID NO: 114 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 114. The extracellular domain of the BVDV-3 E2 protein may contain a sequence of SEQ ID NO: 113 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 113. The IBRV gB protein may contain a sequence of SEQ ID NO: 117 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 117.The transmembrane demembrane region of the IBRV gB protein may contain a sequence of SEQ ID NO: 118 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 118. The IBRV gD protein may contain a sequence of SEQ ID NO: 115 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 115. The extracellular region of the IBRV gD protein may contain SEQ ID NO: 116 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 116.

[0137] Fusion proteins involving BVDV proteins can comprise two or three of the following: BVDV-1 E2 protein or its extracellular domain, BVDV-2 E2 protein or its extracellular domain, and BVDV-3 E2 protein or its extracellular domain. These two or three portions can be linked by a suitable linker. For example, the linker is (GGGGS). n n = 1-6, preferably 2, 3 or 4, more preferably 3. Alternatively, the linker can also be a self-cleaving peptide, preferably a P2A, T2A, E2A or F2A self-cleaving peptide.

[0138] Fusion proteins involving IBRV proteins can comprise the IBRV gB protein or its transmembrane demembrane region and the IBRV gD protein or its extracellular domain, which can be linked by any suitable linker. For example, the linker is (GGGGS). n n = 1-6, preferably 2, 3 or 4, more preferably 3. Alternatively, the linker can also be a self-cleaving peptide, preferably a P2A, T2A, E2A or F2A self-cleaving peptide.

[0139] In addition to immunogenic proteins, fusion proteins may also contain molecular chaperones. The molecular chaperone portion may be bovine IgG Fc protein, T4 Folden protein, or Ft protein. Bovine IgG Fc protein may contain a sequence of SEQ ID NO: 107 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 107. T4 Folden protein may contain a sequence of SEQ ID NO: 105 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 105. The Ft protein may contain a sequence of SEQ ID NO: 108 or having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 108. The molecular chaperone may be located at any position within the fusion protein. Those skilled in the art can appropriately place the molecular chaperone within the fusion protein. Preferably, the molecular chaperone may be located at the C-terminus of the immunogenic protein. In some embodiments, each immunogenic protein is linked to one molecular chaperone. In some embodiments, all immunogenic proteins are linked to one molecular chaperone. In some embodiments, the immunogenic proteins and the molecular chaperone are linked via a connector. The molecular chaperone portion may be bovine IgG Fc protein, T4 Folden protein, or Ft protein. Studies have shown that appropriate surface modifications can enable nanoparticles to stably reside in the circulatory system. Ferritin (Ft) is characterized by its large surface area and hollow spherical structure. It can self-assemble with various expressed fusion proteins to form nanoparticles of 24 identical polypeptides, enhancing antigen stability. This allows for effective capture by dendritic cells and macrophages, promoting antigen recognition and uptake by antigen-presenting cells, thereby inducing a strong immune response. Ferritin has wide applications in vaccine development; fusion expression of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein with ferritin can produce potent neutralizing antibodies. Ft is currently used in subunit vaccines, but its application in mRNA vaccines has not yet been reported. This invention also demonstrates that Ft has a significant role in cellular immunity (see Example 6 for details).

[0140] IgG Fc interacts with FcRn on the surface of immune cells, participating in the transport of IgG across mucosal surfaces and enhancing the immunogenicity of fc fusion proteins in mammals. Proteins fused with IgG Fc can form stable dimers through disulfide bonds in the Fc hinge region, increasing the half-life and stability of recombinant proteins, thereby improving cellular, mucosal, and humoral immune responses. Novel IgG Fc-mediated vaccines have been successfully applied to influenza A virus (HA-HuFc) and classical swine fever virus (CSFV-E2Fc).

[0141] Fusion proteins may also contain signal peptides. Signal peptides are typically located at the N-terminus of the fusion protein. When a fusion protein contains two or more immunogenic moieties, it may also contain two or more signal peptides. For example, each immunogenic protein may contain one signal peptide. Secretory signal peptides may include, but are not limited to, bovine IL2 signal peptide, tPA signal peptide, bovine IL6 signal peptide, or bovine IgG heavy chain signal peptide. The bovine IL2 signal peptide may contain a sequence of SEQ ID NO: 39 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39. The tPA signal peptide may contain a sequence of SEQ ID NO: 40 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40. The bovine IgG heavy chain signal peptide may comprise SEQ ID NO: 41 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41. The bovine IL6 signal peptide comprises SEQ ID NO: 98 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 98.

[0142] The fusion protein may also include a tag protein (V5 tag protein and / or Myc tag protein) and / or a Qα amino acid sequence at its N-terminus and / or C-terminus. The tag protein (V5 tag protein and / or Myc tag protein) and / or Qα amino acid sequence can be directly linked. The insertion positions of the tag protein (V5 tag protein and / or Myc tag protein) and / or Qα amino acid sequence are known to those skilled in the art. For example, the Qα amino acid sequence can be directly linked to the N-terminus of the immunogenic portion of the fusion protein (e.g., the illustrative amino acid sequence below). The tag protein can be directly linked to the Qα amino acid sequence. The Qα amino acid sequence may comprise SEQ ID NO: 103 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0143] Exemplary amino acid sequences (in the sequences below, the function of each sequence is indicated in parentheses after each sequence segment)

[0144] SEQ ID NO: 1IL2sp-BVDV-1 E2 extracellular domain amino acid sequence

[0145] SEQ ID NO: 2IL2sp-BVDV-1 E2 extracellular domain + bovine IgG Fc amino acid sequence

[0146] SEQ ID NO: 3IL2sp-BVDV-1 E2 extracellular domain + Ft amino acid sequence

[0147] SEQ ID NO: 4IL2sp-BVDV-1 E2 full-length amino acid sequence

[0148] SEQ ID NO: 5IL2sp-BVDV-1 E2 full-length + bovine IgG Fc amino acid sequence

[0149] SEQ ID NO: 6IL2sp-BVDV-1 E2 full-length + Ft amino acid sequence

[0150] SEQ ID NO: 7IL2sp-BVDV-2 E2 extracellular domain amino acid sequence

[0151] SEQ ID NO: 8IL2sp-BVDV-2 E2 extracellular domain + bovine IgG Fc amino acid sequence

[0152] SEQ ID NO: 9IL2sp-BVDV-2 E2 extracellular domain + Ft amino acid sequence

[0153] SEQ ID NO: 10IL2sp-BVDV-2 E2 full-length amino acid sequence

[0154] SEQ ID NO: 11IL2sp-BVDV-2 E2 full-length + bovine IgG Fc amino acid sequence

[0155] SEQ ID NO: 12IL2sp-BVDV-2 E2 full-length + Ft amino acid sequence

[0156] SEQ ID NO: 13IL2sp-BVDV-3 E2 extracellular domain amino acid sequence

[0157] SEQ ID NO: 14IL2sp-BVDV-3 E2 extracellular domain + bovine IgG Fc amino acid sequence

[0158] SEQ ID NO: 15IL2sp-BVDV-3 E2 extracellular domain + Ft amino acid sequence

[0159] SEQ ID NO: 16IL2sp-BVDV-3 E2 full-length amino acid sequence

[0160] SEQ ID NO: 17IL2sp-BVDV-3 E2 full-length + bovine IgG Fc amino acid sequence

[0161] SEQ ID NO: 18IL2sp-BVDV-3 E2 full-length + Ft amino acid sequence

[0162] SEQ ID NO: 19IL2sp-BVDV-1 E2 extracellular domain + BVDV-2 E2 extracellular domain + Ft amino acid sequence

[0163] SEQ ID NO: 20IL2sp-BVDV-2 E2 extracellular domain + BVDV-1 E2 extracellular domain + Ft amino acid sequence

[0164] SEQ ID NO: 21IL2sp-BVDV-1 E2 extracellular domain + BVDV-2 E2 extracellular domain + BVDV-3 E2 extracellular domain + Ft amino acid sequence

[0165] SEQ ID NO: 22IL2sp-BVDV-1 E2 extracellular domain + Ft-P2A-IL2sp-BVDV-2 E2 extracellular domain + Ft amino acid sequence

[0166] SEQ ID NO: 23IL2sp-BVDV-2 E2 extracellular domain + Ft-P2A-IL2sp-BVDV-1 E2 extracellular domain + Ft amino acid sequence

[0167] SEQ ID NO: 24IL2sp-BVDV-1 E2 extracellular domain + Ft-P2A-IL2sp-BVDV-2 E2 extracellular domain + Ft-P2A-IL2sp-BVDV-3 E2 extracellular domain + Ft amino acid sequence

[0168] SEQ ID NO: 25IL2sp-IBRV gB transmembrane demembrane amino acid sequence

[0169] SEQ ID NO: 26IL2sp-IBRV gB full-length amino acid sequence

[0170] SEQ ID NO: 27IL2sp-IBRV gD extracellular domain amino acid sequence

[0171] SEQ ID NO: 28IL2sp-IBRV gD full-length amino acid sequence

[0172] SEQ ID NO: 29IL2sp-IBRV gD extracellular domain-P2A-IBRV gB transmembrane demembrane amino acid sequence

[0173] SEQ ID NO: 30IL2sp-IBRV gD full-length-P2A-IBRV gB full-length amino acid sequence

[0174] SEQ ID NO: 31IL2sp-IBRV gB transmembrane region-P2A-IBRV gD extracellular domain amino acid sequence

[0175] SEQ ID NO: 32IL2sp-IBRV gB full-length-P2A-IBRV gD full-length amino acid sequence

[0176] SEQ ID NO: 33IL2sp-IBRV gD extracellular domain + IBRV gB transmembrane demembrane amino acid sequence

[0177] SEQ ID NO: 34IL2sp-IBRV gB transmembrane region +IBRV gD extracellular domain amino acid sequence

[0178] SEQ ID NO: 35IL2sp-IBRV gD full-length + IBRV gB full-length amino acid sequence

[0179] SEQ ID NO: 36IL2sp-IBRV gB full-length + IBRV gD full-length amino acid sequence

[0180] SEQ ID NO: 135BVD-3 complete amino acid sequence

[0181] SEQ ID NO: 136IBRV-3 complete amino acid sequence

[0182] Nucleic acid

[0183] This article provides isolated nucleic acids, which are DNA or RNA molecules and may be double-stranded, single-stranded, or partially double-stranded. The isolated nucleic acid encodes the fusion protein described herein. The isolated nucleic acid may contain elements that regulate the expression of the fusion protein, such as enhancer, promoter, and / or terminator sequences. These sequences may be modified or unmodified. Elements regulating the expression of the fusion protein may also be absent from the isolated nucleic acid. The nucleic acid described herein may encode a fusion protein involving BVDV protein or a fusion protein involving IBRV protein.

[0184] The nucleic acids of particular interest in this invention are mRNA molecules, such as those containing a read frame encoding an immunogenic protein (from BVDV or from IBRV). The mRNA molecules described herein can be mRNA molecules involving BVDV or mRNA molecules involving IBRV proteins.

[0185] mRNA molecules

[0186] Messenger RNA (mRNA) is any RNA that encodes a protein and can be translated to produce the protein-encoded RNA in vitro, in vivo, in situ, or ex vivo. Unless otherwise stated, the nucleic acid sequences in this application may be described as “T” in a representative DNA sequence, but in the case of sequences representing RNA (e.g., mRNA), “T” will be replaced by “U”. Therefore, any DNA indicated herein by sequence number also discloses a corresponding RNA (e.g., mRNA) sequence complementary to the DNA, wherein each “T” in the DNA sequence is replaced by a “U”.

[0187] mRNA molecules can be synthetic and modified. mRNA can be chemically modified. mRNA molecules can be chemically synthesized or transcribed in vitro. mRNA molecules can be placed on a vector. The vector can be a viral vector, a bacterial vector, or a eukaryotic expression vector. In one embodiment, the vector is a plasmid. In some instances, mRNA molecules can be delivered to cells via transfection, electroporation, or transduction (e.g., adenovirus or lentivirus transduction).

[0188] Exemplary mRNA molecules

[0189] SEQ ID NO: 132BVD-3 complete nucleotide sequence

[0190] SEQ ID NO: 133IBRV-3 complete nucleotide sequence

[0191] Chemical modification

[0192] In some embodiments, the nucleic acid (e.g., mRNA) comprises RNA having a read frame encoding an immunogenic protein, wherein the nucleic acid comprises nucleotides and / or nucleosides that may be standard (unmodified) or modified as known in the art. In some embodiments, the nucleotides and nucleosides of the nucleic acid (e.g., mRNA) comprise modified nucleotides or nucleosides. These modified nucleotides and nucleosides may be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. These modifications may include modifications at the sugar, backbone, or nucleobase portions of the nucleotides and / or nucleosides as recognized in the art.

[0193] In some implementations, the nucleic acid (e.g., mRNA) may comprise standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, non-naturally occurring nucleotides and nucleosides, or any combination thereof.

[0194] In some embodiments, nucleic acids (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) comprise different types of standard and / or modified nucleotides and nucleosides. In some embodiments, specific regions of the nucleic acid contain one, two, or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0195] In some implementations, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms exhibit reduced degradation in cells or organisms, relative to unmodified nucleic acids containing standard nucleotides and nucleosides.

[0196] In some implementations, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms may exhibit reduced immunogenicity (e.g., reduced innate response) in cells or organisms, relative to unmodified nucleic acids containing standard nucleotides and nucleosides.

[0197] In some implementations, nucleic acids (e.g., mRNA) comprise non-natural modified nucleotides introduced during or after nucleic acid synthesis to achieve a desired function or property. Modifications can be present at nucleotide linkages, purine or pyrimidine bases, or sugars. Modifications can be introduced chemically or at any other location at the end of the chain or in the chain using polymerases. Any region of the nucleic acid can be chemically modified.

[0198] Nucleic acids (e.g., mRNA) can contain modified nucleosides and nucleotides. A “nucleoside” is a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof combined with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate ester group. Modified nucleotides can be synthesized by any useful method, such as chemical, enzymatic, or recombinant methods, to include one or more modified or non-natural nucleosides. Nucleic acids can contain one or more linked nucleoside regions. These regions can have variable backbone linkages. The linkage can be a standard phosphodiester linkage, in which case the nucleic acid will contain the nucleotide region.

[0199] Modified nucleotide base pairings encompass not only standard adenosylthymine, adenosyluracil, or guanosine cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides containing non-standard or modified bases. In nucleic acids, for example, those with at least one chemical modification, the arrangement of hydrogen bond donors and acceptors allows hydrogen bonding to occur between non-standard and standard bases or between two complementary non-standard base structures. An example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil.

[0200] In some embodiments, the nucleic acid (e.g., mRNA) contains uridine at one or more or all uridine sites. In some embodiments, the mRNA is uniformly modified (e.g., completely modified, modified throughout the entire sequence) for a specific modification. In some embodiments, the nucleic acid can be uniformly modified with methylpseuuridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methylpseuuridine. Similarly, the nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacing it with modified residues (e.g., the modified residues described above).

[0201] In some embodiments, the RNA is modified RNA, wherein the uracil, cytosine, adenine, or guanine nucleotide contains a modifying group. The modifying group may be selected from at least one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 5-methylcytosine, 5-methoxycytosine, N1-methylcytosine, 2-thiouridine, 5-methoxyuridine, or N1-methyladenosine, N1-methylguanine, N1-methylguanine, and isoguanine.

[0202] In some embodiments, the mRNA molecule is a modified mRNA, the modification including the conversion of uracil nucleoside to pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 5-methoxyuridine; and / or, the conversion of cytosine nucleoside to 5-methylcytosine, 5-methoxycytosine, N1-methylcytosine; and / or, the conversion of adenine nucleoside to N1-methyladenosine; and / or, the conversion of adenine nucleoside to N1-methylguanine, N1-methylguanine, isoguanine.

[0203] Typical mRNA sequence

[0204] An mRNA molecule is provided comprising a polynucleotide sequence comprising any one of SEQ ID NO: 42-77, 121-130, and 132-133, or any one or more degenerate sequence variants thereof. Those skilled in the art will understand that this invention covers variant sequences of SEQ ID NO: 42-77, 121-130, and 132-133. For example, the reading frame encoding an immunogenic protein in the mRNA molecule can vary. For example, the reading frame of an immunogenic protein can vary considerably based on codon degeneracy. A degenerate sequence variant refers to a variant nucleotide sequence obtained by altering the nucleotide sequence while retaining the amino acid sequence of the encoded protein. Similarly, those skilled in the art can make appropriate modifications to other elements while retaining the original function.

[0205] mRNA molecule preparation methods

[0206] Methods for preparing and purifying mRNA molecules are known and disclosed in the art. mRNA molecules can be prepared using only in vitro transcription (IVT) enzyme synthesis. Methods for preparing IVT polynucleotides are known in the art and described in WO 2013 / 151666, WO 2013 / 151668, etc. Purification methods include purifying RNA transcripts including polyA tails by contacting a sample with a surface linked to a plurality of thymidines or their derivatives and / or a plurality of uracils or their derivatives (polyT / U) under conditions that allow RNA transcripts to bind to said surface, and eluting the purified RNA transcripts from said surface (WO 2014 / 152031); using ion (e.g., anion) exchange chromatography, which allows for the separation of longer RNAs up to 10,000 nucleotides in length via a scalable method (WO 2014 / 144767); and subjecting the modified mRNA sample to DNase treatment (WO 2014 / 152030).

[0207] In some embodiments, a method for preparing mRNA molecules is provided, comprising (1) transcribing a downstream DNA sequence of a promoter using an RNA polymerase to synthesize mRNA, using linear double-stranded DNA containing a promoter sequence as a template and ATP, GTP, CTP, or N1-Me-pUTP as substrates; and (2) capping the synthesized mRNA using a one-step chemical method with a capping analogue CAP m7Gppp(2'OMeA)pG or CAP5 m7G(5')vppp(5')(2'OMeA)pG. In some embodiments, the promoter is a T7 promoter. In some embodiments, the RNA polymerase is a T7 RNA polymerase.

[0208] During mRNA processing, characteristic structural features of mature mRNA, such as the 5' cap and Poly-A tail, are typically added to the transcribed (immature) mRNA.

[0209] During the in vitro synthesis of mRNA molecules, 5' capping of polynucleotides can be performed simultaneously using chemical RNA cap analogs to produce 5' guanosine cap structures: 5'-guanosine cap structures: 3'-O-Me-m7G(5')ppp(5')G[ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G. In some embodiments described herein, the following kits are used for capping: CAP m7Gppp(2'OMeA)pG or CAP5m7G(5')vppp(5')(2'OMeA)pG. 5' capping of mRNA can also be performed post-transcriptionally using vaccinia virus capping enzymes to produce Cap 0 structures. The Cap 1 structure can be generated using both vaccinia virus capping enzyme and 2'-O methyltransferase to produce m7G(5')ppp(5')G 2'O-methyl. The Cap 2 structure can be generated from the Cap 1 structure, followed by 2'-O methylation of the 5' penultimate nucleotide using 2'-O methyltransferase. The Cap 3 structure can be generated from the Cap 2 structure, followed by 2'-O methylation of the 5' penultimate nucleotide using 2'-O methyltransferase. The 3' Poly-A tail is typically an extension of the adenine nucleotide added to the 3' end of the transcribed mRNA. In some embodiments, it can include up to approximately 400 adenine nucleotides.

[0210] Elements of mRNA molecules

[0211] In some implementations, in addition to the read frame encoding immunogenic proteins and molecular chaperones, the mRNA molecule also contains a 5'UTR and a 3'UTR, as well as a 5' cap structure or a 3' Poly-A tail. The 5'UTR and 3'UTR are typically transcribed from genomic DNA and are elements of immature mRNA.

[0212] When mRNA is engineered to encode immunogenic proteins, it may contain one or more of these untranslated regions (UTRs). Wild-type untranslated regions of nucleic acids are transcribed but not translated. In mRNA, the 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon; while the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal. UTRs may play a regulatory role in the stability of nucleic acid molecules and translation. A variety of 5' UTR and 3' UTR sequences are known and available in the art. The 5' UTR is the mRNA region 5' directly upstream of the start codon (the first codon of the mRNA transcript translated by ribosomes). The 5' UTR does not encode proteins (it is non-coding). Native 5' UTRs have features that play a role in translation initiation. They possess features such as the Kozak sequence, which are well known to be involved in the ribosome-initiated translation of many genes. It is also known that 5' UTRs form secondary structures involved in elongation factor binding.

[0213] The 3'UTR is the mRNA region directly downstream (3') of a stop codon (the codon that transmits the translation termination signal in the mRNA transcript). The 3'UTR does not encode proteins (it is non-coding). Strains containing adenosine and uridine are known to be embedded in natural or wild-type 3'UTRs. These AU-rich features are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three categories (Chen et al., 1995): Class I AREs contain several scattered copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonmers. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III AREs are less clearly defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples of this category. Most proteins that bind to AREs are known to disrupt messenger stability, while members of the ELAV family, particularly HuR, have been shown to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule will result in HuR binding, thereby stabilizing the messenger in vivo. The introduction, removal, or modification of AU-rich elements (AREs) in the 3'UTR can be used to modulate the stability of polynucleotides (e.g., mRNA). When engineering a particular nucleic acid, one or more copies of an ARE can be introduced to make the nucleic acid of this disclosure less stable, thereby reducing translation and reducing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability, thereby increasing the translation and production of the resulting protein. Those skilled in the art will understand that the 5'UTR can be used with any desired 3'UTR sequence.

[0214] A poly-A tail is an mRNA region containing multiple consecutive adenosine monophosphates (ATPs) located downstream of the 3' UTR, for example, directly downstream (i.e., 3'). A poly-A tail can contain 10 to 300 ATPs. In some embodiments, the poly-A tail contains 10 to 400 ATPs (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400). Poly-A tails can be used to protect mRNA from enzymatic degradation, for example, in the cytoplasm, and can facilitate transcription termination and / or the export of mRNA from the nucleus for translation.

[0215] Formulations or compositions

[0216] Formulations or compositions containing nucleic acids (e.g., mRNA molecules) are known in the art and are described, for example, in WO 2013 / 090648. For example, compositions or formulations may be, but are not limited to, nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipids, lipid complexes, liposomes, polymers, carbohydrates (including monosaccharides), cationic lipids, fibrin gels, fibrin hydrogels, fibrin glues, fibrin binders, fibrinogen, thrombin, rapidly eliminating lipid nanoparticles (reLNPs), and combinations thereof.

[0217] vaccine

[0218] This document also provides for nucleic acid vaccines. Nucleic acid vaccines can be mRNA vaccines. In some embodiments, an mRNA vaccine may contain the same mRNA molecule or multiple different mRNA molecules. In some embodiments, the mRNA in an mRNA vaccine may contain the same read frame of an immunogenic protein. In some embodiments, the mRNA in an mRNA vaccine may contain the same read frame of an immunogenic protein, and the mRNA molecule may be the same. In some embodiments, the mRNA in an mRNA vaccine may contain the same read frame of an immunogenic protein, and the mRNA molecules may be different. That is, an mRNA vaccine contains multiple different mRNA molecules that encode the same immunogenic protein. In some embodiments, the mRNA in an mRNA vaccine may contain different immunogenic protein read frames. Nucleic acid vaccines may contain mRNA molecules of any one or more of the polynucleotide sequences or degenerate sequence variants of SEQ ID NO: 42-77, 121-130, and 132-133.

[0219] In some embodiments, two or more different mRNAs can be formulated into the same lipid nanoparticle. In some embodiments, two or more different RNAs encoding antigens can be formulated into separate lipid nanoparticles. The lipid nanoparticles can then be combined and administered as a single vaccine composition (e.g., containing multiple RNAs encoding multiple antigens), or they can be administered alone.

[0220] Vaccine administration

[0221] In some implementations, the compositions, formulations, or vaccines described herein may be administered to a subject (e.g., a mammalian subject, such as a human subject). The dosage is an effective amount to enable the nucleic acid to be translated in vivo to produce an immunogenic protein. An "effective amount" is based at least in part on the target tissue, target cell type, means of administration, physical characteristics of the RNA (e.g., length, nucleotide composition, and / or degree of nucleoside modification), other components of the vaccine, and other determinants such as the subject's age, weight, height, sex, and general health condition. Typically, an effective amount of vaccine provides an induced or enhanced immune response based on the antigen produced in the subject's cells.

[0222] The term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use in vivo or in vitro. A "pharmaceutically acceptable carrier" will not cause undesirable physiological effects when administered to or to a subject. A carrier in a pharmaceutical composition must also be "acceptable" in the sense that it is compatible with and can stabilize the active ingredient. One or more solubilizers may be used as drug carriers to deliver the active agent. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible mediators, adjuvants, additives, and diluents to achieve compositions usable as dosage forms. Other examples of carriers include colloidal silica, magnesium stearate, cellulose, and sodium lauryl sulfate. Further suitable drug carriers and diluents, as well as the pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

[0223] In some embodiments, the vaccine described herein can be used to treat or prevent HPV infection. The vaccine can be administered prophylactically or therapeutically to healthy individuals as part of an active immunization program, or during the early stages of infection, either in the incubation period or during active infection after the onset of symptoms. In some embodiments, the vaccine can treat subjects already infected with HPV. In some embodiments, the amount of RNA provided to cells, tissues, or subjects can be an amount effective for immunoprophylaxis or treatment.

[0224] Vaccines can be administered in combination with other prophylactic or therapeutic compounds. As a non-limiting example, the prophylactic or therapeutic compound can be an adjuvant or a booster. An adjuvant can be a molecular adjuvant comprising an amino acid sequence or a variant thereof from any one of SEQ ID NO: 37, 38, 95-97, 137. Alternatively, the adjuvant can be a nucleic acid, preferably mRNA, encoding the molecular adjuvant. The mRNA encoding the molecular adjuvant can be encapsulated in lipid particles along with mRNA encoding immunogenicity.

[0225] As used herein, when referring to a prophylactic composition such as a vaccine, the term "booster" refers to an additional administration of the prophylactic (vaccine) composition. In exemplary embodiments, the time interval between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year. In some embodiments, the vaccine may be administered intramuscularly, intranasally, or intradermally.

[0226] The present invention provides the following sequence.

[0227] Table 1. Description of SEQ ID NO

[0228] Example

[0229] To make the above-mentioned objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the appendix and specific embodiments.

[0230] Example 1: Design of BVDV mRNA and IBRV mRNA

[0231] This invention uses bovine host to optimize the codon design of the BVDV E2 antigen sequence, IBRV gB and gD antigen sequences, and molecular adjuvant sequences. Based on the protein amino acid sequences (SEQ ID NO: 1–41), corresponding coding nucleotide sequences (SEQ ID NO: 42–82) are generated. A T7 promoter, 5'UTR, and start codon are sequentially added to the 5' end of the nucleotide sequences; a Qα sequence, stop codon, 3'UTR, and polyA are sequentially added to the 3' end of the nucleotide sequences before gene synthesis. The relevant plasmids are constructed using conventional molecular biology methods in this field. Sequence descriptions are shown in Table 1.

[0232] Example 2: Preparation of BVDV mRNA-LNP formulation

[0233] This invention uses linear double-stranded DNA containing the T7 promoter sequence in Table 2 as a template. The reaction system is as follows: 10 μl of linear double-stranded DNA (0.5 μg / μl), 10 μl of T7 RNA polymerase, 10 μl of T7 RNA polymerase buffer, 0.2 μl of inorganic pyrophosphatase, 5 μl of RNase inhibitor, 14.8 μl of WFI, 10 μl each of reaction NTPs (ATP, GTP, CTP, N1-Me-pUTP) and CAP5 [m7G(5')vppp(5')(2'OMeA)pG] (Catalog No.: CAP5011, Jiangsu Shenji Biotechnology Co., Ltd.) (structural formula is...). 10 μl of the mixture was reacted at 37°C for 150 minutes to synthesize an in vitro transcribed mRNA stock solution with a 5' cap and a 3' polyA tail.

[0234] mRNA stock solution was encapsulated using the following lipid formulation (heptadecane-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate (SM-102), DSPC, cholesterol, DMG-PEG2000 ethanol). The lipids and mRNA were mixed in 20 mM sodium citrate buffer (pH 4.0) at a volume ratio of 1:3 (lipids:mRNA) at a flow rate of 12 ml / min using a nanomedicine preparation system (INano, Myanna (Shanghai) Instruments Technology Co., Ltd.). TM The sample solution was prepared by mixing (L / L+). The collected sample solution was diluted 10-fold in 10mM Tris 0.1% NaCl buffer, then passed through a 100kDa Pall ultrafiltration tube, centrifuged and ultrafiltered to concentrate the solution, removing the ethanol content. Finally, it was adjusted to the appropriate concentration with 10mM Tris 0.1% NaCl buffer.

[0235] Table 2. Description of BVDV mRNA vaccine sequences

[0236] Example 3: Immunization regimen for mice using BVDV mRNA-LNP formulation

[0237] Female BALB / c mice aged 6-8 weeks were randomly divided into groups of 5. Mice were immunized intramuscularly with the BVD-1-16 (SEQ ID NO: 42-64) mRNA-LNP preparation prepared as in Example 2 at a dose of 5 μg / mouse. A control group (containing no mRNA molecules) and a group receiving the BVDV-IBRV bivalent inactivated vaccine (Jinyu Baoling) were established, with each mouse receiving 100 μl of the vaccine. A booster immunization was performed 14 days after the initial immunization. Serum was collected from the orbital vein at 7 and 28 days after the initial immunization for ELISA testing as described in Example 4 and neutralizing antibody testing as described in Example 5. Spleen cells were collected 28 days after the initial immunization for Elispot (enzyme-linked immunospot) assay as described in Example 6.

[0238] Example 4: Detection of antibody binding after immunization of mice with BVDV mRNA-LNP formulation

[0239] The internally prepared eukaryotic BVDV-1 E2 and BVDV-2 E2 proteins were diluted to 0.0625 μg / ml and 0.5 μg / ml, respectively, and added to 96-well plates, 100 μl per well, and incubated overnight at 4°C. After washing 5 times with PBST, 100 μl of 2% BSA in PBS buffer was added to each well, and the plates were blocked at 37°C for 1 hour, followed by washing 3 times with PBST. Serum samples were diluted 1:1000 with diluent, 100 μl per well, and incubated at 37°C for 1 hour, followed by washing 5 times with PBST. 100 μl of HRP-labeled goat anti-mouse IgG secondary antibody (1:10000 dilution) was added to each well, and the plates were incubated at 37°C for 1 hour, followed by washing 3 times with PBST. 100 μl of TMB single-component chromogenic solution was added to each well, and the plates were incubated at room temperature for 10 minutes, followed by adding 50 μl of stop solution. Finally, the OD values ​​were read using a microplate reader. 450nm -OD 630nm The results, as shown in Table 3, indicate that 28 days after the initial immunization, the binding antibody levels induced by BVD-1 to 16 mRNA-LNP preparations remained high. This suggests that all 16 mRNA-LNP preparations can elicit a high level of humoral immune response.

[0240] Table 3. Detection of binding antibodies in mice immunized with BVDV mRNA-LNP formulation

[0241] Example 5. Detection of neutralizing antibodies in mice immunized with BVDV mRNA-LNP formulation

[0242] The separated serum was inactivated at 56°C for 30 minutes. The inactivated serum was serially diluted 1:2 starting at DMEM medium, for a total of 12 dilutions. Each dilution was performed in quadruple replicates. 50 μl of the diluted serum was added to each well of a 96-well cell plate. BVDV was diluted to 200 TCID using DMEM medium. 50 / ml. Add an equal volume of the diluted virus to a 96-well cell plate containing serum. Incubate the serum-virus mixture in a 37°C, 5% CO2 incubator for 1 hour. Then add 5 × 10⁶ ml of the diluted virus to each well. 5 100 μl of MDBK cells per ml were added and cultured in the cell culture incubator for 48-72 hours. Cytopathic effects were observed. Cytopathic effects were then recorded daily. Bovine serum neutralizing antibody titers were calculated using the Reed-Muench method. BVDV neutralization results are shown in Table 4. 28 days after the second immunization, the BVDV neutralizing antibody titer induced by the inactivated vaccine in calves was only 573.47, while the BVDV neutralizing antibody titers induced by calves immunized with BVD-3, BVD-6, BVD-9, BVD-12, and BVD-15 mRNA-LNP preparations were higher than those in the other mRNA vaccine groups, indicating that the Ft protein plays an important role in the production of BVDV neutralizing antibodies.

[0243] Table 4. Detection of neutralizing antibodies in mice immunized with BVDV mRNA-LNP formulation

[0244] Example 6: Cellular Immunoassay of BVDV mRNA-LNP Formulation

[0245] Four weeks after the initial immunization of mice, spleen cells were collected, and the level of IFN-γ secreted by the immunized mouse spleen cells was evaluated using the Elispot (enzyme-linked immunospot assay) kit (catalog number: 2210007). The steps were as follows: After collecting mouse spleen cells, the spleen cell concentration was adjusted to 1×10⁻⁶. 6 Cells / ml. Spleen cells were added to Elispot plates at a volume of 100 μl per well. The purified BVDV-1 E2 protein was diluted to 10 μg / ml with complete culture medium (1640 cell culture medium containing 10% FBS), and 100 μl was added to each well of the Elispot plate. The plates were incubated at 37°C for 48-72 h in a 5% CO2 incubator. After incubation, 100 μl of biotin-conjugated antibody was added to each well, and the plates were incubated at 37°C for 1 h. After washing, 100 μl of HRP-conjugated Streptavidin working solution was added to each well. The plates were incubated at 37°C for 1 h. After washing, 100 μl of AEC chromogenic solution was added to each well for color development. After color development, images were created and analyzed using an ELISA dot imaging analyzer.

[0246] The results are shown in Table 5. Compared with inactivated vaccines, 16 BVDV mRNA-LNPs can significantly stimulate mouse spleen cells to secrete IFN-γ, while the mRNA vaccine group containing Ft protein can induce mouse spleen cells to secrete higher levels of IFN-γ, indicating that Ft protein plays an important role in inducing high-level cellular immune responses by BVDV mRNA vaccines.

[0247] Table 5. Detection of IFN-γ secretion in mice immunized with BVDV mRNA-LNP preparation

[0248] Example 7. Preparation of IBRV mRNA-LNP formulation

[0249] This invention uses linear double-stranded DNA containing the T7 promoter sequence in Table 6 as a template. The reaction system is as follows: 10 μl of linear double-stranded DNA (0.5 μg / μl), 10 μl of T7 RNA polymerase, 10 μl of T7 RNA polymerase buffer, 0.2 μl of inorganic pyrophosphatase, 5 μl of RNase inhibitor, 14.8 μl of WFI, 10 μl each of reaction NTPs (ATP, GTP, CTP, N1-Me-pUTP) and CAP5 [m7G(5')vppp(5')(2'OMeA)pG] (Catalog No.: CAP5011, Jiangsu Shenji Biotechnology Co., Ltd.) (structural formula is...). 10 μl of the mixture was reacted at 37°C for 150 minutes to synthesize an in vitro transcribed mRNA stock solution with a 5' cap and a 3' polyA tail.

[0250] mRNA stock solution was encapsulated using the following lipid formulation (heptadecane-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate (SM-102), DSPC, cholesterol, DMG-PEG2000 ethanol). The lipids and mRNA were mixed in 20 mM sodium citrate buffer (pH 4.0) at a volume ratio of 1:3 (lipids:mRNA) at a flow rate of 12 ml / min using a nanomedicine preparation system (INano, Myanna (Shanghai) Instruments Technology Co., Ltd.). TM The sample solution was prepared by mixing (L / L+). The collected sample solution was diluted 10-fold in 10mM Tris 0.1% NaCl buffer, then passed through a 100kDa Pall ultrafiltration tube, centrifuged and ultrafiltered to concentrate the solution, removing the ethanol content. Finally, it was adjusted to the appropriate concentration with 10mM Tris 0.1% NaCl buffer.

[0251] Table 6. IBRV mRNA vaccine sequence description

[0252] Example 8. Immunization regimen for IBRV mRNA-LNP formulation in mice

[0253] Female BALB / c mice aged 6-8 weeks were divided into groups of five. IBRV-1-10 (SEQ ID NO: 68-77) mRNA-LNP preparations were administered to mice via intramuscular injection at a dose of 5 μg / mouse. A control group (containing no mRNA molecules) and a group receiving BVDV-IBRV bivalent inactivated vaccine (Jinyu Baoling) were also established, with each mouse receiving 100 μl of the vaccine. A booster immunization was performed 14 days after the initial immunization. Serum was collected from the orbital vein at 7 and 28 days after the initial immunization for ELISA testing (Example 9) and neutralizing antibody testing (Example 10). Spleen cells were collected from mice 28 days after the initial immunization for Elispot (enzyme-linked immunospot) assay.

[0254] Example 9. Detection of antibody binding after immunization of mice with IBRV mRNA-LNP formulation

[0255] IBRV gD protein expressed in eukaryotic cells was diluted to 0.25 μg / ml and added to 96-well plates (100 μl per well). The plates were incubated overnight at 4°C. After washing five times with PBST, 100 μl of 2% BSA in PBS buffer was added to each well, and the plates were blocked at 37°C for 1 hour, followed by three washes with PBST. Serum samples were diluted 1:1000 with diluent, 100 μl per well, and incubated at 37°C for 1 hour, followed by five washes with PBST. 100 μl of HRP-labeled goat anti-mouse IgG secondary antibody (1:10000 dilution) was added to each well, and the plates were incubated at 37°C for 1 hour, followed by three washes with PBST. 100 μl of TMB single-component chromogenic solution was added to each well, and the plates were incubated at room temperature for 10 minutes. 50 μl of stop solution was then added, and the OD values ​​were read using a microplate reader. 450nm -OD 630nm The results, shown in Table 7, indicate that 28 days after the initial immunization, all 10 mRNA-LNP formulations elicited high levels of gD protein-binding antibodies. These results suggest that all 10 IBRV mRNA-LNP formulations can elicit high levels of humoral immune responses.

[0256] Table 7. Detection of antibody binding in mice immunized with IBRV mRNA-LNP formulation.

[0257] Example 10. Detection of neutralizing antibodies in mice immunized with IBRV mRNA-LNP formulation

[0258] The separated serum was inactivated at 56°C for 30 minutes. The inactivated serum was then serially diluted 1:2 starting at DMEM medium, for a total of 12 dilutions. Each dilution was performed in quadruple replicates. 50 μl of the diluted serum was added to each well of a 96-well cell plate. IBRV was then diluted to 4000 TCID using DMEM medium. 50 / ml. Add an equal volume of the diluted virus to a 96-well cell plate containing serum. Incubate the serum-virus mixture in a 37°C, 5% CO2 incubator for 1 hour. Then add 3 × 10⁻⁶ ml of the diluted virus to each well. 5 100 μl of MDBK cells per ml were added and cultured in a cell culture incubator for 48-72 hours. Cytopathic effects were observed. Cytopathic effects were then recorded daily. Bovine serum neutralizing antibody titers were calculated using the Reed-Muench method, with the highest serum dilution capable of inhibiting 50% cytopathic effects defined as the neutralizing antibody titer. The results are shown in Table 8. IBRV-3 and IBRV-4 could stimulate mice to produce IBRV neutralizing antibody titers of approximately 800, significantly higher than other mRNA and inactivated vaccines, indicating that the two mRNA vaccines with the "gD protein-P2A-gB protein" structural design were the most effective.

[0259] Table 8. Detection of neutralizing antibodies in mice immunized with IBRV mRNA-LNP formulation.

[0260] Example 11. Cellular Immunological Evaluation of IBRV mRNA-LNP Formulation

[0261] Four weeks after the initial immunization of mice, spleen cells were collected, and the level of IFN-γ secreted by the immunized mouse spleen cells was evaluated using the Elispot (enzyme-linked immunospot assay) kit (catalog number: 2210007). The steps were as follows: After collecting mouse spleen cells, the spleen cell concentration was adjusted to 1×10⁻⁶. 6 Cells / ml. Spleen cells were added to Elispot plates at a volume of 100 μl per well. The purified IBRV gD protein was diluted to 10 μg / ml with complete culture medium (1640 cell culture medium containing 10% FBS), and 100 μl was added to each well of the Elispot plate. The plates were incubated at 37°C for 48-72 h in a 5% CO2 incubator. After incubation, 100 μl of biotin-conjugated antibody was added to each well, and the plates were incubated at 37°C for 1 h. After washing, 100 μl of HRP-conjugated Streptavidin working solution was added to each well, and the plates were incubated at 37°C for 1 h. After washing, 100 μl of AEC chromogenic solution was added to each well for color development. After color development, images were created and analyzed using an ELISA dot imaging analyzer.

[0262] The results are shown in Table 9. Compared with the LNP immunization group, spleen cells of mice immunized with all 10 IBRV mRNA-LNPs could secrete IFN-γ upon stimulation by IBRV gD protein, indicating that these 10 mRNA-LNPs could induce significant T cell responses in mice. Among them, mRNA vaccines containing gB protein, such as IBRV-3 to IBRV-10, could elicit higher levels of cellular immune responses. The "gD protein-P2A-gB protein" structural scheme elicited the highest level of cellular immunity.

[0263] Table 9. Detection of IFN-γ secretion in mice immunized with IBRV mRNA-LNP preparation

[0264] Example 12. Preparation of BVDV-IBRV bivalent mRNA vaccine

[0265] First, according to the BVDV and IBRV sequences in Table 10 and the molecular adjuvant BMA-1 (SEQ ID NO: 37) and the indicated mass ratio, the mRNA stock solution of the required sequence was premixed and then encapsulated with LNPs in the same way as in Example 2, and named BAI-1~20 mRNA vaccine.

[0266] Table 10. Information on parallel encapsulation of BVDV and IBRV mRNA

[0267] Example 13. BVDV-IBRV bivalent mRNA vaccine immunization program for calves

[0268] Calves around 5 months of age were divided into groups of 5. First, the calves were immunized initially with 100 μg / calves of 20 mRNA-LNP preparations (BAI-1–20) administered intramuscularly. Simultaneously, a control group with empty LNP and a group with a BVDV-IBRV bivalent inactivated vaccine (Jinyu Baoling) were established, with each calf receiving 2 ml of the vaccine intramuscularly. A booster immunization was administered 28 days after the initial immunization. On day 56 after the initial immunization, serum was collected intravenously for BVDV and IBRV neutralizing antibody testing.

[0269] Example 14. Detection of BVDV neutralizing antibodies after immunization of cattle with BVDV-IBRV mRNA-LNP formulation

[0270] The method was the same as in Example 5. The results of BVDV neutralization detection are shown in Table 11. 56 days after the first immunization, the titer of BVDV neutralizing antibodies induced by the inactivated vaccine in calves was only 171, while the titers of BVDV neutralizing antibodies induced by the mRNA-LNP preparation in calves were all higher than those induced by the inactivated vaccine. Among them, the mRNA vaccines encapsulated in parallel with the molecular adjuvant BMA-1 (BAI-1, BAI-2, BAI-3, BAI-4, BAI-5, BAI-11, BAI-12, BAI-13, BAI-14 and BAI-15) were significantly higher than those without the molecular adjuvant BMA-1 encapsulation (BAI-6, BAI-7, BAI-8, BAI-9, BAI-10, BAI-16, BAI-17, BAI-18, BAI-19 and BAI-20), indicating that the molecular adjuvant significantly enhances the level of BVDV neutralizing antibodies induced by the mRNA vaccine.

[0271] Table 11. Detection of BVDV neutralizing antibodies in bovine serum immunized with BVDV-IBRV mRNA-LNP preparations

[0272] Example 15. Detection of IBRV neutralizing antibodies after immunization of bovine animals with BVDV-IBRV mRNA-LNP formulation

[0273] The method was the same as in Example 10. The IBRV neutralization test results are shown in Table 12. 56 days after the first immunization, the IBRV neutralizing antibody titer induced by inactivated vaccine immunization in calves was only 21, while the IBRV neutralizing antibody titer induced by mRNA-LNP immunization in calves reached over 100, which was significantly higher than that of inactivated vaccine. Among them, the mRNA vaccines encapsulated in parallel with the molecular adjuvant BMA-1 (BAI-1, BAI-2, BAI-3, BAI-4, BAI-5, BAI-11, BAI-12, BAI-13, BAI-14 and BAI-15) were significantly higher than those without the encapsulated molecular adjuvant BMA-1 (BAI-6, BAI-7, BAI-8, BAI-9, BAI-10, BAI-16, BAI-17, BAI-18, BAI-19 and BAI-20), indicating that the molecular adjuvant significantly enhances the level of IBRV neutralizing antibodies induced by the mRNA vaccine.

[0274] Table 12. Detection of IBRV neutralizing antibodies in bovine serum immunized with BVDV-IBRV mRNA-LNP preparation

[0275] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A fusion protein comprising, from N- to C-terminus, a signal peptide, an immunogenic protein, and optionally a chaperone moiety, wherein: the signal peptide is a bovine IL2 signal peptide, a tPA signal peptide, a bovine IL6 signal peptide, or a bovine IgG heavy chain signal peptide; the immunogenic protein comprises one or more of a BVDV-1 E2 protein or an ectodomain thereof, a BVDV-2 E2 protein or an ectodomain thereof, and a BVDV-3 E2 protein or an ectodomain thereof, or, the immunogenic protein comprises one or more of an IBRV gB protein or a transmembrane domain-deleted version thereof, and an IBRV gD protein or an ectodomain thereof; the chaperone moiety is a bovine IgG Fc protein, a T4 Folden protein, or an Ft protein; preferably, wherein the immunogenic protein comprises two or three of a BVDV-1 E2 protein or an ectodomain thereof, a BVDV-2 E2 protein or an ectodomain thereof, and a BVDV-3 E2 protein or an ectodomain thereof, or the immunogenic protein comprises two of an IBRV gB protein or a transmembrane domain-deleted version thereof, and an IBRV gD protein or an ectodomain thereof; preferably, wherein: the bovine IL2 signal peptide comprises SEQ ID NO: 39 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 39; the tPA signal peptide comprises SEQ ID NO: 40 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 40; the bovine IL6 signal peptide comprises SEQ ID NO: 98 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 98; the bovine IgG heavy chain signal peptide comprises SEQ ID NO: 41 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 41; preferably, wherein: the BVDV-1 E2 protein comprises SEQ ID NO: 110 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 110; the BVDV-1 E2 protein ectodomain comprises SEQ ID NO: 109 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 109; the BVDV-2 E2 protein comprises SEQ ID NO: 111 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 111; the BVDV-2 E2 protein ectodomain comprises SEQ ID NO: 112 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 112; the BVDV-3 E2 protein comprises SEQ ID NO: 113 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 113; the BVDV-3 E2 protein ectodomain comprises SEQ ID NO: 114 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 114; the IBRV gB protein comprises SEQ ID NO: 115 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 115; the IBRV gB protein transmembrane domain-deleted version comprises SEQ ID NO: 116 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 116; the IBRV gD protein comprises SEQ ID NO: 117 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 117; and the IBRV gD protein ectodomain comprises SEQ ID NO: 118 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

118. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ the BVDV-2 E2 protein comprises SEQ ID NO: 112 or a sequence at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 112; the BVDV-2 E2 protein ectodomain comprises SEQ ID NO: 111 or a sequence at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 111; the BVDV-3 E2 protein comprises SEQ ID NO: 114 or a sequence at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 114; the BVDV-3 E2 protein ectodomain comprises SEQ ID NO: 113 or a sequence at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 113; the IBRV gB protein comprises SEQ ID NO: 117 or a sequence at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 117; the IBRV gB protein transmembrane region comprises SEQ ID NO: 118 or a sequence at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 118; the IBRV gD protein comprises SEQ ID NO: 115 or a sequence at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 115; the IBRV gD protein ectodomain comprises SEQ ID NO: 116 or a sequence at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 116; Preferably, wherein: the bovine IgG Fc protein comprises SEQ ID NO: 107 or a sequence at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 107; The T4 Folden protein comprises SEQ ID NO: 105 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 105; The Ft protein comprises SEQ ID NO: 108 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 108; Preferably, the fusion protein has any one of the following structures: Signal peptide - [immunogenic protein] x ; Signal peptide - [immunogenic protein - optional linker - chaperone] x ; or Signal peptide - [immunogenic protein] x - optional linker - chaperone; the immunogenic protein is one of a BVDV-1 E2 protein or an ectodomain thereof, a BVDV-2 E2 protein or an ectodomain thereof, and a BVDV-3 E2 protein or an ectodomain thereof, wherein x is 1, 2, or 3; when x is 2 or 3, each unit within [...] is connected by a linker; Preferably, wherein the linker is (GGGGS) n n = 1-6, preferably 2, 3 or 4, more preferably 3; or the linker is a self-cleaving peptide, preferably a P2A, T2A, E2A, or F2A self-cleaving peptide; Preferably, the fusion protein comprises any one of the following structures: IL2sp-BVDV-1 E2 ectodomain; IL2sp-BVDV-1 E2 ectodomain-linker-bovine IgG Fc; IL2sp-BVDV-1 E2 ectodomain-linker-Ft protein; IL2sp-BVDV-1 E2 protein; IL2sp-BVDV-1 E2 protein-linker-bovine IgG Fc; IL2sp-BVDV-1 E2 protein-linker-Ft protein; IL2sp-BVDV-2 E2 ectodomain; IL2sp-BVDV-2 E2 ectodomain-linker-bovine IgG Fc; IL2sp-BVDV-2 E2 ectodomain-linker-Ft protein; IL2sp-BVDV-2 E2 protein; IL2sp-BVDV-2 E2 protein-linker-bovine IgG Fc; IL2sp-BVDV-2 E2 protein-linker-Ft protein; IL2sp-BVDV-3 E2 ectodomain; IL2sp-BVDV-3 E2 ectodomain-linker-bovine IgG Fc; IL2sp-BVDV-3 E2 ectodomain-linker-Ft protein; IL2sp-BVDV-3 E2 protein; IL2sp-BVDV-3 E2 protein-linker-bovine IgG Fc; IL2sp-BVDV-3 E2 protein-linker-Ft protein; IL2sp-BVDV-1 E2 ectodomain-linker-BVDV-2 E2 ectodomain-linker-Ft protein; IL2sp-BVDV-2 E2 ectodomain-linker-BVDV-1 E2 ectodomain-linker-Ft protein; IL2sp-BVDV-1 E2 ectodomain-linker-BVDV-2 E2 ectodomain-linker-BVDV-3 E2 ectodomain-linker-Ft protein; IL2sp-BVDV-1 E2 ectodomain-linker-Ft-P2A-BVDV-2 E2 ectodomain-linker-Ft protein; IL2sp-BVDV-2 E2 ectodomain-linker-Ft-P2A-BVDV-1 E2 ectodomain-linker-Ft protein; IL2sp-BVDV-1 E2 ectodomain-linker-Ft-P2A-BVDV-2 E2 ectodomain-linker-Ft-P2A-BVDV-3 E2 ectodomain-linker-Ft protein; Preferably, wherein the fusion protein has any one of the following structures: signal peptide - immunogenic protein; signal peptide - immunogenic protein - optional linker - immunogenic protein; the immunogenic protein is one or more of IBRV gB protein or a transmembrane domain-deleted thereof and IBRV gD protein or an ectodomain thereof; Preferably, wherein the linker is (GGGGS) n n = 1-6, preferably 2, 3 or 4, more preferably 3; or the linker is a self-cleaving peptide, preferably a P2A, T2A, E2A or F2A self-cleaving peptide; Preferably, wherein the fusion protein has any one of the following structures: IL2sp-IBRV gB transmembrane domain-deleted; IL2sp-IBRV gB protein; IL2sp-IBRV gD ectodomain; IL2sp-IBRV gD protein; IL2sp-IBRV gD ectodomain-P2A-IBRV gB transmembrane domain-deleted; IL2sp-IBRV gD protein-P2A-IBRV gB protein; IL2sp-IBRV gB transmembrane domain-deleted-P2A-IBRV gD ectodomain; IL2sp-IBRV gB protein-P2A-IBRV gD protein; IL2sp-IBRV gD ectodomain-linker-IBRV gB transmembrane domain-deleted; IL2sp-IBRV gB transmembrane domain-deleted-linker-IBRV gD ectodomain; IL2sp-IBRV gD protein-linker-IBRV gB protein; IL2sp-IBRV gB protein-linker-IBRV gD protein; the fusion protein further comprises a Qa amino acid sequence and / or a tag protein, for example a V5 tag protein and / or a Myc tag protein, preferably the Qa amino acid sequence comprises SEQ ID NO: 103 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

2. The fusion protein of claim 1, comprising an amino acid sequence of any one of SEQ ID NOs: 1-36 and 135-136 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 1-36 and 135-136.

3. An isolated nucleic acid encoding the fusion protein of claim 1 or 2; Preferably, the nucleic acid is a single-stranded or double-stranded DNA molecule or an RNA molecule; Preferably, wherein the RNA molecule is an mRNA molecule; Preferably, the mRNA comprises one or more of the following elements: a 5' cap, a 5' UTR, a 3' URT, and a Poly-A tail; Preferably, the 5' cap comprises a compound of the structure of Formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer, or isotopically enriched variant thereof: wherein, is a single bond or null, X1is selected from O, S, CH2, CH2CH2, CH=CH, CH=CHO, CH2O, OCH2, CH2CH2O, OCH2CH2, a three-membered ring alkyl, R1, R2, R3and R4are each independently halogen, OH, unsubstituted or O-C 1-3 unsubstituted or O-C 1-3 unsubstituted or O-C 1-3 unsubstituted or O-C 1-3 unsubstituted or O-C B1and B2are each independently selected from a natural, modified, or non-natural nucleobase, More preferably, the compound of formula (I) is Preferably, the 5' UTR comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 83, 85, 87, 89, 91, or 93, or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polynucleotide sequence of any one of SEQ ID NOs: 83, 85, 87, 89, 91, or 93; the 3' URT comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 84, 86, 88, 90, 92, or 94, or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polynucleotide sequence of any one of SEQ ID NOs: 84, 86, 88, 90, 92, or 94; Preferably, the nucleic acid comprises a polynucleotide sequence of any one of SEQ ID NOs: 42-77 and 132-133, or a degenerate sequence variant thereof; Preferably, wherein the nucleic acid is an mRNA molecule, uracil, cytosine, adenine, or guanine nucleotides of the mRNA molecule contain a modification group, the modification group comprises at least one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 5-methylcytosine, 5-methoxy cytosine, N1-methylcytosine, 2-thiouridine, 5-methoxyuridine, or N1-methyladenosine, N1-methylguanine, N1-methylguanine, isoguanine; Preferably, wherein the mRNA molecule is a modified mRNA, the modification comprises conversion of uracil nucleosides to pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 5-methoxyuridine; and / or, conversion of cytosine nucleosides to 5-methylcytosine, 5-methoxy cytosine, N1-methylcytosine; and / or, conversion of adenine nucleosides to N1-methyladenosine; and / or, conversion of adenine nucleosides to N1-methylguanine, N1-methylguanine, isoguanine.

4. A vector comprising the nucleic acid of claim 3.

5. A cell comprising the fusion protein of claim 1 or 2, the nucleic acid of claim 3, or the vector of claim 4.

6. A composition comprising one or more of the fusion protein according to claim 1 or 2, the nucleic acid of claim 3, or the vector of claim 4.

7. A vaccine composition comprising one or more of the mRNA molecules described in claim 3; Preferably, wherein the vaccine composition is a BVDV-IBRV combination mRNA vaccine composition; Preferably, wherein the vaccine composition is a BVDV-IBRV combination mRNA vaccine composition; Preferably, the vaccine composition comprises an mRNA molecule against BVDV and an mRNA molecule against IBRV, the mRNA molecule against BVDV comprises one or more of SEQ ID NO. 42- SEQ ID NO. 65, the mRNA molecule against IBRV comprises one or more of SEQ ID NO. 66- SEQ ID NO. 77; Preferably, the vaccine composition comprises 1 : 100-100: 1 of the mRNA molecule against BVDV and the mRNA molecule against IBRV; Preferably, the vaccine composition comprises a lipid nanoparticle carrying the mRNA molecule; Preferably, the vaccine composition comprises an adjuvant; Preferably, the adjuvant is a molecular adjuvant or a nucleic acid, preferably an mRNA, encoding the molecular adjuvant, Preferably, the molecular adjuvant is IL-2, GM-CSF, CD40L or a combination or fusion thereof; Preferably, the IL-2 comprises the amino acid sequence of SEQ ID NO: 95 or a sequence of SEQ ID NO: 95 having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; Preferably, the GM-CSF comprises the amino acid sequence of SEQ ID NO: 96 or a sequence of SEQ ID NO: 96 having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; Preferably, the CD40L comprises the amino acid sequence of SEQ ID NO: 97 or a sequence of SEQ ID NO: 97 having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; Preferably, the fusion is a fusion of two or three of IL-2, GM-CSF and CD40L connected by a linker, preferably the linker is a self-cleaving peptide, preferably a P2A, T2A, E2A or F2A self-cleaving peptide; Preferably, the molecular adjuvant is a molecular adjuvant comprising an amino acid sequence of any one of SEQ ID NOs: 37, 38, 95-97 and 137 or a sequence having at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 37, 38, 95-97 and 137 or a nucleic acid, preferably a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 134, encoding the molecular adjuvant.

8. Use of the fusion protein of claim 1 or 2, the nucleic acid of claim 3, the vector of claim 4, the composition of claim 6 or the vaccine composition of claim 7 for the manufacture of a medicament for the treatment or prevention of bovine viral diarrhea virus infection and / or infectious bovine rhinotracheitis virus infection, or for the treatment or prevention of bovine viral diarrhea-mucosal disease and / or bovine rhinotracheitis.

9. Use of the nucleic acid according to claim 3 in an mRNA vaccine.

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