PEDV mRNA vaccine and application thereof

Abstract

The invention relates to a PEDV mRNA vaccine and application thereof. The invention provides a mutant PEDV S protein variant, a fusion protein of an S protein and a molecular chaperone, and a fusion protein of the S protein variant and the molecular chaperone. Also provided are mRNA vaccines encoding S proteins, S protein variants, S protein-chaperone fusion proteins, mRNA of S protein variant-chaperone fusion proteins, and optionally molecular adjuvants, and uses thereof.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a PEDV mRNA vaccine and its application. Background Technology

[0002] Porcine epidemic diarrhea (PED) is caused by PEDV, which is highly contagious. PEDV infection can cause clinical symptoms such as vomiting, diarrhea and dehydration. Piglets are most susceptible, with a morbidity rate of 100% and a mortality rate of 75% to 100% in piglets aged 1 to 7 days.

[0003] Genetic analysis of recent PEDV strain sequences has revealed that the GII group of variant PEDV strains accounts for over 90% of the current prevalent strains, including GII-a and GII-b groups. The proportion of GII-a group is increasing annually, and the S-INDEL strain also exists. Different subgroups of PEDV strains exhibit varying pathogenicity and severity. Variant strains are highly pathogenic, with a mortality rate as high as 100% in piglets under 7 days old, characterized by congestion and thinning of the small intestinal wall, severe diarrhea, and dehydration. Classical strains are less virulent, resulting in milder intestinal villus lesions and milder diarrhea in infected pigs. Compared to GII strains, the S-INDEL strain exhibits similar morbidity, reduced pathological damage, less diarrhea, and a lower mortality rate.

[0004] Existing PEDV vaccines include inactivated vaccines, subunit vaccines, live attenuated vaccines, DNA vaccines, and vector vaccines. my country has launched its first attenuated inactivated vaccine based on a PEDV GII variant strain (AJ1102 strain), but its protective efficacy has significantly decreased due to continuous PEDV mutations. PEDV subunit vaccines elicit good humoral immune responses but fail to stimulate significant cellular immunity, thus hindering the development of PEDV subunit vaccines. In 2015, my country also launched a combined diarrheal trivalent (PEDV, TGEV, and PoRV) live attenuated vaccine. However, live attenuated vaccines have drawbacks such as high transportation and storage costs and the risk of virulence reversion. DNA vaccines are typically administered directly in the form of recombinant plasmids, allowing for long-term stable expression of antigens within host cells. However, long-term presence may also lead to integration into the host cell genome; therefore, the safety of DNA vaccines requires further verification. Viral vector vaccines use genetic engineering biotechnology to insert the PEDV S gene into a vector, which then expresses the antigen protein. However, there are currently few viral vectors that can be effectively and safely used in pigs. This is because viral vectors may have strong side effects, such as redness, swelling, heat, pain at the injection site, or allergies. In addition, recombinant vector vaccines still have the possibility of gene recombination with other corresponding viruses in the body.

[0005] mRNA vaccines are the third generation of vaccines, following inactivated vaccines, live attenuated vaccines, subunit vaccines, and viral vector vaccines. Compared with traditional vaccines, they not only have better immunization effects but also have a much shorter research and development and production cycle, and have been effectively used in many infectious diseases. Summary of the Invention

[0006] This invention is partly based on the inventors’ discovery that mRNAs encoding PEDVI, GIIa, GIIb and GIIc type S proteins, S protein variants, S protein-molecular chaperone fusion proteins, S protein variant-molecular chaperone fusion proteins, and optional molecular adjuvants can stimulate the production of binding antibodies, neutralizing antibodies and cellular immune responses in breast milk and mice.

[0007] The first aspect of the present invention provides a fusion protein comprising porcine epidemic diarrhea virus (PEDV) S protein or a variant thereof, and a molecular chaperone fused directly or indirectly thereto; the molecular chaperone is selected from T4 Folden, porcine IgG Fc, Ft, recombinant humanized type III collagen Rh3C, HIV gp41 6HB, GCN4 or Qα.

[0008] In one implementation, the S protein variant is stabilized in the pre-fusion conformation.

[0009] In one embodiment, the variant comprises one or more mutations selected from K893P, R894P, A969P, A1032P, D1076P, and I1077P; the amino acid position of the mutation is based on the amino acid sequence number shown in any one of the parental SEQ ID NO: 17-20.

[0010] In one embodiment, the S protein variant has D1076P and I1077P mutations.

[0011] In one embodiment, the S protein or S protein variant further comprises a deleted or replaced signal peptide.

[0012] In one embodiment, the replacement signal peptide is a signal peptide selected from porcine IL-2 signal peptide, tPA signal peptide, porcine IgG heavy chain signal peptide, porcine IL-6 signal peptide and porcine IL-10 signal peptide, replacing the natural signal peptide in the parent.

[0013] In one embodiment, the alternative signal peptide comprises a signal peptide sequence selected from any one of SEQ ID NO: 46-50 or a signal peptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 46-50.

[0014] In one embodiment, the variant comprises an amino acid sequence shown in any one of SEQ ID NO: 13-16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 13-16.

[0015] In one embodiment, the S protein comprises an amino acid sequence shown in any one of SEQ ID NO: 17-20 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 17-20.

[0016] In one embodiment, the molecular chaperone is selected from the amino acid sequence shown in any one of SEQ ID NO:39-45 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO:39-45.

[0017] In one embodiment, the fusion protein comprises an amino acid sequence selected from any one of SEQ ID NO: 1-12 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-12.

[0018] In one embodiment, the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NO: 51-58, 127-130 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 51-58, 127-130.

[0019] In one embodiment, the fusion protein is further fused with an HA tag, the HA tag comprising an amino acid sequence as shown in SEQ ID NO: 147.

[0020] A second aspect of the present invention provides a nucleic acid encoding the fusion protein described in the first aspect of the present invention.

[0021] In one embodiment, the nucleic acid comprises a nucleotide sequence selected from any one of SEQ ID NO: 59-70 or a degenerate sequence thereof, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 59-70.

[0022] In one embodiment, the nucleic acid comprises the nucleotide sequence shown in any one of SEQ ID NO: 135-146 or its degenerate sequence, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 135-146.

[0023] In one embodiment, the nucleic acid comprises a nucleotide sequence or degenerate sequence shown in any one of SEQ ID NO: 119-126, 131-134 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 119-126, 131-134.

[0024] A third aspect of the invention provides a nucleic acid comprising a polynucleotide encoding a porcine epidemic diarrhea virus (PEDV) S protein or a variant of the S protein.

[0025] In one implementation, the S protein variant is stabilized in the pre-fusion conformation.

[0026] In one embodiment, the S protein variant comprises one or more mutations selected from K893P, R894P, A969P, A1032P, D1076P, and I1077P.

[0027] In one embodiment, the position of the mutated amino acid is determined according to the amino acid sequence number shown in any one of the parental SEQ ID NO: 17-20.

[0028] In one embodiment, the S protein variant has D1076P and I1077P mutations.

[0029] In one embodiment, the S protein or S protein variant further comprises a deleted or replaced signal peptide.

[0030] In one embodiment, the replacement signal peptide is a signal peptide selected from porcine IL-2 signal peptide, tPA signal peptide, porcine IgG heavy chain signal peptide, porcine IL-6 signal peptide and porcine IL-10 signal peptide, replacing the natural signal peptide in the parent.

[0031] In one embodiment, the alternative signal peptide comprises a signal peptide sequence selected from any one of SEQ ID NO: 46-50 or a signal peptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 46-50.

[0032] In one embodiment, the nucleic acid encoding the substituted signal peptide is selected from any one of the nucleic acid sequences described in SEQ ID NO: 104-108 or its degenerate sequence, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 104-108.

[0033] In one implementation, the PEDV subtype is selected from GIIa, GIIb, GIIc and GI.

[0034] In one embodiment, the S protein comprises an amino acid sequence shown in any one of SEQ ID NO: 17-20 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 17-20.

[0035] In one embodiment, the nucleic acid encoding the S protein comprises the nucleotide sequence of any one of SEQ ID NO: 75-78 or its degenerate sequence, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 75-78.

[0036] In one embodiment, the S protein variant comprises an amino acid sequence shown in any one of SEQ ID NO: 13-16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 13-16.

[0037] In one embodiment, the S protein variant encodes a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NO: 71-74 or its degenerate sequence, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 71-74.

[0038] In one embodiment, the S protein or S protein variant is fused with a molecular chaperone to form a fusion protein.

[0039] In one implementation, the fusion is either a direct fusion or an indirect fusion via connectors.

[0040] In one embodiment, the linker is selected from 2A peptide and GS linker.

[0041] In one embodiment, the GS connector is selected from (GGGGS)n, (GGGS)n; preferably, n is 2, 3, 4, 5 or 6.

[0042] In one embodiment, the molecular chaperone is selected from one or more of T4 Folden (T4 fibritin), porcine IgG Fc, Ft, recombinant humanized type III collagen Rh3C, HIV gp41 6HB, GCN4, and Qα.

[0043] In one embodiment, the molecular chaperone is selected from the amino acid sequence shown in any one of SEQ ID NO:39-45 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO:39-45.

[0044] In one embodiment, the molecular chaperone encoding nucleic acid comprises a nucleotide sequence selected from any one of SEQ ID NO: 97-103 or a degenerate sequence thereof, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 97-103.

[0045] In one embodiment, the fusion protein comprises an amino acid sequence selected from any one of SEQ ID NO: 1-12 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-12.

[0046] In one embodiment, the nucleic acid encoding the fusion protein comprises a nucleotide sequence selected from any one of SEQ ID NO: 59-70 or a degenerate sequence thereof, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 59-70; preferably, the fusion protein comprises an amino acid sequence selected from any one of SEQ ID NO: 51-58, 127-130 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 51-58, 127-130.

[0047] In one embodiment, the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in any one of SEQ ID NO: 135-146 or its degenerate sequence, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 135-146.

[0048] In one embodiment, the fusion protein is further fused with an HA tag, the HA tag comprising an amino acid sequence as shown in SEQ ID NO: 147.

[0049] In one embodiment, the nucleic acid described in the second or third aspect of the present invention is DNA or RNA.

[0050] In one embodiment, the nucleic acid described in the second or third aspect of the present invention is mRNA, circular RNA, or self-replicating RNA.

[0051] In one embodiment, the RNA described in the second or third aspect of the present invention further comprises a 5'UTR, KOZAK, 3'UTR, polyA, and / or a cap structure.

[0052] In one embodiment, the RNA described in the second or third aspect of the present invention is modified RNA, wherein uracil, cytosine, adenine or guanine nucleotides contain modifying groups.

[0053] In one embodiment, the modifying group is 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.

[0054] In one embodiment, the mRNA molecule is a modified mRNA, the modification including the conversion of uracil 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.

[0055] In one embodiment, the nucleic acid described in the second or third aspect of the present invention is mRNA.

[0056] In one embodiment, the 5'UTR comprises a nucleotide sequence shown in any one of SEQ ID NO: 109, 111, 113, 115 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 109, 111, 113, 115.

[0057] In one embodiment, the 3'UTR comprises a nucleotide sequence shown in any one of SEQ ID NO: 110, 112, 114, 116 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 110, 112, 114, 116.

[0058] In one embodiment, the length of the polyA is 50-200 nt; preferably 100-155 nt.

[0059] In one embodiment, the polyA comprises a nucleotide sequence shown in any one of SEQ ID NO: 117-118 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 117-118.

[0060] In one embodiment, the nucleic acid comprises a nucleotide sequence or degenerate sequence shown in any one of SEQ ID NO: 119-126, 131-134 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 119-126, 131-134.

[0061] In one embodiment, the nucleic acid has an alternative polyA, 5'UTR, and / or 3'UTR relative to the nucleotide sequence or degenerate sequence shown in any one of SEQ ID NO: 119-126, 131-134. Preferably, the polyA may be substituted between SEQ ID NO: 117 and SEQ ID NO: 118.

[0062] In one embodiment, the cap structure is a compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer, or isotopic variant thereof:

[0063]

[0064] in,

[0065] --- indicates a single key or that the key does not exist.

[0066] X1 is selected from O, S, CH2, CH2CH2, CH=CH, CH=CHO, CH2O, OCH2, CH2CH2O, OCH2CH2, and tricyclic alkyl groups.

[0067] 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,

[0068] B1 and B2 are each independently selected from natural, modified, or non-natural nucleoside bases.

[0069] Preferably, the compound of formula (I) is any one of the following:

[0070]

[0071]

[0072] A fourth aspect of the present invention provides a nucleic acid composition, characterized in that it comprises the nucleic acid and molecular adjuvant described in the second or third aspect of the present invention.

[0073] In one embodiment, the molecular adjuvant is selected from TGF-β-P2A-BAFF (SMA-6), CD40L-P2A-APRIL, IL-10-P2A-IL-2, GM-CSF-P2A-IL-2, GM-CSF-P2A-CD40L, GM-CSF-P2A-IL-18, IL-17-P2A-IL-21 (SMA-7), IL-17, IL-21, IL-10, IL-2, GM-CSF, IL-18, CCL28, CD40L, BAFF, APRIL, and TGF-β.

[0074] In one embodiment, the molecular adjuvant is derived from pigs.

[0075] In one embodiment, the molecular adjuvant comprises an amino acid sequence as described in any one of SEQ ID NO: 21-38 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 21-38.

[0076] In one embodiment, the molecular adjuvant encoding nucleic acid comprises the nucleotide sequence shown in any one of SEQ ID NO: 79-96 or its degenerate sequence, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 79-96.

[0077] In one embodiment, the composition is a vaccine composition.

[0078] The fifth aspect of the present invention provides a vector comprising the nucleic acid described in the second and third aspects of the present invention or the nucleic acid composition described in the fourth aspect of the present invention; preferably, the vector is an expression vector.

[0079] A sixth aspect of the present invention provides a cell comprising the carrier described in the fifth aspect of the present invention.

[0080] In one embodiment, the cell is a eukaryotic cell or a prokaryotic cell.

[0081] The seventh aspect of the present invention provides a pharmaceutical composition comprising the fusion protein of the first aspect of the present invention, or the nucleic acid of the second or third aspect of the present invention, or the nucleic acid composition of the fourth aspect of the present invention, or the carrier of the fifth aspect of the present invention, or the cell of the sixth aspect of the present invention.

[0082] In one embodiment, the delivery carrier of the pharmaceutical composition includes lipid nanoparticles (LNP), viral vectors such as AAV, polymeric materials such as polyethyleneimine (PEI), polyurethane (PBAE), chitosan, or polypeptides such as cell-penetrating peptides.

[0083] In one embodiment, the lipid nanoparticles (LNPs) comprise ionizable lipids, DSPC, cholesterol, and DMG-PEG2000 ethanol.

[0084] In one embodiment, the lipid nanoparticles (LNPs) are targeted LNPs (tLNPs).

[0085] In one embodiment, the pharmaceutical composition is formulated into a dosage form suitable for intramuscular or subcutaneous injection, oral administration, or inhalation.

[0086] The eighth aspect of the present invention provides the use of the fusion protein described in the first aspect of the present invention, or the nucleic acid described in the second or third aspect of the present invention, or the nucleic acid composition described in the fourth aspect of the present invention, or the vector described in the fifth aspect of the present invention, or the cell described in the sixth aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention in the preparation of a medicament for the prevention or treatment of porcine epidemic diarrhea virus (PEDV) infection or diseases caused by PEDV infection.

[0087] In one implementation, the drug is a vaccine.

[0088] In one implementation, the disease is porcine epidemic diarrhea.

[0089] The main advantages of this invention are: the PEDV mRNA vaccine of this invention can stimulate sows to produce colostrum containing high levels of PEDV neutralizing antibodies, and the level of neutralizing antibodies is significantly higher than that of commercially available PEDV inactivated vaccines; in addition, by utilizing the high inclusiveness of the LNP mRNA vaccine delivery system, the mRNA sequence of PEDV antigen and the mRNA of cytokines that enhance cellular immunity and mucosal immunity are co-encapsulated and administered simultaneously, further enhancing the cellular immunity and mucosal immunity of the PEDV vaccine, thereby providing a new weapon for the prevention and control of PEDV. Detailed Implementation

[0090] 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.

[0091] 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.

[0092] The term "swine epidemic diarrhea virus" or "PEDV" belongs to the genus *Alphavirus* of the family Coronaviridae. It causes acute diarrhea, vomiting, and dehydration in newborn piglets, resulting in high mortality. PEDV has a single-stranded, positive-sense RNA genome, approximately 28 kb in size (excluding polyA), encoding four structural proteins: the S protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein, 16 non-structural proteins (nsp1-nsp16), and one accessory protein, ORF3. The S protein is crucial for the interaction between the virus and specific host cell receptors. This interaction mediates viral binding and entry into cells, as well as the formation of syncytial bodies, and induces the production of neutralizing antibodies. The S protein is divided into S1 (aa1-726) and S2 (aa727-1386) subunits. The N-terminal S1 subunit contains a receptor-binding domain, while the C-terminal S2 subunit is responsible for membrane fusion. Based on the homology of the S gene of PEDV or its N-terminal hypervariable region S1 region sequence, PEDV can be divided into two genotypes—G1 and G2. G1 is further divided into Gla and Glb subtypes, and G2 is further divided into G2a, G2b and G2c subtypes.

[0093] The term "pre-fusion conformation" refers to the structural protein of a virus before membrane fusion occurs, prior to infection of host cells. Typically, the wild-type PEDV S protein is a metastable protein before fusion. Its conformation undergoes a discontinuous, gradual, and irreversible conformational change after viral infection and membrane fusion, transforming into a lower-energy stable conformation, known as the "post-fusion conformation." Mutations of amino acid residues such as proline and cysteine ​​at specific sites in the S protein, as well as splicing substitutions, can enhance the stability of the pre-fusion trimer conformation and antigen. When the mutant S protein in its pre-fusion conformation is used as a vaccine or vaccine component, it can induce higher levels of neutralizing antibodies in immunized animals, exhibiting increased immunogenicity. It is applicable to various PEDV strains, including classic strain 1 (1a and / or 1b) and / or variant strain 2 (2a, 2b, and / or 2c).

[0094] The term "protein variant" refers to a compound that has sequence homology with a wild-type protein or polypeptide. Protein variants described in this application may include proteins with altered amino acid sequences obtained through insertion, deletion, modification, and / or substitution of one or more amino acid residues, while retaining at least one biological activity of the parent wild-type sequence. For example, the variant may have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the parent protein. Insertions, deletions, or substitutions in the variant may be conserved or non-conserved. The variant may be naturally occurring or non-naturally occurring. Non-naturally occurring variants may be generated using techniques known in the art.

[0095] The term "molecular chaperone," also known as chaperone proteins, refers to a class of proteins that assist in intracellular molecular assembly and protein folding. Common molecular chaperones include T4 Folden (T4-Fibritin), porcine IgG Fc, Ft, recombinant humanized type III collagen Rh3C, HIV gp41 6HB, GCN4, and Qα. Among them, T4 Folden, recombinant humanized type III collagen Rh3C, HIV gp41 6HB, and GCN4 are trimerizing domains, which can stabilize the trimerizing conformation of antigen proteins; porcine IgG Fc is a dimerizing domain, derived from the constant region of porcine IgG Fc, which can enable antigen proteins to form a dimer structure; ferritin (Ft) is derived from Helicobacter pylori, and Ft self-assembles with various expressed fusion proteins to form nanoparticles of 24 identical polypeptides; Qα plays a role in maintaining mRNA stability.

[0096] The term "oligomery domain" includes, but is not limited to, polypeptide sequences that can be used to increase the stability of oligomeric envelope proteins, such as those used to increase the stability of PEDVS protein trimers. Oligomery domains can be used to increase the stability of homo-oligomeric peptides and hetero-oligomeric peptides. Oligomery domains are well known in the art and include "trimery domains".

[0097] The term “trimerization domain” refers to the oligomerization domain of a stable trimerized polypeptide. Examples of trimerization domains include, but are not limited to, the “foldon” trimerization domain of T4-fibritin (i.e., “T4 Folden”), the coiled-coil trimerization domain obtained from GCN4 (Yang et al. (2002) J.Virol. 76:4634), and the core structure HIV gp41 6HB derived from the HIV gp41 protein.

[0098] The term "fusion protein" refers to a protein formed by covalently linking two protein parts that do not exist together under natural conditions. In this article, "fusion protein" can refer to PEDVI type S protein-molecular chaperone fusion protein, PEDVI type S protein variant-molecular chaperone fusion protein, GIIa type S protein-molecular chaperone fusion protein, GIIa type S protein variant-molecular chaperone fusion protein, GIIb type S protein variant-molecular chaperone fusion protein, GIIb type S protein variant-molecular chaperone fusion protein, GIIc type S protein-molecular chaperone fusion protein, and GIIc type S protein variant-molecular chaperone fusion protein.

[0099] The term "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 the growing peptide chain across this membrane for processing. ER processing produces a mature protein, in which the signal peptide is typically cleaved from the precursor protein by the host cell's ER-resident signal peptidase, or remains unclew and acts as a membrane anchor. Signal peptides can also promote protein targeting to the cell membrane. Secretory signal peptides can be located at the N-terminus of the fusion protein. The type of secretory signal peptide is not particularly limited, as long as it can guide the secretion of the synthesized fusion protein. Secretory signal peptides can include, but are not limited to, IgG signal peptides, IL-2 signal peptides, tPA signal peptides, Ig kappa signal peptides, and SigMHC signal peptides.

[0100] The term "helper epitope" refers to all epitopes that can assist T cell activation, including PADRE and IL-1β epitopes. PADRE is a short peptide sequence of 13 amino acids that can bind to different DR molecules, be presented on the cell surface, and thereby activate CD4+ T helper cells. PADRE's ability to induce T cell responses is more than 1000 times that of natural epitopes. In vivo, PADRE can immunely activate helper T cells (Th1) to assist CTL activation and can also activate helper T cells (Th2) to assist B cells in secreting specific antibodies. The IL-1β epitope is a short linear peptide sequence of 9 amino acids. Interleukin-1 plays an important role in enhancing cellular and humoral immune responses to foreign antigens, and the IL-1β epitope can enhance immune responses without producing adverse inflammatory effects.

[0101] 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 may 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 may 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 may 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.

[0102] The terms “non-natural” or “engineered” refer to proteins, peptides, nucleic acids, or strains that are not normally present in nature, including at least one modified form.

[0103] The term "isolated nucleic acid" refers to nucleic acid fragments that are not naturally occurring as fragments and do not exist in their natural state. The term "isolated" is also used to refer to polypeptides, proteins, and / or host cells isolated from other cellular proteins, and is intended to include purified and recombinant polypeptides. In other embodiments, the term "isolated" means isolated from components, cells, and other substances that are normally naturally associated with cells, tissues, polynucleotides, peptides, polypeptides, or proteins. "Non-naturally occurring" polynucleotides, peptides, polypeptides, or proteins do not need to be "isolated" to distinguish them from their naturally occurring counterparts.

[0104] The term "mRNA" refers to messenger RNA, a transcript that can be transcribed using a DNA template and may encode a peptide or protein. mRNA is typically single-stranded but may also contain its own complementary sequence. Generally, mRNA contains a 5' cap, a 5'-UTR, a coding region, a 3'-UTR, and a polyA tail. In the context of this invention, mRNA is preferably produced from a DNA template via in vitro transcription (IVT). In vitro transcription methods are known to those skilled in the art, and various in vitro transcription kits are commercially available.

[0105] The term "circular RNA" refers to a closed circular RNA molecule formed by covalent bonds. Circular RNA molecules can be formed by the connection of the 5' end upstream and the 3' end downstream of a linear RNA molecule. Circular RNA possesses protein translation activity and can also be called "circular mRNA."

[0106] The term "self-replicating RNA (srRNA)," also known as "self-amplifying RNA (saRNA)," refers to RNA that, in addition to expressing the target protein, carries a sequence capable of expressing RNA polymerase (called RNA-dependent RNA polymerase, RdRP). This RNA polymerase, once generated, can use the saRNA as a template to produce more copies of saRNA. Sequence-wise, saRNA contains conventional mRNA sequence elements (such as the 5'UTR, protein-coding region (CDS), 3'UTR, and poly(A) tail), plus an additional coding region: a non-structural protein (nsp) gene sequence downstream of the 5'UTR, and a subgenomic promoter sequence (SGP) upstream of the target protein coding sequence. This allows for the expression of RNA replicase within the cell, enabling self-replication. The saRNA backbone is derived from alphaviruses, flaviviruses, lentiviruses, and other viruses.

[0107] The term "sequence identity" refers to the degree to which two sequences (amino acid sequences) 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.

[0108] 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.

[0109] The term "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.

[0110] The term "5' untranslated region" (5'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.

[0111] The term "3' untranslated region" (3'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.

[0112] The term "Poly-A" 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., 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. The Poly-A tail can be used to protect mRNA, for example, from enzymatic degradation in the cytoplasm, and facilitates transcription termination and / or the export of mRNA from the nucleus for translation.

[0113] The term "KOZAK" refers to a nucleic acid sequence, usually GCCACC, located after the 5' cap structure of eukaryotic mRNA. It can bind to translation initiation factors and mediate the translation initiation of mRNA containing the 5' cap structure.

[0114] The term "ionizable lipid" refers to an amphiphilic molecule (e.g., a lipid or lipidoid, such as a 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. In some embodiments, the ionizable lipid is SM-102.

[0115] The term "pharmaceutically acceptable" means a molecule or composition that, when administered to a recipient, is harmless to the recipient or whose benefits to the recipient outweigh any adverse effects. Regarding carriers or excipients used to formulate compositions as disclosed herein, pharmaceutically acceptable carriers or excipients must be compatible with the other components of the composition and be harmless to the recipient or whose benefits to the recipient outweigh any adverse effects.

[0116] The term "pharmaceutically acceptable carrier" refers to 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 drug 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 other components of the formulation and is not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) excipients, such as cocoa butter and suppository waxes; (8) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (9) glycols, such as propylene glycol; and (10) polyols, such as glycerol and sorbitol. (11) Mannitol and polyethylene glycol (PEG); (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide and aluminum hydroxide; (15) Alginate; (16) Pyrothermic water; (17) Isotonic saline; (18) Ringer's solution; (19) pH buffer solution; (20) Polyesters, polycarbonates and / or polyanhydrides; (21) Fillers, such as peptides and amino acids; (22) Serum components, such as serum albumin, HDL and LDL; (23) C2-C12 alcohols, such as ethanol; and (24) Other non-toxic and compatible substances used in pharmaceutical formulations. In the context of this document, those skilled in the art may select a carrier suitable for acceptance on a pharmaceutically acceptable protein or nucleic acid as described herein.

[0117] The term "molecular adjuvant" refers to an auxiliary substance that can enhance the body's immune response to antigens or alter the type of immune response. The molecular adjuvants discussed in this article include interleukins (IL-2, IL-10, IL-17, IL-18, IL-21), chemokines (CCL28), tumor necrosis factor (TGF-β, BAFF, APRIL, CD40L, etc.), colony-stimulating factor (GM-CSF), combinations of the above cytokines, fusion proteins of the same type of cytokines, and combinations of different types of cytokines. The functions of these molecular adjuvants are shown in Table a below:

[0118]

[0119] PEDV S protein variants and their encoded nucleic acids

[0120] This document provides a PEDV S protein variant having one or more amino acid mutations relative to the parent. The mutation location is selected from one or more of K893P, R894P, A969P, A1032P, D1076P, and I1077P. In some embodiments, the mutation location is a double mutation of D1076P and I1077P, which may be simply referred to herein as the "2P" mutation. In some embodiments, the mutation stabilizes the S protein in its pre-fusion state.

[0121] Those skilled in the art will understand that the parental S protein can be any suitable S protein. For example, the parental S protein can be a wild-type S protein or a variant of the S protein that exists in the prior art.

[0122] In some embodiments, the parental S protein may originate from the PEDV GI or GII genotype, and the parental S protein may originate from the GIIa, GIIb, or GIIc subpopulations. The full-length sequence of the GIIa S protein is shown in SEQ ID NO:17, the full-length sequence of the GIIb S protein is shown in SEQ ID NO:18, the full-length sequence of the GIIc S protein is shown in SEQ ID NO:19, and the full-length sequence of the GI S protein is shown in SEQ ID NO:20.

[0123] For the purposes of this invention, the amino acid sequence of any one of SEQ ID NO:17-20 is used to determine the corresponding amino acid residues in another PEDV S protein. The amino acid sequence of the other PEDV S protein is aligned with any one of SEQ ID NO:17-20, and based on this alignment, the Niederman-Onsch algorithm (Niederman and Onsch, 1970, Journal of Molecular Biology 48:443-453) implemented in the Nieder program of the EMBOSS package (EMBOSS: European Open Software Suite for Molecular Biology, Rice et al., 2000, Trends in Genetics 16:276-277) (preferably version 5.0.0 or higher) is used to determine the amino acid position number corresponding to any amino acid residue in the mature polypeptide of any one of SEQ ID NO:17-20. For example, parameters that can be used include a nick opening penalty of 10, a nick extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0124] In some embodiments, the mutation position of the PEDV S protein variant is numbered according to the parental amino acid sequence shown in SEQ ID NO: 17, and the mutation is a "2P" mutation, the sequence of which is shown in SEQ ID NO: 13.

[0125] GIIa S full length (Prefusion) SEQ ID NO: 13

[0126] pp SADVQVDRLITGRLSALNAFVAQTLTKYTEVQASRKLAQQKVNECVKSQSQRYGFCGGDGEHIFSLVQAAPQGLLFLHTVLVPGDFVDVIIAAGLCVNDEIALTLREPGLVLFTHELQNHTATEYFVSSRRMFEPRKPTVSDFVQIESCVVTYV NLTRDQLPDVIPDYIDVNKTLDEILASLPNRTGPSFPLDVFNATYLNLTGEIADLEQRSESLRNTTEELQSLIYNINNTLVDLEWLNRVETYIKWPWWVWLIIFIVLIFVVSLLVFCCISTGCCGCCGCCCACFSGCCRGPRLQPYEVFEKVHVQ

[0127] In some embodiments, the mutation position of the PEDV S protein variant is numbered according to the parental amino acid sequence shown in SEQ ID NO: 18, and the mutation is a "2P" mutation, the sequence of which is shown in SEQ ID NO: 14.

[0128] GIIb S full length (Prefusion) SEQ ID NO:14

[0129] PP SADVQVDRLITGRLSALNAFVAQTLTKYTEVQASRKLAQQKVNECVKSQSQRYGFCGGDGEHIFSLVQAAPQGLLFLHTVLVPGDFVDVIIAAGLCVNDEIALTLREPGLVLFTHELQNHTATEYFVSSRRMFEPRKPTVSDFVQIESCVVTYV NLTRDQLPDVIPDYIDVNKTLDEILASLPNRTGPSLPLDVFNATYLNLTGEIADLEQRSESLRNTTEELQSLIYNINNTLVDLEWLNRVETYIKWPWWVWLIIFIVLIFVVSLLVFCCISTGCCGCCGCCCACFSGCCRGPRLQPYEVFEKVHVQ

[0130] In some embodiments, the mutation position of the PEDV S protein variant is numbered according to the parental amino acid sequence shown in SEQ ID NO: 19, and the mutation is a "2P" mutation, the sequence of which is shown in SEQ ID NO: 15.

[0131] GIIc S full length (Prefusion) SEQ ID NO:15

[0132] pp SADVQVDRLITGRLSALNAFVSQTLTKYTEVQASRKLAQQKVNECVKSQSQRYGFCGGDGEHIFSLVQAAPQGLLFLHTVLVPGDFVDVIIAAGLCVNDEIALTLREPGLVLFTHELQNHTATEYFVSSRRMFEPRKPTVSDFVQIESCVVTYV NLTRDQLPDVIPDYIDVNKTLDEILASLPNRTGPSLPLDVFNATYLNLTGEIADLEQRSESLRNTTEELQSLIYNINNTLVDLEWLNRVETYIKWPWWVWLIIFIVLIFVVSLLVFCCISTGCCGCCSCCCACFSGCCRGPRLQPYEVFEKVHVQ

[0133] In some embodiments, the mutation position of the PEDV S protein variant is numbered according to the parental amino acid sequence shown in SEQ ID NO: 20, and the mutation is a "2P" mutation. The sequence of the PEDV S protein variant is shown in SEQ ID NO: 16.

[0134] GI S full length (Prefusion) SEQ ID NO:16

[0135] pp SADVQVDRLITGRLSALNAFVAQTLTKYTEVQASRKLAQQKVNECVKSQSQRYGFCGGDGEHIFSLVQAAPQGLLFLHTVLVPGDFVDVIIAAGLCVNDEIALTLREPGLVLFTHELQNHTATEYFVSSRRMFEPRKPTVSDFVQIESCVVTYV NLTRDQLPDVIPDYIDVNKTLDEILASLPNRTGSSLPLDVFNATYLNLTGEIADLEQRSESLRNTTEELQSLIYNINNTLVDLEWLNRVETYIKWPWWVWLIIFIVLIFVVSLLVFCCISTGCCGCCGCCCACFSGCCRGPRLQPYEVFEKVHVQ

[0136] In some embodiments, the S protein variant further comprises a deleted or replaced signal peptide.

[0137] In some embodiments, the replacement signal peptide is a signal peptide selected from porcine IL-2 signal peptide, tPA signal peptide, porcine IgG heavy chain signal peptide, porcine IL-6 signal peptide, and porcine IL-10 signal peptide, replacing the natural signal peptide in the parent.

[0138] This document also provides a nucleic acid encoding the aforementioned S protein variant. In some embodiments, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 71 or a degenerate sequence thereof; in some embodiments, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 72 or a degenerate sequence thereof; in some embodiments, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 73 or a degenerate sequence thereof; in some embodiments, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 74 or a degenerate sequence thereof.

[0139] Fusion protein

[0140] This article provides fusion proteins of PEDV S protein or S protein variants with molecular chaperones.

[0141] In some implementations, the fusion is either a direct fusion or an indirect fusion via connectors.

[0142] In some embodiments, the linker is selected from 2A peptide and GS linker.

[0143] In some embodiments, the molecular chaperone is selected from one or more of T4 Folden, porcine IgG Fc, Ft, recombinant humanized type III collagen Rh3C, HIV gp41 6HB, GCN4, and Qα.

[0144] In some embodiments, the molecular chaperone is T4 Folden.

[0145] SEQ ID NO:39: Amino acid sequence of T4 Folden

[0146] GYIPEAPRDGQAYVRKDGEWVFLSTFLSPA (SEQ ID NO:39).

[0147] In some embodiments, the molecular chaperone is GCN4.

[0148] SEQ ID NO:44: Amino acid sequence of GCN4

[0149] RMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGR (SEQ ID NO: 44).

[0150] In some embodiments, the molecular chaperone is Ft.

[0151] SEQ ID NO:41: Amino acid sequence of Ft

[0152] LSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO: 41).

[0153] In some embodiments, the fusion protein is a full-length GIIa S (Prefusion)-(G3S)2-T4folden, which includes the amino acid sequence shown in SEQ ID NO:1.

[0154] In some embodiments, the fusion protein is the full-length GIIb S (Prefusion)-(G3S)2-T4folden, which includes the amino acid sequence shown in SEQ ID NO:2.

[0155] In some embodiments, the fusion protein is the full-length GIIc S (Prefusion)-(G3S)2-T4folden, which includes the amino acid sequence shown in SEQ ID NO:3.

[0156] In some embodiments, the fusion protein is a full-length GIS (Prefusion)-(G3S)2-T4 folden, which includes the amino acid sequence shown in SEQ ID NO:4.

[0157] In some embodiments, the fusion protein is full-length GIIa S (Prefusion)-(G3S)2-GCN4, which includes the amino acid sequence shown in SEQ ID NO:5.

[0158] In some embodiments, the fusion protein is GIIb S full-length (Prefusion)-(G3S)2-GCN4, which includes the amino acid sequence shown in SEQ ID NO:6.

[0159] In some embodiments, the fusion protein is GIIc S full-length (Prefusion)-(G3S)2-GCN4, which includes the amino acid sequence shown in SEQ ID NO:7.

[0160] In some embodiments, the fusion protein is GIS full-length (Prefusion)-(G3S)2-GCN4, which includes the amino acid sequence shown in SEQ ID NO:8.

[0161] In some embodiments, the fusion protein is full-length GIIa S (Prefusion)-(G3S)2-Ft, which includes the amino acid sequence shown in SEQ ID NO:9.

[0162] In some embodiments, the fusion protein is full-length GIIb S (Prefusion)-(G3S)2-Ft, which includes the amino acid sequence shown in SEQ ID NO:10.

[0163] In some embodiments, the fusion protein is GIIc S full-length (Prefusion)-(G3S)2-Ft, which includes the amino acid sequence shown in SEQ ID NO:11.

[0164] In some embodiments, the fusion protein is full-length GIS (Prefusion)-(G3S)2-Ft, which includes the amino acid sequence shown in SEQ ID NO:12.

[0165] In some embodiments, the fusion protein further includes a molecular chaperone Qα and an optional HA tag.

[0166] In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NO: 51-58, 127-130.

[0167] In some embodiments, the fusion protein is the full-length GIIa S (Prefusion)-(G3S)2-T4folden-Qα, which includes the amino acid sequence shown in SEQ ID NO:51.

[0168] GYIPEAPRDGQAYVRKDGEWVFLSTFLSPA QPRFAAA

[0169] In some embodiments, the fusion protein is the full-length GIIb S (Prefusion)-(G3S)2-T4folden-Qα, which includes the amino acid sequence shown in SEQ ID NO:52.

[0170] GYIPEAPRDGQAYVRKDGEWVFLSTFLSPA QPRFAAA

[0171] In some embodiments, the fusion protein is the full-length GIIc S (Prefusion)-(G3S)2-T4folden-Qα, which includes the amino acid sequence shown in SEQ ID NO:53.

[0172] GYIPEAPRDGQAYVRKDGEWVFLSTFLSPA QPRFAAA

[0173] In some embodiments, the fusion protein is the full-length GIS (Prefusion)-(G3S)2-T4folden-Qα, which includes the amino acid sequence shown in SEQ ID NO:54.

[0174] GYIPEAPRDGQAYVRKDGEWVFLSTFLSPA QPRFAAA

[0175] In some embodiments, the fusion protein is the full-length GIIa S (Prefusion)-(G3S)2-GCN4, which includes the amino acid sequence shown in SEQ ID NO:55.

[0176] RMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGR QPRFAAA

[0177] In some embodiments, the fusion protein is the full-length GIIb S (Prefusion)-(G3S)2-GCN4-Qα, which includes the amino acid sequence shown in SEQ ID NO:56.

[0178] RMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGR QPRFAAA

[0179] In some embodiments, the fusion protein is the full-length GIIc S (Prefusion)-(G3S)2-GCN4-Qα, which includes the amino acid sequence shown in SEQ ID NO:57.

[0180] RMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGR QPRFAAA

[0181] In some embodiments, the fusion protein is the full-length GIS (Prefusion)-(G3S)2-GCN4-Qα, which includes the amino acid sequence shown in SEQ ID NO:58.

[0182] RMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGR QPRFAAA

[0183] In some embodiments, the fusion protein may further include an HA tag in addition to any one of SEQ ID NO: 51-58, 127-130; preferably, the HA tag is directly attached to the C-terminus of the fusion protein; preferably, the sequence of the HA tag is as shown in SEQ ID NO: 147.

[0184] The present invention also provides a nucleic acid encoding the above-mentioned fusion protein.

[0185] In some embodiments, the nucleic acid encodes the fusion protein described in any one of SEQ ID NO: 1-12. Preferably, the nucleic acid comprises the nucleotide sequence shown in any one of SEQ ID NO: 59-70 or a degenerate sequence thereof.

[0186] In some embodiments, the nucleic acid encodes the fusion protein described in any one of SEQ ID NOs: 51-58, 127-130. Preferably, the nucleic acid comprises the nucleotide sequence shown in any one of SEQ ID NOs: 135-146 or a degenerate sequence thereof.

[0187] In some embodiments, the nucleic acid encoding a fusion protein obtained by further fusing an HA tag based on any one of SEQ ID NO: 51-58, 127-130.

[0188] Nucleic acid

[0189] This article provides an isolated nucleic acid, which is a DNA or RNA molecule, 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.

[0190] The nucleic acids of particular interest in this invention are mRNA molecules, such as those containing a read frame encoding an immunogenic protein.

[0191] mRNA molecules

[0192] 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”.

[0193] 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).

[0194] Chemical modification

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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).

[0204] 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.

[0205] 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.

[0206] mRNA molecule preparation methods

[0207] 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) enzymes. 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 multiple thymidines or their derivatives and / or multiple 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 (WO2014 / 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).

[0208] 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.

[0209] 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.

[0210] 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, capping is performed using the following kits: CAP m7Gppp(2'OMeA)pG or CAP5m7G(5')vppp(5')(2'OMeA)pG. 5' capping of mRNA can also be performed post-transcriptionally using a vaccinia virus capping enzyme to produce the Cap 0 structure. 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.

[0211] Elements of mRNA molecules

[0212] In some implementations, in addition to the read frame encoding the immunogenic protein, 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] In some implementations, the Poly-A tail contains a polynucleotide sequence of SEQ ID NO:117 or 118.

[0217] In some embodiments, the 5'UTR contains the polynucleotide sequence shown in any one of SEQ ID NO:109,111,113,115 or a variant polynucleotide sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of any one of SEQ ID NO:109,111,113,115.

[0218] In some embodiments, the 3'UTR contains the polynucleotide sequence shown in any one of SEQ ID NO:110, 112, 114, 116 or a variant polynucleotide sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of any one of SEQ ID NO:110, 112, 114, 116.

[0219] In some embodiments, the 5'UTR and 3'UTR are selected from combinations of the following sequences or variant polynucleotide sequences: SEQ ID NO: 109 and 110; SEQ ID NO: 111 and 112; SEQ ID NO: 113 and 114; SEQ ID NO: 115 and 116.

[0220] Typical mRNA sequence

[0221] In some embodiments, the mRNA of the present invention comprises an open reading frame containing a nucleotide sequence shown in any one of SEQ ID NO: 135-146 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 135-146.

[0222] In some embodiments, the open reading frame further includes a nucleotide sequence encoding an HA tag; preferably, the nucleotide sequence encoding the HA tag is directly linked to the 3' end of SEQ ID NO: 135-146.

[0223] In some embodiments, the mRNA of the present invention comprises the nucleotide sequence described in any one of SEQ ID NO: 119-126, 131-134 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 119-126, 131-134.

[0224] In some embodiments, the mRNA comprises the nucleotide sequence shown in any one of SEQ ID NO: 119-126.

[0225] In some embodiments, the mRNA encodes the full-length GIIa S (Prefusion)-(G3S)2-T4folden-Qα-HA tag, which contains the nucleotide sequence shown in SEQ ID NO: 119.

[0226]

[0227] In some embodiments, the mRNA encodes the full-length GIIb S (Prefusion)-(G3S)2-T4folden Qα-HA tag, which contains the nucleotide sequence shown in SEQ ID NO: 120.

[0228]

[0229] In some embodiments, the mRNA encodes the full-length GIIc S (Prefusion)-(G3S)2-T4folden Qα-HA tag, which contains the nucleotide sequence shown in SEQ ID NO: 121.

[0230]

[0231] In some embodiments, the mRNA encodes the full-length GIS (Prefusion)-(G3S)2-T4 foldenQα-HA tag, which contains the nucleotide sequence shown in SEQ ID NO: 122.

[0232]

[0233]

[0234] In some embodiments, the mRNA encodes the full-length GIIb S (Prefusion)-(G3S)2-GCN4-Qα-HA tag, which contains the nucleotide sequence shown in SEQ ID NO: 124.

[0235]

[0236] In some embodiments, the mRNA encodes the full-length GIIc S (Prefusion)-(G3S)2-GCN4-Qα-HA tag, which contains the nucleotide sequence shown in SEQ ID NO: 125.

[0237]

[0238] In some embodiments, the mRNA encodes the full-length GIS (Prefusion)-(G3S)2-GCN4-Qα-HAtag, which contains the nucleotide sequence shown in SEQ ID NO: 126.

[0239]

[0240] In some embodiments, the mRNA of the present invention further comprises a 5' cap structure.

[0241] Nucleic acid combination

[0242] This article provides a composition of the above-mentioned nucleic acid encoding the fusion protein and the nucleic acid encoding the molecular adjuvant.

[0243] In some embodiments, the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in any one of SEQ ID NO:59-70.

[0244] In some embodiments, the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in any one of SEQ ID NO:135-146.

[0245] In some embodiments, the nucleic acid encoding the fusion protein comprises the nucleotide sequence shown in any one of SEQ ID NO: 119-126 and SEQ ID NO: 131-134.

[0246] In some embodiments, the molecular adjuvant is selected from one or more of porcine TGF-β-P2A-porcine BAFF (SMA-6), porcine CD40L-P2A-porcine APRIL, porcine IL-10-P2A-porcine IL-2, porcine GM-CSF-P2A-porcine IL-2, porcine GM-CSF-P2A-porcine CD40L, porcine GM-CSF-P2A-porcine IL-18, porcine IL-17-P2A-porcine IL-21 (SMA-7), porcine IL-17, porcine IL-21, porcine IL-10, porcine IL-2, porcine GM-CSF, porcine IL-18, porcine CCL28, porcine CD40L, porcine BAFF, porcine APRIL, and porcine TGF-β.

[0247] In some embodiments, the molecular adjuvant comprises the amino acid sequence shown in any one of SEQ ID NO: 21-38.

[0248] In some embodiments, the nucleic acid encoding the molecular adjuvant comprises the nucleotide sequence or a degenerate sequence thereof shown in any one of SEQ ID NO: 79-96.

[0249] In some embodiments, the molecular adjuvant is SMA-6, whose amino acid sequence is SEQ ID NO:21; and the nucleic acid sequence encoding SMA-6 is SEQ ID NO:79.

[0250] SMA-6 amino acid sequence: SEQ ID NO: 21

[0251] MPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGDVPPGPLPEAVLALYNSTRDRVAGESVEPEPEPEADYYAKEVTRVLMVESGNQIYDKFKGTPHSLYMLFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNDSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLTRREAIEGFRLSAHCSCDSKDNTLHVEINGFNSGRRGDLATIHGMNRPFLLLMATPLERAQHLHSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCSGSGATNFSLLKQAGDVEENPGPYESTGEQSRLSCLSTREEMKMKETVPILPQKESPSVRISKDGKLLVVTLLLALLSCFLTGIFAPPAPRESSSIQSNRSKRAAQDAEETVTQDCLQLIADSDMPTIRKGAYTFVPWLLSFKRGRALGEKENKTVVKETGYFFIYGQVLYTDNTFAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPREDAKISRDGDGTFFGALKLL

[0252] SMA-6 nucleic acid sequence: SEQ ID NO:79

[0253]

[0254] Formulations or compositions

[0255] Formulations or compositions containing nucleic acids (e.g., mRNA molecules) are known in the art and are described, for example, in WO2013 / 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.

[0256] vaccine

[0257] This document also provides information on 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 read frames of immunogenic proteins. Nucleic acid vaccines may contain mRNA molecules containing any one or more of SEQ ID NO: 119-126, 131-134 (e.g., 2-35, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35) of a nucleic acid sequence or a variant sequence.

[0258] In some embodiments, two or more different mRNAs may be formulated into the same lipid nanoparticle. In some embodiments, two or more different RNAs encoding antigens may be formulated into separate lipid nanoparticles. The lipid nanoparticles may then be combined and administered as a single vaccine composition (e.g., containing multiple RNAs encoding multiple antigens), or they may be administered alone.

[0259] Vaccine administration

[0260] In some embodiments, the compositions, formulations, or vaccines described herein may be administered to a subject (e.g., a mammalian subject, such as a pig). 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.

[0261] 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.

[0262] In some embodiments, the vaccine described herein can be used to treat or prevent PEDV infection. The vaccine can be administered prophylactically or therapeutically to healthy individuals as part of an active immunization program, or during the incubation period or during active infection after the onset of symptoms. In some embodiments, the vaccine can treat subjects already infected with PEDV. In some embodiments, the amount of RNA provided to cells, tissues, or subjects can be an amount effective for immunoprophylaxis or treatment.

[0263] Vaccines may be administered in combination with other prophylactic or therapeutic compounds. As a non-limiting example, the prophylactic or therapeutic compound may be an adjuvant or a booster. 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 administration of 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.

[0264] In some implementations, the mRNA molecules or vaccines described herein are administered at doses of 1 μg, 2 μg, 5 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or more. In some implementations, the mRNA molecules or vaccines described herein are in unit dose form, each unit dose may contain 1 μg, 2 μg, 5 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 μg, etc. The mRNA molecule or vaccine described herein may be in doses of g, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg or more.

[0265] Example

[0266] The invention will be more readily understood by referring to the following examples, which are only used to illustrate certain aspects and embodiments of the invention and are not intended to limit the invention.

[0267] Material

[0268] Unless otherwise stated, all reagents used in this embodiment are commercially available or conventional materials.

[0269] Example 1: PEDV mRNA vaccine design

[0270] Using pigs as the host, codon optimization was performed on the PEDV S antigen and molecular adjuvant sequences. Corresponding coding nucleotide sequences were generated based on the antigen amino acid sequence (Tables 1 and 3), and corresponding coding nucleotide sequences were generated based on the molecular adjuvant amino acid sequence (Table 4). A T7 promoter, a 5' UTR (Table 6), and a start codon were sequentially added to the 5' end of each nucleotide sequence. A Qα sequence (SEQ ID NO: 103), a stop codon, a 3' UTR (Table 6), and a polyA (Table 7) were sequentially added to the 3' end of each nucleotide sequence before gene synthesis. The relevant plasmids were constructed using conventional molecular biology methods in this field. Specific sequence information is shown in Tables 1-8 below.

[0271] Table 1 S protein antigen

[0272] PEDV S protein amino acids Nucleic acid GIIa S full length (Prefusion) SEQ ID NO:13 SEQ ID NO:71 GIIb S full length (Prefusion) SEQ ID NO:14 SEQ ID NO:72 GIIc S Full Length (Prefusion) SEQ ID NO:15 SEQ ID NO:73 GI S full length (Prefusion) SEQ ID NO:16 SEQ ID NO:74 GIIa S full length (Wild type) SEQ ID NO:17 SEQ ID NO:75 GIIb S full length (Wild type) SEQ ID NO:18 SEQ ID NO:76 GIIc S full length (Wild type) SEQ ID NO:19 SEQ ID NO:77 GI S full length (Wild type) SEQ ID NO:20 SEQ ID NO:78

[0273] Table 2 Molecular Chaperones

[0274]

[0275]

[0276] Table 3 S protein-molecular chaperone fusion protein

[0277]

[0278] Table 4 Molecular Adjuvants

[0279]

[0280]

[0281] Table 5 Signal peptide sequences

[0282] signal peptide amino acids Nucleic acid porcine IL-2 signal peptide SEQ ID NO:46 SEQ ID NO:104 tPA signal peptide SEQ ID NO:47 SEQ ID NO:105 porcine IgG heavy chain signal peptide SEQ ID NO:48 SEQ ID NO:106 porcine IL-6 signal peptide SEQ ID NO:49 SEQ ID NO:107 porcine IL-10 signal peptide SEQ ID NO:50 SEQ ID NO:108

[0283] Table 6 UTR

[0284] UTR Nucleic acid 5'UTR(Homo sapiens hemoglobin subunit beta) SEQ ID NO:109 3'UTR(Homo sapiens alpha-1-globin) SEQ ID NO:110 5'UTR(Sus_scrofa HBB) SEQ ID NO:111 3'UTR(Sus_scrofa HBA) SEQ ID NO:112 5'UTR (Homo sapiens ribosomal protein L32) SEQ ID NO:113 3'UTR (Homo sapiens serum albumin) SEQ ID NO:114 5'UTR (Sus_scrofa THRAP3) SEQ ID NO:115 3'UTR (Sus_scrofa THRAP3) SEQ ID NO:116

[0285] Table 7 polyA

[0286] polyA name Sequence polyA SEQ ID NO:117 polyA-1 SEQ ID NO:118

[0287] Table 8 Complete Antigen Construction

[0288]

[0289] In Table 8 above, “ORF nucleic acid” encodes the amino acid sequence of SEQ ID NO: 51-58, 127-130; “full-length nucleic acid” encodes the amino acid sequence of SEQ ID NO: 51-58, 127-130 with the HA tag further fused at the C-terminus. “Full-length nucleic acid” also includes the 5'UTR, KOZAK, 3'UTR and polyA sequences.

[0290] Example 2: Preparation of PEDV mRNA-LNP formulation

[0291] This invention uses linear double-stranded DNA containing the T7 promoter sequence listed in Table 9 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 10 μl of CAP5 [m7G(5')vppp(5')(2'OMeA)pG]. The reaction is carried out at 37°C for 150 minutes to synthesize mRNA stock solution with a 5' cap and a 3' polyA tail through in vitro transcription.

[0292] Table 9. Description of PEDV mRNA vaccine sequences

[0293]

[0294]

[0295] Note: 2P in the table represents the double mutation of D1076P and I1077P.

[0296] LNP encapsulation was performed on the stock mRNA solution based on the PEDV S protein antigen sequence in Table 10. The encapsulation steps are as follows:

[0297] mRNA stock solution was encapsulated using the following lipid formulation (ionizable lipid (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 (Shanghai) Instruments Technology Co., Ltd.). TMThe samples were prepared by mixing (L / L+). The collected sample solution was diluted 10-fold in DPBS buffer and then passed through a 100kDa Pall ultrafiltration tube. After centrifugation and ultrafiltration, the ethanol content in the sample solution was removed. Finally, the solution was adjusted to the appropriate concentration with 10mM Tris 0.1% NaCl (pH 7.4) buffer. The mRNA vaccine names are shown in Table 10.

[0298] Table 10. PEDV mRNA sequence encapsulation instructions

[0299]

[0300]

[0301] Note: 2P in the table represents the D1076P and I1077P double mutation.

[0302] Example 3. Immunization regimen for mice using PEDV mRNA-LNP formulation

[0303] Female BALB / c mice aged 6-8 weeks were randomly divided into groups of 5. Mice were immunized intramuscularly with the PEDV mRNA-LNP preparation (Table 10) at a dose of 10 μg / mice, and with the PEDV inactivated vaccine (Wuhan Keqian Biotechnology) at a dose of 100 μl / mice. An LNP control group was also included. A booster immunization was administered 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. Spleen cells were collected 28 days after the initial immunization for Elispot assay.

[0304] Example 4. Detection of antibody binding in mice immunized with PEDV mRNA-LNP formulation

[0305] The eukaryotically expressed PEDV S1 protein was diluted to 2.5 μg / ml 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 plate was 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 plate was 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 plate was incubated at room temperature for 10 minutes, followed by adding 50 μl of stop solution. Finally, the OD was read using a microplate reader. 450nm -OD 630nmThe results are shown in Table 11. Twenty-eight days after the initial immunization, the PEDV mRNA vaccine effectively stimulated mice to produce antibodies binding to PEDVIIa, IIb, IIc, and type I S proteins, with little difference compared to the S protein binding antibodies stimulated by the inactivated vaccine.

[0306] Table 11. Detection of binding antibodies in mice immunized with PEDV mRNA-LNP formulation

[0307]

[0308]

[0309] Note: 2P in the table represents the D1076P and I1077P double mutation.

[0310] Example 5. Detection of neutralizing antibodies in mice immunized with PEDV mRNA-LNP formulation

[0311] 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. PEDV was then diluted to 200 TCID using DMEM medium. 50 The diluted virus was added in equal volumes to 96-well cell culture plates containing serum. The serum-virus mixture was incubated at 37°C, 5% CO2 for 1 hour. Then, the virus-serum mixture was added to VERO cells and incubated at 37°C, 5% CO2 for 1.5 hours. The cell supernatant was discarded, and the cells were washed three times with PBS. Maintenance medium containing trypsin (10 μg / ml) was added, and the cells were incubated at 37°C, 5% CO2 for 72 hours. Cytopathic effects were recorded daily. Serum neutralizing antibody titers were calculated using the Reed-Muench method.

[0312] The results are shown in Table 12. Among the four groups of PEDV mRNA vaccines (GIIa, GIIb, GIIc, and GI) targeting the four PEDV subtypes, the neutralizing antibody levels induced by the mRNA vaccine containing only the S(WT) protein were similar to those induced by the inactivated vaccine, but lower than those induced by the S(2P) mRNA vaccine with the S-2P mutation. Furthermore, the neutralizing antibody levels induced by the S(2P)-T4 and S(2P)-GCN4 mRNA vaccines with the addition of trimerized T4 Folden and GCN4 molecular chaperones to the C-terminus of the S protein were higher than those induced by the S(2P) mRNA vaccine without the addition of T4 Folden and GCN4. These results indicate that the S protein with the 2P mutation significantly aids in the stimulation of neutralizing antibodies, and that the S protein with the addition of trimerized T4 Folden and GCN4 to the C-terminus significantly aids in the stimulation of neutralizing antibodies.

[0313] Table 12. Detection of neutralizing antibodies in mice immunized with PEDV mRNA-LNP formulation

[0314]

[0315]

[0316] Note: 2P in the table represents the D1076P and I1077P double mutation.

[0317] Example 6. Evaluation of cellular immunity in mice immunized with PEDV mRNA-LNP formulation

[0318] 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. 6Cells / ml. Spleen cells were added to Elispot plates at a volume of 100 μl per well. Purified PEDV S1 and PEDV N proteins were diluted to 10 μg / ml and 5 μg / ml, respectively, 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 cell culture incubator. After incubation, 100 μl of biotinylated antibody was added to each well, and the plates were incubated at 37°C for 1 h. After washing, 100 μl of 1×Streptavidin-HRP 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. The results are shown in Table 13. Compared with the PEDV inactivated vaccine, the PEDV mRNA vaccine induced a high level of cellular immune response in mice.

[0319] Table 13. Detection of IFN-γ secretion in mice immunized with PEDV mRNA-LNP preparation

[0320]

[0321]

[0322] Note: 2P in the table represents the D1076P and I1077P double mutation.

[0323] Example 7. Preparation of PEDV mRNA-LNP formulation

[0324] According to the PEDV S protein sequence in Table 14, the mRNA stock solution containing the desired sequence was premixed with molecular adjuvants SMA-6 and SMA-7 in the indicated mass ratios, and then encapsulated using LNP. The encapsulation steps are as follows:

[0325] mRNA stock solution was encapsulated using the following lipid formulation (ionizable lipid (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 (Shanghai) Instruments Technology Co., Ltd.). TMThe samples were prepared by mixing (L / L+). The collected sample solution was diluted 10-fold in DPBS buffer and then passed through a 100kDa Pall ultrafiltration tube. After centrifugation and ultrafiltration, the ethanol content in the sample solution was removed. Finally, the solution was adjusted to the appropriate concentration with 10mM Tris 0.1% NaCl (pH 7.4) buffer. The mRNA vaccine names are shown in Table 7.

[0326] Table 14. Description of PEDV mRNA sequence parallel encapsulation

[0327]

[0328]

[0329] Note: 2P in the table represents the D1076P and I1077P double mutation.

[0330] Example 8. PEDV mRNA-LNP formulation immunization program for sows

[0331] Sows with one month to go into farrowing were divided into groups of three. The PEDV mRNA-LNP formulation (Table 7) was administered to sows via intramuscular injection at a dose of 100 μg / sow, while piglets were immunized with the PEDV inactivated vaccine at a dose of 1 ml / sow via intramuscular injection. An LNP control group was also established. A booster immunization was administered 14 days after the initial vaccination. Colostrum was collected at farrowing for IgA-binding antibody and neutralizing antibody detection, and anterior vena cava blood was collected for IgG-binding antibody detection.

[0332] Example 9. Detection of IgG binding antibodies in serum of sows immunized with PEDV mRNA-LNP formulation

[0333] The eukaryotically expressed PEDV S1 protein was diluted to 2.5 μg / ml 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 plate was 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-pig IgG secondary antibody (1:10000 dilution) was added to each well and the plate was 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 plate was incubated at room temperature for 10 minutes, followed by adding 50 μl of stop solution. Finally, the OD was read using a microplate reader. 450nm -OD 630nmThe results are shown in Table 8. In the serum of sows before farrowing, PEDV mRNA vaccine could effectively stimulate the production of IgG binding antibodies against PEDVIIb type S protein, and the difference between the S protein binding antibodies stimulated by the inactivated vaccine and those stimulated by the inactivated vaccine was small.

[0334] Table 15. Detection of S protein IgG binding antibody in serum of sows immunized with PEDV mRNA-LNP preparation

[0335]

[0336] Note: 2P in the table represents the D1076P and I1077P double mutation.

[0337] Example 10. Detection of IgA-binding antibodies in colostrum of sows immunized with PEDV mRNA-LNP formulation

[0338] The eukaryotically expressed PEDV S1 protein was diluted to 2.5 μg / ml 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 plate was blocked at 37°C for 1 hour, followed by washing 3 times with PBST. Colostrum samples were diluted 1:100 with dilution buffer, 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-porcine IgA secondary antibody (1:10000 dilution) was added to each well and the plate was 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 plate was 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, shown in Table 9, indicate that compared to the S(2P)-T4 mRNA vaccine and the S(2P)-GCN4 mRNA vaccine encapsulated alone, and the S(2P)-T4 mRNA vaccine and the S(2P)-GCN4 mRNA vaccine encapsulated with the molecular adjuvant SMA-7, the S(2P)-T4 mRNA vaccine and the S(2P)-GCN4 mRNA vaccine encapsulated with the molecular adjuvant SMA-6 induced significantly increased levels of S protein-specific IgA. These results suggest that the molecular adjuvant SMA-6 significantly aids in IgA secretion.

[0339] Table 16. Detection of S protein IgA binding antibody in colostrum of sows immunized with PEDV mRNA-LNP preparation

[0340]

[0341] Note: 2P in the table represents the D1076P and I1077P double mutation.

[0342] Example 11. Detection of neutralizing antibodies in colostrum after immunization of sows with PEDV mRNA-LNP formulation

[0343] Sow colostrum 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 culture plate. PEDV was diluted to 200 TCID using DMEM medium. 50 The diluted virus was added in equal volumes to 96-well cell culture plates containing serum. The serum-virus mixture was incubated at 37°C, 5% CO2 for 1 hour. Then, the virus-serum mixture was added to VERO cells and incubated at 37°C, 5% CO2 for 1.5 hours. The cell supernatant was discarded, and the cells were washed three times with PBS. Maintenance medium containing trypsin (10 μg / ml) was added, and the cells were incubated at 37°C, 5% CO2 for 72 hours. Cytopathic effects were recorded daily. Serum neutralizing antibody titers were calculated using the Reed-Muench method. The results are shown in Table 10. Compared with the S(2P)-T4 mRNA vaccine and S(2P)-GCN4 mRNA vaccine encapsulated alone, and the S(2P)-T4 mRNA vaccine and S(2P)-GCN4 mRNA vaccine encapsulated with the molecular adjuvant SMA-7 in parallel, the S(2P)-T4 mRNA vaccine and S(2P)-GCN4 mRNA vaccine encapsulated with the molecular adjuvant SMA-6 induced significantly higher levels of PEDV neutralizing antibodies in sow colostrum. These results indicate that the molecular adjuvant SMA-6 significantly helps to increase the level of neutralizing antibodies in sow colostrum.

[0344] Table 17. Detection of neutralizing antibodies in colostrum from sows immunized with PEDV mRNA-LNP formulation

[0345]

[0346] Note: 2P in the table represents the D1076P and I1077P double mutation.

[0347] 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, characterized in that, It contains porcine epidemic diarrhea virus (PEDV) S protein or a variant thereof, and a molecular chaperone fused directly or indirectly with it; the molecular chaperone is selected from T4 Folden, porcine IgG Fc, Ft, recombinant humanized type III collagen Rh3C, HIV gp41 6HB, GCN4 or Qα; Preferably, the S protein variant is stabilized in its pre-fusion conformation; Preferably, the variant comprises one or more mutations selected from K893P, R894P, A969P, A1032P, D1076P, and I1077P; the amino acid position of the mutation is based on the amino acid sequence number shown in any one of the parental SEQ ID NO: 17-20. Preferably, the S protein variant has D1076P and I1077P mutations; Preferably, the S protein or S protein variant further comprises a deleted or replaced signal peptide; Preferably, the replacement signal peptide is a signal peptide selected from porcine IL-2 signal peptide, tPA signal peptide, porcine IgG heavy chain signal peptide, porcine IL-6 signal peptide and porcine IL-10 signal peptide to replace the natural signal peptide in the parent. Preferably, the alternative signal peptide comprises a signal peptide sequence selected from any one of SEQ ID NO: 46-50 or a signal peptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 46-50; Preferably, the variant comprises the amino acid sequence shown in any one of SEQ ID NO: 13-16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 13-16; Preferably, the S protein comprises the amino acid sequence shown in any one of SEQ ID NO: 17-20 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 17-20; Preferably, the molecular chaperone is selected from the amino acid sequence shown in any one of SEQ ID NO:39-45 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO:39-45; Preferably, the fusion protein comprises an amino acid sequence selected from any one of SEQ ID NO: 1-12 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-12; Preferably, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NO: 51-58, 127-130 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 51-58, 127-130; Preferably, the fusion protein is further fused with an HA tag, the HA tag comprising an amino acid sequence as shown in SEQ ID NO:

147.

2. Nucleic acid, characterized in that, Encoding the fusion protein of claim 1; preferably, the nucleic acid comprises a nucleotide sequence selected from any one of SEQ ID NO: 59-70 or a degenerate sequence thereof, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 59-70; preferably, the nucleic acid comprises a nucleotide sequence selected from any one of SEQ ID NO: 135-146 or a degenerate sequence thereof, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 135-146.

3. Nucleic acid, characterized in that, Contains a polynucleotide encoding the porcine epidemic diarrhea virus (PEDV) S protein or a variant of the S protein; said S protein variant is stable in the pre-fusion conformation; Preferably, the S protein variant comprises a variant selected from K893P, R894P, A969P, A1032P, and D1076P. One or more mutations in I1077P; Preferably, the position of the mutated amino acid is numbered according to the amino acid sequence number shown in any one of the parental SEQ ID NO: 17-20; Preferably, the S protein variant has D1076P and I1077P mutations; Preferably, the S protein or S protein variant further comprises a deleted or replaced signal peptide; Preferably, the replacement signal peptide is a signal peptide selected from porcine IL-2 signal peptide, tPA signal peptide, porcine IgG heavy chain signal peptide, porcine IL-6 signal peptide and porcine IL-10 signal peptide to replace the natural signal peptide in the parent. Preferably, the alternative signal peptide comprises a signal peptide sequence selected from any one of SEQ ID NO: 46-50 or a signal peptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 46-50; Preferably, the nucleic acid encoding the substituted signal peptide is selected from the nucleic acid sequence described in any one of SEQ ID NO: 104-108 or its degenerate sequence, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 104-108; Preferably, the subtype of PEDV is selected from GIIa, GIIb, GIIc and GI.

4. The nucleic acid according to claim 3, wherein, The S protein comprises the amino acid sequence shown in any one of SEQ ID NO: 17-20 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 17-20; And / or the nucleic acid encoding the S protein comprises the nucleotide sequence of any one of SEQ ID NO: 75-78 or its degenerate sequence or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 75-78; And / or the S protein variant comprises an amino acid sequence shown in any one of SEQ ID NO: 13-16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 13-16; And / or the S protein variant encodes a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NO: 71-74 or its degenerate sequence or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 71-74.

5. The nucleic acid according to claim 3 or 4, wherein the S protein or S protein variant is fused with a molecular chaperone to form a fusion protein; Preferably, the fusion is a direct fusion or an indirect fusion via connectors; Preferably, the linker is selected from 2A peptide and GS linker; Preferably, the GS connector is selected from (GGGGS)n or (GGGS)n; preferably, n is 2, 3, 4, 5 or 6. Preferably, the molecular chaperone is selected from one or more of T4 Folden (T4 fibritin), porcine IgG Fc, Ft, recombinant humanized type III collagen Rh3C, HIV gp41 6HB, GCN4 and Qα; Preferably, the molecular chaperone is selected from the amino acid sequence shown in any one of SEQ ID NO:39-45 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO:39-45; Preferably, the molecular chaperone's encoding nucleic acid comprises a nucleotide sequence selected from any one of SEQ ID NO: 97-103 or its degenerate sequence, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 97-103; Preferably, the fusion protein comprises an amino acid sequence selected from any one of SEQ ID NO: 1-12 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-12; Preferably, the nucleic acid encoding the fusion protein comprises a nucleotide sequence selected from any one of SEQ ID NO: 59-70 or a degenerate sequence thereof, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 59-70; preferably, the fusion protein comprises an amino acid sequence selected from any one of SEQ ID NO: 51-58, 127-130 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 51-58, 127-130; preferably, the nucleic acid encoding the fusion protein comprises a nucleotide sequence selected from any one of SEQ ID NO: 135-146 or a degenerate sequence thereof, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 59-7 ... NO: 135-146 has a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity; Preferably, the fusion protein is further fused with an HA tag, the HA tag comprising an amino acid sequence as shown in SEQ ID NO:

147.

6. The nucleic acid according to any one of claims 2-5, wherein it is DNA or RNA; Preferably, the nucleic acid is mRNA, circular RNA, or self-replicating RNA; Preferably, the RNA further comprises a 5'UTR, KOZAK, 3'UTR, polyA, and / or a cap structure; Preferably, the RNA is modified RNA, wherein uracil, cytosine, adenine or guanine nucleotides contain modifying groups; Preferably, the modifying group is 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. Preferably, 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.

7. The nucleic acid according to claim 6, wherein the nucleic acid is mRNA; Preferably, the 5'UTR contains a nucleotide sequence shown in any one of SEQ ID NO: 109, 111, 113, 115 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 109, 111, 113, 115; Preferably, the 3'UTR contains a nucleotide sequence shown in any one of SEQ ID NO: 110, 112, 114, 116 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 110, 112, 114, 116; Preferably, the polyA comprises the nucleotide sequence shown in any one of SEQ ID NO: 117-118 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 117-118; Preferably, the nucleic acid comprises the nucleotide sequence or degenerate sequence shown in any one of SEQ ID NO: 119-126, 131-134 or the nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 119-126, 131-134; Preferably, the cap structure is a compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer, or isotopic variant thereof: in, It is a single key or does not exist. X1 is selected from O, S, CH2, CH2CH2, CH=CH, CH=CHO, CH2O, OCH2, CH2CH2O. OCH2CH2, a three-membered cycloalkyl group, 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, B1 and B2 are each independently selected from natural, modified, or non-natural nucleoside bases. Preferably, the compound of formula (I) is any one of the following:

8. A nucleic acid composition, characterized in that, The invention comprises the nucleic acid and molecular adjuvant as described in any one of claims 2-7; preferably, the molecular adjuvant is selected from TGF-β-P2A-BAFF (SMA-6), CD40L-P2A-APRIL, IL-10-P2A-IL-2, GM-CSF-P2A-IL-2, GM-CSF-P2A-CD40L, GM-CSF-P2A-IL-18, IL-17-P2A-IL-21 (SMA-7), IL-17, IL-21, IL-10, IL-2, GM-CSF, IL-18, CCL28, CD40L, BAFF, APRIL, and TGF-β; Preferably, the molecular adjuvant is derived from pigs; Preferably, the molecular adjuvant comprises the amino acid sequence described in any one of SEQ ID NO: 21-38 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 21-38; Preferably, the nucleic acid encoding the molecular adjuvant comprises the nucleotide sequence shown in any one of SEQ ID NO: 79-96 or its degenerate sequence, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 79-96; Preferably, the composition is a vaccine composition.

9. A vector comprising the nucleic acid of any one of claims 2-7 or the nucleic acid composition of claim 8; preferably, the vector is an expression vector.

10. A cell comprising the carrier of claim 9; preferably, the cell is a eukaryotic cell or a prokaryotic cell.

11. A pharmaceutical composition comprising the fusion protein of claim 1 or the nucleic acid of any one of claims 2-7 or the nucleic acid composition of claim 8 or the carrier of claim 9 or the cell of claim 10; preferably, the delivery carrier of the pharmaceutical composition comprises lipid nanoparticles (LNP), viral vectors such as AAV, polymeric materials such as polyethyleneimine (PEI), polyurethane (PBAE), chitosan or polypeptides such as cell-penetrating peptides. Preferably, the lipid nanoparticles (LNPs) comprise ionizable lipids, DSPC, cholesterol, and DMG-PEG2000 ethanol; Preferably, the lipid nanoparticles (LNPs) are targeted LNPs (tLNPs); Preferably, the pharmaceutical composition is formulated into a dosage form suitable for intramuscular or subcutaneous injection, oral administration, or inhalation.

12. Use in the preparation of a medicament for the prevention or treatment of porcine epidemic diarrhea virus (PEDV) infection or disease caused by PEDV infection, according to the fusion protein of claim 1, the nucleic acid of any one of claims 2-7, the nucleic acid composition of claim 8, the vector of claim 9, the cell of claim 10, or the pharmaceutical composition of claim 11; preferably, the medicament is a vaccine; preferably, the disease is porcine epidemic diarrhea.

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