Novel protein multimerization tag and use thereof

Abstract

A truncated σ1 protein or a variant thereof, a fusion protein and trimeric protein containing the truncated σ1 protein or the variant thereof, and a nucleic acid molecule and vector containing a nucleotide sequence encoding the truncated σ1 protein. The present invention further relates to a vaccine, kit and pharmaceutical composition containing the truncated σ1 protein or the variant thereof, the trimeric protein, the nucleic acid molecule or the vector.

Description

A novel protein polymerization tag and its application

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application 202410691896.6, filed on May 30, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the fields of genetic engineering and molecular biology, specifically to truncated σ1 protein or variants thereof, fusion proteins and trimeric proteins comprising said truncated σ1 protein or variants thereof, and nucleic acid molecules and vectors comprising their nucleotide sequences. This application also relates to vaccines, kits, and pharmaceutical compositions comprising said truncated σ1 protein or variants thereof, trimeric proteins, nucleic acid molecules, or vectors. Background Technology

[0004] Studies have shown that the structural proteins of many viruses, such as SARS-CoV-2, respiratory syncytial virus (RSV), and influenza virus, which can serve as important vaccine targets, are trimeric in their natural state. Therefore, restoring the native trimeric structure of viral structural proteins in recombinant subunit vaccines within eukaryotic or prokaryotic systems is crucial for the immunogenicity of these vaccines. Currently, structural domains that can assist in the formation of trimers from target proteins (also known as trimer tags) include T4-Foldon derived from T4 phage Fibritin, the leucine zipper-like GCN4 tag derived from yeast transcription factors, and the Trimer-Tag developed by Clover Biopharmaceuticals. T4-Foldon is the C-terminal domain of the T4 phage Fibritin protein, composed of 27 amino acids (YIPEAPRDGQAYVRKDGEWVLLSTFLG). GCN4 is derived from the C-terminal sequence of yeast transcription activators, composed of approximately 33-35 amino acids. Trimer-Tag contains amino acid residues 1156-1406 (251 amino acids) of human type I (α) collagen, formed by three α polypeptide chains. T4-Foldon and GCN4 have small molecular weights, but their trimerization stability is insufficient; Trimer-Tag has a large molecular weight, which may affect the structure and function of the fusion protein, and may also trigger unintended immune responses against the trimerization tag.

[0005] Reoviridae is a family of double-stranded RNA viruses, its scientific name derived from the first letters of "respiratory enteric orphan virus". Mammalian orthoreovirus (MRV) is a member of the genus *Orthoreoviru* within the family Reoviridae, possessing an icosahedral double-layered capsid structure. MRVs are widely distributed in the respiratory and digestive tracts of mammals, exhibiting a broad host range; various MRV strains have been isolated from multiple mammals. MRVs primarily comprise four serotypes: serotype 1 (Lang, T1L), serotype 2 (Jones, T2J), serotype 3 (Dearing, T3D), and serotype 4 (Ndelle, T4N). The MRV genome consists of 10 double-stranded RNA molecules, named according to molecular weight as three groups of 10 segments: large (L1, L2, L3), medium (M1, M2, M3), and small (S1, S2, S3, S4), encoding 8 structural proteins and 3 non-structural proteins. The σ1 protein exhibits a highly extended stalk / sphere structure, protruding from the viral particle surface as long filaments with heads and tails. σ1 is a homotrimeric structure, comprising an N-terminal α-helix and a C-terminal β-helix domain.

[0006] Given the potential shortcomings of existing trimer tags (e.g., T4-Foldo, GCN4, Trimer-Tag), providing a novel trimer tag would have significant application potential. Summary of the Invention

[0007] This invention designs and constructs a series of trimerizing tags based on the N-terminal α-helical domain of the σ1 protein of mammalian ororeoviruses. By fusing the trimerizing tag sequence designed in this invention with the target protein (e.g., the S extracellular domain of the spike protein of SARS-CoV-2 and the hemagglutinin HA protein of seasonal influenza H3 virus), trimerizing proteins with higher expression levels and better stability can be successfully obtained.

[0008] Truncated σ1 protein or its variants

[0009] Therefore, in a first aspect, this application provides a truncated σ1 protein or a variant thereof, wherein, compared with the σ1 protein of wild-type Orthoreoviru virus, the truncated σ1 protein or a variant thereof: (i) is truncated by 1 to 120 amino acids at the N-terminus, or (ii) is truncated by 1 to 140 amino acids at the C-terminus, or (iii) is truncated by 1 to 140 amino acids at both the N-terminus and C-terminus.

[0010] The variant, compared to the truncated σ1 protein, has one or more (e.g., conserved substitutions), deletions, or additions of amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); and the variant substantially retains the biological function of the sequence from which it originates.

[0011] In some embodiments, the truncated σ1 protein or its variants are capable of polymerizing themselves or other proteins or peptides linked to them (e.g., dimerization, trimerization, tetramerization, pentamerization).

[0012] In some embodiments, the truncated σ1 protein or a variant thereof is capable of forming an α-helical structure.

[0013] In some embodiments, the truncated σ1 protein or its variants are able to increase the stability (e.g., thermal stability) of themselves or other proteins or peptides compared to wild-type σ1 protein.

[0014] In some embodiments, the truncated σ1 protein or its variants can increase the proportion of unstable conformations of other proteins or peptides (e.g., pre-F protein of RSV F protein) compared to wild-type σ1 protein.

[0015] In some embodiments, the truncated σ1 protein or its variants are able to increase the protein expression levels of themselves or other proteins or peptides compared to wild-type σ1 protein.

[0016] In some embodiments, the truncated σ1 protein or its variants are able to increase the immunogenicity of themselves or other proteins or peptides compared to wild-type σ1 protein.

[0017] In some embodiments, the truncated σ1 protein or its variants have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence from which it is derived.

[0018] wild σ1 protein

[0019] The native σ1 protein exhibits high sequence conservation among different viruses in the genus *Orthoreoviru*. Furthermore, the sequence identity of the σ1 protein is even higher among different strains of mammalian *Orthoreoviru*. For example, the σ1 protein shares more than 80% sequence identity (e.g., more than 90%) with each of the following strains: Lang (T1L) type 1, Jones (T2J) type 2, Dearing (T3D) type 3, and Ndelle (T4N) type 4. The sequence identity among these native σ1 proteins is known in the art and can be referenced, for example, to data published in Genebank and PDB.

[0020] Given the conservation of the σ1 protein sequence, those skilled in the art can readily compare the amino acid positions between different natural σ1 proteins to identify the corresponding amino acid positions of σ1 proteins in different Ororeoviruses or different strains of the same virus. For example, in almost all identified natural σ1 proteins, the α-helical portion is located at nearly the same amino acid position. Therefore, the conservation of the natural σ1 protein sequence allows it to be used as a reference σ1 protein for comparison of amino acids at specific positions within the σ1 protein.

[0021] As used herein, when referring to the amino acid sequence of the σ1 protein of wild-type Orthoreoviru viruses, the sequence shown in SEQ ID NO: 1 is used for description. For example, the statement "position 20 of the wild-type σ1 protein" refers to the 20th amino acid residue of the protein shown in SEQ ID NO: 1. However, those skilled in the art will understand that there may be multiple versions of the wild-type σ1 protein, which have substantially the same primary structure (i.e., amino acid sequence) and higher-order structure (i.e., spatial structure), and substantially the same biological function, but may still have minor differences in amino acid sequence from one another. Therefore, in this application, the wild-type σ1 protein is not limited to the protein shown in SEQ ID NO: 1, but is intended to cover all known wild-type σ1 proteins. Therefore, in this application, the term "wild-type σ1 protein" should include various naturally occurring, biologically functional σ1 proteins, including, for example, the wild-type σ1 protein shown in SEQ ID NO: 1 and its naturally occurring variants. Furthermore, when describing the amino acid positions of the wild-type σ1 protein, it includes not only the specific amino acid position in SEQ ID NO: 1, but also the corresponding amino acid position in its natural variant. For example, the statement "position 20 of the wild-type σ1 protein" includes the 20th amino acid residue of SEQ ID NO: 1, and the corresponding amino acid position in its natural variant. According to this application, the statement "corresponding amino acid position" refers to the amino acid position at the equivalent position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity.

[0022] In some embodiments, the orthoreoviruses are selected from mammalian orthoreovirus, avian orthoreovirus, neoavian orthoreovirus, piscine orthoreovirus, reptilian orthoreovirus, testudine orthoreovirus, or any combination thereof.

[0023] In some embodiments, the mammalian ororeovirus is selected from Lang (T1L) strain 1, Jones (T2J) strain 2, Dearing (T3D) strain 3, Ndelle (T4N) strain 4, or any combination thereof.

[0024] In some implementations, the wild-type σ1 protein is derived from the T3D strain.

[0025] In some embodiments, the wild-type σ1 protein comprises, or is composed of, sequences selected from, the following:

[0026] (i) The sequence shown in SEQ ID NO:1;

[0027] (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) compared to the sequence shown in SEQ ID NO: 1;

[0028] (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO: 1.

[0029] truncated σ1 protein

[0030] Compared to wild-type σ1 protein, the truncated σ1 protein of this application can be truncated only from the N-terminus, only from the C-terminus, or from both ends (i.e., N-terminus and C-terminus), or any one or more amino acids in the middle of the σ1 protein can be omitted. With these truncation methods, as long as the truncated σ1 protein can still form an α-helix structure, or still possess the activity or function of polymerizing itself or other proteins or peptides linked to it (e.g., dimerization, trimerization, tetramerization, pentamerization), it is acceptable.

[0031] Therefore, in some embodiments, the truncated σ1 protein is truncated at the N-terminus by 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, or 110-120 amino acids compared to the wild-type σ1 protein.

[0032] In some embodiments, the truncated σ1 protein is truncated at the N-terminus by 13, 38, 50, 93, or 100 amino acids compared to the wild-type σ1 protein.

[0033] In some implementations, the above truncation occurs at the N-terminus of the wild-type σ1 protein.

[0034] In some embodiments, the truncated σ1 protein is truncated at the C-terminus by 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, or 130-140 amino acids compared to the wild-type σ1 protein.

[0035] In some embodiments, the truncated σ1 protein is truncated by 280-300, 300-320, 320-340, 340-360, 360-380, 380-400, or 400-420 amino acids at the C-terminus compared to the wild-type σ1 protein.

[0036] In some embodiments, the truncated σ1 protein is truncated at the C-terminus by 300, 303, 340, 349, 353, 358, 359, 365, 367, 378, 372, 390, or 407 amino acids compared to the wild-type σ1 protein.

[0037] In some implementations, the above truncation occurs at the C-terminus of the wild-type σ1 protein.

[0038] In some embodiments, the truncated σ1 protein is truncated by 30-40 amino acids at the N-terminus and 350-370 amino acids at the C-terminus compared to the wild-type σ1 protein.

[0039] In some implementations, the N-terminus is truncated by 38 amino acids, and the C-terminus is truncated by 367 amino acids.

[0040] In some implementations, the N-terminus is truncated by 38 amino acids, and the C-terminus is truncated by 359 amino acids.

[0041] In some embodiments, the truncated σ1 protein is truncated by 40-60 amino acids at the N-terminus and 350-360 amino acids at the C-terminus compared to the wild-type σ1 protein.

[0042] In some implementations, the N-terminus is truncated by 50 amino acids, and the C-terminus is truncated by 353 amino acids.

[0043] In some embodiments, the truncated σ1 protein is truncated by 90-110 amino acids at the N-terminus and 300-310 amino acids at the C-terminus compared to the wild-type σ1 protein.

[0044] In some implementations, the N-terminus is truncated by 93 amino acids, and the C-terminus is truncated by 300 amino acids.

[0045] In some implementations, the N-terminus is truncated by 100 amino acids, and the C-terminus is truncated by 303 amino acids.

[0046] In some embodiments, the truncated σ1 protein is truncated by 13 amino acids at the N-terminus and 378 amino acids at the C-terminus compared to the wild-type σ1 protein.

[0047] In some embodiments, the truncated σ1 protein comprises, or consists of, sequences selected from, the following:

[0048] (i) The sequence shown in any one of SEQ ID NO:2-15;

[0049] (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) compared to the sequence shown in any one of SEQ ID NO: 2-15;

[0050] (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with any of the sequences shown in SEQ ID NO: 2-15;

[0051] Among them, the sequences in (ii) and (iii) basically retain the biological functions of the sequences from which they originated;

[0052] In some embodiments, the truncated σ1 protein comprises, or consists of, sequences selected from, the following:

[0053] (i) The sequence shown in SEQ ID NO:10;

[0054] (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) compared to the sequence shown in SEQ ID NO: 10;

[0055] (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO: 10;

[0056] Among them, the sequences in (ii) and (iii) basically retain the biological functions of the sequences from which they originated.

[0057] truncated variants of σ1 protein

[0058] In some embodiments, the truncated σ1 protein or a variant thereof, as described above, comprises, compared to the wild-type σ1 protein, an amino acid substitution selected from (i) cavity-filling mutations; (ii) electrostatic mutations; or any combination thereof.

[0059] In some embodiments, the variant contains amino acid substitutions at positions selected from one or more of the following: positions corresponding to the 20th, 25th, 27th, 28th, 29th, 31st, 33rd, 34th, 36th, 38th, 42nd, 43rd, 47th, 48th and / or 53rd positions of the wild-type σ1 protein.

[0060] In some embodiments, the variant contains an amino acid substitution at position 53, corresponding to the wild-type σ1 protein.

[0061] Cavity filling mutation

[0062] In some embodiments, the variant comprises one or more cavity-filling mutations. In some embodiments, such cavity-filling mutations contribute to the stability of the trimer formed by the variant itself or by a fusion protein containing it. The term "cavity-filling mutation" refers to the substitution of amino acid residues in the wild-type σ1 protein by amino acids intended to fill the internal cavity of the mature σ1 protein. The cavity formed by the wild-type σ1 protein can be identified by methods known in the art, such as by examining the crystal structure of the σ1 protein or a fusion protein containing it, or by using computational protein design software such as BioLuminate™ [BioLuminate, Schrodinger LLC, New York, 2015], Discovery Studio™ [Discovery Studio Modeling Environment, Accelrys, San Diego, 2015], MOE™ [Molecular Operating Environment, Chemical Computing Group Inc., Montreal, 2015], and Rosetta™ [Rosetta, University of Washington, Seattle, 2015]).

[0063] In some embodiments, the amino acids that can be substituted typically include small aliphatic amino acids (e.g., Gly, Ala, and Val) or small polar amino acids (e.g., Ser and Thr). Accordingly, examples of amino acids that can be substituted include large aliphatic amino acids (Ile, Leu, and Met) or large aromatic amino acids (His, Phe, Tyr, and Trp).

[0064] electrostatic mutation

[0065] In some embodiments, the variants include one or more electrostatic mutations. In some embodiments, such electrostatic mutations contribute to the stability of the trimer formed by the variant itself or a fusion protein containing it. The term "electrostatic mutation" refers to a mutation that reduces ionic repulsion between amino acid residues that are close to each other in the folded structure of a protein or increases ionic attraction between said amino acid residues. Since hydrogen bonding is a special case of ionic attraction, electrostatic mutations can increase hydrogen bonding between such close amino acid residues. Typically, introducing an electrostatic mutation will increase the Tm value of the σ1 protein or its fusion protein.

[0066] Unfavorable electrostatic interactions following the formation of a trimer configuration can be identified using methods known in the art, such as by examining the crystal structure of the σ1 protein in a trimer configuration or a fusion protein containing it, or by using computational protein design software (e.g., BioLuminate™ [BioLuminate, Schrodinger LLC, New York, 2015], Discovery Studio™ [Discovery Studio Modeling Environment, Accelrys, San Diego, 2015], MOE™ [Molecular Operating Environment, Chemical Computing Group Inc., Montreal, 2015.], and Rosetta™ [Rosetta, University of Washington, Seattle, 2015.]).

[0067] conservative replacement

[0068] In some embodiments, the amino acid substitution is a conservative substitution. The term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological activity of a protein / peptide containing an amino acid sequence.

[0069] In some implementations, providing a table of conserved substitutions for functionally similar amino acids is well known to those skilled in the art. The following six groups are examples of amino acids that are considered to have conserved substitutions for each other:

[0070] 1) Substitutions between alanine (A), serine (S), and threonine (T);

[0071] 2) Substitution between aspartic acid (D) and glutamic acid (E);

[0072] 3) Substitution between asparagine (N) and glutamine (Q);

[0073] 4) Substitution between arginine (R) and lysine (K);

[0074] 5) Substitutions among isoleucine (I), leucine (L), methionine (M), and valine (V); and

[0075] 6) Substitutions between phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0076] In some embodiments, the amino acid at position 20, corresponding to the wild-type σ1 protein, is E or N.

[0077] In some embodiments, the amino acid substitution at position 20 of the variant, corresponding to the wild-type σ1 protein, is an A-to-E or N substitution.

[0078] In some embodiments, the amino acid at position 25 corresponding to the wild-type σ1 protein is I.

[0079] In some embodiments, the amino acid substitution at position 25 of the variant, corresponding to the wild-type σ1 protein, is an S-to-I substitution.

[0080] In some embodiments, the amino acid at position 27 corresponding to the wild-type σ1 protein is D or R.

[0081] In some embodiments, the amino acid substitution at position 27 of the variant, corresponding to the wild-type σ1 protein, is an I-to-D or R substitution.

[0082] In some embodiments, the amino acid at position 28 corresponding to the wild-type σ1 protein in the variant is I or L.

[0083] In some embodiments, the amino acid substitution at position 28 of the variant, corresponding to the wild-type σ1 protein, is an H-to-I or L substitution.

[0084] In some embodiments, the amino acid at position 29 of the variant, corresponding to the wild-type σ1 protein, is E.

[0085] In some embodiments, the amino acid substitution at position 29 of the variant, corresponding to the wild-type σ1 protein, is an S-to-E substitution.

[0086] In some embodiments, the amino acid at position 31 corresponding to the wild-type σ1 protein in the variant is E or R.

[0087] In some embodiments, the amino acid substitution at position 31 of the variant, corresponding to the wild-type σ1 protein, is a T-to-E or R substitution.

[0088] In some embodiments, the amino acid at position 33 corresponding to the wild-type σ1 protein is Q, E, or S.

[0089] In some embodiments, the amino acid substitution at position 33 of the variant, corresponding to the wild-type σ1 protein, is an L-to-Q, E, or S substitution.

[0090] In some embodiments, the amino acid K at position 34, corresponding to the wild-type σ1 protein, is present in the variant.

[0091] In some embodiments, the amino acid substitution at position 34 of the variant, corresponding to the wild-type σ1 protein, is an R-to-K substitution.

[0092] In some embodiments, the amino acid at position 36 corresponding to the wild-type σ1 protein in the variant is E, K, or R.

[0093] In some embodiments, the amino acid substitution at position 36 of the variant, corresponding to the wild-type σ1 protein, is a substitution from T to E, K, or R.

[0094] In some embodiments, the amino acid at position 38 of the variant, corresponding to the wild-type σ1 protein, is R.

[0095] In some embodiments, the amino acid substitution at position 38 of the variant, corresponding to the wild-type σ1 protein, is a G-to-R substitution.

[0096] In some embodiments, the amino acid at position 42 corresponding to the wild-type σ1 protein is I.

[0097] In some embodiments, the amino acid substitution at position 42 of the variant, corresponding to the wild-type σ1 protein, is an A-to-I substitution.

[0098] In some embodiments, the variant has an amino acid D or E at position 43 corresponding to the wild-type σ1 protein.

[0099] In some embodiments, the amino acid substitution at position 43 of the variant, corresponding to the wild-type σ1 protein, is an N-to-D or E substitution.

[0100] In some embodiments, the amino acid at position 47, corresponding to the wild-type σ1 protein, is E or Q.

[0101] In some embodiments, the amino acid substitution at position 47 of the variant, corresponding to the wild-type σ1 protein, is an I-to-E or Q substitution.

[0102] In some embodiments, the variant has an amino acid D or Q at position 48 corresponding to the wild-type σ1 protein.

[0103] In some embodiments, the amino acid substitution at position 48 of the variant, corresponding to the wild-type σ1 protein, is an A-to-D or Q substitution.

[0104] In some embodiments, the amino acid at position 53 corresponding to the wild-type σ1 protein is I.

[0105] In some embodiments, the amino acid substitution at position 53 of the variant, corresponding to the wild-type σ1 protein, is an R-to-I substitution.

[0106] In some embodiments, the variant comprises, or consists of, sequences selected from, the following:

[0107] (i) The sequence shown in any one of SEQ ID NO: 16-36;

[0108] (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) compared to the sequence shown in any one of SEQ ID NO:16-36;

[0109] (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with any of the sequences shown in SEQ ID NO: 16-36;

[0110] Among them, the sequences in (ii) and (iii) basically retain the biological functions of the sequences from which they originated.

[0111] In some embodiments, the variant comprises, or consists of, sequences selected from, the following:

[0112] (i) The sequence shown in SEQ ID NO:18;

[0113] (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) compared to the sequence shown in SEQ ID NO:18;

[0114] (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO:18;

[0115] Among them, the sequences in (ii) and (iii) basically retain the biological functions of the sequences from which they originated.

[0116] Fusion protein

[0117] In a second aspect, this application provides a fusion protein comprising a truncated σ1 protein or a variant thereof as described in the first aspect, and additional proteins or peptides (e.g., heterologous proteins or peptides).

[0118] In some embodiments, the additional protein or polypeptide is linked to the truncated σ1 protein or a variant thereof, with or without a linker.

[0119] In some embodiments, the truncated σ1 protein or a variant thereof is located at the C-terminus or N-terminus of another protein or polypeptide.

[0120] Typically, a truncated σ1 protein or its variants are located at the C-terminus of another protein or polypeptide. It can directly bind to the other protein or polypeptide. Optionally, a truncated σ1 protein or its variants are linked via linkers (such as amino acid linkers, e.g., sequences G, GG, GGG, GS, SAIG, GGPG, GPGGG, GGGPG, EAAAK, and PAAAK). Linkers can also be longer linkers (e.g., including repetitive sequences GG, GSG).

[0121] In some embodiments, the fusion protein comprising the truncated σ1 protein or a variant thereof also includes a protease cleavage site, such as a thrombin site, for removing the truncated σ1 protein or a variant thereof from another protein or peptide.

[0122] In some embodiments, the additional protein or polypeptide is immunogenic in mammals.

[0123] In some embodiments, the additional protein or polypeptide is derived from a virus (e.g., a viral membrane surface protein).

[0124] In some embodiments, the additional protein or polypeptide is a target protein of a disease or a fragment thereof (e.g., a target protein of a tumor or a fragment thereof).

[0125] In some embodiments, the additional protein or polypeptide is selected from the spike protein of SARS-CoV-2, the hemagglutinin protein of influenza virus, the glycoprotein of rabies virus, the F protein of respiratory syncytial virus, the envelope protein of HIV, the Gp protein of Ebola virus, the F protein of paramyxovirus, the extracellular domain of human ACE2 (ACE2), the SINV E2 protein, the Lassa virus glycoprotein (LASV G), the human parainfluenza virus 3F protein (HPIV3 F), or any combination thereof.

[0126] In some embodiments, the fusion protein is in the form of a polymer (e.g., a dimer, trimer, tetramer, pentamer, hexamer, hepter, octamer), a monomer, or a mixture of both.

[0127] In some embodiments, the fusion protein is in the form of a mixture of multiple polymers, such as a mixture of trimers, tetramers, hexamers, and octamers.

[0128] In some embodiments, the fusion protein further comprises a signal peptide, a tag, or any combination thereof.

[0129] signal peptide

[0130] To enhance protein production or secretion, the fusion protein of this application may contain a signal peptide.

[0131] In some embodiments, the signal peptide is located at one end (e.g., the N-terminus) of the fusion protein.

[0132] Label

[0133] In some embodiments, the fusion protein of the present invention may include a tag to facilitate the expression, detection, tracing, and / or purification of the fusion protein. Such epitope tags are well known to those skilled in the art, and examples include, but are not limited to, His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to select an appropriate epitope tag according to the desired purpose (e.g., purification, detection, or tracing).

[0134] In some embodiments, the tag is selected from multihistidine tags, antigen or epitope tags, enzyme tags, or any combination thereof.

[0135] In some embodiments, the tag is located at the end (e.g., the C-terminus) of the fusion protein.

[0136] In some exemplary embodiments, the tag is a multihistidine tag, i.e., a 6×His-tag (HHHHHH). In some exemplary embodiments, the tag is linked to the multihistidine tag via an enzyme cleavage site.

[0137] In some embodiments, the additional protein or polypeptide is linked to the signal peptide and / or tag, with or without a linker.

[0138] In some embodiments, the connector has a sequence as shown in SEQ ID NO:42 or SEQ ID NO:43.

[0139] In some embodiments, the fusion protein has a sequence as shown in any one of SEQ ID NO:37-41.

[0140] Trimeric protein

[0141] In a third aspect, this application provides a trimeric protein comprising the truncated σ1 protein or a variant thereof as described in the first aspect.

[0142] In some embodiments, the trimer protein comprises three polypeptide chains, each polypeptide chain comprising: a truncated σ1 protein or a variant thereof as described in the first aspect, and an additional protein or polypeptide.

[0143] In some implementations, the other proteins or peptides in each polypeptide chain may be the same or different.

[0144] In some implementations, the other proteins or peptides in each polypeptide chain are the same.

[0145] In some embodiments, the additional protein or polypeptide is as defined above.

[0146] Nucleic acid molecules

[0147] It is readily understood that the nucleic acid molecules can be used to clone or express the truncated σ1 protein of the present invention or its variants, fusion proteins, or trimer proteins. In some cases, to improve efficiency, the nucleotide sequence of the nucleic acid molecules can be codon-optimized according to cell preferences.

[0148] Therefore, in a fourth aspect, this application provides a nucleic acid molecule comprising a nucleotide sequence encoding the truncated σ1 protein or a variant thereof as described in the first aspect, or the fusion protein as described in the second aspect, or the trimeric protein as described in the third aspect.

[0149] In some implementations, the nucleotide sequence may or may not be codon-optimized based on the host cell's codon preference.

[0150] In some embodiments, the nucleic acid molecule is DNA, or an RNA (mRNA) product transcribed from said DNA, or a mixture of both.

[0151] The nucleic acid molecules in this application may include DNA, cDNA, and RNA sequences.

[0152] carrier

[0153] Vectors for expressing the truncated σ1 protein or its variants, fusion proteins, or trimer proteins of this application in insect or mammalian cells are well known in the art. These vectors can be cloning vectors or expression vectors. In some preferred embodiments, the vectors of the present invention can be, for example, plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and viral vectors, etc. Viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0154] In some preferred embodiments, the vectors of this application are capable of expressing or used to express the truncated σ1 protein or its variants, fusion proteins, or trimers of the present invention. In some preferred embodiments, the vectors of the present invention are capable of expressing or used to express the truncated σ1 protein or its variants, fusion proteins, or trimers of the present invention in a subject (e.g., a mammal, such as a human).

[0155] The vector of this application may contain a variety of elements, including but not limited to one or more of the following: origin of replication; optional marker gene; one or more expression control elements, such as transcription control elements (e.g., promoter, enhancer, terminator) and / or one or more translation signals; and signal sequences or leader sequences for targeting the secretion pathway in selected host cells.

[0156] In a fifth aspect, this application provides a carrier comprising the nucleic acid molecules described in the fourth aspect.

[0157] In some implementations, the vector is a viral vector.

[0158] In some embodiments, the viral vector is selected from: influenza virus vector, reverse transcriptase virus vector, adenovirus vector, adeno-associated virus vector, herpesvirus vector, poxvirus vector, baculovirus vector, papillomavirus vector, or papillomavirus vector.

[0159] host cells

[0160] The truncated σ1 protein or variants thereof provided in this application can be prepared by conventional methods known in the art, such as by expression in recombinant host cells using suitable vectors. Host cells include, for example, insect cells, mammalian cells, avian cells, bacterial cells, and yeast cells. Examples of insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, and Schneider S2 cells. Examples of mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Avian cells include, for example, chicken embryonic stem cells, chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts, and duck cells. Insect cell expression systems, such as baculovirus vector systems, are known to those skilled in the art and described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Avian cell expression systems are also known to those skilled in the art and described, for example, in U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168 and 6,500,668. Similarly, bacterial and mammalian cell expression systems are also known to those skilled in the art and described, for example, in Yeast Genetic Engineering (edited by Barr et al., 1989), Butterworth, London.

[0161] In a sixth aspect, this application provides a host cell comprising the truncated σ1 protein or a variant thereof as described in the first aspect, or the fusion protein as described in the second aspect, or the trimer protein as described in the third aspect, or the nucleic acid molecule as described in the fourth aspect, or the vector as described in the fifth aspect.

[0162] In some embodiments, the host cell is selected from prokaryotic cells (e.g., Escherichia coli cells) or eukaryotic cells.

[0163] In some embodiments, the eukaryotic cells are mammalian cells, such as mouse cells or human cells.

[0164] In some embodiments, the truncated σ1 protein or its variants, fusion proteins, or trimer proteins are displayed on the surface of the host cell's cell membrane.

[0165] Preparation method

[0166] The methods used for expressing and purifying the truncated σ1 protein or its variants as described in the first aspect of this application, the fusion protein as described in the second aspect, or the trimeric protein as described in the third aspect are common in the art and can be found in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 200; and Ausubel et al., Short Protocols in Molecular Biology, 4th Edition, John Wiley & Sons, Inc., 999.

[0167] Therefore, in a seventh aspect, this application provides a method for expressing or producing the truncated σ1 protein or a variant thereof as described in the first aspect, or the fusion protein or the trimer protein as described in the second aspect, or the trimer protein as described in the third aspect, the method comprising culturing the host cell as described in the fifth aspect under conditions that allow protein expression, and optionally, recovering or purifying the truncated σ1 protein or a variant thereof, or the fusion protein or the trimer protein expressed therein.

[0168] vaccine

[0169] The vaccine provided in this application is not limited to protein form, nucleic acid form, or a mixture of both. Furthermore, the nucleic acid can be selected from DNA, cDNA, RNA, or any combination thereof.

[0170] In the eighth aspect, this application provides a vaccine comprising one or more of the following (1) to (6):

[0171] (1) The truncated σ1 protein or a variant thereof as described in the first aspect;

[0172] (2) The fusion protein described in the second aspect;

[0173] (3) The trimeric protein described in the third aspect;

[0174] (4) The nucleic acid molecules described in the fourth aspect;

[0175] (5) The carrier described in the fifth aspect;

[0176] (6) The host cell described in the sixth aspect.

[0177] In some implementations, the vaccine also contains adjuvants and / or buffer solutions.

[0178] In some embodiments, the adjuvant is selected from metal salts, 3-D-monophosphoryl lipid A (MPL), saponins, oil and water emulsions, liposomes, nanoparticles, or any combination thereof.

[0179] In some embodiments, the fusion protein in the vaccine is in the form of a polymer (e.g., a dimer, trimer, or tetramer), a monomer, or a mixture.

[0180] The vaccine provided in this application can be administered using standard routes of administration. Non-limiting administration methods include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration. A single dose can be given to the subject, or one or more booster doses. If a booster vaccination is performed, it is typically administered to the same individual at a time between 1 week and 10 years after the first dose (referred to in such cases as the "primitive vaccination"), for example, between 2 weeks and 6 months.

[0181] The vaccine provided in this application can also be used in conjunction with one or more other vaccines. For example, in adults, it can be used with influenza vaccines, Prevnar vaccines, tetanus vaccines, diphtheria vaccines, and pertussis vaccines. In children, the vaccine provided in this application can be used in conjunction with any other vaccines indicated for pediatric patients.

[0182] Reagent test kit

[0183] In a ninth aspect, this application provides a kit comprising an immunogen component, wherein the immunogen component comprises one or more of the following (1) to (7):

[0184] (1) The truncated σ1 protein or a variant thereof as described in the first aspect;

[0185] (2) The fusion protein described in the second aspect;

[0186] (3) The trimeric protein described in the third aspect;

[0187] (4) The nucleic acid molecules described in the fourth aspect;

[0188] (5) The carrier described in the fifth aspect;

[0189] (6) The host cell described in the sixth aspect.

[0190] (7) The vaccine described in the eighth aspect.

[0191] In some embodiments, the kit further includes a carrier component capable of displaying the immunogen component.

[0192] In some embodiments, the carrier component is selected from: nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), polymerized pedestals, virus-like particles (VLPs), or any combination thereof.

[0193] In some embodiments, the immunogen and carrier components in the kit are provided separately or as a complex.

[0194] In some embodiments, the immunogen component is in the form of a polymer (e.g., a dimer, trimer, or tetramer), a monomer, or a mixture.

[0195] In some implementations, the immunogenic components in the kit are provided in the form of proteins or nucleic acids.

[0196] In some embodiments, the carrier component in the kit is provided in the form of a protein or nucleic acid.

[0197] In some embodiments, the VLP is assembled from proteins derived from RSV, hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

[0198] Pharmaceutical Composition

[0199] In a tenth aspect, this application provides a pharmaceutical composition comprising:

[0200] (i) Selected from any one or more of the following (1) to (8):

[0201] (1) The truncated σ1 protein or a variant thereof as described in the first aspect;

[0202] (2) The fusion protein described in the second aspect;

[0203] (3) The trimeric protein described in the third aspect;

[0204] (4) The nucleic acid molecules described in the fourth aspect;

[0205] (5) The carrier described in the fifth aspect;

[0206] (6) The host cell described in the sixth aspect;

[0207] (7) The vaccine described in the eighth aspect;

[0208] (8) The reagent kit described in aspect nine; and

[0209] (ii) Pharmaceutically acceptable carriers and / or excipients, buffers, adjuvants, or any combination thereof.

[0210] In some preferred embodiments, the pharmaceutically acceptable carrier and / or excipient is selected from pH adjusters (including but not limited to phosphate buffers), surfactants (including but not limited to cationic, anionic, or nonionic surfactants, such as Tween-80), adjuvants, ionic strength enhancers (including but not limited to sodium chloride), diluents, excipients, media for containing or administering therapeutic agents, and any combination thereof.

[0211] In some preferred embodiments, the pharmaceutically acceptable carrier may be a sterile liquid, such as water and oil, including petroleum-derived, animal-, plant-derived, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some preferred embodiments, the pharmaceutically acceptable carrier is selected from water, saline solution, aqueous dextrose, glycerol, and any combination thereof.

[0212] In some preferred embodiments, the pharmaceutically acceptable excipient may be selected from starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, milk powder, glycerin, propylene, ethylene glycol, water, ethanol, and any combination thereof.

[0213] In some preferred embodiments, the pharmaceutical composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder (e.g., lyophilized powder), sustained-release formulation, etc.

[0214] The pharmaceutical compositions of the present invention can be administered by various suitable methods. Suitable methods of administration include, but are not limited to, parenteral administration, such as intravenous, intradermal, subcutaneous, oral, nasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In some preferred embodiments, the pharmaceutical compositions are formulated into pharmaceutical preparations suitable for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans according to conventional procedures.

[0215] Typically, pharmaceutical compositions intended for injection (e.g., intravenous administration, such as by bolus or continuous infusion) are sterile and isotonic. If desired, such pharmaceutical compositions may also contain solubilizers and local anesthetics such as ergotamine to reduce pain at the injection site. Furthermore, pharmaceutical compositions intended for injection may contain preservatives. In some preferred embodiments, pharmaceutical compositions intended for injection may also be available in unit dose form (e.g., stored in ampoules or in multi-dose containers).

[0216] In some embodiments, the pharmaceutical compositions of the present invention may contain additional active ingredients, such as additional vaccines, antiviral agents, and / or monoclonal antibodies.

[0217] In some embodiments, the truncated σ1 protein or variant thereof described in the first aspect, or the fusion protein described in the second aspect, or the nucleic acid molecule described in the third aspect, or the vector described in the fourth aspect, or the host cell described in the fifth aspect, or the vaccine described in the seventh aspect, or the immunogenic composition described in the eighth aspect, or the kit described in the ninth aspect, may be administered simultaneously, separately, or sequentially with the additional active ingredient.

[0218] use

[0219] In another aspect, this application provides the use of the truncated σ1 protein or variant thereof described in the first aspect, or the fusion protein described in the second aspect, or the trimeric protein described in the third aspect, or the nucleic acid molecule described in the fourth aspect, or the vector described in the fifth aspect, or the host cell described in the sixth aspect, or the vaccine described in the eighth aspect, or the kit described in the ninth aspect in the preparation of a pharmaceutical composition for inducing an immune response in a subject against a target protein or fragment thereof of a virus or disease (e.g., a target protein or fragment thereof of a tumor).

[0220] In some embodiments, the immune response includes inducing the subject to produce antibodies (e.g., neutralizing antibodies) against target proteins or fragments thereof of a virus or disease (e.g., target proteins or fragments thereof of a tumor).

[0221] In some implementations, the subject is a mammal, such as a mouse or a human.

[0222] In some embodiments, the fusion protein or trimer protein or other proteins or peptides in the vaccine are derived from target proteins of the virus or disease or fragments thereof (e.g., target proteins of tumors or fragments thereof).

[0223] On the other hand, this application provides the use of the truncated σ1 protein or variant thereof described in the first aspect, or the fusion protein described in the second aspect, or the trimeric protein described in the third aspect, or the nucleic acid molecule described in the fourth aspect, or the vector described in the fifth aspect, or the host cell described in the sixth aspect, or the vaccine described in the eighth aspect, or the kit described in the ninth aspect in the preparation of a pharmaceutical composition for the prevention and / or treatment of viral infection or disease and / or symptoms caused by viral infection.

[0224] In some implementations, the subject is a mammal, such as a mouse or a human.

[0225] In some embodiments, the fusion protein or trimer protein or other proteins or peptides in the vaccine are derived from the virus (e.g., viral membrane surface proteins).

[0226] In some embodiments, the virus is selected from SARS-CoV-2, influenza virus, rabies virus, respiratory syncytial virus, HIV, Ebola virus, paramyxovirus, Sindbis virus, Lassa virus, human parainfluenza virus, or any combination thereof.

[0227] method

[0228] On the other hand, this application provides a method for inducing antibodies against target proteins or fragments thereof of viruses or diseases (e.g., target proteins or fragments thereof of tumors), the method comprising administering an effective amount of the fusion protein described in the second aspect, or the trimer protein described in the third aspect, or the nucleic acid molecule described in the fourth aspect, or the vector described in the fifth aspect, or the host cell described in the sixth aspect, or the vaccine described in the eighth aspect, or the kit described in the ninth aspect, either in cells in vitro or in a subject.

[0229] In some embodiments, the administration method includes intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration.

[0230] In some implementations, the subject is a mammal, such as a mouse or a human.

[0231] In some embodiments, the fusion protein or trimer protein or other proteins or peptides in the vaccine are derived from the virus (e.g., viral membrane surface proteins) or tumor target proteins or fragments thereof.

[0232] In some embodiments, the virus is selected from SARS-CoV-2, influenza virus, rabies virus, respiratory syncytial virus, HIV, Ebola virus, paramyxovirus, Sindbis virus, Lassa virus, human parainfluenza virus, or any combination thereof.

[0233] In some implementations, the tumor is a solid tumor.

[0234] On the other hand, this application provides a method for detecting in vitro whether a subject has a viral infection or a disease (e.g., a tumor), the method comprising: contacting a biological sample obtained from the subject with the fusion protein described in the second aspect or the trimer protein described in the third aspect; and detecting the presence of a complex formed by the fusion protein or trimer protein and an antibody.

[0235] In some implementations, the subject is a mammal, such as a mouse or a human.

[0236] In some embodiments, the fusion protein or trimer protein or other proteins or peptides in the vaccine are derived from the virus (e.g., viral membrane surface proteins) or tumor target proteins or fragments thereof.

[0237] In some embodiments, the virus is selected from SARS-CoV-2, influenza virus, rabies virus, respiratory syncytial virus, HIV, Ebola virus, paramyxovirus, Sindbis virus, Lassa virus, human parainfluenza virus, or any combination thereof.

[0238] In some implementations, the tumor is a solid tumor.

[0239] In some embodiments, the biological sample is selected from whole blood, serum, plasma, or any combination thereof.

[0240] On the other hand, this application provides a method for screening candidate drugs capable of inhibiting viral infection or disease (e.g., tumor) cells, the method comprising contacting the host cells with the candidate drug before, simultaneously with, or after contacting the fusion protein of the second aspect or the trimer protein of the third aspect with the host cells.

[0241] In some embodiments, the virus is selected from SARS-CoV-2, influenza virus, rabies virus, respiratory syncytial virus, HIV, Ebola virus, paramyxovirus, or any combination thereof.

[0242] In some implementations, the tumor is a solid tumor.

[0243] Terminology Definition

[0244] In this article, the term "Orthoreovirus" is also referred to as the genus Riovirus, which is a family of viruses in the family Reoviridae. Viruses in this genus typically infect vertebrates, such as humans, birds, monkeys, and sheep. These viruses possess double-stranded RNA and belong to the ribovirus domain. Some representative species in the genus Orthoreovirus include, but are not limited to, Mammalian orthoreovirus, Avian orthoreovirus, Baboon orthoreovirus, Neoavian orthoreovirus, Piscine orthoreovirus, Reptilian orthoreovirus, and Testudine orthoreovirus.

[0245] In this paper, the term "Mammalian orthoreovirus (MRV)" refers to viruses belonging to the Reoviridae family. MRVs are widely distributed in the respiratory and digestive tracts of mammals, with a broad host range. Different MRV strains have been isolated from various mammals. MRVs mainly include four serotypes: serotype 1 Lang (T1L), serotype 2 Jones (T2J), serotype 3 Dearing (T3D), and serotype 4 Ndelle (T4N). Their σ1 protein amino acid sequences share high homology (e.g., 80%, 90%). To date, several MRV strains have been isolated, and the amino acid sequences of their T3D σ1 proteins have been reported. For example, an example sequence of the T3D σ1 protein is provided in SEQ ID NO:1 (PDB:6GAP). In this paper, MRV encompasses naturally occurring MRVs as well as MRVs generated through genetic drift, artificial synthesis, and / or recombinant processes, and also includes variants derived from naturally occurring MRVs.

[0246] In this document, the term "σ1 protein" refers to a protein in the S1 genome segment of the genus *Orthoreovirus*, primarily responsible for cell attachment. In some embodiments, the σ1 protein is derived from a genome segment of mammalian orthoreovirus 3. In this document, σ1 protein encompasses naturally occurring σ1 protein and its homologs. In this document, "homologous protein" refers to proteins that share significant similarity in amino acid sequences across different organisms or within the same organism, and that perform the same or similar functions. Homologous proteins exhibit species diversity and a common evolutionary origin. In some embodiments, homologs of the σ1 protein share at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the σ1 protein.

[0247] As used herein, the terms “wild,” “wild-type,” or “natural” are used interchangeably. When these terms are used to describe nucleic acid molecules, peptides, or proteins, they mean that the nucleic acid molecule, peptide, or protein exists in nature, is found in nature, and has not undergone any artificial modification or processing. As used herein, wild-type σ1 protein refers to naturally occurring, biologically active σ1 protein.

[0248] As used in this article, the term “multimerization” or “multiplying” refers to the formation of multimers of proteins or polypeptides.

[0249] As used herein, the term “immunogenicity” refers to the ability of a substance, in the presence or absence of an adjuvant, to elicit, trigger, stimulate, or induce an immune response against a specific antigen in a human or animal.

[0250] As used herein, the term "immunogen" or "immunogen component" refers to a substance that is immunogenic. In some embodiments, symptoms or illness caused by a pathogen are prevented (or mitigated or improved) by inhibiting the replication of the pathogen after a subject has been exposed to it. In this document, "immunogen" or "immunogen component" should be understood to encompass substances (e.g., vaccines) intended for administration to a subject or group of subjects to elicit a protective or palliative immune response against a pathogen.

[0251] As used herein, the term "immune response" refers to the response of immune system cells, such as B cells, T cells, or monocytes, to a stimulus. An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen-specific neutralizing antibodies. An immune response can also be a T cell response, such as a CD4+ response or a CD8+ response. In some embodiments, the response is specific to a particular antigen (i.e., an "antigen-specific response"). If the antigen is derived from a pathogen, the antigen-specific response is a "pathogen-specific response." A "protective immune response" refers to an immune response that inhibits the harmful functions or activities of a pathogen, reduces pathogen infection, or alleviates symptoms (including death) arising from pathogen infection. In this document, immune response encompasses all of the above.

[0252] As used herein, the term "adjuvant" refers to an agent that enhances the production of an immune response in a nonspecific manner. Common adjuvants include suspensions of minerals (alum, aluminum hydroxide, aluminum phosphate) onto which antigens are adsorbed, as well as emulsions; said emulsions may include water-in-oil and oil-in-water (and their variants, including double emulsions and reversible emulsions), lipoglycosides, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll-like receptor agonists (especially TLR2, TLR4, TLR7 / 8, and TLR9 agonists), and various combinations of the above components.

[0253] As used herein, the term "mutation" refers to the presence of a missing, added, or substituted amino acid residue in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide. In the specification (particularly the examples), the substitution of an amino acid at a specific position in the protein sequence is expressed as "(amino acid residue in wild-type protein)(amino acid position)(amino acid residue in engineered protein)". For example, "I14D" means that amino acid I at position 14 of the amino acid sequence of the reference protein is substituted with amino acid D.

[0254] As used herein, the term "corresponding position" refers to the amino acid position in the two sequences being compared when performing an optimal alignment, i.e., when the two sequences are aligned to obtain the highest percentage identity. For example, the statement "corresponding to positions 20, 25, 27, 28, 29, 31, 33, 34, 36, 38, 42, 43, 47, 48, and 53 of the wild-type σ1 protein in SEQ ID NO:1" means that when a sequence is optimally aligned with SEQ ID NO:1, i.e., when a sequence is aligned with SEQ ID NO:1 to obtain the highest percentage identity, the amino acid position in the compared sequence that is equivalent to positions 20, 25, 27, 28, 29, 31, 33, 34, 36, 38, 42, 43, 47, 48, and 53 of the wild-type σ1 protein in SEQ ID NO:1.

[0255] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0256] As used herein, the term "antigen" refers to a molecule that can be recognized by antibodies. Examples of antigens include those containing antigenic determinants, such as peptides, lipids, polysaccharides, and nucleic acids that are recognized by immune cells.

[0257] As used in this article, the term "C-terminal truncated by X amino acids" means that the X consecutive amino acids at the very end of the C-terminus are truncated. Similarly, the term "N-terminal truncated by X amino acids" means that the X consecutive amino acids at the very end of the N-terminus are truncated.

[0258] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0259] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0260] As is known to those skilled in the art, codons exhibit degeneracy. That is, during protein translation, each amino acid can correspond to one or more codons, for example, up to six codons. Different species show significant differences in their use of degenerate codons encoding a particular amino acid, exhibiting different preferences. This preference phenomenon is known as "codon bias." Therefore, as used herein, the term "codon bias" refers to the situation where a species prefers to use certain specific codons to encode amino acids. Optimizing the sequence of nucleic acid molecules according to codon bias can be particularly advantageous in certain situations, for example, it may help improve the expression level of the protein encoded by the nucleic acid molecule. For example, when using E. coli (or human cells) to express a protein or fragment thereof, optimizing the nucleic acid sequence encoding the protein or fragment thereof against the codon bias of E. coli (or human cells) would be potentially advantageous.

[0261] As used herein, the term "virus-like particle (VLP)" is a multimeric particle whose structure may be similar to or dissimilar to that of a natural virus particle. In some embodiments, a VLP is a natural virus particle. In some embodiments, a VLP is a virus-like particle assembled from proteins. It has been demonstrated that some viral proteins (e.g., capsid proteins, surface proteins, envelope proteins) can spontaneously form VLPs after recombinant expression in a suitable expression system (e.g., RSV, HBV, HEV, HPV).

[0262] As used herein, the term “pharmaceutical acceptable” means something recognized in the pharmaceutical industry as suitable for use in animals, and particularly in humans. As used herein, the term “pharmaceutical acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters (including, but not limited to, phosphate buffers), surfactants (including, but not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80), adjuvants, ionic strength enhancers (including, but not limited to, sodium chloride), diluents, excipients, media for containing or administering therapeutic agents, and any combination thereof.

[0263] As used herein, pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including petroleum-derived, animal-, plant-based, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Physiological saline is a preferred carrier when administering pharmaceutical compositions intravenously. Saline solutions, as well as aqueous dextran and glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions.

[0264] Pharmaceutically acceptable excipients, as used herein, may include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the pharmaceutical composition may also contain a wetting agent, or an emulsifier such as sodium hyaluronate, or a pH buffer. The pharmaceutical composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc.

[0265] As used herein, the term "subject" refers to mammals, including but not limited to humans, rodents (mice, rats, guinea pigs), dogs, horses, cattle, cats, pigs, monkeys, chimpanzees, etc. Preferably, the subject is a human.

[0266] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for disease prevention is an amount sufficient to prevent, stop, or delay the onset of disease; an effective amount for disease treatment is an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0267] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological activity of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0268] Beneficial effects of the invention

[0269] Compared with the prior art, the truncated σ1 protein or its variants provided in this application have at least one or more of the following features: (1) it can polymerize itself or other proteins or peptides linked to it (e.g., dimerization, trimerization, tetramerization, pentamerization); (2) it can increase the stability of itself or other proteins or peptides (e.g., thermal stability); (3) it can increase the proportion of unstable conformations of other proteins or peptides (e.g., pre-F of RSV F protein); (4) it can increase the protein expression level of itself or other proteins or peptides (up to about 5-fold); (5) it can enhance the immunogenicity of antigen proteins and induce the production of more efficient and broader-spectrum neutralizing antibodies; (6) it can induce its own non-trimeric proteins (e.g., ACE2) to form a multimeric structure.

[0270] In summary, the truncated σ1 protein or its variants provided in this application exhibit excellent polymerization (e.g., trimerization) capabilities, making them suitable for various vaccine platforms, such as nucleic acid vaccines, recombinant protein vaccines, viral vector vaccines, and particulate vaccines. Therefore, the truncated σ1 protein or its variants provided in this application have significant potential in inducing immune responses in subjects.

[0271] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings. However, those skilled in the art will understand that the following embodiments and drawings are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of preferred embodiments. Attached Figure Description

[0272] Figure 1 shows the Fd3 amino acid sequence and its relative position in the structure of the ororeovirus T3Dσ1 protein.

[0273] Figures 2A and 2B show the SDS-PAGE and SEC-HPLC results of the purified StriFK-Fd3 fusion protein, respectively. In Figure 2A, lane Mr: molecular weight label; lane 1: StriFK-Fd3 supernatant sample; lane 2: fraction flowing through the Ni-6FF column; lane 3: fraction eluted with 30 mM imidazole; lane 4: fraction eluted with 250 mM imidazole.

[0274] Figures 3A and 3B show the SEC-HPLC peak chromatograms and natural gel results of the purified StriFK-Fd6 to Fd13 fusion protein, respectively. Figure 3C shows the SDS-PAGE results of the purified StriFK-Fd9 to Fd13 fusion protein.

[0275] Figures 4A and 4B show the SDS-PAGE and SEC-HPLC results of the purified StriFK-Fd15 to Fd19 fusion protein, respectively. In Figure 4A, lane Mr: molecular weight label; lane 1: supernatant; lane 2: fraction flowing through the Ni-6FF column; lane 3: fraction eluted with 30 mM imidazole; lane 4: fraction eluted with 250 mM imidazole.

[0276] Figure 5 shows the SEC-HPLC peak diagram of the purified StriFK-Fd1301 to Fd1321 fusion protein.

[0277] Figure 6 shows the DSF results of the purified StriFK-Fd1301 to Fd1321 fusion protein.

[0278] Figure 7 shows the results of Western blot (WB) and ELISA (Figure 7B) detection of the supernatant from the expression of StriFK-Fd1303 and StriFK fusion proteins. Mr: molecular weight marker; lanes T1 / T2 / T3 represent three replicate samples; red arrows indicate the position of the target protein band.

[0279] Figure 8 shows the detection results of different fusion protein expression levels. The protein yields of the Fd1303 fusion antigen (HA (Figure 8A), RSV F (Figure 8B), ACE2 (Figure 8C), RABV G (Figure 8D), SINV E2 (Figure 8E), LASV G, HPIV3 F, BA.5S (Figure 8F)) in CHO cells were compared with those of the T4-Foldon assay. Mr: Molecular weight marker; lanes T1 / T2 / T3 represent three replicate samples; red arrows indicate the location of the target protein band.

[0280] Figure 9 shows the antibody titer detection results of mice immunized with the fusion proteins HA-Fd1303 and HA-T4 (Figure 9A) and BA.5S-Fd1303 and BA.5S-T4 (Figure 9B).

[0281] Figure 10 shows the design and characterization results of the recombinant ACE2 protein. Figure 10A shows the structural schematics and SDS-PAGE results of ACE2-Ig, HexaACE2-Ig, and TriACE2-Ig. Figure 10B shows the SEC-HPLC results of TriACE2-Ig and ACE2-Ig. Figure 10C shows the analytical ultracentrifugation AUC results of the trimer TriACE2-Ig. Figure 10D shows the single-particle cryo-electron microscopy (cryo-EM) results.

[0282] Figure 11 shows the results of detection of the broad-spectrum neutralizing activity of the ACE2 recombinant protein against SARS-CoV-2 variants and SARS-CoV.

[0283] Sequence information

[0284] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0285] Table 1: Sequence Description Detailed Implementation

[0286] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0287] Unless otherwise specified, the experiments and methods described in the embodiments are performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in this invention can be found in Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by FM. Ausubel et al., (1987)); the *Methods in Enzymology* series (academic publishing company): *PCR 2: A PRACTICAL APPROACH* (edited by MJ. MacPherson, BD. Hames, and GR. Taylor, (1995)); and *Animal Cell Culture*. CULTURE (edited by R.R. Freshney (1987)).

[0288] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0289] Example 1. Expression and analysis of StriFK-Fd3, a fusion protein of the SARS-CoV-2 spike (S) protein and the Fd3 locator derived from ororeovirus 3.

[0290] The EIRBsMie-StriFK plasmid (for details, please refer to: 2021-A recombinant spike protein subunit vaccine confers protective immunity against SARS-CoV-2 infection and transmission in hamsters Sci Transl Med. 2021 Aug11; 13(606):eabg1143.) is a mammalian cell expression vector for the extracellular trimer of the spike protein S region of the SARS-CoV-2 prototype strain, previously constructed by our laboratory. This plasmid expression cassette contains the extracellular region of the SARS-CoV-2 S protein (MN908947.3, corresponding to the S gene coding frame aa1-aa1207 sequence, codon optimized), and the original S protein's Furin protease cleavage site RRAR (aa682-aa685) is mutated to GSAS, preventing the endogenous cellular Furin enzyme from acting on the Spike protein and cleaving it into S1 and S2 proteins, thus preserving the integrity of the Spike protein. The C-terminus of the S protein extracellular region coding frame is fused with the classic trimeric protein tag T4-Foldon (GSG-YIPEAPRDGQAYVRKDGEWVLLSTFLG, where GSG is the N-terminal linker sequence), followed by a polyhistidine peptide (SLE-HHHHHHHH, SLE-8×His, where SLE is the linker sequence) to facilitate affinity chromatography purification of the target protein. In mammalian cells, such as CHO and 293 cells, transfection with the EIRBsMie-StriFK plasmid can express and purify the soluble SARS-CoV-2 S spike protein extracellular region trimeric protein (StriFK-T4). In the EIRBsMie-StriFK plasmid, the N-terminal GS of T4-Foldon is encoded by the BamHI (GGATCC) restriction site, and the C-terminal LE is encoded by the XhoI (CTCGAG) restriction site. Therefore, T4-Foldon can be replaced by double digestion with BamHI / XhoI.

[0291] Based on the known crystal structure of the ororeovirus T3Dσ1 protein (PDB:6GAP, amino acid sequence as shown in SEQ ID NO:1), we hypothesized that its N-terminal α-helical domain (named Fd3, 143aa, sequence as shown in SEQ ID NO:2) may induce the formation of a trimer, possessing the potential to serve as a novel trimeric protein tag (Figure 1). Using the EIRBsMie-StriFK plasmid as a vector, the T4-Foldon sequence was replaced with the synthetic sequence Fd3 (SEQ ID NO:2) by double digestion with BamHI and XhoI, yielding the EIRBsMie-StriFK-Fd3 plasmid (SEQ ID NO:37). The EIRBsMie-StriFK-Fd3 expression plasmid was transfected into CHO cells (ExpiCHO cells) according to the following steps to express the recombinant protein StriFK-Fd3.

[0292] (1) Using 3×10 6 The cell density of ExpiCHO cells was determined by adding an appropriate amount of ExpiCHO culture medium. TM Expression Medium was cultured in Erlenmeyer flasks and placed in a constant temperature shaker at 37°C, 8% CO2, and appropriate rotation speed for 24 hours until the cell density reached 6 × 10⁶ cells / mL. 6 The density.

[0293] (2) Using the ExpiFectamine CHO Transfection Kit, the EIRBsMie-StriFK-Fd3 plasmid was transfected into the cells. After culturing under the same conditions for 17-24 hours, the feed and expression enhancer provided in the kit were added. The cells were then placed in a constant temperature shaker at 32°C, 5% CO2, and appropriate rotation speed for 6 days.

[0294] (3) After 6 days of culture, the expression cell suspension was collected, centrifuged at 12,000 rpm at room temperature for 30 min, the supernatant was collected and dialyzed into PBS, and then filtered through a 0.22 μm filter membrane.

[0295] (4) The supernatant sample dialyzed into PBS was purified by medium-pressure Ni affinity chromatography, washed with 15 mM or 30 mM imidazole solution to remove impurities, and eluted with 150 mM or 250 mM imidazole solution to remove the target protein. The obtained target protein was dialyzed into PBS buffer and stored at -20°C.

[0296] (5) The purity and molecular weight of the StriFK-Fd3 fusion protein were analyzed (Figure 2). The SDS-PAGE results (Figure 2A) showed that the molecular weight of the StriFK-Fd3 fusion protein monomer was slightly less than 250 kDa. The SEC-HPLC results (Figure 2B) showed that the purity of the StriFK-Fd3 fusion protein was greater than 95% and the molecular weight was about 669 kDa, which was about 3 times that of the monomer in the SDS-PAGE results. This proves that the fusion expression of the Fd3 sequence can enable the extracellular region of the SARS-CoV-2 Spike protein to form a trimer.

[0297] Example 2. Construction and molecular evaluation of Fd3 truncated sequence fusion expression plasmid

[0298] Although Fd3 can induce the fusion protein to form a trimer, its full length is 143 amino acids, and its large molecular weight may affect the structure and function of the target protein. To obtain a trimer tag with a shorter sequence and smaller molecular weight, we first designed a truncation from the C-terminus, namely Fd6-Fd13 (see Table 2 for specific truncation methods). The amino acid sequences of Fd6 (13aa), Fd7 (30aa), Fd8 (76aa), Fd9 (71aa), Fd10 (62aa), Fd11 (55aa), Fd12 (48aa), and Fd13 (42aa) are shown in SEQ ID NO:3 to SEQ ID NO:10. Using the EIRBsMie-StriFK-Fd3 expression plasmid as a template, different fragments truncated from the C-terminus of the Fd3 sequence, from Fd6 to Fd13, were obtained by PCR. These fragments were cloned into the StriFK-Fd3 expression plasmid via BamHI and XhoI restriction sites, resulting in the expression plasmids EIRBsMie-StriFK-Fd6, EIRBsMie-StriFK-Fd7, EIRBsMie-StriFK-Fd8, EIRBsMie-StriFK-Fd9, EIRBsMie-StriFK-Fd10, EIRBsMie-StriFK-Fd11, EIRBsMie-StriFK-Fd12, and EIRBsMie-StriFK-Fd13.

[0299] The constructed EIRBsMie-StriFK-Fd6 to EIRBsMie-StriFK-Fd13 expression plasmids were expressed and purified as fusion proteins using the same method as in Example 1. SEC-HPLC results (Figure 3) showed that the three fusion proteins, StriFK-Fd6, StriFK-Fd7, and StriFK-Fd8, could all form trimers, while StriFK-Fd7 and StriFK-Fd6 showed bimodal peaks, indicating a slight decrease in trimer stability (Figure 3A). The proteins StriFK-Fd9, StriFK-Fd10, StriFK-Fd11, StriFK-Fd12, and StriFK-Fd13 could all form relatively homogeneous trimers with a molecular weight of approximately 669 kDa (Figure 3B). The natural gum results for StriFK-Fd9 to Fd13 also showed (Figure 3C) that their molecular weights ranged from 480 to 720 kDa, and they were in trimer form. Among them, Fd13 had the shortest sequence, consisting of only 42 amino acids.

[0300] In addition, we designed five truncated molecules, namely Fd15 to Fd19, based on the coiled-coil structure region of the original Fd3 sequence (see Table 2 for specific truncation methods). Among them, Fd15, Fd16, and Fd18 are located at the N-terminus of the Fd3 sequence, and Fd17 and Fd19 are located at the C-terminus of the Fd3 sequence. The amino acid sequences of Fd15 (28 aa), Fd16 (35 aa), Fd17 (40 aa), Fd18 (30 aa), and Fd19 (30 aa) are shown in SEQ ID NO:11 to SEQ ID NO:15. Using the EIRBsMie-StriFK-Fd3 expression plasmid as a template, truncated fragments from different regions of the Fd3 sequence, Fd15-Fd19, were obtained by PCR. These fragments were cloned into the EIRBsMie-StriFK-Fd3 expression plasmid via BamHI and XhoI restriction sites, resulting in the expression plasmids EIRBsMie-StriFK-Fd15, EIRBsMie-StriFK-Fd16, EIRBsMie-StriFK-Fd17, EIRBsMie-StriFK-Fd18, and EIRBsMie-StriFK-Fd19.

[0301] The constructed StriFK-Fd15 to Fd19 expression plasmids were purified and expressed as fusion proteins using the same method as in Example 1. SDS-PAGE results (Figure 4A) showed that StriFK-Fd15 and StriFK-Fd18 had low protein expression levels, while StriFK-Fd16, StriFK-Fd17, and StriFK-Fd19 showed normal protein expression. SEC-HPLC results (Figure 4B) showed that only StriFK-Fd17 protein exhibited a single peak with a purity greater than 95% and a molecular weight of approximately 669 kDa, indicating that the Fd17 sequence (40 aa) could induce the formation of a homogeneous trimer in the S protein. The other four S fusion proteins showed distinct bimodal peaks, containing both trimers and monomers, indicating a decrease in the trimerizing ability of the truncated sequences Fd15 (28 aa), Fd16 (35 aa), Fd18 (30 aa), and Fd19 (30 aa).

[0302] Therefore, Fd13 was selected as a candidate trimerization base for further research.

[0303] Table 2. Truncation methods for Fd6-Fd13 and Fd15-Fd19

[0304] Example 3. Construction and molecular evaluation of Fd13 sequence mutation-modified sequence fusion expression plasmid.

[0305] To further improve the stability of the Fd13 molecule, we combined structural biology methods to select key sites that form hydrophobic interactions or salt bridges to modify Fd13, designing a total of 21 single-site / combination mutations (specific mutation sites are shown in Table 3), namely Fd1301-Fd1321. Using the EIRBsMie-StriFK-Fd13 expression plasmid as a template, we obtained fragments of Fd13 with different mutations, Fd1301 to Fd1321, by PCR. These fragments were cloned into the StriFK-Fd13 expression plasmid after BamHI and XhoI restriction sites, resulting in StriFK-Fd1301 to StriFK-Fd1321 expression plasmids. The amino acid sequences of Fd1301 to Fd1321 are shown in SEQ ID NO:16 to SEQ ID NO:36.

[0306] The constructed expression plasmids StriFK-Fd1301 to StriFK-Fd1321 were expressed and purified as fusion proteins using the same method as in Example 1. SDS-PAGE results (Figure 5) showed that, except for StriFK-Fd1319 which could not be expressed normally, the other 20 groups of Fd13 mutant S fusion proteins all showed a single peak and a molecular weight of approximately 669 kDa, maintaining a uniform trimer form. Trimeric thermal stability was analyzed using the DSF method. Protein was added to a 96-well PCR plate at a concentration of 200 μg / mL, mixed with fluorescent dye, and placed in a quantitative real-time PCR instrument. Protein buffer PBS was used as a blank control. Each detection group was repeated in triplicate, with the temperature range set from 10℃ to 90℃. Fluorescence intensity was measured every 0.5℃. DSF results (Figure 6) showed that the trimer S protein had two different midpoint denaturation temperatures, Tm1 and Tm2. Previous studies have shown that the thermal stability of S protein trimers gradually increases with increasing Tm1 values, while the effect of Tm2 changes on the properties of the S protein remains unclear. Analysis of the Tm1 values ​​of all S protein trimers (Table 4) shows that the Tm1 value of StriFK is 44℃, while the Tm1 value of SHP (S protein containing 6 proline mutations) constructed in our laboratory earlier is 47℃, an increase of approximately 3℃, indicating that SHP has higher thermal stability, which is largely consistent with previously reported results. In addition, the Tm1 values ​​of StriFK-Fd13 and StriFK-Fd17 are 44℃ and 44.5℃, respectively, with thermal stability comparable to or slightly improved compared to StriFK. Of the 21 designed Fd13 mutations, most had no effect on or only a small increase in the Tm1 value of the S protein trimer. The StriFK-Fd1303 mutation had the highest Tm1 value, approximately 45.5 °C, which was 1.5 °C higher than StriFK and StriFK-Fd13. This indicates that Fd1303 (R40I mutation) can enhance the thermal stability of the S protein trimer, superior to Fd13 and the T4 tag. Therefore, these truncated mutants maintain stable trimerization ability while also showing a suitable improvement in thermal stability.

[0307] Table 3. Mutation sites contained in Fd1301-Fd1321

[0308] Table 4. Thermal stability (Tm) analysis of S proteins fused with Fd1301-Fd1321 tags.

[0309] Example 4. Expression and analysis of StriFK-Fd1303, a fusion protein of the SARS-CoV-2 S protein and the Fd1303 locator.

[0310] The EIRBsMie-StriFK-Fd1303 and EIRBsMie-StriFK expression plasmids were expressed via ExpiCHO. TM ExpiCHO-S cells were transfected using the expression system kit. 10 mL of each of the two expression plasmids was transfected, with three replicates. Culture supernatants were collected 7 days after transfection. Western blotting results (Figure 7A) showed that the bands of the StriFK-Fd1303 and StriFK fusion proteins were slightly smaller than 250 kDa, indicating a certain increase in the expression level of StriFK-Fd1303 compared to the StriFK group. The expression level of the extracellular trimeric protein (45C3 / 85F7-HRP) of the SARS-CoV-2S spike protein used in Example 1 was detected using a double antibody sandwich assay. Supernatant analysis results (Figure 7B) showed that the supernatant expression level of the StriFK-Fd1303 group was significantly increased by approximately 2.3 times compared to the StriFK group.

[0311] Example 5. Effects of Fd1303 on the expression levels of various fusion proteins

[0312] Using EIRBsMie-StriFK-Fd1303 and EIRBsMie-StriFK plasmids as vectors, the target sequences were directly inserted into the following domains after XbaⅠ and BamHI restriction sites: influenza virus hemagglutinin (HA) extracellular domain (SEQ ID NO:44), respiratory syncytial virus pre-fusion conformation F protein head domain (RSV F) (SEQ ID NO:45), human ACE2 extracellular domain (ACE2) (SEQ ID NO:46), rabies virus glycoprotein G extracellular domain (RABV G) (SEQ ID NO:47), Sindbis virus E2 protein extracellular domain (SINV E2) (SEQ ID NO:48), Lassa virus glycoprotein extracellular domain (LASV G) (SEQ ID NO:49), human parainfluenza virus 3F protein extracellular domain (HPIV3 F) (SEQ ID NO:50), and SARS-CoV-2 Omicron BA.5 spike protein extracellular domain (BA.5S) (SEQ ID NO:48). NO:51), obtained HA-Fd1303 and HA-T4, EIRBsMie-RSV F-Fd1303 and EIRBsMie-RSV F-T4, EIRBsMie-ACE2-Fd1303 and EIRBsMie-ACE2-T4, EIRBsMie-RABV G-Fd1303 and EIRBsMie-RABV G-T4, EIRBsMie-SINV E2-Fd1303 and EIRBsMie-SINV E2-T4, EIRBsMie-LASV G-Fd1303 and EIRBsMie-LASV G-T4, EIRBsMie-HPIV3 F-Fd1303 and EIRBsMie-HPIV3 F-T4, EIRBsMie-BA.5S-Fd1303 and EIRBsMie-BA.5S-T4 expression plasmids.

[0313] All the above expression plasmids were processed via ExpiCHO. TM The expression system kit was used to transfect ExpiCHO-S cells, and the culture supernatant was collected 7 days after transfection. The results showed (Figure 8A-E) that the yield of Fd1303 fusion protein in CHO supernatant was significantly increased by 1.4-4.8 times compared with T4-Foldon fusion protein.

[0314] The expression supernatants of HA-Fd1303 and HA-T4 were purified in one step using a Ni column to obtain the HA-Fd1303 and HA-T4 fusion proteins. SDS-PAGE results (Figure 8A) showed that the bands of the HA-Fd1303 and HA-T4 fusion proteins were located between 75 and 100 kDa, and the expression level of HA-Fd1303 was higher than that of HA-T4.

[0315] Expression supernatants from LASV G-Fd1303, LASV G-T4, HPIV3 F-Fd1303, HPIV3 F-T4, and BA.5S-Fd1303 and BA.5S-T4 were purified in one step using a Ni column to obtain LASV G, HPIV3 F, and BA.5S fusion proteins. The results (Figure 8F) showed that the molecular weight of the LASV G, HPIV3 F, and BA.5S fusion proteins on Native-PAGE was 3 times higher than that on SDS-PAGE, indicating that both Fd1303 and T4-Foldon tags can induce appropriate trimerization.

[0316] Example 6. Effect of Fd1303 on vaccine antigen immunogenicity

[0317] To evaluate the immunogenicity of the Fd1303 fusion protein, we used the HA-Fd1303 and HA-T4, BA.5S-Fd1303 and BA.5S-T4 proteins prepared in Example 5 as immunogens in mice for our experiments.

[0318] Mouse immunization:

[0319] Purified HA-Fd1303, HA-T4, BA.5S-Fd1303, and BA.5S-T4 proteins were mixed with an equal volume of Freund's adjuvant and emulsified. Six- to eight-week-old female BALB / c mice were immunized via bilateral inguinal subcutaneous injection at multiple sites, with a booster immunization two weeks after the initial immunization. Serum samples were collected from mice before and after immunization for subsequent testing.

[0320] Combined with antibody detection:

[0321] The ELISA method was used to detect HA or spike-specific binding antibodies in the serum of immunized mice.

[0322] Untagged HA and Spike proteins were diluted separately with 50 mM CB buffer (NaHCO3 / Na2CO3 buffer, final concentration 50 mM, pH 9.6) at pH 9.6 to a final concentration of 2 μg / mL. 100 μL of coating buffer was added to each well of a 96-well microplate, and the plates were coated at 2–8°C for 16–24 hours, followed by coating at 37°C for 2 hours. The plates were washed once with PBST washing buffer (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), and then 200 μL of blocking buffer (20 mM Na2HPO4 / NaH2PO4 buffer solution at pH 7.4 containing 20% ​​fetal bovine serum and 1% casein) was added to each well. The plates were then blocked at 37°C for 2 hours; the blocking buffer was discarded. After drying, the plates were stored in aluminum foil bags at 2–8°C for later use.

[0323] Serum samples were diluted 1:100 in the first well and serially diluted 10-fold. 100 μL of the diluted sample was added to each well of the coated ELISA plate, and the plate was incubated at 37°C for 1 hour. The plate was then washed five times with PBST buffer, and 100 μL of GAM-HRP reaction solution was added to each well. The plate was incubated at 37°C for 30 minutes. After completing the enzyme-labeled reaction, the plate was washed five times with PBST buffer, and 50 μL of TMB chromogenic reagent (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) was added to each well. The plate was incubated at 37°C for 15 minutes. After completing the colorimetric reaction, 50 μL of stop solution (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) was added to each well of the plate, and the OD450 / 630 values ​​of each well were measured using an ELISA reader.

[0324] Neutralizing antibody testing:

[0325] Serum SARS-CoV-2BA.5 pseudovirus neutralizing antibodies were detected using a novel coronavirus pseudovirus neutralizing antibody detection method based on lentivirus (Lvpp) vector.

[0326] One day in advance, huACE2-H1299 cells were seeded onto 96-well fluorescent black plates, 6000 cells per well. Serum samples were serially diluted 30-fold starting from 30-fold and then 3-fold in DMEM containing 2% FBS, for a total of 7 dilutions, with a volume of 60 μL. The virus was diluted using the same diluent to obtain approximately 1200 pseudovirus particles per well, with a volume of 60 μL. The serum and diluted virus were mixed and incubated at 37°C for 1 hour. The cell supernatant was discarded, and 100 μL of the virus-serum mixture was added. After incubation at 37°C for 48 hours, the infected cells were fluorescently imaged using a confocal high-content imaging system. The number of fluorescently positive cells was quantitatively analyzed using Columbus software, and statistical calculations were performed to obtain the corresponding neutralizing antibody titers in the serum.

[0327] Hemagglutination inhibition (HAI) test:

[0328] The hemagglutination inhibition assay was used to detect the HI titer of immune serum against influenza virus A / H3N2.

[0329] Serum was pretreated with receptor-destroying enzyme (RDE) at 37°C for 18 hours, followed by heat inactivation at 56°C for 30 minutes to eliminate nonspecific inhibitors. The treated serum was first diluted 1:5 with PBS buffer, and then serially diluted 2-fold (1:5 to 1:640) in 96-well plates. Influenza virus was mixed with the diluted serum at a 1:1 ratio and incubated at room temperature for 1 hour, followed by the addition of 0.75% guinea pig erythrocytes. The HI titer was defined as the reciprocal of the highest serum dilution showing complete inhibition of hemagglutination after 45 minutes of incubation at room temperature.

[0330] Immunological evaluation in mice (Figures 9A and B) showed high levels of Anti-HA and Anti-Spike binding antibodies, respectively, in the serum of mice immunized with HA-rFd1303 and BA.5S-rFd1303, as well as high titers of neutralizing antibodies (nAbs) against the homologous influenza A virus H3N2 and SARS-CoV-2 BA.5 pseudovirus. Furthermore, under the same immunization dose conditions, the nAb titer induced by the Fd1303 fusion immunogen was comparable to that induced by the control T4-Foldon fusion immunogen. These results indicate that the recombinant vaccine immunogen fused with the Fd1303 tag possesses good immunogenicity.

[0331] Example 7. Fd1303 induces the formation of a trimer structure from its own non-trimeric protein ACE2.

[0332] The EIRBsMie-ACE2-Fc expression plasmid previously constructed in the laboratory contains the full-length amino acid sequence of the extracellular domain of human ACE2 from amino acid residues 1 to 740 (with an additional H345L mutation introduced to eliminate the peptidase activity of the ACE2 extracellular domain, but without affecting the binding activity to SARS-CoV and SARS-CoV-2 spike proteins), and fuses an hIgG1 Fc domain fragment to the C-terminus, i.e., the EIRBsMie-ACE2-Ig expression plasmid. Using the EIRBsMieACE2-Fc plasmid as a vector, after BamHI and PacⅠ restriction sites, an hIgG1 Fc domain fragment (SEQ ID NO:52) was inserted into the C-terminus of the extracellular domain (SEQ ID NO:46) of human ACE2, and the Fc region contained the L309C mutation. Then, the IgM tail sequence (SEQ ID NO:53) was ligated to obtain the EIRBsMie-HexaACE2-Ig expression plasmid. Using the EIRBsMieACE2-Fc plasmid as a vector, the hIgG1 Fc domain fragment was inserted into the C-terminus of the extracellular domain of human ACE2 after digestion with BamHI and PacⅠ enzyme sites, and then the Fd1303 sequence was ligated to obtain the EIRBsMie-TriACE2-Ig expression plasmid.

[0333] The constructed EIRBsMie-ACE2-Ig, EIRBsMie-HexaACE2-Ig, and EIRBsMie-TriACE2-Ig expression plasmids were used to express the fusion proteins according to the same method as in Example 1. The Fc-tagged fusion proteins were purified using MabSelect SuRe affinity chromatography material.

[0334] Schematic diagrams of the structures of ACE2-Ig, HexaACE2-Ig, and TriACE2-Ig are shown in Figure 10A. Reduced SDS-PAGE results (Figure 10A) show that the target bands of ACE2-Ig, HexaACE2-Ig, and TriACE2-Ig proteins are approximately 130-180 kDa. SEC-HPLC results (Figure 10B) show that the molecular weight of the trimer peak of the TriACE2-Ig protein is greater than 669 kDa.

[0335] Analytical ultracentrifugation AUC results (Figure 10C) showed that the sedimentation coefficient of the main peak of the TriACE2-Ig protein sample was 16.1 S, corresponding to a molecular weight of 787 kDa, which is basically consistent with the expected molecular weight of the trimeric ACE-Ig. In addition, the AUC results showed several other additional peaks: 4.870 S (1.05%), 8.640 S (2.55%), 13.017 S (9.44%), 20.483 S (4.44%), and 24.016 S (7.72%), corresponding to molecular weights of 131, 311, 575, 1130, and 1440 kDa, respectively. These may represent half of ACE2-Ig, ACE2-Ig monomer, dimer, tetramer, and hexamer, respectively.

[0336] Single-particle cryo-electron microscopy analysis (Figure 10D) showed that TriACE2 mainly constituted a typical trimeric bouquet structure. Some tetrameric, hexammeric, and octamer bouquet structures were also observed, along with some impurity particles. 2D classification results showed that in the TriACE2-Ig protein sample, the proportions of ACE2-Ig trimers, tetramers, hexamers, and octamers were 63.7%, 11.1%, 9.8%, and 1.6%, respectively.

[0337] The above results indicate that the TriACE2-Ig recombinant protein exists primarily in a trimer form, with small amounts of other polymeric components. Therefore, Fd1303 can induce its own non-trimeric proteins (e.g., ACE2) to form multimeric structures, particularly trimer structures.

[0338] Example 8. Fd13O3 can enhance the highly efficient and broad-spectrum neutralizing activity of trimer TriACE2-Ig.

[0339] To investigate whether the TriACE2-Ig recombinant protein can completely avoid immune escape caused by SARS-CoV-2 virus mutations and achieve broad-spectrum inhibition, we also evaluated its neutralizing activity against different variant strains based on a lentiviral (Lvpp) vector-based coronavirus pseudovirus neutralization detection method.

[0340] The results of pseudovirus neutralization assay (Figure 11) showed that, firstly, the neutralizing activities (IC50 values) of TriACE2-Ig, HexaACE2-Ig, and ACE2-Ig recombinant proteins against SARS-CoV-2D614G, Alpha, Beta, Gamma, Delta, Omicron BA.1, and SARS-CoV (constructed in our laboratory, referencing our published literature Small Methods. 2021 Feb 15; 5(2): 2001031.; Cell Rep Methods. 2024 Sep 16; 4(9): 100856.) were 1.2-2.9 ng / mL, 1.2-10.0 ng / mL, and 10.5-112.6 ng / mL, respectively. Furthermore, we compared the neutralizing activities of TriACE2-Ig and ACE2-Ig against 12 Omicron mutants, including BA.2, BA.4 / 5, BQ.1.1, CH.1.1, XBB.1.5, XBB.1.5.10, XBB.1.16, EG.5.1, CM.8.1, HK.3, BA.2.86, and JN.1 (constructed in our laboratory, referencing our published literature Small Methods. 2021 Feb 15; 5(2):2001031.; Cell Rep Methods. 2024 Sep 16; 4(9):100856.). The IC50 values ​​of the trimeric TriACE2-Ig ranged from approximately 1.0 to 7.8 ng / mL, while those of ACE2-Ig ranged from approximately 35.7 to 180.2 ng / mL. Overall, the mean IC50 value of TriACE2-Ig against all tested variants (19 pseudoviruses) was 2.9 ± 1.7 ng / mL, which was 20.9-fold lower than that of ACE2-Ig (59.7 ± 42.5 ng / mL, 19 pseudoviruses) and 1.5-fold lower than that of HexaACE2-Ig (4.4 ± 3.1 ng / mL, 7 pseudoviruses). These results indicate that trimeric TriACE2-Ig has more efficient and broader-spectrum neutralizing activity. Therefore, Fd1303 can enhance the efficient and broad-spectrum neutralizing activity of trimeric TriACE2-Ig.

[0341] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A truncated σ1 protein or a variant thereof, wherein, Compared with the σ1 protein of wild Orthoreoviru viruses, the truncated σ1 protein or its variants are: (i) truncated by 1 to 120 amino acids at the N-terminus, (ii) truncated by 1 to 140 amino acids at the C-terminus, or (iii) truncated by 1 to 140 amino acids at both the N-terminus and C-terminus. The variant, compared to the truncated σ1 protein, has one or more (e.g., conserved substitutions), deletions, or additions of amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); and the variant substantially retains the biological function of the sequence from which it originates.

2. The truncated σ1 protein or a variant thereof as described in claim 1, having one or more of the following characteristics: (1) It can polymerize itself or other proteins or peptides linked to it (e.g., dimerization, trimerization, tetramerization, pentamerization); (2) It can form an α-helical structure; (3) Compared with wild σ1 protein, it can increase the stability (e.g., thermal stability) of itself or other proteins or peptides. (4) Compared with wild σ1 protein, it can increase the proportion of unstable conformations of other proteins or peptides (e.g., pre-F of RSV F protein); (5) Compared with wild σ1 protein, it can increase the protein expression level and / or immunogenicity of itself or other proteins or peptides. (6) The truncated σ1 protein or its variants have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence from which it is derived.

3. The truncated σ1 protein or a variant thereof as described in claim 1 or 2, wherein, The viruses of the Orthoreovirus genus are selected from Mammalian orthoreovirus, Avian orthoreovirus, Neoavian orthoreovirus, Piscine orthoreovirus, Reptilian orthoreovirus, Testudine orthoreovirus, or any combination thereof; Preferably, the mammalian ororeovirus is selected from Lang (T1L) strain 1, Jones (T2J) strain 2, Dearing (T3D) strain 3, Ndelle (T4N) strain 4, or any combination thereof; Preferably, the wild-type σ1 protein is derived from the T3D strain; Preferably, the wild-type σ1 protein comprises, or is composed of, sequences selected from, the following: (i) The sequence shown in SEQ ID NO:1; (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) compared to the sequence shown in SEQ ID NO: 1; (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO:

1.

4. The truncated σ1 protein or a variant thereof according to any one of claims 1-3, wherein the truncated σ1 protein, compared with the wild-type σ1 protein, has one or more features selected from the following: (1) N-terminus truncated by 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110 or 110-120 amino acids; (2) The N-terminus is truncated by 13, 38, 50, 93 or 100 amino acids; (3) The C-terminus is shortened by 280-300, 300-320, 320-340, 340-360, 360-380, 380-400 or 400-420 amino acids; (4) The C-terminus is shortened by 300, 303, 340, 349, 353, 358, 359, 365, 367, 378, 372, 390 or 407 amino acids; (5) The N-terminus is truncated by 30-40 amino acids, and the C-terminus is truncated by 350-370 amino acids; For example, the N-terminus is truncated by 38 amino acids, and the C-terminus is truncated by 367 amino acids; For example, the N-terminus is truncated by 38 amino acids, and the C-terminus is truncated by 359 amino acids; (6) The N-terminus is truncated by 40-60 amino acids, and the C-terminus is truncated by 350-3600 amino acids; For example, the N-terminus is truncated by 50 amino acids, and the C-terminus is truncated by 353 amino acids; (7) The N-terminus is truncated by 90-110 amino acids, and the C-terminus is truncated by 300-310 amino acids; For example, the N-terminus is truncated by 93 amino acids, and the C-terminus is truncated by 300 amino acids; For example, the N-terminus is truncated by 100 amino acids, and the C-terminus is truncated by 303 amino acids; Preferably, the truncated σ1 protein is truncated by 13 amino acids at the N-terminus and 378 amino acids at the C-terminus compared to the wild-type σ1 protein.

5. The truncated σ1 protein or a variant thereof according to any one of claims 1-4, wherein the truncated σ1 protein comprises, or is composed of, sequences selected from, or is composed of sequences selected from: (i) The sequence shown in any one of SEQ ID NO:2-15; (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) compared to the sequence shown in any one of SEQ ID NO: 2-15; (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with any of the sequences shown in SEQ ID NO: 2-15; in, The sequences in (ii) and (iii) largely retain the biological functions of the sequences from which they originated; Preferably, the truncated σ1 protein comprises, or is composed of, sequences selected from, the following sequences. composition: (i) The sequence shown in SEQ ID NO:10; (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) compared to the sequence shown in SEQ ID NO: 10; (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO: 10; Among them, the sequences in (ii) and (iii) basically retain the biological functions of the sequences from which they originated.

6. The truncated σ1 protein or a variant thereof according to any one of claims 1-5, wherein, compared with the wild-type σ1 protein, the variant comprises an amino acid substitution selected from (i) a cavity-filling mutation; (ii) an electrostatic mutation; or any combination thereof; Preferably, the amino acid substitutions in the variant are selected from one or more of the following positions: positions 20, 25, 27, 28, 29, 31, 33, 34, 36, 38, 42, 43, 47, 48 and / or 53 corresponding to the wild-type σ1 protein; Preferably, the variant contains an amino acid substitution at position 53, corresponding to the wild-type σ1 protein.

7. The truncated σ1 protein or a variant thereof according to any one of claims 1-6, wherein the variant has one or more features selected from the following: (1) The amino acid at position 20, corresponding to the wild-type σ1 protein, of the variant is either E or N; Preferably, the amino acid substitution at position 20 is an A-to-E or N substitution; (2) The amino acid at position 25 corresponding to the wild-type σ1 protein in the variant is I; Preferably, the amino acid substitution at position 25 is an S-to-I substitution; (3) The amino acid at position 27 corresponding to the wild-type σ1 protein in the variant is D or R; Preferably, the amino acid substitution at position 27 is a substitution from I to D or R; (4) The amino acid at position 28 corresponding to the wild-type σ1 protein in the variant is I or L; Preferably, the amino acid substitution at position 28 is an H-to-I or L substitution; (5) The amino acid at position 29 corresponding to the wild-type σ1 protein in the variant is E; Preferably, the amino acid substitution at position 29 is an S-to-E substitution; (6) The amino acid at position 31 corresponding to the wild-type σ1 protein in the variant is E or R; Preferably, the amino acid substitution at position 31 is a substitution from T to E or R; (7) The amino acid at position 33 corresponding to the wild-type σ1 protein in the variant is Q, E or S; Preferably, the amino acid substitution at position 33 is an L-to-Q, E, or S substitution; (8) The amino acid K at position 34 of the variant, corresponding to the wild-type σ1 protein, is K; Preferably, the amino acid substitution at position 34 is an R-to-K substitution; (9) The amino acid at position 36 corresponding to the wild-type σ1 protein in the variant is E, K, or R; Preferably, the amino acid substitution at position 36 is a substitution from T to E, K, or R; (10) The amino acid at position 38 corresponding to the wild-type σ1 protein in the variant is R; Preferably, the amino acid substitution at position 38 is a G-to-R substitution; (11) The amino acid at position 42 corresponding to the wild-type σ1 protein in the variant is I; Preferably, the amino acid substitution at position 42 is an A-to-I substitution; (12) The amino acid at position 43 corresponding to the wild-type σ1 protein in the variant is D or E; Preferably, the amino acid substitution at position 43 is an N-to-D or E substitution; (13) The amino acid at position 47, corresponding to the wild-type σ1 protein, of the variant is either E or Q; Preferably, the amino acid substitution at position 47 is a substitution from I to E or Q; (14) The amino acid at position 48, corresponding to the wild-type σ1 protein, is D or Q in the variant; Preferably, the amino acid substitution at position 48 is a substitution from A to D or Q; (15) The amino acid at position 53 corresponding to the wild-type σ1 protein in the variant is I; Preferably, the amino acid substitution at position 53 is an R-to-I substitution; Preferably, the variant comprises, or consists of, sequences selected from, the following: (i) The sequence shown in any one of SEQ ID NO: 16-36; (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) compared to the sequence shown in any one of SEQ ID NO:16-36; (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with any of the sequences shown in SEQ ID NO: 16-36; in, The sequences in (ii) and (iii) largely retain the biological functions of the sequences from which they originated; Preferably, the variant comprises, or consists of, sequences selected from, the following: (i) The sequence shown in SEQ ID NO:18; (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) compared to the sequence shown in SEQ ID NO:18; (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO:18; Among them, the sequences in (ii) and (iii) basically retain the biological functions of the sequences from which they originated.

8. A fusion protein comprising a truncated σ1 protein or a variant thereof as described in any one of claims 1-7, and additional proteins or peptides (e.g., heterologous proteins or peptides); Preferably, the additional protein or polypeptide is linked to the truncated σ1 protein or a variant thereof, with or without a linker; Preferably, the truncated σ1 protein or its variants are located at the C-terminus or N-terminus of another protein or polypeptide; Preferably, the additional protein or polypeptide is immunogenic in mammals; Preferably, the additional protein or polypeptide is obtained from a virus (e.g., a viral membrane surface protein); Preferably, the additional protein or polypeptide is a target protein of a disease or a fragment thereof (e.g., a target protein of a tumor or a fragment thereof); Preferably, the additional protein or polypeptide is selected from the spike protein of SARS-CoV-2, the hemagglutinin protein of influenza virus, the glycoprotein of rabies virus, the F protein of respiratory syncytial virus, the envelope protein of HIV, the Gp protein of Ebola virus, the F protein of paramyxovirus, the extracellular domain of human ACE2 (ACE2), the SINV E2 protein, the Lassa virus glycoprotein (LASV G), the human parainfluenza virus 3F protein (HPIV3 F), or any combination thereof; Preferably, the fusion protein is in the form of a polymer (e.g., a dimer, trimer, tetramer, pentamer, hexamer, hepter, octamer), a monomer, or a mixture of both; Preferably, the fusion protein is in the form of a mixture of multiple polymers, such as a mixture of trimers, tetramers, hexamers, and octamers.

9. The fusion protein of claim 8, wherein, The fusion protein also includes a signal peptide, a tag, or any combination thereof; Preferably, the signal peptide is located at one end (e.g., the N-terminus) of the fusion protein; Preferably, the tag is located at the end (e.g., the C-terminus) of the fusion protein; Preferably, the tag is selected from multiple histidine tags, antigen or epitope tags, enzyme tags, or any combination thereof; Preferably, the additional protein or polypeptide is linked to the signal peptide and / or tag, with or without a linker; Preferably, the connector has a sequence as shown in SEQ ID NO:42 or SEQ ID NO:43; Preferably, the fusion protein has a sequence as shown in any one of SEQ ID NO:37-41.

10. A trimeric protein comprising the truncated σ1 protein or a variant thereof as described in any one of claims 1-7; Preferably, the trimer protein comprises three polypeptide chains, wherein each polypeptide chain comprises: a truncated σ1 protein or a variant thereof as described in any one of claims 1-7, and additional proteins or polypeptides; Preferably, the other proteins or polypeptides in each polypeptide chain are the same or different; Preferably, the other proteins or polypeptides in each polypeptide chain are the same; Preferably, the additional protein or polypeptide is as defined in claim 8.

11. A nucleic acid molecule comprising a nucleotide sequence encoding a truncated σ1 protein or a variant thereof as described in any one of claims 1-7, a fusion protein as described in claim 8 or 9, or a trimeric protein as described in claim 10; Preferably, the nucleotide sequence is codon-optimized or not optimized according to the codon preference of the host cell; Preferably, the nucleic acid molecule is DNA, or an RNA (mRNA) product transcribed from the DNA, or a mixture of both.

12. A vector comprising the nucleic acid molecule of claim 11; Preferably, the vector is a viral vector; Preferably, the viral vector is selected from: influenza virus vector, reverse transcriptase virus vector, adenovirus vector, adeno-associated virus vector, herpesvirus vector, poxvirus vector, baculovirus vector, papillomavirus vector, or papillomavirus vector.

13. A host cell comprising a truncated σ1 protein or a variant thereof as described in any one of claims 1-7, or a fusion protein as described in claim 8 or 9, or a trimer protein as described in claim 10, or a nucleic acid molecule as described in claim 11, or a vector as described in claim 12; Preferably, the host cell is selected from prokaryotic cells or eukaryotic cells; Preferably, the eukaryotic cell is a mammalian cell, such as a mouse cell or a human cell; Preferably, the truncated σ1 protein or its variants, fusion proteins, or trimer proteins are displayed on the surface of the host cell's cell membrane.

14. A method for expressing or producing a truncated σ1 protein or a variant thereof as claimed in any one of claims 1-7, or a fusion protein as claimed in claim 8 or 9, or a trimeric protein as claimed in claim 10, the method comprising culturing a host cell as claimed in claim 13 under conditions that allow protein expression, and optionally, recovering or purifying the truncated σ1 protein or a variant thereof, or a fusion protein, or a trimeric protein expressed therein.

15. A vaccine comprising one or more of the following (1) to (6): (1) The truncated σ1 protein or a variant thereof as described in any one of claims 1-7; (2) The fusion protein according to claim 8 or 9; (3) The trimeric protein of claim 10; (4) The nucleic acid molecule according to claim 11; (5) The carrier according to claim 12; (6) The host cell according to claim 13; Preferably, the vaccine further comprises an adjuvant and / or a buffer solution; Preferably, the adjuvant is selected from metal salts, 3-D-monophosphoryl lipid A (MPL), saponins, oil and water emulsions, liposomes, nanoparticles, or any combination thereof; Preferably, the fusion protein in the vaccine is in the form of a polymer (e.g., a dimer, trimer, or tetramer), a monomer, or a mixture.

16. A kit comprising an immunogen component, wherein the immunogen component comprises one or more of the following (1) to (7): (1) The truncated σ1 protein or a variant thereof as described in any one of claims 1-7; (2) The fusion protein according to claim 8 or 9; (3) The trimeric protein of claim 10; (4) The nucleic acid molecule according to claim 11; (5) The carrier according to claim 12; (6) The host cell according to claim 13; (7) The vaccine according to claim 15; Preferably, the kit further comprises a carrier component capable of displaying the immunogen component; Preferably, the carrier component is selected from: nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), polymerized pedicles, virus-like particles (VLPs), or any combination thereof; Preferably, the immunogen component and carrier component in the kit are provided separately or as a complex; Preferably, the immunogen component is in the form of a polymer (e.g., a dimer, trimer, or tetramer), a monomer, or a mixture; Preferably, the immunogen components in the kit are provided in the form of proteins or nucleic acids; Preferably, the carrier component in the kit is provided in the form of protein or nucleic acid; Preferably, the VLP is assembled from proteins obtained from RSV, hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

17. A pharmaceutical composition comprising: (i) Selected from any one or more of the following (1) to (8): (1) The truncated σ1 protein or a variant thereof as described in any one of claims 1-7; (2) The fusion protein according to claim 8 or 9; (3) The trimeric protein of claim 10; (4) The nucleic acid molecule according to claim 11; (5) The carrier according to claim 12; (6) The host cell according to claim 13; (7) The vaccine according to claim 15; (8) The kit according to claim 16; and (ii) Pharmaceutically acceptable carriers, excipients, buffers, adjuvants, or any combination thereof; Preferably, the pharmaceutical composition may also contain additional active ingredients; for example, additional vaccines, antiviral agents and / or monoclonal antibodies.

18. Use of the truncated σ1 protein or a variant thereof as described in any one of claims 1-7, or the fusion protein as described in claim 8 or 9, or the trimeric protein as described in claim 10, or the nucleic acid molecule as described in claim 11, or the vector as described in claim 12, or the host cell as described in claim 13, or the vaccine as described in claim 15, or the kit as described in claim 16, in the preparation of a pharmaceutical composition for inducing an immune response in a subject against a target protein or fragment thereof of a virus or disease (e.g., a target protein or fragment thereof of a tumor); Preferably, the immune response includes inducing the subject to produce antibodies (e.g., neutralizing antibodies) against target proteins or fragments of viruses or diseases (e.g., target proteins or fragments of tumors); Preferably, the subject is a mammal, such as a mouse or a human; Preferably, the fusion protein or trimer protein or other proteins or peptides in the vaccine are derived from target proteins of the virus or disease or fragments thereof (e.g., target proteins of tumors or fragments thereof).

19. Use of the truncated σ1 protein or a variant thereof as described in any one of claims 1-7, or the fusion protein as described in claim 8 or 9, or the trimeric protein as described in claim 10, or the nucleic acid molecule as described in claim 11, or the vector as described in claim 12, or the host cell as described in claim 13, or the vaccine as described in claim 15, or the kit as described in claim 16, in the preparation of a pharmaceutical composition for the prevention and / or treatment of viral infection or disease and / or symptoms caused by viral infection; Preferably, the subject is a mammal, such as a mouse or a human; Preferably, the fusion protein or trimer protein or other proteins or peptides in the vaccine are derived from the virus (e.g., viral membrane surface proteins). Preferably, the virus is selected from SARS-CoV-2, influenza virus, rabies virus, respiratory syncytial virus, HIV, Ebola virus, paramyxovirus, Sindbis virus, Lassa virus, human parainfluenza virus, or any combination thereof.

20. A method for inducing antibodies against a target protein or fragment thereof (e.g., a target protein or fragment thereof of a tumor) of a virus or disease, the method comprising administering, in vitro in cells or in a subject, an effective amount of the fusion protein of claim 8 or 9, or the trimer protein of claim 10, or the nucleic acid molecule of claim 11, or the vector of claim 12, or the host cell of claim 13, or the vaccine of claim 15, or the kit of claim 16; Preferably, the method of application includes intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration; Preferably, the subject is a mammal, such as a mouse or a human; Preferably, the fusion protein or trimer protein or other proteins or peptides in the vaccine are derived from the virus (e.g., viral membrane surface proteins) or tumor target proteins or fragments thereof. Preferably, the virus is selected from SARS-CoV-2, influenza virus, rabies virus, respiratory syncytial virus, HIV, Ebola virus, paramyxovirus, Sindbis virus, Lassa virus, human parainfluenza virus, or any combination thereof; Preferably, the tumor is a solid tumor.

21. A method for detecting in vitro whether a subject has a viral infection or a disease (e.g., a tumor), the method comprising: Contact the biological sample obtained from the subject with the fusion protein of claim 8 or 9 or the trimer protein of claim 10; In addition, the presence of a complex formed by the fusion protein or the trimer protein and the antibody is detected; Preferably, the subject is a mammal, such as a mouse or a human; Preferably, the fusion protein or trimer protein or other proteins or peptides in the vaccine are derived from the virus (e.g., viral membrane surface proteins) or tumor target proteins or fragments thereof. Preferably, the virus is selected from SARS-CoV-2, influenza virus, rabies virus, respiratory syncytial virus, HIV, Ebola virus, paramyxovirus, Sindbis virus, Lassa virus, human parainfluenza virus, or any combination thereof; Preferably, the tumor is a solid tumor; Preferably, the biological sample is selected from whole blood, serum, plasma, or any combination thereof.

22. A method for screening candidate drugs capable of inhibiting cells infected with a virus or disease (e.g., tumor), the method comprising contacting the host cells with the candidate drug before, simultaneously with, or after contacting the fusion protein of claim 8 or 9 or the trimer protein of claim 10 with the host cells.

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