Mutant slam protein or active fragment thereof

Abstract

The problem addressed is to provide a new host cell that can be infected by a wide variety of morbilliviruses.　Provided is a mutant SLAM protein or an active fragment thereof that contains a mutant V domain derived from a V domain of a human SLAM protein and has an ancestral amino acid residue and / or an ancestral amino acid deletion.

Description

Mutant SLAM protein or active fragment thereof

[0001] The present invention relates to a mutant SLAM protein or an active fragment thereof, a method for producing morbillivirus, a method for producing a vaccine against morbillivirus infection, a method for measuring the titer of morbillivirus, and the like.

[0002] Morbilliviruses are viruses of the genus Morbillivirus in the family Paramyxoviridae, and pose a major threat to a variety of host animals, including humans and livestock.

[0003] Each morbillivirus has a different host range. Measles virus (MV) primarily infects humans, cetacean morbillivirus (CeMV) infects cetaceans, and canine distemper virus (CDV) infects a wide range of carnivores. Phocid distemper virus (PDV) infects seals, rinderpest virus (RPV) infects cattle, and peste des petits ruminants (PPRV) infects small ruminants such as sheep and goats. Furthermore, new morbilliviruses have recently been discovered in bats, pigs, and cats, suggesting that the morbilliviruses that infect mammals are diverse.

[0004] Morbillivirus infection begins with the binding of a viral receptor-binding protein (RBP) known as hemagglutinin (H) to a receptor on the target cell, while another viral surface glycoprotein, the fusion (F) protein, promotes membrane fusion between the viral envelope and the target cell membrane.

[0005] Morbilliviruses are known to use signaling lymphocyte activation molecule (SLAM) expressed on immune cells and nectin-4 expressed on epithelial cells as receptors. While nectin-4 has a highly conserved amino acid sequence, the amino acid sequences of SLAM proteins are diverse (Non-Patent Document 1). Previous studies have suggested that the diversity of SLAM proteins may significantly contribute to determining the host range of morbilliviruses.

[0006] Cross-species transmission of certain morbilliviruses has been confirmed, including fatal CDV outbreaks in primates. Despite the importance of cross-species transmission, the extent to which species differences in SLAM proteins act as a barrier to morbillivirus host range remains unclear.

[0007] Takeda M. et al., 2020, Curr Opin Virol 41: 38-45.

[0008] The objective of the present invention is to provide a new host cell that can be infected with a wide variety of morbilliviruses.

[0009] To clarify how species differences in SLAM proteins function as a barrier to morbillivirus host range, we infected Vero cells expressing SLAM proteins from various host animals with various morbilliviruses, and found that morbillivirus infectivity is highly dependent on species differences in SLAM proteins.

[0010] Based on these results, the present inventors conceived the possibility of creating new host cells that can be infected by a wide variety of morbilliviruses by modifying the amino acid sequence of the SLAM protein. Such host cells would enable the production of diverse morbilliviruses for research analysis and vaccine production by infecting them with various morbilliviruses, including morbilliviruses newly isolated from nature, and would also enable the easy evaluation of morbillivirus titers.

[0011] To solve the above-mentioned problems, the present inventors performed sequence analysis of SLAM proteins derived from various mammals, including humans, cattle, sheep, dolphins, seals, and dogs, and deduced the amino acid sequence of the common ancestor of these mammals. Vero cells expressing a new SLAM protein variant (hereinafter sometimes referred to as the "ancestral SLAM protein") with this amino acid sequence were generated and subjected to DSP assays, cytopathic effect (CPE) analysis, and plaque assays. As a result, they found that this ancestral SLAM protein functions as a receptor for all morbilliviruses, including measles virus (MV), peste des petits ruminants virus (PPRV), cetacean morbillivirus (CeMV), bat morbillivirus (MBaMV), canine distemper virus (CDV), and phocine distemper virus (PDV). This finding led to the completion of the present invention.

[0012] The present invention is based on the above findings and provides the following. [1] A mutant SLAM protein or an active fragment thereof, comprising a mutant V domain derived from the V domain of a human SLAM protein and having an ancestral amino acid residue and / or an ancestral amino acid deletion, wherein the ancestral amino acid residue and / or the ancestral amino acid deletion is: (a) a Leu residue at a position corresponding to position 21 of SEQ ID NO: 1; (b) a Gly residue at a position corresponding to position 28 of SEQ ID NO: 1; (c) a Leu residue at a position corresponding to position 29 of SEQ ID NO: 1; (d) an Arg residue at a position corresponding to position 38 of SEQ ID NO: 1; (e) a Ser residue at a position corresponding to position 42 of SEQ ID NO: 1; (f) a Ser residue at a position corresponding to position 49 of SEQ ID NO: 1; (g) a Leu residue at a position corresponding to position 63 of SEQ ID NO: 1; (h) a Pro residue at a position corresponding to position 70 of SEQ ID NO: 1; (i) a Gly residue at a position corresponding to position 71 of SEQ ID NO: 1; (j) a Lys residue at a position corresponding to position 75 of SEQ ID NO: 1; (k) a Lys residue at a position corresponding to position 76 of SEQ ID NO: 1. The mutant SLAM protein or an active fragment thereof, comprising one or more selected from the group consisting of: (l) a Leu residue at a position corresponding to position 83 of SEQ ID NO: 1; (m) a Gly residue at a position corresponding to position 86 of SEQ ID NO: 1; (n) a Ser residue at a position corresponding to position 88 of SEQ ID NO: 1; (o) an amino acid deletion at a position corresponding to position 91 of SEQ ID NO: 1; (p) a Glu residue at a position corresponding to position 93 of SEQ ID NO: 1; (q) a Gly residue at a position corresponding to position 95 of SEQ ID NO: 1; (r) a His residue at a position corresponding to position 99 of SEQ ID NO: 1; (s) a Ser residue at a position corresponding to position 104 of SEQ ID NO: 1; (t) an Arg residue at a position corresponding to position 106 of SEQ ID NO: 1; (u) a Leu residue at a position corresponding to position 108 of SEQ ID NO: 1; (v) a Glu residue at a position corresponding to position 124 of SEQ ID NO: 1; (w) a His residue at a position corresponding to position 130 of SEQ ID NO: 1; and (x) a Lys residue at a position corresponding to position 136 of SEQ ID NO: 1.[2] The mutant SLAM protein or active fragment thereof according to [1], wherein the ancestral amino acid residues and / or ancestral amino acid deletions include 21 or more selected from the group consisting of (a) to (x). [3] The mutant SLAM protein or active fragment thereof according to [1], wherein the mutant V domain includes all of (a) to (x). [4] The mutant SLAM protein or an active fragment thereof according to [1], wherein the mutant V domain comprises: (i) an amino acid sequence consisting of positions 21 to 152 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38; (ii) an amino acid sequence in which 1 to 13 amino acid residues have been deleted, substituted, or added in any amino acid sequence consisting of positions 21 to 152 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38; or (iii) an amino acid sequence that is 90% or more identical to the amino acid sequence consisting of positions 21 to 152 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38. [5] (I) an amino acid sequence consisting of positions 21 to 333 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39; (II) an amino acid sequence in which 1 to 13 amino acid residues are deleted, substituted, or added in any amino acid sequence consisting of positions 21 to 333 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39; (III) an amino acid sequence having 90% or more identity to the amino acid sequence consisting of positions 21 to 333 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39; or (IV) an amino acid sequence in which an arbitrary amino acid sequence is added to the N-terminus and / or C-terminus of any of the amino acid sequences (I) to (III). [6] A mutant SLAM protein or an active fragment thereof according to [1], comprising:(1) A variant V domain derived from the V domain of a bovine, dolphin, or seal SLAM protein, comprising an ancestral amino acid residue and / or an ancestral amino acid deletion, wherein the ancestral amino acid residue and / or the ancestral amino acid deletion is: (a) a Leu residue at a position corresponding to position 21 of SEQ ID NO:1; (b) a Gly residue at a position corresponding to position 28 of SEQ ID NO:1; (c) a Leu residue at a position corresponding to position 29 of SEQ ID NO:1; (d) an Arg residue at a position corresponding to position 38 of SEQ ID NO:1; (e) a Ser residue at a position corresponding to position 42 of SEQ ID NO:1; (f) a Ser residue at a position corresponding to position 49 of SEQ ID NO:1; (g) a Leu residue at a position corresponding to position 63 of SEQ ID NO:1; (h) a Pro residue at a position corresponding to position 70 of SEQ ID NO:1; (i) a Gly residue at a position corresponding to position 71 of SEQ ID NO:1; (j) a Lys residue at a position corresponding to position 75 of SEQ ID NO:1; (k) a Lys residue at a position corresponding to position 76 of SEQ ID NO:1. (l) a Leu residue at a position corresponding to position 83 of SEQ ID NO: 1, (m) a Gly residue at a position corresponding to position 86 of SEQ ID NO: 1, (n) a Ser residue at a position corresponding to position 88 of SEQ ID NO: 1, (o) an amino acid deletion at a position corresponding to position 91 of SEQ ID NO: 1, (p) a Glu residue at a position corresponding to position 93 of SEQ ID NO: 1, (q) a Gly residue at a position corresponding to position 95 of SEQ ID NO: 1, (r) a His residue at a position corresponding to position 99 of SEQ ID NO: 1, (s) a Ser residue at a position corresponding to position 104 of SEQ ID NO: 1, (t) an Arg residue at a position corresponding to position 106 of SEQ ID NO: 1, (u) a Leu residue at a position corresponding to position 108 of SEQ ID NO: 1, (v) a Glu residue at a position corresponding to position 124 of SEQ ID NO: 1, (w) a His residue at a position corresponding to position 130 of SEQ ID NO: 1, and (x) a Lys residue at a position corresponding to position 136 of SEQ ID NO: 1, or (2) A mutant V domain derived from the V domain of an ovine SLAM protein and having 23 or more ancestral amino acid residues and / or ancestral amino acid deletions selected from the group consisting of (a) to (x) above; or(3) A mutant SLAM protein or an active fragment thereof, comprising a mutant V domain derived from the V domain of a canine SLAM protein and having 21 or more ancestral amino acid residues and / or ancestral amino acid deletions selected from the group consisting of (a) to (x), or (4) a mutant V domain derived from the V domain of a mouse SLAM protein and having 15 or more ancestral amino acid residues and / or ancestral amino acid deletions selected from the group consisting of (a) to (x). [7] A nucleic acid encoding the mutant SLAM protein or an active fragment thereof according to any of [1] to [6]. [8] A host cell comprising the nucleic acid according to [7] in an expressible state. [9] The host cell according to [8], selected from the group consisting of Vero cells, MDCK cells, CHO cells, HEK293 cells, and BHK-21 cells.

[10] The host cell according to [9], which is infected with a morbillivirus.

[11] The host cell according to

[10] , wherein the morbillivirus is selected from the group consisting of measles virus (MV), canine distemper virus (CDV), seal distemper virus (PDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), cetacean morbillivirus (CeMV), feline morbillivirus (FeMV), bat morbillivirus (MBaMV), and porcine morbillivirus (PoMV).

[12] The host cell according to

[10] , which is a live cell or a fixed cell.

[13] A method for producing a morbillivirus, comprising: an infection step of infecting the host cell according to [8] with a morbillivirus; a growth step of culturing the host cell infected with the morbillivirus to grow the morbillivirus; and a recovery step of recovering the morbillivirus from the culture medium after the growth step.

[14] A method for producing a vaccine against morbillivirus infection, comprising: an infection step of infecting the host cell according to [8] with a morbillivirus, a growth step of culturing the host cell infected with the morbillivirus to grow the morbillivirus, a recovery step of recovering the morbillivirus from the culture medium after the growth step, and a formulation step of formulating the recovered morbillivirus as a vaccine.

[15] A method for measuring the titer of a morbillivirus, comprising:The method includes: an infection step of infecting the host cell according to [8] with a morbillivirus; a culture step of culturing the host cell after the infection step; and a titer determination step of evaluating the host cell after the culture step and determining the titer of the morbillivirus based on the results of the evaluation.

[16] A method for generating an amino acid sequence of a universal viral receptor capable of binding to a viral receptor-binding protein derived from two or more viruses and mediating infection by the two or more viruses, or a candidate sequence thereof, comprising: a deducing step of deducing the amino acid sequence of an ancestral viral receptor common to a first and second viral receptor based on the amino acid sequence of a first viral receptor that binds to a first viral receptor-binding protein derived from a first virus and mediates infection by the first virus, and the amino acid sequence of a second viral receptor that is an orthologous protein of the first viral receptor and binds to a second viral receptor-binding protein derived from a second virus and mediates infection by the second virus; an identifying step of aligning the amino acid sequence of the ancestral viral receptor with the amino acid sequence of the first or second viral receptor to identify ancestral amino acid residues and / or ancestral amino acid deletions contained only in the amino acid sequence of the ancestral viral receptor;

[17] The method according to

[16] , wherein the viral receptor is selected from the group consisting of SLAM protein, nectin 4, ACE2 protein, DPP4 protein, ephrin B2 protein, and ephrin B3 protein.

[18] A method for producing a universal cell or a candidate cell thereof that can be infected with two or more viruses, the method comprising: an introduction step of introducing into a host cell a nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of the universal viral receptor or the candidate sequence thereof, generated using the method according to

[16] , in an expressible state.This specification includes the disclosure of Japanese Patent Application No. 2024-097856, from which the present application claims priority.

[0013] According to the present invention, new host cells are provided that can be infected with a wide variety of morbilliviruses.

[0014] Figure 1 shows the results of observing the formation of multinucleated giant cells after infection of Vero cells expressing SLAM proteins from various animal species with various morbilliviruses. The animal species shown on the left (human, dolphin, bat, dog, and seal) indicate the origin of the SLAM protein expressed in the SLAM-expressing Vero cells, while (-) indicates Vero cells not transfected with the SLAM protein-encoding gene. The morbilliviruses shown at the top of the figure (MV, CeMV, MBaMV, CDV, and PDV) indicate the viruses used to infect the SLAM-expressing Vero cells, and Mock indicates a control uninfected with a morbillivirus. Figure 2 shows the results of neural red staining of plaques formed after infection of Vero cells expressing SLAM proteins from various animal species with various morbilliviruses. The animal species shown on the left and the morbilliviruses shown at the top of the figure are the same as in Figure 1 (and the following figures). Figure 3 shows the quantification of plaque number and plaque size. Figure 3A shows the results of measuring the number of plaques shown in Figure 2. The relative levels of plaque counts (based on the value of 1 in cells expressing SLAM from the original host animal of each virus) are shown based on the gradient shown on the right side of the figure. Figure 3B shows the results of measuring the plaque size of the plaques shown in Figure 2 (actual measurements are shown in mm). The plaque size (diameter) values ​​(mm) are shown based on the gradient shown on the right side of the figure. Figure 4 shows the results of a DSP assay. 293CD4D / DSP1-7 cells expressing SLAM proteins from various animal species were mixed 1:1 with 293FT / DSP8-11 cells expressing H and F proteins from various morbilliviruses, and luciferase activity was quantified two days later. Figure 5 shows the relative levels of luciferase activity quantified in Figure 4. The relative levels of luciferase activity are shown based on the gradient shown on the right side of the figure. Figure 6 shows the results of the predicted amino acid sequence of the ancestral SLAM protein. Figure 6A shows a phylogenetic tree constructed based on the amino acid sequences of SLAM proteins from various mammals.Figure 6B shows the amino acid sequence alignment from the signal peptide to the V domain between the human SLAM proteins, ancSLAM8, and ancSLAM9. Figure 7 shows the amino acid sequence alignment from the signal peptide to the V domain between the human SLAM proteins, ancSLAM8, and ancSLAM9, as well as SLAM proteins derived from cows, sheep, dolphins, seals, dogs, and mice. Figure 8 shows the results of a DSP assay evaluating whether the SLAM proteins derived from humans, dolphins, and bats, as well as ancSLAM8 and ancSLAM9, function as receptors for various morbilliviruses (MV, PPRV, CeMV, MBaMV, CDV, and PDV). Figure 9 shows the results of observing the formation of multinucleated giant cells in ancSLAM8-expressing Vero cells infected with morbilliviruses (MV, CDV, CeMV, MBaMV, and PDV). Figure 10 shows the results of plaque assays using ancSLAM8-expressing Vero cells and Vero cells expressing SLAM proteins derived from each morbillivirus host animal species. Figure 11 shows the results of plaque assays using ancSLAM8-expressing Vero cells, human SLAM8-expressing Vero cells, dolphin SLAM8-expressing Vero cells, and bat SLAM8-expressing Vero cells infected with MV, CeMV, or MBaMV. Figure 12 shows the alignment of the amino acid sequences from the signal peptide to the V domain between various mutant ancSLAM8, ancSLAM8, and human SLAM proteins. Figure 13 shows the results of DSP assays evaluating whether various mutant ancSLAM8 and human SLAM proteins function as receptors for various morbilliviruses (MV, CeMV, and CDV). Figure 14 shows the results of DSP assays evaluating whether various mutant ancSLAM8 and human SLAM proteins function as receptors for various morbilliviruses (MV, CeMV, CDV, PDV, MBaMV, and PPRV).

[0015] 1. Mutant SLAM Protein or Active Fragment Thereof 1-1. Overview A first aspect of the present invention is a mutant SLAM protein or an active fragment thereof.

[0016] 1-2. Definitions of Terms Terms frequently used in this specification are defined below.

[0017] As used herein, "morbillivirus" refers to viruses of the genus Morbillivirus in the family Paramyxoviridae. Specific examples of morbilliviruses include measles virus (MV), canine distemper virus (CDV), seal distemper virus (PDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), cetacean morbillivirus (CeMV), feline morbillivirus (FeMV), bat morbillivirus (MBaMV), and porcine morbillivirus (PoMV). Morbillivirus infection generally begins when a viral receptor-binding protein (RBP) known as hemagglutinin (H) binds to a viral receptor on target cells. Morbilliviruses are known to utilize SLAM proteins expressed on immune cells and nectin-4 expressed on epithelial cells as viral receptors.

[0018] As used herein, "SLAM (signaling lymphocyte-activating molecule)" or "SLAM protein" refers to a viral receptor expressed on target cells, such as immune cells, to which morbilliviruses bind during infection. Because the amino acid sequence of SLAM proteins is diverse, it has been suggested that they may significantly contribute to determining the host range of morbilliviruses. SLAM proteins are transmembrane proteins that contain an IgV domain (often abbreviated as "V domain") and an IgC2 domain (sometimes abbreviated as "C2 domain" herein) in the extracellular region, as well as a motif called an ITSM (immunoreceptor tyrosine-based switch motif) in the intracellular domain. The V domain in the extracellular region is involved in binding to viral receptor-bound proteins (RBPs) on the viral surface. Unless otherwise specified, the N-terminal signal peptide is not included in the SLAM protein or V domain. The SLAM protein and its V domains herein may be derived from any species, including any mammal, such as humans, cows, dolphins, seals, sheep, dogs, or mice. The SLAM protein herein is also known as "SLAMF1" in humans.The amino acid sequence containing the human wild-type SLAM protein and the signal peptide of its V domain is shown in SEQ ID NOs: 10 and 1, the amino acid sequence containing the dolphin wild-type SLAM protein and the signal peptide of its V domain is shown in SEQ ID NOs: 11 and 6, the amino acid sequence containing the bat wild-type SLAM protein and the signal peptide of its V domain is shown in SEQ ID NOs: 12, the amino acid sequence containing the dog wild-type SLAM protein and the signal peptide of its V domain is shown in SEQ ID NOs: 13 and 8, the amino acid sequence containing the seal wild-type SLAM protein and the signal peptide of its V domain is shown in SEQ ID NOs: 14 and 7, the amino acid sequence containing the bovine wild-type SLAM protein and the signal peptide of its V domain is shown in SEQ ID NOs: 15 and 4, the amino acid sequence containing the sheep wild-type SLAM protein and the signal peptide of its V domain is shown in SEQ ID NOs: 16 and 5, and the amino acid sequence containing the mouse wild-type SLAM protein and the signal peptide of its V domain is shown in SEQ ID NOs: 17 and 9. In addition, in the human wild-type SLAM protein shown in SEQ ID NO: 10, the sequence from positions 1 to 20 is the signal peptide, the sequence from positions 21 to 153 is the V domain, the sequence from positions 154 to 236 is the C2 domain, the sequence from positions 237 to 263 is the transmembrane domain, and the sequence from positions 264 to 335 is the intracellular domain.

[0019] As used herein, "mutant SLAM protein" refers to a SLAM protein consisting of a mutant amino acid sequence derived from the amino acid sequence of a wild-type SLAM protein. Also, as used herein, "mutant V domain" refers to a V domain consisting of a mutant amino acid sequence derived from the amino acid sequence of the wild-type V domain constituting a wild-type SLAM protein.

[0020] As used herein, the term "ancestral SLAM protein" refers to a SLAM protein encoded by a putative ancestral gene of a gene encoding a SLAM protein in an extant biological species. An ancestral SLAM protein is preferably one that is possessed or presumed to have been possessed by an ancestral species shared by two or more (e.g., three or more, four or more, five or more, or six or more) species, genera, and / or orders. For example, a SLAM protein may be possessed or presumed to have been possessed by an ancestral species shared by primates, Carnivora, and Cetacea.

[0021] As used herein, the term "ancestral amino acid residue" refers to an amino acid residue of a protein encoded by a putative ancestral gene. Ancestral amino acid residues are preferably those that were or are presumed to have been present in an ancestral species shared by two or more (e.g., three or more, four or more, five or more, or six or more) species, genera, and / or orders. Examples of such residues include those that were or are presumed to have been present in an ancestral species shared by Primates and the Order Carnivora and Order Cetacea.

[0022] As used herein, the term "ancestral amino acid deletion" refers to an amino acid deletion contained in a protein encoded by a putative ancestral gene. The ancestral amino acid deletion is preferably one that was or is presumed to have been present in an ancestral species shared by two or more (e.g., three or more, four or more, five or more, or six or more) species, genera, and / or orders. For example, an ancestral amino acid deletion may be one that was or is presumed to have been present in an ancestral species shared by Primates and the Order Carnivora and Order Cetacea.

[0023] As used herein, the term "active fragment" of a mutant SLAM protein refers to a fragment of the mutant SLAM protein that has the activity of binding to morbillivirus and mediating its infection, e.g., a fragment having at least 50%, 60%, 70%, 80%, or 90% of the activity of the mutant SLAM protein, or an activity equivalent to or greater than that. An example of an active fragment is a fragment containing the extracellular region of the mutant SLAM protein, including the V domain and C2 domain, the transmembrane domain, and the intracellular region containing the ITSM. The active fragment may also be an N-terminal fragment, a C-terminal fragment, or a fragment lacking amino acid residues at the N and C termini of the mutant SLAM protein. The amino acid length of the polypeptide constituting the fragment is not particularly limited, but may be, for example, a region of at least 230, 280, 290, 300, 310, 320, or 330 consecutive amino acids in the mutant SLAM protein.

[0024] As used herein, "plurality" refers to, for example, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, 2 to 3, or 2.

[0025] As used herein, "amino acid identity" refers to the percentage (%) of matching amino acid residues in the total number of amino acid residues when the amino acid sequences of two polypeptides being compared are aligned by inserting appropriate gaps into one or both sequences as needed to maximize the number of identical amino acid residues. Alignment of two amino acid sequences to calculate amino acid identity can be performed using known programs such as Blast, FASTA, and ClustalW. "Nucleotide identity" is calculated in a similar manner.

[0026] As used herein, "(amino acid) substitution" refers to substitution within a conservative amino acid group that has similar properties, such as charge, side chain, polarity, and aromaticity, among the 20 amino acids that constitute natural proteins. Examples include substitutions within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because they are known to be less likely to cause changes in the properties of polypeptides.

[0027] 1-3. Configuration The mutant SLAM protein or active fragment thereof of the present invention comprises a mutant V domain. In the mutant SLAM protein or active fragment thereof of the present invention, the mutant V domain comprises one or more ancestral amino acid residues and / or ancestral amino acid deletions.

[0028] In the mutant SLAM protein or active fragment thereof of the present invention, when the mutant V domain contains an ancestral amino acid residue, the ancestral amino acid residue is selected from the following (a) to (n) and (p) to (x): (a) a Leu residue at a position corresponding to position 21 of SEQ ID NO: 1, (b) a Gly residue at a position corresponding to position 28 of SEQ ID NO: 1, (c) a Leu residue at a position corresponding to position 29 of SEQ ID NO: 1, (d) an Arg residue at a position corresponding to position 38 of SEQ ID NO: 1, (e) a Ser residue at a position corresponding to position 42 of SEQ ID NO: 1, (f) a Ser residue at a position corresponding to position 49 of SEQ ID NO: 1, (g) a Leu residue at a position corresponding to position 63 of SEQ ID NO: 1, (h) a Pro residue at a position corresponding to position 70 of SEQ ID NO: 1, (i) a Gly residue at a position corresponding to position 71 of SEQ ID NO: 1, (j) a Lys residue at a position corresponding to position 75 of SEQ ID NO: 1, (k) a Lys residue at a position corresponding to position 76 of SEQ ID NO: 1, (l) a Leu residue at a position corresponding to position 83 of SEQ ID NO: 1. (m) a Gly residue at a position corresponding to position 86 of SEQ ID NO:1, (n) a Ser residue at a position corresponding to position 88 of SEQ ID NO:1, (p) a Glu residue at a position corresponding to position 93 of SEQ ID NO:1, (q) a Gly residue at a position corresponding to position 95 of SEQ ID NO:1, (r) a His residue at a position corresponding to position 99 of SEQ ID NO:1, (s) a Ser residue at a position corresponding to position 104 of SEQ ID NO:1, (t) an Arg residue at a position corresponding to position 106 of SEQ ID NO:1, (u) a Leu residue at a position corresponding to position 108 of SEQ ID NO:1, (v) a Glu residue at a position corresponding to position 124 of SEQ ID NO:1, (w) a His residue at a position corresponding to position 130 of SEQ ID NO:1, and (x) a Lys residue at a position corresponding to position 136 of SEQ ID NO:1.

[0029] As used herein, "a position corresponding to position X in SEQ ID NO: 1" refers to a position in the mutant V domain that corresponds to position X in the amino acid sequence shown in SEQ ID NO: 1 when the amino acid sequence of the mutant V domain is compared with the amino acid sequence shown in SEQ ID NO: 1 (the amino acid sequence from the signal peptide to the V domain in human wild-type SLAM protein). For example, when alignment is performed by inserting appropriate gaps into one or both amino acid residues as necessary to maximize the number of identical amino acid residues, the position corresponds to position X in the amino acid sequence shown in SEQ ID NO: 1. Furthermore, as used herein, "a mutant V domain contains a Y residue at a position corresponding to position X in SEQ ID NO: 1" refers to a position in the mutant V domain that corresponds to position X in the amino acid sequence shown in SEQ ID NO: 1 when the amino acid sequence of the mutant V domain is compared with the amino acid sequence shown in SEQ ID NO: 1 (the amino acid sequence from the signal peptide to the V domain in human wild-type SLAM protein).

[0030] Furthermore, in the mutant SLAM protein or active fragment thereof of the present invention, when the mutant V domain contains an ancestral amino acid deletion, the ancestral amino acid deletion is the following (o): (o) an amino acid deletion at a position corresponding to position 91 of SEQ ID NO: 1. As used herein, the phrase "the mutant V domain contains an amino acid deletion at a position corresponding to position X of SEQ ID NO: 1" means that, when the amino acid sequence of the mutant V domain is compared with the amino acid sequence shown in SEQ ID NO: 1 (the amino acid sequence from the signal peptide to the V domain in human wild-type SLAM protein), the mutant V domain lacks an amino acid residue at a position corresponding to position X of the amino acid sequence shown in SEQ ID NO: 1.

[0031] In the mutant SLAM protein or active fragment thereof of the present invention, the mutant V domain contains one or more ancestral amino acid residues and / or ancestral amino acid deletions. For example, the mutant V domain contains one or more ancestral amino acid residues or at least one ancestral amino acid deletion. The number of ancestral amino acid residues and / or ancestral amino acid deletions contained in the mutant V domain is not particularly limited as long as it is one or more, and may be, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more, preferably 21 or more, 22 or more, or 23 or more. More preferably, the mutant V domain contains all of the above (a) to (x).

[0032] In one embodiment, the variant V domain comprises 21 or more ancestral amino acid residues and / or ancestral amino acid deletions, e.g., the variant V domain comprises all of (a) to (x) above except for (1), (m), and (n).

[0033] In one embodiment, the variant V domain comprises 22 or more ancestral amino acid residues and / or ancestral amino acid deletions, for example, the variant V domain comprises all of (a) to (x) above except for (b) and (c), all of (a) to (x) above except for (j) and (k), all of (a) to (x) above except for (h) and (i), or all of (a) to (x) above except for (p) and (q).

[0034] In one embodiment, the variant V domain comprises 23 or more ancestral amino acid residues and / or ancestral amino acid deletions. For example, the variant V domain comprises all of (a) to (x) above except (b), all of (a) to (x) above except (c), all of (a) to (x) above except (g), all of (a) to (x) above except (w), or all of (a) to (x) above except (x).

[0035] In the mutant SLAM protein or active fragment thereof of the present invention, the mutant V domain can be derived from any mammalian species, including, but not limited to, humans, cows, dolphins, seals, sheep, dogs, and mice.

[0036] In one embodiment, the variant V domain is derived from the V domain of a human SLAM protein, and in this embodiment, the variant V domain comprises one or more selected from the group consisting of (a) to (x) above, and preferably 21 or more, 22 or more, 23 or more, or all of them.

[0037] In a further embodiment, the variant V domain is: (i) an amino acid sequence consisting of positions 21 to 152 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38; (ii) an amino acid sequence in which a plurality of amino acid residues (for example, 1 to 30, 1 to 20, or 1 to 15, preferably 1 to 13, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) have been deleted, substituted, or added in any amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38; or (iii) An amino acid sequence having 60% or more, 70% or more, 80% or more, or 85% or more, preferably 90% or more, 91% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence consisting of positions 21 to 152 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38. Regarding (iii) above, in the Examples described below, there is a difference of 13 amino acids between the V domain of ancSLAM8 (L83P / G86A / S88P) and the V domain of ancSLAM9, and based on the fact that 119 of the 132 amino acids constituting the V domain are identical, it is preferable that the identity is 90% or more.

[0038] In one embodiment, the variant V domain is derived from the V domain of a bovine, dolphin, or seal SLAM protein. Because the V domains of wild-type bovine, dolphin, and seal SLAM proteins contain 21 ancestral amino acid residues and / or ancestral amino acid deletions selected from the group consisting of (a) to (x) above, in this embodiment, the variant V domain preferably contains 22 or more, 23 or more, or all of the above selected from the group consisting of (a) to (x).

[0039] In one embodiment, the variant V domain is derived from the V domain of an ovine SLAM protein. Since the V domain of a wild-type ovine SLAM protein contains 22 ancestral amino acid residues and / or ancestral amino acid deletions selected from the group consisting of (a) to (x) above, in this embodiment, the variant V domain preferably contains 23 or more, or all, of the group consisting of (a) to (x) above.

[0040] In one embodiment, the variant V domain is derived from the V domain of a canine SLAM protein. Because the V domain of a wild-type canine SLAM protein contains 20 ancestral amino acid residues and / or ancestral amino acid deletions selected from (a) to (x) above, in this embodiment, the variant V domain preferably contains 21 or more, 22 or more, 23 or more, or all of the above selected from the group consisting of (a) to (x).

[0041] In one embodiment, the mutant V domain is derived from the V domain of a mouse SLAM protein. Because the V domain of a wild-type mouse SLAM protein contains 14 ancestral amino acid residues and / or ancestral amino acid deletions selected from the group consisting of (a) to (x) above, in this embodiment, the mutant V domain preferably contains 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, or all of the above selected from the group consisting of (a) to (x).

[0042] In the mutant SLAM protein or active fragment thereof of the present invention, the amino acid sequence other than the mutant V domain is not particularly limited and may be derived from any mammalian species, for example, the amino acid sequence other than the mutant V domain may be derived from the same mammalian species as the V domain is derived from, such as a human, cow, dolphin, seal, sheep, dog, or mouse.

[0043] For example, the mutant SLAM protein or active fragment thereof of the present invention is derived from a human, dolphin, bat, dog, seal, cow, sheep, or mouse, and consists of or contains an amino acid sequence that has 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence from position 21 onwards in any amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 17 (the full-length amino acid sequence excluding the signal peptide sequence of the wild-type SLAM protein derived from each animal species).

[0044] In one embodiment, the mutant SLAM protein or active fragment thereof of the present invention is: (I) an amino acid sequence consisting of positions 21 to 333 of any amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39 (the full-length amino acid sequence excluding the signal peptide sequence of the mutant SLAM protein); (II) an amino acid sequence in which a plurality of amino acid residues (e.g., 1 to 30, 1 to 20, or 1 to 15, preferably 1 to 13, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) have been deleted, substituted, or added in any amino acid sequence consisting of positions 21 to 333 of any amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39; (III) an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence consisting of positions 21 to 333 of any amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39; or (IV) an amino acid sequence in which an arbitrary amino acid sequence is added to the N-terminus and / or C-terminus of any of the amino acid sequences (I) to (III). The arbitrary amino acid sequence in (IV) above is not particularly limited. Examples include tag sequences, fluorescent proteins, and ubiquitination sequences. The number of amino acids to be added is not limited, and may be 1 to 500, 10 to 400, 50 to 300, or 100 to 200.

[0045] As used herein, a "tag sequence" refers to a short peptide consisting of several to approximately 100 amino acid residues that can be used to label proteins and is used for protein detection and purification. Various types of tag peptides have been developed in the field, and any tag peptide may be used. Specific examples of tag peptides include FLAG, HA, His, and Myc. The number of amino acids in a tag sequence may be, for example, 50, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0046] 1-4. Effects The mutant SLAM proteins or active fragments thereof of the present invention can bind to viral receptor-binding proteins on the viral surface of a wider range of morbilliviruses and mediate their infection, compared to the human wild-type SLAM protein. Furthermore, they can bind to viral receptor-binding proteins on the viral surface of a wider range of morbilliviruses and mediate their infection, compared to the wild-type SLAM proteins derived from cattle, dolphins, seals, sheep, dogs, and mice.

[0047] The mutant SLAM proteins or active fragments thereof of the present invention can mediate infection by multiple morbilliviruses in addition to measles virus (MV), such as canine distemper virus (CDV), phocine distemper virus (PDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), cetacean morbillivirus (CeMV), feline morbillivirus (FeMV), bat morbillivirus (MBaMV), and / or porcine morbillivirus (PoMV), or all morbilliviruses.

[0048] 2. Nucleic Acids Encoding Mutant SLAM Proteins or Active Fragments Thereof The second aspect of the present invention is nucleic acids encoding mutant SLAM proteins or active fragments thereof.

[0049] The "nucleic acid encoding a mutant SLAM protein or an active fragment thereof" of the present invention may be any nucleic acid encoding any of the mutant SLAM proteins or active fragments thereof described in the first aspect. The base sequence of such a nucleic acid is not limited. Examples include a codon-optimized base sequence and a base sequence with an initiation codon (ATG) added to the 5' end.

[0050] An example of the nucleic acid of the present invention is a gene expression vector containing a nucleic acid encoding a mutant SLAM protein or an active fragment thereof in an expressible state. The gene expression vector contains a nucleic acid encoding the mutant SLAM protein or an active fragment thereof and a promoter, and is capable of expressing the mutant SLAM protein or an active fragment thereof in cells. In addition to the nucleic acid and promoter components, the gene expression vector may also contain components such as a drug resistance gene, an intron, an enhancer, a terminator, a replication origin, and / or a polyA signal, as necessary.

[0051] As used herein, the term "gene expression vector" refers to a vector that contains a gene or gene fragment (hereinafter referred to as "gene, etc.") in an expressible state and includes an expression unit that can control the expression of the gene, etc. The gene expression vector may be a plasmid vector or a viral vector. The plasmid vector may be a commercially available expression vector for mammalian cells, such as Promega's pSI vector, or a shuttle vector that can replicate between mammalian cells and bacteria such as E. coli.

[0052] As used herein, "in an expressible state" refers to the placement of a gene to be expressed downstream of a promoter under the control of the promoter. Known vectors include plasmid vectors and viral vectors, and any of these vectors can be used. Generally, a plasmid vector that is easy to manipulate for genetic recombination is sufficient.

[0053] As used herein, a "promoter" refers to a gene expression regulatory region that can control the expression of a gene or the like located downstream (on the 3' end) in a cell into which a gene expression vector has been introduced. The promoter may be either a constitutively active promoter or an inducible promoter.

[0054] 3. Host Cells A third aspect of the present invention is a host cell comprising, in an expressible state, a nucleic acid encoding a mutant SLAM protein or an active fragment thereof.

[0055] Herein, the type of host cell is not limited. The host cell may be any cell in which a mutant SLAM protein or an active fragment thereof is expressed on the cell membrane, such as a eukaryotic cell. Preferred host cells are cells that can be infected by morbilliviruses, such as mammalian cells, in which a viral receptor-binding protein on the viral surface of the morbillivirus binds to a mutant SLAM protein or an active fragment thereof on the host cell. The mammalian cell is not limited and may be, for example, a human, bovine, dolphin, seal, sheep, dog, or mouse cell. Specific examples of mammalian cells include Vero cells, MDCK cells, CHO cells, HEK293 cells, and BHK-21 cells.

[0056] In one embodiment, the host cell of this aspect is infected with a morbillivirus. The morbillivirus infecting the host cell of this aspect is, for example, measles virus (MV), canine distemper virus (CDV), seal distemper virus (PDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), cetacean morbillivirus (CeMV), feline morbillivirus (FeMV), bat morbillivirus (MBaMV), or porcine morbillivirus (PoMV). The host cell infected with a morbillivirus may be either a live cell or a fixed cell.

[0057] In one embodiment, the host cell of this aspect is a live cell. Live cells infected with a morbillivirus can be used as cells for vaccine production, as well as for potency testing of live vaccines (e.g., process control testing, quality control testing), and production of viral antigens. Live cells not infected with a morbillivirus can also be used for diagnosis of morbillivirus infections and pathogen surveillance based on virus detection.

[0058] In one embodiment, the host cells of this aspect are fixed cells. For example, by placing the fixed cells on a substrate such as a slide, plates for enzyme-linked immunosorbent assay (ELISA) or slides for antibody tests can be prepared. For safety reasons, it is preferable that infectious viruses in the fixed cells are inactivated.

[0059] 4. Method for Producing Morbillivirus A fourth aspect of the present invention is a method for producing morbillivirus. The method for producing morbillivirus of this aspect includes, as essential steps, an infection step, a propagation step, and a recovery step. Each step will be described below.

[0060] (Infection step) In this embodiment, the "infection step" refers to a step of infecting the host cells of the third embodiment with a morbillivirus. The infection method in this step is not particularly limited as long as it allows the morbillivirus to come into contact with the host cells. For example, infection can be achieved by adding a morbillivirus suspension to a culture solution of the host cells and incubating for a certain period of time or longer.

[0061] (Growth Step) In this embodiment, the "growth step" refers to a step of culturing host cells infected with a morbillivirus to grow the morbillivirus.

[0062] Culture conditions for this step may be those known in the art that are appropriate for the type and origin of the host cells. Typically, culture is carried out in 5% CO2 at 30 to 40°C (e.g., 37°C). The culture method is not limited, and may be, for example, adherent culture, suspension culture, or floating culture.

[0063] The type of medium used in this step is not limited as long as it is capable of maintaining the host cells as viable cells. For example, the medium may be any medium commonly used in cell culture and known in the art. The medium may be any of basal medium, serum-free medium, low-serum medium, and serum-supplemented medium, but typically may be a basal medium, such as a standard cell culture medium. Specific examples include Eagle Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F10 (Ham's Nutrient Mixture F10), Ham's Nutrient Mixture F12 (Ham's Nutrient Mixture F12), M199 medium, High Performance Medium 199 (High Performance Medium 199), Roswell Park Memorial Institute-1640 (RPMI-1640), and Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F12 (DMEM / F12).

[0064] The culture time in this step varies depending on the type of host cells and the morbillivirus that has infected the host cells, but is 1 hour to 1 week, 2 hours to 72 hours, 3 hours to 36 hours, or 4 hours to 24 hours to achieve sufficient proliferation of the morbillivirus.

[0065] (Recovery Step) In this embodiment, the "recovery step" is a step of recovering morbillivirus from the culture medium after the proliferation step.

[0066] The recovery method in this step is not limited as long as it can separate the culture supernatant containing the morbillivirus from the culture solution after the growth step. For example, the supernatant containing the morbillivirus can be obtained by centrifugation, filtration, sedimentation, decantation, or a combination thereof. All of these methods can basically be performed according to conventional methods in the field. When recovering by filtration, a filter with a pore size that allows the morbillivirus to pass through but does not allow the host cells cultured in the growth step to pass through can be used.

[0067] The morbillivirus produced by the method for producing a morbillivirus of this embodiment can be used, for example, to produce a viral antigen. The produced viral antigen can be used in various test reagents, etc.

[0068] 5. Method for Producing a Vaccine Against Morbillivirus Infections A fifth aspect of the present invention is a method for producing a vaccine against morbillivirus infections.

[0069] As used herein, the term "morbillivirus infection" refers to an infection caused by a morbillivirus, including, for example, measles, canine distemper virus infection, seal distemper virus infection, rinderpest, peste des petit ruminants, cetacean morbillivirus infection, feline morbillivirus infection, bat morbillivirus infection, and porcine morbillivirus infection.

[0070] Examples of "vaccine" as used herein include live vaccines and inactivated vaccines.

[0071] The vaccine production method of this embodiment includes, as essential steps, an infection step, a proliferation step, a recovery step, and a formulation step. The infection step, proliferation step, and recovery step of this embodiment are similar to the infection step, proliferation step, and recovery step of the fourth embodiment, and therefore will not be described here.

[0072] In one embodiment, the morbillivirus used in this aspect is an attenuated virus.

[0073] Furthermore, the vaccine production method of the present invention may include, in addition to the above-mentioned steps, a step of inactivating the morbillivirus recovered after the recovery step. Morbillivirus may be inactivated using, for example, formalin, ultraviolet light, or β-propiolactone.

[0074] (Formulation step) In this embodiment, the "formulation step" refers to a step of isolating and / or purifying the morbillivirus recovered in the recovery step, if necessary, and then formulating the morbillivirus as a vaccine. The specific method for formulation is not limited, and for example, the morbillivirus may be formulated by mixing it with a pharmaceutically acceptable carrier or adjuvant (e.g., aluminum hydroxide gel, aluminum phosphate gel, etc.).

[0075] 6. Method for measuring titer of morbillivirus The sixth aspect of the present invention is a method for measuring titer of morbillivirus. The titer measurement method of this aspect includes, as essential steps, an infection step, a culture step, and a titer determination step.

[0076] (Infection step) In this embodiment, the "infection step" refers to a step of infecting the host cells of the third embodiment with a morbillivirus. The infection method in this step is not particularly limited as long as it allows the morbillivirus to come into contact with the host cells. For example, infection can be achieved by adding a morbillivirus suspension to a culture medium of the host cells and incubating for a certain period of time or longer. In this step, using an appropriately diluted morbillivirus suspension for infection may sometimes improve the efficiency of evaluation in the titer determination step described below. For example, a suspension diluted 2-fold to 10,000-fold, 10-fold to 1,000-fold, or 100-fold to 500-fold can be used. Alternatively, a dilution series obtained by diluting the morbillivirus suspension can also be used in this step.

[0077] (Culturing step) In this embodiment, the "culturing step" refers to a step of culturing host cells after the infection step. The culturing in the culturing step can be performed in accordance with the configuration of the proliferation step described above, and therefore a detailed description thereof will be omitted here. The culturing time in this step can be, for example, 1 hour to 1 week, 2 hours to 72 hours, 3 hours to 36 hours, 4 hours to 24 hours, 5 hours to 12 hours, etc., depending on the specific evaluation method in the titer determination step described below.

[0078] (Titer Determination Step) In this embodiment, the "titer determination step" is a step of evaluating the host cells after the culture step and determining the titer of the morbillivirus based on the results of the evaluation.

[0079] The method for evaluating host cells in this step is not limited as long as it allows for measuring the number of host cells infected with a morbillivirus. For example, the formation of multinucleated giant cells can be observed as a cytopathic effect (CPE) in the host cells, and the virus titer can be determined by quantitatively evaluating the number and / or size of the multinucleated giant cells. Plaque formation can also be evaluated using adherent cells (e.g., Vero cells) cultured in an adherent culture. For example, the virus titer can be determined by staining the plaques after plaque formation with a dye such as neutral red and then measuring the number and / or size of the plaques.

[0080] 7. Method for generating an amino acid sequence of a universal virus receptor or a candidate sequence thereof The seventh aspect of the present invention is a method for generating an amino acid sequence of a universal virus receptor or a candidate sequence thereof that can bind to viral receptor-binding proteins derived from two or more viruses and mediate infection by the two or more viruses. The method of this aspect includes, as essential steps, a prediction step, an identification step, and a generation step.

[0081] As used herein, the term "viral receptor" refers to a receptor that is expressed on the cell membrane of a target cell infected by a virus and mediates viral infection by binding to a viral receptor-binding protein on the viral surface. Specific examples of viral receptors include SLAM protein and nectin 4, which mediate morbillivirus infection, ACE2 protein and DPP4 protein, which mediate coronavirus infection, and ephrin B2 protein and ephrin B3 protein, which mediate henipavirus infection.

[0082] As used herein, "viral surface receptor-binding protein (RBP)" refers to a molecule that exists on the surface of a virus and mediates viral infection by binding to a viral receptor on a target cell that the virus infects. Specific examples of viral receptor-binding proteins include hemagglutinin, which mediates morbillivirus infection by binding to SLAM protein, and S protein, which mediates coronavirus infection.

[0083] As used herein, the term "universal viral receptor" refers to a viral receptor that can bind to viral receptor-binding proteins derived from two or more viruses and mediate infection by those two or more viruses. In particular, a viral receptor that can mediate viral infection by binding to viral receptor-binding proteins derived from more types of viruses than naturally occurring wild-type viral receptors is preferred, and a viral receptor that can mediate viral infection by binding to viral receptor-binding proteins derived from three or more, four or more, five or more, six or more, or seven or more viruses is preferred.

[0084] As used herein, the term "ancestral viral receptor" refers to a viral receptor encoded by a putative ancestral gene. The ancestral viral receptor is preferably one that is possessed or presumed to have been possessed by an ancestral species shared by two or more (e.g., three or more, four or more, five or more, or six or more) species, genera, and / or orders. Examples include viral receptors that are possessed or presumed to have been possessed by an ancestral species shared by primates, Carnivora, and Cetacea.

[0085] As used herein, the term "orthologous protein" refers to a protein encoded by a homologous gene that arose from a common ancestral gene when species diverged during the course of biological evolution.

[0086] (Deducation step) In this aspect, the "deducation step" is a step of deducing the amino acid sequence of a common ancestral viral receptor for the first and second viral receptors, based on the amino acid sequence of a first viral receptor that binds to a first viral receptor-binding protein derived from a first virus to mediate infection by the first virus, and the amino acid sequence of a second viral receptor that is an orthologous protein of the first viral receptor and binds to a second viral receptor-binding protein derived from a second virus to mediate infection by the second virus.

[0087] The specific estimation method in this step is not limited. For example, estimation can be performed by aligning the amino acid sequence of the first viral receptor with the amino acid sequence of the second viral receptor and determining one of the amino acid residues at a position where the amino acid residues differ as the ancestral amino acid residue. This determination may also be performed by further aligning the amino acid sequence of a third viral receptor, which is an orthologous protein descended from the ancestral viral receptor common to the first and second viral receptors, and determining the same amino acid residue as the amino acid residue at the corresponding position in this third viral receptor as the ancestral amino acid residue. Alternatively, further aligning the amino acid sequence of a fourth viral receptor that diverged before the time when the ancestral viral receptor common to the first and second viral receptors appeared in evolution, and determining the same amino acid residue as the amino acid residue at the corresponding position in this fourth viral receptor as the ancestral amino acid residue. Furthermore, by aligning the amino acid sequences of the first and second viral receptors, at positions where the amino acid residues differ, another amino acid that is not present in either the first or second viral receptor but that can mutate into amino acid residues in the first and second viral receptors during the course of biological evolution may be determined as the ancestral amino acid residue.

[0088] In one embodiment, this step involves estimating the amino acid sequence of an ancestral viral receptor based on maximum likelihood. As used herein, "maximum likelihood" refers to a method for estimating the amino acid sequence of an ancestral protein at a specific branching point in the process of biological evolution, in which ancestral amino acid residues are estimated so as to maximize the probability (likelihood) that an ancestral amino acid residue will mutate to an amino acid residue at the corresponding position in an existing protein. By applying this method to all amino acid residues that make up the amino acid sequence, it is possible to estimate the ancestral amino acid sequence with the highest probability.

[0089] In the maximum likelihood method, an amino acid substitution rate matrix is ​​used, which indicates the probability that each amino acid will mutate to a specific amino acid. Although the amino acid substitution rate matrix is ​​not limited in this specification, an amino acid substitution rate matrix composed of substitution rates empirically determined by analyzing the amino acid sequences of various proteins is often used. An example of such an amino acid substitution rate matrix is ​​the Jones-Taylor-Thornton (JTT) model.

[0090] (Identification step) In this embodiment, the "identification step" is a step of aligning the amino acid sequence of an ancestral viral receptor with the amino acid sequence of a first or second viral receptor to identify ancestral amino acid residues and / or ancestral amino acid deletions that are contained only in the amino acid sequence of the ancestral viral receptor.

[0091] (Generation process) In this aspect, the "generation process" refers to a process of generating an amino acid sequence in which all or part of ancestral amino acid residues and / or ancestral amino acid deletions have been introduced into the amino acid sequence of a first or second viral receptor, as the amino acid sequence of a universal viral receptor or a candidate sequence thereof that binds to both viral receptor-binding proteins derived from the first and second viruses.

[0092] 8. Method for Producing Universal Cells or Candidate Cells Therefor The eighth aspect of the present invention is a method for producing universal cells or candidate cells therefor that can be infected with two or more viruses. The method of this aspect includes an introduction step as an essential step and a selection step as a selective step.

[0093] As used herein, the term "universal cells" refers to cells that can be infected by two or more viruses. In particular, cells that can be infected by more types of viruses than naturally occurring cells are preferred, and cells that can be infected by three or more, four or more, five or more, six or more, or seven or more viruses are preferred.

[0094] (Introduction step) In this aspect, the "introduction step" refers to a step of introducing a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of a universal viral receptor or a candidate sequence thereof, generated using the method of the seventh aspect, into a host cell in an expressible state. The introduction method is not limited, and any gene introduction method (transformation method) known in the art may be used. Examples include lipofection, electroporation, the calcium phosphate method, and the DEAE-Dextran method.

[0095] (Selection Step) In this embodiment, the "selection step" refers to a step of selecting universal cells from host cells into which nucleic acid has been introduced in the introduction step. In this step, host cells are infected with two or more viruses and the infection efficiency is measured to select cells that can be infected with two or more viruses, preferably cells that can be infected with more types of viruses than naturally occurring cells, for example, cells that can be infected with three or more, four or more, five or more, six or more, or seven or more viruses, thereby selecting universal cells from candidate cells.

[0096] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0097] Example 1: Verification of the effectiveness of different SLAM proteins in infection with various morbilliviruses (Objective) Vero cells expressing SLAM proteins derived from various host animals are infected with various morbilliviruses to evaluate the infection efficiency.

[0098] (Methods and Results) (1) Generation of SLAM-Expressing Cells. Expression plasmids were constructed by cloning genes encoding human SLAM protein (SEQ ID NO: 10), dolphin SLAM protein (SEQ ID NO: 11), bat SLAM protein (SEQ ID NO: 12), dog SLAM protein (SEQ ID NO: 13), or seal SLAM protein (SEQ ID NO: 14), each of which contains an N-terminal signal peptide, into the pCAGGS vector or its derivatives. SLAM-expressing Vero cells obtained by transfecting each expression plasmid into Vero cells (hereinafter, Vero cells expressing human-derived SLAM proteins may be referred to as "human SLAM-expressing Vero cells") were cultured in DMEM medium supplemented with 7.5% fetal calf serum (FCS) in the presence of antibiotics. Note that non-human SLAM proteins contain a hemagglutinin (HA) epitope further N-terminally than the signal peptide.

[0099] (2) Cytopathic Effect (CPE) Vero cells expressing SLAM were infected with various morbilliviruses. The morbilliviruses used were recombinant wild-type measles virus (MV) (IC323-EGFP strain) expressing EGFP, cetacean morbillivirus (CeMV) muc strain, bat morbillivirus (MBaMV) expressing EGFP, canine distemper virus (CDV) Ac96I strain, and phocine distemper virus (PDV) 982A strain. Specifically, subconfluent monolayers of SLAM-expressing Vero cells were infected with 1,000 PFU of each morbillivirus and monitored daily for 4 days for cytopathic effect (CPE) and the formation of multinucleated giant cells.

[0100] Figure 1 shows the results of CPE observations 24 hours after infection with morbilliviruses. CeMV and CDV induced the formation of multinucleated cells in Vero cells expressing SLAM proteins from dolphins, bats, dogs, and seals at 24 hours postinfection. PDV induced the formation of multinucleated cells in these four cell lines at 96 hours postinfection (data not shown). MV induced the formation of multinucleated cells in Vero cells expressing SLAM proteins from dolphins, dogs, and seals, but not in bat SLAM-expressing Vero cells. MV induced the formation of multinucleated cells in human SLAM-expressing Vero cells, whereas other morbilliviruses did not. MBaMV induced the formation of multinucleated cells in bat SLAM-expressing Vero cells, but not in Vero cells expressing SLAM proteins from other animal species.

[0101] (3) Plaque Assay: Subconfluent monolayers of SLAM-expressing Vero cells were incubated for 1 hour with serial dilutions of each morbillivirus (containing 5000 PFU, 500 PFU, and 50 PFU of virus, respectively) prepared at 1:10 dilutions. DMEM medium supplemented with 1% methylcellulose and 7.5% FCS (DMEM / MC / FCS) was then added to the cells. Four days after infection, DMEM / MC / FCS containing neutral red was added to the culture medium. The following day, the number of neutral red-stained plaques was counted and the plaque size was measured.

[0102] Figure 2 shows the results of plaque staining, and Figure 3A and 3B show the quantification of plaque number and size, respectively. MV efficiently formed plaques in Vero cells expressing human, dolphin, dog, and seal SLAM proteins, but had low plaque formation ability in bat SLAM-expressing Vero cells (Figure 2). MV formed a higher number of plaques in Vero cells expressing dolphin, dog, and seal SLAM proteins than in human SLAM-expressing Vero cells (Figure 3A), and the plaque size was particularly large in dolphin SLAM-expressing Vero cells (Figure 3B). Similarly, CeMV, CDV, and PDV formed plaques in Vero cells expressing dolphin, dog, seal, and bat SLAM proteins, but not in human SLAM-expressing Vero cells (Figure 2). Furthermore, among morbilliviruses, MBaMV formed plaques only in bat SLAM-expressing Vero cells, but CeMV, CDV, and PDV also formed plaques in bat SLAM-expressing Vero cells.

[0103] Example 2: Cell fusion assay (Objective) Morbillivirus infection is known to be initiated by the binding of hemagglutinin (H), a viral receptor-binding protein (RBP), to the viral receptor on the target cell. At the same time, another viral surface glycoprotein, the fusion (F) protein, promotes fusion of the viral envelope with the target cell membrane. Therefore, we conducted a cell fusion assay based on dual split proteins (DSPs) (hereinafter sometimes referred to as "DSP assays") to verify whether SLAM proteins from various animal species function as viral receptors for the H and F proteins derived from various morbilliviruses.

[0104] (Methods and Results) 293CD4D / DSP1-7 cells were transfected with plasmids encoding SLAM proteins (containing signal peptides but no N-terminal HA tag) from various animal species (human, bovine, ovine, dolphin, bat, canine, and harbor seal). 293FT / DSP8-11 cells were transfected with plasmids encoding the H and F proteins of various morbilliviruses (MV, peste des petits ruminants virus (PPRV), CeMV, MBaMV, CDV, and PDV). 24 hours after transfection, the cells were resuspended in fresh DMEM / MC / FCS medium, and the 293CD4D / DSP1-7 and 293FT / DSP8-11 cells were mixed at a 1:1 ratio and seeded into 96-well plates. After 2 days of incubation, luciferase activity was quantified using the Renilla-Glo luciferase assay system (Promega). Note that in the DSP assay, luciferase expression is induced in cells formed by fusion of 293CD4 / DSP1-7 and 293FT / DSP8-11 cells, so higher luciferase activity indicates a higher number of fused cells.

[0105] The quantitative results of luciferase activity are shown in Figures 4 and 5. The results of the DSP assay were similar to those of the infection assay in Example 1. Furthermore, the SLAM proteins derived from bovine and ovine animals functioned as receptors for MV, PPRV, CeMV, CDV, and PDV. The human SLAM protein did not function as a receptor for PPRV, as is the case with other morbilliviruses that infect animals other than humans.

[0106] Example 3: Prediction of the sequence of an ancestral SLAM protein (Objective) The amino acid sequence of an ancestral SLAM protein is predicted based on the amino acid sequences of SLAM proteins derived from various mammalian species.

[0107] (Methods and Results) The amino acid sequences of SLAM proteins (each containing an N-terminal signal peptide) from human (NP_003028.1; SEQ ID NO: 10), cow (Bos taurus) (AAI14834.1; SEQ ID NO: 15), sheep (Ovis aries) (ABB58749.1; SEQ ID NO: 16), dolphin (Tursiops truncatus) (XP_004327894.1; SEQ ID NO: 11), seal (Phoca largha) (BAH10672.1; SEQ ID NO: 14), dog (Canis lupus) (AAK61857.1; SEQ ID NO: 13), and mouse (Mus musculus) (AAF22231.1; SEQ ID NO: 17) were obtained from NCBI (accession numbers for each species are shown in parentheses). Protein sequences were aligned using Clustal Omega. Then, using the parameters of the Jones-Taylor-Thornton (JTT) model, a phylogenetic tree was constructed using the neighbor-joining method in MEGA X (Fig. 6A). Furthermore, using the parameters of the Jones-Taylor-Thornton (JTT) model, the predicted amino acid sequence of the ancestral SLAM protein was reconstructed using PAML.

[0108] The amino acid sequence of the ancestral SLAM protein (including a signal peptide at the N-terminus) estimated based on the amino acid sequences of SLAM proteins derived from humans, cows, sheep, dolphins, seals, and dogs is shown in SEQ ID NO: 18, and this ancestral SLAM protein (including a signal peptide at the N-terminus) is referred to as "ancSLAM8." The sequence from the signal peptide to the V domain in ancSLAM8 is shown in SEQ ID NO: 2 (the sequence from positions 1 to 20 in SEQ ID NO: 2 is the signal peptide).

[0109] The amino acid sequence of an ancestral SLAM protein (containing a signal peptide at the N-terminus) estimated based on the amino acid sequences of SLAM proteins derived from cows, sheep, dolphins, seals, and dogs is shown in SEQ ID NO: 19, and this ancestral SLAM protein (containing a signal peptide at the N-terminus) is referred to as "ancSLAM9." The sequence from the signal peptide to the V domain in ancSLAM9 is shown in SEQ ID NO: 3 (the sequence from positions 1 to 20 in SEQ ID NO: 3 is the signal peptide).

[0110] The amino acid sequence of ancSLAM8 is a predicted ancestral SLAM protein sequence for animal species, including humans, that existed before the divergence of Carnivora (dogs and seals) and Cetacea (dolphins, cattle, and sheep) from primates (humans). In contrast, the amino acid sequence of ancSLAM9 is a predicted ancestral SLAM protein sequence for animal species other than humans, that existed before the divergence of Carnivora (dogs and seals) from Cetacea (dolphins, cattle, and sheep).

[0111] Figure 6B shows the results of an alignment of the amino acid sequences from the signal peptide to the V domain (the sequence from positions 1 to 20 in each amino acid sequence in the figure is the signal peptide) between human SLAM proteins, ancSLAM8, and ancSLAM9.

[0112] In ancSLAM8 and ancSLAM9, the 21st amino acid residue from the N-terminus is a Leu residue, the 28th amino acid residue from the N-terminus is a Gly residue, the 29th amino acid residue from the N-terminus is a Leu residue, the 38th amino acid residue from the N-terminus is an Arg residue, the 42nd amino acid residue from the N-terminus is a Ser residue, the 49th amino acid residue from the N-terminus is a Ser residue, the 63rd amino acid residue from the N-terminus is a Leu residue, the 70th amino acid residue from the N-terminus is a Pro residue, the 71st amino acid residue from the N-terminus is a Gly residue, the 75th amino acid residue from the N-terminus is a Lys residue, the 76th amino acid residue from the N-terminus is a Lys residue, the 83rd amino acid residue from the N-terminus is a Leu residue, the 86th amino acid residue from the N-terminus is a Gly residue, and the 88th amino acid residue from the N-terminus is a The 93rd amino acid residue from the N-terminus is replaced with a Glu residue, the 95th amino acid residue from the N-terminus is replaced with a Gly residue, the 99th amino acid residue from the N-terminus is replaced with a His residue, the 104th amino acid residue from the N-terminus is replaced with a Ser residue, the 106th amino acid residue from the N-terminus is replaced with an Arg residue, the 108th amino acid residue from the N-terminus is replaced with a Leu residue, the 124th amino acid residue from the N-terminus is replaced with a Glu residue, the 130th amino acid residue from the N-terminus is replaced with a His residue, and the 136th amino acid residue from the N-terminus is replaced with a Lys residue (these amino acid substitutions are sometimes referred to as ancestral amino acid substitutions of ancSLAM8), and the 91st amino acid residue from the N-terminus is deleted (this amino acid deletion is sometimes referred to as the ancestral amino acid deletion of ancSLAM8 and ancSLAM9).

[0113] In ancSLAM9, in addition to the amino acid substitutions and deletions mentioned above, the 23rd amino acid residue from the N-terminus is replaced with a Cys residue, the 27th amino acid residue from the N-terminus is replaced with a Glu residue, the 37th amino acid residue from the N-terminus is replaced with a Gly residue, the 48th amino acid residue from the N-terminus is replaced with an Ala residue, the 51st amino acid residue from the N-terminus is replaced with a Gly residue, the 53rd amino acid residue from the N-terminus is replaced with a Ser residue, the 68th amino acid residue from the N-terminus is replaced with a Glu residue, the 84th amino acid residue from the N-terminus is replaced with a Pro residue, the 94th amino acid residue from the N-terminus is replaced with an Asn residue, the 109th amino acid residue from the N-terminus is replaced with a Phe residue, and the 151st amino acid residue from the N-terminus is replaced with a Ser residue (these amino acid substitutions, in addition to the ancestral amino acid substitutions of ancSLAM8, are sometimes referred to as the ancestral amino acid substitutions of ancSLAM9).

[0114] Figure 7 shows the results of an alignment of the amino acid sequences from the signal peptide to the V domain (the sequence from positions 1 to 20 in each amino acid sequence in the figure is the signal peptide) between the human SLAM proteins, ancSLAM8, and ancSLAM9, as well as SLAM proteins derived from cows, sheep, dolphins, seals, dogs, and mice.

[0115] <Example 4: Measurement of activity of ancestral SLAM proteins> (Purpose) We will evaluate whether ancSLAM8 and ancSLAM9, which have amino acid sequences predicted to be ancestral SLAM proteins, function as viral receptors for various morbilliviruses using DSP assays and plaque assays.

[0116] (Methods and Results) (1) DSP Assay DSP assays were performed on ancSLAM8 and ancSLAM9. 293CD4D / DSP1-7 cells transfected with plasmids encoding SLAM proteins derived from humans, dolphins, and bats, as well as ancSLAM8 and ancSLAM9, were mixed at a 1:1 ratio with 293FT / DSP8-11 cells transfected with plasmids encoding the H and F proteins of various morbilliviruses (MV, PPRV, CeMV, MBaMV, CDV, and PDV), and luciferase assays were performed using the same method as in Example 2.

[0117] The results of the luciferase assay are shown in Figure 8. It was revealed that both ancSLAM8 and ancSLAM9 function as receptors for all morbilliviruses, including MV, PPRV, CeMV, MBaMV, CDV, and PDV.

[0118] (2) Verification of Cytopathic Effect (CPE) Using a method similar to that described in (1) of Example 1, an expression plasmid was constructed by cloning the gene encoding ancSLAM8 into the pCAGGS vector or a derivative thereof. Vero cells expressing ancSLAM8 were generated by introducing this expression plasmid into Vero cells (hereinafter, ancSLAM8-expressing Vero cells may be referred to as "ancSLAM8-expressing Vero cells"). Subconfluent monolayers of ancSLAM8-expressing Vero cells were then infected with morbilliviruses (MV, CDV, CeMV, MBaMV, and PDV) using a method similar to that described in (2) of Example 1, and the formation of multinucleated giant cells was observed as a cytopathic effect (CPE).

[0119] The results are shown in Figure 9. All morbilliviruses induced the formation of multinucleated giant cells. These results demonstrate that ancSLAM8 functions as a receptor for all morbilliviruses, including MV, CeMV, MBaMV, CDV, and PDV.

[0120] (3) Plaque Assay Using the same method as in Example 1(3), plaque assays were performed on ancSLAM8-expressing Vero cells and Vero cells expressing SLAM proteins derived from the host animal species of each morbillivirus.

[0121] The results of plaque staining are shown in Figure 10. It was revealed that ancSLAM8 functions as a receptor for all morbilliviruses, including MV, CeMV, MBaMV, CDV, and PDV, similar to the SLAM proteins derived from the host animal species of each morbillivirus.

[0122] Figure 11 shows the results of plaque assays in which ancSLAM8-expressing Vero cells, human SLAM8-expressing Vero cells, dolphin SLAM8-expressing Vero cells, and bat SLAM8-expressing Vero cells were infected with MV, CeMV, or MBaMV. The human SLAM protein functions as a receptor for MV but not for CeMV or MBaMV. The dolphin SLAM protein functions as a receptor for MV and CeMV but not for MBaMV. The bat SLAM protein functions as a receptor for CeMV and MBaMV but barely functions as a receptor for MV. In contrast to these results, ancSLAM8 was demonstrated to universally function as a receptor for all morbilliviruses, including MV, CeMV, MBaMV, CDV, and PDV.

[0123] <Example 5: Necessity of ancestral amino acid residues and ancestral amino acid deletions> (Purpose) We will create a mutant (hereinafter referred to as "mutant ancSLAM8") in which some of the ancestral amino acid substitutions in ancSLAM8 have been reverted to amino acid residues in human SLAM, and verify whether its function as a receptor for various morbilliviruses is maintained.

[0124] (Methods and Results) The mutant ancSLAM8 in which the 63rd amino acid residue from the N-terminus of ancSLAM8 was changed from Leu to Val was designated ancSLAM9(L63V). The amino acid sequence from the signal peptide to the V domain of ancSLAM8(L63V) is shown in SEQ ID NO: 22, and the full-length amino acid sequence of ancSLAM8(L63V) (including the signal peptide) is shown in SEQ ID NO: 23.

[0125] The mutant ancSLAM8, in which the 70th amino acid residue from the N-terminus of ancSLAM8 was changed from Pro to Leu and the 71st amino acid residue from Gly to Glu, was designated ancSLAM8(P70L / G71E). The amino acid sequence from the signal peptide to the V domain of ancSLAM8(P70L / G71E) is shown in SEQ ID NO: 32, and the full-length amino acid sequence of ancSLAM8(P70L / G71E) (including the signal peptide) is shown in SEQ ID NO: 33.

[0126] The mutant ancSLAM8, in which the 75th amino acid residue from the N-terminus of ancSLAM8 was changed from Lys to Glu and the 76th amino acid residue from Lys to Asn, was designated ancSLAM8(K75E / K76N). The amino acid sequence from the signal peptide to the V domain of ancSLAM8(K75E / K76N) is shown in SEQ ID NO: 30, and the full-length amino acid sequence of ancSLAM8(K75E / K76N) (including the signal peptide) is shown in SEQ ID NO: 31.

[0127] The mutant ancSLAM8 in which the 83rd amino acid residue from the N-terminus of ancSLAM8 was changed from Leu to Pro, the 86th amino acid residue from the N-terminus was changed from Gly to Ala, and the 88th amino acid residue from the N-terminus was changed from Ser to Pro was designated ancSLAM8(L83P / G86A / S88P). The amino acid sequence from the signal peptide to the V domain of ancSLAM8(L83P / G86A / S88P) is shown in SEQ ID NO:36, and the full-length amino acid sequence of ancSLAM8(L83P / G86A / S88P) (including the signal peptide) is shown in SEQ ID NO:37.

[0128] The mutant ancSLAM8 in which the 92nd amino acid residue from the N-terminus of ancSLAM8 was changed from Glu to Gly and the 94th amino acid residue from Gly to Arg was designated ancSLAM8(E92G / G94R). The amino acid sequence from the signal peptide to the V domain of ancSLAM8(E92G / G94R) is shown in SEQ ID NO: 34, and the full-length amino acid sequence of ancSLAM8(E92G / G94R) (including the signal peptide) is shown in SEQ ID NO: 35.

[0129] The mutant ancSLAM8, in which the 129th amino acid residue from the N-terminus of ancSLAM8 was changed from His to Arg, is called ancSLAM8(H129R). The amino acid sequence from the signal peptide to the V domain of ancSLAM8(H129R) is shown in SEQ ID NO: 24, and the full-length amino acid sequence of ancSLAM8(H129R) (including the signal peptide) is shown in SEQ ID NO: 25.

[0130] The mutant ancSLAM8, in which the 135th amino acid residue from the N-terminus of ancSLAM8 was changed from Lys to Arg, is called ancSLAM8(K135R). The amino acid sequence from the signal peptide to the V domain of ancSLAM8(K135R) is shown in SEQ ID NO: 20, and the full-length amino acid sequence of ancSLAM8(K135R) (including the signal peptide) is shown in SEQ ID NO: 21.

[0131] The mutant ancSLAM8, in which the 28th amino acid residue from the N-terminus of ancSLAM8 was changed from Gly to Arg, is called ancSLAM8(G28R). The amino acid sequence from the signal peptide to the V domain of ancSLAM8(G28R) is shown in SEQ ID NO: 26, and the full-length amino acid sequence of ancSLAM8(G28R) (including the signal peptide) is shown in SEQ ID NO: 27.

[0132] The mutant ancSLAM8, in which the 29th amino acid residue from the N-terminus of ancSLAM8 was changed from Leu to Met, is called ancSLAM8(L29M). The amino acid sequence from the signal peptide to the V domain of ancSLAM8(L29M) is shown in SEQ ID NO: 38, and the full-length amino acid sequence of ancSLAM8(L29M) (including the signal peptide) is shown in SEQ ID NO: 39.

[0133] A mutant ancSLAM8 in which the 28th amino acid residue from the N-terminus of ancSLAM8 was changed from Gly to Arg and the 29th amino acid residue from the N-terminus was changed from Leu to Met was designated ancSLAM8(G28R / L29M). The amino acid sequence from the signal peptide to the V domain of ancSLAM8(G28R / L29M) is shown in SEQ ID NO: 28, and the full-length amino acid sequence of ancSLAM8(G28R / L29M) (including the signal peptide) is shown in SEQ ID NO: 29.

[0134] Figure 12 shows the results of an alignment of the amino acid sequences from the signal peptide to the V domain between the above mutant ancSLAM8, ancSLAM8, and human SLAM protein.

[0135] A DSP assay was performed on the mutant ancSLAM8. 293CD4D / DSP1-7 cells transfected with plasmids encoding the mutant ancSLAM8, human SLAM proteins, and ancSLAM8 and ancSLAM9 were mixed at a 1:1 ratio with 293FT / DSP8-11 cells transfected with plasmids encoding the H and F proteins of various morbilliviruses (MV, PPRV, CeMV, MBaMV, CDV, and PDV), and a luciferase assay was performed as in Example 2.

[0136] The results of the luciferase assay are shown in Figures 13 and 14. The mutant ancSLAM8 was found to function as a viral receptor for all morbilliviruses used in the experiment. These results demonstrate that the function as a viral receptor for various morbilliviruses is maintained even when one to three of the ancestral amino acid substitutions in ancSLAM8 are reverted to amino acid residues in the human SLAM protein. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A mutant SLAM protein or an active fragment thereof, comprising a mutant V domain derived from the V domain of a human SLAM protein and having an ancestral amino acid residue and / or an ancestral amino acid deletion, wherein the ancestral amino acid residue and / or the ancestral amino acid deletion is: (a) a Leu residue at a position corresponding to position 21 of SEQ ID NO:1; (b) a Gly residue at a position corresponding to position 28 of SEQ ID NO:1; (c) a Leu residue at a position corresponding to position 29 of SEQ ID NO:1; (d) an Arg residue at a position corresponding to position 38 of SEQ ID NO:1; (e) a Ser residue at a position corresponding to position 42 of SEQ ID NO:1; (f) a Ser residue at a position corresponding to position 49 of SEQ ID NO:1; (g) a Leu residue at a position corresponding to position 63 of SEQ ID NO:1; (h) a Pro residue at a position corresponding to position 70 of SEQ ID NO:1; (i) a Gly residue at a position corresponding to position 71 of SEQ ID NO:1; (j) a Lys residue at a position corresponding to position 75 of SEQ ID NO:1; (k) a Lys residue at a position corresponding to position 76 of SEQ ID NO:

1. The mutant SLAM protein or an active fragment thereof, comprising one or more selected from the group consisting of: (l) a Leu residue at a position corresponding to position 83 of SEQ ID NO: 1; (m) a Gly residue at a position corresponding to position 86 of SEQ ID NO: 1; (n) a Ser residue at a position corresponding to position 88 of SEQ ID NO: 1; (o) an amino acid deletion at a position corresponding to position 91 of SEQ ID NO: 1; (p) a Glu residue at a position corresponding to position 93 of SEQ ID NO: 1; (q) a Gly residue at a position corresponding to position 95 of SEQ ID NO: 1; (r) a His residue at a position corresponding to position 99 of SEQ ID NO: 1; (s) a Ser residue at a position corresponding to position 104 of SEQ ID NO: 1; (t) an Arg residue at a position corresponding to position 106 of SEQ ID NO: 1; (u) a Leu residue at a position corresponding to position 108 of SEQ ID NO: 1; (v) a Glu residue at a position corresponding to position 124 of SEQ ID NO: 1; (w) a His residue at a position corresponding to position 130 of SEQ ID NO: 1; and (x) a Lys residue at a position corresponding to position 136 of SEQ ID NO:

1.

2. A mutant SLAM protein or an active fragment thereof described in claim 1, wherein the ancestral amino acid residues and / or ancestral amino acid deletions include 21 or more selected from the group consisting of (a) to (x).

3. A mutant SLAM protein or an active fragment thereof according to claim 1, wherein the mutant V domain comprises all of (a) to (x).

4. The mutant SLAM protein or active fragment thereof according to claim 1, wherein the mutant V domain comprises: (i) an amino acid sequence consisting of positions 21 to 152 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38; (ii) an amino acid sequence in which 1 to 13 amino acid residues have been deleted, substituted, or added in any amino acid sequence consisting of positions 21 to 152 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38; or (iii) an amino acid sequence that is 90% or more identical to the amino acid sequence consisting of positions 21 to 152 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.

5. (I) an amino acid sequence consisting of positions 21 to 333 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39; (II) an amino acid sequence in which 1 to 13 amino acid residues are deleted, substituted, or added in any amino acid sequence consisting of positions 21 to 333 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39; (III) an amino acid sequence having 90% or more identity to the amino acid sequence consisting of positions 21 to 333 in any amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39; or (IV) an amino acid sequence in which an arbitrary amino acid sequence is added to the N-terminus and / or C-terminus of any of the amino acid sequences (I) to (III). The mutant SLAM protein or an active fragment thereof according to claim 1, comprising:

6. A mutant SLAM protein or an active fragment thereof, (1) comprising a mutant V domain derived from the V domain of a bovine, dolphin, or seal SLAM protein and having an ancestral amino acid residue and / or an ancestral amino acid deletion, wherein the ancestral amino acid residue and / or the ancestral amino acid deletion is: (a) a Leu residue at a position corresponding to position 21 of SEQ ID NO:1; (b) a Gly residue at a position corresponding to position 28 of SEQ ID NO:1; (c) a Leu residue at a position corresponding to position 29 of SEQ ID NO:1; (d) an Arg residue at a position corresponding to position 38 of SEQ ID NO:1; (e) a Ser residue at a position corresponding to position 42 of SEQ ID NO:1; (f) a Ser residue at a position corresponding to position 49 of SEQ ID NO:1; (g) a Leu residue at a position corresponding to position 63 of SEQ ID NO:1; (h) a Pro residue at a position corresponding to position 70 of SEQ ID NO:1; (i) a Gly residue at a position corresponding to position 71 of SEQ ID NO:1; (j) a Lys residue at a position corresponding to position 75 of SEQ ID NO:

1. (k) a Lys residue at a position corresponding to position 76 of SEQ ID NO: 1, (l) a Leu residue at a position corresponding to position 83 of SEQ ID NO: 1, (m) a Gly residue at a position corresponding to position 86 of SEQ ID NO: 1, (n) a Ser residue at a position corresponding to position 88 of SEQ ID NO: 1, (o) an amino acid deletion at a position corresponding to position 91 of SEQ ID NO: 1, (p) a Glu residue at a position corresponding to position 93 of SEQ ID NO: 1, (q) a Gly residue at a position corresponding to position 95 of SEQ ID NO: 1, (r) a His residue at a position corresponding to position 99 of SEQ ID NO: 1, (s) a Ser residue at a position corresponding to position 104 of SEQ ID NO: 1, (t) an Arg residue at a position corresponding to position 106 of SEQ ID NO: 1, (u) a Leu residue at a position corresponding to position 108 of SEQ ID NO: 1, (v) a Glu residue at a position corresponding to position 124 of SEQ ID NO: 1, (w) a His residue at a position corresponding to position 130 of SEQ ID NO: 1, and (x) a Lys residue at a position corresponding to position 136 of SEQ ID NO: 1, or(2) A mutant V domain derived from the V domain of a sheep SLAM protein and having 23 or more ancestral amino acid residues and / or ancestral amino acid deletions selected from the group consisting of (a) to (x); (3) A mutant V domain derived from the V domain of a canine SLAM protein and having 21 or more ancestral amino acid residues and / or ancestral amino acid deletions selected from the group consisting of (a) to (x); or (4) A mutant SLAM protein or an active fragment thereof derived from the V domain of a mouse SLAM protein and having 15 or more ancestral amino acid residues and / or ancestral amino acid deletions selected from the group consisting of (a) to (x).

7. A nucleic acid encoding a mutant SLAM protein or an active fragment thereof according to any one of claims 1 to 6.

8. A host cell comprising the nucleic acid of claim 7 in an expressible state.

9. The host cell of claim 8, which is selected from the group consisting of Vero cells, MDCK cells, CHO cells, HEK293 cells, and BHK-21 cells.

10. The host cell of claim 9, which is infected with a morbillivirus.

11. The host cell of claim 10, wherein the morbillivirus is selected from the group consisting of measles virus (MV), canine distemper virus (CDV), seal distemper virus (PDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), cetacean morbillivirus (CeMV), cat morbillivirus (FeMV), bat morbillivirus (MBaMV), and porcine morbillivirus (PoMV).

12. The host cell of claim 10, which is a live cell or a fixed cell.

13. A method for producing a morbillivirus, comprising: an infection step of infecting a host cell according to claim 8 with a morbillivirus; a growth step of culturing the host cell infected with the morbillivirus to grow the morbillivirus; and a recovery step of recovering the morbillivirus from the culture medium after the growth step.

14. A method for producing a vaccine against morbillivirus infection, comprising: an infection step of infecting a host cell according to claim 8 with a morbillivirus; a growth step of culturing the host cell infected with the morbillivirus to grow the morbillivirus; a recovery step of recovering the morbillivirus from the culture medium after the growth step; and a formulation step of formulating the recovered morbillivirus as a vaccine.

15. A method for measuring the titer of a morbillivirus, comprising: an infection step of infecting a host cell described in claim 8 with a morbillivirus; a culture step of culturing the host cell after the infection step; and a titer determination step of evaluating the host cell after the culture step and determining the titer of the morbillivirus based on the results of the evaluation.

16. A method for generating an amino acid sequence or candidate sequences of a universal viral receptor capable of binding to viral receptor-binding proteins derived from two or more viruses and mediating infection by the two or more viruses, comprising: a deducing step of deducing the amino acid sequence of an ancestral viral receptor common to a first and second viral receptor based on the amino acid sequence of a first viral receptor that binds to a first viral receptor-binding protein derived from a first virus and mediates infection by the first virus, and the amino acid sequence of a second viral receptor that is an orthologous protein of the first viral receptor and binds to a second viral receptor-binding protein derived from a second virus and mediates infection by the second virus; an identifying step of aligning the amino acid sequence of the ancestral viral receptor with the amino acid sequence of the first or second viral receptor to identify ancestral amino acid residues and / or ancestral amino acid deletions contained only in the amino acid sequence of the ancestral viral receptor; and The method includes a generating step of generating an amino acid sequence in which all or part of the ancestral amino acid residues and / or ancestral amino acid deletions have been introduced into the amino acid sequence of the first or second viral receptor, as the amino acid sequence of a universal viral receptor or a candidate sequence thereof that binds to both viral receptor-binding proteins derived from the first and second viruses.

17. The method of claim 16, wherein the viral receptor is selected from the group consisting of SLAM protein, nectin 4, ACE2 protein, DPP4 protein, ephrin B2 protein, and ephrin B3 protein.

18. A method for producing a universal cell or a candidate cell thereof that can be infected by two or more viruses, the method comprising an introduction step of introducing into a host cell a nucleic acid containing a base sequence encoding the amino acid sequence of the universal virus receptor or a candidate sequence thereof, generated using the method of claim 16, in an expressible state.

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