Mutants of coxsackievirus a16 and virus-like particles thereof

Abstract

The present disclosure relates to the field of biological medicine, and provides a mutant of coxsackievirus A16 and a virus-like particle thereof, wherein the mutant has at least one amino acid mutation site in the following regions compared with wild-type coxsackievirus A16: (1) a canyon region of a virus capsid; (2) a channel region at a dihedral axis; or (3) a channel region at a quasi-trihedral axis. The mutant and the virus-like particle are obtained by optimizing and modifying structural proteins VP0, VP1 and VP3 constituting the virus capsid by using computational biology and structural biology methods, co-expressing and co-assembling the structural proteins into the virus-like particle in vivo in a Hansenula yeast expression system, and then purifying the virus-like particle by serial chromatography. The mutation scheme of the coxsackievirus A16 virus-like particle in the present disclosure can significantly improve the antigen immunogenicity compared with the unmutated virus-like particle, and has clinical application value.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biological medicine, and in particular, the present disclosure relates to a mutant of Coxsackievirus A16 and a virus-like particle thereof. BACKGROUND

[0002] Hand-foot-mouth disease (HFMD) is an acute infectious disease caused by multiple human enteroviruses (HEVs) and has broken out and spread globally. HFMD mainly occurs in children under 5 years old, and the main clinical symptoms are fever and rash or herpes on the hands, feet, and mouth, etc. Generally, it is self-healing after 2 weeks. However, a small number of severe patients can have serious neurological complications (such as aseptic meningitis, acute flaccid myelitis, and myocarditis, etc.), and even death in severe cases. Coxsackievirus (CV) is one of the main pathogens causing HFMD, and HFMD caused by CVA16 has broken out and spread in many places around the world. At present, there is no specific treatment drug for CVA16, and vaccine is the most effective technical means to prevent CVA16 infection and control its spread.

[0003] At present, the research direction of CVA16 vaccine mainly focuses on two technical routes of whole virus inactivated vaccine and recombinant VLP vaccine. A good VLP vaccine should have the characteristics of high safety, good immunogenicity, low price, and easy preservation, etc. The immunogenicity of the vaccine is an important point in the field of vaccine research. However, in the research of enterovirus VLP, the main difficulty at present is that the structural instability leads to low immunogenicity. Therefore, structure-based immunogen screening is crucial for effective vaccine development. SUMMARY

[0004] Technical problems solved:

[0005] One aspect of the present disclosure is to solve the problem of low immunogenicity caused by the unstable structure of enterovirus virus-like particles in the prior art, and to provide a mutant of Coxsackievirus A16 and a virus-like particle thereof.

[0006] Specifically, through structural analysis and computational simulation, the present disclosure analyzes the conformational changes and motion patterns in the uncoating process of Coxsackievirus A16, and predicts the main residue sites that control the uncoating motion of Coxsackievirus A16. The present inventors found that these residue sites are mainly located in the canyon region of the virus capsid, the channel region at the dihedral axis, and the channel region at the quasi-trihedral axis, and then introduced mutations in these regions to stabilize the conformation. Based on this strategy, the present disclosure provides multiple mutation schemes in the embodiments to stabilize the Coxsackievirus A16 virus-like particle in the pre-swelling conformation, thereby solving the above technical problems.

[0007] Technical Solution

[0008] A mutant of coxsackievirus A16 (CVA16) having at least one amino acid mutation site in the following regions compared to wild-type coxsackievirus A16:

[0009] (1) canyon region of the viral capsid; or

[0010] (2) channel region at the two-fold axis; or

[0011] (3) channel region at the quasi-three-fold axis.

[0012] In some embodiments, the amino acid mutation described above can be substitution of an amino acid residue.

[0013] In some embodiments, the mutant described above can have at least one amino acid mutation site compared to the amino acid sequence of the structural protein of wild-type coxsackievirus A16, the mutation being substitution of an amino acid residue, at the following positions:

[0014] (1) any of positions 115-123 in the VP0 sequence; or

[0015] (2) any of positions 155-159 in the VP0 sequence; or

[0016] (3) position 314 in the VP0 sequence; or

[0017] (4) any of positions 137-154 in the VP3 sequence; or

[0018] (5) any of positions 175-188 in the VP3 sequence; or

[0019] (6) any of positions 228-238 in the VP3 sequence; or

[0020] (7) position 110 in the VP3 sequence; or

[0021] (8) any of positions 170-177 in the VP1 sequence; or

[0022] (9) any of positions 222-225 in the VP1 sequence; or

[0023] (10) positions 119 and 158 in the VP1 sequence.

[0024] In some embodiments, the mutant can have at least one amino acid mutation site compared to the amino acid sequence of the structural protein of wild-type coxsackievirus A16 at:

[0025] (a) at least one selected from the positions (1), (2) or (3); or

[0026] (b) at least one selected from the positions (4), (5), (6) or (7); or

[0027] (c) at least one selected from the positions (8), (9) or (10).

[0028] In some embodiments, the amino acid mutation site can be selected from:

[0029] (1) at least one of the positions 115, 118, 123, 155, 158, 159, 314 of the VP0 sequence; or

[0030] (2) at least one of the positions 110, 137, 138, 140, 141, 149, 152, 154, 175, 176, 177, 180, 188, 228, 230, 238 of the VP3 sequence; or

[0031] (3) at least one of the positions 119, 158, 170, 173, 175, 176, 177, 222 or 225 of the VP1 sequence.

[0032] In some embodiments, the amino acid mutation site can be:

[0033] (1) the position 158 of the VP0 sequence; or

[0034] (2) the positions 118, 123 and 158 of the VP0 sequence; or

[0035] (3) the positions 155, 158 and 159 of the VP0 sequence; or

[0036] (4) amino acid positions 158 in the VP0 sequence, 176, 180 and 228 in the VP3 sequence; or

[0037] (5) amino acid positions 158 in the VP0 sequence, 158 and 177 in the VP1 sequence; or

[0038] (6) amino acid positions 115, 158 and 314 in the VP0 sequence, 137, 149 and 154 in the VP3 sequence; or

[0039] (7) amino acid positions 158 in the VP0 sequence, 176, 230 and 238 in the VP3 sequence, 158, 170, 176 and 177 in the VP1 sequence; or

[0040] (8) amino acid positions 155, 158 and 159 in the VP0 sequence; or

[0041] (9) amino acid positions 155 and 158 in the VP0 sequence; or

[0042] (10) amino acid positions 158 in the VP0 sequence, 180 in the VP3 sequence; or

[0043] (11) amino acid positions 158 in the VP0 sequence, 175, 177 and 180 in the VP3 sequence; or

[0044] (12) amino acid positions 158 in the VP0 sequence, 152 in the VP3 sequence; or

[0045] (13) amino acid positions 158 in the VP0 sequence, 110 in the VP3 sequence; or

[0046] (14) amino acid positions 158 in the VP0 sequence, 119 in the VP1 sequence; or

[0047] (15) amino acid positions 158 in the VP0 sequence, 110, 152 and 180 in the VP3 sequence; or

[0048] (16) amino acid positions 138 and 152 in the VP3 sequence; or

[0049] (17) amino acid positions 175, 177 and 180 in the VP3 sequence; or

[0050] (18) amino acid positions 119, 173 and 175 in the VP1 sequence; or

[0051] (19) amino acid positions 188 on the VP3 sequence, 222 and 225 on the VP1 sequence; or

[0052] (20) amino acid positions 140 and 141 on the VP3 sequence.

[0053] In some embodiments, the above-mentioned amino acid mutation sites can be:

[0054] (1) the 158th amino acid on the VP0 sequence is substituted with C, W or F; or

[0055] (2) the 118th amino acid on the VP0 sequence is substituted with C, the 123rd amino acid is substituted with C and the 158th amino acid is substituted with C; or

[0056] (3) the 155th amino acid on the VP0 sequence is substituted with M, the 158th amino acid is substituted with M and the 159th amino acid is substituted with M; or

[0057] (4) the 158th amino acid on the VP0 sequence is substituted with M, the 176th amino acid on the VP3 sequence is substituted with M, the 180th amino acid is substituted with M and the 228th amino acid is substituted with F; or

[0058] (5) the 158th amino acid on the VP0 sequence is substituted with M, the 158th amino acid on the VP1 sequence is substituted with F and the 177th amino acid is substituted with R; or

[0059] (6) the 115th amino acid on the VP0 sequence is substituted with L, the 158th amino acid is substituted with F, the 314th amino acid is substituted with M, the 137th amino acid on the VP3 sequence is substituted with V, the 149th amino acid is substituted with V and the 154th amino acid is substituted with V; or

[0060] (7) the 158th amino acid on the VP0 sequence is substituted with F, the 176th amino acid on the VP3 sequence is substituted with R, the 230th amino acid is substituted with Y, the 238th amino acid is substituted with P, the 158th amino acid on the VP1 sequence is substituted with F, the 170th amino acid is substituted with R, the 176th amino acid is substituted with W and the 177th amino acid is substituted with R; or

[0061] (8) the 155th amino acid on the VP0 sequence is substituted with L, the 158th amino acid is substituted with F and the 159th amino acid is substituted with Y; or

[0062] (9) the 155th amino acid on the VP0 sequence is substituted with L and the 158th amino acid is substituted with F; or

[0063] (10) the 158th amino acid on the VP0 sequence is substituted with F, the 180th amino acid on the VP3 sequence is substituted with L; or

[0064] (11) the 158th amino acid on the VP0 sequence is substituted with F, the 175th amino acid on the VP3 sequence is substituted with M, the 177th amino acid is substituted with F, and the 180th amino acid is substituted with L; or

[0065] (12) the 158th amino acid on the VP0 sequence is substituted with F, and the 152th amino acid on the VP3 sequence is substituted with I; or

[0066] (13) the 158th amino acid on the VP0 sequence is substituted with F, and the 110th amino acid on the VP3 sequence is substituted with L; or

[0067] (14) the 158th amino acid on the VP0 sequence is substituted with F, and the 119th amino acid on the VP1 sequence is substituted with W; or

[0068] (15) the 158th amino acid on the VP0 sequence is substituted with F, the 110th amino acid on the VP3 sequence is substituted with L, the 152th amino acid is substituted with I, and the 180th amino acid is substituted with L; or

[0069] (16) the 138th amino acid on the VP3 sequence is substituted with I, and the 152th amino acid is substituted with M; or

[0070] (17) the 175th amino acid on the VP3 sequence is substituted with M, the 177th amino acid is substituted with F, and the 180th amino acid is substituted with M; or

[0071] (18) the 119th amino acid on the VP1 sequence is substituted with L, the 173rd amino acid is substituted with F, and the 175th amino acid is substituted with L; or

[0072] (19) the 188th amino acid on the VP3 sequence is substituted with L, the 222nd amino acid on the VP1 sequence is substituted with Y, and the 225th amino acid is substituted with M; or

[0073] (20) the 140th amino acid on the VP3 sequence is substituted with V, and the 141st amino acid is substituted with V.

[0074] In some more specific embodiments, the amino acid sequence of VP0, VP1 or VP3 of the above mutants can be as shown in SEQ ID Nos: 4-69, or a sequence having 90% and above identity thereto and having the same or similar biological function.

[0075] In some embodiments, the amino acid sequences of the wild-type structural proteins VP0, VP1 and VP3 of coxsackievirus A16 described above are shown in SEQ ID Nos: 1-3, and the nucleotide sequences encoding the amino acid sequences described above are shown in SEQ ID Nos: 70-72. The mutation sites of the VP0 sequence, the VP1 sequence or the VP3 sequence described in the present disclosure are all based on the amino acid sequences described above.

[0076] Another aspect of the present disclosure is to provide a virus-like particle assembled from the mutant described above.

[0077] Another aspect of the present disclosure is to provide an isolated polynucleotide encoding the mutant described above, or VP0, VP1 or VP3 of the mutant.

[0078] In some embodiments, the isolated polynucleotide described above can be codon-optimized.

[0079] In some specific embodiments, the sequence of the isolated polynucleotide described above is shown in SEQ ID Nos: 73-138.

[0080] Another aspect of the present disclosure is to provide a vector comprising the isolated polynucleotide described above, or the mutant described above.

[0081] In some embodiments, the vector described above can be a protein expression vector, a gene delivery vector or a protein delivery vector.

[0082] Another aspect of the present disclosure is to provide a host cell comprising the mutant described above, the isolated polynucleotide described above, or the vector described above.

[0083] In some embodiments, the host cell described above can be an E. coli cell, a yeast cell, an insect cell or a mammalian cell. In some more specific embodiments, the host cell can be a Hansenula polymorpha cell.

[0084] Another aspect of the present disclosure is to provide a recombinant protein vaccine comprising the virus-like particle described above, and optionally comprising an adjuvant.

[0085] In some embodiments, the adjuvant described above can be at least one selected from an aluminum adjuvant, an oil-water emulsion adjuvant, an AS01 adjuvant system, an AS03 adjuvant system, an AS04 adjuvant system, MF59, CpG, an immunostimulatory substance or an immunomodulatory substance.

[0086] Another aspect of the present disclosure is to provide a nucleic acid vaccine comprising the polynucleotide described above.

[0087] Another aspect of the present disclosure is to provide a liposome complex comprising the polynucleotide described above.

[0088] Another aspect of the present disclosure is to provide a pharmaceutical composition comprising the mutant described above, the virus-like particle described above, the isolated polynucleotide described above, the vector described above, the host cell described above, the vaccine described above, or the liposome complex described above, and a pharmaceutically acceptable carrier, delivery medium, excipient, stabilizer, diluent, or a combination thereof.

[0089] Another aspect of the present disclosure is to provide a method of eliciting an immune response against coxsackievirus A16 in a subject or treating coxsackievirus A16 infection in a subject, the method comprising administering to the subject an effective dose of the recombinant protein vaccine described above, the nucleic acid vaccine described above, the liposome complex described above, or the pharmaceutical composition described above.

[0090] Advantages:

[0091] The coxsackievirus A16 mutant and the virus-like particle thereof in the present disclosure are obtained by optimizing and modifying the structural proteins VP0, VP1, and VP3 that constitute the viral capsid using computational biology and structural biology methods, and then co-expressing and co-assembling the structural proteins into virus-like particles in a Hansenula yeast expression system, followed by series chromatography purification. The mutation scheme of the coxsackievirus A16 virus-like particle in the present disclosure can significantly improve the immunogenicity of the antigen compared to the unmutated version, and has clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0092] Figure 1 FIG. 4 is a graph showing the SDS-PAGE detection results of the recombinant coxsackievirus A16 virus-like particle in Example 3 of the present disclosure;

[0093] Figure 2 FIG. 5 is a graph showing the Western-blot detection results of the recombinant coxsackievirus A16 virus-like particle in Example 3 of the present disclosure;

[0094] Figure 3 FIG. 6 is a graph showing the transmission electron microscope observation results of the recombinant coxsackievirus A16 virus-like particle in Example 4 of the present disclosure, wherein A-I are the transmission electron microscope results of the wild type (M0), M1, M2, M3, M4, M5, M9, M10, and M11, respectively;

[0095] Figure 4 FIG. 7 is a graph showing the binding activity of the recombinant coxsackievirus A16 virus-like particle to murine monoclonal antibodies in Example 4 of the present disclosure;

[0096] Figure 5Figures showing serum specific IgG antibody titers and neutralizing antibody titers for different protocols in Example 5 of the disclosure, wherein A is a graph of specific IgG antibody titers and B is a graph of antibody titers.

[0097] Sequence Listing.

[0098] DETAILED DESCRIPTION

[0099] DETAILED DESCRIPTION / DETAILED DESCRIPTION.

[0100] The present application discloses a mutant of coxsackievirus A16 and a virus-like particle thereof, and those skilled in the art can refer to the content herein to appropriately improve the process parameters for implementation. It needs to be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are all regarded as included in the present application, and the relevant personnel can obviously make changes or appropriate changes and combinations to the content described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0101] In the present disclosure, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art, unless otherwise specified. Unless otherwise explicitly stated, throughout the specification and claims, the term "comprise" or its variants such as "contain" or "include" will be understood to encompass the stated elements or components, without excluding other elements or components. The terms "a" and "an" include plural referents. The term "plurality" means two or more. The terms "such as", "for example", and the like are intended to indicate exemplary embodiments, and are not intended to limit the scope of the present disclosure.

[0102] In the present disclosure, when a range of values is provided, it should be understood that, unless the context clearly dictates otherwise, the endpoints of the range are included and each intermediate value and any other specified or intervening value or smaller range within the stated range is encompassed.

[0103] In the present disclosure, the term "about" generally means a variation of 0.5% to 10% above or below the stated value, for example, a variation of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the stated value.

[0104] In the present disclosure, “one implementation,” “one embodiment,” “some implementations,” “certain embodiments,” “related embodiments,” “certain embodiment,” “certain embodiments,” “additional embodiments,” or “further embodiments,” or “further implementations,” or “another embodiment,” “other embodiments,” means that at least one feature or characteristic described in relation to the embodiment is included in at least some implementations. Thus, the above phrases do not necessarily all refer to the same embodiment. In addition, particular features can be combined in any suitable manner in one or more implementations.

[0105] In the present disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Definitions of common terms in molecular biology can be found in Lewin’s GENES, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Publisher: Jones & Bartlett Learning. Definitions of common terms in biochemistry can be found in Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: W. H. Freeman. Definitions of common terms in cell biology can be found in Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Publisher: Garland Science. Definitions of common terms in genetics can be found in Genetics: Analysis of Genes and Genomes, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Publisher: Jones & Bartlett Learning.

[0106] Unless otherwise indicated, the experimental techniques in this text employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found, for example, in the following standard textbooks: Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook; and the like.

[0107] Definitions:

[0108] The term "Coxsackievirus A16" (CVA16) in the present disclosure belongs to the member of Enterovirus of Picornaviridae, which is a single-stranded positive RNA virus with 27-30 nm in diameter, no envelope, and icosahedral structure. The full-length of the genome is about 7400 bp, which is mainly composed of non-coding regions (Untranslated Regions, UTRs) at both ends and an open reading frame (ORF). The ORF mainly encodes a polyprotein of about 200 KDa, which is cleaved into precursor proteins P1, P2 and P3 by the hydrolytic protease produced by itself. P1 mainly encodes viral structural proteins VP0, VP1 and VP3, and VP0 can be further hydrolyzed into VP2 and VP4 during maturation. The four structural proteins VP1-VP4 form a structural unit, and sixty structural units are assembled into a virus capsid. VP1, VP2 and VP3 are located on the surface of the virus capsid, among which VP1 has an important cell receptor binding site and a specific neutralization site, and is also the main basis for serotyping of enteroviruses. VP4 is located inside the capsid and is connected with viral RNA. P2 and P3 mainly encode seven non-structural proteins: 2A, 2B, 2C, 3A, 3B, 3C and 3D, which are related to functions such as replication and transcription of viral RNA, cleavage of viral polyprotein, and assembly of viral particles. The UTRs at both ends of the RNA mainly contain the initiation signals for polypeptide translation and RNA synthesis. The spatial structure of the CVA16 virus capsid has high symmetry, containing 6 five-fold rotational axes (five-fold axes, or five-fold axes), 10 three-fold rotational axes (three-fold axes, or three-fold axes) and 15 two-fold rotational axes (two-fold axes, or two-fold axes). The five-fold axis is surrounded by VP1 from five adjacent structural units, and the interior of VP1 contains a hydrophobic pocket combined with a lipid molecule, which can stabilize the structure of the virus capsid, and a canyon is formed below the hydrophobic pocket, which is symmetrically distributed around the five-fold axis on the surface of the virus capsid. The three-fold axis is composed of VP2 and VP3 from adjacent structural units. The two-fold axis is formed by the interaction of VP2 from adjacent structural units, mainly by the face-to-face contact of the alpha-helices of the two adjacent VP2. In the vicinity of the two-fold axis, there is also a quasi-three-fold axis formed by the interaction of VP1, VP2 and VP3 from adjacent structural units. In the present disclosure, the mutant of Coxsackievirus A16 and the virus-like particle thereof refer to the mutant of the peptide chain formed by the structural proteins VP0, VP1 and VP3 of Coxsackievirus A16 and the virus-like particle assembled by the above proteins.

[0109] The term "wild type" in the present disclosure, which can also be referred to as "native" or "natural", refers to the absence of non-naturally occurring mutations. In some embodiments, exemplary examples of wild type coxsackievirus A16 structural proteins include the sequences shown in SEQ ID Nos. 1-3.

[0110] The term "mutation" in the present disclosure refers to a change in a sequence (e.g., a nucleotide or amino acid sequence) relative to the corresponding sequence (i.e., the non-mutated sequence) of a native, wild type, standard, or reference form. Amino acid mutations generally include substitutions, deletions or insertions of amino acid residues. Mutations can be artificially made or naturally occurring. In some embodiments, the mutation refers to a substitution of an amino acid residue in a peptide chain. These mutations are considered to be key to achieving the objectives of the present disclosure.

[0111] The term "site" in the present disclosure refers to a position within a peptide, polypeptide or polynucleotide that can be modified, altered or derivatized within the polypeptide or polynucleotide-based molecule. Typically, sites are arranged or named in numerical order.

[0112] The terms "peptide", "polypeptide" and "protein" in the present disclosure can be used interchangeably and generally refer to peptides and proteins that are covalently linked by peptide bonds of amino acids. The term "protein" encompasses purified natural products, or products that can be produced in part or in whole using recombinant or synthetic techniques. The terms "peptide" and "protein" can refer to aggregates of proteins, such as dimers or other multimers, fusion proteins, protein variants or derivatives thereof. The term also includes modifications of proteins, such as proteins modified by glycosylation, acetylation, phosphorylation, pegylation, ubiquitination, etc. Proteins can include amino acids that are not encoded by nucleic acid codons. Proteins can have amino acid sequences of sufficient length to produce higher levels of tertiary and / or quaternary structure.

[0113] The term "virus-like particle (VLP)" or "pseudovirus" in the present disclosure refers to a multi-protein structure composed of corresponding native viral structural proteins, but lacking all or part of the viral genome, particularly the replication and infectious components of the viral genome, and thus is not replicative and infectious. The multi-protein structure highly mimics its corresponding native viral particle in morphology and size, and can form spontaneously upon recombinant expression of the viral structural proteins. VLPs are highly structured protein particles that are self-assembled from one or more structural proteins of a virus. They are between 20-150 nanometers in diameter and maintain the native conformation of the viral antigenic proteins. VLPs can self-assemble into icosahedral, rod-shaped or spherical structures, etc. VLPs can be artificially obtained by recombinant expression in prokaryotic cell lines, yeast cell systems, plant cells, animal cells, etc.

[0114] The term "identity" or "sequence identity" in the present disclosure refers to the complete identity of nucleotide or amino acid residues at the same position of two sequences. It is a fundamental and important concept in bioinformatics, and is usually used to compare the degree of similarity between two nucleic acid sequences or protein sequences. The value of sequence identity is usually expressed as a percentage, which reflects the degree of consistency between sequences.

[0115] In some embodiments of the present disclosure, "having 90% or more identity and having the same or similar biological function" generally means that the actual sequence and the sequence described in the present disclosure can be at least 90%, at least 91%, at least 92, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, and the different parts can be due to artificially or non-artificially introduced substitution / insertion / deletion mutations, for example, substitution of conservative amino acids. Artificially introduced mutations can be based on specific purposes, such as easier protein expression, etc. However, the introduction of these different parts is not enough to change the biological function of the original protein represented by the amino acid sequence, for example, immunogenicity. In some embodiments, some conservative amino acids are replaced by point mutations to obtain "conservative amino acid substitution variants". Changes therein result in some amino acids being replaced by other amino acids that are similar in chemical properties and / or functions. Conservative substitution tables that provide chemically and / or functionally similar amino acids are well known in the art. Typical examples of mutually conservative substitutions, for example, (1) alanine (A), glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); (6) phenylalanine (F), tyrosine (Y), tryptophan (W); (7) serine (S), threonine (T); (8) cysteine (C), methionine (M).

[0116] The term "isolated" in the present disclosure refers to a substance or entity that is separated from its natural environment or the environment in which it existed before separation, and is separated from other components. The separation ratio can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%. Isolated substances can have different levels of purity relative to the substances before they are separated.

[0117] The term "vector" in the present disclosure is a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector are expressed in the host cell. The vector is well known to those skilled in the art, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as lambda phage or M13 phage, and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site.

[0118] The term "host cell" in the present disclosure is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

[0119] The term "protein delivery vehicle" in the present disclosure refers to a certain protein that can act as a carrier for an epitope peptide, i.e., it can insert an epitope peptide at a specific position (e.g., inside the protein, N-terminal or C-terminal) so that the epitope peptide can be presented and thus recognized by an antibody or the immune system.

[0120] The term "immunogenicity" in the present disclosure refers to the ability of a substance to elicit, provoke, stimulate or induce an immune response against a specific antigen in an animal, in the presence or absence of an adjuvant.

[0121] The term "adjuvant" in the present disclosure refers to a non-specific immune enhancer that, when mixed with an antigen, can enhance the immune response of the body to the antigen or change the type of immune response, including but not limited to aluminum adjuvants such as aluminum hydroxide, Freund's complete adjuvant, Freund's incomplete adjuvant, etc.

[0122] The term "pharmaceutically acceptable carrier" in the present disclosure can be any pharmaceutically acceptable additive, such as physiological saline, cell culture medium, glucose, water for injection, glycerol, amino acids and their combinations, stabilizers, surfactants, preservatives, isotonic agents, etc.

[0123] Optimization of codons:

[0124] Codon optimization is an advanced technique that enhances the expression level of proteins in organisms by improving the translation efficiency of target genes. In organisms, due to the existence of codon degeneracy, multiple codons can encode the same amino acid, which leads to the possibility of multiple mRNA sequences for a specific amino acid sequence. However, different organisms or cells have their own bias in choosing these synonymous codons, which is called codon bias. Therefore, when a heterologous gene is expressed in a host cell, its codons may not match the optimal codon usage frequency of the host cell, affecting the expression level of the protein. Through codon optimization, synonymous codons that better match the host cell bias can be selected, thereby improving the translation efficiency of the protein.

[0125] Various methods can be used to optimize codons. For example, the steps of some methods are as follows: (1) analyze the codon usage frequency of the host cell: first, the codon bias of the host cell needs to be understood, that is, which codons have a high frequency of use in the host cell. This can be obtained by consulting relevant literature or databases. (2) design optimization scheme: according to the codon usage frequency of the host cell, design the optimization scheme. This includes selecting which synonymous codons to replace in the heterologous gene and how to adjust the GC content, etc. (3) implement optimization: use gene synthesis or site-directed mutagenesis and other techniques to apply the optimization scheme to the heterologous gene. (4) verify the optimization effect: verify whether the expression level of the optimized gene in the host cell has been improved through experiments. This can be evaluated by measuring protein concentration, enzyme activity, etc.

[0126] Embodiment:

[0127] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below in combination with specific embodiments.

[0128] Example 1: Analysis of conformational transition of coxsackievirus A16 virus particles and design of scheme

[0129] The conformational motion patterns of the uncoating process of Coxsackievirus A16 were simulated by using the package developed by computational structural biology. Then the main residue sites controlling the uncoating motion of Coxsackievirus A16 were predicted. The calculation results showed that these key residue sites were mainly located in: (1) the canyon region of the virus capsid. Experimental studies found that the binding of the virus capsid canyon region to the cell receptor led to the collapse of the VP1 hydrophobic pocket and the extrusion of the lipid molecule ligand at the hydrophobic pocket, triggering the entire virus capsid uncoating motion process. The key sites in this region identified in this study may mediate the coupling between the collapse of the hydrophobic pocket and the overall expansion motion. (2) the channel region at the dihedral axis. These key residues control the opening of the channel and the release of viral RNA; structural biology studies have shown that the opening of the dihedral axis channel is a distinct feature of the expansion of the Coxsackievirus A16 virus capsid, and is the release channel of viral RNA from the inside of the capsid to the host cell. (3) the channel region at the quasi-trihedral axis. These key residues control the opening of the quasi-trihedral axis channel. Structural biology studies have shown that the uncoating motion of the virus capsid will lead to the rearrangement of the conformation at the quasi-trihedral axis, and the N-terminus of VP4 and VP1 extends from the channel to the outside of the capsid. These key residues play an important role in the formation of the viral RNA release channel. According to the above results, the predicted key residue sites were mutated, and the mutation design is shown in Table 1.

[0130] Table 1 Mutation design

[0131]

[0132] Example 2: Construction and expression of Coxsackievirus A16 virus-like particle expression plasmid

[0133] The nucleotide sequence encoding the Coxsackievirus A16 virus-like particle was codon optimized according to the codon bias and tRNA abundance of Hansenula polymorpha. The optimized nucleotide sequence is shown as SEQ ID NO. 4-69. After constructing the yeast expression plasmid, it was transformed into the Hansenula polymorpha deficient strain to construct a recombinant yeast strain. Positive high-expression recombinant Coxsackievirus A16 virus-like particle yeast strains were obtained by ELISA screening.

[0134] Example 3: Purification and identification of Coxsackievirus A16 virus-like particles

[0135] The positive yeast strain obtained was cultured and the supernatant was harvested after disruption. After series chromatography purification, recombinant Coxsackievirus A16 virus-like particles were obtained. The obtained virus-like particles were identified, and the SDS-PAGE detection results are shown as follows: Figure 1As shown, 4 band positions correspond to the theoretical molecular weight of each target protein, band 1 is VP0, the molecular weight is about 35-40 KDa; band 2 is VP1, the molecular weight is about 30-35 KDa; band 3 is VP1 degradation band, the molecular weight is about 30 KDa; band 4 is VP3, the molecular weight is about 25-30 KDa. After the purified recombinant coxsackievirus A16 virus-like particles were subjected to SDS-PAGE electrophoresis and then electrotransferred to a PVDF membrane, Western-blot identification was performed using coxsackievirus A16 rabbit polyclonal antibody (dilution: 1000 times), and the results are shown in Figure 2 As shown, bands appeared at the expected positions.

[0136] Example 4: Detection of physicochemical properties and biological activity of coxsackievirus A16 virus-like particles

[0137] The obtained virus-like particles were subjected to physicochemical property analysis, and mutant M3 was taken as an example for illustration. The purified recombinant coxsackievirus A16 virus-like particles were subjected to molecular exclusion chromatography analysis using a TSK-GEL G3000 chromatographic column, and the protein purity was greater than 85%, as shown in Table 2. Transmission electron microscopy was used to observe the recombinant coxsackievirus A16 virus-like particles, and it was found that the particles were uniform in size and good in morphology, with a diameter of about 30-40 nm, as shown in Figure 3 .

[0138] Table 2 SEC-HPLC purity results of recombinant coxsackievirus A16 virus-like particles

[0139]

[0140] The purified recombinant coxsackievirus A16 virus-like particles were subjected to 2-fold serial horizontal dilution with a coating solution starting from 10 µg / ml for 12 gradients, 100 µl / well, coated onto a 96-well enzyme-labeled plate at 4°C for 8-12 h, with a blank well as a negative control. After washing the plate with PBST solution, a blocking solution was added, and incubated at 37°C for 2 h. After washing the plate with PBST solution, coxsackievirus A16 virus mouse monoclonal antibody diluted to 1 µg / ml was added, 100 µl / well, and incubated at 37°C for 1 h. After washing the plate with PBST solution, diluted horseradish peroxidase-labeled goat anti-mouse IgG antibody was added, 100 µl / well, and incubated at 37°C for 1 h. After washing the plate with PBST solution, color developing solution A and B were added in sequence, and color developed at room temperature for 5-10 min, and termination solution C was added. The cut-off value was determined by reading the values at double wavelengths (absorbance 450 nm and 630 nm) on an enzyme-labeled instrument, and the protein concentration-absorbance value curve was drawn. The results of antibody binding activity are shown in Figure 4 As shown, the results showed that the recombinant coxsackievirus A16 virus-like particles could bind to coxsackievirus A16 virus mouse monoclonal antibody, and had good biological activity.

[0141] Example 5: Evaluation of immunological effects of recombinant coxsackievirus A16 virus-like particles with different mutation schemes in mice in vivo

[0142] According to the animal experiment scheme shown in Table 3, the recombinant coxsackievirus A16 virus-like particles with different schemes were mixed and adsorbed with aluminum hydroxide, and then 2 μg / dose / each / 0.5 ml was intraperitoneally injected into BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF level, female, 6-8 weeks old), 8 mice per group. The mice were immunized with the recombinant coxsackievirus A16 virus-like particles with different schemes for 1 time at 0w, 1w and 3w, respectively, and blood was collected at 4w to separate serum. The specific IgG antibody and neutralizing antibody levels in the serum were detected by ELISA method and virus micro-neutralization test method, respectively, and the detection results are shown in Tables 4 and Figure 5

[0143] Table 3 Animal experiment scheme for immunological effects of different mutations

[0144]

[0145] Table 4 Geometric mean titers of specific IgG antibody and neutralizing antibody in serum of mice with different mutations

[0146]

[0147] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A mutant of Coxsackievirus A16 (CVA16), characterized in that, The amino acid sequences of the mutants VP0, VP3, and VP1 are as follows: (1) Shown as SEQ ID No:4, SEQ ID No:5, SEQ ID No:6; (2) Shown as SEQ ID No:7, SEQ ID No:8, SEQ ID No:9; (3) SEQ ID No:10, SEQ ID No:11, SEQ ID No:12; (4) SEQ ID No:13, SEQ ID No:14, SEQ ID No:15; (5) SEQ ID No:16, SEQ ID No:17, SEQ ID No:18; (6) SEQ ID No:28, SEQ ID No:29, SEQ ID No:30; (7) As shown in SEQ ID No:31, SEQ ID No:32, and SEQ ID No:33; or (8) As shown in SEQ ID No:34, SEQ ID No:35, and SEQ ID No:

36.

2. A virus-like particle, characterized in that, The virus-like particles are assembled from the mutant as described in claim 1.

3. An isolated polynucleotide, characterized in that, The isolated polynucleotide encodes the mutant as described in claim 1.

4. The isolated polynucleotide according to claim 3, characterized in that, The isolated polynucleotides were codon-optimized.

5. The isolated polynucleotide according to claim 3 or 4, characterized in that, The sequences encoding VP0, VP3, and VP1, which are separated from each other, are as follows: (1) Shown as SEQ ID No:73, SEQ ID No:74, SEQ ID No:75; (2) SEQ ID No:76, SEQ ID No:77, SEQ ID No:78; (3) Shown as SEQ ID No:79, SEQ ID No:80, SEQ ID No:81; (4) Shown as SEQ ID No:82, SEQ ID No:83, SEQ ID No:84; (5) SEQ ID No:85, SEQ ID No:86, SEQ ID No:87; (6) SEQ ID No:97, SEQ ID No:98, SEQ ID No:99; (7) As shown in SEQ ID No:100, SEQ ID No:101, and SEQ ID No:102; or (8) As shown in SEQ ID No:103, SEQ ID No:104, and SEQ ID No:

105.

6. A carrier, characterized in that, The vector comprises the isolated polynucleotide as described in claim 3, 4 or 5, or the mutant as described in claim 1.

7. The carrier according to claim 6, characterized in that, The vector is a protein expression vector, a gene delivery vector, or a protein delivery vector.

8. A host cell, characterized in that, The host cell contains the mutant as described in claim 1, the isolated polynucleotide as described in claim 3, 4 or 5, or the vector as described in claim 6 or 7.

9. A recombinant protein vaccine, characterized in that, The recombinant protein vaccine comprises virus-like particles as described in claim 2, and optionally includes an adjuvant.

10. The recombinant protein vaccine according to claim 9, characterized in that, The adjuvant is selected from at least one of aluminum adjuvants, oil-water emulsion adjuvants, AS01 adjuvant system, AS03 adjuvant system, AS04 adjuvant system, MF59, CpG, immunostimulatory substances, or immunomodulatory substances.

11. A nucleic acid vaccine, characterized in that, The nucleic acid vaccine comprises the polynucleotides as described in claim 3, 4 or 5.

12. A liposome complex, characterized in that, The liposome complex comprises the polynucleotide as described in claim 3, 4 or 5.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the mutant of claim 1, the virus-like particle of claim 2, the isolated polynucleotide of claim 3, 4 or 5, the vector of claim 6 or 7, the host cell of claim 8, the vaccine of any one of claims 9 to 11, or the liposome complex of claim 12, and pharmaceutically acceptable carriers, delivery media, excipients, stabilizers, diluents or combinations thereof.

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