Mutants of enterovirus 71 and virus-like particles thereof

Abstract

The disclosure provides a mutant of enterovirus 71 and a virus-like particle thereof, and relates to the field of biological medicine.The virus-like particle of enterovirus 71 in the disclosure is obtained by modifying the structural genes VP0, VP1 and VP3 of the virus capsid protein by using computational structural biology, and completing self-assembly in vivo by a Hansenula polymorpha expression system.The mutant of enterovirus 71 and the virus-like particle thereof in the disclosure can significantly improve immunogenicity compared with the unmodified one, and have a high clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biological medicine, in particular, the present disclosure relates to a mutant of enterovirus 71 and a virus-like particle thereof. BACKGROUND

[0002] Hand-foot-mouth disease (HFMD) is a common infectious disease in children, mainly caused by enterovirus A, enterovirus D, coxsackievirus A and coxsackievirus B, etc. EV71 (Enterovirus 71, EV71) is one of the main pathogens leading to high morbidity and mortality in infants.

[0003] There is no specific treatment for HFMD caused by EV71 infection, and vaccination is an economical and effective measure to control EV71 infection. At present, three kinds of EV71 inactivated vaccines have been marketed in China. With the marketing and wide application of EV71 inactivated vaccine, the prevention and control of HFMD caused by EV71 has achieved remarkable results, and the related pathogen spectrum structure has also changed to some extent, with the alternation of dominant strains of EV71, CVA16, CVA10, CVA6, etc. Therefore, the development of a multivalent vaccine targeting the main HFMD-related viruses is the trend of the times, and EV71 is an indispensable component of the multivalent HFMD vaccine. VLP vaccine is a relatively mature vaccine development technology route, which has the advantages of high safety, similar antigen form to natural virus, strong immunogenicity, large yield, easy enrichment and purification, etc., and has good development prospects. At present, most EV71 VLP vaccines are in the preclinical research stage. The recombinantly expressed EV71 VLP spontaneously undergoes a process of conformational transition from uncoating to swelling, which destroys the neutralizing epitopes on the surface, and makes the VLP change from the non-swollen conformation with strong immunogenicity to the swollen post-conformation with significantly reduced immunogenicity, which is one of the main technical bottlenecks limiting the development of VLP vaccine. Therefore, maintaining the non-swollen conformation of EV71 VLP becomes an important means to solve the above problems. SUMMARY

[0004] Technical problems solved:

[0005] An aspect of the present disclosure is to provide a new mutant of enterovirus 71 and a virus-like particle thereof to solve the problem of low immunogenicity caused by unstable virus-like particle structure of enterovirus 71.

[0006] Technical solution:

[0007] The mutant of enterovirus 71 (EV71) has at least one amino acid mutation site in the following positions compared with the amino acid sequence of the structural protein of the wild-type enterovirus 71, and the mutation is substitution of amino acid residues:

[0008] (1) at least one of the 155th amino acid position, the 158th amino acid position, the 159th amino acid position, the 149th amino acid position, the 122th amino acid position, the 84th amino acid position, the 114th amino acid position, the 115th amino acid position, the 265th amino acid position in the VP0 sequence; or

[0009] (2) at least one of the 236th amino acid position, the 186th amino acid position, the 180th amino acid position, the 177th amino acid position, the 149th amino acid position, the 110th amino acid position, the 229th amino acid position, the 232th amino acid position in the VP3 sequence; or

[0010] (3) at least one of the 117th amino acid position, the 163th amino acid position, the 247th amino acid position, the 284th amino acid position, the 293th amino acid position, the 200th amino acid position, the 222th amino acid position, the 225th amino acid position, the 45th amino acid position, the 136th amino acid position, the 154th amino acid position, the 198th amino acid position, the 234th amino acid position in the VP1 sequence.

[0011] In some embodiments, the above-mentioned amino acid mutation positions can be:

[0012] (1) the 155th, the 158th and the 159th amino acid positions in the VP0 sequence; or

[0013] (2) the 158th amino acid position in the VP0 sequence; or

[0014] (3) the 149th in the VP0 sequence, the 236th in the VP3 sequence, the 117th, the 163th and the 247th amino acid positions in the VP1 sequence; or

[0015] (4) the 155th and the 159th amino acid positions in the VP0 sequence; or

[0016] (5) the 159th amino acid position in the VP0 sequence; or

[0017] (6) the 122th, the 155th and the 159th amino acid positions in the VP0 sequence; or

[0018] (7) the 84th, the 122th and the 159th amino acid positions in the VP0 sequence; or

[0019] (8) the 284th and the 293th amino acid positions in the VP1 sequence; or

[0020] (9) amino acid positions 159 in the VP0 sequence and 186 in the VP3 sequence; or

[0021] (10) amino acid positions 159 in the VP0 sequence and 180 in the VP3 sequence; or

[0022] (11) amino acid positions 159 in the VP0 sequence, 177 in the VP3 sequence and 200 in the VP1 sequence; or

[0023] (12) amino acid positions 159 in the VP0 sequence and 149 in the VP3 sequence; or

[0024] (13) amino acid positions 114, 115 and 159 in the VP0 sequence; or

[0025] (14) amino acid positions 159 in the VP0 sequence and 110 in the VP3 sequence; or

[0026] (15) amino acid positions 159 in the VP0 sequence, 229 and 232 in the VP3 sequence; or

[0027] (16) amino acid positions 159 in the VP0 sequence and 222 in the VP1 sequence; or

[0028] (17) amino acid positions 159 in the VP0 sequence and 225 in the VP1 sequence; or

[0029] (18) amino acid positions 159 and 265 in the VP0 sequence; or

[0030] (19) amino acid positions 180 in the VP3 sequence, 45 and 136 in the VP1 sequence; or

[0031] (20) amino acid positions 180 in the VP3 sequence, 45, 136, 154, 198 and 234 in the VP1 sequence; or

[0032] (21) amino acid positions 155, 159 in the VP0 sequence, 284 and 293 in the VP1 sequence.

[0033] In some more specific embodiments, the above-mentioned amino acid mutation positions can be:

[0034] (1) the amino acid at position 155 in the VP0 sequence is substituted with L, the amino acid at position 158 is substituted with F and the amino acid at position 159 is substituted with Y; or

[0035] (2) the amino acid at position 158 in the VP0 sequence is substituted with W; or

[0036] (3) the amino acid at position 149 in the VP0 sequence is substituted with L, the amino acid at position 236 in the VP3 sequence is substituted with M, the amino acid at position 117 in the VP1 sequence is substituted with C, the amino acid at position 163 is substituted with I, and the amino acid at position 247 is substituted with A; or

[0037] (4) the amino acid at position 155 in the VP0 sequence is substituted with L and the amino acid at position 159 is substituted with Y; or

[0038] (5) the amino acid at position 159 in the VP0 sequence is substituted with Y; or

[0039] (6) the amino acid at position 122 in the VP0 sequence is substituted with W, the amino acid at position 155 is substituted with L, and the amino acid at position 159 is substituted with Y; or

[0040] (7) the amino acid at position 84 in the VP0 sequence is substituted with W, the amino acid at position 122 is substituted with W, and the amino acid at position 159 is substituted with Y; or

[0041] (8) the amino acid at position 284 in the VP1 sequence is substituted with S and the amino acid at position 293 is substituted with T; or

[0042] (9) the amino acid at position 155 in the VP0 sequence is substituted with M and the amino acid at position 159 is substituted with M; or

[0043] (10) the amino acid at position 155 in the VP0 sequence is substituted with M, the amino acid at position 158 is substituted with L, and the amino acid at position 159 is substituted with M; or

[0044] (11) the amino acid at position 159 in the VP0 sequence is substituted with M and the amino acid at position 186 in the VP3 sequence is substituted with W; or

[0045] (12) the amino acid at position 159 in the VP0 sequence is substituted with M and the amino acid at position 180 in the VP3 sequence is substituted with M; or

[0046] (13) the amino acid at position 159 in the VP0 sequence is substituted with M, the amino acid at position 177 in the VP3 sequence is substituted with Y, and the amino acid at position 200 in the VP1 sequence is substituted with I; or

[0047] (14) the amino acid at position 159 in the VP0 sequence is substituted with M and the amino acid at position 149 in the VP3 sequence is substituted with M; or

[0048] (15) the amino acid at position 114 in the VP0 sequence is substituted with I, the amino acid at position 115 is substituted with V, and the amino acid at position 159 is substituted with M; or

[0049] (16) the amino acid at position 159 in the VP0 sequence is substituted with M and the amino acid at position 110 in the VP3 sequence is substituted with M; or

[0050] (17) the 159th amino acid in the VP0 sequence is substituted with M, the 229th amino acid in the VP3 sequence is substituted with M, and the 232nd amino acid in the VP3 sequence is substituted with M; or

[0051] (18) the 159th amino acid in the VP0 sequence is substituted with M, and the 222nd amino acid in the VP1 sequence is substituted with M; or

[0052] (19) the 159th amino acid in the VP0 sequence is substituted with M, and the 225th amino acid in the VP1 sequence is substituted with F; or

[0053] (20) the 159th amino acid in the VP0 sequence is substituted with M, and the 265th amino acid in the VP0 sequence is substituted with M; or

[0054] (21) the 180th amino acid in the VP3 sequence is substituted with L, the 45th amino acid in the VP1 sequence is substituted with I, and the 136th amino acid in the VP1 sequence is substituted with L; or

[0055] (22) the 180th amino acid in the VP3 sequence is substituted with L, the 45th amino acid in the VP1 sequence is substituted with I, the 136th amino acid in the VP1 sequence is substituted with L, the 154th amino acid in the VP1 sequence is substituted with I, the 198th amino acid in the VP1 sequence is substituted with I, and the 234th amino acid in the VP1 sequence is substituted with L; or

[0056] (23) the 155th amino acid in the VP0 sequence is substituted with M, the 159th amino acid in the VP0 sequence is substituted with M, the 284th amino acid in the VP1 sequence is substituted with S, and the 293rd amino acid in the VP1 sequence is substituted with T; or

[0057] (24) the 158th amino acid in the VP0 sequence is substituted with C; or

[0058] (25) the 158th amino acid in the VP0 sequence is substituted with F.

[0059] In some more specific embodiments, the amino acid sequence of the VP0, VP1 or VP3 of the mutant described above is as shown in SEQ ID Nos: 4-78, or a sequence having 90% and above identity thereto and having the same or similar biological function.

[0060] Another aspect of the present disclosure is to provide a virus-like particle assembled from the mutant described above.

[0061] Another aspect of the present disclosure is to provide an isolated polynucleotide encoding the mutant described above, or the VP0, VP1 or VP3 of the mutant.

[0062] In some embodiments, the isolated polynucleotide described above can be codon-optimized.

[0063] In some embodiments, the sequence of the isolated polynucleotide is set forth in SEQ ID Nos: 82-156.

[0064] Another aspect of the present disclosure is to provide a vector comprising the mutant, the isolated polynucleotide, or the vector.

[0065] In some embodiments, the vector can be a protein expression vector, a gene delivery vector, or a protein delivery vector.

[0066] Another aspect of the present disclosure is to provide a host cell comprising the mutant, the isolated polynucleotide, or the vector.

[0067] In some embodiments, the host cell 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.

[0068] Another aspect of the present disclosure is to provide a recombinant protein vaccine comprising the virus-like particle, and optionally, an adjuvant.

[0069] In some embodiments, the adjuvant can be at least one selected from an aluminum adjuvant, an oil-in-water emulsion adjuvant, an AS01 adjuvant system, an AS03 adjuvant system, an AS04 adjuvant system, MF59, CpG, an immunostimulatory substance, or an immunomodulatory substance.

[0070] Another aspect of the present disclosure is to provide a nucleic acid vaccine comprising the polynucleotide.

[0071] Another aspect of the present disclosure is to provide a liposome complex comprising the polynucleotide.

[0072] Another aspect of the present disclosure is to provide a pharmaceutical composition comprising the mutant, the virus-like particle, the isolated polynucleotide, the vector, the host cell, the vaccine, or the liposome complex, and a pharmaceutically acceptable carrier, a delivery medium, an excipient, a stabilizer, a diluent, or a combination thereof. The pharmaceutical composition can be a vaccine composition.

[0073] Another aspect of the present disclosure is to provide a method of eliciting an immune response against enterovirus 71 or treating enterovirus 71 infection in a subject, the method comprising administering to the subject an effective amount of the recombinant protein vaccine, the nucleic acid vaccine, the liposome complex, or the pharmaceutical composition.

[0074] Beneficial effects:

[0075] The enterovirus 71 virus-like particles in the present disclosure are obtained by using computational structural biology to modify the structural genes VP0, VP1, VP3 of the virus capsid protein, and completing self-assembly in vivo through a Hansenula yeast expression system. The enterovirus 71 mutant and the virus-like particles thereof in the present disclosure can significantly improve immunogenicity compared with those before modification, and have a high clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 Figure 4 is a SDS-PAGE detection result diagram of the recombinant enterovirus 71 virus-like particles in Example 3 of the present disclosure, wherein M26630, M26619 represent Marker, A represents T24, Q represents T25, and RF represents negative control-irrelevant protein;

[0077] Figure 2 Figure 5 is a Western-blot detection result diagram of the recombinant enterovirus 71 virus-like particles in Example 3 of the present disclosure, wherein A is a detection result using Anti EV71-VP0 antibody, B is a detection result using Anti EV71-VP1, and C is a detection result using Anti EV71-VP3, wherein M26630, M26619 represent Marker, A represents T24, Q represents T25, and RF represents negative control-irrelevant protein;

[0078] Figure 3 Figure 6 is a transmission electron microscope observation result diagram of the recombinant enterovirus 71 virus-like particles in Example 4 of the present disclosure, wherein A-R respectively represent wild type (T0), T24, T25, T1, T2, T4, T5, T7, T8, T10, T11, T12, T13, T14, T15, T16, T20, and T23;

[0079] Figure 4 、 Figure 5 、 Figure 6 Figure 7 is a result diagram of the binding activity of the recombinant enterovirus 71 virus-like particles in Example 4 of the present disclosure with different monoclonal antibodies, wherein Figure 4 Figure 7A is a result of enterovirus 71 virus D6 mouse monoclonal antibody, Figure 5 Figure 7B is a result of enterovirus 71 virus A9 mouse monoclonal antibody, Figure 6 Figure 7C is a result of enterovirus 71 virus 10F0 mouse monoclonal antibody, wherein A represents T24 and Q represents T25;

[0080] Figures 7 to 18 Figure 8 is a result diagram of the specific IgG antibody titer and neutralizing antibody titer of the serum of the mice immunized by different schemes in Example 5 of the present disclosure.

[0081] SEQUENCE DESCRIPTION

[0082] DETAILED DESCRIPTION DETAILED DESCRIPTION / DETAILED DESCRIPTION.

[0083] The present application discloses a mutant of enterovirus 71 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 replacements 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.

[0084] 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 the 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.

[0085] In the present disclosure, when a range of values is provided, it should be understood that unless the context clearly indicates 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.

[0086] In the present disclosure, the term "about" generally means a variation of 0.5-10% above or below the specified numerical value, for example, a variation within the range 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 specified numerical value.

[0087] 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 of the disclosure. Thus, the above phrases are not necessarily all referring to the same embodiment in various places in the present disclosure. In addition, a particular feature or characteristic can be combined with any suitable implementation in one or more embodiments.

[0088] In the present disclosure, unless otherwise stated, the 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.

[0089] Unless otherwise indicated, the experimental techniques utilized herein are according to conventional methods of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, well known and within the skill of the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Cellular Biology (TCP Press, 1999); Gait, ed., Oligonucleotide Synthesis: Methods and Applications (1984); and Wu, ed., Method in Enzymology: Molecular Biology of Incision Repair (1996).

[0090] Definitions:

[0091] The term "Enterovirus 71" (EV71) in the present disclosure belongs to the genus Enterovirus of the family Picornaviridae, which is a non-enveloped virus. The viral nucleic acid is single-stranded positive-sense RNA, with a genome length of about 7.4 kb, containing an open reading frame (ORF) and two highly structured non-coding regions (Untranslated Regions, UTRs) at the ends, i.e., 5' UTR and 3' UTR. The ORF can be divided into three regions: P1, P2, and P3. Among them, P1 encodes four viral structural proteins VP1~VP4, with molecular weights of 32 kD, 28 kD, 27 kD, and 8 kD, respectively, which are enterovirus capsid proteins; P2 and P3 encode seven non-structural proteins, P2 proteolysis forms viral specific proteases 2A and 2BC proteins, and 2BC is further cleaved into 2B and 2C; P3 is initially cleaved into 3AB and 3CD, and further proteolysis forms 3A, 3B, 3C, and 3D proteins. The seven non-structural proteins are related to functions such as viral RNA replication, transcription, and viral particle assembly, among which the 3C protein has the activity of serine protease and cysteine protease, which can catalyze the cleavage of precursor proteins and form mature structural proteins and non-structural proteins.

[0092] 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 Enterovirus 71 structural proteins are the sequences shown in SEQ ID No. 1-3.

[0093] The term "mutation" in the present disclosure refers to a change in a sequence (such as 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. An amino acid mutation generally includes substitution, deletion, or insertion of an amino acid residue. The mutation can be artificially manufactured or naturally formed. In some embodiments, the mutation refers to the substitution of an amino acid residue in a peptide chain. These mutations are considered to be key to achieving the purposes of the present disclosure.

[0094] 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 molecule based on the polypeptide or polynucleotide. Typically, sites are arranged or named in numerical order.

[0095] 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 partially or entirely 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, and the like. 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.

[0096] The term "virus-like particle (VLP)" or "pseudovirus" in the present disclosure refers to a multi-protein structure composed of corresponding natural 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 natural virus particle in morphology and size, and can form spontaneously after recombinant expression of the structural proteins of the virus. 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.

[0097] 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 in two sequences. It is a fundamental and important concept in bioinformatics, and is often 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.

[0098] In some embodiments of the disclosure, "having 90% or more identity and having the same or similar biological function" generally means that the actual sequence 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 to the sequence described in the disclosure, 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, for example, easier expression of proteins, etc. But 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).

[0099] 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 isolation, 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 isolated.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] Optimization of codons:

[0107] 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.

[0108] 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 the codons in the heterologous gene, and how to adjust the GC content, etc. (3) implement optimization: use gene synthesis or site-directed mutagenesis and other technologies 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 is improved through experiments. This can be evaluated by measuring protein concentration, enzyme activity, etc.

[0109] Embodiment:

[0110] 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.

[0111] Example 1: Enterovirus 71 virus particle scheme design.

[0112] EV71 in the whole replication cycle, the virus particle structure will be different transformation. From the immunological point of view, the mature virus particles have strong immunogenicity, can induce high titer of neutralizing antibody, however, the swollen A particles and empty capsid virus particles induced neutralizing antibody levels decreased significantly. Similarly, the phenomenon of recombinant VLP also occurs, so the stable EV71 VLP structure in the immune dominant state is the purpose of this design. Using the method of structural biology on EV71 virus subunit structure for saturation mutation scanning, calculate the influence of the mutation on the stability of the overall structure, screening the key mutation amino acid which enhances the stability, these sites are mainly located in the RNA release channel region of the secondary axis and quasi three axis region. Part of the key amino acid sites are obtained by consensus sequence alignment, which exist in the non-interface region. By mutating the key amino acid sites to enhance the hydrophobic interaction of the intermolecular binding interface, further enhance the stability of the capsid structure, the design of the 25 kinds of EV71 virus VLP mutation scheme as shown in Table 1.

[0113] Table 1 Mutation design scheme

[0114]

[0115] Example 2: Construction and expression of enterovirus 71 virus-like particle expression plasmid.

[0116] According to the codon bias and tRNA abundance of Hansenula polymorpha, the nucleotide sequence encoding the enterovirus 71 virus-like particle was optimized. The optimized nucleotide sequence is shown as SEQ ID No: 82-156. After constructing the yeast expression plasmid, it was transformed into the Hansenula polymorpha deficient strain to construct a recombinant yeast strain. The positive high expression of the recombinant enterovirus 71 virus-like particle was screened by ELISA.

[0117] Example 3: Purification and identification of enterovirus 71 virus-like particles.

[0118] The positive yeast strain was cultured and the supernatant was harvested after disruption. After series chromatography purification, the recombinant enterovirus 71 virus-like particle was obtained. The obtained virus-like particle was identified. The SDS-PAGE detection result is shown in Figure 1 The molecular weight of band 1 is about 35-40 KDa, band 2 is VP1 with a molecular weight of about 30-35 KDa, and band 3 is VP3 with a molecular weight of about 25-30 KDa. After SDS-PAGE electrophoresis of the purified recombinant enterovirus 71 virus-like particle, the PVDF membrane was electroblotted, and the enterovirus 71 VP0 / VP1 / VP3 rabbit polyclonal antibody (dilution: 1000 times) was used for Western-blot identification. The results are shown in Figure 2As shown, the bands appeared at the expected positions.

[0119] Example 4: Detection of physicochemical properties and biological activity of the recombinant enterovirus 71 virus-like particles.

[0120] The obtained virus-like particles were subjected to physicochemical property analysis. The purified recombinant enterovirus 71 virus-like particles were subjected to molecular exclusion chromatography analysis using a TSK-GEL G3000 chromatographic column, and the results are shown in Table 2. The recombinant enterovirus 71 virus-like particles were observed using transmission electron microscopy, and the particles were uniform in size and good in morphology, with a diameter of about 30-40 nm, and the results are shown in Figure 3 Table 2. SEC-HPLC purity results of recombinant enterovirus 71 virus-like particles

[0121] Table 2. SEC-HPLC purity results of recombinant enterovirus 71 virus-like particles

[0122]

[0123] Table 3. Dynamic light scattering results of recombinant enterovirus 71 virus-like particles

[0124]

[0125] The purified recombinant enterovirus 71 virus-like particles were subjected to 2-fold serial transverse dilution in 12 gradients starting from 10 µg / ml using the coating solution, 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, blocking solution was added, and incubated at 37°C for 2 h; after washing the plate with PBST solution, enterovirus 71 virus D6 / A9 / 10F0 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, goat anti-mouse IgG antibody labeled with horseradish peroxidase 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 min, and stop solution C was added; the double-wavelength (OD450 nm and 630 nm) reading was performed on an enzyme-labeled instrument to determine the Cut-off value, and the protein concentration-absorbance value curve was plotted. The results showed that the recombinant enterovirus 71 virus-like particles could bind to different neutralizing mouse monoclonal antibodies, and had good biological activity, and the results are shown in Figure 4 、 Figure 5 、 Figure 6

[0126] Example 5: Evaluation of the immunological effect of recombinant enterovirus 71 virus-like particles with different mutation schemes in mice. ​

[0127] According to the animal experiment scheme shown in Table 4, Table 5 and Table 6, the different mutant recombinant enterovirus 71 virus-like particles were mixed and adsorbed with aluminum hydroxide, and then were intraperitoneally injected into BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF level, female, 6-8 weeks old) at 2 μg / dose / 0.5 mL / each, 10 mice per group, 2 times of immunization with an interval of three weeks, and blood was collected 2 weeks after immunization. The serum specific IgG antibody and neutralizing antibody levels were detected by ELISA method and virus micro-neutralization test method, respectively, and the detection results are shown in Table 7, Table 8 and Table 9. Figures 7 to 18 and Table 7, Table 8, Table 9.

[0128] Table 4 Animal experiment scheme for evaluating immunological effects of different mutations

[0129]

[0130] Table 5 Animal experiment scheme for evaluating immunological effects of different mutations

[0131]

[0132] Table 6 Animal experiment scheme for evaluating immunological effects of different mutations

[0133]

[0134] Table 7 Geometric mean titer of serum specific IgG antibody and neutralizing antibody of mice after immunization with different mutations

[0135]

[0136] Table 8 Geometric mean titer of serum specific IgG antibody and neutralizing antibody of mice after immunization with different mutations

[0137]

[0138] Table 9 Geometric mean titer of serum specific IgG antibody and neutralizing antibody of mice after immunization with different mutations

[0139]

[0140] 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 enterovirus 71 (EV71), characterized in that, The amino acid sequences of the mutants VP0, VP3, and VP1 are as follows: (1) Shown as SEQ ID No:13, SEQ ID No:14, SEQ ID No:15; (2) Shown as SEQ ID No:34, SEQ ID No:35, SEQ ID No:36; (3) SEQ ID No: 43, SEQ ID No: 44, SEQ ID No: 45; (4) As shown in SEQ ID No:46, SEQ ID No:47, and SEQ ID No:48; or (5) SEQ ID No:70, SEQ ID No:71, SEQ ID No:

72.

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:91, SEQ ID No:92, SEQ ID No:93; (2) SEQ ID No: 112, SEQ ID No: 113, SEQ ID No: 114; (3) As shown in SEQ ID No: 121, SEQ ID No: 122, and SEQ ID No: 123; (4) As shown in SEQ ID No:124, SEQ ID No:125, and SEQ ID No:126; or (5) As shown in SEQ ID No: 148, SEQ ID No: 149, and SEQ ID No:

150.

6. A carrier, characterized in that, The vector comprises the isolated polynucleotide as described in any one of claims 3 to 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 any one of claims 3 to 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, AS04 adjuvant system or CpG.

11. The recombinant protein vaccine according to claim 10, characterized in that, The oil-water emulsion adjuvant is either the AS03 adjuvant system or MF59.

12. A nucleic acid vaccine, characterized in that, The nucleic acid vaccine comprises the polynucleotides as described in any one of claims 3 to 5.

13. A liposome complex, characterized in that, The liposome complex comprises the polynucleotide as described in any one of claims 3 to 5.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the mutant as claimed in claim 1, the virus-like particle as claimed in claim 2, the isolated polynucleotide as claimed in any one of claims 3 to 5, the vector as claimed in claim 6 or 7, the host cell as claimed in claim 8, the vaccine as claimed in any one of claims 9 to 12, or the liposome complex as claimed in claim 13, and a pharmaceutically acceptable carrier and / or stabilizer.

15. The pharmaceutical composition according to claim 14, characterized in that, The carrier includes a delivery medium, an excipient, a diluent, or a combination thereof.

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