Mutant of enterovirus 71 and virus-like particle thereof

Abstract

The invention provides a mutant of enterovirus 71 and virus-like particles thereof, and relates to the field of biological medicines. The enterovirus 71 type virus-like particle is obtained by modifying structural genes VP0, VP1 and VP3 of virus capsid protein by utilizing computational structure biology and completing self-assembly in vivo through a hansenula polymorpha expression system. Compared with the unmodified enterovirus 71 type mutant and the virus-like particles thereof, the enterovirus 71 type mutant and the virus-like particles thereof have the advantages that the immunogenicity can be obviously improved, and the clinical application prospect is relatively high.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine, and in particular, to mutants of enterovirus type 71 and virus-like particles thereof. Background Art

[0002] Hand, foot and mouth disease (HFMD) is a common infectious disease in children, mainly caused by pathogens such as enterovirus A, D, coxsackievirus A and coxsackievirus B. Among them, EV71 (Enterovirus71, EV71) is one of the main pathogens that causes high morbidity and mortality in infants and young children.

[0003] While there is no specific treatment for HFMD caused by EV71 infection, vaccination is a cost-effective measure to control EV71 infection. Currently, three inactivated EV71 vaccines are commercially available in China. With the launch and widespread use of inactivated EV71 vaccines, significant progress has been made in the prevention and control of HFMD caused by EV71. The pathogen spectrum has also shifted, with a shift in the predominant strains of EV71, CVA16, CVA10, and CVA6. Therefore, the development of multivalent vaccines that effectively target the major hand, foot, and mouth disease-associated viruses is a pressing need, and EV71 remains an essential component of these multivalent HFMD vaccines. VLP vaccines, as a mature vaccine development technology, offer advantages such as high safety, antigenic morphology similar to the native virus, strong immunogenicity, high yield, and ease of enrichment and purification, holding great promise for future development. Currently, most EV71 VLP vaccines are in the preclinical development stage. Recombinantly expressed EV71 VLPs spontaneously undergo a conformational uncoating process, destroying neutralizing epitopes on their surface. This process causes the VLP to transition from a highly immunogenic, non-expanded conformation to a significantly less immunogenic, expanded conformation. This is a major technical bottleneck limiting VLP vaccine development. Therefore, maintaining the non-expanded conformation of EV71 VLPs is an important approach to addressing this issue. Summary of the Invention

[0004] Technical issues solved:

[0005] One aspect of the present invention is to address the problem that the virus-like particles of enterovirus 71 have unstable structures and thus low immunogenicity, and provide a new mutant of enterovirus 71 and its virus-like particles.

[0006] Technical solution:

[0007] A mutant of enterovirus 71 (EV71), wherein the mutant has at least one amino acid mutation site at the following position compared to the amino acid sequence of a structural protein of wild-type enterovirus 71, wherein the mutation is a substitution of an amino acid residue:

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

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

[0010] (3) At least one of the amino acid positions 117, 163, 247, 284, 293, 200, 222, 225, 45, 136, 154, 198, and 234 on the VP1 sequence.

[0011] In some embodiments, the amino acid mutation site may be:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0028] (17) amino acid position 159 in the VP0 sequence and amino acid position 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 and 45 and 136 in the VP1 sequence; or

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

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

[0033] In some more specific embodiments, the amino acid mutation sites may be:

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

[0035] (2) The 158th amino acid in the VP0 sequence is replaced by W; or

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

[0037] (4) The 155th amino acid in the VP0 sequence is substituted by L and the 159th amino acid is substituted by Y; or

[0038] (5) The 159th amino acid in the VP0 sequence is replaced by Y; or

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

[0040] (7) The 84th amino acid in the VP0 sequence is substituted by W, the 122nd amino acid is substituted by W, and the 159th amino acid is substituted by Y; or

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

[0042] (9) The 155th amino acid in the VP0 sequence is substituted by M and the 159th amino acid is substituted by M; or

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

[0044] (11) The 159th amino acid in the VP0 sequence is substituted by M and the 186th amino acid in the VP3 sequence is substituted by W; or

[0045] (12) The 159th amino acid in the VP0 sequence is substituted by M and the 180th amino acid in the VP3 sequence is substituted by M; or

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

[0047] (14) The 159th amino acid in the VP0 sequence is substituted by M and the 149th amino acid in the VP3 sequence is substituted by M; or

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

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

[0050] (17) The amino acid at position 159 in the VP0 sequence is substituted by M, the amino acid at position 229 in the VP3 sequence is substituted by M, and the amino acid at position 232 is substituted by M; or

[0051] (18) The amino acid at position 159 in the VP0 sequence is substituted by M and the amino acid at position 222 in the VP1 sequence is substituted by M; or

[0052] (19) The amino acid at position 159 in the VP0 sequence is substituted by M and the amino acid at position 225 in the VP1 sequence is substituted by F; or

[0053] (20) The amino acid at position 159 in the VP0 sequence is substituted by M and the amino acid at position 265 in the VP0 sequence is substituted by M; or

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

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

[0056] (23) The amino acid at position 155 in the VP0 sequence is substituted by M, the amino acid at position 159 is substituted by M, the amino acid at position 284 in the VP1 sequence is substituted by S, and the amino acid at position 293 is substituted by T; or

[0057] (24) The 158th amino acid in the VP0 sequence is replaced by C; or

[0058] (25) The 158th amino acid in the VP0 sequence was replaced by F.

[0059] In some more specific embodiments, the amino acid sequence of VP0, VP1 or VP3 of the above-mentioned mutant is as shown in SEQ ID No: 4-78, or a sequence that has 90% or more identity thereto and has the same or similar biological function.

[0060] Another aspect of the present disclosure provides a virus-like particle, which is assembled from the mutant.

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

[0062] In some embodiments, the isolated polynucleotide may be codon-optimized.

[0063] In some specific embodiments, the sequence of the isolated polynucleotide is shown as SEQ ID No: 82-156.

[0064] Another aspect of the present disclosure provides a vector comprising the isolated polynucleotide or the mutant.

[0065] In some embodiments, the above-mentioned vector may be a protein expression vector, a gene delivery vector or a protein delivery vector.

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

[0067] In some embodiments, the host cell may be an Escherichia coli cell, a yeast cell, an insect cell, or a mammalian cell. In some more specific embodiments, the host cell may be a Hansenula cell.

[0068] Another aspect of the present disclosure is to provide a recombinant protein vaccine, which includes the above-mentioned virus-like particles and optionally an adjuvant.

[0069] In some embodiments, the adjuvant may be at least one selected from aluminum adjuvant, oil-water emulsion adjuvant, AS01 adjuvant system, AS03 adjuvant system, AS04 adjuvant system, MF59, CpG, immunostimulatory substances or immunoregulatory substances.

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

[0071] Another aspect of the present disclosure provides a liposome complex comprising the above-mentioned polynucleotide.

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

[0073] Another aspect of the present disclosure provides a method for eliciting an immune response against enterovirus 71 in a subject or treating enterovirus 71 infection in a subject, by administering an effective dose of the above-mentioned recombinant protein vaccine, the above-mentioned nucleic acid vaccine, the above-mentioned liposome complex or the above-mentioned pharmaceutical composition to the subject.

[0074] Beneficial effects:

[0075] The EV71 virus-like particles disclosed herein are derived by modifying the structural genes VP0, VP1, and VP3 of the viral capsid protein using computational structural biology, and then self-assembling in vivo using a Hansenula expression system. The EV71 mutants and their virus-like particles disclosed herein have significantly improved immunogenicity compared to their unmodified counterparts and have promising clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0077] Figure 2 This is a graph showing the Western-blot detection results of the recombinant enterovirus 71 virus-like particles in Example 3 of the present disclosure, wherein A represents the detection result using the Anti EV71-VP0 antibody, B represents the detection result using the Anti EV71-VP1 antibody, and C represents the detection result using the Anti EV71-VP3 antibody. M26630 and M26619 represent markers, A represents T24, Q represents T25, and RF represents a negative control - an unrelated protein.

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

[0079] Figure 4 、 Figure 5 、 Figure 6 This is a graph showing the binding activity of the recombinant enterovirus 71 virus-like particles with different monoclonal antibodies in Example 4 of the present disclosure, wherein: Figure 4 This is the result of enterovirus 71 virus D6 mouse monoclonal antibody, Figure 5 This is the result of enterovirus 71 virus A9 mouse monoclonal antibody. Figure 6 These are the results of enterovirus 71 virus 10F0 mouse monoclonal antibody, where A represents T24 and Q represents T25;

[0080] Figures 7 to 18 This is a graph showing the results of specific IgG antibody titers and neutralizing antibody titers in mouse serum after immunization with different regimens in Example 5 of the present disclosure.

[0081] Sequence description.

[0082] DETAILED DESCRIPTION

[0083] DETAILED DESCRIPTION OF THE INVENTION / SPECIFIC EMBODIMENTS.

[0084] The present invention discloses a mutant of enterovirus 71 and a virus-like particle thereof. Those skilled in the art may refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications obvious to those skilled in the art are considered to be included in the present invention, and relevant persons will obviously be able to modify or appropriately change and combine the contents described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0085] In this disclosure, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly indicated, throughout the specification and claims, the term "comprise" or its variations such as "comprises" or "comprising" will be understood to include the elements or components stated without excluding other elements or other components. The terms "a", "an", and "the" include plural referents. The term "multiple" refers to two or more. The terms "such as", "for example", etc. are intended to refer to exemplary embodiments and are not intended to limit the scope of this disclosure.

[0086] In this disclosure, when a range of values ​​is provided, it is understood that the endpoints are included in the range and that each intervening value between the upper and lower limits of the range and any other specified value or intervening value in the stated range and any smaller range between the specified values ​​are encompassed unless the context clearly dictates otherwise.

[0087] In this disclosure, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 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 a specified value.

[0088] Throughout this disclosure, references to "one embodiment," "an example," "some embodiments," "specific embodiments," "related embodiments," "an example," "some examples," "additional embodiments," or "further embodiments," "further implementations," or "another embodiment," "other examples" mean that at least one feature or characteristic description is included in connection with an embodiment. Thus, references to these phrases in various places throughout this disclosure are not necessarily referring to the same embodiment. Furthermore, particular features may be combined in any suitable manner in one or more embodiments.

[0089] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. For definitions of common terms in molecular biology, see Lewin's Genes, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Publisher: Jones & Bartlett Learning. For definitions of common terms in biochemistry, see Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: WH Freeman. For definitions of common terms in cell biology, see Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Publisher: Garland Science. For definitions of common terms in genetics, see Genetics: Analysis of Genes and Genomes, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Publisher: Jones & Bartlett Learning.

[0090] Unless otherwise specified, the experimental techniques herein employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard books such as Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook, etc.

[0091] definition:

[0092] The term "Enterovirus 71" (EV71) as used herein refers to a non-enveloped virus belonging to the genus Enterovirus in the family Picornaviridae. The viral nucleic acid is a single-stranded positive-strand RNA genome approximately 7.4 kb in length, consisting of an open reading frame (ORF) and two highly structured untranslated regions (UTRs) at either end: the 5' and 3' UTRs. The ORF is divided into three regions: P1, P2, and P3. P1 encodes four viral structural proteins, VP1 to VP4, with molecular weights of 32 kD, 28 kD, 27 kD, and 8 kD, respectively, which are enteroviral capsid proteins. P2 and P3 encode seven non-structural proteins. P2 is proteolytically cleaved to form the virus-specific protease 2A and 2BC, which is further cleaved into 2B and 2C. P3 is initially cleaved into 3AB and 3CD, which are further cleaved into 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 them, the 3C protein has serine protease and cysteine ​​protease activities, which can catalyze the cleavage of precursor proteins and form mature structural proteins and non-structural proteins.

[0093] The term "wild type" in this disclosure, which may also be referred to as "native" or "natural", refers to a state in which no non-natural mutations have been introduced. In some embodiments, exemplary examples of wild-type Enterovirus 71 structural proteins are shown in SEQ ID Nos. 1-3.

[0094] The term "mutation" as used herein refers to a change in a sequence (e.g., a nucleotide or amino acid sequence) relative to the corresponding sequence in its native, wild-type, standard, or reference form (i.e., the non-mutated sequence). Amino acid mutations generally include substitutions, deletions, or insertions of amino acid residues. Mutations can be artificial or naturally occurring. In some embodiments, the mutation refers to the substitution of amino acid residues within a peptide chain. These mutations are considered key to achieving the objectives of this disclosure.

[0095] The term "site" in this disclosure refers to a position within a peptide, polypeptide or polynucleotide that can be modified, altered or derivatized within a molecule based on the polypeptide or polynucleotide. Typically, sites are arranged or named in numerical order.

[0096] The terms "peptide," "polypeptide," and "protein" in this disclosure are used interchangeably and generally refer to peptides and proteins of amino acids covalently linked by peptide bonds. 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 terms also include modifications of proteins, for example, proteins modified by glycosylation, acetylation, phosphorylation, pegylation, ubiquitination, and the like. Proteins may include amino acids that are not encoded by nucleic acid codons. Proteins may have amino acid sequences that are long enough to produce higher levels of tertiary and / or quaternary structure.

[0097] The term "virus-like particle (VLP)" or "pseudovirus" as used herein refers to a multiprotein structure composed of the structural proteins of a corresponding native virus, but lacking all or part of the viral genome, particularly the replicative and infectious components of the viral genome, and therefore lacking replication and infectivity. This multiprotein structure closely mimics its corresponding native viral particle in morphology and size and can spontaneously form following recombinant expression of viral structural proteins. VLPs are highly structured protein particles self-assembled from one or more viral structural proteins. Their diameter ranges from 20 to 150 nanometers, preserving the native conformation of viral antigenic proteins. VLPs can self-assemble into structures such as icosahedral, rod-shaped, or spherical. VLPs can be artificially produced through recombinant expression in prokaryotic cell lines, yeast cell systems, plant cells, animal cells, and the like.

[0098] The term "identity" or "sequence identity" as used herein refers to the exact same nucleotide or amino acid residues at the same position in two sequences. It is a fundamental and important concept in bioinformatics, commonly used to compare the similarity between two nucleic acid or protein sequences. Sequence identity is typically expressed as a percentage, reflecting the degree of sequence identity.

[0099] In some embodiments of the present disclosure, "having more than 90% identity and having the same or similar biological function" generally means that 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%, or at least 99% of the actual sequence and the sequence described in the present disclosure may be identical, and the different parts thereof may be due to substitution / insertion / deletion mutations introduced artificially or non-artificially, for example, substitutions of conservative amino acids. Artificially introduced mutations may be based on specific purposes, such as making it easier to express proteins, etc. However, the introduction of these different parts is not sufficient 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, thereby obtaining "conservative amino acid substitution variants". The changes therein result in some amino acids being replaced by other amino acids with similar chemical properties and / or functions. It is well known in the art to provide conservative substitution tables of amino acids with similar chemical properties and / or functions. Typical examples of mutually conservative substitutions include, 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).

[0100] The term "isolated" as used herein refers to a substance or entity that has been separated from its natural environment or the environment in which it existed prior to separation and from other components. The separation 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%, or at least 90%. Isolated substances may have varying levels of purity relative to their pre-isolation counterparts.

[0101] The term "vector" as used herein refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing the protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell through transformation, transduction, or transfection, enabling expression of the genetic material it carries in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Vectors may also contain an origin of replication.

[0102] The term "host cell" in this disclosure refers to 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.

[0103] The term "protein delivery vector" in the present disclosure refers to a protein that can serve as a carrier of an epitope peptide, that is, it can insert an epitope peptide at a specific position (e.g., inside the protein, at the N-terminus or C-terminus) so that the epitope peptide can be presented, thereby enabling the epitope peptide to be recognized by antibodies or the immune system.

[0104] The term "immunogenicity" in this disclosure refers to the ability of a substance to elicit, trigger, stimulate or induce an immune response against a specific antigen in an animal, with or without an adjuvant.

[0105] The term "adjuvant" in the present disclosure refers to a nonspecific immunopotentiator that can enhance the body's immune response to the antigen or change the type of immune response after mixing with the antigen, including but not limited to aluminum adjuvants such as aluminum hydroxide, Freund's complete adjuvant, Freund's incomplete adjuvant, etc.

[0106] The term "pharmaceutically acceptable carrier" in the present disclosure can be any pharmaceutically acceptable additive, for example, physiological saline, cell culture medium, glucose, water for injection, glycerol, amino acids and combinations thereof, stabilizers, surfactants, preservatives, isotonic agents, etc.

[0107] Codon optimization:

[0108] Codon optimization is an advanced technology that effectively enhances protein expression in vivo by improving the translation efficiency of target genes. In vivo, due to codon degeneracy—that is, multiple codons can encode the same amino acid—multiple different mRNA sequences may exist for a given amino acid sequence. However, different organisms or cells have their own biases in the selection of these synonymous codons, which is known as codon bias. Therefore, when a heterologous gene is expressed in a host cell, its codon usage may not match the host cell's optimal codon usage frequency, affecting protein expression levels. Codon optimization can select synonymous codons that better match the host cell's bias, thereby improving protein translation efficiency.

[0109] There are many methods that 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, it is necessary to understand the codon bias of the host cell, that is, which codons are used more frequently in the host cell. This can be obtained by consulting relevant literature or databases. (2) Design an optimization plan: Design an optimization plan based on the codon usage frequency of the host cell. This includes selecting which synonymous codons to replace the codons in the heterologous gene and how to adjust the GC content. (3) Implement optimization: Use techniques such as gene synthesis or site-directed mutagenesis to apply the optimization plan to the heterologous gene. (4) Verify the optimization effect: Verify experimentally whether the expression level of the optimized gene in the host cell is improved. This can be evaluated by measuring indicators such as protein concentration and enzyme activity.

[0110] Example:

[0111] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0112] Example 1: Design of enterovirus 71 virus particles.

[0113] Throughout the EV71 replication cycle, the virion undergoes various structural transformations. From an immunological perspective, mature virions exhibit strong immunogenicity, inducing high titers of neutralizing antibodies. However, expanded A particles and empty capsid virions induce significantly lower levels of neutralizing antibodies. This phenomenon also occurs with recombinantly expressed VLPs, therefore stabilizing the EV71 VLP structure in an immunodominant state is the goal of this design. Using structural biology methods, a saturation mutation scan was performed on a subunit of the EV71 virus. The effects of mutations on overall structural stability were calculated, and key amino acid mutations that enhance stability were identified. These sites are primarily located in the RNA release channel region of the secondary axis and the quasi-tertiary axis. Some key amino acid sites, identified through consensus sequence alignment, are located in non-interface regions. By mutating key amino acid sites to enhance hydrophobic interactions at the intermolecular binding interface and further enhance capsid structural stability, 25 EV71 VLP mutation schemes were designed, as shown in Table 1.

[0114] Table 1 Mutation design scheme

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

[0116] The nucleotide sequence encoding the EV71 virus-like particles was codon-optimized based on Hansenula codon bias and tRNA abundance. The optimized nucleotide sequences are shown in SEQ ID Nos: 82-156. Yeast expression plasmids were constructed and transformed into defective Hansenula cells to construct recombinant yeast strains. Emulsifier-based ELISA screening was performed to identify yeast strains that highly expressed the recombinant EV71 virus-like particles.

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

[0118] The obtained positive yeast strain was expanded and then crushed to harvest the supernatant. After a series of chromatography purification, the recombinant enterovirus 71 virus-like particles were obtained. The obtained virus-like particles were identified, and the SDS-PAGE test results were as follows: Figure 1 As shown, the positions of the four bands correspond to the theoretical molecular weights of the target proteins. Band 1 is VP0, with a molecular weight of approximately 35-40 KDa; band 2 is VP1, with a molecular weight of approximately 30-35 KDa; and band 3 is VP3, with a molecular weight of approximately 25-30 KDa. The purified recombinant EV71 virus-like particles were electrophoresed by SDS-PAGE and then transferred to a PVDF membrane. Western-blot identification was performed using rabbit polyclonal antibodies against EV71 VP0 / VP1 / VP3 (dilution: 1000-fold). The results are shown in Figure 2. Figure 2As shown, a band can be seen at the expected position.

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

[0120] The physical and chemical properties of the obtained virus-like particles were analyzed. The purified recombinant enterovirus 71 virus-like particles were subjected to molecular exclusion chromatography using a TSK-GEL G3000 column. The results are shown in Table 2. The recombinant enterovirus 71 virus-like particles were observed using a transmission electron microscope. The particles were uniform in size and well-shaped, with a diameter of approximately 30 to 40 nm. Figure 3 Dynamic light scattering was used to analyze the recombinant enterovirus 71 virus-like particles. The proteins were present in the form of particles with a diameter of approximately 30 to 50 nm and good uniformity. The results are shown in Table 3.

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

[0122] Table 3 Dynamic light scattering results of recombinant enterovirus 71 virus-like particles

[0123] Purified recombinant EV71 VLPs were serially diluted two-fold starting at 10 µg / ml using coating buffer in 12 dilutions at 100 µl / well and coated onto a 96-well microplate. The plates were incubated at 4°C for 8-12 h, with blank wells serving as negative controls. The plates were washed with PBST and then blocked with blocking buffer at 37°C for 2 h. After washing with PBST, 100 µl / well of mouse monoclonal antibodies against EV71 D6 / A9 / 10F0 diluted to 1 µg / ml were added, each well incubated at 37°C for 1 h. After washing with PBST, 100 µl / well of diluted horseradish peroxidase-conjugated goat anti-mouse IgG antibody was added, each well incubated at 37°C for 1 h. After washing with PBST, chromogen solutions A and B were added sequentially, and color was developed at room temperature for 5 min. Stop solution C was then added. Dual wavelength readings (OD 450 nm and 630 nm) were performed on a microplate reader to determine the cut-off value, and a protein concentration-absorbance 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. Figure 4 、 Figure 5 、 Figure 6 shown.

[0124] Example 5: Evaluation of the immunological effects of recombinant enterovirus 71 virus-like particles with different mutation schemes in mice.

[0125] According to the animal experimental schemes shown in Tables 4, 5, and 6, recombinant enterovirus 71 virus-like particles with different mutations were mixed with aluminum hydroxide for adsorption and then intraperitoneally injected into BALB / c mice (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., SPF-grade, female, 6-8 weeks old) at a dose of 2µg / dose / 0.5mL / mouse. 10 mice / group were immunized twice, three weeks apart. Blood was collected 2 weeks after immunization. Serum specific IgG antibody and neutralizing antibody levels were measured using ELISA and virus microneutralization test, respectively. The test results are shown in the figure below. Figures 7 to 18 As shown in Table 7, Table 8 and Table 9.

[0126] Table 4 Animal experimental scheme 1 for evaluating the immunological effects of different mutations

[0127] Table 5 Animal experimental scheme 2 for evaluating the immunological effects of different mutations

[0128] Table 6 Animal experimental scheme 3 for evaluating the immunological effects of different mutations

[0129] Table 7 Geometric mean titers of specific IgG antibodies and neutralizing antibodies in the serum of mice immunized with different mutations1

[0130] Table 8 Geometric mean titers of specific IgG antibodies and neutralizing antibodies in the serum of mice immunized with different mutations

[0131] Table 9 Geometric mean titers of specific IgG antibodies and neutralizing antibodies in the serum of mice immunized with different mutations

[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A mutant of enterovirus 71 (EV71), characterized in that Compared with the amino acid sequence of the structural protein of wild-type enterovirus 71, the mutant has at least one amino acid mutation site at the following position, and the mutation is a substitution of the amino acid residue: (1) at least one of amino acid positions 155, 158, 159, 149, 122, 84, 114, 115, and 265 in the VP0 sequence; or (2) at least one of amino acid position 236, amino acid position 186, amino acid position 180, amino acid position 177, amino acid position 149, amino acid position 110, amino acid position 229, and amino acid position 232 of the VP3 sequence; or (3) At least one of the amino acid positions 117, 163, 247, 284, 293, 200, 222, 225, 45, 136, 154, 198, and 234 on the VP1 sequence.

2. The mutant according to claim 1, characterized in that The mutant according to claim 1, wherein the amino acid mutation site is: (1) amino acid positions 155, 158, and 159 in the VP0 sequence; or (2) amino acid position 158 in the VP0 sequence; or (3) amino acid positions 149 in the VP0 sequence, 236 in the VP3 sequence, 117, 163, and 247 in the VP1 sequence; or (4) amino acid positions 155 and 159 in the VP0 sequence; or (5) amino acid position 159 in the VP0 sequence; or (6) amino acid positions 122, 155, and 159 in the VP0 sequence; or (7) amino acid positions 84, 122, and 159 in the VP0 sequence; or (8) amino acid positions 284 and 293 in the VP1 sequence; or (9) amino acid position 159 in the VP0 sequence and amino acid position 186 in the VP3 sequence; or (10) amino acid position 159 in the VP0 sequence and amino acid position 180 in the VP3 sequence; or (11) amino acid position 159 in the VP0 sequence, amino acid position 177 in the VP3 sequence, and amino acid position 200 in the VP1 sequence; or (12) amino acid position 159 in the VP0 sequence and amino acid position 149 in the VP3 sequence; or (13) amino acid positions 114, 115, and 159 in the VP0 sequence; or (14) amino acid position 159 in the VP0 sequence and amino acid position 110 in the VP3 sequence; or (15) amino acid positions 159 in the VP0 sequence and 229 and 232 in the VP3 sequence; or (16) amino acid position 159 in the VP0 sequence and amino acid position 222 in the VP1 sequence; or (17) amino acid position 159 in the VP0 sequence and amino acid position 225 in the VP1 sequence; or (18) amino acid positions 159 and 265 in the VP0 sequence; or (19) amino acid positions 180 in the VP3 sequence and 45 and 136 in the VP1 sequence; or (20) amino acid positions 180 in the VP3 sequence and 45, 136, 154, 198 and 234 in the VP1 sequence; or (21) Amino acid positions 155 and 159 in the VP0 sequence, and positions 284 and 293 in the VP1 sequence.

3. The mutant according to claim 2, characterized in that The amino acid mutation sites are: (1) The amino acid at position 155 is substituted by L, the amino acid at position 158 is substituted by F, and the amino acid at position 159 is substituted by Y in the VP0 sequence; or (2) The 158th amino acid in the VP0 sequence is replaced by W; or (3) The 149th amino acid in the VP0 sequence is substituted by L, the 236th amino acid in the VP3 sequence is substituted by M, the 117th amino acid in the VP1 sequence is substituted by C, the 163rd amino acid is substituted by I, and the 247th amino acid is substituted by A; or (4) The 155th amino acid in the VP0 sequence is substituted by L and the 159th amino acid is substituted by Y; or (5) The 159th amino acid in the VP0 sequence is replaced by Y; or (6) The amino acid at position 122 in the VP0 sequence is substituted by W, the amino acid at position 155 is substituted by L, and the amino acid at position 159 is substituted by Y; or (7) The 84th amino acid in the VP0 sequence is substituted by W, the 122nd amino acid is substituted by W, and the 159th amino acid is substituted by Y; or (8) The amino acid at position 284 in the VP1 sequence is substituted by S and the amino acid at position 293 is substituted by T; or (9) The 155th amino acid in the VP0 sequence is substituted by M and the 159th amino acid is substituted by M; or (10) The amino acid at position 155 is substituted by M, the amino acid at position 158 is substituted by L, and the amino acid at position 159 is substituted by M in the VP0 sequence; or (11) The 159th amino acid in the VP0 sequence is substituted by M and the 186th amino acid in the VP3 sequence is substituted by W; or (12) The 159th amino acid in the VP0 sequence is substituted by M and the 180th amino acid in the VP3 sequence is substituted by M; or (13) The 159th amino acid in the VP0 sequence is substituted by M, the 177th amino acid in the VP3 sequence is substituted by Y, and the 200th amino acid in the VP1 sequence is substituted by I; or (14) The 159th amino acid in the VP0 sequence is substituted by M and the 149th amino acid in the VP3 sequence is substituted by M; or (15) The 114th amino acid in the VP0 sequence is substituted by I, the 115th amino acid is substituted by V, and the 159th amino acid is substituted by M; or (16) The amino acid at position 159 in the VP0 sequence is substituted by M and the amino acid at position 110 in the VP3 sequence is substituted by M; or (17) The 159th amino acid in the VP0 sequence is substituted by M, the 229th amino acid in the VP3 sequence is substituted by M, and the 232nd amino acid in the VP3 sequence is substituted by M; or (18) The amino acid at position 159 in the VP0 sequence is substituted by M and the amino acid at position 222 in the VP1 sequence is substituted by M; or (19) The amino acid at position 159 in the VP0 sequence is substituted by M and the amino acid at position 225 in the VP1 sequence is substituted by F; or (20) The amino acid at position 159 in the VP0 sequence is substituted by M and the amino acid at position 265 in the VP0 sequence is substituted by M; or (21) The 180th amino acid in the VP3 sequence is substituted by L, the 45th amino acid in the VP1 sequence is substituted by I, and the 136th amino acid is substituted by L; or (22) The 180th amino acid in the VP3 sequence is substituted by L, the 45th amino acid in the VP1 sequence is substituted by I, the 136th amino acid is substituted by L, the 154th amino acid is substituted by I, the 198th amino acid is substituted by I, and the 234th amino acid is substituted by L; or (23) The amino acid at position 155 in the VP0 sequence is substituted by M, the amino acid at position 159 is substituted by M, the amino acid at position 284 in the VP1 sequence is substituted by S, and the amino acid at position 293 is substituted by T; or (24) The 158th amino acid in the VP0 sequence is replaced by C; or (25) The 158th amino acid in the VP0 sequence was replaced by F.

4. The mutant according to claim 1, characterized in that The amino acid sequence of VP0, VP1 or VP3 of the mutant is shown in SEQ ID No: 4-78, or a sequence that has 90% or more identity thereto and has the same or similar biological function.

5. A virus-like particle, characterized in that The virus-like particle is assembled from the mutant according to any one of claims 1 to 4.

6. An isolated polynucleotide, characterized in that The isolated polynucleotide encodes the mutant according to any one of claims 1 to 4, or VP0, VP1 or VP3 of the mutant.

7. The isolated polynucleotide according to claim 6, characterized in that The isolated polynucleotide is codon-optimized.

8. The isolated polynucleotide according to claim 6 or 7, characterized in that The sequences of the isolated polynucleotides are shown in SEQ ID No: 82-156.

9. A carrier, characterized in that The vector comprises the isolated polynucleotide according to claim 6, 7 or 8, or the mutant according to any one of claims 1 to 4.

10. The carrier according to claim 9, characterized in that The vector is a protein expression vector, a gene delivery vector or a protein delivery vector.

11. A host cell, characterized in that The host cell comprises the mutant according to any one of claims 1 to 4, the isolated polynucleotide according to claim 6, 7 or 8, or the vector according to claim 9 or 10.

12. A recombinant protein vaccine, characterized in that: The recombinant protein vaccine comprises the virus-like particle according to claim 5 and optionally comprises an adjuvant.

13. The recombinant protein vaccine according to claim 12, characterized in that The adjuvant is at least one selected from aluminum adjuvant, oil-water emulsion adjuvant, AS01 adjuvant system, AS03 adjuvant system, AS04 adjuvant system, MF59, CpG, immunostimulatory substances or immunoregulatory substances.

14. A nucleic acid vaccine, characterized in that The nucleic acid vaccine comprises the polynucleotide as described in claim 6, 7 or 8.

15. A liposome complex, characterized in that: The liposome complex comprises the polynucleotide as claimed in claim 6, 7 or 8.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a mutant according to any one of claims 1 to 4, a virus-like particle according to claim 5, an isolated polynucleotide according to claim 6, 7 or 8, a vector according to claim 9 or 10, a host cell according to claim 11, a vaccine according to any one of claims 12 to 14, or a liposome complex according to claim 15, and a pharmaceutically acceptable carrier, delivery vehicle, excipient, stabilizer, diluent or a combination thereof.

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