A poliomyelitis vaccine and its preparation method

Abstract

The present invention discloses a polio vaccine and a preparation method thereof. Immunization with the vaccine made of the polio virus-like particles disclosed in the present invention can produce high-level neutralizing antibodies against polio type I, type II and type III, and has broad-spectrum neutralizing activity against currently widely prevalent strains. The recombinant polio vaccine disclosed in the present invention does not rely on live viruses, and the vaccine production and testing process does not contain live viruses, has no biosafety risks, and will not cause derivative cases such as OPV vaccines. At the same time, there is no high-level facility requirement required for IPV vaccine production and testing, which reduces production and quality control costs and can improve vaccine accessibility in low- and middle-income countries.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine, and in particular, to a poliomyelitis vaccine and a preparation method thereof. Background Art

[0002] Poliomyelitis is an acute infectious disease caused by polio virus infection. After the virus invades the nervous system, it damages the motor nerve cells. In severe cases, flaccid paralysis of the limbs occurs. It is more common in children, so it is also called poliomyelitis. This is a disease that fears the whole world. It has a sudden onset and can cause lifelong paralysis. There is currently no effective drug to treat polio virus infection, and prevention is mainly through vaccine immunization.

[0003] The two currently used vaccines, oral attenuated live vaccine (OPV) and inactivated vaccine (IPV), have played a huge role in the prevention and treatment of polio. The global poliovirus prevention and control campaign has achieved great success, making poliovirus expected to become the second virus completely eliminated by humans after smallpox. However, since OPV and IPV need to use live viruses in the vaccine production and testing process, there is also a biosafety risk of virus leakage. Therefore, the use of genetic engineering technology to develop new vaccines that are not contagious is of great significance for improving the production capacity of polio vaccines and promoting the process of completely eliminating polio worldwide. At present, there are many studies on recombinant polio vaccines worldwide, but they are all trapped in the low expression of structural proteins, the inability to guarantee the activity of 3CD enzyme cleavage, or the difficulty in stabilizing the antigen structure. The obtained antigens are mostly in the form of inactive proteins. Therefore, solving the above problems has become a top priority in this field. Summary of the invention

[0004] Technical issues solved:

[0005] One aspect of the present disclosure is to provide a polio vaccine and a method for preparing the same in response to the problems of weak immunogenicity, low activity, instability, etc. of recombinant polio antigens in the prior art.

[0006] Specifically, the present invention successfully prepared polio virus-like particles (VLPs) by using new polio type I, II and III structural protein sequences and expression strategies. Experimental verification shows that the polio virus-like particles and their compositions can solve technical problems such as weak immunogenicity, low activity and instability of recombinant polio antigens.

[0007] Technical solutions provided:

[0008] A poliovirus-like particle, wherein the virus-like particle is:

[0009] (1) Poliovirus type I virus-like particles,

[0010] It includes VP0-I as shown in SEQ ID No.1, VP1-I as shown in SEQ ID No.2, and VP3-I as shown in SEQ ID No.3, or an amino acid sequence having more than 90% identity with the amino acid sequences shown in SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3 and having the same or similar biological function; or

[0011] (2) polio virus-like particles type II,

[0012] It includes VP0-II as shown in SEQ ID No.4, VP1-II as shown in SEQ ID No.5, and VP3-II as shown in SEQ ID No.6, or an amino acid sequence having more than 90% identity with the amino acid sequences shown in SEQ ID No.4, SEQ ID No.5, and SEQ ID No.6 and having the same or similar biological function; or

[0013] (3) polio virus-like particles type III,

[0014] It includes VP0-III with an amino acid sequence as shown in SEQ ID No.7, VP1-III with an amino acid sequence as shown in SEQ ID No.8, and VP3-III with an amino acid sequence as shown in SEQ ID No.9, or an amino acid sequence that has more than 90% identity with the amino acid sequences shown in SEQ ID No.7, SEQ ID No.8, and SEQ ID No.9 and has the same or similar biological function.

[0015] In some embodiments of the present disclosure, the above-mentioned poliovirus-like particles may also contain other polypeptides, and the addition of the polypeptides does not affect the biological activity of the poliovirus-like particles. The biological activity is mainly immunogenicity. Examples of the other polypeptides include, but are not limited to, tags for protein purification, localization or quantification, such as affinity tags (e.g., glutathione S transferase tags, maltose binding protein MBP tags, SUMO tags, thioredoxin Trx tags), epitope tags (e.g., histidine His tags, FLAG tags, hemagglutinin HA tags, Myc tags), fluorescent tags (e.g., green fluorescent protein GFP tags, orange fluorescent protein OFP tags, red fluorescent protein RFP tags, yellow fluorescent protein YFP tags); signal peptides for secretory expression of proteins in host cells; polypeptides for other specific functions, such as polypeptides for enhancing stability, solubility, signal transduction and other functions.

[0016] In the present disclosure, in order to better achieve the purpose of the present disclosure, the amino acid sequences of VP0, VP1, and VP3 in the above three types of polio virus-like particles have at least one modification compared with the corresponding wild type, and the modification can be the deletion or replacement of amino acids.

[0017] In other embodiments of the present disclosure, the virus-like particle may be:

[0018] (1) Poliovirus type I virus-like particles,

[0019] It consists of VP0-I as shown in SEQ ID No.1, VP1-I as shown in SEQ ID No.2 and VP3-I as shown in SEQ ID No.3, or an amino acid sequence that has more than 90% identity with the amino acid sequences shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 and has the same or similar biological function; or

[0020] (2) polio virus-like particles type II,

[0021] It consists of VP0-II as shown in SEQ ID No.4, VP1-II as shown in SEQ ID No.5, and VP3-II as shown in SEQ ID No.6, or an amino acid sequence that has more than 90% identity with the amino acid sequences shown in SEQ ID No.4, SEQ ID No.5, and SEQ ID No.6 and has the same or similar biological function; or

[0022] (3) polio virus-like particles type III,

[0023] It consists of VP0-III with an amino acid sequence as shown in SEQ ID No.7, VP1-III with an amino acid sequence as shown in SEQ ID No.8 and VP3-III with an amino acid sequence as shown in SEQ ID No.9, or an amino acid sequence that has more than 90% identity with the amino acid sequences shown in SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9 and has the same or similar biological function.

[0024] Another aspect of the present disclosure is to provide a composition of polio virus-like particles, the composition comprising at least two selected from the above-mentioned polio type I virus-like particles, polio type II virus-like particles or polio type III virus-like particles. In some embodiments of the present disclosure, the above-mentioned composition may include, for example, (1) the polio type I virus-like particles and the polio type II virus-like particles; (2) the polio type II virus-like particles and the polio type III virus-like particles; (3) the polio type I virus-like particles and the polio type III virus-like particles. Similarly, the polio virus-like particles in the above-mentioned composition may also contain other polypeptides, and the addition of the polypeptides does not affect the biological activity of the polio virus-like particles.

[0025] In some embodiments of the present disclosure, the mass ratio of polio type I virus-like particles, polio type II virus-like particles and polio type III virus-like particles in the above composition can be 1:0.2:1 to 1:2:6. For example, 1:0.2:1, 1:0.5:1, 1:1:1, 1:2:3, 1:2:4, 1:2:5, 1:2:6, 1:0.2:2, 1:0.2:5, 1:0.2:6, 1:0.5:2, 1:0.5:5, 1:0.5:6, 1:1:2, 1:1:4, 1:1:6, etc.

[0026] In some embodiments of the present disclosure, the content of polio type I virus-like particles in the above-mentioned composition can be 0.5μg to 45μg, the content of polio type II virus-like particles can be 0.2μg to 45μg, and the content of polio type III virus-like particles can be 0.5μg to 45μg. For example, the content of polio type I virus-like particles can be 0.5μg, 0.8μg, 1μg, 2μg, 5μg, 10μg, 15μg, 20μg, 25μg, 30μg, 35μg, 40μg, 45μg, etc.; the content of polio type II virus-like particles can be 0.2μg, 0.4μg, 0.5μg, 0.8μg, 1μg, 2 μg, 5μg, 10μg, 15μg, 20μg, 25μg, 30μg, 35μg, 40μg, 45μg, etc.; the content of polio type III virus-like particles can be 0.5μg, 0.8μg, 1μg, 2μg, 5μg, 10μg, 15μg, 20μg, 25μg, 30μg, 35μg, 40μg, 45μg, etc.

[0027] Another aspect of the present disclosure is to provide a method for producing polio virus-like particles, comprising:

[0028] Step 1) cloning the genes of VP0-I, VP1-I and VP3-I into the same or different vectors, respectively, wherein the genes of VP0-I, VP1-I and VP3-I have independent open reading frames; or

[0029] The genes of VP0-II, VP1-II and VP3-II are cloned into the same or different vectors, respectively, and the genes of VP0-II, VP1-II and VP3-II have independent open reading frames; or

[0030] The genes of VP0-III, VP1-III and VP3-III are cloned into the same or different vectors, respectively, and the genes of VP0-III, VP1-III and VP3-III have independent open reading frames;

[0031] Step 2) transferring the vector obtained in step 1) into a host cell for expression and assembly, and purifying the expression product to obtain the polio virus-like particles.

[0032] In the present disclosure, the above-mentioned poliovirus-like particle expression strategy can be widely used in the preparation of poliovirus-like particles, and can also be specifically used in the preparation of poliovirus-like particles in the present disclosure.

[0033] In some embodiments of the present disclosure, the genes of VP0, VP1 and VP3 in the above method can be cloned into three different vectors, or cloned into the same vector, or cloned two by two into one vector and independently expressed in host cells and assembled into virus-like particles.

[0034] The above-mentioned host cell can be a host cell capable of expressing the poliovirus-like particle protein described in the present disclosure, including a cultured prokaryotic cell or a cultured eukaryotic cell. Examples of the host cell include, but are not limited to, Hansen yeast cells, Escherichia coli cells, Pichia pastoris cells, Saccharomyces cerevisiae cells, mammalian cells (e.g., CHO cells, HEK293 cells, NIH-3T3 cells, 293-T cells, Vero cells, HeLa cells), insect cells (e.g., Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, HIGH FIVE cells), avian cells (e.g., chicken embryo cells, duck cells). In one embodiment of the present disclosure, the host cell is a Hansen yeast cell.

[0035] For industrial-scale production, in some embodiments of the present disclosure, the expression in the above method can be performed on a large scale using a biological fermentation method.

[0036] Another aspect of the present disclosure is to provide a method for producing a composition of polio virus-like particles, comprising:

[0037] Step 1) cloning the genes of VP0-I, VP1-I and VP3-I into the same or different vectors, respectively, wherein the genes of VP0-I, VP1-I and VP3-I have independent open reading frames; or

[0038] The genes of VP0-II, VP1-II and VP3-II are cloned into the same or different vectors, respectively, and the genes of VP0-II, VP1-II and VP3-II have independent open reading frames; or

[0039] The genes of VP0-III, VP1-III and VP3-III are cloned into the same or different vectors, respectively, and the genes of VP0-III, VP1-III and VP3-III have independent open reading frames;

[0040] Step 2) transferring the vector obtained in step 1) into a host cell for expression and assembly, and purifying the expression product to obtain the poliovirus-like particles;

[0041] Step 3) at least two of the polio type I virus-like particles, polio type II virus-like particles or polio type III virus-like particles obtained in step 2) are mixed in proportion.

[0042] Another aspect of the present disclosure is to provide an isolated polynucleotide comprising a nucleic acid sequence encoding the above amino acid sequence.

[0043] In one embodiment of the present disclosure, the polynucleotides encoding the amino acid sequences of polio type I virus-like particles VP0-I, VP1-I and VP3-I are shown as SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12, respectively; the polynucleotides encoding the amino acid sequences of polio type II virus-like particles VP0-II, VP1-II and VP3-II are shown as SEQ ID No.13, SEQ ID No.14 and SEQ ID No.15, respectively; the polynucleotides encoding the amino acid sequences of polio type III virus-like particles VP0-III, VP1-III and VP3-III are shown as SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18, respectively.

[0044] Another aspect of the present disclosure is to provide an isolated vector, wherein the vector comprises the above-mentioned polynucleotide,

[0045] Among them, the polynucleotides encoding the amino acid sequences of polio type I virus-like particles VP0-I, VP1-I and VP3-I are respectively operably connected to the promoter, the polynucleotides encoding the amino acid sequences of polio type II virus-like particles VP0-II, VP1-II and VP3-II are respectively operably connected to the promoter, and the polynucleotides encoding the amino acid sequences of polio type III virus-like particles VP0-III, VP1-III and VP3-III are respectively operably connected to the promoter.

[0046] Another aspect of the present disclosure is to provide an isolated host cell, wherein the host cell comprises the above polynucleotide or the above vector;

[0047] Preferably, in some embodiments of the present disclosure, the host cell may be a Hansenula cell, an Escherichia coli cell, a Pichia pastoris cell, a Saccharomyces cerevisiae cell, a mammalian cell (e.g., a CHO cell, a HEK293 cell, a NIH-3T3 cell, a 293-T cell, a Vero cell, a HeLa cell), an avian cell (e.g., a chicken embryo cell, a duck cell), or an insect cell (e.g., a Sf9 cell, a Sf21 cell, a Tn5 cell, a Schneider S2 cell, a HIGH FIVE cell);

[0048] More preferably, in one embodiment of the present disclosure, the host cell is a Hansenula cell.

[0049] Another aspect of the present disclosure is to provide a recombinant protein vaccine, which includes the above-mentioned polio virus-like particles, or a combination of the above-mentioned polio virus-like particles, and optionally includes an adjuvant.

[0050] In some embodiments of the present disclosure, 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 immunomodulatory substances. Further, in some embodiments of the present disclosure, the adjuvant is aluminum hydroxide. Further, in some embodiments of the present disclosure, the content of aluminum hydroxide in each dose of the recombinant protein vaccine is 0 μg to 500 μg. Further, in one embodiment of the present disclosure, the content of aluminum hydroxide in each dose of the recombinant protein vaccine is 300 μg.

[0051] Another aspect of the present disclosure is to provide a genetically engineered vector vaccine, which includes the above-mentioned vector.

[0052] Another aspect of the present disclosure is to provide a nucleic acid vaccine comprising the above-mentioned polynucleotide.

[0053] Another aspect of the present disclosure provides a liposome complex, wherein the liposome complex comprises the above-mentioned polynucleotide.

[0054] Another aspect of the present disclosure is to provide a pharmaceutical composition, which includes the above-mentioned vaccine or the above-mentioned liposome complex, and a pharmaceutically acceptable carrier, delivery medium, excipient, stabilizer, diluent or a combination thereof.

[0055] Another aspect of the present disclosure is to provide the use of the above-mentioned polio virus-like particles, the above-mentioned composition, the above-mentioned isolated polynucleotide, the above-mentioned isolated vector, the above-mentioned isolated host cell, the above-mentioned vaccine, the above-mentioned liposome complex or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing or treating a disease caused by polio virus, wherein the polio virus is at least one selected from polio type I virus, polio type II virus or polio type III virus.

[0056] Another aspect of the present disclosure is to provide a method for detecting polio-specific antibodies in a sample, comprising:

[0057] Step 1) making the polio virus-like particles carry a detectable label;

[0058] Step 2) contacting the sample in solution with the labeled poliovirus-like particles obtained in step 1);

[0059] Step 3) detecting polio virus-like particles in the immune complex formed in the above steps.

[0060] Another aspect of the present disclosure is to provide a kit, which includes the above-mentioned polio virus-like particles or the above-mentioned composition.

[0061] The present disclosure also provides use of the above-mentioned kit in detecting poliomyelitis-specific antibodies in a sample.

[0062] Beneficial effects:

[0063] Vaccination using the polio virus-like particles disclosed in the present invention can produce high-level neutralizing antibodies against polio types I, II and III, and has broad-spectrum neutralizing activity against currently prevalent strains. The recombinant polio vaccine disclosed in the present invention does not rely on live viruses, and the vaccine production and testing process does not contain live viruses, has no biosafety risks, and will not cause derivative cases such as OPV vaccines. At the same time, there is no need for high-level facilities required for IPV vaccine production and testing, which reduces production and quality control costs and can improve vaccine accessibility in low- and middle-income countries. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 It is a schematic diagram of the 18S-MU yeast expression plasmid for recombinantly expressing VP1 or P1 protein in Example 1 and Comparative Examples 1 and 2 of the present disclosure;

[0065] Figure 2 It is a schematic diagram of the 25S-DML yeast expression plasmid for recombinantly expressing VP0 and VP3 proteins or 3CD proteins in Example 1 and Comparative Examples 1 and 2 of the present disclosure;

[0066] Figure 3 This is a graph showing the SDS-PAGE results of the VLP proteins obtained in Example 1 of the present disclosure, wherein lanes 1 and 2 are polio type I VLP proteins, lanes 3 and 4 are polio type II VLP proteins, and lanes 5 and 6 are polio type III VLP proteins;

[0067] Figure 4 The Western-blot result of the VLP protein obtained in Example 1 of the present disclosure is shown, wherein lanes 1, 4 and 7 are negative controls, lanes 2 and 3 are polio type I VLP proteins, lanes 5 and 6 are polio type II VLP proteins, and lanes 8 and 9 are polio type III VLP proteins;

[0068] Figure 5 This is a transmission electron microscopy observation result diagram of poliomyelitis type I, type II and type III VLP proteins obtained in Example 1 of the present disclosure;

[0069] Figure 6 This is a graph showing the results of neutralizing antibody detection in BALB / c mice of the eight recombinant poliomyelitis vaccines in different formulations in Example 3 of the present disclosure;

[0070] Figure 7 This is a graph showing the results of neutralizing antibody detection in Wistar rats after 2 injections of the recombinant poliomyelitis vaccines of 6 different combinations in Example 3 of the present disclosure were given 1 month apart;

[0071] Figure 8 This is a graph showing the results of neutralizing antibody detection in Wistar rats after 2 injections of the recombinant poliomyelitis vaccines of 6 different combinations in Example 3 of the present disclosure at an interval of 2 months;

[0072] Fig. 9 This is a graph showing the results of neutralizing antibody detection in Wistar rats after 3 injections of the recombinant poliomyelitis vaccines of 7 different combinations in Example 3 of the present disclosure;

[0073] Fig.10 This is a graph showing the results of neutralizing antibody detection in SD rats after three injections of the recombinant poliomyelitis vaccines in two different combinations in Example 3 of the present disclosure;

[0074] Fig.11 This is a graph showing the results of neutralizing antibody detection in cynomolgus monkeys after three injections of the recombinant polio vaccines of two different combinations in Example 3 of the present disclosure;

[0075] Fig.12 This is a graph showing the results of cross-neutralizing antibody detection of three batches of vaccines in Example 4 of the present disclosure;

[0076] Fig.13 is a graph showing the neutralizing antibody test results of the effects of different adjuvant contents on the immunogenicity of the vaccine in Example 5 of the present disclosure;

[0077] Fig.14 1 is a transmission electron microscopy observation result diagram of poliomyelitis type II and type III VLPs obtained by using the "P1+3CD" scheme in Comparative Example 1 of the present disclosure;

[0078] Fig.15 It is a graph showing the neutralizing antibody detection results of the "P1+3CD" VLP scheme and the "VP0+VP1+VP3" VLP scheme in Comparative Example 1 of the present disclosure;

[0079] Fig.16 1 is a graph showing the results of SDS-PAGE electrophoresis of VLPs obtained in Comparative Example 2 of the present disclosure, wherein lanes 1, 3 and 5 are graphs showing the results of poliomyelitis type I, type II and type III mVLP detection obtained in Example 1, and lanes 2, 4 and 6 are graphs showing the results of poliomyelitis type I, type II and type III wVLP detection obtained in Comparative Example 2;

[0080] Fig.17 It is a transmission electron microscopy test result diagram of poliomyelitis type I, type II and type III wVLP obtained in comparative example 2 of the present disclosure;

[0081] Fig.18 It is a graph of the neutralizing antibody detection results of wVLP and mVLP in Comparative Example 2 of the present disclosure.

[0082] Sequence Description

[0083] Sequence Listing

[0084]

[0085]

[0086] DETAILED DESCRIPTION

[0087] The present invention discloses a poliomyelitis vaccine and a method for preparing the same. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the same. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention, and relevant personnel can obviously 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.

[0088] In the present invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "include" or its transformations such as "include" or "include" etc. will be understood to include the elements or components stated, without excluding other elements or other components. The term "one (a)" ("a", "an" and "the") includes plural indicators. The term "multiple (multiple)" refers to two (kinds) or more (kinds). The terms "such as", "for example" etc. are intended to refer to exemplary embodiments, and are not intended to limit the scope of the present disclosure.

[0089] In the present disclosure, when a range of values ​​is provided, it should be understood that the endpoints are included in the range and each intervening value between the upper and lower limits of the range and any other specified value or intervening value in the specified range and any value in the smaller range between the specified values ​​are included unless the context clearly dictates otherwise.

[0090] In this disclosure, the term "about" generally refers to variations 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.

[0091] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. The definition 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. The definition of common terms in biochemistry can be found in Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: WH Freeman. The definition 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. The definition 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.

[0092] 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 the following: Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook, etc.

[0093] definition:

[0094] The term "poliovirus" in the present disclosure belongs to the Picornaviridae, Enterovirus genus, Enterovirus type C. It is a non-enveloped single-stranded positive-strand RNA virus with a genome length of about 7.4Kb. The genome contains only one open reading frame, encoding one polyprotein. Under the action of protease, the polyprotein is hydrolyzed to produce three fragments, VP0, VP1 and VP3, which are then assembled into the empty shell structure of the virus particle. In the presence of genomic RNA, VP0 is further hydrolyzed into VP2 and VP4, thereby completing the assembly of mature virus particles. The diameter of the virus particle is 27-30nm, and the compact and dense particle structure is the key for the virus particle to bind to the virus receptor and invade the cell. The expanded structure transformed after heating does not have infectivity and neutralization activity. The poliovirus mainly enters the host cell by endocytosis by binding to the polio receptor PVR (CD155) on the host cell membrane. During this process, the surface conformation of the polio virus changes accordingly, from a fluffy state to a compact state, and the corresponding Canyon domain binds to the virus receptor. As a result, the polio virus has two antigenic forms, namely, the compact particle structure D antigen with immune neutralization activity and the fluffy particle structure C antigen without immune neutralization activity.

[0095] At present, the serotypes of poliovirus include type I, type II and type III. The poliovirus-like particles described in the present disclosure can be derived from wild-type poliovirus strains, such as Salk strain, Saukett strain, Mahoney strain; or can be derived from attenuated poliovirus strains, such as Sabin 1 strain, Sabin 2 strain, Sabin 3 strain.

[0096] The term "virus-like particles" (VLPs) in the present disclosure refers to hollow particles containing one or more structural proteins of a certain virus, which have no viral nucleic acid and cannot replicate autonomously, but are identical or similar in morphology to real virus particles, commonly known as pseudoviruses. VLPs are highly structured protein particles self-assembled by one or more structural proteins of a virus. Its diameter is between 20 and 150 nanometers, maintaining the natural conformation of viral antigen proteins. VLPs can self-assemble into structures such as icosahedrons, rods or spheres. 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 fact that the nucleotides or amino acid residues at the same site of two sequences are exactly the same. It is a basic and important concept in bioinformatics and is usually used to compare the similarity between two nucleic acid sequences or protein sequences. The numerical value of sequence identity is usually expressed as a percentage, which reflects the degree of consistency between sequences.

[0098] 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%, at least 99% of the actual sequence and the sequence described in the present disclosure may be identical, and the different parts may be due to substitution / insertion / deletion mutations introduced artificially or non-artificially, and the artificially introduced mutations may be based on specific purposes, such as easier protein expression, 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, such as immunogenicity.

[0099] The term "open reading frame" (ORF) in the present disclosure refers to a continuous sequence of bases starting from the start codon (usually AUG) of the mRNA sequence and ending at the stop codon (usually UAA, UAG, UGA). This sequence has the potential to encode protein on DNA or mRNA.

[0100] The term "isolated" in this disclosure refers to a substance or entity that is separated from its natural environment or the environment that existed before separation and is separated from other components. The separation ratio can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%. The isolated substances may have different purity levels relative to the substances before their separation.

[0101] The term "vector" in the present disclosure is a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, 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 it carries are expressed 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 (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages 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, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[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 "pharmaceutically acceptable carrier" in the present disclosure may 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, and the like.

[0104] Codon Optimization:

[0105] Codon optimization is an advanced technology that effectively 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, that is, multiple codons can encode the same amino acid, which leads to the possibility of multiple different mRNA sequences for a specific amino acid sequence. However, different organisms or cells have their own preferences when selecting 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, thereby 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.

[0106] There are many methods that can be used to optimize codons. For example, the steps of some methods are: (1) Analyze the codon usage frequency of the host cell: First, you need 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 choosing which synonymous codons to replace the codons in the heterologous gene, and how to adjust the GC content, etc. (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.

[0107] In some embodiments of the present disclosure, codon optimization is performed on the VP0, VP1 and VP3 genes of the poliovirus for the Hansen yeast expression system, wherein the polynucleotides encoding the amino acid sequences of polio type I virus-like particles VP0-I, VP1-I and VP3-I are shown as SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12, respectively; the polynucleotides encoding the amino acid sequences of polio type II virus-like particles VP0-II, VP1-II and VP3-II are shown as SEQ ID No.13, SEQ ID No.14 and SEQ ID No.15, respectively; the polynucleotides encoding the amino acid sequences of polio type III virus-like particles VP0-III, VP1-III and VP3-III are shown as SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18, respectively.

[0108] Recombinant polio vaccine:

[0109] The recombinant polio vaccine described in the present disclosure is a recombinant protein vaccine. It includes the polio virus-like particles produced using recombinant protein technology. When the recombinant polio vaccine is a monovalent vaccine, it may include polio type I virus-like particles, polio type II virus-like particles or polio type III virus-like particles; when the recombinant polio vaccine is a bivalent vaccine, it may include any two selected from polio type I virus-like particles, polio type II virus-like particles or polio type III virus-like particles; when the recombinant polio vaccine is a trivalent vaccine, it may include polio type I virus-like particles, polio type II virus-like particles and polio type III virus-like particles. Since the polio virus-like particles are non-infectious, they do not need to be inactivated before being used as a vaccine.

[0110] The recombinant polio vaccine described in the present disclosure may also include an adjuvant. Non-limiting examples of the adjuvant include, for example, aluminum adjuvants (e.g., aluminum hydroxide, aluminum phosphate), oil-water emulsion adjuvants, AS01 adjuvant system, AS03 adjuvant system, AS04 adjuvant system, MF59, CpG, immunostimulatory substances, immunomodulatory substances, etc. In one embodiment of the present disclosure, the adjuvant is aluminum hydroxide.

[0111] The present disclosure also provides a recombinant polio vaccine preparation, wherein each dose of the recombinant polio vaccine preparation includes about 0.2 μg to 60 μg of the polio type I virus-like particles, about 0.1 μg to 60 μg of the polio type II virus-like particles, or about 0.2 μg to 60 μg of the polio type III virus-like particles. Further, in order to achieve a better effect, in some embodiments of the present disclosure, each dose of the recombinant polio vaccine preparation includes about 0.5 μg to 45 μg of the polio type I virus-like particles, about 0.2 μg to 45 μg of the polio type II virus-like particles, or about 0.5 μg to 45 μg of the polio type III virus-like particles.

[0112] The present disclosure also provides a recombinant polio vaccine preparation, wherein each dose of the recombinant polio vaccine preparation includes at least two selected from about 0.2 μg to 60 μg of the polio type I virus-like particles, about 0.1 μg to 60 μg of the polio type II virus-like particles, or about 0.2 μg to 60 μg of the polio type III virus-like particles. Further, in order to achieve a better effect, in some embodiments of the present disclosure, each dose of the recombinant polio vaccine preparation includes at least two selected from about 0.5 μg to 45 μg of the polio type I virus-like particles, about 0.2 μg to 45 μg of the polio type II virus-like particles, or about 0.5 μg to 45 μg of the polio type III virus-like particles.

[0113] In some embodiments of the present disclosure, the above-mentioned recombinant polio vaccine preparation is in the form of an oral preparation or an intramuscular injection.

[0114] In some embodiments of the present disclosure, the above-mentioned recombinant polio vaccine can also be used in combination with other vaccines, for example, BCG, DTP3 vaccine, measles vaccine, mumps vaccine, rubella vaccine, hepatitis B vaccine, Japanese encephalitis vaccine, meningococcal vaccine, etc.

[0115] The present disclosure also provides a method for preventing or treating poliomyelitis, the method comprising: administering an effective dose of the above-mentioned recombinant poliomyelitis vaccine to a subject. The administration can be oral, intramuscular, intraperitoneal or subcutaneous.

[0116] Example:

[0117] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0118] Example 1: Obtaining recombinant poliomyelitis VLP protein

[0119] 1) Obtaining gene sequences:

[0120] For poliomyelitis type I, such as VP0 with an amino acid sequence as shown in SEQ ID No.1, VP1 with an amino acid sequence as shown in SEQ ID No.2, and VP3 with an amino acid sequence as shown in SEQ ID No.3, poliomyelitis type II, such as VP0 with an amino acid sequence as shown in SEQ ID No.4, VP1 with an amino acid sequence as shown in SEQ ID No.5, and VP3 with an amino acid sequence as shown in SEQ ID No.6, and poliomyelitis type III, such as VP0 with an amino acid sequence as shown in SEQ ID No.7, VP1 with an amino acid sequence as shown in SEQ ID No.8, and VP3 with an amino acid sequence as shown in SEQ ID No.9, gene sequences were optimized according to the codon preference of Hansenula codons. The optimized gene sequences of poliomyelitis type I VP0, VP1 and VP3 are shown in SEQ ID No.10, SEQ ID No.11, and SEQ ID No.12, respectively, and the optimized gene sequences of poliomyelitis type II VP0, VP1 and VP3 are shown in SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23, SEQ ID No.24, SEQ ID No.25, SEQ ID No.26, SEQ ID No.27, SEQ ID No.28, SEQ ID No.29, SEQ ID No.30, SEQ ID No.31, SEQ ID No.32, SEQ ID No.33, SEQ ID No.34, SEQ ID The optimized gene sequences of VP0, VP1 and VP3 of poliomyelitis type III are shown in SEQ ID No. 16, SEQ ID No. 17 and SEQ ID No. 18 respectively. The VP0, VP1 and VP3 genes of poliomyelitis type I, type II or type III are synthesized in their entirety.

[0121] 2) Construction of expression vector:

[0122] The VP1 gene of poliomyelitis type I, type II or type III was inserted into the Hansenula expression vector 18S-MU (eg Figure 1 The expression vector uses the MOX gene promoter and terminator as the promoter and terminator of the target gene, the URA3 gene as the marker gene, the 18S-rDNA gene as the homologous recombination integration arm, and amp as the resistance gene. The VP0 and VP3 genes of poliomyelitis type I, II or III are respectively inserted into the Hansen yeast expression vector 25S-DML (as shown in Figure 2As shown), the expression vector contains two independent open reading frames, one of which uses the MOX gene promoter and terminator as the promoter and terminator of the target gene, and the other uses the DAS gene promoter and terminator as the promoter and terminator of the target gene. The expression vector uses the LEU3 gene as a marker gene, the 25S-rDNA gene as a homologous recombination integration arm, and amp as a resistance gene.

[0123] 3) Obtaining recombinant protein:

[0124] HP-MU plasmid with VP1 gene inserted and HP-DL plasmid with VP0 and VP3 genes inserted were electrotransformed into uracil and leucine double auxotrophic host bacteria (NVSI-HP-105) lacking URA3 and LEU2 genes to construct recombinant bacteria, and ELISA method was used to screen positive yeast strains with high expression of target protein to obtain recombinant strains. After high-density fermentation culture and serial chromatography purification, VLP proteins of poliomyelitis type I, type II or type III were obtained.

[0125] Protein identification: SDS-PAGE was used to detect the target protein (such as Figure 3 As shown in Figure 2, it can be seen that the molecular weight of the target band is basically consistent with the theoretical value. Western-blot detection was performed using specific antibodies against VP1, VP2, VP3 and VP4 (as shown in Figure 2). Figure 4 As shown in the figure, the target band can be specifically bound to the corresponding antibody; the purity of the target protein detected by HPLC is higher than 95%; the protein is granular and the hydrated particle size is about 40-60nm with good uniformity; the protein morphology is observed under transmission electron microscopy (as shown in the figure). Figure 5 The results show that the virus particles have a structure with a particle size of 30-50 nm and good morphology, which confirms that the three structural proteins VP0, VP1 and VP3 can spontaneously assemble VLPs in yeast.

[0126] Example 2: Preparation of recombinant poliomyelitis vaccine

[0127] The polio type I, type II or type III VLP protein with a certain protein content prepared in Example 1 is fully mixed with a certain amount of aluminum hydroxide adjuvant to prepare an adsorbed monovalent stock solution. The three types of adsorbed monovalent stock solutions are mixed in a certain proportion to prepare a vaccine semi-finished product, which is packaged according to a certain volume to obtain a finished vaccine product.

[0128] Example 3: Comparison of immunogenicity of recombinant poliomyelitis vaccines with different compatibility ratios

[0129] Recombinant polio vaccines with different compatibility ratios and contents were prepared and the vaccine immunogenicity was studied in various animal strains, as follows:

[0130] 1) Evaluation of immunogenicity in BALB / c mice

[0131] According to the experimental scheme shown in Table 1, BALB / c mice were inoculated with 8 recombinant poliomyelitis vaccines of different proportions and contents. The sera after immunization were subjected to microneutralization tests using pseudoviruses against three serotypes of polio type I-Mahoney, polio type II-MEF-1 and polio type III-Saukett. The results of neutralizing antibody detection are shown in Tables 2 and Figure 6 As shown, it can be seen that 8 recombinant polio vaccines with different combinations can produce good neutralizing activity against polio types I, II and III in BALB / c mice, and there is no statistical difference between the groups within the same serotype.

[0132] Table 1. In vivo immunogenicity evaluation protocol in BALB / c mice

[0133]

[0134]

[0135] Table 2. Neutralizing antibody GMT results in BALB / c mice

[0136]

[0137] 2) Evaluation of immunogenicity in Wistar rats

[0138] In Wistar rats, we conducted several studies on the interval between immunizations and the number of immunizations for recombinant polio vaccines with different ratios and contents, as follows:

[0139] ①2 injections - 1 month interval between immunizations

[0140] According to the experimental scheme shown in Table 3, 6 recombinant polio vaccines with different compatibility ratios and contents were inoculated in Wistar rats, and the sera after immunization were subjected to microneutralization tests using pseudoviruses against three serotypes of polio type I-Mahoney, polio type II-MEF-1 and polio type III-Saukett. The results of neutralizing antibody detection are shown in Tables 4 and Figure 7 As shown, it can be seen that the six recombinant polio vaccines with different combinations can produce good neutralizing activity against polio types I, II and III in Wistar rats, and there is no statistical difference between the groups within the same serotype.

[0141] Table 3. In vivo immunogenicity evaluation test plan for Wistar rats (2 injections - 1 month interval between immunizations)

[0142]

[0143] Table 4. Neutralizing antibody GMT in Wistar rats (2 injections - 1 month interval between immunizations)

[0144]

[0145] ②2 injections - 2 months between immunizations

[0146] According to the experimental scheme shown in Table 5, 6 recombinant polio vaccines with different compatibility ratios and contents were inoculated in Wistar rats, and the sera after immunization were subjected to microneutralization tests using pseudoviruses against three serotypes of polio type I-Mahoney, polio type II-MEF-1 and polio type III-Saukett. The results of neutralizing antibody detection are shown in Tables 6 and Figure 8 As shown, it can be seen that the six recombinant polio vaccines with different combinations can produce good neutralizing activity against polio types I, II and III in Wistar rats, and there is no statistical difference between the groups within the same serotype.

[0147] Table 5. In vivo immunogenicity evaluation test plan for Wistar rats (2 injections - 2 months between immunizations)

[0148]

[0149]

[0150] Table 6. Neutralizing antibody GMT in Wistar rats (2 injections - 2 months interval between immunizations)

[0151]

[0152] ③3 injections - 1 month interval between immunizations

[0153] According to the test scheme shown in Table 7, 7 recombinant polio vaccines with different compatibility ratios and contents were inoculated in Wistar rats, and inactivated polio vaccine (sIPV) was used as a positive control. The sera after immunization were subjected to microneutralization tests using pseudoviruses against three serotypes of polio type I-Mahoney, polio type II-MEF-1 and polio type III-Saukett. The results of neutralizing antibody detection are shown in Tables 8 and Fig. 9 As shown, it can be seen that the seven recombinant polio vaccines with different combinations can produce good neutralizing activity against polio types I, II and III in Wistar rats, and there is no statistical difference among the groups of recombinant polio vaccines within the same serotype.

[0154] Table 7. In vivo immunogenicity evaluation test plan for Wistar rats (3 injections)

[0155]

[0156]

[0157] Table 8. Neutralizing antibody GMT in Wistar rats (3 injections)

[0158]

[0159] 3) Evaluation of immunogenicity in SD rats

[0160] According to the test scheme shown in Table 9, SD rats were inoculated with two recombinant poliomyelitis vaccines of different compatibility ratios and contents. The sera after immunization were subjected to microneutralization tests using pseudoviruses against three serotypes of polio type I-Mahoney, polio type II-MEF-1 and polio type III-Saukett. The results of neutralizing antibody detection are shown in Tables 10 and Fig.10 As shown, it can be seen that the two recombinant polio vaccines with different combinations can produce good neutralizing activity against polio types I, II and III in SD rats, and there is no statistical difference between the groups within the same serotype.

[0161] Table 9. In vivo immunogenicity evaluation test plan in SD rats

[0162]

[0163]

[0164] Table 10. Neutralizing antibody GMT in SD rats

[0165]

[0166] 4) Evaluation of immunogenicity in cynomolgus monkeys

[0167] According to the experimental scheme shown in Table 11, two recombinant polio vaccines with different compatibility ratios and contents were inoculated in cynomolgus monkeys. The sera after immunization were subjected to microneutralization tests using pseudoviruses against three serotypes of polio type I-Mahoney, polio type II-MEF-1 and polio type III-Saukett. The results of neutralizing antibody detection are shown in Tables 12 and Fig.11 As shown, it can be seen that the two recombinant polio vaccines with different combinations can produce good neutralizing activity against polio types I, II and III in crab-eating macaques, and there is no statistical difference between the groups within the same serotype.

[0168] Table 11. In vivo immunogenicity evaluation test plan for cynomolgus monkeys

[0169]

[0170] Table 12. Neutralizing Antibodies GMT in Cynomolgus Monkeys

[0171]

[0172] Example 4: Evaluation of cross neutralization of recombinant polio vaccine

[0173] According to the experimental scheme shown in Table 13, three batches of recombinant poliomyelitis vaccines were inoculated in Wistar rats, and the sera after immunization were subjected to microneutralization tests using multiple pseudoviruses against three serotypes of polio type I (Mahoney, Sabin1, VDPV-1, XJ-WPV-1), polio type II (MEF-1, Sabin2, AWB-2, VDPV-2) and polio type III (Saukett, Sabin3, AWB-3, AS0-3). The results of neutralizing antibody detection are shown in Tables 14 and Fig.12 As shown, it can be seen that the recombinant polio vaccine can produce good neutralizing activity against the current major representative epidemic strains and has good immunogenicity.

[0174] Table 13. Cross-neutralization assay protocol

[0175]

[0176] Table 14. Cross-neutralizing antibody GMT values

[0177]

[0178] Example 5: Effects of different adjuvant contents on recombinant poliomyelitis vaccines

[0179] The effects of different adjuvant contents (no adjuvant, 100 μg / dose, 300 μg / dose, 500 μg / dose) on the immunogenicity of recombinant polio vaccine were evaluated in Wistar rats. Animal immunization was performed according to the test scheme shown in Table 15. The sera after immunization were subjected to microneutralization test using pseudoviruses against three serotypes of polio type I-Mahoney, polio type II-MEF-1 and polio type III-Saukett. The results of neutralizing antibody detection are shown in Tables 16 and Fig.13 As shown, it can be seen that the absence of adjuvant and the addition of different amounts of adjuvant can stimulate the production of a certain level of neutralizing antibodies in rats, and the addition of adjuvant can greatly increase the level of neutralizing antibodies.

[0180] Table 15. Experimental scheme with different adjuvant contents

[0181]

[0182] Table 16. Neutralizing antibody GMT values ​​at different adjuvant contents

[0183]

[0184] Comparative Example 1: Comparison of recombinant poliomyelitis VLP expression strategies

[0185] VLPs were obtained by co-expression of P1 protein and 3CD protein. Specifically, the genes encoding P1 protein and 3CD protein were optimized according to the codon preference of Hansenula, and the optimized P1 target gene (polio type I P1 gene sequence is shown in SEQ ID No.19, polio type II P1 gene sequence is shown in SEQ ID No.20, and polio type III P1 gene sequence is shown in SEQ ID No.21) replaced the VP1 gene in the 18S-MU plasmid, and the optimized 3CD protein target gene (polio type I 3CD gene sequence is shown in SEQ ID No.22, polio type II 3CD gene sequence is shown in SEQ ID No.23, and polio type III 3CD gene sequence is shown in SEQ ID No.24) replaced the VP0 gene in the 25S-DML plasmid, and the reading frame expressing the VP3 protein was knocked out. The plasmids containing P1 and 3CD genes were co-transformed into the uracil and leucine double auxotrophic host bacteria (NVSI-HP-105) lacking the URA3 and LEU2 genes to construct recombinant bacteria. The ELISA method was used to screen the positive yeast strains with high expression of the target protein, and the recombinant strains were obtained. The target protein was obtained after high-density fermentation culture and series of chromatography purification. After repeated experiments, VLP proteins were obtained for types II and III, but not for type I, which may be related to the low efficiency of 3CD enzyme cleavage and the unsuccessful assembly of particles. The obtained VLP proteins were observed by transmission electron microscopy (such as Fig.14 As shown), VLPs with a diameter of 30-50 nm were formed.

[0186] The VLP proteins obtained by the "P1+3CD" scheme for poliomyelitis type II and type III and the VLP proteins obtained by the "VP0+VP1+VP3" co-expression scheme were mixed with aluminum hydroxide adjuvant at a certain dose, and BALB / c mice were immunized and blood was collected according to the test scheme shown in Table 17. The sera after immunization were subjected to microneutralization tests against polio type II-MEF-1 and polio type III-Saukett serotype pseudoviruses. The neutralizing antibody test results are shown in Tables 18 and 19. Fig.15 As shown, it can be seen that the neutralizing antibody GMT values ​​of the "VP0+VP1+VP3" expression strategy of type II and type III VLPs are higher than those of the "P1+3CD" scheme, and the difference is statistically significant.

[0187] Table 17. Experimental scheme for evaluating immunogenicity of VLPs with different expression strategies

[0188]

[0189] Table 18. GMT values ​​of neutralizing antibodies in VLPs with different expression strategies

[0190]

[0191] Comparative Example 2: Comparison of proteins with different sequence structures

[0192] The polio virus itself has D antigen and C antigen distinction, and the immunogenicity of VLPs composed of different VP0, VP1 and VP3 amino acid sequences is different. The same technical solution as in Example 1 was used to reconstruct VLPs composed of different VP0, VP1 and VP3 amino acid sequences, as follows:

[0193] For poliomyelitis type I, such as VP0 with an amino acid sequence as shown in SEQ ID No.25, VP1 with an amino acid sequence as shown in SEQ ID No.26, and VP3 with an amino acid sequence as shown in SEQ ID No.27, poliomyelitis type II, such as VP0 with an amino acid sequence as shown in SEQ ID No.28, VP1 with an amino acid sequence as shown in SEQ ID No.29, and VP3 with an amino acid sequence as shown in SEQ ID No.30, and poliomyelitis type III, such as VP0 with an amino acid sequence as shown in SEQ ID No.31, VP1 with an amino acid sequence as shown in SEQ ID No.32, and VP3 with an amino acid sequence as shown in SEQ ID No.33, gene sequences were optimized according to the codon preference of Hansenula codons. The optimized gene sequences of poliomyelitis type I VP0, VP1 and VP3 are shown in SEQ ID No.34, SEQ ID No.35, and SEQ ID No.36, respectively, and the optimized gene sequences of poliomyelitis type II VP0, VP1 and VP3 are shown in SEQ ID No.37, SEQ ID No.38, SEQ ID No.39, SEQ ID As shown in No.39, the optimized gene sequences of poliomyelitis type III VP0, VP1 and VP3 are shown in SEQ ID No.40, SEQ ID No.41 and SEQ ID No.42, respectively. The above poliomyelitis type I, type II or type III VP0, VP1 and VP3 genes are fully synthesized. A yeast expression plasmid is constructed according to the expression vector construction method shown in Example 1, and the yeast expression plasmid is electrotransformed into a uracil and leucine double auxotrophic host bacteria (NVSI-HP-105) lacking the URA3 and LEU2 genes to construct a recombinant bacterium. The ELISA method is used to screen positive yeast strains with high expression of the target protein to obtain a recombinant strain. The target protein is obtained after high-density fermentation culture and a series of chromatographic purifications, and the target protein is electrophoresed by SDS-PAGE (as shown in Fig.16As shown in the figure, it can be seen that the molecular weight of the target band is basically consistent with the theoretical value; the purity of the target protein detected by HPLC is higher than 95%; the protein is granular and the hydrated particle size is about 40-60nm with good uniformity as detected by dynamic light scattering; the protein morphology is observed under transmission electron microscopy (as shown in the figure Fig.17 As shown), it presents a virus particle structure with a particle size between 30-50nm and good morphology.

[0194] The VLP obtained in Comparative Example 2 was named wVLP, and the VLP obtained in Example 1 was named mVLP. The wVLP and mVLP were mixed with aluminum hydroxide adjuvant at a certain dose, and Wistar rats were immunized and blood was collected according to the test scheme shown in Table 19. The sera after immunization were subjected to microneutralization tests using pseudoviruses against three serotypes of polio type I-Mahoney, polio type II-MEF-1, and polio type III-Saukett. The neutralizing antibody test results are shown in Tables 20 and 21. Fig.18 As shown, it can be seen that the neutralizing antibody GMT values ​​of the mVLP regimen for polio types I, II and III are all higher than those of the wVLP regimen, among which types II and III have statistical differences.

[0195] Table 19. Experimental scheme for evaluating immunogenicity of VLPs obtained from different structural protein sequences

[0196]

[0197]

[0198] Table 20. GMT values ​​of VLP neutralizing antibodies obtained with different structural protein sequences

[0199]

[0200] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A poliovirus-like particle, characterized in that: The virus-like particle is: (1) Poliovirus type I virus-like particles, It consists of VP0-I as shown in the amino acid sequence of SEQ ID No.1, VP1-I as shown in the amino acid sequence of SEQ ID No.2, and VP3-I as shown in the amino acid sequence of SEQ ID No.3; or (2) Poliovirus type II virus-like particles, It consists of VP0-II as shown in the amino acid sequence of SEQ ID No.4, VP1-II as shown in the amino acid sequence of SEQ ID No.5, and VP3-II as shown in the amino acid sequence of SEQ ID No.6; or (3) Poliovirus type III virus-like particles, It consists of VP0-III with an amino acid sequence as shown in SEQ ID No.7, VP1-III with an amino acid sequence as shown in SEQ ID No.8 and VP3-III with an amino acid sequence as shown in SEQ ID No.

9.

2. A composition of poliovirus-like particles, characterized in that: The composition comprises at least two selected from the polio type I virus-like particles, polio type II virus-like particles or polio type III virus-like particles as described in claim 1.

3. The composition according to claim 2, characterized in that The mass ratio of polio type I virus-like particles, polio type II virus-like particles and polio type III virus-like particles in the composition is 1:0.2:1-1:2:

6.

4. The composition according to claim 2, characterized in that The content of polio type I virus-like particles in the composition is 0.5 μg to 45 μg, the content of polio type II virus-like particles is 0.2 μg to 45 μg, and the content of polio type III virus-like particles is 0.5 μg to 45 μg.

5. A method for producing poliovirus-like particles, characterized in that: include: Step 1) Cloning the genes of VP0-I, VP1-I and VP3-I described in claim 1 into the same or different vectors, respectively, wherein the genes of VP0-I, VP1-I and VP3-I have independent open reading frames; or The genes of VP0-II, VP1-II and VP3-II described in claim 1 are cloned into the same or different vectors, respectively, and the genes of VP0-II, VP1-II and VP3-II have independent open reading frames; or The genes of VP0-III, VP1-III and VP3-III described in claim 1 are cloned into the same or different vectors, respectively, and the genes of VP0-III, VP1-III and VP3-III have independent open reading frames; Step 2) transferring the vector obtained in step 1) into a host cell for expression and assembly, and purifying the expression product to obtain the polio virus-like particles.

6. The method according to claim 5, characterized in that Step 2) The expression method is biological fermentation.

7. A method for producing a composition of poliovirus-like particles, characterized in that: include: Step 1) Cloning the genes of VP0-I, VP1-I and VP3-I described in claim 1 into the same or different vectors, respectively, wherein the genes of VP0-I, VP1-I and VP3-I have independent open reading frames; The genes of VP0-II, VP1-II and VP3-II described in claim 1 are cloned into the same or different vectors, respectively, and the genes of VP0-II, VP1-II and VP3-II have independent open reading frames; and The genes of VP0-III, VP1-III and VP3-III described in claim 1 are cloned into the same or different vectors, respectively, and the genes of VP0-III, VP1-III and VP3-III have independent open reading frames; Step 2) transferring the vector obtained in step 1) into a host cell for expression and assembly, and purifying the expression product to obtain the poliovirus-like particles; Step 3) at least two of the polio type I virus-like particles, polio type II virus-like particles or polio type III virus-like particles obtained in step 2) are mixed in proportion.

8. An isolated polynucleotide, characterized in that The polynucleotide comprises a nucleic acid sequence encoding the amino acid sequence as set forth in claim 1.

9. The polynucleotide according to claim 8, characterized in that The polynucleotides encoding the amino acid sequences of polio type I virus-like particles VP0-I, VP1-I and VP3-I are shown as SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12, respectively; the polynucleotides encoding the amino acid sequences of polio type II virus-like particles VP0-II, VP1-II and VP3-II are shown as SEQ ID No.13, SEQ ID No.14 and SEQ ID No.15, respectively; the polynucleotides encoding the amino acid sequences of polio type III virus-like particles VP0-III, VP1-III and VP3-III are shown as SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18, respectively.

10. A separated vector, characterized in that The vector comprises the polynucleotide according to claim 8 or 9, Among them, the polynucleotides encoding the amino acid sequences of polio type I virus-like particles VP0-I, VP1-I and VP3-I are respectively operably connected to the promoter, the polynucleotides encoding the amino acid sequences of polio type II virus-like particles VP0-II, VP1-II and VP3-II are respectively operably connected to the promoter, and the polynucleotides encoding the amino acid sequences of polio type III virus-like particles VP0-III, VP1-III and VP3-III are respectively operably connected to the promoter.

11. An isolated host cell, characterized in that The host cell comprises the polynucleotide according to claim 8 or 9, or the vector according to claim 10.

12. The host cell according to claim 11, characterized in that The host cell is a Hansenula cell, an Escherichia coli cell, a Pichia pastoris cell, a Saccharomyces cerevisiae cell, a mammalian cell, an avian cell or an insect cell.

13. The host cell according to claim 12, characterized in that The host cell is a Hansenula cell.

14. A recombinant protein vaccine, characterized in that: The recombinant protein vaccine comprises the poliovirus-like particle according to claim 1, or the combination of poliovirus-like particles according to claim 2, 3 or 4, and optionally comprises an adjuvant.

15. The recombinant protein vaccine according to claim 14, 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.

16. The recombinant protein vaccine according to claim 15, characterized in that The adjuvant is aluminum hydroxide.

17. The recombinant protein vaccine according to claim 16, characterized in that The content of aluminum hydroxide in each dose of the recombinant protein vaccine is 0 μg to 500 μg.

18. The recombinant protein vaccine according to claim 17, characterized in that The content of aluminum hydroxide in each dose of the recombinant protein vaccine is 300 μg.

19. A genetically engineered vector vaccine, characterized in that: The genetically engineered vector vaccine comprises the vector as claimed in claim 10.

20. A nucleic acid vaccine, characterized in that: The nucleic acid vaccine comprises the polynucleotide according to claim 8 or 9.

21. A liposome complex, characterized in that: The liposome complex comprises the polynucleotide of claim 8 or 9.

22. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the vaccine as described in any one of claims 14 to 20, or the lipid complex as described in claim 21, and a pharmaceutically acceptable carrier, delivery medium, excipient, stabilizer, diluent or a combination thereof.

23. Use of the poliovirus-like particle according to claim 1, the composition according to claim 2, 3 or 4, the isolated polynucleotide according to claim 8 or 9, the isolated vector according to claim 10, the isolated host cell according to any one of claims 11 to 13, the vaccine according to any one of claims 14 to 20, the liposome complex according to claim 21 or the pharmaceutical composition according to claim 22 in the preparation of a medicament for preventing or treating a disease caused by poliovirus, characterized in that: The poliovirus is at least one selected from polio type I virus, polio type II virus or polio type III virus.

24. A method for detecting poliomyelitis-specific antibodies in a sample, characterized in that: include: Step 1) making the poliovirus-like particles described in any one of claims 1 to 4 carry a detectable label; Step 2) contacting the sample in solution with the labeled poliovirus-like particles obtained in step 1); Step 3) Detecting polio virus-like particles in the immune complex formed in the above steps.

25. A kit, characterized in that The kit comprises the poliovirus-like particle according to claim 1, or the composition according to claim 2, 3 or 4.

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