Polio vaccine and method for preparing same

Abstract

Provided are a polio vaccine and a method for preparing same. Immunization with vaccines made from poliovirus-like particles can induce high levels of neutralizing antibodies against poliovirus types I, II, and III, exhibiting broad-spectrum neutralizing activity against widely prevalent strains at present. The recombinant polio vaccine does not rely on live viruses. There are no live viruses involved in the vaccine production and testing processes, posing no biosafety risks or risks of inducing OPV vaccine-derived conditions. Also, the vaccine requires no high-grade facilities needed for the production and testing of IPV vaccines, which reduces the costs of production and quality control and improves the accessibility of the vaccine in mid- and low-income countries.

Description

A polio vaccine and its preparation method

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202411721937.8, filed on November 28, 2024, entitled "A Polio Vaccine and a Method for Preparing the Same", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of biomedicine, and in particular, to a polio vaccine and a method for preparing the same. Background Technology

[0004] Poliomyelitis is an acute infectious disease caused by the poliovirus. After the virus invades the nervous system, it damages motor nerve cells, and in severe cases, it can cause flaccid paralysis of the limbs. It is more common in children, hence the name polio. It is a disease that terrifies the world. It has a sudden onset and can cause lifelong paralysis. Currently, there is no effective drug to treat poliovirus infection, and prevention is mainly achieved through vaccination.

[0005] Currently used vaccines, oral live attenuated vaccine (OPV) and inactivated vaccine (IPV), have played a significant role in polio prevention and control. Global efforts to combat polio have achieved remarkable success, making polio a potential second virus to be completely eradicated after smallpox. However, the use of live viruses in the production and testing of OPV and IPV vaccines poses a biosafety risk of virus leakage. Therefore, developing novel, non-infectious vaccines using genetic engineering technology is crucial for increasing polio vaccine production capacity and advancing the global eradication of polio. Currently, numerous studies on recombinant polio vaccines exist worldwide, but they are hampered by low structural protein expression levels, unreliable 3CD enzyme activity, or unstable antigen structures, resulting in mostly inactive protein forms of the obtained antigens. Therefore, addressing these issues has become a pressing priority in this field. Summary of the Invention

[0006] Technical problems to be solved:

[0007] One aspect of this disclosure is to provide a polio vaccine and its preparation method, addressing the problems of weak immunogenicity, low activity, and instability of recombinant polio antigens in the prior art.

[0008] Specifically, this disclosure successfully prepared polio virus-like particles (VLPs) using novel structural protein sequences and expression strategies for polio types I, II, and III. Experimental verification shows that the polio virus-like particles and their compositions can solve the technical problems of weak immunogenicity, low activity, and instability of recombinant polio antigens.

[0009] Technical solutions provided:

[0010] A polio virus-like particle, wherein the virus-like particle is:

[0011] (1) Poliovirus type I-like particles

[0012] It includes amino acid sequences such as 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 amino acid sequences that have 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 have the same or similar biological functions; or

[0013] (2) Poliovirus type II-like particles

[0014] It includes amino acid sequences such as 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 amino acid sequences that have 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 have the same or similar biological functions; or

[0015] (3) Poliovirus type III-like particles

[0016] It includes amino acid sequences such as VP0-III shown in SEQ ID No. 7, VP1-III shown in SEQ ID No. 8, and VP3-III shown in SEQ ID No. 9, or amino acid sequences that have 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 have the same or similar biological functions.

[0017] In some embodiments of this disclosure, the poliovirus-like particles may further contain other polypeptides, the addition of which does not affect the biological activity of the poliovirus-like particles. The biological activity is primarily immunogenicity. Examples of these 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 the secretory expression of proteins in host cells; and polypeptides for other specific functions, such as polypeptides for enhancing stability, solubility, signal transduction, etc.

[0018] In this disclosure, in order to better achieve the purpose of this disclosure, the amino acid sequences of VP0, VP1, and VP3 in the three types of poliovirus-like particles mentioned above have at least one modification compared with the corresponding wild type. This modification can be the deletion or substitution of an amino acid.

[0019] In other embodiments of this disclosure, the virus-like particles may be:

[0020] (1) Poliovirus type I-like particles

[0021] It is composed of amino acid sequences such as 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 amino acid sequences that have 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 have the same or similar biological functions; or

[0022] (2) Poliovirus type II-like particles

[0023] It is composed of amino acid sequences such as 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 amino acid sequences that have 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 have the same or similar biological functions; or

[0024] (3) Poliovirus type III-like particles

[0025] It is composed of amino acid sequences such as VP0-III as shown in SEQ ID No. 7, VP1-III as shown in SEQ ID No. 8, and VP3-III as shown in SEQ ID No. 9, or amino acid sequences that have 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 have the same or similar biological functions.

[0026] Another aspect of this disclosure is to provide a composition of poliovirus-like particles, the composition comprising at least two selected from the above-described poliovirus-like particles type I, poliovirus-like particles type II, or poliovirus-like particles type III. In some embodiments of this disclosure, the composition may include, for example, (1) the poliovirus-like particles type I and the poliovirus-like particles type II; (2) the poliovirus-like particles type II and the poliovirus-like particles type III; or (3) the poliovirus-like particles type I and the poliovirus-like particles type III. Similarly, the poliovirus-like particles in the above composition may also contain other polypeptides, the addition of which does not affect the biological activity of the poliovirus-like particles.

[0027] In some embodiments of this disclosure, the mass ratio of poliovirus type I, poliovirus type II, and poliovirus type III particles in the above composition can be from 1:0.2:1 to 1:2:6. For example, ratios such as 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, and 1:1:6 are used.

[0028] In some embodiments of this disclosure, the content of poliovirus type I (PCV1)-like particles in the above-described composition can be 0.5 μg to 45 μg, the content of PCV2 (PCV2)-like particles can be 0.2 μg to 45 μg, and the content of PCV3 (PCV3)-like particles can be 0.5 μg to 45 μg. For example, the content of PCV1-like particles can be 0.5 μg, 0.8 μg, 1 μg, 2 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, etc.; the content of PCV2-like particles can be 0.2 μg, 0.4 μg, 0.5 μg, 0.8 μg, 1 μg, 2 μg, etc. The concentrations of poliovirus type III (PVIII)-like particles can be 0.5 μg, 0.8 μg, 1 μg, 2 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, etc.

[0029] Another aspect of this disclosure is to provide a method for generating poliovirus-like particles, comprising:

[0030] Step 1) Clone the genes of VP0-I, VP1-I, and VP3-I into the same or different vectors, respectively, wherein each gene of VP0-I, VP1-I, and VP3-I has an independent open reading frame; or

[0031] The genes of VP0-II, VP1-II, and VP3-II were cloned into the same or different vectors, each of which has an independent open reading frame; or

[0032] The genes of VP0-III, VP1-III and VP3-III were cloned into the same or different vectors, and the genes of VP0-III, VP1-III and VP3-III each had an independent open reading frame;

[0033] Step 2) The vector obtained in Step 1) is transferred to the host cell for expression and assembly. The expression product is purified to obtain the poliovirus-like particles.

[0034] In this disclosure, the above-described expression strategy for poliovirus-like particles can be widely used in the preparation of poliovirus-like particles, or it can be specifically used in the preparation of poliovirus-like particles in this disclosure.

[0035] In some embodiments of this disclosure, the genes of VP0, VP1 and VP3 in the above method can be cloned into three different vectors, or into the same vector, or cloned in pairs into one vector and simultaneously expressed independently in the host cell and assembled into virus-like particles.

[0036] The host cell described above can be a host cell capable of expressing the poliovirus-like particle protein described in this disclosure, including cultured prokaryotic cells or cultured eukaryotic cells. Examples of the host cell include, but are not limited to, Hansenula polymorpha 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), and avian cells (e.g., chicken embryo cells, duck cells). In one embodiment of this disclosure, the host cell is a Hansenula polymorpha cell.

[0037] For industrial-scale production, in some embodiments of this disclosure, the expression in the above method can be expressed on a large scale using a bio-fermentation method.

[0038] Another aspect of this disclosure is a method for providing a composition for producing poliovirus-like particles, comprising:

[0039] Step 1) Clone the genes of VP0-I, VP1-I, and VP3-I into the same or different vectors, respectively, wherein each gene of VP0-I, VP1-I, and VP3-I has an independent open reading frame; or

[0040] The genes of VP0-II, VP1-II, and VP3-II were cloned into the same or different vectors, each of which has an independent open reading frame; or

[0041] The genes of VP0-III, VP1-III and VP3-III were cloned into the same or different vectors, and the genes of VP0-III, VP1-III and VP3-III each had an independent open reading frame;

[0042] Step 2) The vector obtained in Step 1) is transferred into the host cell for expression and assembly. The expression product is purified to obtain the poliovirus-like particles.

[0043] Step 3) Mix at least two of the poliovirus type I, poliovirus type II, or poliovirus type III particles obtained in step 2) in a certain proportion.

[0044] Another aspect of this disclosure is to provide an isolated polynucleotide comprising a nucleic acid sequence encoding the above-described amino acid sequence.

[0045] In one embodiment of this disclosure, the polynucleotides encoding the amino acid sequences of poliovirus type I virus-like particles VP0-I, VP1-I, and VP3-I are shown in SEQ ID No. 10, SEQ ID No. 11, and SEQ ID No. 12, respectively; the polynucleotides encoding the amino acid sequences of poliovirus type II virus-like particles VP0-II, VP1-II, and VP3-II are shown in SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15, respectively; and the polynucleotides encoding the amino acid sequences of poliovirus type III virus-like particles VP0-III, VP1-III, and VP3-III are shown in SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 18, respectively.

[0046] Another aspect of this disclosure is to provide an isolated vector containing the aforementioned polynucleotides.

[0047] The polynucleotides encoding the amino acid sequences of poliovirus type I (PVI)-like particles VPO-I, PV1-I, and PV3-I are operatively linked to promoters, the polynucleotides encoding the amino acid sequences of poliovirus type II (PVI)-like particles VPO-II, PV1-II, and PV3-II are operatively linked to promoters, and the polynucleotides encoding the amino acid sequences of poliovirus type III (PVI)-like particles VPO-III, PV1-III, and PV3-III are operatively linked to promoters.

[0048] Another aspect of this disclosure is to provide an isolated host cell containing the aforementioned polynucleotide or the aforementioned vector;

[0049] Preferably, in some embodiments of this disclosure, the host cell may be Hansenula polymorpha 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), avian cells (e.g., chicken embryo cells, duck cells), or insect cells (e.g., Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, HIGH FIVE cells);

[0050] More preferably, in one embodiment of this disclosure, the host cell is a Hansenula polymorpha cell.

[0051] Another aspect of this disclosure is to provide a recombinant protein vaccine comprising the above-described poliovirus-like particles, or a composition of the above-described poliovirus-like particles, and optionally including an adjuvant.

[0052] In some embodiments of this disclosure, the adjuvant is at least one selected from aluminum adjuvants, oil-water emulsion adjuvants, AS01 adjuvant systems, AS03 adjuvant systems, AS04 adjuvant systems, MF59, CpG, immunostimulatory substances, or immunomodulatory substances. Further, in some embodiments of this disclosure, the adjuvant is aluminum hydroxide. Further, in some embodiments of this disclosure, the aluminum hydroxide content in each dose of the recombinant protein vaccine is from 0 μg to 500 μg. Further, in one embodiment of this disclosure, the aluminum hydroxide content in each dose of the recombinant protein vaccine is 300 μg.

[0053] Another aspect of this disclosure is to provide a genetically engineered vector vaccine comprising the aforementioned vector.

[0054] Another aspect of this disclosure is to provide a nucleic acid vaccine comprising the aforementioned polynucleotides.

[0055] Another aspect of this disclosure is to provide a liposome complex comprising the aforementioned polynucleotide.

[0056] Another aspect of this disclosure is to provide a pharmaceutical composition comprising the above-described vaccine or the above-described liposome complex, and a pharmaceutically acceptable carrier, delivery medium, excipient, stabilizer, diluent, or combination thereof.

[0057] Another aspect of this disclosure is the use of the above-described poliovirus-like particles, the above-described composition, the above-described isolated polynucleotides, the above-described isolated vectors, the above-described isolated host cells, the above-described vaccine, the above-described liposome complex, or the above-described pharmaceutical composition in the preparation of a medicament for the prevention or treatment of diseases caused by poliovirus, wherein the poliovirus is at least one selected from poliovirus type I, poliovirus type II, or poliovirus type III.

[0058] Another aspect of this disclosure is to provide a method for detecting polio-specific antibodies in a sample, comprising:

[0059] Step 1) To attach a detectable tag to the above-mentioned poliovirus-like particles;

[0060] Step 2) Contact the sample in solution with the tagged poliovirus-like particles obtained in Step 1).

[0061] Step 3) Detect poliovirus-like particles in the immune complexes formed in the above steps.

[0062] Another aspect of this disclosure is to provide a kit comprising the above-described poliovirus-like particles or the above-described composition.

[0063] This disclosure also provides the use of the above-described kit in detecting polio-specific antibodies in samples. Beneficial effects:

[0064] Immunization using the poliovirus-like particle vaccine disclosed herein can produce high levels of neutralizing antibodies against polio types I, II, and III, exhibiting broad-spectrum neutralizing activity against currently prevalent strains. The recombinant polio vaccine of this disclosure does not rely on live virus; the vaccine production and testing processes are virus-free, posing no biosafety risks and preventing the occurrence of derivative cases as seen with OPV vaccines. Furthermore, it eliminates the need for high-level facilities required for IPV vaccine production and testing, reducing production and quality control costs and improving vaccine accessibility in low- and middle-income countries. Attached Figure Description

[0065] Figure 1 is a schematic diagram of the 18S-MU yeast expression plasmids that recombinantly express VP1 or P1 protein in Example 1 and Comparative Examples 1 and 2 of this disclosure.

[0066] Figure 2 is a schematic diagram of the 25S-DML yeast expression plasmids that recombinantly express VP0 and VP3 proteins or 3CD proteins in Example 1 and Comparative Examples 1 and 2 of this disclosure.

[0067] Figure 3 is an SDS-PAGE result diagram of VLP protein obtained in Example 1 of this disclosure, where channels 1 and 2 are polio type I VLP protein, channels 3 and 4 are polio type II VLP protein, and channels 5 and 6 are polio type III VLP protein.

[0068] Figure 4 shows the Western blot results of VLP protein obtained in Example 1 of this disclosure, where channels 1, 4 and 7 are negative controls, channels 2 and 3 are polio type I VLP protein, channels 5 and 6 are polio type II VLP protein, and channels 8 and 9 are polio type III VLP protein.

[0069] Figure 5 shows the transmission electron microscopy observation results of VLP proteins of poliomyelitis type I, II and III obtained in Embodiment 1 of this disclosure;

[0070] Figure 6 shows the results of neutralizing antibody detection in BALB / c mice for eight different combinations of recombinant polio vaccines in Example 3 of this disclosure.

[0071] Figure 7 shows the results of neutralizing antibody detection in Wistar rats after two immunizations with recombinant polio vaccines of six different combinations in Example 3 of this disclosure, administered one month apart.

[0072] Figure 8 shows the results of neutralizing antibody detection in Wistar rats after two immunizations with recombinant polio vaccines of six different combinations in Example 3 of this disclosure, administered at two-month intervals.

[0073] Figure 9 shows the results of neutralizing antibody detection in Wistar rats after three immunizations with recombinant polio vaccines of seven different combinations in Example 3 of this disclosure.

[0074] Figure 10 shows the results of neutralizing antibody detection in SD rats after three immunizations with recombinant polio vaccines of two different formulations in Example 3 of this disclosure.

[0075] Figure 11 shows the results of neutralizing antibody detection in cynomolgus monkeys after three doses of recombinant polio vaccine with two different compatibility methods in Example 3 of this disclosure.

[0076] Figure 12 shows the cross-neutralizing antibody detection results of three batches of vaccines in Example 4 of this disclosure;

[0077] Figure 13 is a graph showing the results of neutralizing antibody detection on the effect of different adjuvant contents on vaccine immunogenicity in Example 5 of this disclosure;

[0078] Figure 14 is a transmission electron microscopy observation of type II and type III poliomyelitis VLP obtained using the “P1+3CD” scheme in Comparative Example 1 of this disclosure;

[0079] Figure 15 shows the results of neutralizing antibody detection in the “P1+3CD” VLP protocol and the “VP0+VP1+VP3” VLP protocol in Comparative Example 1 of this disclosure;

[0080] Figure 16 shows the VLPSDS-PAGE electrophoresis detection results obtained in Comparative Example 2 of this disclosure, where channels 1, 3 and 5 are the mVLP detection results of poliomyelitis type I, II and III obtained in Example 1, and channels 2, 4 and 6 are the wVLP detection results of poliomyelitis type I, II and III obtained in Comparative Example 2.

[0081] Figure 17 is a transmission electron microscopy (WVLP) image of poliomyelitis type I, II and III obtained in Comparative Example 2 of this disclosure.

[0082] Figure 18 shows the detection results of neutralizing antibodies against wVLP and mVLP in Comparative Example 2 of this disclosure.

[0083] Sequence Description

[0084] sequence list Detailed Implementation

[0085] This invention discloses a polio vaccine and its preparation method. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the vaccine. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately change and combine the content described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0086] In this invention, unless otherwise stated, 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 "comprising" or its variations such as "including" or "comprising of," etc., shall be understood to include the stated elements or components without excluding other elements or other components. The term "a," "an," and "the" includes plural indicators. The term "a plurality of" 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.

[0087] In this disclosure, when a range of values ​​is provided, it should be understood that, unless the context otherwise explicitly indicates otherwise, the range includes endpoints and each intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range and any value within a smaller range between specified values.

[0088] In this disclosure, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

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

[0090] Unless otherwise specified, the experimental techniques used in this paper employ standard techniques from 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* and *Cell Biology: A Laboratory Handbook*.

[0091] definition:

[0092] The term "poliovirus" in this disclosure belongs to the Picornaviridae family, Enterovirus genus, and Enterovirus type C. It is a non-enveloped, single-stranded, positive-sense RNA virus with a genome length of approximately 7.4 kb. The genome contains only one open reading frame encoding a single polyprotein. This polyprotein is hydrolyzed by proteases to produce three fragments: VP0, VP1, and VP3, which then assemble into the empty shell structure of the viral particle. In the presence of genomic RNA, VP0 is further hydrolyzed into VP2 and VP4, thus completing the assembly of the mature viral particle. The viral particle has a diameter of 27-30 nm. Its compact and dense particle structure is crucial for binding to viral receptors and invading cells; the expanded structure that transforms after heating does not possess infectivity or neutralizing activity. Poliovirus primarily enters host cells through endocytosis by binding to the polio receptor PVR (CD155) on the host cell membrane. During this process, the surface conformation of the poliovirus undergoes a corresponding change, transforming from a swollen state to a compact state, allowing it to bind to the viral receptor via the corresponding Canyon domain. This results in the existence of two antigenic forms of the poliovirus: the compact granular D antigen, which has immune-neutralizing activity, and the swollen granular C antigen, which has no immune-neutralizing activity.

[0093] Currently, poliovirus serotypes include type I, type II, and type III. The poliovirus-like particles described in this disclosure can be derived from wild-type poliovirus strains, such as the salk strain, the Saukett strain, and the Mahoney strain; or from attenuated poliovirus strains, such as the Sabin 1 strain, the Sabin 2 strain, and the Sabin 3 strain.

[0094] The term "virus-like particle" (VLP) in this disclosure refers to a hollow particle containing one or more structural proteins of a virus. It lacks viral nucleic acid, cannot replicate autonomously, but is morphologically identical or similar to a true viral particle; commonly known as a pseudovirus. VLPs are highly structured protein particles that are self-assembled from one or more structural proteins of a virus. Their diameter ranges from 20 to 150 nanometers, maintaining the native conformation of viral antigen proteins. VLPs can self-assemble into icosahedral, rod-shaped, or spherical structures. VLPs can be artificially obtained through recombinant expression in prokaryotic cell lines, yeast cell systems, plant cells, animal cells, etc.

[0095] The term "identity" or "sequence identity" in this disclosure refers to the complete similarity of two sequences at the same site, consisting of identical nucleotide or amino acid residues. It is a fundamental and important concept in bioinformatics, commonly used to compare the degree of similarity between two nucleic acid or protein sequences. Sequence identity is typically expressed as a percentage, reflecting the degree of consistency between sequences.

[0096] In some embodiments of this disclosure, "having more than 90% identity and having the same or similar biological function" generally means that the actual sequence and the sequence described in this disclosure can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. The differences can be due to artificially or non-artificially introduced substitution / insertion / deletion mutations. Artificially introduced mutations can be based on a specific purpose, such as facilitating protein expression. However, the introduction of these differences is insufficient to alter the biological function of the original protein represented by the amino acid sequence, such as immunogenicity.

[0097] The term "open reading frame" (ORF) in this disclosure refers to a continuous sequence of bases that begins at a start codon (usually AUG) and ends at a stop codon (usually UAA, UAG, or UGA) in an mRNA sequence. This sequence, on DNA or mRNA, has the potential to encode a protein.

[0098] The term "separated" in this disclosure refers to a substance or entity that has been removed from its natural environment or the environment in which it existed before separation and is separate 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%, or at least 90%. Separated substances may have different purity levels relative to their pre-separated counterparts.

[0099] The term "vector" as used in this disclosure refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain a replication initiation site.

[0100] The term "host cell" in this disclosure refers to a cell in which nucleic acid molecules have been introduced using molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and accelerated introduction of naked DNA via electroporation, lipid transfection, and particle gun techniques.

[0101] The term "pharmaceutically acceptable carrier" in this disclosure can refer to any pharmaceutically permissible additive, such as physiological saline, cell culture medium, glucose, water for injection, glycerol, amino acids and combinations thereof, stabilizers, surfactants, preservatives, isotonic agents, etc.

[0102] Codon optimization:

[0103] Codon optimization is an advanced technique that effectively enhances protein expression levels in organisms by improving the translation efficiency of target genes. In organisms, codon degeneracy—meaning multiple codons can encode the same amino acid—leads to multiple different mRNA sequences for a given amino acid sequence. However, different organisms or cells exhibit their own biases in selecting these synonymous codons, a phenomenon known as codon bias. Therefore, when a foreign gene is expressed in a host cell, its codons may not match the optimal codon usage frequency of the host cell, thus affecting protein expression levels. Codon optimization allows the selection of synonymous codons that better match the host cell's bias, thereby improving protein translation efficiency.

[0104] Codons can be optimized using various methods. For example, some methods involve the following steps: (1) Analyzing the codon usage frequency in host cells: First, it is necessary to understand the codon bias in host cells, i.e., which codons are used more frequently in host cells. This can be obtained by consulting relevant literature or databases. (2) Designing optimization schemes: Based on the codon usage frequency in host cells, design optimization schemes. This includes selecting which synonymous codons to replace codons in heterologous genes, and how to adjust GC content, etc. (3) Implementing optimization: Using techniques such as gene synthesis or site-directed mutagenesis, apply the optimization scheme to heterologous genes. (4) Verifying the optimization effect: Verify through experiments whether the expression level of the optimized gene in host cells has been improved. This can be evaluated by measuring indicators such as protein concentration and enzyme activity.

[0105] In some embodiments of this disclosure, codon optimization was performed on the VP0, VP1, and VP3 genes of poliovirus for the Hansenula polymorpha expression system. Specifically, the polynucleotides encoding the amino acid sequences of poliovirus type I (PVI)-like particles VP0-I, VP1-I, and VP3-I are shown in SEQ ID No. 10, SEQ ID No. 11, and SEQ ID No. 12, respectively; the polynucleotides encoding the amino acid sequences of poliovirus type II (PVI)-like particles VP0-II, VP1-II, and VP3-II are shown in SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15, respectively; and the polynucleotides encoding the amino acid sequences of poliovirus type III (PVI)-like particles VP0-III, VP1-III, and VP3-III are shown in SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 18, respectively.

[0106] Recombinant polio vaccine:

[0107] The recombinant polio vaccine described in this disclosure is a recombinant protein vaccine. It comprises poliovirus-like particles (PVMPs) produced using recombinant protein technology. When the recombinant polio vaccine is a monovalent vaccine, it may include poliovirus-like particles (PVMPs), poliovirus-like particles (PVMPs), or poliovirus-like particles (PVMPs). When the recombinant polio vaccine is a bivalent vaccine, it may include any two selected from poliovirus-like particles (PVMPs), poliovirus-like particles (PVMPs), or poliovirus-like particles (PVMPs). When the recombinant polio vaccine is a trivalent vaccine, it may include poliovirus-like particles (PVMPs), poliovirus-like particles (PVMPs), and poliovirus-like particles (PVMPs). Because the poliovirus-like particles are non-infectious, they do not need to be inactivated before being used as a vaccine.

[0108] The recombinant polio vaccine described in this disclosure may further include an adjuvant. Non-limiting examples of the adjuvant include, for instance, aluminum adjuvants (e.g., aluminum hydroxide, aluminum phosphate), oil-water emulsion adjuvants, AS01 adjuvant systems, AS03 adjuvant systems, AS04 adjuvant systems, MF59, CpG, immunostimulants, immunomodulatory substances, etc. In one embodiment of this disclosure, the adjuvant is aluminum hydroxide.

[0109] This disclosure also provides a recombinant polio vaccine formulation, each dose of which comprises approximately 0.2 μg to 60 μg of poliovirus-like particles (PVLP), approximately 0.1 μg to 60 μg of poliovirus-like particles (PVLP), or approximately 0.2 μg to 60 μg of poliovirus-like particles (PVLP). Further, to achieve better results, in some embodiments of this disclosure, each dose of the recombinant polio vaccine formulation comprises approximately 0.5 μg to 45 μg of poliovirus-like particles (PVLP), approximately 0.2 μg to 45 μg of poliovirus-like particles (PVLP), or approximately 0.5 μg to 45 μg of poliovirus-like particles (PVLP).

[0110] This disclosure also provides a recombinant polio vaccine formulation, each dose of which includes at least two selected from about 0.2 μg to 60 μg of poliovirus-like particles (PVLP), about 0.1 μg to 60 μg of poliovirus-like particles (PVLP), or about 0.2 μg to 60 μg of poliovirus-like particles (PVLP). Further, for better results, in some embodiments of this disclosure, each dose of the recombinant polio vaccine formulation includes at least two selected from about 0.5 μg to 45 μg of poliovirus-like particles (PVLP), about 0.2 μg to 45 μg of poliovirus-like particles (PVLP), or about 0.5 μg to 45 μg of poliovirus-like particles (PVLP).

[0111] In some embodiments of this disclosure, the dosage form of the above-mentioned recombinant polio vaccine preparation is an oral preparation or an intramuscular injection.

[0112] In some embodiments of this disclosure, the recombinant polio vaccine described above can also be used in combination with other vaccines, such as BCG, DPT, measles, mumps, rubella, hepatitis B, Japanese encephalitis, and meningococcal vaccines.

[0113] This disclosure also provides a method for preventing or treating polio, the method comprising administering an effective dose of the above-described recombinant polio vaccine to a subject. The administration may be oral, intramuscular, intraperitoneal, or subcutaneous.

[0114] Example:

[0115] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0116] Example 1: Obtaining recombinant polio VLP protein

[0117] 1) Obtaining the gene sequence:

[0118] For poliomyelitis type I (VP0, VP1, and VP3 as shown in SEQ ID No. 1; poliomyelitis type II (VP0, VP1, and VP3 as shown in SEQ ID No. 5; and VP3 as shown in SEQ ID No. 6), and poliomyelitis type III (VP0, VP1, and VP3 as shown in SEQ ID No. 7, VP1, and VP3 as shown in SEQ ID No. 8; and VP3 as shown in SEQ ID No. 9), gene sequences were optimized according to the codon preference of *Hansenula polymorpha*. The optimized gene sequences of VP0, VP1, and VP3 for poliomyelitis type I are shown in SEQ ID No. 10, SEQ ID No. 11, and SEQ ID No. 12, respectively; and the optimized gene sequences of VP0, VP1, and VP3 for poliomyelitis type II are shown in SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15, respectively. As shown in No. 15, the optimized gene sequences of VP0, VP1, and VP3 for polio 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 for polio types I, II, or III were synthesized using the whole genome.

[0119] 2) Construction of the expression carrier:

[0120] The VP1 gene for poliotypes I, II, or III was inserted into the *Hansenula polymorpha* expression vector 18S-MU (Figure 1). This 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 integrator arm, and amp as the resistance gene. The VP0 and VP3 genes for poliotypes I, II, or III were inserted into the *Hansenula polymorpha* expression vector 25S-DML (Figure 2). This vector contains two independent open reading frames: one using the MOX gene promoter and terminator as the promoter and terminator of the target gene, and the other using the DAS gene promoter and terminator. This vector uses the LEU3 gene as the marker gene, the 25S-rDNA gene as the homologous recombination integrator arm, and amp as the resistance gene.

[0121] 3) Obtaining recombinant proteins:

[0122] Recombinant bacteria were constructed by electroporating the HP-MU plasmid containing the VP1 gene and the HP-DL plasmid containing the VP0 and VP3 genes into a uracil- and leucine auxotrophic host bacterium (NVSI-HP-105) lacking the URA3 and LEU2 genes. ELISA was used to screen for yeast strains with high expression of the target protein to obtain recombinant strains. After high-density fermentation and serial chromatography purification, VLP proteins for poliotypes I, II, or III were obtained.

[0123] Protein identification: SDS-PAGE was used to detect the target protein (as shown in Figure 3), and the molecular weight of the target band was basically consistent with the theoretical value. Western blot detection was performed using specific antibodies against VP1, VP2, VP3, and VP4 (as shown in Figure 4), and the target bands could specifically bind to the corresponding antibodies. The purity of the target protein was detected by HPLC, and the purity was higher than 95%. Dynamic light scattering detection showed that the protein was granular, with a hydrated particle size of about 40-60 nm and good uniformity. The protein morphology was observed under transmission electron microscopy (as shown in Figure 5), showing a viral particle structure with a particle size of 30-50 nm and good morphology, confirming that the three structural proteins VP0, VP1, and VP3 can spontaneously assemble into VLPs in yeast.

[0124] Example 2: Preparation of Recombinant Polio Vaccine

[0125] The polio type I, II or III VLP protein with a certain protein content prepared in Example 1 is thoroughly 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 then packaged into a certain volume to obtain the finished vaccine product.

[0126] Example 3: Comparison of immunogenicity of recombinant polio vaccines with different formulation ratios

[0127] Multiple recombinant polio vaccines with different formulation ratios and concentrations were prepared, and immunogenicity studies were conducted in various animal strains, as detailed below:

[0128] 1) Immunogenicity evaluation in BALB / c mice

[0129] According to the experimental protocol shown in Table 1, BALB / c mice were inoculated with eight different ratios and concentrations of recombinant polio vaccine. After immunization, the serum was subjected to a micro-neutralization test using pseudoviruses targeting three serotypes: polio I-Mahoney, polio II-MEF-1, and polio III-Saukett. The results of neutralizing antibody detection are shown in Table 2 and Figure 6. It can be seen that all eight different combinations of recombinant polio vaccine can produce good neutralizing activity against polio I, II, and III in BALB / c mice, and there are no statistically significant differences between groups within the same serotype.

[0130] Table 1. Immunogenicity Evaluation Protocol in BALB / c Mice

[0131] Table 2. Results of neutralizing antibody GMT in BALB / c mice

[0132] 2) Immunogenicity evaluation in Wistar rats

[0133] In Wistar rats, several studies were conducted on recombinant polio vaccines with different ratios and concentrations, including immunization intervals and number of injections. Details are as follows:

[0134] ① 2 doses - 1 month interval between immunizations

[0135] According to the experimental protocol shown in Table 3, six different ratios and concentrations of recombinant polio vaccine were administered to Wistar rats. After immunization, the serum was subjected to a micro-neutralization test using pseudoviruses targeting polio type I (Mahoney), polio type II (MEF-1), and polio type III (Saukett). The results of the neutralizing antibody detection are shown in Table 4 and Figure 7. It can be seen that all six different combinations of recombinant polio vaccine produced good neutralizing activity against polio types I, II, and III in Wistar rats, with no statistically significant differences between groups within the same serotype.

[0136] Table 3. In vivo immunogenicity evaluation protocol for Wistar rats (2 doses - 1-month interval between immunizations)

[0137] Table 4. Neutralizing antibody GMT in Wistar rats (2 doses - 1 month interval between immunizations)

[0138] ② 2 doses - immunization interval 2 months

[0139] According to the experimental protocol shown in Table 5, six different ratios and concentrations of recombinant polio vaccine were administered to Wistar rats. After immunization, the serum was subjected to a micro-neutralization test using pseudoviruses targeting poliotypes I (Mahoney), II (MEF-1), and III (Saukett). The results of the neutralizing antibody detection are shown in Table 6 and Figure 8. It can be seen that all six different combinations of recombinant polio vaccine produced good neutralizing activity against poliotypes I, II, and III in Wistar rats, with no statistically significant differences between groups within the same serotype.

[0140] Table 5. Wistar rat in vivo immunogenicity evaluation test protocol (2 doses - 2-month interval between immunizations)

[0141] Table 6. Neutralizing antibody GMT in Wistar rats (2 doses - 2-month interval between immunizations)

[0142] ③ 3 doses - immunization interval of 1 month

[0143] According to the experimental protocol shown in Table 7, seven different ratios and concentrations of recombinant polio vaccine were administered to Wistar rats. Inactivated polio vaccine (sIPV) was used as a positive control. Post-immunization serum was subjected to a micro-neutralization test using pseudoviruses targeting poliotypes I (Mahoney), II (MEF-1), and III (Saukett). The results of neutralizing antibody detection are shown in Table 8 and Figure 9. It can be seen that all seven different combinations of recombinant polio vaccine produced good neutralizing activity against poliotypes I, II, and III in Wistar rats. There were no statistically significant differences in the recombinant polio vaccine among the groups within the same serotype.

[0144] Table 7. Wistar rat in vivo immunogenicity evaluation test protocol (3 injections)

[0145] Table 8. Neutralizing antibody GMT in Wistar rats (3 injections)

[0146] 3) Immunogenicity evaluation in SD rats

[0147] According to the experimental protocol shown in Table 9, SD rats were inoculated with two recombinant polio vaccines with different ratios and contents. After immunization, the serum was subjected to a micro-neutralization test using pseudoviruses against three polio types: polio I-Mahoney, polio II-MEF-1, and polio III-Saukett. The results of the neutralizing antibody detection are shown in Table 10 and Figure 10. It can be seen that both 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 statistically significant difference between groups within the same polio type.

[0148] Table 9. Immunogenicity Evaluation Protocol in SD Rats

[0149] Table 10. Neutralizing antibody GMT in SD rats

[0150] 4) Immunogenicity evaluation in cynomolgus monkeys

[0151] According to the experimental protocol shown in Table 11, two recombinant polio vaccines with different ratios and concentrations were inoculated into cynomolgus monkeys. After immunization, the serum was subjected to a micro-neutralization test using pseudoviruses against three serotypes: polio type I (Mahoney), polio type II (MEF-1), and polio type III (Saukett). The results of the neutralizing antibody detection are shown in Table 12 and Figure 11. It can be seen that both recombinant polio vaccines with different ratios can produce good neutralizing activity against polio types I, II, and III in cynomolgus monkeys, and there are no statistically significant differences between groups within the same serotype.

[0152] Table 11. Immunogenicity Evaluation Protocol in Cynomolgus Monkeys

[0153] Table 12. Neutralizing antibody GMT in cynomolgus monkeys

[0154] Example 4: Cross-neutralization evaluation of recombinant polio vaccine

[0155] According to the experimental protocol shown in Table 13, three batches of recombinant polio vaccine were inoculated into Wistar rats. After immunization, the serum was subjected to micro-neutralization tests using multiple pseudoviruses against three serotypes: 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, ASO-3). The results of neutralizing antibody detection are shown in Table 14 and Figure 12. It can be seen that the recombinant polio vaccine can produce good neutralizing activity against the current major representative circulating strains and has good immunogenicity.

[0156] Table 13. Cross-neutralization test protocol

[0157] Table 14. GMT values ​​of cross-neutralizing antibodies

[0158] Example 5: Effect of different adjuvant contents on recombinant polio vaccine

[0159] The effects of different adjuvant concentrations (no adjuvant, 100 μg / dose, 300 μg / dose, 500 μg / dose) on the immunogenicity of recombinant polio vaccine were evaluated in Wistar rats. Animals were immunized according to the experimental protocol shown in Table 15. After immunization, serum was subjected to a micro-neutralization test using pseudoviruses against three poliotypes: Mahoney (polio type I), MEF-1 (polio type II), and Saukett (polio type III). The results of neutralizing antibody detection are shown in Table 16 and Figure 13. It can be seen that both no adjuvant and the addition of different adjuvant concentrations can stimulate the production of a certain level of neutralizing antibodies in rats. The addition of adjuvants can significantly increase the level of neutralizing antibodies.

[0160] Table 15. Test schemes for different adjuvant contents

[0161] Table 16. GMT values ​​of neutralizing antibodies with different adjuvant contents

[0162] Comparative Example 1: Comparison of Recombinant Polio VLP Expression Strategies

[0163] VLPs were obtained by co-expressing P1 and 3CD proteins. Specifically, the genes encoding P1 and 3CD proteins were sequenced according to the codon preference of *Hansenula polymorpha*. The optimized P1 target gene (the P1 gene sequence for poliotype I is shown in SEQ ID No. 19, the P1 gene sequence for poliotype II is shown in SEQ ID No. 20, and the P1 gene sequence for poliotype III is shown in SEQ ID No. 21) replaced the VP1 gene in the 18S-MU plasmid. The optimized 3CD protein target gene (the 3CD gene sequence for poliotype I is shown in SEQ ID No. 22, the 3CD gene sequence for poliotype II is shown in SEQ ID No. 23, and the 3CD gene sequence for poliotype III 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. A plasmid containing the P1 and 3CD genes was co-transformed into a uracil- and leucine auxotrophic host bacterium (NVSI-HP-105) lacking the URA3 and LEU2 genes to construct a recombinant strain. ELISA was used to screen for yeast strains highly expressing the target protein, and the recombinant strains were obtained. The target protein was then obtained after high-density fermentation and serial chromatography purification. Through repeated experiments, VLP protein was obtained from types II and III, but not from type I, possibly due to low 3CD restriction enzyme efficiency and unsuccessful particle assembly. Transmission electron microscopy (TEM) observation of the obtained VLP protein (Figure 14) showed the formation of VLPs with a diameter of 30-50 nm.

[0164] VLP proteins obtained from poliotypes II and III using the "P1+3CD" protocol and the "VP0+VP1+VP3" co-expression protocol were mixed with aluminum hydroxide adjuvant at specific doses. BALB / c mice were immunized and blood was collected according to the experimental protocol shown in Table 17. Post-immunization serum was subjected to a micro-neutralization test using pseudoviruses of poliotype II-MEF-1 and poliotype III-Saukett serotypes. The results of neutralizing antibody detection are shown in Table 18 and Figure 15. It can be seen that the GMT values ​​of neutralizing antibodies from the "VP0+VP1+VP3" expression strategy for both type II and type III VLPs were higher than those from the "P1+3CD" protocol, and the differences were statistically significant.

[0165] Table 17. VLP Immunogenicity Evaluation Protocols with Different Expression Strategies

[0166] Table 18. GMT values ​​of neutralizing antibodies against VLPs expressed using different strategies

[0167] Comparative Example 2: Comparison of proteins with different sequence structures

[0168] The poliovirus itself has D antigen and C antigen differentiation. VLPs composed of different VP0, VP1, and VP3 amino acid sequences have different immunogenicity. VLPs targeting different VP0, VP1, and VP3 amino acid sequences were reconstructed using the same technical solution as in Example 1, as follows:

[0169] For poliomyelitis type I (VP0, VP1, and VP3 as shown in SEQ ID No. 25, SEQ ID No. 26, and VP3 as shown in SEQ ID No. 27), for poliomyelitis type II (VP0, VP1, VP1, and VP3 as shown in SEQ ID No. 28, SEQ ID No. 29, and VP3 as shown in SEQ ID No. 30), and for poliomyelitis type III (VP0, VP1, VP1, and VP3 as shown in SEQ ID No. 33, SEQ ID No. 31, VP1, and VP3 as shown in SEQ ID No. 33), gene sequences were optimized according to the codon preference of *Hansenula polymorpha*. The optimized gene sequences of VP0, VP1, and VP3 for poliomyelitis type I are shown in SEQ ID No. 34, SEQ ID No. 35, and SEQ ID No. 36, respectively; and the optimized gene sequences of VP0, VP1, and VP3 for poliomyelitis type II are shown in SEQ ID No. 37, SEQ ID No. 38, and SEQ ID No. 39, respectively. As shown in No. 39, the optimized gene sequences of VP0, VP1, and VP3 for polio type III are shown in SEQ ID No. 40, SEQ ID No. 41, and SEQ ID No. 42, respectively. The VP0, VP1, and VP3 genes for polio types I, II, or III were synthesized in their entirety. Yeast expression plasmids were constructed according to the expression vector construction method shown in Example 1, and electroporated into a uracil- and leucine auxotrophic host bacterium (NVSI-HP-105) lacking the URA3 and LEU2 genes to construct recombinant bacteria. ELISA was used to screen for yeast strains with high expression of the target protein to obtain recombinant strains. The target protein was obtained after high-density fermentation and a series of chromatographic purifications. SDS-PAGE was used to detect the target protein (as shown in Figure 16), and the molecular weight of the target band was basically consistent with the theoretical value. HPLC purity was found to be higher than 95%. Dynamic light scattering analysis showed that the protein was granular with a hydrated particle size of approximately 40-60 nm and good uniformity. Transmission electron microscopy (as shown in Figure 17) revealed the protein morphology to be a viral particle structure with a particle size of 30-50 nm and good morphology.

[0170] The VLP obtained in Comparative Example 2 was named wVLP, and the VLP obtained in Example 1 was named mVLP. wVLP and mVLP were mixed with aluminum hydroxide adjuvant at certain doses, and Wistar rats were immunized and blood was collected according to the experimental protocol shown in Table 19. After immunization, the serum was subjected to a micro-neutralization test using pseudoviruses against three serotypes: polio I-Mahoney, polio II-MEF-1, and polio III-Saukett. The results of neutralizing antibody detection are shown in Table 20 and Figure 18. It can be seen that the neutralizing antibody GMT values ​​of the mVLP protocol for polio I, II, and III were all higher than those of the wVLP protocol, with statistical differences between polio II and III.

[0171] Table 19. Immunogenicity evaluation assays for VLPs obtained from different structural protein sequences

[0172] Table 20. GMT values ​​of VLP neutralizing antibodies obtained from different structural protein sequences

[0173] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A poliovirus-like particle, characterized in that, The virus-like particles are: (1) Poliovirus type I-like particles It includes amino acid sequences such as 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 amino acid sequences that have 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 have the same or similar biological functions; or (2) Poliovirus type II-like particles It includes amino acid sequences such as 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 amino acid sequences that have 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 have the same or similar biological functions; or (3) Poliovirus type III-like particles It includes amino acid sequences such as VP0-III shown in SEQ ID No. 7, VP1-III shown in SEQ ID No. 8, and VP3-III shown in SEQ ID No. 9, or amino acid sequences that have 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 have the same or similar biological functions.

2. The poliovirus-like particle according to claim 1, characterized in that, The virus-like particles are: (1) Poliovirus type I-like particles It is composed of amino acid sequences such as 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 amino acid sequences that have 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 have the same or similar biological functions; or (2) Poliovirus type II-like particles It is composed of amino acid sequences such as 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 amino acid sequences that have 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 have the same or similar biological functions; or (3) Poliovirus type III-like particles It is composed of amino acid sequences such as VP0-III as shown in SEQ ID No. 7, VP1-III as shown in SEQ ID No. 8, and VP3-III as shown in SEQ ID No. 9, or amino acid sequences that have 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 have the same or similar biological functions.

3. A composition of poliovirus-like particles, characterized in that, The composition comprises at least two selected from the poliovirus-like particles (PVL-1), poliovirus-like particles (PVL-2), or poliovirus-like particles (PVL-3) as described in claim 1 or 2.

4. The composition according to claim 3, characterized in that, The mass ratio of poliovirus type I, poliovirus type II, and poliovirus type III in the composition is 1:0.2:1 to 1:2:

6.

5. The composition according to claim 3, characterized in that, The composition contains 0.5 μg to 45 μg of poliovirus type I, 0.2 μg to 45 μg of poliovirus type II, and 0.5 μg to 45 μg of poliovirus type III.

6. A method for generating poliovirus-like particles, characterized in that, include: Step 1) Clone the genes of VPO-I, VP1-I, and VP3-I as described in claim 1 or 2 into the same or different vectors, wherein each gene of VPO-I, VP1-I, and VP3-I has an independent open reading frame; or The genes of VP0-II, VP1-II, and VP3-II as described in claim 1 or 2 are cloned into the same or different vectors, wherein each gene of VP0-II, VP1-II, and VP3-II has an independent open reading frame; or The genes of VP0-III, VP1-III and VP3-III as described in claim 1 or 2 are cloned into the same or different vectors, wherein the genes of VP0-III, VP1-III and VP3-III each have an independent open reading frame; Step 2) The vector obtained in Step 1) is transferred to the host cell for expression and assembly. The expression product is purified to obtain the poliovirus-like particles.

7. The method according to claim 6, characterized in that, The expression method described in step 2) is biological fermentation.

8. A method for producing a composition of poliovirus-like particles, characterized in that, include: Step 1) The genes of VPO-I, VP1-I and VP3-I as described in claim 1 or 2 are cloned into the same or different vectors, wherein the genes of VPO-I, VP1-I and VP3-I each have an independent open reading frame; The genes of VP0-II, VP1-II, and VP3-II as described in claim 1 or 2 are cloned into the same or different vectors, wherein each gene of VP0-II, VP1-II, and VP3-II has an independent open reading frame; and The genes of VP0-III, VP1-III and VP3-III as described in claim 1 or 2 are cloned into the same or different vectors, wherein the genes of VP0-III, VP1-III and VP3-III each have an independent open reading frame; Step 2) The vector obtained in Step 1) is transferred into the host cell for expression and assembly. The expression product is purified to obtain the poliovirus-like particles. Step 3) Mix at least two of the poliovirus type I, poliovirus type II, or poliovirus type III particles obtained in step 2) in a certain proportion.

9. An isolated polynucleotide, characterized in that, The polynucleotide comprises a nucleic acid sequence encoding the amino acid sequence as described in claim 1 or 2.

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

11. A separate carrier, characterized in that, The carrier contains the polynucleotide as described in claim 9 or 10. The polynucleotides encoding the amino acid sequences of poliovirus type I (PVI)-like particles VPO-I, PV1-I, and PV3-I are operatively linked to promoters, the polynucleotides encoding the amino acid sequences of poliovirus type II (PVI)-like particles VPO-II, PV1-II, and PV3-II are operatively linked to promoters, and the polynucleotides encoding the amino acid sequences of poliovirus type III (PVI)-like particles VPO-III, PV1-III, and PV3-III are operatively linked to promoters.

12. An isolated host cell, characterized in that, The host cell contains the polynucleotide as described in claim 9 or 10, or the vector as described in claim 11; Preferably, the host cell is a Hansenula polymorpha cell, an Escherichia coli cell, a Pichia pastoris cell, a Saccharomyces cerevisiae cell, a mammalian cell, a avian cell, or an insect cell; More preferably, the host cell is a Hansenula polymorpha cell.

13. A recombinant protein vaccine, characterized in that, The recombinant protein vaccine comprises poliovirus-like particles as described in claim 1 or 2, or a composition of poliovirus-like particles as described in claim 3, 4 or 5, and optionally includes an adjuvant.

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

15. The recombinant protein vaccine according to claim 14, characterized in that, The adjuvant is aluminum hydroxide; Preferably, the aluminum hydroxide content in each dose of the recombinant protein vaccine is from 0 μg to 500 μg; More preferably, the recombinant protein vaccine contains 300 μg of aluminum hydroxide per dose.

16. A genetically engineered vector vaccine, characterized in that, The genetically engineered vector vaccine includes the vector as described in claim 11.

17. A nucleic acid vaccine, characterized in that, The nucleic acid vaccine comprises the polynucleotides as described in claim 9 or 10.

18. A liposome complex, characterized in that, The liposome complex comprises the polynucleotide as described in claim 9 or 10.

19. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a vaccine as described in any one of claims 13 to 17, or a lipid complex as described in claim 18, and a pharmaceutically acceptable carrier, delivery medium, excipient, stabilizer, diluent, or combination thereof.

20. The use of the poliovirus-like particles of claim 1 or 2, the composition of claim 3, 4 or 5, the isolated polynucleotide of claim 9 or 10, the isolated vector of claim 11, the isolated host cell of claim 12, the vaccine of any one of claims 13 to 17, the liposome complex of claim 18, or the pharmaceutical composition of claim 19 in the preparation of a medicament for the prevention or treatment of diseases caused by poliovirus, characterized in that, The poliovirus is selected from at least one of poliovirus type I, poliovirus type II, or poliovirus type III.

21. A method for detecting polio-specific antibodies in a sample, characterized in that, include: Step 1) attach a detectable tag to the poliovirus-like particles described in any one of claims 1 to 5; Step 2) Contact the sample in solution with the tagged poliovirus-like particles obtained in Step 1). Step 3) Detect poliovirus-like particles in the immune complexes formed in the above steps.

22. A reagent kit, characterized in that, The kit includes poliovirus-like particles as described in claim 1 or 2, or the composition as described in claim 3, 4 or 5.

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