Recombinant chikungunya virus vaccine as well as preparation method and application thereof

Abstract

The invention relates to a recombinant chikungunya virus vaccine as well as a preparation method and application thereof, in particular to a recombinant protein which comprises chikungunya virus protein or a variant thereof. The chikungunya fever virus serving as a main target of a neutralizing antibody is optimized, new nanoparticle protein is formed and used for preparing the vaccine, and the vaccine can stimulate an organism to generate the neutralizing antibody aiming at the chikungunya fever virus and prevent in-vivo infection and has a good neutralizing effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a recombinant chikungunya virus vaccine, its preparation method, and its application. Background Technology

[0002] Chikungunya fever is an acute infectious disease caused by the chikungunya virus (CHIKV) and transmitted through the bite of Aedes mosquitoes (primarily Aedes aegypti and Aedes albopictus). After a mosquito bite, the E2 protein on the surface of the virus recognizes host cell receptors, while the E1 protein mediates the fusion of the viral envelope with the cell membrane. Subsequently, viral RNA is released into the cytoplasm for replication. Its main clinical features are high fever, severe joint pain, and rash, significantly impacting the patient's quality of life. The virus exhibits broad cell and tissue tropism, replicating in most cell types except B and T cells, and spreads throughout the body via severe viremia, primarily causing joint inflammation. Although chikungunya fever is often self-limiting, it can leave serious sequelae, especially affecting the elderly and children.

[0003] Currently, there is no specific antiviral drug for chikungunya fever, and clinical treatment mainly involves symptomatic and supportive care. Therefore, vaccination is considered the most effective strategy for providing lasting immunity and preventing the disease. However, the development of a chikungunya virus vaccine faces multiple challenges. First, the virus has high genetic diversity and its surface proteins mutate rapidly, making it easy for the immune system to evade, thus reducing the protective efficacy of the vaccine. An ideal vaccine must be able to effectively prevent all major lineages of the virus, which increases the difficulty of development.

[0004] Globally, vaccine development has made some progress, but safe and effective products available for widespread use remain scarce. Various technological approaches have been employed in the development of chikungunya vaccines, including inactivated vaccines, viral vector vaccines, and nucleic acid vaccines (such as mRNA and DNA vaccines). There is still room for improvement in existing vaccine technologies. Currently, there is an urgent need to develop a novel vaccine for chikungunya that is highly safe, immunogenic, provides broad-spectrum protection, and is cost-effective. Summary of the Invention

[0005] To overcome the above-mentioned shortcomings, this invention provides a technical solution comprising a recombinant protein including a chikungunya virus protein variant, a preparation method thereof, and its application in the preparation of a chikungunya virus nanoparticle vaccine. Specifically, In a first aspect, the present invention provides a recombinant protein comprising the protein sequence of SEQ ID No: 2 or a variant thereof, wherein the protein sequence of the variant comprises a mutation at any of the positions 324, 479, 737, and 966 of SEQ ID No: 2.

[0006] Preferably, the mutation includes any one or more of the following: 1) R324N; 2) S479F; 3) L737R; 4) K966N.

[0007] Preferably, the protein sequence of the variant further includes mutations at any of the following positions in SEQ ID No: 2: 116, 197, 237, 243, 293, 878, 963, 1105.

[0008] Preferably, the mutation further includes any one or more of the following: 1) E116D; 2) S197F; 3) R237N; 4) V243L; 5) E293D; 6) K878Q; 7) V963L; 8) L1105R.

[0009] In one specific implementation, the mutations include R237N, V243L, R324N, S479F, L737R, and K966N.

[0010] In one specific implementation, the mutations include E116D, R324N, S479F, K878Q, V963L, and L1105R.

[0011] In one specific implementation, the mutations include S197F, E293D, L737R, and K966N.

[0012] Preferably, the protein sequence of the variant includes: (A1), as shown in any of SEQ ID No: 3-5, (A2) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of (A1); (A3) and any one of (A1)-(A2) that has more than 80% identity and the same function.

[0013] In this article, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0014] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0015] The aforementioned recombinant proteins form recombinant nanoparticle proteins.

[0016] In a second aspect, the present invention provides a nucleotide molecule that encodes any of the recombinant proteins described above.

[0017] Preferably, the nucleotide molecule comprises DNA and / or RNA, such as recombinant DNA, or mRNA.

[0018] More preferably, the nucleotide molecule comprises: (B1), as shown in any of SEQ ID No: 7-10, The complementary, degenerate, or transcribed sequences of (B2) and (B1), (B3) A DNA molecule or mRNA that has more than 75% identity with the DNA molecule or mRNA defined in (B1) or (B2) and encodes the corresponding protein in the fusion protein.

[0019] Those skilled in the art can readily mutate the nucleotide sequence encoding the recombinant protein of the present invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity to the nucleotide sequence encoding the recombinant protein, as long as they encode the recombinant protein and have the same function, are derived from and equivalent to the nucleotide sequence of the present invention.

[0020] The term "identity" refers to the sequence similarity to the compared nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with any of the nucleotide sequences shown in SEQ ID Nos: 7-10 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0021] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0022] In a third aspect, the present invention provides a carrier comprising any of the nucleotide molecules described above.

[0023] Preferably, the vector further includes regulatory factors, such as promoters that initiate transcription of the polypeptide or recombinant protein-encoding gene sequence, and may also include terminators that terminate transcription of the polypeptide or recombinant protein-encoding gene sequence. Furthermore, the vector may also include enhancer sequences.

[0024] The vectors mentioned in this article refer to vectors that can carry exogenous DNA, mRNA or target genes into host cells for amplification and expression. The vectors can be cloning vectors or expression vectors, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage, etc.), granules (i.e., Cos plasmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0025] In a fourth aspect, the present invention provides a host cell comprising any of the aforementioned nucleotide molecules or carriers.

[0026] The host cell (also called the recipient cell) described herein may be a plant cell or an animal cell. The term "host cell" can be understood not only to refer to a specific recipient cell, but also to the offspring of such a cell, which may not necessarily be identical to the original parent cell due to natural, accidental, or intentional mutations and / or alterations, but are still included within the scope of the host cell. Suitable host cells are those known in the art, including: plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc., but not limited to these; animal cells such as mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), young hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., African clawed frog (Xenopus laevis) cells or giant salamander (Andrias davidianus) cells), fish cells (e.g., grass carp, carp, rainbow trout or catfish cells), insect cells (e.g., Sf21 cells or Sf-9 cells), etc., but not limited to these.

[0027] Preferably, the host cell can also be a microorganism, which may be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. The bacteria may be derived from, but are not limited to, *Escherichia sp.*, *Erwinia sp.*, *Agrobacterium sp.*, *Flavobacterium sp.*, *Alcaligenes sp.*, *Pseudomonas sp.*, or *Bacillus sp.*, for example, *Escherichia coli*, *Bacillus subtilis*, or *Bacillus pumilus*.

[0028] In a fifth aspect, the present invention provides a medicament comprising any of the recombinant proteins, nucleotide molecules, vectors, or host cells described above.

[0029] Preferably, the drug is a vaccine; more preferably, the vaccine further includes an adjuvant and / or a vaccine delivery system.

[0030] More preferably, the adjuvant may be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response against the antigen co-inoculated with it. The adjuvants described herein may be those known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic compounds, quinine, saponins, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokine and nucleic acid adjuvants (such as monocyte clone stimulating factor, leukocyte cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid carriers, etc.), and emulsion adjuvants (such as Freund's adjuvant). The adjuvant may be a pharmaceutically acceptable adjuvant.

[0031] As is well known to those skilled in the art, in order to enhance the immunogenicity of antigen proteins, in addition to adding compounds with immunomodulatory effects as adjuvants, gene combinations can be adjusted to express them into particulate structures; or they can be aggregated in vitro and encapsulated in liposomes or microspheres.

[0032] The vaccine delivery system described herein can be a substance capable of carrying antigens to the body's immune system, where they can be stored and exert their antigenic effects for an extended period. The vaccine delivery system described herein can be an aluminum salt gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system, or a nano-adjuvant vaccine delivery system.

[0033] Furthermore, the drug may also include one or more pharmaceutically acceptable carriers.

[0034] The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, humectant, disintegrant, absorption enhancer, adsorbent, surfactant, or lubricant, but is not limited thereto.

[0035] The vaccine for preventing infection described in this invention may be an intramuscular liquid injection, an intravenous liquid injection, an intranasal liquid injection, an intradermal liquid injection, or a subcutaneous liquid injection.

[0036] In a sixth aspect, the present invention provides a method for preparing the above-mentioned recombinant protein, the above-mentioned host cell, or the above-mentioned drug, the method comprising introducing the above-mentioned nucleotide molecule or vector encoding the recombinant protein into a host cell and culturing the host cell.

[0037] Preferably, the preparation method includes screening for monoclonal cell lines with high and stable expression levels.

[0038] More preferably, the screening method includes screening methods conventional in the art, such as adding screening reagents, comparing the biological activity of clonal pools using ELISA, etc.

[0039] More preferably, the method for preparing the drug further includes adding the adjuvant, vaccine delivery system and / or pharmaceutically acceptable carrier described in the fifth aspect.

[0040] In a seventh aspect, the present invention provides an application of any of the above-mentioned recombinant proteins, nucleotide molecules, vectors, host cells, or drugs, said application comprising any of the following: (1) Use in the preparation of products for the prevention and / or treatment of diseases caused by chikungunya virus infection; (2) Application in the preparation of products for inducing an immune response to chikungunya virus antigen; (3) Use in the prevention and / or treatment of diseases caused by chikungunya virus infection; (4) Application in inducing an immune response to chikungunya virus antigen.

[0041] The products described in this article may be reagents, drugs, or vaccines.

[0042] The products described in (1) and (2) may be vaccines or antibodies against chikungunya virus, and the antibodies include full-length antibodies or antigen-binding fragments (such as Fab fragments, Fv fragments, Fab′ fragments, F(ab′)2 fragments, single-chain antibodies (ScFv), nanobodies (single-domain antibodies), bispecific antibodies or minimum recognition units (MRUs), etc., but not limited to these).

[0043] Furthermore, the chikungunya virus antibody may be a neutralizing antibody that specifically binds to the chikungunya virus. The neutralizing antibody may be a high-titer neutralizing antibody against multiple circulating strains of chikungunya virus.

[0044] In the above applications, the diseases / symptoms caused by the chikungunya virus infection include, but are not limited to, acute chikungunya fever, chronic arthritis / arthritis, rash, and complications that may affect multiple systems.

[0045] Preferably, the acute chikungunya fever may include symptoms such as fever, joint pain, rash, headache, muscle pain, nausea, vomiting, fatigue and / or swollen lymph nodes.

[0046] Preferably, the chronic arthritis / arthritis can manifest as joint pain, stiffness, swelling and / or limited mobility lasting for weeks, months or even years, and some patients may develop rheumatoid arthritis-like disease.

[0047] More preferably, the complications that may affect multiple systems may include ocular complications (such as conjunctivitis), neurological complications, cardiovascular complications, and / or digestive symptoms.

[0048] More preferably, the neurological complications may include meningitis, encephalitis, meningoencephalitis, peripheral neuropathy, optic neuritis, and / or Guillain-Barré syndrome.

[0049] More preferably, the cardiovascular complications may include myocarditis.

[0050] More preferably, the digestive symptoms may include loss of appetite, nausea, vomiting, and / or abdominal pain.

[0051] Preferred products for the prevention and / or treatment of diseases caused by chikungunya virus infection include vaccines.

[0052] In a ninth aspect, the present invention also provides a method for generating an immune response, the method comprising administering any of the above-described vaccines to a subject.

[0053] In the above method, administering the vaccine to the subject can induce an immune response against chikungunya virus in the subject. This immune response can be a cellular immune response, a humoral immune response, or a combination of both.

[0054] The cellular immune response may include B cell immune response and T cell immune response.

[0055] The subjects described in this article may be humans or non-human animals.

[0056] Furthermore, the non-human animal may be a non-human mammal.

[0057] The non-human mammal may be any one of the following, but is not limited to: mouse, rat, guinea pig, hamster, pig, dog, sheep, monkey, rabbit, cat, cow, horse.

[0058] The subjects mentioned in this article include, but are not limited to, healthy subjects, symptomatic infected subjects, asymptomatic infected subjects, or recovered subjects (subjects who have recovered after infection).

[0059] The administration methods described herein include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral or nasal spray, or nebulized inhalation.

[0060] In summary, compared with the prior art, the beneficial effects of the present invention are as follows: The recombinant protein of this invention optimizes the sequence of the chikungunya virus protein based on existing technologies, thereby increasing the effect of inducing the body to produce antibodies. The recombinant protein of this application has a better neutralizing effect than existing technologies, and the vaccine of this invention can be used for effective prevention or treatment of chikungunya virus. Attached Figure Description

[0061] Figure 1SDS-PAGE electrophoresis results of recombinant nanoparticle protein solution: Lane 1 is the marker, Lane 2 is CHIKV37997, Lane 3 is CHIKV93, and Lane 4 is CHIKV107.

[0062] Figure 2 SDS-PAGE electrophoresis results of recombinant nanoparticle protein solution: lane 1 is marker, lane 2 is CHIKV135, and lane 3 is CHIKV176.

[0063] Figure 3 Electron micrographs of the purified products of recombinant nanoparticle proteins CHIKV37997, CHIKV93, CHIKV107, CHIKV135, and CHIKV176 (from top left to bottom right).

[0064] Figure 4 The titer test results generated in ELISA, AE correspond to CHIKV37997, CHIKV93, CHIKV107, CHIKV135, and CHIKV176, respectively. Detailed Implementation

[0065] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the examples are listed below; unless otherwise specified, all others are commercially available.

[0067] Example 1: Design, preparation and purification of recombinant nanoparticle proteins I. Design of Recombinant Nanoparticle Proteins CHIKV37997 (SEQ ID No: 1) CHIKV37997 nucleic acid sequence (SEQ ID No: 6) Among them, CHIKV37997 is the control sequence of the overseas vaccine VIMKUNYA.

[0068] CHIKV93 (SEQ ID No: 2) CHIKV93 nucleic acid sequence (SEQ ID No: 7) CHIKV107 (SEQ ID No: 3) CHIKV107 nucleic acid sequence (SEQ ID No: 8) CHIKV135 (SEQ ID No: 4) CHIKV135 nucleic acid sequence (SEQ ID No: 9) CHIKV176 (SEQ ID No: 5) CHIKV176 nucleic acid sequence (SEQ ID No: 10) II. Preparation of Recombinant Nanoparticle Proteins Construction of recombinant plasmids Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize plasmids containing the protein-coding gene sequences of the aforementioned recombinant nanoparticles.

[0069] 2. Expression of recombinant nanoparticle proteins Electroporation and expression of CHIKV37997, CHIKV93, CHIKV107, CHIKV135, and CHIKV176 plasmids were performed in CHO K1Q cells (Kangsheng Biopharmaceutical Co., Ltd., catalog number A14101). Immunogenicity data were compared, and cell lines with high expression were screened.

[0070] The specific steps are as follows: The recombinant plasmid was transfected into CHO K1Q cells using the EBXP-F1 electroporator from Suzhou Yida Biotechnology Co., Ltd. The specific electroporation steps are as follows: 1. Remove the buffer, cell culture medium, and D-PBS 30 minutes before electroporation and allow them to return to room temperature.

[0071] 2. Cell collection and counting: After the cells are evenly suspended, they are placed in centrifuge tubes for counting.

[0072] 3. Centrifugation: Take the required culture medium and place the cells into a new centrifuge tube, place it in a centrifuge (Suzhou Guofei Laboratory Instruments Co., Ltd., item number: TDL-5A), and centrifuge at 1000 rpm for 5 min.

[0073] 4. DPBS washing: Discard the supernatant culture medium, obtain the desired cells, add 1 mL of D-PBS (Thermo Fisher Gibco, catalog number: 2334304) to resuspend the cells, and centrifuge at 1000 rpm for 5 min.

[0074] 5. Mix DNA, cells, and buffer: Discard D-PBS, add the required amount of electroporation buffer (Suzhou Yida Biotechnology Co., Ltd., catalog number: H10305) and 10 μg of plasmid, and gently pipette to mix.

[0075] 6. Electroporation: Add the cell suspension containing plasmids to the H1 electroporation cuvette (Suzhou Yida Biotechnology Co., Ltd., catalog number: H10201) at a ratio of 200 μl + DNA volume / cuvette. Insert the electroporation cuvette into the base and perform electroporation according to the electroporation conditions shown in Table 1.

[0076] Table 1. Electrostatic Discharge Conditions

[0077] 7. Add the electroporated cells to a T25 flask (Wuxi Nais Life Science & Technology Co., Ltd., catalog number: 707003) containing 10 mL of CD04 medium (Kangsheng Biopharmaceutical Co., Ltd., catalog number: A11004) and incubate for 48 hours.

[0078] The specific steps for culturing and screening cell clones are as follows: Cells were sampled from the T25 flasks and their viability was monitored using a cell counter (Sanofi, model: Countess II FL). When the viability was above 70%, 10,000 cells per well were seeded into 96-well plates and cultured in CD04 medium containing 25 mM MSX (Sigma, catalog number: M5379-1G). Positive clones were selected using ELISA. The cells were then expanded to 125 mL shake flasks (Wuxi Nice Life Science Co., Ltd., catalog number: 781011). After approximately 5-7 days of culture in the 125 mL shake flasks, when the viability decreased to between 50-80%, the supernatant was collected for ELISA detection.

[0079] The ELISA detection method is as follows: The supernatant was diluted 10-fold, 100-fold, 1000-fold, and 10000-fold and coated. A 1000-fold diluted E2 protein antibody (Sinobio, E2 / Envelope 2 Antibody, Catalog No.: 40440-T46) was used as the primary antibody, and goat anti-rabbit IgG-HRP (ABCam, Catalog No.: AB6721) was used as the secondary antibody. Signal readings were performed using a microplate reader (Shanghai Kehua, Catalog No.: RD-SH-012), and the sample with the strongest signal was selected as the highest expression sample. The supernatant from the highest expression sample was harvested for further purification.

[0080] III. Purification of Recombinant Nanoparticle Proteins Following the method described in the literature "Cryo-EM Structure of Chikungunya Virus in Complex with the Mxra8 Receptor," the supernatant of the expression cell line culture medium was purified using a Q Sepharose XL (GE Healthcare, GE17-5072-01). The specific purification steps were as follows: The selected cell supernatant culture medium was centrifuged at 8000 rpm for 20 minutes and filtered through a 0.45 μm filter membrane (Jinteng, catalog number: JTSF 025013 / 014) to obtain approximately 100 mL of solution. Equilibration buffer was added to a final volume of 200 mL. The Q Sepharose XL column was equilibrated with equilibration buffer, and the sample was loaded using an A1 (salt-free buffer) pump at a flow rate of 1.5 mL / min. After loading, the column was washed with equilibration buffer until the absorbance returned to its pre-loading level and stabilized. A gradient elution was performed using eluent (20 mM Tris, 0.5 M NaCl, pH 8.5) at a flow rate of 2 mL / min for 50 min to achieve the gradient change in the sample loading ratio from 0% to 100% using a B pump (containing salt eluent). The elution peaks were collected. The eluent was then concentrated by ultrafiltration to obtain a recombinant nanoparticle protein solution, which was aliquoted and used for SDS-PAGE and Western blot analysis.

[0081] The specific steps for SDS-PAGE analysis are as follows: Add 20 μL of 5× protein to 80 μL of recombinant nanoparticle protein solution, treat at 95℃ for 10 min, and then centrifuge. Take 15 μL of the supernatant for SDS-PAGE analysis, and stain to observe protein expression.

[0082] The SDS-PAGE electrophoresis results of the recombinant nanoparticle protein solution are as follows: Figure 1 , 2 As shown. The results indicate that CHIKV37997, CHIKV93, and CHIKV107 ( Figure 1 From left to right, corresponding to lanes 2-4), CHIKV135, CHIKV176 ( Figure 2 From left to right, corresponding to lanes 2-3, the expected bands appeared at the 50kd and 38kd positions, and the purity met the standard.

[0083] Example 2: Nanoparticle morphology analysis of recombinant nanoparticle proteins The purified products of recombinant nanoparticle proteins CHIKV37997, CHIKV93, CHIKV107, CHIKV135, and CHIKV176 prepared in Example 1 were negatively stained. The specific negative staining procedure was as follows: An ultrathin carbon membrane was pre-vacuumed for 3 minutes using a Harrick Basic Plasma Cleaner PDC-32G-2 instrument, followed by glow discharge at a medium setting for 30 seconds, and then removed. 4 μl of sample was pipetted onto the carbon membrane, placed horizontally for 1 minute, and then blotted dry with filter paper. Then, 7 μl of 2% uranium acetate was added, placed for 1 minute, blotted dry with filter paper, and after several minutes, the negatively stained purified samples were observed using a FEI Tecnai Arctica TEM D683 transmission electron microscope.

[0084] The results are as follows Figure 3 As shown, the results indicate that the purified products of recombinant nanoparticle proteins CHIKV37997, CHIKV93, CHIKV107, CHIKV135, and CHIKV176 (from top left to bottom right) all showed regular nanoparticles under an electron microscope. Transmission electron microscopy analysis revealed clear nanoparticle morphology and good particle integrity.

[0085] Example 3 Immunogenicity Study of Recombinant Nanoparticle Protein I. Immunity 1. Experimental Materials and Methods Experimental materials: 6-8 week old female Balb / c mice (Speford (Beijing) Biotechnology Co., Ltd., catalog number: B201-02).

[0086] Experimental methods: Sixty-four 6-8 week old Balb / c mice were randomly divided into 8 groups of 8 mice each. The treatment methods for each group were as follows: CHIKV37997 protein: Two intramuscular injections were administered in the thigh on day 0 and day 21, with each injection consisting of 1 μg of CHIKV37997 protein, 50 μg of aluminum hydroxide adjuvant (Croda, model: AJV3012 / 0250 / VP05), and 100 μl of PBS buffer (Solarbio, catalog number P1020).

[0087] CHIKV93 protein: Two intramuscular injections were administered in the thigh on day 0 and day 21, with each injection consisting of 1 μg of CHIKV93 protein, 50 μg of aluminum hydroxide adjuvant (Croda, model: AJV3012 / 0250 / VP05), and 100 μl of PBS buffer (Solarbio, catalog number P1020).

[0088] CHIKV107 protein: Two intramuscular injections were administered in the thigh on day 0 and day 21, with each injection consisting of 1 μg of CHIKV107 protein, 50 μg of aluminum hydroxide adjuvant (Croda, model: AJV3012 / 0250 / VP05), and 100 μl of PBS buffer (Solarbio, catalog number P1020).

[0089] CHIKV135 protein: Two intramuscular injections were administered in the thigh on day 0 and day 21, with each injection consisting of 1 μg of CHIKV135 protein, 50 μg of aluminum hydroxide adjuvant (Croda, model: AJV3012 / 0250 / VP05), and 100 μl of PBS buffer (Solarbio, catalog number P1020).

[0090] CHIKV176 protein: Two intramuscular injections were administered in the thigh on day 0 and day 21, with each injection consisting of 1 μg of CHIKV176 protein, 50 μg of aluminum hydroxide adjuvant (Croda, model: AJV3012 / 0250 / VP05), and 100 μl of PBS buffer (Solarbio, catalog number P1020).

[0091] II. Detection of antibodies in serum using ELISA method On day 28 post-immunization (approximately 6 weeks), mouse serum was collected for ELISA analysis. The specific steps of the ELISA analysis are as follows: 200 ng of CHIKV protein was used for coating each well. Mouse serum was used as the primary antibody and serially diluted at 250, 1250, 6250, 31250, 156250, 781250, and 3906250 times. The secondary antibody was mouse secondary antibody (Cell Signaling Technology, catalog number: 7076S). The signal was read using an ELISA reader (Shanghai Kehua, catalog number: RD-SH-012).

[0092] The titer of mouse serum after two immunizations was measured by ELISA as follows: Figure 4 As shown in the figure. The results indicate that CHIKV37997, CHIKV93, CHIKV107, CHIKV135, and CHIKV176 (A, B, C, D, and E, respectively) all produced ELISA binding titers. Among them, CHIKV107, CHIKV135, and CHIKV176 had higher mean ELISA binding titers, reaching 92160, 101120, and 104960, respectively, which were significantly different from CHIKV37997 and CHIKV93 (mean values ​​of 23680 and 44160).

[0093] III. Mouse serum neutralization test Pseudoviruses were prepared using 293T cells and culture medium provided by Beacon. For transient chemical transfection, reagents were prepared as follows: 45 μg plasmid and 180 μg PEI were added dropwise to every 30 mL of cell suspension while shaking. The mixture was then incubated in a shaker for 48 hours, after which the pseudoviruses were harvested (the preparation and titration methods for the pseudoviruses are described in the literature: Preparation and application of chikungunya pseudovirus containing double reporter genes). The titer of the cultured CHIKV pseudoviruses was determined using Vero cells and the Alex488 immunofluorescence assay, and the titer was 1.2E+04 PFU / mL.

[0094] Eight mouse serum samples from each of the above groups were selected and diluted with 1xPBS. Starting with a 40-fold dilution, the serum was serially diluted 3-fold to a final volume of 29-160-fold. This was mixed with an equal volume of 200 pfu of virus solution and incubated at 37°C for 1 hour. 200 μl of each sample was seeded into a VERO cell plate, with three replicates per mouse serum sample. The plates were incubated at 37°C for 22 hours. After incubation, the culture medium was removed, and 100 μl of 4% cell fixative was added to each well. 300 μl of 5% milk was added to each well, and the plates were incubated at 37°C for 0.5 hours. Primary antibody working solution (Sinobio, E2 / Envelope 2 Antibody, Catalog No.: Cat: 40440-T46, 500-fold dilution) and anti-human IgG Alex488 conjugated secondary antibody (Invitrogen) (1:500 dilution, diluted with PBS) were used. After incubation for 1 hour, unbound secondary antibody was washed away. Fluorescence was read using a CQ1 instrument. The readings were converted to percentages. That is, (fluorescence count in neutralizing wells - fluorescence count in blank control wells) / (fluorescence count in virus control wells - fluorescence count in blank control wells). Based on the percentage, calculate the serum dilution corresponding to a 50% reduction in fluorescence, and take the average of two replicates as the half-maximal protective neutralizing titer of that serum. Neutralizing antibody results were plotted using GraphPad Prism 8 software, and significant differences were analyzed using an unpaired t-test.

[0095] The results are shown in Table 2. The results indicate that CHIKV37997, CHIKV93, CHIKV107, CHIKV135, and CHIKV176 in this invention all exhibited pseudovirus neutralizing protective effects. Among them, CHIKV107, CHIKV135, and CHIKV176 had higher geometric mean titers (GMT) of neutralizing antibodies, reaching 3542, 2970, and 2617, respectively, showing significant differences from CHIKV37997 (P values ​​were 0.0002, ***; 0.0017, **; and 0.0021, **, respectively).

[0096] Table 2 Neutralization titers

[0097] The preferred embodiments of the present invention have been described in detail above. The present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0098] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0099] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A recombinant protein, characterized in that, The recombinant protein comprises the protein sequence of SEQ ID No: 2 or a variant thereof, wherein the protein sequence of the variant comprises a mutation at any of the positions 324, 479, 737, and 966 of SEQ ID No:

2.

2. The recombinant protein according to claim 1, characterized in that, The mutations mentioned include any one or more of the following: 1) R324N; 2) S479F; 3) L737R; 4) K966N.

3. The recombinant protein according to any one of claims 1-2, characterized in that, The protein sequence of the variant further includes mutations at any of the following positions in SEQ ID No: 2: 116, 197, 237, 243, 293, 878, 963, and 1105. Preferably, the mutations include one or more of the following: 1) E116D; 2) S197F; 3) R237N; 4) V243L; 5) E293D; 6) K878Q; 7) V963L; 8) L1105R.

4. The recombinant protein according to any one of claims 1-3, characterized in that, The protein sequence of the variant includes: (A1), as shown in any of SEQ ID No: 3-5, (A2) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of (A1); (A3) and any one of (A1)-(A2) that has more than 80% identity and the same function.

5. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the recombinant protein according to any one of claims 1-4.

6. A carrier, characterized in that, The carrier comprises the nucleotide molecule as described in claim 5.

7. A host cell, characterized in that, The host cell comprises the nucleotide molecule of claim 5 or the vector of claim 6.

8. A drug, characterized in that, The drug comprises any one of the recombinant proteins of claims 1-4, the nucleotide molecule of claim 5, the vector of claim 6, or the host cell of claim 7. Preferably, the drug is a vaccine. More preferably, the vaccine further comprises an adjuvant and / or a vaccine delivery system.

9. A method for preparing the recombinant protein of any one of claims 1-4, the host cell of claim 7, or the drug of claim 8, characterized in that, The preparation method includes introducing a nucleotide sequence encoding any one of the recombinant proteins of claims 1-4, a nucleotide molecule of claim 5, or a vector of claim 6 into a host cell, and culturing the host cell.

10. The use of any one of the recombinant proteins of claims 1-4, the nucleotide molecule of claim 5, the vector of claim 6, the host cell of claim 7, or the drug of claim 8, wherein the use comprises any one of the following: (1) Use in the preparation of products for the prevention and / or treatment of diseases caused by chikungunya virus infection; (2) Application in the preparation of products for inducing immune responses to chikungunya virus antigens.

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