Virus-like particle, preparation thereof and application of virus-like particle as vaccine

Abstract

The invention belongs to the field of biological products and the field of nucleic acid vaccines, and particularly relates to a virus (oropravirus)-like particle, preparation and application of the virus (oropravirus)-like particle as a vaccine. The virus-like particles are Oropers virus-like particles, and the virus-like particles contain proteins encoded by M genome segments of OROV strains or proteins encoded by gene segments of the M genome segments and N proteins of the OROV strains; wherein the corresponding sequence can also be a consistency sequence with the similarity of 75% with the corresponding sequence. Meanwhile, the particle VLP is used as a serum binding antibody of an antigen and an evaluation method of a T cell reaction, and the method can be used for detecting a serum lifting method after vaccine immunization. The invention further provides five nucleic acid vaccines for coding different functional domains of the OROV and combinations thereof, the immunogenicity and T cell reaction induced by the nucleic acid vaccines under different design strategies are displayed, strong body fluid and cell reaction is still kept 154 days after immunization, and it is proved that the nucleic acid vaccines can induce lasting and efficient immune reaction, and the immunogenicity and the T cell reaction are not influenced. The method can be used as a technical reserve and has huge clinical significance and application scenarios.

Description

Technical Field

[0001] This invention belongs to the fields of biological products and nucleic acid vaccines, specifically relating to a virus (oropecie virus)-like particle, its preparation, and its application as a vaccine. Background Technology

[0002] Oropouche virus (OROV) belongs to the genus Orthobunyavirus of the family Peribunyaviridae in the order Bunyavirales. It is an arbovirus that primarily uses the paracrine midge (Culicoides paraensis) as its intermediate host. OROV is a segmented, single-stranded negative-sense RNA virus with a genome consisting of three segments, named L (Large segment), M (Middle segment), and S (Smallsegment) according to their length. The L segment encodes RNA-dependent RNA polymerase (RdRp), and the M segment encodes a polyprotein. This polyprotein is cleaved by the host protease into two glycoproteins (Gn / Gc) and a non-structural protein (NSm). The Gn and Gc glycoproteins play important roles in viral infection and invasion, including receptor recognition and membrane fusion, while the role of NSm is unknown. The S fragment encodes the nucleocapsid protein (NP) and the non-structural protein NSs. The NSs of OROV have an interferon-antagonistic effect and are considered important virulence factors related to infection and immune escape. The N protein of Bunyaviridae members is one of the major viral antigens, and recombinant N proteins have been used for the serological diagnosis of several Bunyaviridae members, including Hantavirus, Puma virus, and Crimean-Congo hemorrhagic fever virus. OROV is an enveloped virus, approximately 100 nm in diameter. Its interior consists of ribonucleoprotein complexes formed by the interaction of the nucleocapsid protein and the RNA genome, with attached RdRp. The exterior is a double-membrane structure with spike structures formed by trimerized Gn / Gc heteroglycoprotein dimers.

[0003] Currently, no vaccines or drugs targeting OroV have been approved for clinical trials, and related laboratory research results are scarce. Only attenuated VSV-OROV chimeric virus is currently a candidate vaccine. This attenuated VSV-OROV chimeric virus vaccine can induce high titers of neutralizing antibodies in BALB / c mice and effectively reduce the degree of viremia and the viral load of OroV in brain tissue during OroV challenge experiments. However, this vaccine is constructed based on the OroV prototype strain BeAn19991, and its neutralizing effect against currently circulating strains has not been evaluated.

[0004] Based on the above technical background, the purpose of this invention is to design a variety of nucleic acid vaccines against Oropche virus and to develop a serological detection method based on Oropche virus-like particles, thereby further improving the methodology and etiological research technology system related to Oropche virus. Summary of the Invention

[0005] The purpose of this invention is to provide a virus (oropche virus)-like particle, its preparation, and its application as a vaccine.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A virus-like particle, wherein the virus-like particle is an Oropic virus-like particle, the virus-like particle containing a protein encoded by the M genome fragment of the Oropic virus strain, or a protein encoded by a gene fragment containing the M genome fragment and the N protein of the Oropic virus strain; wherein the corresponding sequence may also be a sequence with greater than 75% sequence similarity.

[0008] The M genome fragment of the OROV virus strain is shown in SEQ ID 01, and the sequence with greater than 75% similarity to it is shown in SEQ ID 09; the gene fragment of the N protein is shown in SEQ ID 03.

[0009] The M genome fragment has an N-terminus of the Gn glycoprotein with an HA tag attached and a C9 tag added to the C-terminus of the Gc protein, the sequence of which is SEQ ID 05; the N protein has a C-terminus of the FLAG tag attached, the sequence of which is SEQ ID 07.

[0010] A method for preparing the aforementioned virus-like particles:

[0011] The aforementioned virus-like particles were constructed onto the gene fragments and then onto plasmids (pcDNA3.1) to obtain recombinant plasmids.

[0012] The recombinant plasmid was co-transfected into eukaryotic expression cells, the cell supernatant was collected, and the bottom precipitate was collected and dissolved after ultracentrifugation using a sucrose pad.

[0013] Furthermore, the method for obtaining OROV virus-like particles by expressing the coding gene in eukaryotic cells using a eukaryotic expression system involves: cloning the coding gene of the protein or mutant into a baculovirus recombinant vector to construct a recombinant baculovirus recombinant vector; co-transfecting insect cells with the recombinant baculovirus recombinant vector and baculovirus DNA to obtain recombinant baculovirus; infecting insect hosts or insect cells with the recombinant baculovirus, culturing the infected insect cells or insect hosts to express the corresponding antigen, and purifying the virus, etc.

[0014] An application of the aforementioned virus-like particles: the application of the Oropec virus-like particles in vaccine immunization and immunization evaluation.

[0015] The oropuchie virus-like particles are used in an enzyme-linked immunosorbent assay (ELISA) to detect their application in vaccines.

[0016] The virus-like particles are used as an enzyme-linked immunosorbent assay (ELISA) to detect vaccine-induced serum antibody levels.

[0017] An application of the aforementioned virus-like particles, specifically the application of the Oropec virus-like particles in the preparation of nucleic acid vaccines.

[0018] One application of the aforementioned virus-like particles, wherein the oropecie virus-like particles are used as probes in antibody reactions. This includes, but is not limited to, colloidal gold test strip reactions.

[0019] An oropucian virus nucleic acid vaccine, wherein the immunogen sequence of the nucleic acid vaccine contains an M genome fragment of oropucian virus, or a gene fragment containing an M genome fragment of oropucian virus and an N protein; wherein the M genome fragment of oropucian virus may also be a sequence with one or more amino acid substitutions, deletions or insertions, or homologous sequences with sequence similarity greater than 75%.

[0020] The immunogen sequence in the nucleic acid vaccine contains a nucleic acid sequence encoding the genes of the oropeceutical virus Gn protein, Gc protein, or M protein and N protein; wherein, the M genome fragment of the oropeceutical virus may also be a sequence with one or more amino acid substitutions, deletions or insertions, or homologous sequences with sequence similarity greater than 75%.

[0021] The nucleic acid vaccine encoded by the genes of the oropeceutical virus Gn protein, Gc protein, M protein and N protein has the encoded amino acid sequences SEQ ID 12, SEQ ID 14, SEQ ID 16 and SEQ ID 18, respectively.

[0022] The nucleic acid sequence includes the following elements sequentially from 5' to 3': a 5' cap structure, a 5' UTR sequence, an immunogen sequence in the nucleic acid vaccine, a 3' UTR sequence, and a polyadenylate tail sequence.

[0023] The recombinant host cell for the recombinant expression vector is selected from prokaryotic cells, yeast, or eukaryotic cells, and may further be selected from Escherichia coli, Rhodococcus rubrum, Bacillus subtilis, and yeast. The recombinant expression vector described in the third aspect of the present invention is transformed into the host cell to obtain the corresponding genetically engineered bacteria.

[0024] In this invention, no limitation is made on the administration method of the provided pharmaceutical composition, which may include, for example, injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid dosage form, oral liquid dosage form), rectal, inhalation, implantation, and topical (e.g., ocular) administration. Non-limiting examples of parenteral administration preparations include, but are not limited to, injectable solutions, injectable dry powders, injectable suspensions, and injectable emulsions.

[0025] The main advantages of this invention lie in its high immunogenicity, broad-spectrum potential, safety, and synergistic application of multiple antigens. In terms of application, this invention has clear clinical application prospects and can be rapidly transformed into a preventative product against Oropec virus (OROV), effectively addressing the public health crisis caused by OROV. This research, through innovative mRNA and VLP technologies, has successfully constructed a highly efficient, safe, and rapidly producible OROV vaccine and a VLP-based serological detection method, possessing both scientific breakthrough and practical application value, providing important technological reserves for responding to the current epidemic and future emerging infectious diseases. Attached Figure Description

[0026] Figure 1 a represents a plasmid map of virus-like particles provided in an embodiment of the present invention, wherein... Figure 1 From top to bottom, in a are OROV-M-pcDNA3.1(+), OROV-N-pcDNA3.1(+), OROV-M(AM0088)-pcDNA3.1(+), OROV-M-pFastBac and OROV-N-pFastBac.

[0027] Figure 1 b is a plasmid map of the oropeceutical virus nucleic acid vaccine provided in this embodiment of the invention, wherein... Figure 1 From top to bottom, b represents OROV-M-pUC57, OROV-N-pUC57, OROV-Gn-pUC57, and OROV-Gc-pUC57.

[0028] Figure 1c shows the immunoblotting verification diagram of the in vitro expression of oropeceutical virus-like particles provided in this embodiment of the invention; where the left side represents the Gn domain of the M protein with an HA tag, and the right side represents the N protein with a FLAG tag. Immunoblotting hybridization verifies the presence of glycoprotein and N protein in the VLP. The expression of glycoprotein is detected using an HA-tagged antibody, and the expression of N protein is detected using a FLAG-tagged antibody. Red arrows indicate the corresponding specific bands.

[0029] Figure 2 This is an electron micrograph of virus-like particles provided in an embodiment of the present invention. The image was taken using a transmission electron microscope, with a scale bar of 100 nm.

[0030] Figure 3 This invention provides schematic diagrams of various constructed nucleic acid vaccine forms and in vitro nucleic acid expression verification diagrams. Specifically, a: Four mRNA vaccine template plasmids were constructed: M, Gc, Gn, and N. These four antigenic forms of mRNA-LNP were obtained through in vitro transcription and lipid nanoparticle encapsulation. Based on these four antigens, three single-component vaccines (Gn, Gc, and M) and two two-component vaccines (M / N and Gn / Gc) were prepared. b: Immunofluorescence assays were used to detect expression. N mRNA expression was detected using a FLAG-tagged antibody, M mRNA expression using a HA-tagged antibody, and Gn and Gc mRNA expression using a His-tagged antibody.

[0031] Figure 4 The diagrams provided in this embodiment of the invention show the mouse immunization process and humoral immunity level evaluation for various nucleic acid vaccines, where a: mouse immunization process, b: specific antibodies against virus-like particles in mouse serum on day 42 post-immunization, c: specific antibodies against virus-like particles in mouse serum on day 84 post-immunization, and d: specific antibodies against virus-like particles in mouse serum on day 154 post-immunization.

[0032] Figure 5 The present invention provides a nucleic acid vaccine-induced mouse-specific IgG typing detection method; wherein, a: mouse serum specific IgG1 antibody against virus-like particles, b: mouse serum specific IgG2a antibody against virus-like particles, c: mouse serum specific IgG3 antibody against virus-like particles, and d: ratio of mouse serum specific IgG2a antibody to IgG1 antibody against virus-like particles.

[0033] Figure 6 This is an evaluation diagram of the binding antibody against the AM0088 mutant strain of Oropecie virus induced by the nucleic acid vaccine provided in this embodiment of the invention.

[0034] Figure 7The image shows the T-cell response evaluation of mouse spleen cells to the OROV specific antigen, provided in an embodiment of the present invention. Wherein, a: T-cell response evaluation of mouse spleen cells to OROV virus-like particles; b: T-cell response evaluation of mouse spleen cells to the T-cell peptide of protein N in Figure N.

[0035] Figure 8 The results of the serum neutralization experiment of mouse post-immunization serum against pseudoviruses provided in this embodiment of the invention; wherein, Figure 8 Left: OROV (BeAn19991 strain) - VSV pseudovirus and Figure 8 Right: OROV (AM0088 strain) - VSV pseudovirus. Detailed Implementation

[0036] Throughout the specification and claims, in this invention, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0037] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be fully described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0039] Numerous specific details are set forth in the following detailed description of embodiments. Those skilled in the art will understand that the invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well-known to those skilled in the art are not described in detail in order to highlight the main points.

[0040] Example 1: Preparation of Oropec virus-like particles and verification by Western blotting

[0041] 1. Plasmid preparation method

[0042] 1) The amino acid sequences of oropeceutical virus strain BeAn19991 M (AJE24679.1) and N protein (AJE24680.1) were obtained by searching the GenBank database as SEQ ID 02 and SEQ ID 04, respectively; and the amino acid sequence of oropeceutical virus strain AM0088 M protein (XHP15871.1) was obtained as SEQ ID 10.

[0043] 2) Link the N-terminus of the Gn glycoprotein obtained from the M gene of BeAn19991 strain to the HA tag, and add the C9 tag to the C-terminus of Gc, i.e., the sequence is SEQ ID 05; then link the C-terminus of the N protein obtained from BeAn19991 strain (same sequence as AM0088 strain) to the FLAG tag, i.e., the sequence is SEQ ID 07.

[0044] The amino acid sequence of the M protein of the Oropecie virus strain AM0088 obtained above is SEQ ID 09.

[0045] Meanwhile, the nucleotide sequence of the obtained sequence was optimized by codons to obtain the corresponding nucleotide sequence.

[0046] 3) The optimized nucleotide sequence of the Oropecchiovirus BeAn19991 strain obtained above was used for gene synthesis. Then, the optimized nucleotide sequence was recombinated with either pcDNA3.1(+) or pFastBac plasmid to construct expression plasmids such as OROV-M-pcDNA3.1(+), OROV-N-pcDNA3.1(+), OROV-M-pFastBac, or OROV-N-pFastBac (e.g., Figure 1 (as shown in a).

[0047] 4) Obtain a large number of plasmids by transforming OROV-M-pcDNA3.1(+) or OROV-N-pcDNA3.1 plasmids into TOP10 or DH10Bac Escherichia coli competent cells.

[0048] 2. Preparation of Oropecie virus-like particles for eukaryotic expression system

[0049] 1) Use approximately 1x10 one day in advance. 6 Expi 293F cells or Freestyle 293F cells were seeded at different densities in suspension cell culture flasks.

[0050] 2) Co-transfect the expression plasmids OROV-M-pcDNA3.1(+) and OROV-N-pcDNA3.1(+) at a mass ratio of 1:1 into the cell suspension culture flasks described above.

[0051] 3) Five days later, collect and culture the supernatant.

[0052] 3. Preparation of Oropuch virus-like particles for insect cell expression system

[0053] 1) Inoculate each well of a six-well plate with 1.5 x 10 g of the solution one day in advance. 6 Sf9 cells.

[0054] 2) The expression plasmids OROV-M-pFastBac and OROV-N-pcFastBac were co-transfected into the above Sf9 cells at a mass ratio of 1:1.

[0055] 3) Incubate at 28℃ for 4–6 hours, then replace with fresh serum-free culture medium and continue culturing for 5 days.

[0056] 4) Infect more logarithmically growing Sf9 cells with P0 virus at a 1:50 volume ratio to prepare P1 virus. Observe cytopathic effect (CPE) after 72 hours and collect the supernatant. Further increase the volume of P1 according to this procedure to obtain high-titer (102) P2 / P3. 6 (The above) virus is for backup.

[0057] 5) Select virus P3 with a high titer and an infection density of 2 x 10⁻⁶. 6 / ml High Five cells.

[0058] 6) After culturing at 28℃ for 72 hours, collect the culture supernatant.

[0059] 4. Enrichment and purification of virus-like particles by ultracentrifugation

[0060] After collecting the culture supernatant from the above eukaryotic expression system Oropche virus-like particles (OROVM+N) and insect cell expression system Oropche virus-like particles (OROVVLP), they were subjected to ultracentrifugation with 20% sucrose solution.

[0061] The centrifugation parameters were: 100,000g, 2 hours, 4℃, and the centrifuge rotor was SW 32Ti.

[0062] The precipitate at the bottom of the centrifuge tube was retained, dissolved, and purified oropche virus VLP was obtained.

[0063] Western blotting was used to detect the components of VLP.

[0064] 1) Take 5 μg of purified VLP and perform 10% SDS-PAGE gel electrophoresis.

[0065] 2) Transfer the membrane onto the PVDF membrane.

[0066] 3) Block with TBST containing 5% BSA for 1 hour.

[0067] 4) Incubate with primary antibody overnight at 4℃.

[0068] 5) Incubate with the secondary antibody for 1 hour.

[0069] 6) Wash with TBST after each antibody incubation.

[0070] 7) Add substrate and develop, then capture images using a Bio-Rad ChemiDoc developer (see...). Figure 1 c and Figure 2 ).

[0071] Results and Analysis: In the eukaryotic expression system co-transfected with M genome-encoded proteins and N protein particles, the purified VLP contained OROV glycoprotein and nucleocapsid protein; and... Figure 1 In vitro expression of oropeceutical virus-like particles (VLPs) was validated using Western blotting. Immunoblotting hybridization confirmed the presence of glycoproteins and N protein in the VLPs. HA-tagged antibodies were used to detect glycoprotein expression, and FLAG-tagged antibodies were used to detect N protein expression. Simultaneously, Figure 2 Under electron microscopy, OROV M+N virus-like particles with a spherical three-dimensional morphology of about 80 nm can be seen after negative staining with uranium acetate.

[0072] Example 2: Preparation of nucleic acid vaccine and verification of in vitro expression of mRNA.

[0073] 1. Transcription template preparation and linearization

[0074] 1) According to the gene obtained in Example 1, the N-terminus of the Gn glycoprotein in the M gene (BeAn19991) was linked to the HA tag for expression detection, the sequence of which is SEQ ID 05; the C-terminus of the N protein was linked to the FLAG tag for expression detection, the sequence of which is SEQ ID 07; and the C-terminus of the Gn and Gc proteins were linked to the HIS tag, which are SEQ ID 11 and SEQ ID 13, respectively.

[0075] 2) The sequence obtained in step (1) above is codon optimized, and then the nucleotide sequence is used for gene synthesis. The gene is then constructed into pUC57 as a vector by a company (e.g., Figure 1 As shown in b), template plasmids of OROV-M-pUC57, OROV-N-pUC57, OROV-Gn-pUC57, and OROV-Gc-pUC57 were constructed respectively.

[0076] 3) The above-obtained template plasmids were transformed into TOP10 E. coli competent cells to obtain a large number of plasmids.

[0077] 4) Take 50 μg of each template plasmid and add 5 μL of BspQI restriction endonuclease, react at 37℃ for 2 h to linearize.

[0078] 5) Obtain the target band of the linearized plasmid by electrophoresis, and purify it by gel extraction kit to obtain linearized DNA.

[0079] 2. In vitro transcription (IVT)

[0080] 1) In vitro transcription reaction system: Add T7 RNA polymerase mix, 10×Transcription Buffer, ATP, GTP, CTP, N1-Me-Pseudo UTP according to the method provided in the kit to obtain different linearized DNA templates and RNase-free H2O, and react at 37℃ for 2 hours.

[0081] 2) Add 1 μL of DNase I to each of the above-obtained in vitro transcription reaction systems and react at 37°C for 15 minutes.

[0082] 3) Obtain the initial in vitro transcription product, and then perform enzymatic in vitro capping reaction and RNA purification.

[0083] 2. Enzymatic capping reaction

[0084] The obtained in vitro transcripts were diluted with enzyme-free water to a concentration of 1 μg / μL. The resulting RNA was then denatured at 65°C for 5 minutes to eliminate its secondary structure.

[0085] For the enzymatic capping reaction of in vitro transcribed RNA, RNA, 10× Capping Buffer, SAM (4mM), GTP (10mM), Vaccinia Capping Enzyme, and mRNA Cap2'-O-Methyltransferase were added and reacted at 37°C for 2 hours. The product was then purified into RNA.

[0086] 3. Immunofluorescence assay to verify mRNA expression

[0087] 1) 24 hours before transfection, 293T cells were seeded in six-well tissue culture dishes (0.5×10⁶ / well); and cultured in DMEM containing 10% FBS for 24 hours.

[0088] 2) In 6-well plates, HEK293T cells were transfected with 3 μg of the purified in vitro transcribed mRNA. The Gn / Gc group was transfected with 1.5 μg of Gn mRNA and 1.5 μg of Gc mRNA, while the control group was not transfected with mRNA.

[0089] 3) Remove the transfected cells and discard the culture medium.

[0090] 4) Fix with 4% paraformaldehyde and leave at room temperature for 30 minutes.

[0091] 5) Aspirate the supernatant, add PBS buffer containing 0.05% Triton X-100, and incubate at room temperature for 15 minutes.

[0092] 6) Aspirate the supernatant, add 5% skim milk, and seal at 37°C for 2 hours.

[0093] 7) Aspirate the supernatant, add the primary antibody, and incubate at 37°C for 1.5 hours.

[0094] 8) Aspirate the primary antibody and wash with PBST.

[0095] 9) Aspirate the supernatant, add the secondary antibody, and incubate at 37°C for 45 minutes.

[0096] 10) Aspirate the secondary antibody and wash with PBST.

[0097] 11) Aspirate the supernatant, add diluted DAPI staining solution, and incubate at 37°C for 15 minutes.

[0098] 12) Remove the DAPI staining solution, add 1 mL of PBST, and observe under a fluorescence microscope. Figure 3 Results and Analysis:

[0099] like Figure 3 As shown, M, Gc, Gn, and N mRNAs transcribed in vitro can be expressed normally in vitro. In particular, through immunofluorescence experiments, it was found that under the same transfection and immunofluorescence experimental conditions, there was a significant difference in the mRNA expression efficiency of Gn and Gc, which is speculated to be related to factors such as mRNA sequence and mRNA secondary structure.

[0100] Example 3: Evaluation of humoral immunity using enzyme-linked immunosorbent assay (ELISA) for oropic virus nucleic acid vaccine

[0101] 1) Take 6-8 week old Balb / c mice for mouse immunization experiments (e.g. Figure 4 (as shown in a) Five mice were in each group. Each mouse was immunized intramuscularly three times every 14 days with a dose of 5 μg / mouse. On day 42, day 84 and day 154, peripheral blood of mice was collected to separate serum for the experiment.

[0102] The groups are respectively set as Gc group (i.e., OROV-Gc-pUC57), Gn group (i.e., OROV-Gn-pUC57), Gn / Gc group (i.e., OROV-Gn-pUC57 and OROV-Gc-pUC57 are mixed in a 1:1 mass ratio), M group (i.e., OROV-M-pUC57), M / N group (i.e., OROV-M-pUC57 and OROV-N-pUC57 are mixed in a 1:1 mass ratio), and VLP / Al group (i.e., the virus-like particles OROVVLP prepared in Example 1 are mixed with aluminum phosphate adjuvant in a 1:1 volume ratio).

[0103] 2) The oropche virus-like particles (OROV M+N) (BeAn19991 strain or the mutant strain AM0088 strain) prepared in Example 1 above at a concentration of 1 μg / mL were coated onto an ELISA plate at 4°C overnight.

[0104] 3) After coating, block with blocking buffer (PBS containing 5% BSA) at 37°C for 2 hours.

[0105] 4) Mouse serum was serially diluted 7 times in PBST at a ratio of 1:6, with the first well being diluted 1:100.

[0106] 5) Incubate the diluted serum with the coated ELISA plate at 37°C for 1 hour.

[0107] 6) Wash with PBST 4 times.

[0108] 7) Add the secondary antibody to the ELISA plate and incubate at 37°C for 45 minutes.

[0109] 8) Wash with PBST 4 times.

[0110] 9) Add tetramethylbenzidine (TMB) for color development.

[0111] 10) Terminate the reaction and read the absorbance at 450 nm.

[0112] 11) The ELISA endpoint titer is expressed as the reciprocal of the highest serum dilution factor in which the absorbance value at 450 nm is 2.1 times higher than the background value (see [reference]). Figure 4 ).

[0113] Results and Analysis:

[0114] The titer of oropeceutical virus-specific IgG in the serum of immunized mice was quantitatively detected by ELISA. The endpoint titer was determined using the endpoint titer method, with titers greater than 2.1 times the background OD value recorded as the endpoint titer. The quantitative results (BeAn19991 strain) are as follows: Figure 4As shown, 14 days after the second booster immunization, i.e., day 42 of the immunization schedule, all vaccine groups induced a certain level of binding antibodies. The binding antibody titer in the Gc group was 1,228-fold dilution; the binding antibody titers in the Gn and Gn / Gc groups were 15,094 and 10,548-fold dilutions, respectively; and the binding antibody titers in the M and M / N groups were 44,230 and 129,600-fold dilutions, respectively. The binding antibody titer in the VLP / Al group was 10,549-fold dilution.

[0115] Fifty-six days after the second booster immunization, i.e., day 84 of the immunization schedule, the antibody titers in all groups except Gc showed no significant decrease. Twelve-six days after the second booster immunization, i.e., day 154 of the immunization schedule, the antibody titer in the Gc group decreased to 293-fold dilution, the titers in the Gn and Gn / Gc groups decreased slightly compared to before, and the titers of the OROV-specific binding antibodies in the M and M / N groups still did not show a significant decrease, remaining at 44,230-fold and 129,600-fold dilutions, respectively.

[0116] To further analyze the vaccine-induced humoral immune response, ELISA was used to quantitatively detect the titers of oropeceutical virus (BeAn19991 strain)-specific IgG subtypes: IgG1, IgG2a, and IgG3 in the serum of immunized mice (see [link to ELISA]). Figure 5 ).

[0117] In IgG1, except for the Gc group which induced only a 205-fold dilution of binding antibody titer, all other groups induced IgG1 subtype binding antibodies at titers ranging from 5,151 to 10,549-fold. The M and M / N groups induced IgG2a subtype binding antibodies at titers of approximately 5,151-fold and 21,600-fold dilutions, respectively; all groups also induced IgG3 subtype binding antibodies at titers ranging from approximately 83 to 205-fold. The results indicate that the M / N group induced a stronger and more sustained humoral immune response, with OROV-specific binding antibody titers reaching 129,600-fold dilutions, which could be maintained for more than 120 days after booster immunization. Furthermore, the M / N group induced an immune response predominantly Th1-type dominant.

[0118] Furthermore, since the mutant strain AM0088 shares the same N protein sequence as the BeAn19991 strain, the M protein sequence shared by the BeAn19991 strain was replaced with the M sequence from the mutant strain AM0088 to prepare the corresponding virus-like particles, following the method described above. Moreover, the M / N ratio in the binding antibody levels of the AM0088 virus-like particles showed a significantly stronger effect than that of other groups of immune sera, with the geometric mean titer maintained at 10. 5 The above (e.g.) Figure 6(As shown). It is believed that because the M / N group mRNA vaccine simultaneously delivers mRNA encoding both the full-length OROV glycoprotein and nucleocapsid protein, if both mRNAs are simultaneously delivered to and expressed in the same cell, VLP may be secreted in vivo, thus further enhancing the immune response on top of the mRNA vaccine immunization. This process partially mimics the life cycle of live OROV infection and displays viral antigens in multiple forms, thereby inducing a more comprehensive immune response in mice.

[0119] Example 4: Evaluation of Oropecene virus nucleic acid vaccine by enzyme-linked immunospot assay

[0120] 1) In Example 3 above, the spleen cells of mice were isolated and immunized with the oropecchiovirus nucleic acid vaccine obtained by BeAn19991 strain, and then prepared into a single-cell suspension after erythropoiesis.

[0121] 2) IFN-γ positive cells were detected using the MabTech Mouse IFN-γ ELISpot Flex (ALP) kit. All procedures were performed according to the kit instructions.

[0122] 3) Mouse spleen cells were seeded into ELISPOT plates at a density of 3 × 10⁵ cells per well.

[0123] 4) Stimulate with 2 μg / 100 μL of oropeceutical virus VLP or 1 μg / 100 μL of peptide for 48 hours.

[0124] 5) After incubating the relevant antibodies, use Analyzers count antigen-specific spots (see...) Figure 7 ).

[0125] Results and Analysis:

[0126] To evaluate the cellular immune response to the OROV vaccine (e.g. Figure 7 As shown in the figure, the number of T cells activating and secreting IFN-γ after stimulation with oropeceutical virus VLP or nucleocapsid protein immunodominant epitope peptide N2-3 was detected using ELISPOT. In the Gn / Gc group, few T cells were induced to secrete IFN-γ under stimulation with either VLP or N2-3 peptide. The M group only showed strong cellular immunity under VLP stimulation, inducing a strong IFN-γ response. The M / N group exhibited the best cellular immune response, inducing strong IFN-γ secretion under both VLP and N2-3 peptide stimulation. Furthermore, on day 126 after the second booster immunization, the intensity of the cellular response decreased slightly, but remained close to the intensity on day 7 after the second booster immunization. In conclusion, the mRNA vaccine in the M / N group induced a strong and durable cellular immune response.

[0127] Example 5: Evaluation of humoral immunity to the oropche virus nucleic acid vaccine using the OROV-VSV pseudovirus neutralization assay.

[0128] Fake virus packaging:

[0129] 1) Seed HEK293T cells in advance at a rate of 1,000,000 cells / well in a six-well plate and culture for 24 hours;

[0130] 2) Transfect 293T cells with the above-prepared OROV-M-pcDNA3.1(+) or the pcDNA3.1 eukaryotic expression plasmid containing the M protein of the AM0088 strain (for the construction of the pcDNA3.1 eukaryotic expression plasmid of the M protein of the AM0088 strain, please refer to the above-mentioned construction of OROV-M-pcDNA3.1(+)). Specifically, dilute 5 μg of the corresponding plasmid with 100 μL LMEM and dilute 2 μL of transfection reagent (Vigofect) with 100 μL LMEM. After mixing, let stand at room temperature for 15 minutes and then add to a six-well plate.

[0131] 3) Twelve hours after transfection, wash twice with PBS and add 40 μL of solution containing 10 5 Add the above titer of VSV-G to the cell supernatant and incubate for 2 hours.

[0132] 1) Wash twice with PBS, add 1 μg / ml I1 monoclonal antibody, and incubate in DMEM medium with 10% fetal bovine serum for 24 hours. Collect the supernatant as OROV-VSV pseudovirus. Figure 8 (Left) or OROV-AM0088-VSV fake virus ( Figure 8 right)

[0133] Pseudovirus neutralization test:

[0134] 1) Use Huh7 cells, seeded at a density of 10,000 cells per well in a 96-well plate, and culture for 24 hours.

[0135] 2) Subsequently, a viral solution containing 1 μg / ml of I1 monoclonal antibody was prepared as a diluent for the mouse serum on day 42 in Example 3 above (e.g., Figure 4 (As shown in a) The first well was diluted 200-fold, followed by 10-fold dilutions (for the pseudovirus BeAn19991, such as...). Figure 8 (Left image) or 4 times (for fake viruses like AM0088, such as) Figure 8 (Right figure) Compared to dilution.

[0136] 3) Then aspirate the supernatant of Huh7 cell culture medium and add 100 μL of diluted serum to each well.

[0137] 4) After 12 hours, add 100 μL of culture medium containing 10% fetal bovine serum.

[0138] 5) After 30 hours, lyse the cells, add Renilla luciferase substrate, and read the fluorescence intensity.

[0139] Results and Analysis:

[0140] The average fluorescence intensity of the virus wells was used as the virus infection value to calculate the inhibition rate for each well. An inhibition curve was then fitted using Graphpad (see [link to graphpad]). Figure 8 Quantitative results are as follows: Figure 8 As shown, 14 days after the second booster immunization (day 42 of the immunization schedule), the neutralizing titer of serum from the VLP / A1 immunized group against the BeAn19991 (OROV-VSV) pseudovirus was 184,076-fold dilution. In contrast, the neutralizing titers of serum from the M and M / N immunized groups against the BeAn19991 pseudovirus were 3,746,973-fold and 1,421,954-fold dilutions, respectively, and against the AM0088 pseudovirus were 138,520-fold and 108,064-fold dilutions, respectively. This demonstrates that both the M and M / N nucleic acid vaccines exhibit a strong neutralizing response to the BeAn19991 pseudovirus and can maintain a highly efficient neutralizing level against the AM0088 mutant pseudovirus.

Claims

1. A virus-like particle, characterized in that: The virus-like particle is an Oropouche virus-like particle, and the virus-like particle contains proteins encoded by an M genome segment of an OROV strain, or contains proteins encoded by an M genome segment of an OROV strain and an N protein gene segment; wherein the corresponding sequence can also be a sequence with a sequence similarity of greater than 75%.

2. The virus-like particle according to claim 1, characterized in that: The M genome segment of the OROV strain is an M genome segment of an OROV BeAn19991 strain, and the sequence is shown as SEQ ID 01, and a sequence with a sequence similarity of greater than 75% is shown as SEQ ID 09; the N protein gene segment is shown as SEQ ID 03.

3. The virus-like particle of claim 1, wherein: The N-terminal of the Gn glycoprotein in the M genome segment is connected to an HAtag, and the C-terminal of the Gc is connected to a C9 tag, and the sequence is shown as SEQ ID 05; the C-terminal of the N protein is connected to a FLAG tag, and the sequence is shown as SEQ ID 07.

4. A preparation method of the virus-like particle of claim 1, characterized in that: the gene segments of the virus-like particle in claim 1 are respectively constructed on plasmids to obtain recombinant plasmids; the recombinant plasmids are co-transfected into eukaryotic expression cells, cell supernatants are collected, and the bottom precipitate is collected after using a sucrose pad to perform ultracentrifugation.

5. Use of the virus-like particle according to claim 1, characterized in that: The Oropouche virus-like particle is used for vaccine immunization and immune evaluation.

6. Use of the virus-like particle according to claim 1, characterized in that: The Oropouche virus-like particle is used for preparing a nucleic acid vaccine.

7. Use of the virus-like particle according to claim 1, characterized in that: The Oropouche virus-like particle is used as a probe for detecting antibody reactions.

8. An orthopoxvirus nucleic acid vaccine, characterized in that, The immunogen sequence in the nucleic acid vaccine contains an M genome segment of an Oropouche virus, or contains an M genome segment of an Oropouche virus and an N protein gene segment; wherein the M genome segment of the Oropouche virus can also be a sequence with a sequence similarity of greater than 75% after one or more amino acids are substituted, deleted or inserted.

9. The Orthopoxvirus nucleic acid vaccine of claim 8, wherein, The immunogen sequence in the nucleic acid vaccine contains nucleic acid sequences encoded by genes of Oropouche virus Gn protein, Gc protein, or M protein and N protein; wherein the M genome segment of the Oropouche virus can also be a sequence with a sequence similarity of greater than 75% after one or more amino acids are substituted, deleted or inserted.

10. The Orthopoxvirus nucleic acid vaccine of claim 8 or 9, characterized in that, The nucleic acid sequence comprises the following elements in the order from 5' to 3' direction: a 5' cap structure, a 5' UTR sequence, the immunogen sequence in the nucleic acid vaccine of claim 8 or claim 9, a 3' UTR sequence, and a polyadenylic acid tail sequence.

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