Influenza A virus-like particle for chimeric expression of novel coronavirus RBD protein as well as preparation method and application of influenza A virus-like particle

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to influenza virus-like particles for chimeric expression of new coronavirus RBD protein as well as a preparation method and application of the influenza virus-like particles. The virus-like particle is prepared by embedding fusion protein on the surface of influenza virus M1, and the fusion protein comprises an HA2 segment of HA protein of influenza virus, an extracellular functional region of M2e matrix protein, a conserved region short peptide of nucleoprotein, and RBD tandem protein with mutated five sites of 417, 452, 478, 484 and 501 of new coronavirus; the virus-like particles can be used for preventing specific influenza and new crowns; the preparation method of the virus-like particles is simple and low in cost, the virus-like particles are good in immunogenicity and cross protectiveness, the preparation requirements of large-scale influenza vaccines can be met, the biological safety is high, and the immunity of an organism to various influenza viruses and new coronal viruses can be stimulated.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology, and in particular relates to influenza virus-like particles that chimera express the SARS-CoV-2 RBD protein, as well as a preparation method and application thereof. Background Art

[0002] Due to the high mutability of influenza and COVID-19 viruses, individuals infected with the virus are unable to develop an antibody response to prevent reinfection. Vaccines remain an effective means of protecting humans against influenza and COVID-19. Traditional vaccines are administered intramuscularly, which does not produce effective mucosal immunity and cannot generate immune responses immediately following infection. Therefore, continuous improvement of current vaccine production strategies to enhance the vaccine's protective potency, breadth, and variant coverage is a key focus of current vaccine research. Furthermore, a universal vaccine that provides multiple protections with a single injection, possesses broad-spectrum protective activity, and is administered transmucosally could reduce the number of injections, minimize complications, and enhance immune efficacy.

[0003] The insect baculovirus expression system can simultaneously express multiple structural proteins and automatically assemble to form virus-like particles. Virus-like particles are assembled from specific proteins of the virus but do not contain the viral nucleic acid components. The conformation of the outer surface antigen is similar to that of the original virus, thus causing humoral and cellular immune stimulation. At the same time, it can be inhaled through the nasal cavity and has the advantages of high biosafety, low cost, high yield and good effect. It can be used to develop influenza virus and new coronavirus vaccines. Summary of the Invention

[0004] To achieve cross-protection against influenza virus and novel coronavirus, the present invention provides an influenza virus-like particle (IVLP) chimerically expressing the novel coronavirus RBD, as well as a preparation method and application thereof. By rationally designing the protein fragments of the immunogen, the present invention improves the immunogenicity and cross-protection of the VLP, and can be used for the development and production of vaccines to prevent multiple strains of influenza and novel coronavirus.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: Influenza virus-like particles (VLPs) expressing the SARS-CoV-2 RBD protein are prepared by displaying the fusion protein (TsHA protein) on the surface of the influenza virus M1 protein. The amino acid sequence of the M1 protein is shown in SEQ ID NO:1, and the ORF of the gene encoding the M1 protein is shown in SEQ ID NO:2.

[0006] The fusion protein comprises a tandem protein of the influenza virus HA protein (the HA2 segment of the HA protein), the M2e protein (the extracellular domain of the M2e matrix protein), and a highly conserved sequence of the NP protein (a short peptide from the conserved region of the nucleoprotein), along with the RBD protein with mutations at five sites: 417, 452, 478, 484, and 501. Specifically, the sequence of the fusion protein from N-terminus to C-terminus comprises: RBD1 with five common mutation sites, RBD2 with five common mutation sites, two conserved sequences from the nucleoprotein, five M2e proteins from different subtypes and species, and the HA2 segment of the HA protein. The sequences are connected by a linker peptide. The sequences of RBD1 with the five common mutation sites and RBD2 with the five common mutation sites are identical. Furthermore, the fusion protein contains a bee venom signal peptide (SP) at the N-terminus and the TM (transmembrane region) and CT (intracellular region) of the HA protein at the C-terminus. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 3, and the nucleic acid sequence of the gene encoding the fusion protein is shown in SEQ ID NO: 4.

[0007] The present invention expresses conserved regions of the HA protein, the conserved region M2e of the M2 protein, and the conserved region of the NP protein in tandem, then connects the RBD-RBD of the SARS-CoV-2 virus containing five frequently mutated sites: 417, 452, 478, 484, and 501. This fusion protein is then combined with the transmembrane region (TM) and intracellular region (CT) of the HA protein to recombinantly generate a fusion protein. This fusion protein is then displayed on the surface of influenza virus-like particles to construct a universal virus-like particle. This virus-like particle has the ability to provide specific immunity against both influenza and the SARS-CoV-2, and can produce effective mucosal immunity when administered nasally.

[0008] The method for preparing virus-like particles chimerically expressing the SARS-CoV-2 RBD protein comprises the following steps: Step 1: Clone the influenza virus M1 protein encoding gene into the baculovirus expression plasmid to obtain the recombinant M1 plasmid. The specific steps are as follows: Perform multiple cloning site analysis on the influenza virus M1 protein encoding gene and the gene of the baculovirus expression plasmid. Select two restriction endonucleases provided on the baculovirus expression plasmid but not on the target fragment (M1 protein encoding gene). Design PCR primers for the target fragment. After amplification and double enzyme digestion of the target fragment, recover the target fragment. Insert the target fragment into the cloning site behind the promoter on the baculovirus expression plasmid and transform competent E. coli cells to obtain a recombinant M1 plasmid. The coding gene of the fusion protein was cloned into the baculovirus expression plasmid to obtain the recombinant TsHA plasmid. The specific steps are as follows: The fusion protein encoding gene is obtained through gene synthesis technology. The fusion protein encoding gene and the gene of the baculovirus expression plasmid are subjected to multiple cloning site analysis. Two restriction endonucleases provided on the baculovirus expression plasmid but not on the target fragment (fusion protein encoding gene) are selected. PCR primers for the target fragment are designed. The target fragment is amplified and double-enzyme digested to recover the target fragment. The target fragment is inserted into the cloning site behind the promoter on the baculovirus expression plasmid and transformed into competent Escherichia coli cells to obtain a recombinant TsHA plasmid. The baculovirus expression plasmid is pFastBac1. The competent E. coli cell is DH5α.

[0009] Step 2: Transform the recombinant M1 plasmid and the recombinant TsHA plasmid into competent E. coli cells to obtain recombinant baculovirus plasmids Bacmid-M1 and Bacmid-TsHA, respectively. Use the recombinant baculovirus plasmids Bacmid-M1 and Bacmid-TsHA to transfect insect cells, respectively, to rescue recombinant baculovirus M1 and recombinant baculovirus TsHA. The competent E. coli cells used were DH10Bac, and the insect cells were Sf9 cells. The confluence of the Sf9 insect cells reached over 80% during transfection.

[0010] The specific steps for obtaining recombinant baculovirus are as follows: recombinant M1 plasmid and recombinant TsHA plasmid are added to Escherichia coli competent cells respectively, ice-bathed for 30 minutes, heat-shocked at 42°C for 45 seconds, then ice-bathed for 2 minutes, added with antibiotic-free LB medium, and shaken at 37°C and 200 rpm for 4 hours. 100 µL of the shaken solution is spread on a triple-antibody LB plate containing X-gal and IPTG, and cultured at 37°C for 48 hours. The white spots screened by blue-white spot screening are the recombinant baculovirus plasmid Bacmid.

[0011] Step 3: Mix the recombinant baculovirus M1 and recombinant baculovirus TsHA and inoculate the suspended insect cells. After harvesting and purification, virus-like particles expressing the SARS-CoV-2 RBD protein are obtained. The details are as follows: After recombinant baculovirus M1 and recombinant baculovirus TsHA were mixed at a virus titer ratio of 1:1, the suspended insect cells were inoculated at an MOI of 3, and harvested 3 days later. Virus-like particles expressing the RBD protein of the new coronavirus were obtained after purification. The insect cells used for inoculation of recombinant baculovirus were Sf9 cells, and the concentration of Sf9 insect cells was 2×10 6 / mL or above.

[0012] The virus-like particles were purified by sucrose density gradient centrifugation. The specific process was as follows: after culturing the insect cells inoculated with the recombinant baculovirus for 72 hours, the suspended cell culture medium was collected and centrifuged at 4°C and 4000 rpm for 20 minutes to obtain the supernatant. The supernatant was concentrated by ultracentrifugation at 4°C and 30,000 rpm for 1 hour. The concentrate was resuspended in PBS and dissolved at 4°C overnight. Finally, it was centrifuged at 30,000 rpm for 1 hour in 20%-30%-60% sucrose. After the sucrose removal step, the solution between 30% and 60% gradient was centrifuged at 30,000 rpm for 1 hour. The precipitate was resuspended to obtain the purified virus-like particles.

[0013] The present invention also provides the use of the prepared virus-like particles that chimerally express the SARS-CoV-2 RBD protein in the preparation of vaccines for preventing influenza and / or SARS-CoV-2 infection.

[0014] The beneficial effects of the present invention are: The present invention utilizes the baculovirus insect expression system as an expression vector and utilizes cells as a bioreactor to produce virus-like particles, which has the advantages of high yield and low production cost. The virus-like particles produced have good immunogenicity and cross-protection, can be used for the large-scale preparation of influenza vaccines, and have no problems such as biosafety risks. It can stimulate the body's immunity to multiple influenza viruses and the new coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the gene composition of the fusion protein (TsHA protein) provided in an embodiment of the present invention.

[0016] Figure 2 Figures of indirect immunofluorescence provided in the examples of the present invention. A is an immunofluorescence image of TsHA-VLPs obtained using HA mouse polyclonal antibody as the primary antibody, B is an immunofluorescence image of TsHA-VLPs obtained using RBD rabbit monoclonal antibody as the primary antibody, and C is an immunofluorescence image of TsHA-VLPs obtained using M1 mouse polyclonal antibody as the primary antibody. D is a test image of Sf9 cells free of baculovirus infection obtained using HA mouse polyclonal antibody as the primary antibody, E is a test image of Sf9 cells free of baculovirus infection obtained using RBD rabbit monoclonal antibody as the primary antibody, and F is a test image of Sf9 cells free of baculovirus infection obtained using M1 mouse polyclonal antibody as the primary antibody.

[0017] Figure 3 Protein expression identification provided in the examples of the present invention. A is a Western blot using HA rabbit polyclonal antibody as the primary antibody against TsHA-VLPs, B is a Western blot using RBD rabbit monoclonal antibody as the primary antibody against TsHA-VLPs, and C is a Western blot using M1 mouse polyclonal antibody as the primary antibody against TsHA-VLPs.

[0018] Figure 4Electron micrographs provided in the examples of the present invention: A is an electron micrograph of virus-like particles TsHA-VLPs, and B is an immunoelectron micrograph of virus-like particles TsHA-VLPs.

[0019] Figure 5 Specific IgG antibody detection chart provided in the embodiments of the present invention. A shows the serum-specific antibody level against H3N2. B shows the serum-specific antibody level against H1N1. C shows the serum-specific antibody level against the SARS-CoV-2 (2019-nCoV) Spike RBD protein. D shows the serum-specific antibody level against the SARS-CoV-2 BA.2 (Omicron) Spike RBD protein. E shows the IgG1 and IgG2a antibody levels against H3N2. F shows the IgG1 and IgG2a antibody levels against the SARS-CoV-2 (2019-nCoV) Spike RBD protein.

[0020] Figure 6 Figure 1 shows the specific IgA antibody detection. A shows the level of bronchoalveolar lavage fluid-specific antibodies against H3N2. B shows the level of bronchoalveolar lavage fluid-specific antibodies against H1N1. C shows the level of bronchoalveolar lavage fluid-specific antibodies against the SARS-CoV-2 (2019-nCoV) Spike RBD protein. D shows the level of bronchoalveolar lavage fluid-specific antibodies against the SARS-CoV-2 BA.2 (Omicron) Spike RBD protein.

[0021] Figure 7 The embodiment of the present invention provides a method for detecting the neutralizing antibody titer against SARS-CoV-2 BA.2 (Omicron).

[0022] Figure 8 Figure 1 shows the hemagglutination inhibition antibody titer assay provided by an embodiment of the present invention. A shows the hemagglutination inhibition antibody titer against H3N2. B shows the hemagglutination inhibition antibody titer against H1N1 provided by an embodiment of the present invention.

[0023] Figure 9 Figures showing the results of the challenge protection experiment provided in the embodiments of the present invention. A is a graph showing the weight changes of mice in the H3N2 challenge group. B is a graph showing the survival rate changes of mice in the H3N2 challenge group. C is a graph showing the weight changes of mice in the H1N1 challenge group. D is a graph showing the survival rate changes of mice in the H1N1 challenge group. E is a graph showing the weight changes of mice in the SARS-CoV-2 BA.2 (Omicron) challenge group. F is a graph showing the weight changes of mice in the H3N2 and SARS-CoV-2 BA.2 (Omicron) combined challenge group. G is a graph showing the survival rate changes of mice in the H3N2 and SARS-CoV-2 BA.2 (Omicron) combined challenge group.

[0024] Figure 10 Detection of viral load in tissues of mice challenged with the virus group provided in the embodiments of the present invention. A is the result of viral titer detection in the lungs of mice challenged with H3N2. B is the result of viral titer detection in the nasal turbinates of mice challenged with H3N2. C is the result of viral titer detection in the lungs of mice challenged with H1N1. D is the result of viral titer detection in the nasal turbinates of mice challenged with H1N1. E is the result of viral load detection in the lungs of mice challenged with SARS-CoV-2 BA.2 (Omicron). F is the result of viral load detection in the nasal turbinates of mice challenged with SARS-CoV-2 BA.2 (Omicron). G is the result of influenza virus titer detection in the lungs of mice challenged with H3N2 and SARS-CoV-2 BA.2 (Omicron). H is the result of influenza virus titer detection in the nasal turbinates of mice challenged with H3N2 and SARS-CoV-2 BA.2 (Omicron). I is the result of the detection of the SARS-CoV-2 load in the lungs of mice in the H3N2 and SARS-CoV-2BA.2 (Omicron) combined challenge group. J is the result of the detection of the SARS-CoV-2 load in the nasal turbinates of mice in the H3N2 and SARS-CoV-2BA.2 (Omicron) combined challenge group.

[0025] In the figure, TsHA represents the virus-like particles (TsHA virus-like particles) expressing the SARS-CoV-2 RBD protein provided by the embodiments of the present invention. DETAILED DESCRIPTION

[0026] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] H1N1 influenza virus mouse-adapted strain A / Changchun / 01 / 2009 (H1N1, group 1), H3N2 influenza virus mouse-adapted strain A / bai kal teal / Shanghai / SH-89 / 2013 (H3N2, group 2), and SARS-CoV-2BA.2 were all provided by the laboratory; pFastBac1 was purchased from Invitrogen. E . coli DH5α and E . coliDH10Bac was purchased from Solebao; the fusion protein gene was synthesized and provided by BGI; technical services such as recombinant plasmid gene sequencing and primer synthesis were provided by Sangon Biotechnology; the insect cell transfection kit was purchased from Invitrogen; the nuclease was purchased from Thermo Fisher Scientific; the genome extraction kit was purchased from Beijing Quanshijin Biotechnology; SIM-SF was purchased from Beijing Sino-Bio; antibiotics and other substances were purchased from Solebao; HA rabbit polyclonal antibody was purchased from Beijing Sino-Bio; RBD rabbit monoclonal antibody was purchased from GeneTex; M1 mouse polyclonal antibody was purchased from Suzhou Jieen; BCA protein detection kit was purchased from Thermo; HPR-labeled goat anti-mouse IgA, IgG, IgG1, and IgG2a were purchased from Southern Biotechnology; TMB was purchased from Sigma; 6- to 8-week-old BALB / c female mice were purchased from Beijing Weitonglihua.

[0028] Example 1 S1: RNA extracted from a laboratory-stored H5N1 virus (A / meerkat / S-hanghai / SH-1 / 2012 (H5N1, clade 2.3.2.1; group 1)) was reverse transcribed into cDNA. The M1 gene fragment was amplified by PCR using the cDNA as a template and primers encoding the gene encoding the H5N1 M1 protein. The PCR amplification process was as follows: 95°C for 5 min, 95°C for 30 s, 56°C for 90 s, and 72°C for 1 min, for a total of 30 cycles; based on EcoR Ⅰ and Not The obtained M1 gene fragment was cloned into the plasmid pFastBac1 at the restriction endonuclease I restriction site, transformed into Escherichia coli DH5α competent cells, plated and cultured overnight, positive bacteria were screened, plasmid was extracted, PCR, sequencing and restriction enzyme digestion were performed to obtain the correct pFastBac1-M1 plasmid; The upstream primer sequence for amplifying the M1 gene fragment is: 5'-TCCCCCGGGATGAGTCTTCTAACCGAGGTCGAAA -3'; The downstream primer sequence for amplifying the M1 gene fragment is: 5'-TGCATGCATTCACTTGAATCGCTGCATCTGCACT-3'.

[0029] The amino acid sequence of the M1 protein is shown in SEQ ID NO: 1, and the ORF of the gene encoding the M1 protein is shown in SEQ ID NO: 2.

[0030] S2: Gene fragments of the fusion protein (TsHA protein) were obtained by gene synthesis, and the gene fragments of the fusion protein were amplified using primers encoding the gene of the fusion protein. EcoR Ⅰ and Not The gene fragment of the fusion protein was cloned into the pFastBac1 plasmid using the restriction enzyme cleavage site I to obtain the correct pFastBac1-TsHA plasmid; Figure 1 As shown, the sequence of the fusion protein from N-terminus to C-terminus is composed of: bee venom signal peptide (SP) + RBD with 5 common mutation sites + RBD with 5 common mutation sites + two conserved sequences of NP protein + 5 M2e proteins from different subtypes and species + HA2 protein + TM (transmembrane region) and CT (intracellular region) of influenza virus HA protein, and the sequences are connected by connecting peptides.

[0031] The upstream primer sequence for amplifying the gene fragment of the fusion protein is: 5′-TGAAGTGGTTCGCATCCTC -3′; The downstream primer sequence for amplifying the gene fragment of the fusion protein is: 5'-TGGACAAACCACAACTAGAATG-3'.

[0032] The amino acid sequence of the fusion protein is shown in SEQ ID NO: 3, and the nucleic acid sequence of the gene encoding the fusion protein is shown in SEQ ID NO: 4.

[0033] S3: Construction of recombinant bacmid 10 ng of pFastBac1-M1 plasmid and 10 ng of pFastBac1-TsHA plasmid were added to 100 μL of DH10Bac competent cells, respectively. After ice bathing for 30 minutes, the cells were heat-shocked at 42°C for 45 seconds, then ice bathed for 2 minutes. Antibody-free LB medium was added and shaken at 37°C and 200 rpm for 4 hours. 100 μL of the shaken solution was spread on a triple-antibody LB plate containing X-gal and IPTG, and cultured at 37°C for 48 hours. White spots were selected from the blue-white colonies produced by bacterial growth, and the plasmids were extracted for PCR identification to obtain recombinant bacmids Bacmid-M1 and Bacmid-TsHA, respectively.

[0034] S4: Rescue of recombinant baculovirus Positively identified Bacmid-M1 and Bacmid-TsHA were mixed at a viral titer ratio of 1:1 and transfected into Sf9 cells using the Cellfectin™ II transfection kit at an MOI of 3. After three days of culture at 27°C, the cell culture supernatant P1 was collected and inoculated into new Sf9 cells at a volume ratio of 5%. The culture was continued to amplify the viral virulence. After 4 days of culture, the P2 supernatant was collected and inoculated into new Sf9 cells at a volume ratio of 5%. After another 4 days of culture, the P3 supernatant was collected. DNA in the P3 supernatant was extracted for PCR verification, and the morphological changes of the P3 cells were observed and recorded. The expression of the P3 cells was identified by indirect immunofluorescence.

[0035] Figure 2 A~ Figure 2 C are the test images of Sf9 cells infected with Bacmid-M1 and Bacmid-TsHA respectively, which were identified by indirect immunofluorescence using HA mouse polyclonal antibody, RBD rabbit monoclonal antibody and M1 mouse polyclonal antibody as primary antibodies. Figure 2 D~ Figure 2 E is a control test diagram of Sf9 cells without baculovirus infection obtained by indirect immunofluorescence using HA mouse polyclonal antibody, RBD rabbit monoclonal antibody, and M1 mouse polyclonal antibody as primary antibodies. Figure 2 A~ Figure 2 C found that P3 generation Sf9 cells infected with two recombinant baculoviruses both had strong fluorescence expression, indicating that the virus-like particles expressed the corresponding protein.

[0036] S5: Large-scale expression and purification of virus-like particles The P3 generation baculovirus titer was determined using a Takara rapid baculovirus titer detection kit. The P3 generation baculovirus was inoculated into a large number of suspended Sf9 cells at an MOI of 3 (the concentration of Sf9 insect cells was 2×10 6 / mL or more), the cell culture medium was collected 4 days later, and purified virus-like particles were obtained after a series of steps including removing cell debris → centrifugal concentration → sucrose density gradient centrifugation → sucrose removal.

[0037] (1) Verification of prepared virus-like particles using Western blot Western blot verification was performed using HA rabbit polyclonal antibody as the primary antibody, and it was found that TsHA-VLPs had a band around 120KD. The verification results are as follows Figure 3 As shown in A; Western blot verification was performed using RBD rabbit monoclonal antibody as the primary antibody, and it was found that TsHA-VLPs had a band around 120KD. The verification results are as follows Figure 3 As shown in B; Western blot verification was performed using M1 mouse polyclonal antibody as the primary antibody, and it was found that TsHA-VLPs had a band around 30KD. The verification results are as follows Figure 3 As shown in C. This indicates that TsHA-VLPs were successfully expressed.

[0038] (2) Observation of virus-like particles using transmission electron microscopy TsHA-VLPs were observed under transmission electron microscopy. Figure 4 As shown in A, spherical particles of about 100 nm in size were observed, and there were stem structures around the spheres, indicating that TsHA-VLPs were assembled correctly. TsHA-VLPs were tested by immunoelectron microscopy using antibodies, and the test results were as follows: Figure 4 As shown in B, virus-like particles were observed under an electron microscope to be particles of about 100 nm in size, and the particles were marked with gold particles, indicating that they were assembled into virus-like particles.

[0039] Example 2 1. The specific process of the experiment is as follows: S1: Immunity and Anti-Toxin The protein concentration of influenza virus-like particles was detected using a BCA detection kit. At week 0 and week 3, 50 μL of 10 μg or 5 μg virus-like particle vaccine was dripped into the nose of the experimental group mice, and 50 μL of PBS was dripped into the nose of the mock group mice.

[0040] Two weeks after the second immunization, 10 MLD 50 Virus (H3N2 influenza virus mouse-adapted strain A / baikalteal / Shanghai / SH-89 / 2013 (H3N2, group 2)), 5 MLD 50 Virus (H1N1 influenza virus mouse-adapted strain A / Changchun / 01 / 2009 (H1N1, group 1)), 10 3 PFU SARS-CoV-2BA.2(Omicron), 1 MLD 50 virus (H3N2 influenza virus mouse-adapted strain A / bai kal teal / Shanghai / SH-89 / 2013 (H3N2, group 2)) combined with 10 2 The mock and experimental groups were challenged with PFU SARS-CoV-2BA.2 (Omicron) by intranasal injection. All experimental conditions and procedures complied with the ethical guidelines of the International Association for the Study of Pain.

[0041] S2: Specific IgG antibody detection Blood was collected from the eye sockets of mice at 0, 3, and 5 weeks after immunization. The collected blood was placed at room temperature for 2 hours and then centrifuged at 3,000 rpm for 10 minutes. The upper serum was separated and stored at -80°C.

[0042] ELISA was used to detect influenza virus-specific IgG, IgG1, and IgG2a in serum. 96-well plates were coated with inactivated H1N1 influenza virus mouse-adapted strain A / Changchun / 01 / 2009 (H1N1, group 1) and H3N2 influenza virus mouse-adapted strain A / baikalteal / Shanghai / SH-89 / 2013 (H3N2, group 2) at 5 μg / mL overnight at 4°C; SARS-CoV-2 (2019-nCoV) Spike RBD and SARS-CoV-2 BA.2 (Omicron) Spike were coated with 5 μg / mL of inactivated H1N1 influenza virus mouse-adapted strain A / Changchun / 01 / 2009 (H1N1, group 1) and H3N2 influenza virus mouse-adapted strain A / baikalteal / Shanghai / SH-89 / 2013 (H3N2, group 2) respectively. RBD protein was coated on a 96-well plate at 2 μg / mL at 4°C overnight; the plate was blocked with 5% skim milk at room temperature for 2 h, diluted serum samples were added to the 96-well plate and incubated at 37°C for 1.5 h, washed with PBST, and then incubated with HRP-labeled goat anti-mouse IgG, IgG1, and IgG2a at 37°C for 1 h. After washing with PBST, TMB was added and allowed to act at 25°C for 30 min. The reaction was terminated by adding stop solution, and the results were detected by spectrophotometer at 450 nm.

[0043] S3: Specific IgA antibody detection Mice were sacrificed by cervical dislocation at 0, 3, and 6 weeks after immunization, and bronchoalveolar lavage fluid was collected and stored at -20°C.

[0044] ELISA was used to detect influenza virus-specific IgA in serum. A 96-well plate was coated with inactivated H1N1 influenza virus mouse-adapted strain A / Changchun / 01 / 2009 (H1N1, group 1) and H3N2 influenza virus mouse-adapted strain A / baikal teal / Shanghai / SH-89 / 2013 (H3N2, group 2) at 5 μg / mL overnight at 4°C. SARS-CoV-2 (2019-nCoV) Spike RBD and SARS-CoV-2 BA.2 (Omicron) Spike were also coated with 5 μg / mL of inactivated H1N1 influenza virus mouse-adapted strain A / Changchun / 01 / 2009 (H1N1, group 1) and H3N2 influenza virus mouse-adapted strain A / baikal teal / Shanghai / SH-89 / 2013 (H3N2, group 2) at 5 μg / mL of inactivated H1N1 influenza virus mouse-adapted strain A / baikal teal / Shanghai / SH-89 / 2013 (H3N2, group 2) at 4°C overnight. RBD protein was coated on a 96-well plate at 2 μg / mL at 4°C overnight; the plate was blocked with 5% skim milk at room temperature for 2 h, diluted bronchoalveolar lavage fluid samples were added to the 96-well plate and incubated at 37°C for 1.5 h, washed with PBST, and then incubated with HRP-labeled goat anti-mouse IgA at 37°C for 1 h. After washing with PBST, TMB was added and reacted at 25°C for 30 min. Then, stop solution was added to terminate the reaction, and the results were detected by spectrophotometer at 450 nm.

[0045] S4: Neutralizing Antibody Titer Determination Blood was collected from the eye sockets of mice 3 and 5 weeks after immunization. The collected blood was placed at room temperature for 2 hours and then centrifuged at 4000 rpm for 10 minutes. The upper serum was separated and stored at -20°C.

[0046] A SARS-CoV-2 BA.2 (Omicron) neutralization test was performed in a biosafety level 3 laboratory. SARS-CoV-2 BA.2 (Omicron) was heat-inactivated at 56°C for 30 minutes. Serum was diluted twofold (1:10 to 1:20480) with a fraction containing 1% penicillin-streptomycin and 2% fetal bovine serum albumin and incubated with 100 TCID50 of SARS-CoV-2 at 37°C for 1 hour. The cells were then transferred to pre-plated 96-well plates of Vero E6 cells and incubated at 37°C for 60 hours. After incubation, the cytopathic effect of each well was observed under an electron microscope, and the neutralization titer of the sera from the mouse immunization group was calculated according to the Reed-Menuch method.

[0047] S5: Hemagglutination inhibition antibody titer determination Blood was collected from the eye sockets of mice 3 and 5 weeks after immunization. The collected blood was placed at room temperature for 2 hours and then centrifuged at 4000 rpm for 10 minutes. The upper serum was separated and stored at -20°C.

[0048] In a biosafety level 3 laboratory, hemagglutination inhibition assays were performed using inactivated H1N1 mouse-adapted influenza virus strain A / Changchun / 01 / 2009 (H1N1) and H3N2 mouse-adapted influenza virus strain A / baikal teal / Shanghai / SH-89 / 2013 (H3N2). Receptor-destroying enzyme was diluted with serum in a 4:1 ratio and incubated overnight at 37°C. Inactivated H1N1 mouse-adapted influenza virus strain A / Changchun / 01 / 2009 (H1N1) and H3N2 mouse-adapted influenza virus strain A / baikal teal / Shanghai / SH-89 / 2013 (H3N2) were thawed in ice water, and four units of the corresponding antigen were prepared. Hemagglutination titers were determined using freshly prepared 1% chicken red blood cells. Place freshly prepared four-unit antigen on ice and use within 2 hours. Prepare a 96-well V-shaped hemagglutination plate and use a multichannel pipette to add 25 µL of saline to each well. Add 25 µL of treated mouse sera to the first column, mix thoroughly, aspirate 25 µL, add to the second column, and dilute serially to the tenth column. Discard 25 µL of liquid in the tenth column. Add 25 µL of the prepared four-unit antigen to each well of the 96-well hemagglutination plate from the first to the tenth column. Add 25 µL of saline to the eleventh column as a negative control and the four-unit antigen to the twelfth column as a positive control. Incubate in a 37°C incubator for 30 minutes. After incubation, add 50 µL of 1% chicken red blood cells to each well. Let stand at room temperature for 15 minutes. Observe and record the hemagglutination results of each well in the 96-well hemagglutination plate. Results were determined as follows: 100% nonagglutination was observed if the red blood cells in each well appeared tear-like; 100% agglutination was observed if the red blood cells in each well appeared sandy. The HI titer of the sera from the immunized mice was calculated using the Reed-Menuch method.

[0049] S6: Attack and protection experiment The changes in body weight and survival rate of mice in each group were observed within 14 days after infection in S1.

[0050] S7: Influenza virus titration in lungs or nasal turbinates The lung homogenate or nasal turbinates of mice on the fourth day after infection with influenza virus (H1N1 influenza virus mouse-adapted strain A / Changchun / 01 / 2009 (H1N1, group 1)), (H3N2 influenza virus mouse-adapted strain A / baikal teal / Shanghai / SH-89 / 2013 (H3N2, group 2)) in S1 and influenza virus (H3N2 influenza virus mouse-adapted strain A / bai kal teal / Shanghai / SH-89 / 2013 (H3N2, group 2)) combined with SARS-CoV-2BA.2 (Omicron) were diluted in a 1:10, 1:10 ratio. 2 ,…,1:10 10 After dilution, inoculate chicken embryos. For each dilution, inoculate three 9-day-old SPF-grade chicken embryos. Each chicken embryo is inoculated with 100µL of lung or nasal turbinate homogenate. After sealing with glue, incubate at 37°C for 48 hours. After 48 hours, detect the hemagglutination results of the allantoic fluid. Take 50µL of allantoic fluid from each chicken embryo and mix it with 50µL of 1% chicken red blood cell suspension. Observe and record the results after 30 minutes, and calculate the chicken embryo median infectious dose (EID50) of the lung or nasal turbinate grind fluid of each mouse using the Reed-Muench method.

[0051] S8: Measurement of SARS-CoV-2 load in lungs or nasal turbinates The lungs or nasal turbinates of mice on the fourth day after infection with SARS-CoV-2BA.2 (Omicron) in S1, influenza virus (H3N2 influenza virus mouse-adapted strain A / bai kalteal / Shanghai / SH-89 / 2013 (H3N2, group 2)), and SARS-CoV-2BA.2 (Omicron) were collected and homogenized. Viral RNA was extracted by magnetic bead method. One-step real-time fluorescence PCR technology was used, with the novel coronavirus ORF1ab-specific gene as the target region. Specific primers and probes were designed, and the novel coronavirus nucleic acid in the sample was quantitatively detected by detecting changes in fluorescence signals.

[0052] 2. Experimental results (1) Results of immune response detection induced by TsHA virus-like particles in mice The specific antibody levels in the sera of mice at week 3 and 5 of immunization were detected, respectively. The serum specific antibody levels against inactivated virus (H3N2 influenza virus mouse-adapted strain A / baikal teal / Shanghai / SH-89 / 2013 (H3N2, group 2)), inactivated virus (H1N1 influenza virus mouse-adapted strain A / Changchun / 01 / 2009 (H1N1, group 1)), SARS-CoV-2 (2019-nCoV) Spike RBD protein, and SARS-CoV-2 BA.2 (Omicron) Spike RBD protein were as follows: Figure 5 A. Figure 5 B. Figure 5 C. Figure 5 As shown in D; Figure 5 A~ Figure 5 The level of specific IgG antibodies in the serum of immunized mice in D increased with the increase of immunization weeks, indicating the production of specific antibodies. Figure 5 A~ Figure 5 As shown in D, the high-dose TsHA virus-like particle group stimulated mice to produce significantly higher levels of specific antibodies against inactivated influenza virus and SARS-CoV-2 RBD protein, and had a better broad spectrum. The IgG1 and IgG2a antibody subtypes of the fifth week serum of all immunization groups were detected, as shown in Figure 5 As shown in E, the IgG2a titer of the high-dose TsHA virus-like particle group against the inactivated virus (H3N2 influenza virus mouse-adapted strain A / baikalteal / Shanghai / SH-89 / 2013 (H3N2, group 2)) was higher than the IgG1 titer, indicating that the antibody that produces the specific immune response to the virus is mainly IgG2a; Figure 5 As shown in F, the titer of IgG2a against the inactivated virus (SARS-CoV-2 (2019-nCoV) SpikeRBD) in the high-dose TsHA virus-like particle group was higher than that of IgG1, indicating that the antibodies that produce specific immune responses to the virus are mainly IgG2a.

[0053] The levels of specific antibodies in the bronchoalveolar lavage fluid of mice at 3 and 6 weeks after immunization were detected, respectively. The levels of specific antibodies in the bronchoalveolar lavage fluid against inactivated virus (H3N2 influenza virus mouse-adapted strain A / baikal teal / Shanghai / SH-89 / 2013 (H3N2, group 2), inactivated virus (H1N1 influenza virus mouse-adapted strain A / Changchun / 01 / 2009 (H1N1, group 1), SARS-CoV-2 (2019-nCoV) Spike RBD protein, and SARS-CoV-2 BA.2 (Omicron) Spike RBD protein were as follows: Figure 6 A. Figure 6 B. Figure 6 C. Figure 6 As shown in D. Figure 6 A~ Figure 6 The level of specific IgA antibodies in D increased with the increase of immunization weeks, indicating the production of specific antibodies. Figure 6 A~ Figure 6 As shown in D, the high-dose TsHA virus-like particle group stimulated mice to produce significantly higher levels of specific antibodies against inactivated influenza virus and SARS-CoV-2 RBD protein, and had better broad spectrum.

[0054] (2) Neutralizing antibody detection results induced by TsHA virus-like particles in mice The neutralizing antibody titers against SARS-CoV-2 BA.2 (Omicron) in the mouse serum were tested at 3 and 5 weeks after immunization. Figure 7 As shown, the neutralizing antibody titer after the first immunization was not significantly different from that of the control group, but was significantly higher after the second immunization than that of the control group, and the titer after both immunizations was significantly higher than that after the first immunization. The antibody titer of the high-dose TsHA virus-like particle group was significantly higher than that of the low-dose group.

[0055] (3) Results of hemagglutination inhibition antibody detection induced by TsHA virus-like particles in mice The hemagglutination inhibition antibody titers against inactivated virus (H3N2 influenza virus mouse-adapted strain A / baikalteal / Shanghai / SH-89 / 2013 (H3N2, group 2)) and inactivated virus (H1N1 influenza virus mouse-adapted strain A / Changchun / 01 / 2009 (H1N1, group 1)) in the mouse serum were detected at 3 and 5 weeks after immunization. The test results were as follows: Figure 8 A. Figure 8 As shown in B, the hemagglutination inhibition antibody titer after the second immunization was significantly higher than that of the control group, and the antibody titer of the high-dose TsHA virus-like particle group was significantly higher than that of the low-dose group.

[0056] (4) Protection against virus attack by TsHA virus-like particles The weight change rate and survival rate curves of mice in the H3N2 challenge group are shown in Figure 2. Figure 9 A. Figure 9 As shown in B, the weight change rate and survival rate curves of mice in the H1N1 challenge group are as follows Figure 9 C. Figure 9 As shown in D, the weight change rate of mice in the SARS-CoV-2BA.2 challenge group was as follows Figure 9As shown in E, the weight change rate and survival rate curves of mice in the H3N2 and SARS-CoV-2BA.2 combined challenge group are as follows Figure 9 F. Figure 9 As shown in G. Figure 9 A. Figure 9 C. Figure 9 As can be seen from F, among the weight change rates of mice in each challenge group, the high-dose TsHA virus-like particle group showed less weight loss and recovered earlier. Figure 9 E shows that when SARS-CoV-2BA.2 is infected alone, there is no significant change in the weight of mice. Figure 9 B. Figure 9 D. Figure 9 G shows that the survival rate of mice in each challenge group was 100% in the high-dose TsHA virus-like particle group. The body weight and survival rate test results show that the high-dose TsHA virus-like particle group has a good broad-spectrum protection against different subtypes of influenza and new coronavirus.

[0057] (5) TsHA virus-like particles can inhibit the replication of influenza virus and new coronavirus like Figure 10 A~ Figure 10 As shown in Figure D, the lungs and nasal turbinates of mice were collected on the fourth day after influenza virus (H3N2 influenza virus mouse-adapted strain A / baikal teal / Shanghai / SH-89 / 2013 (H3N2, group 2)) and (H1N1 influenza virus mouse-adapted strain A / Changchun / 01 / 2009 (H1N1, group 1)) challenge, and the virus titers of various types of influenza viruses were measured in 9-day-old SPF chicken embryos. The results showed that the virus titers in the lungs and nasal turbinates of the high-dose TsHA virus-like particle group were significantly lower than those in the low-dose group and the control group, indicating that the high-dose TsHA virus-like particle group can inhibit the replication of influenza virus in the lungs and nasal turbinates of mice.

[0058] like Figure 10 E~ Figure 10 As shown in Figure F, the lungs and nasal turbinates of mice dissected on the fourth day after SARS-CoV-2BA.2 (Omicron) infection were ground, and the viral load in the lungs and nasal turbinates of mice was detected by real-time fluorescence quantitative PCR using primers targeting the ORF 1 a / b gene. The results showed that the number of SARS-CoV-2 copies in the lungs and nasal turbinates of the high-dose TsHA virus-like particles group was significantly lower than that in the low-dose group and the control group, indicating that the high-dose TsHA virus-like particles group can inhibit the replication of SARS-CoV-2 in the lungs and nasal turbinates of mice.

[0059] like Figure 10 G~ Figure 10As shown in Figure 1, mice were dissected on the fourth day after joint infection with the H3N2 influenza virus mouse-adapted strain A / baikal teal / Shanghai / SH-89 / 2013 (H3N2, group 2) and SARS-CoV-2 BA.2 (Omicron), and the influenza virus titer and SARS-CoV-2 load in the lungs and nasal turbinates of the mice were detected. The results showed that the influenza virus titer and SARS-CoV-2 copy number in the lungs and nasal turbinates of the high-dose TsHA virus-like particle group were significantly lower than those in the low-dose group and the control group, indicating that the high-dose TsHA virus-like particle group can inhibit the replication of influenza virus and SARS-CoV-2 in the lungs and nasal turbinates of mice.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Influenza virus-like particles expressing the SARS-CoV-2 RBD protein, characterized in that: The influenza virus-like particles are prepared by displaying a fusion protein on the surface of the influenza virus M1 protein. The fusion protein includes the HA2 segment of the influenza virus HA protein, the extracellular functional region of the M2e matrix protein, a short peptide in the conserved region of the nucleoprotein, and the RBD tandem protein with mutations at five sites: 417, 452, 478, 484, and 501 of the new coronavirus.

2. The virus-like particle according to claim 1, characterized in that The sequence of the fusion protein from N-terminus to C-terminus includes: RBD1 with 5 common mutation sites, RBD2 with 5 common mutation sites, two conserved sequences of the nucleoprotein, 5 M2e proteins from different subtypes and species, and the HA2 segment of the HA protein. The sequences are connected by a connecting peptide. The sequences of RBD1 with 5 common mutation sites and RBD2 with 5 common mutation sites are the same.

3. The virus-like particle according to claim 1, characterized in that The N-terminus of the fusion protein also contains a bee venom signal peptide, and the C-terminus also contains a transmembrane region and an intracellular region of the HA protein.

4. The virus-like particle according to claim 1, characterized in that The amino acid sequence of the M1 protein is shown in SEQ ID NO:

1.

5. The virus-like particle according to claim 1, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO:

3.

6. The method for preparing virus-like particles according to claim 1, characterized in that: include: Step 1: Clone the influenza virus M1 protein encoding gene into a baculovirus expression plasmid to obtain a recombinant M1 plasmid; clone the fusion protein encoding gene into a baculovirus expression plasmid to obtain a recombinant TsHA plasmid; Step 2: The recombinant M1 plasmid and the recombinant TsHA plasmid were transformed into competent Escherichia coli cells to obtain recombinant baculovirus plasmid Bacmid-M1 and recombinant baculovirus plasmid Bacmid-TsHA, respectively; the recombinant baculovirus plasmid Bacmid-M1 and the recombinant baculovirus plasmid Bacmid-TsHA were used to transfect insect cells to rescue the recombinant baculovirus M1 and the recombinant baculovirus TsHA; Step 3: Mix the recombinant baculovirus M1 and recombinant baculovirus TsHA and inoculate the suspended insect cells. After harvesting and purification, virus-like particles expressing the SARS-CoV-2 RBD protein are obtained.

7. The preparation method according to claim 6, characterized in that In step 2, the insect cells are Sf9 cells, and the confluence of the Sf9 insect cells during transfection reaches more than 80%.

8. The preparation method according to claim 6, characterized in that In step 3, the insect cells are Sf9 cells, and the recombinant baculovirus M1 and the recombinant baculovirus TsHA are mixed at a virus titer of 1:1, and the suspended Sf9 insect cells are inoculated at an MOI of 3.

9. The preparation method according to claim 6, characterized in that In step 3, the insect cells are Sf9 cells, and the concentration of Sf9 insect cells when inoculating the recombinant baculovirus is 2×10 6 / mL or above.

10. Use of the virus-like particles expressing the chimeric SARS-CoV-2 RBD protein according to any one of claims 1 to 5 in the preparation of vaccines for preventing influenza and / or SARS-CoV-2 infection.

Images