A beta coronavirus and influenza chimeric antigen, preparation method and application thereof

Abstract

The present invention relates to the field of biomedicine technology, and specifically to SARS-CoV-2 and influenza chimeric antigens, preparation methods and applications thereof. The RBD of the S protein of SARS-CoV-2 is used to replace the head region of the HA protein, and the stem region of the HA protein is connected to construct a chimeric protein, which can prepare chimeric antigens and mRNA vaccines. The antigen prepared by the present invention can stimulate mice to produce very high titers of neutralizing antibodies, and can prevent two important respiratory pathogens, SARS-CoV-2 and influenza, at the same time, greatly reducing the global prevention and control pressure and vaccine immunization costs for the two pathogens.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to SARS-CoV-2 and influenza chimeric antigens, and preparation methods and applications thereof. Background Art

[0002] The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus emerged in late 2019 and spread rapidly around the world, with the World Health Organization declaring a global pandemic on March 11, 2020. This new beta coronavirus has never been seen before, and genomic analysis found that it is similar to severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) to enter cells and infect the human body, causing patients to have clinical symptoms such as fever, dry cough, and muscle pain. A few people also have symptoms such as nasal congestion, sore throat, and diarrhea. Severe patients rapidly progress to acute respiratory distress syndrome, septic shock, difficult-to-correct metabolic acidosis and coagulation dysfunction, and even cause death. According to WHO statistics, as of May 4, 2022, worldwide, more than 510 million people have been infected, of which more than 6.2 million have died.

[0003] The S protein is a glycoprotein on the surface of the SARS-CoV-2 virus, which is mainly responsible for binding to the receptor ACE2 and mediating the fusion between the virus and the host cell membrane. Therefore, the S protein, especially its receptor binding region (RBD), is widely selected as the antigen of the SARS-CoV-2 vaccine. At present, there are several vaccines targeting the RBD region of the S protein that are being or will be approved for clinical use, including protein subunit vaccines (ZF2001, A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS) and mRNA vaccines (ARCoV, A thermostable mRNA vaccine against COVID-19). The study entitled "Efficacy and safety of the RBD-Dimer–Based Covid-19 vaccine ZF2001 in adults" published in NEJM showed that the results of the phase 3 trial of ZF2001 as a three-dose vaccination showed an efficacy of 75.7% in protecting against mild COVID-19 and an efficacy of 87.6% in severe to critical cases, indicating that the vaccine design targeting the RBD antigen is very effective. However, as SARS-CoV-2 spreads on a large scale, the virus itself is also evolving rapidly, with a large number of variants appearing since the end of 2020. Because some variants show increased transmissibility, immune evasion, or enhanced disease, five of them have been declared as variants of concern (VOC) by the WHO – B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta) and B.1.1.529 (Omicron). Therefore, it is urgent and important to develop a safe, effective and broad-spectrum vaccine against various SARS-CoV-2 variants.

[0004] Influenza viruses belong to the family Orthomyxoviridae. They are spherical or filamentous, have an envelope, and their genome is segmented, negative-sense, single-stranded RNA. Influenza viruses can cause acute respiratory infections in humans. They are easily transmitted from person to person and infect people of all ages, posing a serious threat to public health. The symptoms of influenza infection vary depending on the physical condition of the infected person and the virus strain, ranging from asymptomatic infection or mild infection of the upper respiratory tract to high fever, chills, muscle pain, pneumonia, and even death. According to the World Health Organization (WHO), global influenza epidemics cause approximately 3-5 million severe cases and 290,000 to 650,000 deaths each year. Due to their unique genetic structure and antigenic properties, influenza viruses are prone to gene rearrangement and antigenic mutation, thereby escaping previous natural immunity. Governments around the world need to invest a lot of resources every year to monitor influenza epidemics, predict the next year's epidemic strains, and redesign seasonal influenza vaccines based on epidemiological data. These vaccines work well when they are well matched to the circulating virus strains. However, the vaccine strain composition is based on predictions, and mismatches occur relatively frequently. Therefore, the development of an efficient universal influenza virus vaccine that can protect against all influenza viruses is a major area of ​​focus in the research community.

[0005] The HA protein on the surface of influenza virus is responsible for virus adhesion and membrane fusion. It is an important immunogen and the main target of the vaccine. HA consists of two subunits (HA1 and HA2), which are connected by disulfide bonds to form a homotrimeric spike embedded in the viral envelope. From the outside to the inside, it can be divided into the extracellular region, the transmembrane region and the intracellular region. The extracellular region can be further divided into the head region and the stem region. HA1 constitutes the head region, and a small part of HA1 and the extracellular part of HA2 constitute the stem region. The head region contains the receptor binding region, and the antigens of different subtypes vary greatly, while the stem region is relatively conservative. With the discovery of a large number of broad-spectrum neutralizing antibodies against the HA stem (Corti, D., et al. (2011). "A Neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins." Science 333(6044): 850-856.), the design of subunit vaccines based on the modification of the HA stem region has become an important research direction for universal vaccines. There are different transformation strategies, such as amino acid mutation and introduction of trimer tags to stabilize the conformation of the HA stem region (Impagliazzo, A., et al., A stable trimeric influenza hemagglutinin stem as abroadly protective immunogen. Science, 2015. 349(6254): p. 1301-6.; Yassine, HM, et al., Hemagglutinin-stem nanoparticles generate heterosubtypicinfluenza protection. Nat Med, 2015. 21(9): p. 1065-70.). In subsequent immune evaluation, the stabilized trimer stem region protein was found to maintain the natural conformation of many protective epitopes, induce broad-spectrum protective antibodies, and achieve good prevention and protection effects.

[0006] The continued global spread of SARS-CoV-2 and seasonal influenza outbreaks have become a huge threat to human public health security. In order to reduce the prevention and control pressure of the two pathogens and the cost of vaccine immunization, it is crucial to design a safe, efficient, broad-spectrum vaccine that can prevent both pathogens at the same time. Summary of the invention

[0007] The object of the present invention is to provide a SARS-CoV-2 and influenza chimeric antigen, a preparation method and application thereof. Based on the conclusion that both the SARS-CoV-2 RBD protein and the HA stem region of the influenza virus can stimulate protective antibodies against each other, the present invention attempts to connect the SARS-CoV-2 RBD protein and the HA stem region through a connecting segment, and at the same time, to maintain the natural conformation of the HA stem region, mutations are introduced at key amino acid sites. The expressed RBD and HA chimeric proteins are stably expressed and uniform in form. Protein vaccines and mRNA-LNP vaccines are prepared, and the SARS-CoV-2 RBD protein and HA stem region chimeric antigens expressed by the present invention have good immunogenicity. The vaccine prepared from the antigen can stimulate mice to produce very high titers of neutralizing antibodies, and can produce cross-protection against different subtypes of strains.

[0008] The present invention provides a chimeric protein of the RBD protein of SARS-CoV-2 and the HA stem region, characterized in that the RBD of the S protein of SARS-CoV-2 is used to replace the head region of the HA protein, and the stem regions of the HA proteins are connected to construct a chimeric protein.

[0009] Preferably, the prototype strain of SARS-CoV-2, the VOC SARS-CoV-2 variant, especially the S protein of the Delta strain is used to replace the HA protein head region; the HA is derived from the HA sequence of influenza H1N1, H3N2, H5N1, and H7N9 influenza virus strains, preferably influenza A / Victoria / 2570 / 2019.

[0010] More specifically, amino acids 319 to 537 of the S protein of the Delta strain of SARS-CoV-2 are used to replace amino acids 53 to 321 of the HA1 subunit (SEQ ID NO: 9).

[0011] In addition, preferably, the RBD is connected to the stem region of the HA protein via a connecting segment, and preferably the connecting segment is GGGG or GSGGSG.

[0012] Further preferably, it also includes a signal peptide, preferably an influenza A / Victoria / 2570 / 2019 (H1N1) (sequence such as GISAID:EPI1801581) self-signal peptide.

[0013] Also preferably, the chimeric protein further comprises a histidine tag, or comprises a GCN4 trimer tag and a histidine tag.

[0014] More preferably, the amino acid sequence fragment at positions 60-92 of the HA2 subunit (SEQ ID NO: 10) is deleted; or the amino acid sequence fragment at positions 76-89 of the HA2 subunit is replaced by GCN4.

[0015] In a specific embodiment, the amino acid sequence thereof is SEQ ID NO:2 or SEQ ID NO:4.

[0016] Preferably, 4 amino acid site mutations, i.e., K51M, Y94D, N95L and E103L, or 6 amino acid site mutations, i.e., I10T, F63Y, V66I, K68C, F70Y and L73S, are introduced into the HA2 subunit region; and 3 amino acid mutations, i.e., K325C, V338K and I341K, are introduced into the HA1 subunit. More specifically, the amino acid sequence thereof is as shown in SEQ ID NO: 6 or SEQ ID NO: 8.

[0017] The present invention thus provides a nucleic acid encoding said gene, preferably a nucleotide sequence thereof as shown in SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7.

[0018] Preferably, it also includes a non-coding sequence (UTR) at the 5' end and a UTR at the 3' end, and preferably 120 polyadenylic acids (Poly A) are added after the 3' UTR.

[0019] The present invention also provides a recombinant vector containing the nucleic acid, preferably an expression vector.

[0020] The present invention also provides chimeric antigens of beta coronavirus and influenza virus of the chimeric protein.

[0021] The present invention therefore provides mRNA vaccines based on said nucleic acids.

[0022] The chimeric antigens of SARS-CoV-2 RBD protein and HA stem region constructed by the present invention have good immunogenicity. Vaccines prepared from the antigens can stimulate mice to produce very high titers of neutralizing antibodies, and can simultaneously prevent two important respiratory pathogens, SARS-CoV-2 and influenza, greatly reducing the global prevention and control pressure against the two pathogens and the cost of vaccine immunization. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A and 1B This is the strategy for constructing chimeric antigen expression of SARS-CoV-2 RBD protein and HA stem region in Example 1.

[0024] Figure 2 It is the UV absorption graph when the SARS-CoV-2 RBD protein and HA stem region expressed in Example 1 were chimeric and subjected to Superdex200Hiload16 / 60 column (GE) molecular sieve chromatography, and the result graph of SDS-PAGE of the purified protein.

[0025] Figure 3 It is the RBD and HA specific IgG antibody titer induced after mice were immunized with SARS-CoV-2 RBD protein and HA stem region chimeric protein in Example 2. Among them: 2µg, 10µg represent the immune amount of the immune source used for each immunization. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0026] Figure 4 It is the titer of SARS-CoV-2 pseudovirus neutralizing antibody induced after mice were immunized with SARS-CoV-2 RBD protein and HA stem region chimeric protein in Example 2. Among them: 2µg, 10µg represent the immune amount of the immune source used for each immunization. Analysis of significant differences: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0027] Figure 5 Three days after the A / Brisbane / 02 / 2018 (H1N1) challenge in Example 3, the lungs of the mice were removed and the tissue homogenate was used to detect the virus titer (TCID 50 ) Result graph. Among them: 2µg, 10µg represent the immune amount of the immune source used in each immunization. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0028] Fig. 6A The weight changes of mice within 14 days after the A / Brisbane / 02 / 2018 (H1N1) challenge in Example 4. Among them: 2μg and 10μg represent the immune amount of the immune source used for each immunization.

[0029] Figure 6B The survival of mice within 14 days after the A / Brisbane / 02 / 2018 (H1N1) challenge in Example 4. Among them: 2μg, 10μg represent the immune amount of the immune source used for each immunization.

[0030] Figure 7 It is the protective effect of the vaccine on the lung tissue of mice after the H1N1 subtype influenza virus challenge in Example 5. Among them: 2μg, 10μg represent the immune amount of the immune source used for each immunization.

[0031] Figure 8 Three days after the A / Astrakhan / 3212 / 2020 (H5N8) challenge in Example 6, the lungs of the mice were removed and the tissue homogenate was used to detect the virus titer (TCID 50) Result graph. Among them: 2µg, 10µg represent the immune amount of the immune source used in each immunization. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0032] Fig.9A The weight changes of mice within 14 days after A / Astrakhan / 3212 / 2020 (H5N8) challenge in Example 7. Wherein: 2µg, 10µg represent the immune amount of the immune source used for each immunization.

[0033] Fig. 9B The survival of mice within 14 days after A / Astrakhan / 3212 / 2020 (H5N8) challenge in Example 7. Wherein: 2µg, 10µg represent the immune amount of the immune source used for each immunization.

[0034] Fig.10 It is the protective effect of the vaccine on the lung tissue of mice after the H5N8 subtype influenza virus challenge in Example 8. Among them: 2μg, 10μg represent the immune amount of the immune source used for each immunization.

[0035] Fig.11 This is a schematic diagram of the construction of the SARS-CoV-2 RBD protein and HA stem region chimeric antigen mRNA vaccine in Example 9.

[0036] Fig.12 This is a diagram of the immunization and H1N1 subtype influenza virus challenge strategy in Examples 10 to 23.

[0037] Fig.13 The results of Example 11 are as follows: Fig.12 SARS-CoV-2 RBD-specific antibody IgG titer in serum collected according to the immunization strategy in the experiment. Among them: 2µg and 10µg represent the immune amount of the immune source used in each immunization. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0038] Fig.14 The results of Example 12 are as follows: Fig.12 The IgG titer of H1N1 subtype influenza virus-specific antibodies in the serum collected according to the immunization strategy. Among them: 2µg and 10µg represent the immune amount of the immune source used in each immunization. Analysis of significant differences: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0039] Fig.15 The results of Example 13 are as follows. The spleens of mice after the second immunization in Example 10 were collected at week 16 and the spleen cells were stimulated with the SARS-CoV-2 RBD peptide library to detect the production of cytokines (IFN-γ and IL-4). Among them: 2µg and 10µg represent the immune amount of the immune source used for each immunization. Analysis of significant differences: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0040] Fig.16 The results of Example 14 are as follows. The spleens of mice after the second immunization in Example 10 were collected at week 16 and spleen cells were stimulated with the SARS-CoV-2 RBD peptide library. CD4 + and CD8 + Response and interferon-γ (IFN-γ), tumor necrosis factor (TNF-a), interleukin 2 (IL-2), interleukin 4 (IL-4) and interleukin 5 (IL-5) produced results. Among them: 2μg, 10μg represents the immune amount of the immune source used in each immunization. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0041] Fig.17 The results of Example 15 are as follows. The spleens of mice after the second immunization in Example 10 were collected at week 16 and the splenocytes were stimulated with the H1N1 influenza virus HA peptide library to detect the production of cytokines (IFN-γ and IL-4). Among them: 2µg and 10µg represent the immune amount of the immune source used for each immunization. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0042] Fig.18 The results of Example 16 are as follows: the spleens of mice after the second immunization in Example 10 were collected at week 16 and spleen cells were stimulated with H1N1 influenza virus HA peptide library. CD4 + and CD8 + Response and interferon-γ (IFN-γ), tumor necrosis factor (TNF-α), interleukin 2 (IL-2), interleukin 4 (IL-4) and interleukin 5 (IL-5) produced results. Among them: 2μg, 10μg represents the immune amount of the immune source used in each immunization. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0043] Fig.19 The results of Example 17 are as follows: Fig.12 SARS-CoV-2 pseudovirus neutralizing antibody titers in serum collected from the immunization strategy. Among them: 2µg, 10µg represent the immune amount of the immune source used in each immunization. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0044] Fig. 20 In Example 18, Fig.10 Immunization strategy in the second immunization of mice, according to Fig.12 As shown, 20 LD were used on day 42 50 A / Brisbane / 02 / 2018(H1N1) dose challenge. Three days after the challenge, the lungs of the mice were removed and the tissue homogenate was used to detect the virus titer (TCID 50 ) Result graph. Among them: 2µg, 10µg represent the immune amount of the immune source used in each immunization. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0045] Fig.21A In Example 19, Fig.10 Immunization strategy in the second immunization of mice, according to Fig.12 As shown, 20 LD were used on day 42 50 A / Brisbane / 02 / 2018(H1N1) dose challenge. Changes in mouse body weight within 14 days after challenge. Among them: 2µg, 10µg represent the immune dose of the immune source used in each immunization.

[0046] Fig. 21B In Example 19, Fig.10 Immunization strategy in the second immunization of mice, according to Fig.12 As shown, 20 LD were used on day 42 50 A / Brisbane / 02 / 2018(H1N1) dose challenge. Survival of mice within 14 days after challenge. Among them: 2µg, 10µg represent the immune dose of the immunogen used in each immunization.

[0047] Fig. 22 It is the protective effect of the vaccine on the lung tissue of mice after the H1N1 subtype influenza virus challenge in Example 20. Wherein: 2μg, 10μg represent the immune amount of the immune source used for each immunization.

[0048] Fig.23Three days after the A / Astrakhan / 3212 / 2020 (H5N8) challenge in Example 21, the lungs of the mice were removed and the tissue homogenate was used to detect the virus titer (TCID 50 ) Result graph. Among them: 2µg, 10µg represent the immune amount of the immune source used in each immunization. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0049] Fig.24A The weight changes of mice within 14 days after A / Astrakhan / 3212 / 2020 (H5N8) challenge in Example 22. Wherein: 2µg, 10µg represent the immune amount of the immune source used for each immunization.

[0050] Fig. 24B The survival of mice within 14 days after A / Astrakhan / 3212 / 2020 (H5N8) challenge in Example 22. Wherein: 2µg, 10µg represent the immune amount of the immune source used for each immunization.

[0051] Fig.25 The protective effect of the vaccine on the lung tissue of mice after the H5N8 subtype influenza virus challenge in Example 23. Wherein: 2μg, 10μg represent the immune amount of the immune source used for each immunization.

[0052] Fig.26 It is described in Example 24, and H1Delta is purified by molecular sieve chromatography L -Absorbance curve of the complex of TF protein, CB6 Fab and CR9114 Fab.

[0053] Fig. 27 is described in Example 24 of the present invention, H1Delta L -Schematic diagram of the cryo-electron microscopy structure of the complex of TF protein, CB6 Fab and CR9114 Fab. Specific implementation plan

[0054] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments, but they do not constitute a limitation of the present invention.

[0055] Example 1: SARS-CoV-2 RBD Delta Construction, expression and purification of chimeric antigens of protein and HA stem region

[0056] The first design H1Delta S like Figure 1A :

[0057] The S protein RBD of SARS-CoV-2 Delta (SEQ ID NO: 1 is the nucleotide sequence, SEQ ID NO: 2 is the amino acid sequence) replaces the HA protein head region (i.e., amino acids 55-321 in the HA1 region shown in the nucleotide sequence of SEQ ID NO: 9), connected on both sides by a connecting segment (GSGGSG), and at the same time deletes amino acids 60-92 in the HA2 region shown in the nucleotide sequence of SEQ ID NO: 10, and uses the influenza A / Victoria / 2570 / 2019 (H1N1) (sequence such as GISAID: EPI1801581) self-signal peptide. In order to maintain the natural conformation of the HA stem region, 4 amino acid site mutations (K51M, Y94D, N95L and E103L) are introduced into the HA2 region. After adding a nucleotide sequence encoding 6 histidines at the 3' end, a stop codon is added at the 3' end to construct an RBD-HA chimeric protein (SEQ ID NO: 3 is H1Delta S SEQ ID NO: 4 is H1Delta S amino acid sequence).

[0058] The second design H1Delta L like Figure 1B :

[0059] (1) The S protein RBD of SARS-CoV-2 Delta (SEQ ID NO: 1 is the nucleotide sequence, SEQ ID NO: 2 is the amino acid sequence) replaces the HA protein head region (i.e., amino acids 53-321 in the HA1 region shown in the nucleotide sequence of SEQ ID NO: 9), is connected on both sides by a linker (GGGG), and uses the influenza A / Victoria / 2570 / 2019 (H1N1) (sequence such as GISAID: EPI1801581) self-signal peptide. To maintain the natural conformation of the HA stem region, three amino acid mutations (K325C, V338K and I341K) are introduced at HA1, and six amino acid site mutations (I10T, F63Y, V66I, K68C, F70Y and L73S) are introduced in the HA2 region shown in the nucleotide sequence of SEQ ID NO: 10. At the same time, the original virus sequence is replaced with the GCN4 sequence (CMKQIEDKIEEIESK) at sites 76-89 to make the protein more conducive to forming a trimer. After adding a nucleotide sequence encoding six histidine residues to the 3' end, a stop codon was added to the 3' end to construct an RBD-HA chimeric protein (SEQ ID NO: 5 is H1Delta L The nucleotide sequence of SEQ ID NO: 6 is H1Delta L amino acid sequence).

[0060] (2) The design was the same as (1). At the same time, in order to make the protein trimer more stable, a trimer tag (amino acid sequence: YIPEAPRDGQAYVRKDGEWVLLSTFL) was added to the 3' end, followed by a nucleotide sequence encoding 6 histidines. Finally, a stop codon was added to the 3' end to construct the RBD-HA chimeric protein (SEQ ID NO: 7 is H1Delta L -TF nucleotide sequence, SEQ ID NO: 8 is H1Delta L -The amino acid sequence of TF).

[0061] The nucleotide sequence encoding the above construction was inserted into the pcDNA3.1 vector between the EcoRI and XhoI restriction sites, and the Kozak sequence gccacc was contained upstream of the start codon. The plasmid and PEI were incubated with 150mM NaCl respectively and then mixed and transfected into 293F cells for expression. Two days later, the cell suspension was centrifuged at 6500rpm and the supernatant was harvested. The cell supernatant was filtered through a 0.22μm filter membrane to remove cell debris. The cell culture supernatant was hung on a nickel affinity column (Histrap HP) at 4 degrees overnight. The resin was washed with buffer A (20mM Tris, 150mM NaCl, pH8.0) to remove non-specific binding proteins. Finally, the target protein was eluted from the resin with buffer B (20mM Tris, 150mM NaCl, pH 8.0, 300mM imidazole), and the eluate was concentrated to less than 2ml with a 30K cutoff (30 cutoff) concentrator. The target protein was further purified by molecular sieve chromatography using a Superdex20010 / 300 GL (GE) column. The molecular sieve chromatography buffer was 20mM Tris (pH8.0), 150mM NaCl. After molecular sieve chromatography, the samples were collected for SDS-PAGE analysis. The results are shown in the figure. Figure 2 As shown, H1Delta S The construct has only one main peak near the elution volume of 13 mL, and the protein band is between 55-75 kd. L The construct has only one main peak near the elution volume of 11 mL, and the protein band is around 75 kd. L The construct had only one main peak near the elution volume of 11 mL, and the protein band was around 75 kd.

[0062] Example 2: Experiment on immunizing mice with chimeric proteins of SARS-CoV-2 RBD and HA stem regions

[0063] The antigen obtained in Example 1 (H1Delta S , H1Delta Land H1Delta L -TF) was diluted in physiological saline and mixed with adjuvant (Addavax) for emulsification. The immunization grouping and immunization dose of each group are shown in Table 1. The blank part indicates "none", and there are 8 mice in each group. Each mouse received 3 vaccinations on day 0, day 21 and day 42 by thigh muscle injection, with an inoculation volume of 100µl each time (containing 2µg and 10µg immunogen, respectively). 14 days after the 2nd and 3rd immunizations, the mice were bled from the eye sockets. The mouse serum was obtained by centrifugation at 3000rpm for 10 minutes after standing and stored in a refrigerator at -20℃ for specific antibody detection and pseudovirus neutralization detection.

[0064] Table 1

[0065] Group Immunogen Dosage (µg) 1 <![CDATA[H1Delta S ]]> 2 2 <![CDATA[H1Delta S ]]> 10 3 <![CDATA[H1Delta L ]]> 2 4 <![CDATA[H1Delta L ]]> 10 5 <![CDATA[H1Delta L -TF]]> 2 6 <![CDATA[H1Delta L -TF]]> 10 7 PBS

[0066] Detection of specific antibody titer of mouse serum by ELISA experiment:

[0067] (1) The RBD of SARS-CoV-2 Delta The monomeric protein was diluted to 2 µg / ml with ELISA coating solution (Solabo, C1050), 100 µl was added to each well of a 96-well ELISA plate (Coring, 3590), and the plate was incubated at 4 °C for 12 h.

[0068] (2) Pour off the coating solution and use 5% skim milk prepared in PBS as the blocking solution. Add 100µl to each well of the 96-well plate and place at 37°C for 1 hour. After blocking, wash once with PBS solution.

[0069] (3) Dilute mouse serum during blocking. Serum samples were diluted from 100 times. Then 100 µl was added to each well of the ELISA plate. The negative control was added with blocking solution, incubated at 37 degrees for 1 hour, and then washed 4 times with PBST.

[0070] (4) Add HRP-conjugated goat anti-mouse secondary antibody (Abcam, ab6789) diluted 1:2000 with blocking solution, incubate at 37°C for 1 hour, and then wash 4 times with PBST. Add TMB colorimetric solution for color development, add 2M sulfuric acid after 30 minutes to terminate the reaction, and measure the OD450 reading on a microplate reader. The antibody titer value is defined as the highest serum dilution factor with a reaction value greater than 2.1 times the negative control value. When the reaction value of the lowest dilution factor (detection limit) is still less than 2.1 times the background value, the titer of the sample is defined as half of the lowest dilution factor, i.e. 1:50.

[0071] The results are as follows Figure 3As shown in Figure 2, the vaccines constructed by H1DeltaS, H1DeltaL and H1DeltaL-TF induced specific RBD and HA binding antibodies in all mice immunized twice and three times. The average endpoint titer of RBD and HA specific IgG antibodies in mice immunized with a high dose of 10 µg was up to 10 5 In particular, the average endpoint titer of RBD and HA-specific IgG antibodies induced by H1DeltaL-TF vaccine can reach 10 6 about.

[0072] Pseudovirus neutralization assay to detect the neutralization titer of serum against SARS-CoV-2:

[0073] The serum sample obtained in Example 2 was diluted from 20 times and then diluted with an equal volume of 100 TCID 50 The pseudoviruses were mixed and incubated at 37°C for 60 minutes. The mixture was added to a 96-well plate of Vero cells with a growth density of about 90%. After incubation at 37°C for 18 hours, the activity value was detected by a fluorescence detector. The pseudovirus surface carries a spike protein. The pseudovirus infects cells to release DNA and express luciferase, but does not replicate. If neutralizing antibodies are present, the pseudovirus cannot infect cells and does not express luciferase. This method is used to test the neutralization titer of the serum.

[0074] The results are as follows Figure 4 As shown, H1Delta S , H1Delta L and H1Delta L The vaccine constructed by -TF induced neutralizing antibodies in all mice after the second and third immunizations. This result indicates that the vaccine has great potential for clinical development.

[0075] Example 3: H1N1 subtype influenza virus challenge protection experiment

[0076] H1Delta after three immunizations in Example 2 L -TF group mice were given 20 LD 50 The mice were challenged with A / Brisbane / 02 / 2018(H1N1) at a dose of 100 mg / kg. Three days after the challenge, the lungs of the mice were removed and the tissue homogenate was used to detect the virus titer (TCID 50 ), the result is as follows Figure 5 As shown, the amount of virus in the lung tissue of the vaccine group mice was significantly reduced compared with the Placebo control group, indicating that H1Delta L -TF constructed vaccine has very significant protection against influenza H1N1 live virus challenge.

[0077] Example 4: Body weight changes and survival status of mice within 14 days after challenge with H1N1 subtype influenza virus

[0078] H1Delta after three immunizations in Example 2 L -TF group mice were given 20 LD 50 The mice were challenged with A / Brisbane / 02 / 2018(H1N1) at a dose of 100 mg / kg. The mice were weighed and their survival was observed every day for 14 days after the challenge. Fig. 6A and Figure 6B All mice in the Placebo control group died within 8 days, and H1Delta L All mice in the -TF immunization group survived, and their body weight first decreased and then increased. These results show that H1Delta L -TF constructed protein vaccine can provide complete protection against influenza H1N1 virus.

[0079] Example 5: Testing the protective effect of the vaccine on the lung tissue of mice after challenge with H1N1 subtype influenza virus

[0080] The lung tissues of mice challenged with H1N1 subtype influenza virus in Example 3 were fixed with 4% paraformaldehyde, and then stained with hematoxylin-eosin. The changes in the lung tissues were observed by sectioning. The results are as follows: Figure 7 As shown, the lung tissue of the control group mice (i.e., Placebo group) showed severe interstitial pneumonia, alveolitis, inflammatory cell infiltration, and bronchial epithelial cell necrosis. L -The mice in the TF vaccine group were able to significantly alleviate the lung damage caused by the virus attack, showing mild to moderate lung damage, with clear alveoli and less inflammatory cell infiltration. These results show that the vaccine can significantly alleviate the lung damage caused by the H1N1 subtype influenza virus attack.

[0081] Example 6: H5N8 subtype influenza virus challenge protection experiment

[0082] To verify the protective effect of constructing against heterologous influenza, H1Delta L -TF vaccine group received 10LD on day 42 50 A / Astrakhan / 3212 / 2020 (H5N8) dose was used for the challenge, and the experimental method is shown in Example 3. Figure 8 As shown, compared with the Placebo control group, the amount of virus in the lung tissue of the vaccine group mice was significantly reduced, indicating that the constructed protein vaccine has a very significant protection against the live virus attack of heterologous H5N8 subtype influenza virus.

[0083] Example 7: Body weight changes and survival status of mice within 14 days after challenge with H5N8 subtype influenza virus

[0084] H1DeltaL -TF vaccine group received 10 LD on day 42 50 A / Astrakhan / 3212 / 2020 (H5N8) dose challenge was performed, and the experimental method is shown in Example 4. The results are as follows Fig.9A and Fig. 9B All mice in the Placebo control group died within 10 days, while the body weight of the vaccine-immunized group decreased slightly and then recovered. These results show that H1Delta L -TF constructed protein vaccine can provide good protection against heterologous H5N8 subtype influenza virus live virus attack.

[0085] Example 8: Testing the protective effect of the vaccine on the lung tissue of mice after challenge with H5N8 subtype influenza virus

[0086] The lung tissues of mice challenged with H5N8 subtype influenza virus in Example 6 were fixed with 4% paraformaldehyde, and then stained with hematoxylin-eosin. The changes in the lung tissues were observed by sectioning. The results are as follows: Fig.10 As shown, the lung tissues of the control group mice (i.e., the Placebo group) showed severe interstitial pneumonia, alveolitis, inflammatory cell infiltration, and necrosis of bronchial epithelial cells. The immunized group mice greatly alleviated the lung damage caused by the virus attack, showing mild to moderate lung damage, with clear alveoli and less inflammatory cell infiltration. These results show that the vaccine can greatly alleviate the lung damage caused by the heterologous H5N8 subtype influenza virus attack.

[0087] Example 9: Construction and preparation of SARS-CoV-2 RBD protein and HA stem region chimeric antigen mRNA vaccine

[0088] As indicated Fig.11 As shown, the design is the same as Example 2, and a non-coding sequence (UTR) and a 3' UTR are added to the 5' end of the chimeric antigen nucleic acid sequence, and 120 polyadenylic acids (Poly A) are added after the 3' UTR. It is cloned between the XbaI and BamHI-HF restriction sites of the T7 promoter-containing vector pET-3a (pET-3a is from GenScript).

[0089] (1) After plasmid extraction, linearization is performed by enzyme digestion and the digestion product is recovered.

[0090] (2) Add the components of the in vitro transcription reaction (nearshore protein, E131) and incubate at 37°C for 3 hours. After the reaction, add DNaseI and incubate at 37°C for 15 minutes to digest the transcribed DNA template.

[0091] (3) After the mRNA product is purified by lithium chloride, it is heated at 65°C for 10 minutes and then placed on ice for 5 minutes.

[0092] (4) Add the components of the capping reaction (nearshore protein, M082) and incubate at 37°C for 30 min. The mRNA capping product is then purified and recovered by lithium chloride.

[0093] (5) The product obtained in (4) was dissolved in citric acid at pH 4, and the four lipids used to synthesize lipid nanoparticles (LNPs) were dissolved in ethanol; the ratio of the four lipids: ionizable cationic lipid, phosphatidylcholine, cholesterol and PEG-lipid was 50:10:38.5:1.5 mol / mol, and the mRNA to lipid ratio was about 0.05 (wt / wt) encapsulated into mRNA-LNPs vaccine.

[0094] Example 10: Experiment on immunization of mice with chimeric antigen mRNA vaccine of SARS-CoV-2 RBD protein and HA stem region

[0095] The immunization groups and the immunization doses of each group are shown in Table 2, and the blank cells represent "none". S and H1Delta L ) were diluted to the required concentration in DPBS. Then 6-8 week old BALB / c mice (average weight 20-25 g, the same below) were immunized in groups, with 8 mice in each group.

[0096] Immunization strategies such as Fig.12 Each mouse received two vaccinations on day 0 and day 21, each with a 100µl inoculation volume, by thigh muscle injection. On day 19 and day 35, the mice were bled from their eye sockets. Mouse serum was obtained by centrifugation at 3000rpm for 10 minutes after standing, heat-inactivated at 56°C for 30 minutes, and stored in a -20°C refrigerator for specific antibody titer detection and pseudovirus neutralization detection.

[0097] Table 2

[0098] Group Immunogen Dosage (µg) 1 <![CDATA[H1Delta S ]]> 2 2 <![CDATA[H1Delta S ]]> 10 3 <![CDATA[H1Delta L ]]> 2 4 <![CDATA[H1Delta L ]]> 10 5 Placebo

[0099] Example 11: ELISA assay to detect vaccine-induced SARS-CoV-2 specific antibody titers

[0100] The experimental method is shown in Example 2, and the results are as follows Fig.13 As shown, the two constructed mRNA vaccines induced dose-dependent specific RBD binding antibodies in all mice in the first and booster immunizations. The average endpoint titer of RBD-specific IgG antibodies in mice in the high-dose 10µg immunization group increased to >10 6 , which was significantly higher than that of mice in the low-dose 2µg immunization group.

[0101] Example 12: ELISA test to detect vaccine-induced H1N1 subtype influenza virus-specific antibody titers

[0102] The experimental method is shown in Example 2, and the results are as follows Fig.14 As shown, the two constructed mRNA vaccines induced dose-dependent specific H1N1 subtype influenza HA binding antibodies in all mice in the first and booster immunizations. The average endpoint titer of HA-specific IgG antibodies in mice in the high-dose 10µg immunization group increased to >10 6 , which was significantly higher than that of mice in the low-dose 2µg immunization group.

[0103] Example 13: ELISPOT assay to detect vaccine-induced SARS-CoV-2 long-term specific T cell immunity

[0104] In Example 10, three mice were euthanized at week 16 after secondary immunization, and spleens were removed and spleen cells were isolated. Mouse spleen cells were added to an ELISPOT plate (Dakoway) (1 x10 5 cells / well). Then, peptide pools of SARS-CoV-2 RBD protein (2µg / ml) were added to the wells. Phorbol myristate amine (PMA) was used as a positive control. Cells incubated without stimulation were used as negative controls. After 12 hours of incubation, the cells were removed and the plates were treated with biotinylated IFN-γ, IL-4 detection antibody, streptavidin-HRP conjugate, and substrate in sequence. When the intensity of the colored spots was sufficient for visual observation, the samples were thoroughly rinsed with deionized water to terminate the reaction. The number of spots was determined using an automated ELISPOT reader and image analysis software.

[0105] The experimental results are as follows Fig.15 Compared with the control group mice, the immunized group mice produced a significant increase in IFN-γ, while there was no significant increase in IL-4, indicating that the vaccine induced an immune response in the TH1 direction.

[0106] Example 14: Intracellular cytokine staining (ICS) assay to detect vaccine-induced SARS-CoV-2 long-term specific T cell immunity

[0107] Mouse spleen cell isolation is shown in Example 13, and antigen-specific CD4 + and CD8 + The results were as follows: Fig.16 As shown, the vaccine induced the production of IFN-γ, TNF-a and IL-2 in CD4 + T and CD8 +The percentages of T cells increased significantly, and the percentages of T cells producing IL-4 and IL-5 did not change significantly compared with the control group, indicating that the vaccine strongly induced an antiviral response in the TH1 direction.

[0108] Example 15: ELISPOT assay to detect vaccine-induced long-term specific T cell immunity of H1N1 subtype influenza virus

[0109] The experimental method is shown in Example 13, and the results are as follows Fig.17 As shown, compared with the control group mice, the immunized group mice had a significant increase in IFN-γ production, while IL-4 did not increase significantly, indicating that the vaccine induced an immune response in the TH1 direction.

[0110] Example 16: Intracellular cytokine staining (ICS) assay to detect vaccine-induced long-term specific T cell immunity of H1N1 subtype influenza virus

[0111] The experimental method is shown in Example 14, and the results are as follows Fig.18 As shown, the vaccine induced the production of IFN-γ and IL-2 in CD4 + The percentage of T cells increased significantly, producing IFN-γCD8 + The percentage of T cells increased significantly, while the percentage of T cells producing IL-4 and IL-5 did not change significantly compared with the control group, indicating that the vaccine strongly induced TH1 antiviral response.

[0112] Example 17: Pseudovirus Neutralization Experiment of SARS-CoV-2 Delta Variant by Immune Serum

[0113] The experimental method is shown in Example 2, and the results are as follows Fig.19 As shown, both constructed mRNA vaccines induced neutralizing antibodies in all mice in the first and booster immunizations. L Neutralizing antibody IC in high-dose group 50 The average value can reach 10 5 above.

[0114] Example 18: H1N1 subtype influenza virus challenge protection experiment

[0115] The mice after the second immunization in Example 10 were Fig.12 As shown, 20 LD were used on day 42 50 A / Brisbane / 02 / 2018(H1N1) dose challenge. Three days after the challenge, the lungs of the mice were removed and the tissue homogenate was used to detect the virus titer (TCID 50 ), the result is as follows Fig. 20As shown, the amount of virus in the lung tissue of the vaccine group mice was significantly reduced compared with the Placebo control group. Both mRNA vaccines have a very significant protective effect against influenza H1N1 live virus challenge.

[0116] Example 19: Body weight changes and survival status of mice within 14 days after challenge with H1N1 subtype influenza virus

[0117] The mice after the second immunization in Example 10 were Fig.12 As shown, 20 LD were used on day 42 50 A / Brisbane / 02 / 2018(H1N1) dose challenge. Within 14 days after the challenge, the mice were weighed every day and their survival was observed. Fig.21A and Fig. 21B All mice in the Placebo control group died within 6 days, and H1Delta S One mouse died in the low-dose group, and all the other mice survived, with their weight first decreasing and then increasing. These results show that the two constructed mRNA vaccines can provide good protection against influenza H1N1 virus attack.

[0118] Example 20: Testing the protective effect of the vaccine on the lung tissue of mice after challenge with H1N1 subtype influenza virus

[0119] The lung tissues of mice in the H1N1 subtype influenza virus challenge experiment in Example 18 were fixed with 4% paraformaldehyde, and then stained with hematoxylin-eosin. The changes in the lung tissues were observed by sectioning. The results are as follows: Fig. 22 As shown, the lung tissues of the control group mice (i.e., Placebo group) showed severe interstitial pneumonia, alveolitis, inflammatory cell infiltration, and bronchial epithelial cell necrosis. S Or H1Delta L Both can greatly alleviate the lung damage caused by virus attack, showing mild to moderate lung damage, with clear alveoli and less inflammatory cell infiltration. These results show that the vaccine can greatly alleviate the lung damage caused by H1N1 subtype influenza virus attack.

[0120] Example 21: H5N8 subtype influenza virus challenge protection experiment

[0121] To verify the protective effect of constructing against heterologous influenza, H1Delta L The constructed vaccine was administered with 10 LD on day 42. 50 A / Astrakhan / 3212 / 2020 (H5N8) dose challenge, the experimental method is shown in Example 18. The results are as follows Fig.23As shown, compared with the Placebo control group, the amount of virus in the lung tissue of the vaccine group mice was significantly reduced, indicating that the mRNA vaccine constructed in this way has a very significant protection against the live virus attack of the heterologous H5N8 subtype influenza virus.

[0122] Example 22: Body weight changes and survival status of mice within 14 days after challenge with H5N8 subtype influenza virus

[0123] The mice after the second immunization in Example 10 were Fig.12 As shown, 20 LD were used on day 42 50 A / Brisbane / 02 / 2018(H1N1) dose challenge. Within 14 days after the challenge, the mice were weighed every day and their survival was observed. Fig.24A and Fig. 24B All mice in the Placebo control group died within 9 days, and H1Delta L All the vaccine-immunized groups survived, and their weight first decreased and then increased. These results show that the constructed mRNA vaccine can provide good protection against the challenge of heterologous H5N8 subtype influenza virus.

[0124] Example 23: Testing the protective effect of the vaccine on the lung tissue of mice after challenge with H5N8 subtype influenza virus

[0125] The lung tissues of mice in the H5N8 subtype influenza virus challenge experiment in Example 21 were fixed with 4% paraformaldehyde, and then stained with hematoxylin-eosin. The changes in the lung tissues were observed by sectioning. The results are as follows: Fig.25 As shown, the lung tissues of the control group mice (i.e., Placebo group) showed severe interstitial pneumonia, alveolitis, inflammatory cell infiltration, and bronchial epithelial cell necrosis. L Vaccine immunity greatly alleviated the lung damage caused by virus attack, showing mild to moderate lung damage, with clear alveoli and less inflammatory cell infiltration. These results show that the constructed mRNA vaccine can greatly alleviate the lung damage caused by heterologous H5N8 subtype influenza virus attack.

[0126] Example 24: H1Delta L -Structure analysis of the complex of TF protein, CB6 Fab and CR9114 Fab

[0127] According to the method of Example 1, H1Delta was expressed L -TF chimeric protein. After purification, H1Delta was collected L -TF protein peak. H1Delta L-TF protein was mixed with the novel coronavirus antibody CB6 Fab protein and the influenza antibody CR9114 Fab protein and incubated at 4°C for 12 hours. TM H1Delta was purified by molecular sieve chromatography (pH 8.0) using a 200 μL Increase 10 / 300 GL column (GE Healthcare). L -The complex of TF protein, CB6 Fab and CR9114 Fab has a molecular sieve chromatography curve as shown in Fig.26 As shown, one of the eluting peaks is H1Delta L -TF protein, CB6 Fab and CR9114 Fab complex, the other peak is the excess CB6 Fab and CR9114 Fab, which shows that: H1DeltaL-TF protein can bind to CB6 Fab and CR9114Fab and form a complex. The eluate of the complex of H1DeltaL-TF protein, CB6 Fab and CR9114 Fab collected above was concentrated and used for cryo-electron microscopy analysis after concentration, the procedure is as follows:

[0128] Prepare the Quantifoil grid (specification 1.2 / 1.3) for sample preparation in advance and perform glow discharge hydrophilization treatment. L The complex of -TF protein, CB6 Fab and CR9114 Fab was dripped onto the prepared grid, and the grid was quickly inserted into liquid ethane using an automatic sample preparation machine Vitrobot Mark IV to complete sample preparation.

[0129] Data collection was performed using a 300 kV Titan Krios transmission electron microscope (Thermo Fisher) with a K2 direct electron detector camera, and a large number of photos were collected using the Serial-EM automatic collection program. Then, the collected raw data were drift corrected using MotionCor2 software, the images were contrast-transfer-function corrected using CTFFIND4.1 software, and the images were further processed and reconstructed using Relion-3.1 software.

[0130] The cryo-electron microscopy image of the complex of the H1DeltaL-TF protein, CB6 Fab and CR9114 Fab of the present invention is as follows: Fig. 27 As shown by Fig. 27 Visible: In H1Delta L - In the complex of TF protein, CB6 Fab and CR9114 Fab, H1Delta L -TF protein presents a homotrimeric conformation, divided into a head region and a neck region; H1DeltaL -TF protein can bind to CB6 Fab and CR9114 Fab at the same time. CB6 Fab binds to the RBD domain in the head region, and CR9114 Fab binds to the HA2 domain in the neck region. The binding epitopes of the two antibodies are arranged in a staggered manner, and there is no steric hindrance between them, indicating that H1Delta L -The main epitopes of TF protein are fully exposed, which is conducive to activating immune response.

[0131] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A chimeric protein of the RBD protein and the HA stem region of SARS-CoV-2, characterized in that: The RBD of the S protein of SARS-CoV-2 was used to replace the head region of the HA protein and connected to the stem region of the HA protein to construct a chimeric protein; The amino acid sequence of the chimeric protein is shown in SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:

8.

2. A nucleic acid encoding the chimeric protein of claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleotide sequence thereof is shown in SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:

7.

4. The nucleic acid according to claim 2, characterized in that It also includes the 5' end non-coding sequence UTR and the 3' end non-coding sequence UTR.

5. The nucleic acid according to claim 4, characterized in that Poly A is also added after the 3' non-coding sequence UTR.

6. The nucleic acid according to claim 5, characterized in that The polyadenylic acid Poly A is 120 polyadenylic acid Poly A.

7. A recombinant vector containing the nucleic acid according to any one of claims 2 to 6.

8. The recombinant vector according to claim 7, characterized in that It is an expression vector.

9. A chimeric antigen prepared according to the chimeric protein of claim 1.

10. An mRNA vaccine prepared according to the nucleic acid as described in any one of claims 2 to 6.

11. The mRNA vaccine according to claim 10, characterized in that It is a lipid nanoparticle mRNA vaccine.

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