Influenza virus universal nanoparticle vaccine and method of making same

Abstract

The application discloses an influenza virus universal nanoparticle vaccine and a preparation method thereof. The application designs an influenza virus antigen by using an influenza virus matrix protein 2 extracellular domain (M2e) and hemagglutinin (HA), and forms a trimer structure by fusing a foldon motif at the C terminal of the antigen. On this basis, the application prepares an influenza virus antigen multimeric complex through a GvTagOpti / SdCatcher system, and prepares the influenza virus universal nanoparticle vaccine which can be inoculated through the respiratory system and has good immunoprotective effect by taking the obtained multimeric complex as an immunogen. The nanoparticle vaccine disclosed by the application not only has good immunoprotective effect and can prevent infection of different subtypes of influenza viruses after inoculation, but also has a simple preparation method, high safety and can be quickly applied to clinical trials.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More particularly, it relates to a universal nanoparticle vaccine for influenza virus and a preparation method thereof. BACKGROUND

[0002] Influenza virus has the ability to escape from epidemic through antigenic drift, so it is necessary to update the components of seasonal influenza vaccine every year to match the newly epidemic virus, but the immune protection effect of seasonal influenza vaccine is not always as expected. Therefore, there is an urgent need for a universal influenza vaccine that can cause broad cross-protection against different influenza viruses, reduce immunization based on the predicted seasonal major circulating strains, and reduce the threat of influenza virus.

[0003] In addition to finding a suitable immunogen, the immune approach is another important issue in vaccine development. At present, injection vaccination is the universal vaccination method for influenza vaccine, but injection vaccination has the disadvantages of being easy to cause infection and easy to cause adverse reactions. Intranasal vaccination is a more attractive non-invasive vaccination method, which is safer and can be managed by untrained personnel. In addition, since intranasal vaccine is similar to the route of influenza virus invasion of the host, it can stimulate the production of large amounts of secretory IgA (S-IgA) antibodies, providing better protection against influenza virus. However, there are few universal influenza vaccines that can be vaccinated through the respiratory system, and the immune protection effect needs to be improved. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the above-mentioned universal influenza vaccine, and to provide a universal nanoparticle vaccine for influenza virus and a preparation method thereof.

[0005] The first object of the present application is to provide an influenza virus antigen.

[0006] The second object of the present application is to provide an influenza virus antigen multimeric complex.

[0007] The third object of the present application is to provide the use of the antigen or the antigen multimeric complex in the preparation of a drug for resisting influenza virus.

[0008] The fourth object of the present application is to provide a universal nanoparticle vaccine for influenza virus.

[0009] The fifth object of the present application is to provide a preparation method of the universal nanoparticle vaccine for influenza virus.

[0010] The above objects of the present application are achieved by the following technical solutions:

[0011] To provide an influenza virus universal nanoparticle vaccine which can be inoculated through the respiratory system and has good immunoprotective effect, the present application designs an influenza virus antigen by using influenza virus matrix protein 2 ectodomain (M2e) and hemagglutinin (HA), wherein the antigen is a HA-3M2e fusion protein formed by replacing the head domain of hemagglutinin (HA) with three repeated influenza virus matrix protein 2 ectodomains (3M2e), and a trimer structure is formed by connecting a foldon motif at the C-terminal end of the HA-3M2e fusion protein. On the basis of the influenza virus antigen, the present application prepares an influenza virus antigen multimeric complex and an influenza virus universal nanoparticle vaccine by using a GvTagOpti / SdCatcher system (the Gv / Sd system can be referred to in Chinese Patent No. CN113621031A). After immunizing BALB / c mice by the respiratory system, it is found that the prepared influenza virus universal nanoparticle vaccine can induce the production of higher titer of antigen-specific IgG antibodies, and the respiratory system inoculation can protect the mice from the infection of influenza viruses of different subtypes.

[0012] Therefore, the present application protects the influenza virus antigen, in particular, the antigen is a HA-3M2e fusion protein formed by replacing the head domain of hemagglutinin (HA) with three repeated influenza virus matrix protein 2 ectodomains (3M2e).

[0013] As an alternative embodiment, the amino acid sequence of the antigen is shown in SEQ ID NO. 1.

[0014] In order to make the prepared influenza virus antigen have a trimer structure, as an alternative embodiment, a foldon motif is connected at the C-terminal end of the antigen.

[0015] The amino acid sequence of the influenza virus antigen shown in SEQ ID NO. 1 of the present application after connecting the foldon motif is shown in SEQ ID NO. 2.

[0016] In order to facilitate the secretory expression of the influenza virus antigen, a secretory signal peptide is further connected at the N-terminal end of the antigen.

[0017] As an alternative embodiment, the amino acid sequence of the fusion protein obtained by connecting the influenza virus antigen shown in SEQ ID NO. 1 of the present application with the secretory signal peptide, the foldon motif and Gv respectively is shown in SEQ ID NO. 5.

[0018] The application also protects an influenza virus antigen multimeric complex (HA-3M2e-NP protein), which is a fusion protein obtained by fusion expression of an influenza virus antigen with a foldon motif connected at the C terminal and Gv shown in SEQ ID NO. 3, and then connecting the obtained fusion protein with Sd-Ferritin protein shown in SEQ ID NO. 4.

[0019] The application also protects the use of the influenza virus antigen or the antigen multimeric complex in the preparation of a medicine against influenza virus.

[0020] Specifically, the medicine is a universal vaccine against influenza virus. The advantages of the universal vaccine of the application are embodied in two aspects, one is that after vaccination, the subject can be protected from infection with different subtypes of influenza virus, and the other is that the vaccine can be inoculated by injection or by respiratory system.

[0021] Based on the above-mentioned influenza virus antigen multimeric complex, the application also provides a universal nanoparticle vaccine against influenza virus, which is prepared by taking the antigen multimeric complex (HA-3M2e-NP protein) as an antigen.

[0022] The application also provides a preparation method of the universal nanoparticle vaccine against influenza virus, comprising the following steps:

[0023] S1. Expressing a fusion protein of an influenza virus antigen with a foldon motif connected at the C terminal and Gv shown in SEQ ID NO. 3 in a eukaryotic expression system and purifying;

[0024] S2. Expressing Sd-Ferritin protein shown in SEQ ID NO. 4 in a prokaryotic expression system and purifying;

[0025] S3. Incubating the fusion protein obtained in step S1 with the Sd-Ferritin protein obtained in step S2 in a buffer without enzyme to obtain an influenza virus antigen multimeric complex;

[0026] S4. Formulating the influenza virus antigen multimeric complex obtained in step S3 with an adjuvant to obtain a universal nanoparticle vaccine against influenza virus.

[0027] As an alternative embodiment, the adjuvant is a cyclic di-GMP adjuvant, and the mass ratio of the influenza virus antigen multimeric complex to the cyclic di-GMP adjuvant is 5:1-2.

[0028] Specifically, the mass ratio of the influenza virus antigen multimeric complex to the cyclic di-GMP adjuvant is 5:1.

[0029] Specifically, the buffer contains 20mM Tris-HCl and 50mM NaCl.

[0030] The present application has the following advantages:

[0031] The present application designs an influenza virus antigen by using the extracellular domain of influenza virus matrix protein 2 and hemagglutinin, and forms a trimer structure by fusing a foldon motif at the C-terminal of the antigen. On this basis, the present application prepares an influenza virus antigen multimeric complex by a GvTagOpti / SdCatcher system, and prepares an influenza virus universal nanoparticle vaccine which can be inoculated through the respiratory system and has good immunoprotective effect by using the obtained multimeric complex as an immunogen. The nanoparticle vaccine described in the present application not only has good immunoprotective effect and can prevent infection by different subtypes of influenza virus after inoculation, but also has a simple preparation method, high safety, and can be quickly applied to clinical trials. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Figure 1 is a structural schematic diagram of an influenza virus antigen (HA-3M2e fusion protein) and a Sd-Ferritin fusion protein.

[0033] Figure 2 Figure 2 is a schematic diagram of an influenza virus antigen multimeric complex (HA-3M2e-NP protein) assembled by using an influenza virus antigen (HA-3M2e fusion protein) and a Helicobacter pylori ferritin (Ferritin) as a core.

[0034] Figure 3 Figure 3 is an SDS-PAGE diagram of a Sd-Ferritin protein, a HA-3M2e protein, and a HA-3M2e-NP protein.

[0035] Figure 4 Figure 4 is a purification molecular sieve (SEC) diagram of an influenza virus antigen multimeric complex (HA-3M2e-NP protein).

[0036] Figure 5 Figure 5 is a transmission electron microscope (TEM) diagram of an influenza virus antigen multimeric complex (HA-3M2e-NP protein).

[0037] Figure 6 Figure 6 is an immunization strategy diagram for immunizing BALB / c mice with the obtained influenza virus universal nanoparticle vaccine.

[0038] Figure 7 Figure 7 is a detection result of the titer of antigen-specific IgG antibodies in the serum of mice 6 weeks after inoculation (s.c.) and inoculation (i.n.) of the influenza virus universal nanoparticle vaccine through the respiratory system; in the figure, *** indicates a very significant difference, p<0.001.

[0039] Figure 8The weight change curve and survival curve of mice immunized with the universal nanoparticle vaccine of influenza virus after infection with different subtypes of influenza A virus. DETAILED DESCRIPTION

[0040] The present application will be further described by the following description of drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the art.

[0041] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0042] Example 1 Construction of influenza virus antigen multimeric complex

[0043] The structural schematic diagram of the HA-3M2e-Gv fusion protein and the Sd-Ferritin protein required for constructing the influenza virus antigen multimeric complex (HA-3M2e-NP protein) of the present application is shown in Figure 1 As can be seen from the schematic diagram shown in Figure 1 As can be seen from the schematic diagram shown in

[0044] In order to facilitate the secretory expression of the influenza virus antigen HA-3M2e and the construction of the influenza virus antigen multimeric complex (HA-3M2e-NP protein) through the intermolecular isopeptide bond of Sd and Gv, the present application also connects a secretion signal peptide (SP) to the N-terminus of the influenza virus antigen HA-3M2e, and connects a Gv shown in SEQ ID NO. 3 to the C-terminus of the foldon motif. The amino acid sequence of the fusion protein obtained after the influenza virus antigen HA-3M2e is connected with the secretion signal peptide, the foldon motif and the Gv, respectively, is shown in SEQ ID NO. 5.

[0045] The prepared HA-3M2e-foldon-Gv fusion protein and the Sd-Ferritin protein shown in SEQ ID NO. 4 are placed in a buffer (20 mM Tris-HCl, 50 mM NaCl) without any enzyme, and incubated at 25 DEG C overnight. The HA-3M2e-foldon-Gv fusion protein and the Sd-Ferritin protein shown in SEQ ID NO. 4 can be covalently combined through the GvTagOpti / Sdcatcher (Gv / Sd) system (the GvTagOpti / Sdcatcher (Gv / Sd) system can be referred to in Chinese Patent No. CN113621031A), and self-assembled to obtain an influenza virus antigen multimeric complex (HA-3M2e-NP protein), i.e. a nanoparticle, through the spontaneous chemical bond of Gv-Sd. The schematic diagram of the influenza virus antigen HA-3M2e assembled into the influenza virus antigen multimeric complex (HA-3M2e-NP protein) with the Helicobacter pylori ferritin (Ferritin) as the core is shown in Figure 2 Figure 2 It can be known that the HA-3M2e-foldon trimer structure is presented on the surface of the Ferritin nanoparticle, and can be used for preparing an influenza virus universal nanoparticle vaccine.

[0046] Specifically, the preparation method of the influenza virus antigen multimeric complex (HA-3M2e-NP protein) of the present application is as follows:

[0047] 1. Preparation of HA-3M2e-foldon-Gv fusion protein

[0048] Six His-labeled DNA sequences encoding the protein shown in SEQ ID NO. 5 are cloned into a pcDNA3.1 vector, and the expressed plasmid is transfected into CHO-S cells to induce expression. After seven days, centrifugation is performed to remove cell debris, and the supernatant is collected. The clarified supernatant is passed through Ni-NTA agarose microspheres to enrich the His-labeled target protein, and then eluted with an imidazole-containing Tris buffer. The purified protein is concentrated and the buffer is replaced with a conventional Tris buffer. The protein concentration is determined by the BCA assay method.

[0049] 2. Preparation of Sd-Ferritin protein

[0050] ​Sd-Ferritin was expressed and purified in E. coli. Six His-tagged DNA sequences encoding Sd-Ferritin protein were cloned into pET28a vector and the constructs were transformed into BL21 (Takara) cells; the single colonies were expanded in LB medium containing kanamycin under 37℃ condition with shaking; isopropyl-β-D-thiogalactoside (IPTG) was added to the bacterial culture to induce protein expression; after 18 hours of induction, the bacteria expressing the protein were harvested and treated by high-pressure disruption, and the supernatant was collected; the supernatant was incubated with Ni-NTA agarose (GE Healthcare) to enrich the His-tagged Sd-Ferritin protein, and then the protein was eluted with imidazole-containing Tris buffer; the purified protein was concentrated and the buffer was replaced with conventional Tris buffer; the concentration of the Sd-Ferritin protein was determined by BCA assay.

[0051] The prepared HA-3M2e-foldon-Gv fusion protein and Sd-Ferritin protein were incubated in a buffer (20 mM Tris-HCl and 50 mM NaCl) without any enzyme at 25℃ overnight to self-assemble an influenza virus antigen polymeric complex (HA-3M2e-NP protein), i.e., a nanoparticle, through spontaneous chemical bond combination of Gv-Sd.

[0052] The prepared Sd-Ferritin protein, HA-3M2e protein and HA-3M2e-NP protein were detected by SDS-PAGE, and the purity was verified by Coomassie blue staining, and the SDS-PAGE diagram of the Sd-Ferritin protein, HA-3M2e protein and HA-3M2e-NP protein is shown in Figure 3 Figure 3 It can be seen that the Sd-Ferritin protein, influenza virus antigen HA-3M2e and HA-3M2e-NP protein are successfully prepared, and the size is consistent with the expectation.

[0053] Example 2 Characterization of HA-3M2e-NP nanoparticle

[0054] The purity and uniformity of the HA-3M2e-NP protein were verified by molecular sieve (SEC) and transmission electron microscopy (TEM). The prepared HA-3M2e-NP protein (nanoparticle) in Example 1 was separated and collected by SEC, and then concentrated. The purified molecular sieve diagram of the HA-3M2e-NP protein is shown in Figure 4 Figure 4 ​​It can be seen that the obtained HA-3M2e-NP protein has a single peak, indicating that the HA-3M2e-NP protein obtained by the application has a purity higher than 99%.

[0055] The HA-3M2e-NP protein obtained by molecular sieve separation is also observed by transmission electron microscopy (TEM), and the transmission electron microscopy (TEM) image of the HA-3M2e-NP protein is as shown in Figure 5 Figure 5 It can be seen that the HA-3M2e-NP nanoparticle exhibits a sharp spike protruding from the spherical core after the influenza virus antigen is covalently combined with the Ferritin nanoparticle, indicating that through the display strategy of displaying the antigen on the surface of the Ferritin nanoparticle by the method of the application, the candidate antigen is successfully enriched on the surface of the nanoparticle, which can be used for constructing a nanoparticle vaccine.

[0056] Example 3 Immunoprotection experiment

[0057] The influenza virus antigen polycomplex (HA-3M2e-NP protein) obtained by the preparation of Example 1 is formulated with a c-di-GMP adjuvant (the mass ratio of the influenza virus antigen polycomplex to the c-di-GMP adjuvant is 5:1) to obtain a HA-3M2e-NP nanoparticle vaccine, and BALB / c mice are immunized by subcutaneous inoculation (s.c.) and respiratory system inoculation (i.n.) respectively, and the immunoprotection effect of the HA-3M2e-NP nanoparticle vaccine is detected.

[0058] BALB / c mice are immunized by subcutaneous inoculation (s.c.) and respiratory system inoculation (i.n.) respectively, and each mouse is inoculated with a dose of 10 μg, and the immunization strategy is as shown in Figure 6 Figure 6 It can be seen that all the mice are immunized by a double-needle immunization strategy, that is, immunization at weeks 0 and 4; two weeks after the vaccine immunization, the serum of the mouse is collected, and the titer of the antigen-specific IgG antibody in the serum is detected.

[0059] After 6 weeks of subcutaneous inoculation (s.c.) and respiratory system inoculation (i.n.) of the nanoparticle vaccine, the detection result of the titer of the antigen-specific IgG antibody in the serum of the mouse is as shown in Figure 7 Figure 7 It can be seen that, compared with the control group, the HA-3M2e-NP nanoparticle vaccine immunization can induce the production of a higher titer of antigen-specific IgG antibody, wherein the titer of the subcutaneous inoculation is close to 10 6 , and the titer of the respiratory system inoculation is higher than 10 4 .

[0060] ​​​To further explore the in vivo protective ability of the prepared HA-3M2e-NP nanoparticle vaccine against different subtypes of influenza A viruses, the BALB / c mice immunized by the HA-3M2e-NP nanoparticle vaccine through subcutaneous inoculation (s.c.) and respiratory system inoculation (i.n.) respectively in Example 2 were transferred to a biosafety level two laboratory, and live virus challenge tests were performed using H1N1 and H3N2 respectively (the mice were anesthetized with isoflurane, and 1.2x10 3 TCID50 of viruses were used to infect the mice through nose drops), and the body weight changes and survival of the mice were monitored within two weeks after the live virus challenge tests. The body weight change curves and survival curves of the mice after being immunized by the HA-3M2e-NP nanoparticle vaccine and infected by different subtypes of influenza A viruses are shown in Figure 8 As can be seen from the above, Figure 8 whether it is H1N1 or H3N2 virus strain infection, the mice immunized by the HA-3M2e-NP nanoparticle vaccine through respiratory system inoculation only have slight body weight loss, and no death occurs, indicating that the HA-3M2e-NP nanoparticle vaccine through respiratory system inoculation can protect the mice from infection by different subtypes of influenza A viruses.

[0061] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. An influenza virus antigen polymer complex, characterized in that The antigen multimeric complex is obtained by fusing the influenza virus antigen with the amino acid sequence shown in SEQ ID NO.2 with Gv shown in SEQ ID NO.3 to obtain a fusion protein, and then connecting the obtained fusion protein with the Sd-Ferritin protein shown in SEQ ID NO.

4.

2. Use of the antigen multimeric complex according to claim 1 in the preparation of anti-influenza virus drugs.

3. The application according to claim 2, characterized in that: The medicine is a universal influenza virus vaccine.

4. A universal nanoparticle vaccine for influenza virus, characterized in that: The vaccine is prepared using the antigen multimer complex according to claim 1 as an antigen.

5. The method for preparing the universal influenza virus nanoparticle vaccine according to claim 4, characterized in that: The following steps are involved: S1. Expressing the amino acid sequence of influenza virus antigen as shown in SEQ ID NO.2 and the fusion protein of Gv as shown in SEQ ID NO.3 in a eukaryotic expression system and purifying it; S2. Express the Sd-Ferritin protein shown in SEQ ID NO.4 in a prokaryotic expression system and purify it; S3. The fusion protein obtained in step S1 is incubated with the Sd-Ferritin protein obtained in step S2 in an enzyme-free buffer to obtain an influenza virus antigen multimeric complex; S4. The influenza virus antigen multimeric complex obtained in step S3 is prepared with an adjuvant to obtain a universal influenza virus nanoparticle vaccine.

6. The method according to claim 5, characterized in that The adjuvant is a cyclic diguanylate adjuvant.

7. The method according to claim 6, characterized in that The mass ratio of the influenza virus antigen polymer complex to the cyclic diguanylate adjuvant is 5:1-2.

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