CN115850395A - Influenza virus universal nanoparticle vaccine and preparation method thereof - Google Patents
Influenza virus universal nanoparticle vaccine and preparation method thereof Download PDFInfo
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- CN115850395A CN115850395A CN202211058390.9A CN202211058390A CN115850395A CN 115850395 A CN115850395 A CN 115850395A CN 202211058390 A CN202211058390 A CN 202211058390A CN 115850395 A CN115850395 A CN 115850395A
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Abstract
The invention discloses a universal influenza virus nanoparticle vaccine and a preparation method thereof. The invention designs an influenza virus antigen by using an influenza virus matrix protein 2 ectodomain (M2 e) and Hemagglutinin (HA), and forms a trimer structure by fusing foldon motif at the C terminal of the antigen. On the basis, the influenza virus antigen multimeric complex is prepared by a GvTagOpti/SdCatccher system, and the influenza virus universal nanoparticle vaccine which can be inoculated through a respiratory system and has a good immune protection effect is prepared by taking the obtained multimeric complex as an immunogen. The nanoparticle vaccine disclosed by the invention has a good immune protection effect, can be prevented from being infected by different subtype influenza viruses after being inoculated, is simple in preparation method and high in safety, and can be quickly applied to clinical tests.
Description
Technical Field
The invention belongs to the technical field of biological medicines. More particularly, relates to a universal nanoparticle vaccine for influenza virus and a preparation method thereof.
Background
Influenza viruses have the ability to gain escape epidemics through antigenic drift, and therefore, the components of seasonal influenza vaccines need to be updated every year to match the new epidemic, but the immunoprotective effects of seasonal influenza vaccines are not always as expected. Therefore, there is an urgent need for a universal influenza vaccine that can induce broad cross-protection against different influenza viruses, reduce vaccination based on predicting seasonal major circulating strains, and mitigate the threat of influenza viruses.
In addition to finding suitable immunogens, the route of immunization is another important issue in vaccine development. At present, injection vaccination is a general vaccination mode of influenza vaccines, but the injection vaccination has the defects of easy infection, easy adverse reaction and the like. Intranasal vaccination is a more attractive non-invasive vaccination method, which is safer and can be administered by personnel without medical training. In addition, because the intranasal vaccine is similar to the approach of the influenza virus to invade a host, the intranasal vaccine can stimulate the large-scale production of secretory IgA (S-IgA) antibodies and provides more excellent protection against the influenza virus. However, the existing influenza virus universal vaccines which can be inoculated through the respiratory system are few, and the immune protection effect is still to be improved.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing universal influenza vaccine and provides an influenza virus universal nanoparticle vaccine and a preparation method thereof.
It is a first object of the present invention to provide an influenza virus antigen.
The second objective of the invention is to provide an influenza virus antigen polyplex.
The third purpose of the invention is to provide the application of the antigen or the antigen polyplex in preparing the medicine for resisting influenza virus.
The fourth purpose of the invention is to provide a universal nanoparticle vaccine for influenza virus.
The fifth purpose of the invention is to provide a preparation method of the influenza virus universal nanoparticle vaccine.
The above purpose of the invention is realized by the following technical scheme:
in order to provide a universal influenza virus nanoparticle vaccine which can be inoculated through a respiratory system and HAs a good immune protection effect, the invention designs an influenza virus antigen by using an influenza virus matrix protein 2 ectodomain (M2 e) and Hemagglutinin (HA), wherein the antigen is an HA-3M2e fusion protein formed by replacing the head structure domain of the Hemagglutinin (HA) with three repeated influenza virus matrix protein 2 ectodomains (3M 2 e), and a foldon motif is connected to the C terminal of the HA-3M2e fusion protein to form a trimer structure. On the basis of the influenza virus antigen, the influenza virus antigen multimeric complex and the influenza virus universal nanoparticle vaccine are prepared by a GvTagOpti/SdCatccher system (the GvTagOpti/Sdcatcher (Gv/Sd) system can be referred to Chinese patent with publication number CN 113621031A). In addition, after a BALB/c mouse is immunized by a respiratory system inoculation mode, the prepared influenza virus universal nanoparticle vaccine can induce the generation of antigen-specific IgG antibodies with higher titer, and the mouse can be protected from the infection of influenza viruses of different subtypes by respiratory system inoculation.
Thus, the present application protects the influenza virus antigen, in particular, the antigen is an HA-3M2e fusion protein formed by replacing the head domain of Hemagglutinin (HA) with the ectodomain (3M 2 e) of the influenza virus matrix protein 2 in triplicate.
As an alternative embodiment, the amino acid sequence of the antigen is shown in SEQ ID NO. 1.
In order to make the influenza virus antigen have a trimer structure, the C-terminal of the antigen is connected with a foldon motif as an alternative embodiment.
The amino acid sequence of the influenza virus antigen shown in SEQ ID NO.1 after being connected with foldon motif is shown in SEQ ID NO. 2.
In order to facilitate the secretory expression of the influenza virus antigen, the N end of the antigen is also connected with a secretory signal peptide.
As an alternative embodiment, the amino acid sequence of the fusion protein obtained by linking the influenza virus antigen shown in SEQ ID NO.1 of the present invention to the secretion signal peptide, the foldon motif and the Gv is shown in SEQ ID NO. 5.
The invention also provides an influenza virus antigen polyplex (HA-3M 2e-NP protein), which is obtained by fusion expression of an influenza virus antigen with foldon motif connected at C terminal and Gv shown in SEQ ID No.3 to obtain a fusion protein, and then connecting the obtained fusion protein and Sd-Ferritin protein shown in SEQ ID No. 4.
The invention also provides application of the influenza virus antigen or the antigen multimeric complex in preparing a medicament for resisting influenza virus.
Specifically, the medicament is an influenza virus universal vaccine. The universal vaccine has the advantages that the universal vaccine can be prevented from being infected by influenza viruses of different subtypes after inoculation, and can be inoculated through injection and a respiratory system.
Based on the influenza virus antigen polyplex, the invention also provides an influenza virus universal nanoparticle vaccine which is prepared by taking the antigen polyplex (HA-3M 2e-NP protein) as an antigen.
The invention also provides a preparation method of the influenza virus universal nanoparticle vaccine, which comprises the following steps:
s1, expressing fusion protein of an influenza virus antigen with a foldon motif connected to the C end and Gv shown in SEQ ID NO.3 in a eukaryotic expression system and purifying;
s2, expressing the Sd-Ferritin protein shown in SEQ ID NO.4 in a prokaryotic expression system and purifying;
s3, incubating the fusion protein obtained in the step S1 and the Sd-Ferritin protein obtained in the step S2 in a buffer solution without enzyme to obtain an influenza virus antigen multimeric complex;
and S4, preparing the influenza virus antigen polymer compound obtained in the step S3 and an adjuvant to obtain the influenza virus universal nanoparticle vaccine.
As an alternative embodiment, the adjuvant is a cyclic diguanylic acid (c-di-GMP) adjuvant, and the mass ratio of the influenza virus antigen polyplex to the cyclic diguanylic acid adjuvant is 5:1 to 2.
Specifically, the mass ratio of the influenza virus antigen polyplex to the cyclic diguanylic acid adjuvant is 5:1.
specifically, the buffer contained 20mM Tris-HCl and 50mM NaCl.
The invention has the following beneficial effects:
the invention designs an influenza virus antigen by utilizing the extracellular domain of influenza virus matrix protein 2 and hemagglutinin, and forms a trimer structure by fusing foldon motif at the C terminal of the antigen. On the basis, the influenza virus antigen multimeric complex is prepared by a GvTagOpti/SdCatccher system, and the influenza virus universal nanoparticle vaccine which can be inoculated through a respiratory system and has a good immune protection effect is prepared by taking the obtained multimeric complex as an immunogen. The nanoparticle vaccine disclosed by the invention is good in immune protection effect, can be prevented from being infected by influenza viruses of different subtypes after inoculation, is simple in preparation method and high in safety, and can be quickly applied to clinical tests.
Drawings
FIG. 1 is a schematic diagram of the structures of an influenza virus antigen (HA-3M 2e fusion protein) and an Sd-Ferritin fusion protein.
FIG. 2 is a schematic diagram of an influenza virus antigen polyplex (HA-3M 2e-NP protein) assembled by an influenza virus antigen (HA-3M 2e fusion protein) with a helicobacter pylori Ferritin (Ferritin) as a core.
FIG. 3 is an SDS-PAGE image of the Sd-Ferritin protein, HA-3M2e protein and HA-3M2e-NP protein.
FIG. 4 is a purified molecular Sieve (SEC) pattern of influenza virus antigen polyplexes (HA-3M 2e-NP protein).
FIG. 5 is a Transmission Electron Microscope (TEM) image of influenza virus antigen polyplex (HA-3M 2e-NP protein).
FIG. 6 is a schematic diagram of the immunization strategy of BALB/c mice immunized with the influenza virus universal nanoparticle vaccine.
Fig. 7 is the results of the measurement of the titer of antigen-specific IgG antibodies in mouse serum 6 weeks after vaccination by subcutaneous injection (s.c.) and vaccination by respiratory system (i.n.) of the influenza virus universal nanoparticle vaccine; the graph indicates that the difference is very significant, p < 0.001.
Fig. 8 is a weight change curve and a survival curve of mice infected with different subtype influenza a viruses after immunization with the influenza virus universal nanoparticle vaccine.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 construction of influenza Virus antigen polyplexes
The structural schematic diagram of HA-3M2e-Gv fusion protein and Sd-Ferritin protein required by the construction of influenza virus antigen multimeric complex (HA-3M 2e-NP protein) is shown in figure 1. As can be seen from the schematic diagram shown in FIG. 1, the sequence of HA and 3M2e in the influenza virus antigen (i.e., HA-3M2e fusion protein, hereinafter referred to as influenza virus antigen HA-3M2 e) of the present invention is HA1-3M2e-HA1-HA2, and the head domain of Hemagglutinin (HA) is derived from the three repeated ectodomains (3M 2 e) of influenza virus matrix protein 2. Specifically, the amino acid sequence of the influenza virus antigen HA-3M2e constructed by the invention is shown in SEQ ID NO. 1. In order to enable influenza virus antigen HA-3M2e to have a trimer structure in a neutral environment, the C end of the influenza virus antigen HA-3M2e is fused with a foldon motif, and the amino acid sequence of the influenza virus antigen connected with the foldon motif is shown in SEQ ID NO. 2.
In addition, in order to facilitate the secretion expression of the influenza virus antigen HA-3M2e and the construction of an influenza virus antigen multimeric complex (namely HA-3M2e-NP protein) through intermolecular isopeptide bonds of Sd and Gv, the invention also connects a secretion Signal Peptide (SP) at the N end of the influenza virus antigen HA-3M2e, connects Gv shown by SEQ ID NO.3 at the C end of a foldon motif, and the amino acid sequence of the fusion protein obtained after the influenza virus antigen HA-3M2e is respectively connected with the secretion signal peptide, the foldon motif and the Gv is shown by SEQ ID NO. 5.
The prepared HA-3M2e-foldon-Gv fusion protein and Sd-ferrtin protein shown in SEQ ID NO.4 are placed in a buffer solution (20 mM Tris-HCl and 50mM NaCl) without any enzyme, and incubated overnight at 25 ℃, and the HA-3M2e-foldon-Gv fusion protein and the Sd-ferrtin protein shown in SEQ ID NO.4 can be bound by covalent with a GvTagOpti/Sdchecher (Gv/Sd) system (the GvTagOpti/Sdchecher (Gv/Sd) system can be referred to in Chinese patent with publication number CN 113621031A) and bound by spontaneous chemical bond of Gv-Sd to self-assemble an influenza virus antigen multimeric complex (HA-3M 2e-NP protein), namely nanoparticles. A schematic diagram of an influenza virus antigen multimeric complex (HA-3M 2e-NP protein) assembled by influenza virus antigen HA-3M2e with helicobacter pylori Ferritin (Ferritin) as a core is shown in FIG. 2, and as can be seen from FIG. 2, an HA-3M2e-foldon trimer structure is presented on the surface of Ferritin nanoparticles and can be used for preparing an influenza virus universal nanoparticle vaccine.
Specifically, the preparation method of the influenza virus antigen polyplex (HA-3M 2e-NP protein) comprises the following steps:
1. preparation of HA-3M2e-foldon-Gv fusion protein
2. Preparation of Sd-Ferritin protein
Sd-Ferritin is expressed in e.coli (e.coli) and purified. Cloning 6 His-tagged Sd-Ferritin protein-encoding DNA sequences into pET28a vector, and transforming the construct into BL21 (Takara) cells; carrying out shake culture on the amplified monoclonal in an LB culture medium containing kanamycin at the temperature of 37 ℃; adding isopropyl-beta-D-thiogalactoside (IPTG) to the bacterial culture to induce protein expression; after 18 hours of induction expression, harvesting bacteria expressing protein and carrying out high-pressure crushing treatment, and collecting supernatant; incubating the supernatant with Ni-NTA agarose (GE Healthcare) to enrich for His-tagged Sd-Ferritin protein, followed by elution of the protein with imidazole-containing Tris buffer; concentrating the purified protein and replacing the buffer with conventional Tris buffer; the concentration of Sd-Ferritin protein was determined by BCA assay.
Placing the prepared HA-3M2e-foldon-Gv fusion protein and Sd-Ferritin protein into buffer solution (20 mM Tris-HCl and 50mM NaCl) without any enzyme, incubating overnight at 25 ℃, and binding through spontaneous chemical bond of Gv-Sd to obtain influenza virus antigen multimeric complex (HA-3M 2e-NP protein), namely the nanoparticle through self-assembly.
The prepared Sd-Ferritin protein, HA-3M2e protein and HA-3M2e-NP protein are detected by SDS-PAGE, the purity is verified by Coomassie brilliant blue staining, and an SDS-PAGE picture of the Sd-Ferritin protein, the HA-3M2e protein and the HA-3M2e-NP protein is shown in figure 3. As can be seen from FIG. 3, the Sd-Ferritin protein, the HA-3M2e influenza virus antigen and the HA-3M2e-NP protein were successfully prepared according to the present invention, and the sizes of the proteins were consistent with those expected.
Example 2 characterization of HA-3M2e-NP nanoparticles
The invention verifies the purity and uniformity of HA-3M2e-NP protein by using a molecular Sieve (SEC) and a Transmission Electron Microscope (TEM). The HA-3M2e-NP protein (nanoparticle) prepared in example 1 was separated and collected by SEC, and then concentrated. The molecular sieve diagram of the purified HA-3M2e-NP protein is shown in FIG. 4, and as can be seen from FIG. 4, the peak of the obtained HA-3M2e-NP protein is single, which indicates that the HA-3M2e-NP protein with the purity higher than 99% is obtained by the invention.
In addition, the HA-3M2e-NP protein obtained by molecular sieve separation is observed through a Transmission Electron Microscope (TEM), the Transmission Electron Microscope (TEM) image of the HA-3M2e-NP protein is shown in figure 5, and as can be known from figure 5, after the influenza virus antigen is covalently combined with the Ferritin nano-particles, the HA-3M2e-NP nano-particles show spines protruding from spherical cores, which indicates that candidate antigens are successfully enriched on the surfaces of the nano-particles through the display strategy of displaying the antigens on the surfaces of the Ferritin nano-particles by the method disclosed by the invention, and the method can be used for constructing nano-particle vaccines.
Example 3 immunoprotection assay
The influenza virus antigen polyplex (HA-3M 2e-NP protein) prepared in example 1 and cyclic diguanylic acid (c-di-GMP) adjuvant are prepared (the mass ratio of the influenza virus antigen polyplex to the cyclic diguanylic acid adjuvant is 5).
BALB/c mice were immunized by subcutaneous (s.c.) and respiratory (i.n.) vaccination, respectively, at a dose of 10 μ g per mouse, the immunization strategy being shown in fig. 6. As can be seen in fig. 6, all mice were immunized using the strategy of double-needle immunization, i.e., at weeks 0 and 4; two weeks after immunization with the vaccine, serum from the mice was collected and the titer of the antigen-specific IgG antibody in the serum was determined.
The results of measuring the titer of antigen-specific IgG antibodies in mouse serum 6 weeks after subcutaneous (s.c.) and respiratory (i.n.) nanoparticle vaccines are shown in FIG. 7. From FIG. 7, it can be seen that the HA-3M2e-NP nanoparticle vaccine induced higher titer antigen-specific IgG antibody production compared to the control group, where the titer of subcutaneous vaccination was close to 10 6 And the titer of the respiratory inoculation is higher than 10 4 。
To further explore the in vivo protection ability of the HA-3M2e-NP nanoparticle vaccine prepared by the method against different subtypes of influenza A virus, BALB/c mice immunized with the HA-3M2e-NP nanoparticle vaccine respectively by subcutaneous inoculation (s.c.) and respiratory inoculation (i.n.) in example 2 were transferred to a biosafety secondary laboratory, live virus challenge experiments were performed with H1N1 and H3N2 respectively (mice were anesthetized with isoflurane, and 1.2 × 10 3 TCID50 virus infected mice by nasal drip) and mice were monitored for weight change and survival within two weeks after live virus challenge. Body weight change curve and survival curve of mice infected with different subtype influenza A viruses after HA-3M2e-NP nanoparticle vaccine immunizationAs shown in FIG. 8, it can be seen from FIG. 8 that the mice inoculated with the HA-3M2e-NP nanoparticle vaccine via respiratory system showed only slight weight loss and no death, regardless of the infection with H1N1 or H3N2 viral strains, indicating that the HA-3M2e-NP nanoparticle vaccine via respiratory system could protect the mice from different subtypes of influenza A virus.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. An influenza antigen which is a fusion protein formed by replacing the head domain of hemagglutinin with the ectodomain of three repeats of influenza virus matrix protein 2.
2. The antigen of claim 1, wherein the antigen has the amino acid sequence set forth in SEQ ID No. 1.
3. The antigen of claim 1 or 2, wherein the antigen has a foldon motif attached to the C-terminus.
4. An influenza virus antigen polyplex, characterized in that the antigen polyplex is obtained by fusion expression of the antigen of claim 3 and Gv shown in SEQ ID No.3 to obtain a fusion protein, and then the obtained fusion protein is linked to Sd-Ferritin protein shown in SEQ ID No. 4.
5. Use of an antigen according to any one of claims 1 to 3 or an antigen polyplex according to claim 4 for the preparation of a medicament against an influenza virus.
6. The use of claim 5, wherein the medicament is an influenza virus universal vaccine.
7. A universal nanoparticle vaccine for influenza virus, wherein the vaccine is prepared by using the antigen polyplex of claim 4 as an antigen.
8. The method for preparing the universal nanoparticle influenza vaccine of claim 7, comprising the steps of:
s1, expressing the fusion protein of the antigen of claim 3 and Gv shown in SEQ ID NO.3 in a eukaryotic expression system and purifying;
s2, expressing the Sd-Ferritin protein shown in SEQ ID No.4 in a prokaryotic expression system and purifying;
s3, incubating the fusion protein obtained in the step S1 and the Sd-Ferritin protein obtained in the step S2 in a buffer solution without enzyme to obtain an influenza virus antigen multimeric complex;
and S4, preparing the influenza virus antigen polymer compound obtained in the step S3 and an adjuvant to obtain the influenza virus universal nanoparticle vaccine.
9. The method of claim 8, wherein the adjuvant is a cyclic diguanylate adjuvant.
10. The method of claim 9, wherein the mass ratio of the influenza virus antigen polyplex to the cyclodiguanylic acid adjuvant is 5:1 to 2.
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