CN116444684A - Influenza virus vaccine and application thereof - Google Patents
Influenza virus vaccine and application thereof Download PDFInfo
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- CN116444684A CN116444684A CN202310378027.3A CN202310378027A CN116444684A CN 116444684 A CN116444684 A CN 116444684A CN 202310378027 A CN202310378027 A CN 202310378027A CN 116444684 A CN116444684 A CN 116444684A
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- A61P31/12—Antivirals
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- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The invention relates to the field of medicines, and particularly discloses an influenza virus vaccine fusion Protein, which is formed by connecting an HA antigen fragment coded by an influenza HA antigen gene and an M2 extracellular region fragment (M2 e; membrane Protein (M2) Extracellular Region (M2 e) in series and then connecting an Fc fragment coded by a human immunoglobulin gene in series.
Description
Technical Field
The invention relates to the field of medicines, in particular to an influenza virus vaccine and application thereof.
Background
Influenza, abbreviated influenza, is an acute respiratory infectious disease caused by influenza virus, is a seasonal epidemic, and usually enters into the season of epidemic in autumn every year. The peak of the current is reached in winter and spring.
Influenza virus belongs to orthomyxoviridae, is a single-stranded and negative-strand RNA genome virus, and is divided into four types of A (A), B (B), C (C) and D (D) according to different virus nucleoprotein and matrix protein, and can be divided into different subtypes (H subtype and N subtype) according to different Hemagglutinin (HA) and Neuraminidase (NA) in an envelope. The virus A frequently generates antigen variation, has large infectivity and quick transmission, and is a main virus for causing seasonal or pandemic influenza. Currently, the main infections in humans are the Victoria and Yamagata lines in the H1N1, H3N2 subtypes of type A (also known as type A) and in type B (also known as type B). Influenza virus mutation occurs rapidly and new epidemic strains occur every year, and the mutation is mainly carried out in two ways: antigen drift and antigen shift. Antigen drift is a small change caused by a small number of point mutations, which occur in both influenza a and b, and which can keep the virus away from the recognition of the immune system, causing the infected person to become infected again, causing recurrent outbreaks of influenza. Antigen transformation is caused by genetic reassortment of different HA antigens, which can give rise to new subtypes of viruses, and can directly infect humans across species, such as avian influenza, which occurs mainly in influenza a.
Three proteins are present on the surface of influenza virus envelope: HA (hemagglutinin protein), NA (sialidase) and M2 (channel protein). HA is the most abundant influenza surface envelope protein, the major component of viral adhesion receptor cells, and the major antigen for the induction of neutralizing antibody production. The presently found HA subtypes are 18 in number, each containing many different strains, keeping HA similar to the recently prevalent strains is the basis for vaccine production of highly neutralizing antibodies. Therefore, in recent years, the world-guard organization predicts, publishes and distributes main epidemic strains annually, and provides the main epidemic strains for various national disease control departments, so that the vaccine production enterprises can conveniently apply the main epidemic strains. M2 is a third protein except HA and NA on the surface of influenza virus envelope, the molecular weight is small, the outer membrane region of each M2 protein is only 23 amino acids, the selection pressure is small, the variable range is small, and the variation degree is low. In all M2 proteins recorded in NCBI in 2019, only 10 kinds of the proteins are distinguished according to extracellular region sequences, the sequences have high conservation compared with thousands of HA, and experiments prove that the sequences have humoral immunity and cellular immunity activities, so that the sequences are one of antigen choices for developing general vaccines with wide prevention effects.
Influenza viruses are mainly transmitted through droplets, and mucous membranes such as oral cavity, nasal cavity, eyes and the like can be directly or indirectly contacted with objects polluted by viruses to be infected. The influenza virus is generally susceptible to the population, and can directly invade the respiratory system of a human body to cause various serious complications which threaten life, such as viral pneumonia, secondary bacterial pneumonia, acute respiratory distress syndrome, shock, disseminated intravascular coagulation and the like. Severe influenza mainly occurs in high risk groups such as the elderly, young children, obese people, pregnant and lying-in women, and patients with chronic underlying diseases, and also in general groups.
The influenza vaccine can effectively prevent influenza virus infection of corresponding subtype/line, and reduce the risk of serious complications; antibodies with protective levels can be produced 2-4 weeks after influenza vaccination, and antibody titers begin to decay after 6-8 months.
Influenza virus vaccines are classified into two major classes, i.e., inactivated vaccine and attenuated live vaccine, depending on the vaccine species, and there are trivalent (H1N 1, H3N2, B/Victoria) and tetravalent (H1N 1, H3N2, B/Victoria, B/Yamagata) influenza virus vaccines depending on the valence number.
At present, influenza vaccine varieties which are issued in batches in China mainly comprise three product types of tetravalent inactivated/split vaccines, trivalent inactivated/split vaccines and trivalent attenuated live vaccines. The vaccine is subjected to virus inactivation, and then the antigen with HA as a main antigen is extracted as the vaccine, so that the problems of limited yield, less beneficiary population, low immunogenicity and the like exist, the acceptance and popularization of the influenza vaccine are affected, and therefore, the development of the efficient broad-spectrum recombinant vaccine is an important research and development direction of the influenza vaccine.
Disclosure of Invention
The present inventors have found through studies that HA-Fc HAs an effective neutralizing antibody induction ability as an antigen, and that multiple copies of M2 extracellular region (M2 e) in tandem as an antigen can induce humoral immunity and cellular immunity having a broad spectrum of action properties. The HA antigen, the M2 antigen and the Fc tag with synergistic effect are expressed together in series, so that the immune effect of three elements can be utilized simultaneously. Wherein, under the condition that the T/B cell epitope exists on the M2, the neutralizing antibody can be effectively induced to generate. However, the ability of the short peptide epitope to induce antibodies is generally low, the neutralizing antibody titer is low, and in practice, the short peptide epitope needs to be linked to a carrier to induce antibody production effectively. After the M2 extracellular region (M2 e) epitope is connected with HA, the HA can play a role of HA serving as an antigen and a role of M2 carrier, so that the induction capability of M2 serving as the antigen is improved. The sequence conservation of M2 is limited, and a plurality of different M2 can cover more strains in series, so that the insensitivity to influenza strain variation is improved.
Therefore, the invention provides an influenza virus vaccine fusion protein, which is formed by connecting an HA antigen segment coded by an influenza HA antigen gene and an M2 extracellular region segment in series and then connecting an Fc segment coded by a human immunoglobulin gene in series.
Preferably, the C-terminus of the HA antigen fragment is linked to an M2 extracellular region fragment, and the C-terminus of the M2 extracellular region fragment is linked to an Fc fragment.
More specifically, the influenza HA antigen gene is selected from influenza a subtypes H1N1, H3N2, and human pathogenic influenza strains of the victoria and Yamagata lines.
More preferably, the HA antigen gene is derived from the HA gene of an influenza strain, e.g., strain A/Beijing/24/2019 (H1N 1) corresponds to GenBank accession number MN853429.1; preferably, the point mutant R344T encoded protein with the 1 st to 2012 th base sequence is selected, for example, the amino acid sequence of which is shown in SEQ ID NO: 1.
Further preferably, an M2 extracellular region fragment refers to the N-terminal 2-23 amino acid sequence of M2 thereof, preferably 2-6M 2 extracellular region fragments, e.g. 2, 3, 4, 5 or 6; preferably, the 2-6M 2 extracellular region fragments are derived from different or identical M2, e.g. from QID40717, QFR17397, QFR17408, QFR17284, QDW80887, respectively, and in one example the M2 extracellular region fragments have the amino acid sequence as set forth in SEQ ID NO: 2.
In one embodiment, there may or may not be a linker segment between the HA antigen fragment, the M2 extracellular region fragment, and the Fc fragment.
Further preferably, the Fc fragment is derived from human IgG1, and in one example the Fc fragment has the amino acid sequence set forth in SEQ ID NO: 3.
The invention provides nucleic acids, expression vectors and recombinant host cells encoding said influenza virus vaccine fusion proteins, preferably said host cells are HEK293FT cells, MDCK cells, VERO cells, CHO-K1Q cells, HEK293FT cells or other human cells.
The invention also provides an influenza vaccine taking the influenza virus vaccine fusion protein as an active ingredient, and optionally, the influenza vaccine further comprises an adjuvant or pharmaceutically acceptable auxiliary materials.
The invention further provides application of the influenza virus vaccine fusion protein or the influenza vaccine in preparation of medicines for preventing or treating influenza.
The invention has the beneficial effects that: HA is a membrane protein which binds to host receptors, and part of antibodies induced by HA can prevent viruses from binding to the receptors after binding to HA, thus preventing virus invasion, and being effective antigens for inducing neutralizing antibodies. The sequence conservation of M2 endows the immune response induced by the antigen with broad-spectrum virus-binding effect, but the sequence of M2 is short, the neutralizing antibody has weak induction capability, and the effect of the single M2 antigen is limited. The protein antigen combines effective key neutralizing antibody immunogen HA and broad-spectrum immunogen M2e, and can prevent the infection of main epidemic strains and secondary epidemic strains. The tandem M2 in the fusion protein can come from different strains, and the range of the strains can be wider. The fusion protein of the invention HAs higher antigenicity than the HA antigen alone and the M2 antigen alone. Further, the Fc fragment of the present invention can enhance the purification efficiency of the protein and prolong the in vivo half-life of the antigen.
Drawings
FIG. 1, protein HA-M2E-Fc, HA-Fc electrophoresis detection. Wherein M is the protein molecular weight maker,1, 2: HA-M2Ex5-Fc,3, 4: HA-Fc.
FIG. 2, three six week old mice serum binding antibody Elisa assay.
Detailed Description
The following examples and drawings of the present application are merely illustrative of specific embodiments for carrying out the invention and are not to be construed as limiting the invention, as any changes may be made without departing from the principles and spirit of the invention and are within the scope of the invention. The experimental techniques and methods used in the examples of the present application are conventional techniques unless otherwise specified. Materials, reagents, and the like used in the examples of the present application are available from regular commercial sources unless otherwise specified.
Example 1: construction of human influenza HA-M2e-Fc and HA-Fc gene vector
1. The HA gene from influenza A/Beijing/24/2019 (H1N 1) is numbered GenBank: MN853429.1, and the point mutation type R344T of 1-2012 base sequence is selected (the point is the point at which HA0 breaks into HA1 and HA2, the mutation can keep the protein in the HA0 state, namely the pre-fusion conformation, so that the generated antibody can be more aimed at the pre-fusion conformation, or the conformation before the virus does not enter cells, the pre-fusion conformation is favorable for inducing and neutralizing the antibody, and the amino acid sequence of the antibody is shown as SEQ ID NO: 1). N-terminal connection sequence: atgtacaggatgcagctgctctcttgcatcgccctgagtctCgcCcttgtgaccaacagcTACGGCAGCAAGAAAAGGAGACAGAGAAGGCGG the C-terminal is used as signal peptide, the N-terminal 2-23 amino acid sequences of 5M 2 genes are sequentially connected or not connected in series (the amino acid sequence of the M2 extracellular region fragment is shown as SEQ ID NO:2 in the embodiment), and the C-terminal of M2x5 is connected with the Fc fragment of human IgG1 (the amino acid sequence of the Fc fragment is shown as SEQ ID NO:3 in the embodiment). The fusion protein is synthesized and constructed by a gene company at the HindIII-XbaI site of an expression vector pKS001, and is identified by sequencing.
2. PCR cloning is performed by using a primer pair H5SPF/H5SPR in a PCR cloning mode, and the PCR cloning is concretely performed as follows:
H5SPF：gTTTGTGGTGGCcGCcGCTACcGGcGTgCAGTCCGACACATTATGTATAGGTTATCATG
H5SPR：ggcggccaccacaaacaggaacctccaggtccagtccatGGTGGCGGCAAGCTT。
taking the carrier as a template, and adopting a PCR reaction system: 10 mu M H SPF 0.5ul, 10 mu M H SPR 0.5ul, 5x Phusion buffer 4ul, dNTP 1ul, phusion polymerase 1ul and ultra pure water 12.7ul.
The PCR procedure was used as follows:
| step (a) | Program setting |
| 1 | 94℃，4min |
| 2 | 94℃，45sec,45℃,45sec,72℃,5min 30sec,3cycles |
| 3 | 94℃，45sec,65℃,45sec,72℃,5min 30sec,26cycles |
| 4 | 72℃，10min |
After the PCR is finished, adding 1ul of Dpn I enzyme into the reaction product, digesting for 4hrs at 37 ℃, taking 10ul of the product, transforming TOP10 escherichia coli competent cells by adopting a conventional method, screening ampicillin resistant strain clones, carrying out sequencing identification, obtaining an expression vector clone strain with an N-terminal sequence of H5 antibody signal peptide sequence (ATGGA CTGGA CCTGG AGGTT CCTGT TTGTG GTGGC CGCCG CTACC GGCGT GCAGT CC), and extracting plasmids.
Example 2: preparation of HA-M2x5-Fc and HA-Fc fusion proteins by CHO expression System
Cell culture: CHO-K1Q cells were cultured with CD02 medium (available from Sanchen, kanga, zhongshan) supplemented with 4mM Gln at 37℃with 5% CO 2 Shaking table suspension culture at 110 rpm. The cell culture activity reaches more than 98 percent, and the density is 2.0 multiplied by 10 6 The above procedure was used for transfection.
Plasmid big extraction: coli engineering bacteria containing the desired expression vector described in example 1 were cultured on a shake flask basis using LB culture containing ampicillin, plasmids were extracted using endotoxin-free plasmid large extraction kit (purchased from tiangen biochemistry), and plasmids were concentrated by ethanol precipitation.
The plasmid is transformed into CHO cells by an electric shock transformation method, the CHO cells are cultured for 5 days, the culture supernatant is purified by a protein A affinity column, and high-purity target proteins HA-M2Ex5-Fc and HA-Fc are obtained by electrophoresis detection, and the results are shown in figure 1.
Example 3: immunization of mice, preparation of serum, detection of bound antibodies
After mixing the objective protein HA-M2Ex5-Fc prepared in example 2 with a homemade adjuvant (homemade adjuvant, see CN 202110995286.1), BALB/c mice were immunized, female, for 6-8 weeks, and purchased from St Bei Fu (Beijing) Biotechnology Co., ltd. The immunization groups are as follows in table 1:
table 1: group test of mouse immunity dose
Immunization and blood collection procedure: immunization was performed at weeks 0, 3, and 6, with intramuscular injection, 0.1 mL/needle. 3. Blood was collected for 6, 9, 12 and 15 weeks to prepare serum.
Antisera binding potency assay: the fusion proteins HA-M2Ex5-Fc and HA-Fc prepared in example 2 were prepared in a solution of 62.5ng/ml, a section of M2E synthetic short peptide (QDW 80887N-terminal 2-23 amino acids) was prepared in a solution of 1ug/ml, and each solution was added to an ELISA plate of 100 ul/well and coated overnight at 4 ℃. Adding 1% BSA after PBST washing, blocking with PBST solution, blocking at 37 ℃ for 2 hours, discarding blocking solution, adding 100ul of antiserum diluted by gradient of blocking solution, setting two repetitions for each sample, incubating at 37 ℃ for 1.5 hours, adding HRP-labeled goat anti-mouse secondary antibody after PBST washing, incubating at 37 ℃ for 1 hour, adding color development solution for three times after PBST washing, and carrying out light-proof reaction for 15 minutes, and adding 50ul of stop solution to stop the reaction. The enzyme label instrument reads the absorbance at 450nm to be more than 0.1 of the negative sample as the dilution endpoint. The mean value of the blank control was subtracted with EXEL and calculated to give the immunobinding titers. The results are shown in FIG. 2.
As can be seen from the results, the 2ug antigen HA-M2E-Fc fusion protein is close to the upper limit of immunity, and three immunizations can induce the antigen HA-M2E-Fc fusion protein to reach 10 6 Serum antibodies of the above magnitude indicate that the fusion protein has very high immunity induction capability. Specific binding to the antibody was detected with both HA-Fc and an M2e synthetic polypeptide, indicating the ability of each component of the antigen to induce specific antibodies. HA-Fc exhibited a high immune antibody binding capacity but was lower than that of HA-M2E-Fc, showing the ability of the part deleted by deletion of the M2E part antigen to induce antibodies. The singly coated M2Ex1 HAs the capability of binding to the antibody, and the 5-fold effect can fill the difference between the results of HA-M2Ex5-Fc and HA-Fc to a certain extent.
Claims (10)
1. An influenza virus vaccine fusion protein is a fusion protein formed by connecting an HA antigen segment coded by an influenza HA antigen gene and an M2 extracellular region segment in series and then connecting an Fc segment coded by a human immunoglobulin gene in series.
2. The influenza virus vaccine fusion protein of claim 1, wherein the HA antigen fragment is C-terminally linked to the M2 extracellular region fragment and the M2 extracellular region fragment is C-terminally linked to the Fc fragment.
3. The influenza virus vaccine fusion protein of claim 1, wherein the influenza HA antigen gene is selected from influenza a subtypes H1N1, H3N2, and human pathogenic influenza strains of Victoria and Yamagata lines.
4. The influenza virus vaccine fusion protein of claim 1, wherein the HA antigen gene is derived from the HA gene of the influenza strain, e.g. strain a/beijin/24/2019 (H1N 1) corresponds to GenBank number MN853429.1; preferably, the point mutant R344T encoded protein with the 1 st to 2012 th base sequence is selected, for example, the amino acid sequence of which is shown in SEQ ID NO: 1.
5. An influenza virus vaccine fusion protein according to claim 1, wherein the M2 extracellular region fragment refers to the N-terminal 2-23 amino acid sequence of M2, preferably 2-6M 2 extracellular region fragments, such as 2, 3, 4, 5 or 6; preferably, the 2-6M 2 extracellular region fragments are derived from different or identical M2, e.g. from QID40717, QFR17397, QFR17408, QFR17284, QDW80887, respectively, and in one example the M2 extracellular region fragments have the amino acid sequence as set forth in SEQ ID NO: 2.
6. The influenza virus vaccine fusion protein of claim 1, wherein there may or may not be a linker segment between the HA antigen segment, the M2 extracellular region segment, and the Fc segment.
7. Influenza virus vaccine fusion protein according to claim 1, characterized in that the Fc fragment is derived from human IgG1, preferably the NCBI data thereof is encoding 4CDH a.
8. Nucleic acid encoding the influenza virus vaccine fusion protein according to any one of claims 1 to 7, expression vector and recombinant host cell, preferably said host cell is a HEK293FT cell, MDCK cell, VERO cell, CHO-K1Q cell or a human cell.
9. The influenza vaccine of any one of claims 1 to 7, optionally further comprising an adjuvant or pharmaceutically acceptable adjuvant.
10. Use of an influenza virus vaccine fusion protein according to any one of claims 1 to 7 or an influenza vaccine according to claim 9 in the manufacture of a medicament for the prophylaxis or treatment of influenza.
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