A kind of influenza virus vaccine and its application

Abstract

The present invention relates to the field of medicine, and specifically discloses an influenza virus vaccine fusion protein, which is a fusion protein formed by connecting an HA antigen fragment encoded by an influenza HA antigen gene and an M2 extracellular region fragment (M2e; Membrane Protein (M2) Extracellular Region (M2e) in series with an Fc fragment encoded by a human immunoglobulin gene in series. The fusion protein of the present invention has higher antigenicity than a single HA antigen or a single M2 extracellular region fragment antigen, and the Fc fragment can improve protein purification efficiency and prolong the half-life of the antigen in vivo. Therefore, the influenza vaccine provided by the present invention has great advantages and great application value.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to influenza virus vaccines and applications thereof. Background Art

[0002] Influenza, or flu for short, is an acute respiratory infectious disease caused by influenza virus. It is seasonal and usually enters the epidemic season in autumn every year. The epidemic peaks in winter and spring.

[0003] Influenza virus belongs to the family Orthomyxoviridae. It is a single-stranded, negative-strand RNA genome virus. According to the difference in viral nucleoprotein and matrix protein, it is divided into four types: A, B, C, and D. According to the difference in hemagglutinin (HA) and neuraminidase (NA) in its envelope, it can be divided into different subtypes (H subtype and N subtype). Type A virus often undergoes antigenic variation, is highly contagious, and spreads rapidly. It is the main virus that causes seasonal or pandemic influenza. Currently, the main viruses that infect humans are the H1N1 and H3N2 subtypes of type A (also known as type A) and the Victoria and Yamagata strains of type B (also known as type B). The frequency of influenza virus variation is fast, and new epidemic strains occur every year. Its variation mainly occurs in two ways: antigenic drift and antigenic shift. Antigenic drift is a small change caused by a small number of point mutations. It occurs in both type A and type B influenza. It can enable the virus to avoid recognition by the immune system, causing people who have been infected to become infected again and causing repeated influenza outbreaks. Antigenic shift is caused by genetic recombination of different HA antigens, which can produce new subtypes of the virus and can cross species to directly infect humans, such as avian influenza, which mainly occurs in influenza A.

[0004] There are three proteins on the surface of the influenza virus envelope: HA (hemagglutinin protein), NA (sialidase) and M2 (channel protein). HA is the most abundant influenza surface envelope protein, the main component of the virus adhesion receptor cells, and the main antigen that induces the production of neutralizing antibodies. There are 18 HA subtypes discovered so far, each of which contains many different strains. Keeping HA similar to the recent epidemic strains is the basis for the vaccine to produce highly effective neutralizing antibodies. Therefore, in recent years, the WHO predicts, publishes and distributes the main epidemic strains every year, and supplies them to the disease control departments of various countries to facilitate the application of vaccine manufacturers. M2 is the third protein on the surface of the influenza virus envelope besides HA and NA. It has a small molecular weight, and each M2 protein has only 23 amino acids in the extramembrane region. It has low selection pressure, a small variable range, and a low degree of variation. Among all the M2 proteins included in NCBI since 2019, there are only more than 10 types distinguished by the extracellular region sequence. Compared with the thousands of HAs, it is highly conserved. Experiments have shown that this sequence has humoral and cellular immune activity, and is one of the antigen options for developing a universal vaccine with a broad preventive effect.

[0005] Influenza viruses are mainly spread through droplets, and can also be transmitted through direct or indirect contact of mucous membranes such as the mouth, nose, and eyes with virus-contaminated objects. People are generally susceptible to influenza viruses, which directly invade the human respiratory system and can cause viral pneumonia, secondary bacterial pneumonia, acute respiratory distress syndrome, shock, disseminated intravascular coagulation and other serious life-threatening complications. Severe influenza mainly occurs in high-risk groups such as the elderly, young children, obese people, pregnant women and those with chronic underlying diseases, but can also occur in the general population.

[0006] Influenza vaccination can effectively prevent influenza virus infection of the corresponding subtype / lineage and reduce the risk of serious complications. Usually, protective levels of antibodies can be produced 2-4 weeks after influenza vaccination, and the antibody titer begins to decline after 6-8 months.

[0007] According to the type of vaccine, influenza virus vaccines are divided into two categories: inactivated vaccines and live attenuated vaccines. According to the valence, influenza virus vaccines are divided into trivalent (H1N1, H3N2, B / Victoria) and quadrivalent (H1N1, H3N2, B / Victoria, B / Yamagata).

[0008] At present, the influenza vaccines approved by my country are mainly quadrivalent inactivated / split vaccines, trivalent inactivated / split vaccines and trivalent live attenuated vaccines. These vaccines are mainly HA-based antigens extracted after virus inactivation. They have problems such as limited production, small number of beneficiaries and low immunogenicity, which affect the acceptance and promotion of influenza vaccines. Therefore, the development of highly efficient and broad-spectrum recombinant vaccines is an important research and development direction for influenza vaccines. Summary of the invention

[0009] The inventors have found through research that HA-Fc has an effective ability to induce neutralizing antibodies as an antigen, and multiple copies of the tandem M2 extracellular region (M2e) as an antigen can induce humoral immunity and cellular immunity with broad-spectrum properties. The HA antigen, the M2 antigen, and the Fc tag with a synergistic effect are expressed in series, and the immune effects of the three elements can be utilized at the same time. Among them, when there are T / B cell epitopes on M2, neutralizing antibodies can be effectively induced. However, its antibody induction ability as a short peptide epitope itself is usually low, and the neutralizing antibody titer is low. In practice, short peptides need to be connected to a carrier as an antigen to effectively induce antibody production. After the M2 extracellular region (M2e) epitope is connected to HA, HA can play the role of HA as an antigen and as an M2 carrier, thereby improving the induction ability of M2 as an antigen. The sequence conservation of M2 is limited, and the series connection of multiple different M2s can cover more strains and improve the insensitivity to influenza strain mutations.

[0010] Therefore, the present invention provides an influenza virus vaccine fusion protein, which is a fusion protein formed by connecting an HA antigen fragment encoded by an influenza HA antigen gene and an M2 extracellular region fragment in series and then connecting an Fc fragment encoded by a human immunoglobulin gene in series.

[0011] Preferably, the C-terminus of the HA antigen fragment is connected to the M2 extracellular region fragment, and the C-terminus of the M2 extracellular region fragment is further connected to the Fc fragment.

[0012] More specifically, the influenza HA antigen gene is selected from influenza A subtypes H1N1 and H3N2, and influenza B victoria and Yamagata strains that cause human disease.

[0013] More preferably, the HA antigen gene is derived from the HA gene of an influenza strain, for example, strain A / Beijing / 24 / 2019 (H1N1) corresponds to GenBank No.: MN853429.1; preferably, the point mutation R344T encoding protein of its 1st-2012 base sequence is selected, for example, its amino acid sequence is shown in SEQ ID NO: 1.

[0014] Further preferably, the M2 extracellular region fragment refers to the N-terminal 2-23 amino acid sequence of M2, preferably 2-6 M2 extracellular region fragments are connected in series, for example 2, 3, 4, 5 or 6; preferably, the 2-6 M2 extracellular region fragments are derived from different or the same M2, for example, respectively derived from QID40717, QFR17397, QFR17408, QFR17284, QDW80887, and the amino acid sequence of one example of the M2 extracellular region fragment is shown in SEQ ID NO: 2.

[0015] In a specific embodiment, there may or may not be a connecting fragment between the HA antigen fragment, the M2 extracellular region fragment, and the Fc fragment.

[0016] In addition, preferably, the Fc fragment is derived from human IgG1. In one example of the Fc fragment, its amino acid sequence is shown in SEQ ID NO:3.

[0017] The present invention provides nucleic acid encoding the influenza virus vaccine fusion protein, expression vector and recombinant host cell. Preferably, the host cell is HEK293FT cell, MDCK cell, VERO cell, CHO-K1Q cell, HEK293FT cell or other human cell.

[0018] The present invention also provides an influenza vaccine with the influenza virus vaccine fusion protein as an active ingredient, which optionally further comprises an adjuvant or a pharmaceutically acceptable auxiliary material.

[0019] The present invention further provides use of the influenza virus vaccine fusion protein or the influenza vaccine in preparing a medicament for preventing or treating influenza.

[0020] Beneficial effects of the present invention: HA is a membrane protein that binds to host receptors. Some of the antibodies induced by it can prevent viruses from binding to receptors after binding to HA, play a role in preventing virus invasion, and are effective antigens for inducing neutralizing antibodies. The sequence conservation of M2 gives it the ability to induce a broad-spectrum binding effect on viruses, but the sequence of M2 is short and the ability to induce neutralizing antibodies is weak, so the effectiveness of the M2 antigen alone is limited. The protein antigen of the present invention combines the effective key neutralizing antibody immunogen HA and the broad-spectrum immunogen M2e, which can prevent infection by major and minor epidemic strains. The tandem M2 in the fusion protein of the present invention can come from different strains, and a wider range of strains can be taken into account. The fusion protein of the present invention has higher antigenicity than a single HA antigen and a single M2 antigen. Furthermore, the Fc fragment in the present invention can improve the efficiency of protein purification and prolong the half-life of the antigen in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 , protein HA-M2E-Fc, HA-Fc electrophoresis detection. Wherein, M: protein molecular weight maker, 1, 2: HA-M2Ex5-Fc, 3, 4: HA-Fc.

[0022] Figure 2 , Elisa detection of serum binding antibodies in mice vaccinated three times for six weeks. DETAILED DESCRIPTION

[0023] The following examples and drawings of the present application only illustrate specific implementation schemes for implementing the present invention. These schemes and drawings should not be construed as limitations on the present invention. Any changes made without departing from the principles and essence of the present invention fall within the scope of protection of the present invention. The experimental techniques and experimental methods used in the examples of the present application are conventional technical methods unless otherwise specified. The materials, reagents, etc. used in the examples of the present application, unless otherwise specified, can be obtained through regular commercial channels.

[0024] Example 1: Construction of human influenza HA-M2e-Fc and HA-Fc gene vectors

[0025] 1. The HA gene derived from the influenza strain A / Beijing / 24 / 2019 (H1N1), numbered GenBank: MN853429.1, selected the point mutation R344T of the 1-2012 base sequence (this site is the site where HA0 breaks into HA1 and HA2. The mutation will keep the protein in the HA0 state, that is, maintain the pre-fusion conformation, so that the antibodies produced can be more targeted at the pre-fusion conformation, or the conformation before the virus enters the cell. The pre-fusion conformation is conducive to inducing neutralizing antibodies, and its amino acid sequence is shown in SEQ ID NO: 1). The N-terminal connection sequence: atgtacaggatgcagctgctctcttgcatcgccctgagtctCgcCcttgtgaccaacagcTACGGCAGCAAGAAAAGGAGACAGAGAAGGCGG is used as a signal peptide, the C-terminal is sequentially connected or not connected to the N-terminal 2-23 amino acid sequences of 5 M2 genes in series (the amino acid sequence of the M2 extracellular region fragment used in this example is shown in SEQ ID NO: 2), and the C-terminal of M2x5 is connected to the Fc fragment of human IgG1 (the amino acid sequence of the Fc fragment used in this example is shown in SEQ ID NO: 3). The fusion protein was synthesized by Gene Company and constructed at the HindIII-XbaI site of the expression vector pKS001, and it was confirmed by sequencing.

[0026] 2. Use PCR cloning method and primer pair H5SPF / H5SPR for PCR cloning, as follows:

[0027] H5SPF: gTTTGGTGGTGGCcGCcGCTACcGGcGTgCAGTCCGACACATTATGTATAGGTTATCATG

[0028] H5SPR: ggcggccaccacaaacaggaacctccaggtccagtccatGGTGGCGGCAAGCTT.

[0029] Using the above vector as a template, a PCR reaction system was used: 10 μM H5SPF 0.5ul, 10 μM H5SPR 0.5ul, 5xPhusion buffer 4ul, dNTP 1ul, Phusion polymerase 1ul and ultrapure water 12.7ul.

[0030] The PCR program used was as follows:

[0031] step Program Settings 1 94℃,4min 2 94℃,45sec,45℃,45sec,72℃,5min 30sec,3cycles 3 94℃,45sec,65℃,45sec,72℃,5min 30sec,26cycles 4 72℃,10min

[0032] After PCR, 1 ul of Dpn I enzyme was added to the reaction product and digested at 37°C for 4 hrs. 10 ul of the product was taken and transformed into TOP10 Escherichia coli competent cells by conventional methods. Ampicillin-resistant strain clones were screened for sequencing and identification, and an expression vector cloning strain with an N-terminal sequence of the H5 antibody signal peptide sequence (ATGGA CTGGA CCTGG AGGTT CCTGT TTGTG GTGGC CGCCG CTACCGGCGT GCAGT CC) was obtained, and the plasmid was extracted.

[0033] Example 2: Preparation of HA-M2x5-Fc and HA-Fc fusion proteins using the CHO expression system

[0034] Cell culture: CHO-K1Q cells were cultured in CD02 medium (purchased from Zhongshan Kangtianshenghe) supplemented with 4 mM Gln at 37°C and 5% CO. 2 , shaker suspension culture at 110 rpm. Cell culture viability reached over 98% and density was 2.0×10 6 The above is used for transfection.

[0035] Plasmid extraction: The E. coli engineered bacteria containing the target expression vector described in Example 1 were cultured in a shake flask using LB containing ampicillin, and the plasmid was extracted using an endotoxin-free plasmid extraction kit (purchased from Tiangen Biochemical), and the plasmid was concentrated by ethanol precipitation.

[0036] The plasmid was transformed into CHO cells by electroporation and cultured for 5 days. The culture supernatant was purified by protein A affinity column and high-purity target proteins HA-M2Ex5-Fc and HA-Fc were obtained by electrophoresis. The results are shown in Figure 1 .

[0037] Example 3: Immunization of mice, preparation of serum, and detection of binding antibodies

[0038] The target protein HA-M2Ex5-Fc prepared in Example 2 was mixed with a homemade adjuvant (see CN202110995286.1 for homemade adjuvant) and immunized with female BALB / c mice, 6-8 weeks old, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. The immunization groups are shown in Table 1 below:

[0039] Table 1: Mouse immunization dose grouping test

[0040]

[0041] Immunization and blood collection procedures: Immunization was performed at 0, 3, and 6 weeks. In this experiment, intramuscular injection was used, 0.1 mL / injection. Blood was collected at 3, 6, 9, 12, and 15 weeks to prepare serum.

[0042] Antiserum binding titer detection: The fusion proteins HA-M2Ex5-Fc and HA-Fc prepared in Example 2 were prepared into a solution of 62.5ng / ml, and a solution of a synthetic short peptide of M2E (2-23 amino acids at the N-terminus of QDW80887) was prepared into a solution of 1ug / ml, and added to 100ul / well of the ELISA plate respectively, and coated at 4°C overnight. After washing the plate with PBST, 1% BSA was added, and the PBST solution was blocked. The plate was blocked at 37°C for 2h, and the blocking solution was discarded. 100ul of antiserum diluted in a gradient manner with the blocking solution was added. Two replicates were set for each sample, and the plate was incubated at 37°C for 1.5h. After washing the plate with PBST, HRP-labeled sheep anti-mouse secondary antibody was added, and the plate was incubated at 37°C for 1h. The plate was washed three times with PBST, and the color developing solution was added. The reaction was protected from light for 15min. 50ul of the stop solution was added to terminate the reaction. The absorbance at 450nm was read by the ELISA instrument, and the dilution end point was greater than 0.1 of the negative sample. The immune binding titer was calculated by deducting the average value of the blank control using EXEL. The results are shown in Figure 2 .

[0043] The results show that 2ug of antigen HA-M2E-Fc fusion protein is close to the upper limit of immunity, and three immunizations can induce up to 10 6 The above magnitude of serum antibodies indicates that the fusion protein has a high immune induction ability. Specific binding antibodies can be detected with HA-Fc and an M2e synthetic peptide, indicating that each component of the antigen can induce specific antibodies. HA-Fc showed a high immune antibody binding ability, but lower than HA-M2E-Fc, showing the ability to induce antibodies due to the lack of M2E antigen. The M2ex1 coated alone has the ability to bind antibodies, and the 5-fold effect can fill the difference in results between HA-M2Ex5-Fc and HA-Fc to a certain extent.

Claims

1. An influenza virus vaccine fusion protein, which is a fusion protein formed by a HA antigen fragment encoded by an influenza HA antigen gene and an M2 extracellular region fragment in series, and then a Fc fragment encoded by a human immunoglobulin gene in series; The C-terminus of the HA antigen fragment is connected to five M2 extracellular region fragments in series, and the C-terminus of the M2 extracellular region fragment is then connected to the Fc fragment; The HA antigen gene is derived from the HA gene of the influenza strain, and the amino acid sequence encoded by it is shown in SEQ ID NO: 1; The amino acid sequence of the five tandem M2 extracellular region fragments is shown in SEQ ID NO:

2.

2. The influenza virus vaccine fusion protein according to claim 1, characterized in that There may or may not be a connecting fragment between the HA antigen fragment, the M2 extracellular region fragment, and the Fc fragment.

3. The influenza virus vaccine fusion protein according to claim 1, characterized in that The Fc fragment is derived from human IgG1.

4. The influenza virus vaccine fusion protein according to claim 3, characterized in that The amino acid sequence of the Fc fragment is shown in SEQ ID No:

3.

5. A nucleic acid encoding the influenza virus vaccine fusion protein according to any one of claims 1 to 4.

6. An expression vector containing the nucleic acid according to claim 5.

7. A recombinant host cell containing the expression vector according to claim 6.

8. The recombinant host cell according to claim 7, characterized in that It is HEK293FT cell, MDCK cell, VERO cell, CHO-K1Q cell or human cell.

9. An influenza vaccine comprising the influenza virus vaccine fusion protein according to any one of claims 1 to 4 as an active ingredient.

10. The influenza vaccine according to claim 9, characterized in that Adjuvants or pharmaceutically acceptable excipients are also included.

11. Use of the influenza virus vaccine fusion protein according to any one of claims 1 to 4 or the influenza vaccine according to claim 9 or 10 in the preparation of a medicament for preventing or treating influenza.

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