Vaccinia virus vector influenza A broad-spectrum vaccine

Abstract

The invention provides a recombinant vaccinia virus, a broad-spectrum influenza vaccine for expressing an influenza A virus antigen based on a vaccinia virus vector and application of the broad-spectrum influenza vaccine. The recombinant vaccinia virus is obtained by integrating coding sequences of influenza A virus NP, M2, HA and NA antigen proteins into a genome of a vaccinia virus Tian Tan strain. The recombinant vaccinia virus not only has good immunogenicity and cross protection ability, but also has no biological safety risk, provides a new thought and technical means for developing a new generation of universal influenza A virus vaccines, and has significant application prospects and values in the field of public health on the aspect of influenza resistance.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically, to a recombinant vaccinia virus, a broad-spectrum influenza vaccine expressing influenza A virus antigens based on a vaccinia virus vector, and applications thereof. Background Art

[0002] Vaccinia virus (Vaccinia virus) is a large, enveloped DNA virus that was initially used as a vaccine against smallpox. Due to its large genome, ability to accommodate foreign genes, and long history of use, Vaccinia virus is currently widely used as a vector for constructing vaccines against infectious diseases, as well as for developing therapeutic tumor vaccines and oncolytic viruses. The Vaccinia virus Tiantan strain (VTT), a successful smallpox vaccine strain independently developed in China, played a crucial role in the eradication of smallpox.

[0003] Influenza A virus is a highly variable RNA virus with multiple subtypes. Among them, seasonal influenza H1N1 and H3N2 subtype influenza viruses circulate in the human population every year, posing a major threat to human health. Vaccination is a key intervention strategy in the influenza prevention and control system, and its immune prevention efficacy has been widely recognized in the global public health field. Based on the seasonal epidemic characteristics and antigenic variation patterns of influenza viruses, the World Health Organization (WHO) has clearly put forward standardized prevention and control recommendations for annual vaccination. However, the high-frequency antigenic drift characteristics of influenza virus surface glycoproteins can cause antigenic epitope mismatches between vaccine strains and epidemic strains. This immune escape mechanism directly leads to a significant decrease in the titer of neutralizing antibodies in the serum of the population, thereby weakening the immune protection efficacy induced by the vaccine. Therefore, there is an urgent need to develop a new influenza vaccine with high safety, good broad-spectrum protection effect and long-term effective protection.

[0004] Cell-mediated immunity plays a crucial role in clearing influenza A virus infection. T cells can generate immune responses and promote recovery by recognizing conserved epitopes across different influenza A virus subtypes, leading to better clinical outcomes and prognosis. Cytotoxic CD8 T cells recognize viral antigenic peptides presented by HLA class I molecules and generate the broadest immune response against different influenza strains and subtypes. Therefore, influenza vaccines based on CD8 T cell recognition of conserved epitopes have the potential to provide broad immune protection and are receiving increasing attention. Recent research has focused on developing novel vaccine strategies targeting conserved influenza epitopes, such as relatively conserved epitopes of influenza virus hemagglutinin (HA), neuraminidase (NA), ion channel proteins and their extracellular domains (M2e), and nucleoprotein (NP). However, no new broad-spectrum influenza vaccine has yet been successfully developed. Summary of the Invention

[0005] In response to the problems existing in the prior art, the purpose of this application is to provide a novel broad-spectrum influenza vaccine based on vaccinia virus vectors expressing influenza A virus antigens.

[0006] Specifically, this application involves the following aspects:

[0007] 1. A recombinant vaccinia virus, wherein the recombinant vaccinia virus is obtained by integrating the coding sequences of influenza A virus NP, M2, HA and NA antigenic proteins into the genome of the vaccinia virus Tiantan strain.

[0008] 2. The recombinant vaccinia virus according to item 1, wherein the coding sequences of influenza A virus NP and M2 antigen proteins are integrated into the F4L gene position of the genome of the vaccinia virus Tiantan strain, and the coding sequences of influenza A virus HA and NA antigen proteins are integrated into the TK gene position;

[0009] The coding sequences of influenza A virus NP and M2 antigen proteins are connected with their 5' ends facing each other and are respectively connected downstream of different promoters; the coding sequences of influenza A virus HA and NA antigen proteins are connected with their 5' ends facing each other and are respectively connected downstream of different promoters.

[0010] 3. The recombinant vaccinia virus according to item 2, wherein:

[0011] The amino acid sequence of the influenza A virus NP antigen protein is shown in SEQ ID NO: 3;

[0012] The amino acid sequence of the influenza A virus HA antigen protein is shown in SEQ ID NO: 8;

[0013] The amino acid sequence of the influenza A virus NA antigen protein is shown in SEQ ID NO: 10;

[0014] The influenza A virus M2 antigen protein is selected from:

[0015] (i) having the amino acid sequence shown in SEQ ID NO: 5; or

[0016] (ii) comprising one or more engineered M2 proteins based on mutations of a reference influenza A virus M2 antigen protein, the amino acid sequence of which is shown in SEQ ID NO: 5, wherein the mutation is a deletion of amino acids 29 to 31 in the transmembrane region.

[0017] 4. The recombinant vaccinia virus according to item 2 or 3, wherein the coding sequence of the influenza A virus NP antigen protein is linked downstream of the p7.5 promoter, and the coding sequence of the influenza A virus M2 antigen protein is linked downstream of the p11 promoter; and

[0018] The coding sequence of the influenza A virus NA antigen protein is connected to the downstream of the p11 promoter, and the coding sequence of the influenza A virus HA antigen protein is connected to the downstream of the p7.5 promoter.

[0019] 5. The recombinant vaccinia virus according to item 4, wherein the coding sequence of the influenza A virus NP antigen protein, the p7.5 promoter, the p11 promoter, and the coding sequence of the influenza A virus M2 antigen protein are connected in series;

[0020] The sequence after tandem connection is shown in SEQ ID NO: 1.

[0021] 6. The recombinant vaccinia virus according to item 4, wherein the coding sequence of the influenza A virus HA antigen protein, the p7.5 promoter, the p11 promoter, and the coding sequence of the influenza A virus NA antigen protein are connected in series;

[0022] The sequence after tandem connection is shown in SEQ ID NO: 2.

[0023] 7. The method for constructing a recombinant vaccinia virus according to any one of items 1 to 6, wherein the method comprises:

[0024] The coding sequences of influenza A virus NP, M2, HA and NA antigenic proteins were integrated into the genome of vaccinia virus Tiantan strain by homologous recombination;

[0025] Preferably, the coding sequences of influenza A virus NP and M2 antigen proteins are integrated into the F4L gene position of the vaccinia virus Tiantan strain genome by homologous recombination, and the coding sequences of influenza A virus HA and NA antigen proteins are integrated into the TK gene position of the vaccinia virus Tiantan strain genome by homologous recombination.

[0026] 8. An influenza vaccine based on a vaccinia virus vector expressing influenza A virus antigens, wherein the influenza vaccine comprises the recombinant vaccinia virus according to any one of items 1 to 6.

[0027] 9. The influenza vaccine according to item 8, wherein the influenza vaccine is administered by one or more delivery methods selected from the group consisting of respiratory tract aerosol inhalation, nasal drops, oral administration, direct injection, and mucosal administration.

[0028] 10. Use of the recombinant vaccinia virus according to any one of items 1 to 6 in the preparation of an influenza vaccine.

[0029] 11. Use of the recombinant vaccinia virus according to any one of items 1 to 6 or the influenza vaccine according to item 8 or 9 in the preparation of a product for preventing and / or treating influenza.

[0030] 12. Use of the recombinant vaccinia virus according to any one of items 1 to 6 in the preparation of a detection reagent or kit for diagnosing influenza.

[0031] 13. Use of the recombinant vaccinia virus according to any one of items 1 to 6 as an immunogen in the preparation of influenza A virus antibodies.

[0032] Beneficial effects:

[0033] The present application provides a novel broad-spectrum influenza vaccine based on a vaccinia virus vector expressing influenza A virus antigens. By integrating the coding sequences of influenza A virus NP, M2, HA, and NA antigenic proteins into the genome of the Tiantan strain of vaccinia virus, a recombinant vaccinia virus capable of simultaneously expressing NP, M2, HA, and NA antigenic proteins was constructed. After immunizing mice with the recombinant vaccinia virus of the present application, high levels of HA and NP antibody titers, as well as broad-spectrum hemagglutination-inhibiting antibodies, were detected in the mouse serum. Furthermore, antigen-specific T cell immune responses were induced after immunization, and challenge protection experiments further demonstrated cross-type protection.

[0034] The recombinant vaccinia virus of the present application not only has good immunogenicity and cross-protection ability, but also does not pose any biosafety risks. It provides new ideas and technical means for the development of a new generation of universal influenza A virus vaccine, and has significant application prospects and value in combating influenza in the field of public health. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of homologous recombination constructed for the recombinant plasmid pF4L-NPM2.

[0036] Figure 2 Schematic diagram of homologous recombination constructed for the recombinant plasmid pTK-HANA.

[0037] Figure 3 The results of PCR identification of recombinant vaccinia virus TK-HANA / F4L-NPM2. WT indicates wild-type virus, dTF indicates vaccinia virus lacking the TK and F4L regions, rTK indicates recombinant vaccinia virus TK-HANA lacking the TK region and inserting HA and NA, and rF4 indicates recombinant vaccinia virus lacking the F4L region and inserting NP and M2. del29-31 Recombinant vaccinia virus F4L-NPM2 del29-31 , rTF indicates recombinant vaccinia virus TK-HANA / F4L-NPM2.

[0038] Figure 4 After the recombinant vaccinia virus TK-HANA / F4L-NPM2 was infected into Vero cells, the influenza A virus HA, NA, NP, M2 were detected by Western Blot. del29-31 Protein expression results. NC represents the negative control, dTF represents the vaccinia virus lacking the TK and F4L regions, rTK represents the recombinant vaccinia virus TK-HANA lacking the TK region and inserting HA and NA, and rF4 represents the recombinant vaccinia virus lacking the F4L region and inserting NP and M2. del29-31 Recombinant vaccinia virus F4L-NPM2 del29-31 , rTF indicates recombinant vaccinia virus TK-HANA / F4L-NPM2.

[0039] Figure 5A-5B The results of the detection of HA or NP specific antibodies in the serum of mice after intramuscular injection immunization. Figure 5A is the test result of HA-specific antibodies; Figure 5B This is the detection result of NP-specific antibodies.

[0040] Figure 6 Results of hemagglutination inhibition antibody detection in the serum of mice after intramuscular immunization.

[0041] Figure 7 Results of cellular immune response testing after intramuscular injection of immunization in mice.

[0042] Figures 8A-8D The results of monitoring the dynamic changes in weight and survival of mice after intramuscular immunization with influenza A virus A / PR / 8 / 1934 (H1N1) or A / Aichi / 2 / 1968 (H3N2). Figure 8A This is the monitoring result of the dynamic changes in mouse body weight after infection with influenza A virus A / PR / 8 / 1934 (H1N1); Figure 8B This is the monitoring result of the dynamic changes in mouse body weight after challenge with influenza A virus A / Aichi / 2 / 1968(H3N2); Figure 8C The data show the survival of mice after challenge with influenza A virus A / PR / 8 / 1934 (H1N1). Figure 8D These are the results of monitoring the survival of mice after infection with influenza A virus A / Aichi / 2 / 1968 (H3N2).

[0043] Figure 9A-9B Results of the determination of virus titers in the lungs of mice after intramuscular immunization with influenza A virus A / PR / 8 / 1934 (H1N1) or A / Aichi / 2 / 1968 (H3N2). Figure 9AThe results are for the determination of virus titers in mouse lungs after challenge with influenza A virus A / PR / 8 / 1934 (H1N1); Figure 9B These are the results of virus titer determination in mouse lungs after challenge with influenza A virus A / Aichi / 2 / 1968 (H3N2).

[0044] Figures 10A-10E The results are for the detection of HA-specific antibodies in nasal lavage fluid, alveolar lavage fluid and serum of mice after intranasal immunization. Figure 10A The results of the HA-specific antibody (anti-HA sIgA) test in nasal lavage fluid; Figure 10B The results of the HA-specific antibody (anti-HA sIgA) test in the bronchoalveolar lavage fluid; Figure 10C It is the test result of HA-specific antibody (anti-HAIgA) in serum; Figure 10D is the test result of HA-specific antibody (anti-HA IgG) in serum; Figure 10E The results of serum NP-specific antibodies (anti-NP IgG) are shown. IN-dTF indicates intranasal immunization with vaccinia virus dTF lacking the TK and F4L regions; IM-rTF indicates intramuscular immunization with recombinant vaccinia virus TK-HANA / F4L-NPM2; and IN-rTF indicates intranasal immunization with recombinant vaccinia virus TK-HANA / F4L-NPM2.

[0045] Figure 11 These are the results of hemagglutination inhibition antibody detection in the serum of mice after intranasal immunization.

[0046] Figure 12 These are the test results of cellular immune response after intranasal immunization of mice.

[0047] Figures 13A-13D The results show the dynamic changes in body weight and survival of mice after intranasal immunization and challenge with influenza A virus A / PR / 8 / 1934 (H1N1) or A / Aichi / 2 / 1968 (H3N2). Figure 13A This is the monitoring result of the dynamic changes in mouse body weight after infection with influenza A virus A / PR / 8 / 1934 (H1N1); Figure 13B This is the monitoring result of the dynamic changes in mouse body weight after challenge with influenza A virus A / Aichi / 2 / 1968(H3N2); Figure 13C The data show the survival of mice after challenge with influenza A virus A / PR / 8 / 1934 (H1N1). Figure 13D These are the results of monitoring the survival of mice after infection with influenza A virus A / Aichi / 2 / 1968 (H3N2).

[0048] Figures 14A-14BThe results of the determination of virus titers in the lungs of mice after intranasal immunization with influenza A virus A / PR / 8 / 1934 (H1N1) or A / Aichi / 2 / 1968 (H3N2). Figure 14A The results are for the determination of virus titers in mouse lungs after challenge with influenza A virus A / PR / 8 / 1934 (H1N1); Figure 14B These are the results of virus titer determination in mouse lungs after challenge with influenza A virus A / Aichi / 2 / 1968 (H3N2). DETAILED DESCRIPTION

[0049] The present application is further described below with reference to examples. It should be understood that the examples are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0050] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[0051] definition

[0052] The term "mutation" as used herein generally refers to any type of change or modification to a sequence (nucleic acid or amino acid sequence), including deletion, absence, truncation, inactivation, disruption, substitution or translocation of amino acids or nucleotides.

[0053] As used herein, the terms "amino acid" or "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and refer to naturally occurring or synthetic molecules. When "amino acid sequence" is described herein as referring to the amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and similar terms are not intended to limit the amino acid sequence to the complete native amino acid sequence associated with the described protein molecule.

[0054]

[00146] The term "amino acid," as used herein, may be referred to by its name, by its commonly known three letter symbol, or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0055] As used herein, the terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," "polynucleotide," "polynucleotide sequence," "RNA sequence," or "DNA sequence" refer to oligonucleotides, nucleotides, or polynucleotides, and fragments and portions thereof, and refer to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and represent the sense or antisense strand. The sequence may be a non-coding sequence, a coding sequence, or a mixture of the two. The nucleic acid sequences of the present application may be prepared using standard techniques well known to those skilled in the art.

[0056] As used herein, the term "immunogen" is intended to refer to a substance capable of inducing an adaptive immune response in an individual, wherein the adaptive immune response is capable of inducing an immune response that is significant against pathogens that share the same immunological characteristics as the immunogen. An "immunogen" refers to any substance introduced into the body for the purpose of generating an immune response. The substance can be a physiological molecule, such as a protein, or can be encoded by a vector, such as DNA, mRNA, or a virus.

[0057] The term "promoter" as used herein is used in its conventional sense and refers to a nucleotide sequence that initiates transcription of an operably linked nucleotide sequence. A promoter is located near the same chain of the nucleotide sequence that it transcribes. A promoter can be constitutive, inducible, or repressible. A promoter can be naturally occurring or synthetic. The sources from which a promoter can be derived include viruses, bacteria, fungi, plants, insects, and animals. The promoter can be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source). For example, if the vector to be used is a DNA plasmid, the promoter can be endogenous to the plasmid (homologous), or derived from other sources (heterologous).

[0058] The term "tandem" as used herein refers to multiple copies of a polynucleotide sequence arranged continuously and uninterruptedly.

[0059] As used herein, the term "coding sequence" means a sequence of nucleotides (position including stop codons) that corresponds to the amino acid sequence in a protein; the features include conceptual translation of the amino acids.

[0060] The term "recombinant" as used herein means "produced by genetic engineering." Preferably, a "recombinant object" such as a recombinant vaccinia virus in the context of this application does not occur naturally.

[0061] The terms "NP protein" and "NP antigen protein" used in this article are used interchangeably; "M2 protein" and "M2 antigen protein" are used interchangeably; "NA protein" and "NA antigen protein" are used interchangeably; and "HA protein" and "HA antigen protein" are used interchangeably.

[0062] The term "shuttle plasmid" as used herein refers to a type of plasmid vector that has two different replication origins and selection markers and can therefore survive and replicate in two different groups of hosts.

[0063] Recombinant vaccinia virus

[0064] The present application provides a recombinant vaccinia virus, which is obtained by integrating the coding sequences of influenza A virus NP, M2, HA and NA antigen proteins into the genome of the vaccinia virus Tiantan strain.

[0065] The genome information of the vaccinia virus Tiantan strain can be found in the NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov) database, and its GenBank number is JX489139.1.

[0066] This application is based on the Mosaic vaccine design strategy. By analyzing the amino acid sequences of the HA, NA and NP antigen proteins of all known human H1N1 and H3N2 from 2009 to 2021, the Mosaic HA, NA and NP sequences with the highest CTL cell epitope coverage are finally designed, in the hope of improving the T cell immune response of the influenza antigen and forming a broad-spectrum protection effect against different influenza A viruses. In addition, the M2 protein is an ion channel protein of influenza A virus, and its extracellular domain sequence is highly conserved and can induce a broad-spectrum T cell immune response. Therefore, this application also uses the M2 antigen protein of the H1N1 representative strain PR8. This design not only ensures a broad-spectrum immune response against multiple influenza A viruses, but also enhances the targeting effect on the key stages of the virus life cycle. The multivalent recombinant vaccinia virus obtained based on this design and construction provides a powerful tool for the prevention and control of influenza A virus.

[0067] Specifically, the recombinant vaccinia virus is obtained by integrating the coding sequences of influenza A virus NP and M2 antigen proteins at the F4 gene position in the genome of the Tiantan strain of the vaccinia virus, and integrating the coding sequences of influenza A virus HA and NA antigen proteins at the TK gene position.

[0068] Among them, the amino acid sequence of the influenza A virus NP antigen protein is shown in SEQ ID NO: 3; the amino acid sequence of the influenza A virus HA antigen protein is shown in SEQ ID NO: 8; and the amino acid sequence of the influenza A virus NA antigen protein is shown in SEQ ID NO: 10.

[0069] The sequence shown in SEQ ID NO: 3 is as follows:

[0070] MASQGTKRSYEQMETGGERQDTTEIRASVGRMIGGIGRFYIQMCTELKLSDYDGRLIQNSI

[0071] TIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRIDGKWTRELILYDKEEIRRVWRQANN

[0072] GEDATAGLTHIMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVG

[0073] TIAMELIRMIKRGINDRNFWRGENGRRTRVAYERMCNILKGKFQTAAQRAMMDQVRESRNPG

[0074] NAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGHDFEREGYSLVGIDPFKLLQNSQVVSL

[0075] MRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGKKVIPRGKLSTRGVQIASNENVETMDSNT

[0076] LELRSRYWAIRTRSGGNTNQQKASAGQISVQPTFSVQRNLPFERATVMAAFSGNNEGRTSDMR

[0077] TEVIRMMESAKPEDLSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDN;

[0078] The sequence shown in SEQ ID NO:8 is as follows:

[0079] MKQGKTKATKMKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNL

[0080] LEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFI

[0081] DYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPK

[0082] LSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQE

[0083] GRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINT

[0084] SLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYH

[0085] HQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGF

[0086] LDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMES

[0087] VKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSL

[0088] QCRICI;

[0089] The sequence shown in SEQ ID NO:10 is as follows:

[0090] MNPNQKIITIGSVCMTIGMANLILQIGNIISIWVSHSIQIGNQSQIETCNQSVITYENNTWVN

[0091] QTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLE

[0092] CRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTI<^

[0093] GISGPDSGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTIGPSDGQASYKIFRIE

[0094] KGKIVKSVEMNAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGVF

[0095] GDNPRPNDKTGSCCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRKGFEMIWDPNGWTGTD

[0096] NNFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPEENTIWTSGSSISFCGVNS

[0097] DTVGWSWPDGAELPFTIDK.

[0098] Wherein, the influenza A virus M2 antigen protein is selected from:

[0099] (i) having the amino acid sequence shown in SEQ ID NO: 5; or

[0100] (ii) comprising one or more engineered M2 proteins based on mutations of a reference influenza A virus M2 antigen protein, the amino acid sequence of which is shown in SEQ ID NO: 5, wherein the mutation is a deletion of amino acids 29 to 31 in the transmembrane region.

[0101] The amino acid sequence shown in SEQ ID NO:5 is as follows:

[0102] MSLLTEVETPIRNEWGCRCNGSSDPLAIAANIIGILHLILWILDRLFFKCIYRRFKYGLKGGPSTEGVPKSMREEYRKEQQSAVDADDGHFVSIELE.

[0103] With respect to polypeptides as described herein, the term "reference" refers to naturally occurring polypeptides that do not include artificial substitutions, insertions or deletions at one or more amino acid positions, and naturally occurring or synthetic polypeptides that include one or more artificial substitutions, insertions or deletions at one or more amino acid positions. Similarly, with respect to polynucleotides, the term "reference" refers to naturally occurring polynucleotides that do not include artificial substitutions, insertions or deletions at one or more nucleosides, and naturally occurring or synthetic polynucleotides that include one or more artificial substitutions, insertions or deletions at one or more nucleosides. For example, polynucleotides encoding wild-type or parent polypeptides are not limited to naturally occurring polynucleotides, and encompass any polynucleotides encoding wild-type or parent polypeptides.

[0104] In the present application, the amino acids at positions 29 to 31 are determined based on the amino acid sequence shown in SEQ ID NO: 5, and are counted from the N-terminus. The amino acid sequence of the engineered M2 protein with amino acids at positions 29 to 31 deleted in the transmembrane region is shown in SEQ ID NO: 6.

[0105] The sequence shown in SEQ ID NO:6 is as follows:

[0106] MSLLTEVETPIRNEWGCRCNGSSDPLAIIIGILHLILWILDRLFFKCIYRRFKYGLKGGPSTEGVPKSMREEYRKEQQSAVDADDGHFVSIELE.

[0107] The present application does not impose any limitation on the method for preparing the engineered M2 protein, which can be mutated according to conventional methods in the art, such as directed mutagenesis, random mutagenesis, or construction of synthetic oligonucleotides.

[0108] The coding sequences of influenza A virus NP and M2 antigen proteins are connected with their 5' ends facing each other and are respectively connected downstream of different promoters; the coding sequences of influenza A virus HA and NA antigen proteins are connected with their 5' ends facing each other and are respectively connected downstream of different promoters.

[0109] The present application is not intended to limit the specific type of the promoter. Those skilled in the art will understand that as long as the selected promoter is known in the art and can promote the effective expression of the target gene, it meets the requirements of the present application.

[0110] In some embodiments, the coding sequence of the influenza A virus NP antigen protein is connected downstream of the p7.5 promoter, and the coding sequence of the influenza A virus M2 antigen protein is connected downstream of the p11 promoter; and the coding sequence of the influenza A virus NA antigen protein is connected downstream of the p11 promoter, and the coding sequence of the influenza A virus HA antigen protein is connected downstream of the p7.5 promoter.

[0111] In some embodiments, the coding sequence of influenza A virus NP antigen protein, p7.5 promoter, p11 promoter, and the coding sequence of influenza A virus M2 antigen protein are connected in series, and the sequence after series connection is shown in SEQ ID NO: 1.

[0112] The sequence shown in SEQ ID NO: 1 is as follows:

[0113]

[0114] In some embodiments, the coding sequence of influenza A virus HA antigen protein, p7.5 promoter, p11 promoter, and the coding sequence of influenza A virus NA antigen protein are connected in series, and the sequence after series connection is shown in SEQ ID NO: 2.

[0115] The sequence shown in SEQ ID NO: 2 is as follows:

[0116]

[0117] In some embodiments, the recombinant vaccinia virus is obtained by integrating the sequence shown in SEQ ID NO: 1 at the F4 gene position in the genome of the Tiantan strain of the vaccinia virus and integrating the sequence shown in SEQ ID NO: 2 at the TK gene position.

[0118] Method for constructing recombinant vaccinia virus

[0119] The present application provides a method for constructing any of the above-mentioned recombinant vaccinia viruses, comprising: integrating the coding sequences of influenza A virus NP, M2, HA and NA antigen proteins into the genome of the Tiantan strain of vaccinia virus by homologous recombination.

[0120] This application is not intended to limit the specific method for constructing the recombinant vaccinia virus. Those skilled in the art can use any method to integrate the coding sequences of influenza A virus NP, M2, HA and NA antigen proteins into the genome of the Tiantan strain of vaccinia virus.

[0121] Specifically, in the present application, the coding sequences of the influenza A virus NP and M2 antigen proteins are integrated into the F4L gene position of the vaccinia virus Tiantan strain genome by homologous recombination, and the coding sequences of the influenza A virus HA and NA antigen proteins are integrated into the TK gene position of the vaccinia virus Tiantan strain genome by homologous recombination.

[0122] In some embodiments, a shuttle plasmid carrying the homologous left and right arms of the F4L gene is used as a vector to integrate the coding sequences of the influenza A virus NP and M2 antigen proteins into the F4L gene position of the vaccinia virus Tiantan strain genome by homologous recombination; and a shuttle plasmid carrying the homologous left and right arms of the TK gene is used as a vector to integrate the coding sequences of the influenza A virus HA and NA antigen proteins into the TK gene position of the vaccinia virus Tiantan strain genome by homologous recombination.

[0123] The shuttle plasmid may be any suitable plasmid, and may contain any suitable promoter capable of driving the expression of the coding sequence of influenza A virus NP and M2 antigen proteins, or a promoter capable of driving the expression of the coding sequence of influenza A virus HA and NA antigen proteins.

[0124] In some embodiments, the shuttle plasmid carrying the homologous left and right arms of the F4L gene is pF4L, and the pF4L comprises the homologous left and right arms of the F4L gene, promoter P7.5, and promoter P11.

[0125] Among them, the homologous left and right arms of the F4L gene are sequences for homologous recombination between vaccinia virus and shuttle plasmid, the promoter P11 is used to promote the expression of the coding sequence of influenza A virus M2 antigen protein, and the promoter P7.5 is used to promote the expression of the coding sequence of influenza A virus NP antigen protein.

[0126] The method of ligating the coding sequences of influenza A virus M2 and NP antigen proteins into the shuttle plasmid pF4L is conventional in the art. The nucleotide sequence of the obtained recombinant plasmid containing the coding sequences of influenza A virus M2 and NP antigen proteins is shown in SEQ ID NO: 12.

[0127] In some embodiments, the shuttle plasmid carrying the homologous left and right arms of the TK gene is pTK, and the pTK comprises the homologous left and right arms of the TK gene, promoter P7.5, and promoter P11.

[0128] Among them, the homologous left arm and right arm of the TK gene are sequences for homologous recombination between vaccinia virus and shuttle plasmid, the promoter P7.5 is used to initiate the expression of the coding sequence of influenza A virus HA antigen protein, and the promoter P11 is used to initiate the expression of the coding sequence of influenza A virus NA antigen protein.

[0129] The method of ligating the coding sequences of influenza A virus HA and NA antigen proteins into the shuttle plasmid pTK is conventional in the art. The nucleotide sequence of the obtained recombinant plasmid containing the coding sequences of influenza A virus HA and NA antigen proteins is shown in SEQ ID NO: 19.

[0130] Broad-spectrum influenza vaccine

[0131] The present application provides an influenza vaccine based on a vaccinia virus vector expressing influenza A virus antigens, wherein the influenza vaccine comprises any of the above-mentioned recombinant vaccinia viruses.

[0132] In some embodiments, the influenza vaccine further comprises a pharmaceutically acceptable carrier or adjuvant.

[0133] Among them, pharmaceutically acceptable carriers may include non-toxic buffers such as phosphoric acid, citric acid, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; chloride); benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens, such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and nonionic surfactants such as Tween or polyethylene glycol (PEG).

[0134] Examples of adjuvants include, but are not limited to, mineral salts or gels (e.g., aluminum salts, aluminum phosphate gels, or calcium phosphate gels), emulsion / amphiphilic molecule and surfactant-based preparations, particulate adjuvants, pathogen-associated molecules, human endogenous immunomodulatory adjuvants, and inert carriers.

[0135] In some embodiments, the influenza vaccine is administered by one or more delivery methods selected from the group consisting of respiratory aerosol inhalation, nasal drops, oral administration, direct injection, and mucosal administration.

[0136] Direct injection includes, for example, intravenous injection, subcutaneous injection, intradermal injection or intramuscular injection.

[0137] use

[0138] The present application provides the use of any of the above-mentioned recombinant vaccinia viruses in the preparation of influenza vaccines.

[0139] The present application provides the use of any of the above-mentioned recombinant vaccinia viruses or any of the above-mentioned influenza vaccines in the preparation of a product for preventing and / or treating influenza.

[0140] The present application provides the use of any of the above-mentioned recombinant vaccinia viruses in the preparation of a detection reagent or kit for diagnosing influenza.

[0141] The present application also provides the use of any of the above-mentioned recombinant vaccinia viruses as an immunogen in the preparation of influenza A virus antibodies.

[0142] This application constructs a novel broad-spectrum influenza vaccine based on a vaccinia virus vector expressing multivalent conserved antigens of influenza A virus. The vaccine is effective against a variety of influenza A virus strains and, compared with existing influenza vaccines, can provide broader protection, have higher safety and stability, and is expected to overcome the limitations of existing influenza vaccines. Specifically, this application integrates the coding sequences of influenza A virus NP and M2 antigen proteins at the F4 gene position in the genome of the vaccinia virus Tiantan strain, and integrates the coding sequences of influenza A virus HA and NA antigen proteins at the TK gene position, thereby obtaining a recombinant vaccinia virus capable of simultaneously expressing NP, M2, HA, and NA antigen proteins.

[0143] Example

[0144] The present application will be described below in conjunction with specific examples, but the scope of the present application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following examples are all conventional reagents and instruments in this area and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the described scheme and obtain corresponding results based on the embodiments.

[0145] Example 1 Construction of a gene capable of expressing influenza A virus NP and M2 del29-31 Recombinant vaccinia virus F4L-NPM2 protein del29-31

[0146] 1.1 Construction of recombinant plasmid pF4L-NPM2

[0147] NP and M2 of influenza A virus del29-31 After the protein coding sequence was codon-optimized, it was linked to the vector pF4L carrying the homology arms (F3L, F5L) of the F4L region of vaccinia virus (GenBank No. JX489139.1) by molecular cloning methods to obtain the recombinant plasmid pF4L-NPM2 (the nucleotide sequence of which is shown in SEQ ID NO: 12). The constructed recombinant plasmid pF4L-NPM2 mainly includes vaccinia virus P7.5, P11 promoter, NP, M2 del29-31 , and F4L region homology arms (F3L, F5L), the schematic diagram of homologous recombination is shown in Figure 1 shown.

[0148] Among them, the amino acid sequence of influenza A virus NP protein is shown in SEQ ID NO: 3, and the coding sequence of influenza A virus NP protein is shown in SEQ ID NO: 4.

[0149] The sequence shown in SEQ ID NO: 3 is as follows:

[0150] MASQGTKRSYEQMETGGERQDTTEIRASVGRMIGGIGRFYIQMCTELKLSDYDGRLIQNSITIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRIDGKWTRELILYDKEEIRRVWRQANNGEDATAGLTHIMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTIAMELIRMIKRGINDRNFWRGENGRRTRVAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGHDFEREGYSLVGIDPFKLLQNSQVVSLMRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGKKVIPRGKLSTRGVQIASNENVETMDSNTLELRSRYWAIRTRSGGNTNQQKASAGQISVQPTFSVQRNLPFERATVMAAFSGNNEGRTSDMRTEVIRMMESAKPEDLSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDN;

[0151] The sequence shown in SEQ ID NO:4 is as follows:

[0152]

[0153] Among them, influenza A virus M2 del29-31 The amino acid sequence of the protein is shown in SEQ ID NO: 6, influenza A virus M2 del29-31 The coding sequence of the protein is shown in SEQ ID NO:7.

[0154] The sequence shown in SEQ ID NO:6 is as follows:

[0155] MSLLTEVETPIRNEWGCRCNGSSDPLAIIIGILHLILWILDRLFFKCIYRRFKYGLKGGPSTEGVPKSMREEYRKEQQSAVDADDGHFVSIELE;

[0156] The sequence shown in SEQ ID NO:7 is as follows:

[0157] ATGAGTCTTCTAACCGAGGTCGAAACGCCTATCAGAAACGAATGGGGGTGCAGATGCAACGGTTCAAGTGATCCTCTCGCTATTATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTCTTTTTTTCAAATGCAT TTACCGTCGCTTTAAATACGGACTGAAAGGAGGGCCTTCTACGGAAGGAGTGCCAAAGTCTATGAGGGAAGAATATCGAAAGGAACAGCAGAGTGCTGTGGATGCTGACGATGGTCATTTTGTCAGCATAGAGCTAGAGTAA.

[0158] 1.2 Recombinant vaccinia virus F4L-NPM2 del29-31 Acquisition

[0159] 293T cells (5×10 5 cells / well) and cultured overnight. When the cells grew to 60% to 70% confluence, they were transfected with the recombinant plasmid pF4L-NPM2. Four hours after transfection, they were infected with wild-type vaccinia virus. Two hours after infection, the supernatant was discarded and replaced with fresh complete medium. After 48 hours, the cells were harvested and frozen and thawed three times to obtain a virus suspension.

[0160] Virus was purified by plaque formation assay and Vero cells (1×10 6Cells / well) were cultured overnight. When the cells grew to 80% to 90% confluence, the collected virus suspension was diluted 10-fold and added to a six-well plate for infection. After 2 hours of infection, the cells were washed once with PBS and replaced with a low-melting point agar medium with a final concentration of 1%. After 3 days of culture, plaques were picked and identified by PCR using specific primers. After obtaining positive plaques, the method was repeated for multiple rounds of screening until pure recombinant vaccinia virus F4L-NPM2 was obtained. del29-31 The PCR primer sequences are shown in Table 1 below.

[0161] Table 1 Primer sequences used in PCR in Example 1

[0162]

[0163] 1.3 Recombinant vaccinia virus F4L-NPM2 del29-31 Identification

[0164] use Pure recombinant vaccinia virus F4L-NPM2 was extracted using a DNA mini kit (QIAGEN) del29-31 and wild-type vaccinia virus DNA; the extracted viral DNA was amplified and identified by PCR (the results are shown in Figure 3 The sequences of PCR primers are shown in Table 1. At the same time, the PCR products were sequenced to confirm the recombinant vaccinia virus F4L-NPM2 del29-31 Accurately insert influenza A virus NP and M2 into the F4L region del29-31 Protein coding sequence.

[0165] Example 2 Construction of recombinant vaccinia virus TK-HANA capable of expressing influenza A virus HA and NA proteins and simultaneous expression of influenza A virus HA, NA, NP, M2 del29-31 Recombinant vaccinia virus TK-HANA / F4L-NPM2

[0166] 2.1 Construction of recombinant plasmid pTK-HANA

[0167] After codon optimization, the coding sequences of HA and NA proteins of influenza A virus were linked to the vector pTK carrying the TK region homology arms (J1R, J3R) of vaccinia virus (GenBank No. JX489139.1) by molecular cloning methods to obtain the recombinant plasmid pTK-HANA (the nucleotide sequence of which is shown in SEQ ID NO: 19). The constructed recombinant plasmid pTK-HANA mainly includes vaccinia virus P7.5, P11 promoter, HA, NA, and TK region homology arms (J1R, J3R). The schematic diagram of homologous recombination is shown in FIG. Figure 2 shown.

[0168] Among them, the amino acid sequence of influenza A virus HA protein is shown in SEQ ID NO: 8, and the coding sequence of influenza A virus HA protein is shown in SEQ ID NO: 9.

[0169] The sequence shown in SEQ ID NO:8 is as follows:

[0170] ;

[0171] The sequence shown in SEQ ID NO:9 is as follows:

[0172]

[0173] Among them, the amino acid sequence of influenza A virus NA protein is shown in SEQ ID NO: 10, and the coding sequence of influenza A virus NA protein is shown in SEQ ID NO: 11.

[0174] The sequence shown in SEQ ID NO: 10 is as follows:

[0175] MNPNQKIITIGSVCMTIGMANLILQIGNIISIWVSHSIQIGNQSQIETCNQSVITYENNTWVNQTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVI REPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNNILRTQESECACVN GSCFTIMTDGPSDGQASYKIFRIEKGKIVKSVEMNAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQIGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFK YGNGVWIGRTKSISSRKGFEMIWDPNGWTGTDNNFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPEENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK;

[0176] The sequence shown in SEQ ID NO: 11 is as follows:

[0177]

[0178] 2.2 Obtaining recombinant vaccinia virus TK-HANA and recombinant vaccinia virus TK-HANA / F4L-NPM2

[0179] 293T cells (5×10 5 Cells / well) were cultured overnight. When the cells grew to 60% to 70% of the confluence, the recombinant plasmid pTK-HANA was transfected. Four hours after transfection, wild-type vaccinia virus or the obtained recombinant vaccinia virus F4L-NPM2 was infected. del29-31 ; After 2 hours of infection, the supernatant was discarded and replaced with fresh complete medium. After 48 hours, the cells were harvested and frozen and thawed three times to obtain the virus suspension.

[0180] Virus was purified by plaque formation assay and Vero cells (1×10 6 Cells were grown overnight. When the cells reached 80% to 90% confluence, the collected virus suspension was diluted 10-fold and added to a six-well plate for infection. After 2 hours of infection, the cells were washed once with PBS and replaced with a low-melting-point agar medium with a final concentration of 1%. After 3 days of culture, plaques were picked and identified by PCR using specific primers. After obtaining positive plaques, the method was repeated for multiple rounds of screening until pure recombinant vaccinia virus TK-HANA or pure recombinant vaccinia virus TK-HANA / F4L-NPM2 was obtained. The PCR primer sequences are shown in Table 2 below.

[0181] Table 2 Primer sequences used in PCR in Example 2

[0182]

[0183]

[0184] 2.3 Identification of recombinant vaccinia virus TK-HANA and recombinant vaccinia virus TK-HANA / F4L-NPM2

[0185] use Viral DNA of pure recombinant vaccinia virus TK-HANA, pure recombinant vaccinia virus TK-HANA / F4L-NPM2, and wild-type vaccinia virus was extracted using a DNA mini kit (QIAGEN); the extracted viral DNA was amplified and identified by PCR. The PCR primer sequences are shown in Tables 1 and 2, and the PCR identification results are shown in Tables 1 and 2. Figure 3As shown in Figure 2, recombinant vaccinia virus TK-HANA and recombinant vaccinia virus TK-HANA / F4L-NPM2 have been successfully obtained. At the same time, sequencing of the PCR products confirmed that the recombinant vaccinia virus TK-HANA / F4L-NPM2 had influenza A virus HA and NA inserted into the TK region, and influenza A virus NP and M2 accurately inserted into the F4L region. del29-31 Protein coding sequence.

[0186] Furthermore, the obtained recombinant vaccinia virus TK-HANA / F4L-NPM2 was used to infect Vero cells. After 24 hours, the cells were lysed and the expression of influenza A virus HA, NA, NP, M2 was detected by Western Blot. del29-31 Protein expression was detected using the following primary antibodies: anti-HA antibody (Beijing Sino Biological Science Co., Ltd., 86001-RM01), anti-NA antibody (Beijing Sino Biological Science Co., Ltd., 11058-R001), anti-NP antibody (Beijing Sino Biological Science Co., Ltd., 40205-MM16), and anti-M2 antibody (GeneTex, GTX125951). Figure 4 As shown, influenza A virus HA, NA, NP, M2 del29-31 The protein was successfully expressed.

[0187] Example 3: Immunization of mice with recombinant vaccinia virus TK-HANA / F4L-NPM2 by intramuscular injection

[0188] 3.1 Mouse immunization protocol

[0189] BalB / C mice aged 6-8 weeks were divided into 3 groups, with 5 mice in each group: the first group was a blank control group (PBS); the second group was a viral vector control group (dTF), which was infected with vaccinia virus dTF lacking TK and F4L regions (by not carrying HA, NA, NP, M2 del29-31 The third group was the recombinant vaccine group (rTF), which used the TK region deleted and inserted into HA and NA, and the F4L region deleted and inserted into NP and M2. del29-31 The mice were immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2.

[0190] On day 0, the blank control group (PBS) was immunized with 100 μL PBS, and the viral vector control group (dTF) was immunized with 100 μL (1×10 6 PFU / mL)dTF, recombinant vaccine group (rTF) immunization 100μL (1×10 6PFU / mL) recombinant vaccinia virus TK-HANA / F4L-NPM2; serum was collected for antibody detection on the 21st day after immunization, and the harvested mouse serum was inactivated at 56°C for 30 minutes and then stored in a -80°C refrigerator.

[0191] 3.2 Detection of HA or NP-specific antibodies in mouse serum

[0192] 21 days after immunization, the serum of mice in each group was collected for testing. The detection of HA-specific antibodies in mouse serum used HA protein (Influenza A H1N1 (A / California / 04 / 2009) Hemagglutinin / HA0 Protein, Beijing Sino Biological Science and Technology Co., Ltd.) to coat the ELISA plate; the detection of NP-specific antibodies in mouse serum used NP protein (Influenza AH1N1 (A / Victoria / 4897 / 2022) Nucleoprotein / NP Protein, Beijing Sino Biological Science and Technology Co., Ltd.) to coat the ELISA plate. The detection steps are as follows:

[0193] The ELISA plate required for detection (coated with influenza A virus HA protein or NP protein) was first washed 5 times with 300 μL 1× washing buffer, and then the mouse serum to be detected was added with a gradient dilution (4-fold gradient dilution). After incubation at room temperature for 2 hours, the plate was washed 5 times with 1× washing buffer, and anti-mouse IgG antibody-HRP (1:100) was added and incubated at room temperature for 1 hour. After the incubation, the plate was washed 5 times, and the prepared substrate (substrate A and substrate B were mixed in equal volumes at a ratio of 1:1) was added to the ELISA plate and incubated in the dark for 20 minutes. Finally, 50 μL / well stop solution was added, and the optical absorbance value at OD450nm was read within 20 minutes.

[0194] The results are as follows Figure 5A-5B As shown in the figure, no IgG antibodies to HA were detected in the blank control group (PBS) and the viral vector control group (dTF), while HA-specific antibodies could be detected in the serum of mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2 ( Figure 5A ); Similarly, NP-specific antibodies were detected in the serum of mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2 ( Figure 5B ).

[0195] 3.3 Detection of hemagglutination inhibition (HAI) antibodies in mouse serum

[0196] 21 days after immunization, serum from each group of mice was collected for hemagglutination inhibition test to detect the titer of hemagglutination inhibition antibodies against 7 influenza A viruses. The test steps are as follows:

[0197] (1) Preparation of 1% chicken blood red blood cells: fresh turkey blood was centrifuged at 1500 rpm / min at 4°C for 10 minutes, with the lowest acceleration and deceleration rates set to prevent red blood cell rupture, and washed three times with PBS; the supernatant was slowly aspirated with a disposable pipette and discarded; after the final wash, the blood was centrifuged again at 3000 rpm / min for 10 minutes, the supernatant was discarded, and the thick packed red blood cells were retained. An appropriate volume of red blood cells was aspirated and a red blood cell suspension with PBS was prepared to a working concentration of 1%;

[0198] (2) Preparation of 4HA standard virus solution: Calculate the dilution ratio based on the measured virus HA titer: For example, if the HA titer is 26 = 1:64, then dilute 64 / 8 = 8 times to prepare a standard virus solution containing 8 hemagglutination units per 50 uL (4 hemagglutination units per 25 uL);

[0199] (3) 4HA standard virus solution test: Take 100uL of the prepared standard virus solution and place it in the first column of a 96-well U-shaped plate (two replicates), add 50uL / well PBS to columns 2 to 6, and pipette 50uL / well from column 1 to the next column to mix thoroughly. Discard column 5 and use column 6 as a negative control; add 50uL / well 1% chicken blood red blood cells and let it stand for 30 minutes; refer to the negative control wells in column 6. If wells 1, 2, 3, and 4 are "positive" and well 5 is negative, it means that the virus dilution is accurate and can be used for subsequent experiments;

[0200] (4) Serum treatment: serum samples were mixed with receptor-destroying enzyme (RDE) at a ratio of 1:3, reacted at 37°C for 16 hours, and then inactivated at 56°C for 30 minutes. Concentrated chicken red blood cells were used to remove nonspecific agglutination and stored at 4°C until use. At this point, the serum had been diluted 4 times;

[0201] (5) Add 40 μL / well PBS and 10 μL / well treated serum to the first column of a 96-well U-shaped plate, add 25 μL / well PBS to columns 2 to 12, and pipette 25 μL / well from column 1 to the next column. Discard 25 μL / well liquid from column 11. Column 12 serves as a positive control. At this point, the serum in the first well has been diluted 20 times.

[0202] (6) Add 25 μL of diluted 4HA standard virus antigen to each well, mix thoroughly, and incubate at room temperature for 1 hour;

[0203] (7) Add 50 μL of 1% chicken red blood cells to each well and let it stand for 20-30 minutes;

[0204] (8) Determination of results: Tilt the 96-well U-shaped plate at 45° and observe the hemagglutination tear drop reaction. The HAI titer is the reciprocal of the highest dilution of the standard antiserum that completely inhibits hemagglutination. If hemagglutination does not occur in the first well but occurs in the second well, the HAI titer is 1:20, and so on.

[0205] HAI titer>40 indicates good neutralization effect. Figure 6 As shown, no neutralizing antibodies were detected in the blank control group (PBS) and the viral vector control group (dTF), while the serum of mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2 could produce hemagglutination-inhibiting antibodies against 7 influenza A viruses.

[0206] 3.4 Detection of cellular immune response in mice

[0207] 21 days after immunization, spleens of mice in each group were collected, ground, and separated using red blood cell lysis buffer to prepare splenic mononuclear cell suspensions, which were then detected using an ELISPOT kit (mouse IFN-γ pre-coated ELISPOT kit, Dakoway Biotechnology Co., Ltd.). The specific method is as follows:

[0208] Mouse spleen cells were isolated, and the PVDF 96-well plate was removed and washed five times with 200 μL / well PBS. 200 μL / well 1640 complete medium was added and the plate was blocked at 37°C for 2 hours. The medium was discarded and 200 μL / well freshly isolated mouse spleen cells (1×10 6 / mL); then add 10uL of the corresponding stimulus mixture (HA peptide, NA peptide, NP peptide, M2 peptide), the final peptide concentration is 5ug / mL, the total protein concentration is 10ug / mL, and a PMA positive control and a PBS negative control are set at the same time, and incubate for 24 hours; after the incubation, discard the cell suspension, wash 5 times with 200uL PBST / well, and pat dry; add 100uL HRP-Anti-mouse-IFN-γ antibody (diluted with 5% inactivated FBS / PBST) and incubate for 2 hours; wash the plate 5 times with 200uL PBST / well, pat dry; add 100uL / well TMB colorimetric substrate and incubate at room temperature in the dark for 10-30 minutes; after the incubation, discard the substrate, wash 3 times with sterile water, air dry, and use an ELISPOT plate reader system to detect the number of spots.

[0209] Among them, the HA peptide consists of multiple peptide segments including IYSTVASSL (SEQ ID NO: 26), FERFEIFPK (SEQ ID NO: 27), LYQNADAYV (SEQ ID NO: 28), LYEKVRSQL (SEQ ID NO: 29), GLFGAIAGFIEGG (SEQ ID NO: 30), and TYNAELLVL (SEQ ID NO: 31);

[0210] The NA peptide consists of multiple peptide segments: SGSFVQHPELTGL (SEQ ID NO: 32), TFFLTQGAL (SEQ ID NO: 33), TIWTSGSSI (SEQ ID NO: 34), KYNGIITDT (SEQ ID NO: 35), SSISFCGV (SEQ ID NO: 36), and FCGVNSDTV (SEQ ID NO: 37);

[0211] The NP peptide consists of multiple peptide segments: AYERMCNILKGK (SEQ ID NO: 38), TYQRTRALVRTGMDP (SEQ ID NO: 39), RFYIQMCTEL (SEQ ID NO: 40), NDATYQRTRALVRTG (SEQ ID NO: 41), RLIQNSITIERMVLSAFDERRN (SEQ ID NO: 42), KFQTAAQRAMMDQVR (SEQ ID NO: 43), and TYQRTRALV (SEQ ID NO: 44);

[0212] The M2 peptide consists of MSLLTEVETPIRNEW (SEQ ID NO:45), TEVETPIRNEWGCRC (SEQ ID NO:46), TPIRNEWGCRCNGSS (SEQ ID NO:47), NEWGCRCNGSSDPLA (SEQ ID NO:48), CRCNGSSDPLAIIIG (SEQ ID NO:49), GSSDPLAIIIGILHL (SEQ ID NO:50), PLAIIIGILHLILWI (SEQ ID NO:51), IIGILHLILWILDRL (SEQ ID NO:52), LHLILWILDRLFKC (SEQ ID NO:53), LWILDRLFFKCIYRR (SEQ ID NO:54), DRLFFKCIYRRFKYG (SEQ ID NO:55), FKCIYRRFKYGLKGG (SEQ ID NO:56), YRRFKYGLKGGPSTE(SEQ ID NO:57), KYGLKGGPSTEGVPK(SEQ ID NO:58), KGGPSTEGVPKSMRE(SEQ ID NO:59), STEGVPKSMREEYRK(SEQ ID NO:60), VPKSMREEYRKEQQS(SEQ ID NO:61), MREEYRKEQQSAVDA(SEQ ID NO:62), YRKEQQSAVDADDGH(SEQ ID NO:63), QQSAVDADDGHFVSI(SEQ ID NO:64), VDADDGHFVSIELE(SEQ ID NO:65), SLLTEVETPIRNEWGC (SEQ ID NO:66), IYRRFKYGL (SEQ ID NO:67), VETPIRNEW (SEQ ID NO:68). It consists of multiple peptide segments.

[0213] The results are as follows Figure 7 As shown in the results, compared with the blank control group (PBS) and the viral vector control group (dTF), IFNγ expression was detected after immunization with the recombinant vaccinia virus TK-HANA / F4L-NPM2 upon stimulation with HA peptide, NA peptide, NP peptide and M2 peptide, indicating that immunization with the recombinant vaccinia virus TK-HANA / F4L-NPM2 can induce influenza antigen-specific T cell immune response.

[0214] 3.5 Mouse challenge immune protection experiment

[0215] Twenty-one days after immunization, each group of mice was intranasally inoculated with a lethal dose (5MLD50) of influenza A virus A / PR / 8 / 1934 (H1N1) or A / Aichi / 2 / 1968 (H3N2) at 50 μL to establish a challenge model. The dynamic changes in mouse body weight and survival were monitored for 14 consecutive days after challenge. When the individual body weight loss was greater than 20% of the initial value, humane endpoint killing was performed in accordance with animal ethics guidelines.

[0216] The monitoring results of mouse weight changes and survival status are as follows Figures 8A-8D As shown in the figure, both the blank control group (PBS) and the viral vector control group (dTF) mice showed significant weight loss and decreased survival rate; while the weight and survival rate of mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2 did not change significantly compared with normal mice after infection.

[0217] In addition, on day 4 after challenge (peak viral replication period), lung tissue samples were collected from mice aseptically for determination of lung viral titer. The specific method is as follows:

[0218] (1) On the fourth day after infection, the lung tissues of mice were collected and placed in a centrifuge tube containing 1 mL of PBS and magnetic beads. The tissues were lysed by shaking in Tissue-lysis II and centrifuged to obtain the supernatant.

[0219] (2) Subculture well-grown MDCK cells, digest with 0.25% Trypsin-EDTA to single cells and count them, and dilute with virus diluent to 1.5×10 5 / mL, add 100 μL / well cells into a 96-well cell culture plate and culture in a cell culture incubator for 1 hour;

[0220] (3) 150 μL of lung tissue extract was added to the first row of a 96-well plate, with four replicates for each sample. 135 μL of virus dilution was added to the remaining wells, followed by ten-fold serial dilutions. The last row served as a negative control.

[0221] (4) Remove the 96-well MDCK cell plate, wash it three times with 100 μL / well PBS, add 100 μL / well DMEM medium, take 50 μL of tissue dilution solution into the 96-well MDCK cell culture plate, and culture it in a cell culture incubator for 72 hours;

[0222] (5) 50 μL / well of cell supernatant from 96 wells was added to a U-shaped 96-well plate, and 50 μL / well of 1% turkey red blood cell suspension was added sequentially until the last column, gently flicked to mix, and allowed to stand at room temperature for 1 hour;

[0223] (6) Result determination: Refer to the negative cell control well in column 12 and determine “positive” or “negative” based on the agglutination of turkey blood in the U-shaped 96-well plate. Calculate the TCID50 of the virus according to the Reed-Muench method.

[0224] The results are as follows Figure 9A-9B As shown in the figure, after the mice were challenged with the virus, the virus titer in the lungs of the blank control group (PBS) and the virus vector group (dTF) failed to decrease, while the virus titer in the lungs of mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2 was significantly reduced after the challenge.

[0225] The above results showed that the blank control group (PBS) and the viral vector control group (dTF) did not produce good immune protection against the two influenza A viruses, while mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2 could produce good immune protection against the two influenza A viruses.

[0226] Example 4: Intranasal immunization of mice with recombinant vaccinia virus TK-HANA / F4L-NPM2

[0227] This example evaluated the mucosal immune antibodies produced by nasal immunization. The rest of the immunization and protection evaluation methods were the same as those of the intramuscular injection immunization in 3.1-3.5 of Example 3.

[0228] The mice were immunized by intranasal drops and divided into three groups, with 5 mice in each group: the first group was a blank control group (PBS); the second group was a viral vector control group (IN-dTF), which was immunized with vaccinia virus dTF lacking the TK and F4L regions; the third group was a recombinant vaccine group (IN-rTF), which was immunized with HA and NA inserted into the TK region, NP and M2 inserted into the F4L region. del29-31 The mice were immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2.

[0229] 4.1 Detection of HA-specific antibodies in mouse nasal / alveolar lavage fluid and HA or NP-specific antibodies in mouse serum

[0230] Twenty-one days after immunization, nasal lavage and alveolar lavage fluid were collected from each group of mice and coated with HA protein (Influenza AH1N1 (A / California / 04 / 2009) Hemagglutinin / HA0 Protein, Sino Biological Technology Co., Ltd., Beijing) on ​​ELISA plates. The detection steps were as follows:

[0231] The ELISA plate required for detection (coated with influenza A virus HA protein) was first washed 5 times with 300 μL 1× washing buffer, and then the mouse lavage fluid to be tested was added with a gradient dilution (4-fold gradient dilution), incubated at room temperature for 2 hours, and then washed 5 times with 1× washing buffer; anti-mouse IgA antibody-HRP (1:100) was added and incubated at room temperature for 1 hour; after the incubation, the plate was washed 5 times, and the prepared substrate (substrate A and substrate B were mixed in equal volumes at a ratio of 1:1) was added to the ELISA plate and incubated in the dark for 20 minutes; finally, 50 μL / well stop solution was added, and the optical absorbance value at OD450nm was read within 20 minutes.

[0232] The results are as follows Figures 10A-10B As shown, no HA IgA antibodies were detected in the blank control group (PBS) and the viral vector control group (IN-dTF), while HA-specific antibodies were detected in the lavage fluid of mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2.

[0233] Furthermore, the IgA-specific antibodies to HA in the mouse serum were detected and compared with the intramuscular injection immunization, and the detection steps were the same as above.

[0234] The results are as follows Figure 10C As shown in the data, no HA IgA antibodies were detected in the blank control group (PBS) and the viral vector control group (IN-dTF), while HA-specific antibodies were detected in the serum of mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2, and the IgA level in the serum of mice immunized with intranasal drops was higher than that of mice immunized with intramuscular injection, indicating that intranasal immunization with the recombinant vaccinia virus TK-HANA / F4L-NPM2 can effectively induce influenza-specific mucosal immune responses.

[0235] In addition, IgG specific antibodies to HA or NP in mouse serum were detected and compared with intramuscular injection immunization. The detection steps were the same as 3.2 in Example 3.

[0236] The results are as follows Figure 10D-10E As shown, HA- and NP-specific antibodies were detected in the sera of mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2.

[0237] 4.2 Detection of hemagglutination inhibition antibodies in mouse serum

[0238] The results are as follows Figure 11 As shown in the figure, an HAI titer > 40 indicates a good neutralization effect. No neutralizing antibodies were detected in the blank control group (PBS) and the virus vector control group (IN-dTF), while the serum of mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2 could produce hemagglutination inhibition antibodies against 5 influenza A viruses.

[0239] 4.3 Detection of cellular immune response in mice

[0240] The results are as follows Figure 12 As shown in the results, compared with the blank control group (PBS) and the viral vector control group (IN-dTF), IFNγ expression was detected after immunization with the recombinant vaccinia virus TK-HANA / F4L-NPM2 upon stimulation with HA peptide, NA peptide, NP peptide and M2 peptide, indicating that intranasal immunization with the recombinant vaccinia virus TK-HANA / F4L-NPM2 can induce influenza antigen-specific T cell immune responses.

[0241] 4.4 Mouse challenge immune protection experiment

[0242] The monitoring results of mouse weight changes and survival status are as follows Figures 13A-13D As shown in the figure, both the blank control group (PBS) and the viral vector control group (IN-dTF) mice showed significant weight loss and decreased survival rate; while the weight and survival rate of mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2 did not change significantly compared with normal mice after infection.

[0243] In addition, on the 4th day after infection (the peak period of viral replication), mouse lung tissue samples were collected aseptically to measure the lung virus titer.

[0244] The results are as follows Figures 14A-14B As shown in the figure, after the mice were challenged with the virus, the virus titer in the lungs of the blank control group (PBS) and the virus vector group (IN-dTF) failed to decrease, while the virus titer in the lungs of mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2 was significantly reduced after the challenge.

[0245] The above results showed that the blank control group (PBS) and the viral vector control group (IN-dTF) did not produce good immune protection against the two influenza A viruses, while mice immunized with the recombinant vaccinia virus TK-HANA / F4L-NPM2 by intranasal drops could produce good immune protection against the two influenza A viruses.

[0246] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application in any other manner. Any person skilled in the art may utilize the above disclosed technical content to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the present application.

Claims

1. A recombinant vaccinia virus, wherein: The recombinant vaccinia virus is obtained by integrating the coding sequences of influenza A virus NP, M2, HA and NA antigen proteins into the genome of the Tiantan strain of vaccinia virus.

2. The recombinant vaccinia virus according to claim 1, wherein Integrating the coding sequences of influenza A virus NP and M2 antigen proteins at the F4L gene position in the genome of the vaccinia virus Tiantan strain, and integrating the coding sequences of influenza A virus HA and NA antigen proteins at the TK gene position; The coding sequences of influenza A virus NP and M2 antigen proteins are connected with their 5' ends facing each other and are respectively connected downstream of different promoters; the coding sequences of influenza A virus HA and NA antigen proteins are connected with their 5' ends facing each other and are respectively connected downstream of different promoters.

3. The recombinant vaccinia virus according to claim 2, wherein: The amino acid sequence of the influenza A virus NP antigen protein is shown in SEQ ID NO: 3; The amino acid sequence of the influenza A virus HA antigen protein is shown in SEQ ID NO: 8; The amino acid sequence of the influenza A virus NA antigen protein is shown in SEQ ID NO: 10; The influenza A virus M2 antigen protein is selected from: (i) having the amino acid sequence shown in SEQ ID NO: 5; or (ii) comprising one or more engineered M2 proteins based on mutations of a reference influenza A virus M2 antigen protein, the amino acid sequence of which is shown in SEQ ID NO: 5, wherein the mutation is a deletion of amino acids 29 to 31 in the transmembrane region.

4. The recombinant vaccinia virus according to claim 2 or 3, wherein The coding sequence of the influenza A virus NP antigen protein is connected to the downstream of the p7.5 promoter, and the coding sequence of the influenza A virus M2 antigen protein is connected to the downstream of the p11 promoter; and The coding sequence of the influenza A virus NA antigen protein is connected to the downstream of the p11 promoter, and the coding sequence of the influenza A virus HA antigen protein is connected to the downstream of the p7.5 promoter.

5. The recombinant vaccinia virus according to claim 4, wherein The coding sequence of the influenza A virus NP antigen protein, the p7.5 promoter, the p11 promoter, and the coding sequence of the influenza A virus M2 antigen protein are connected in series; The sequence after tandem connection is shown in SEQ ID NO:

1.

6. The recombinant vaccinia virus according to claim 4, wherein The coding sequence of the influenza A virus HA antigen protein, the p7.5 promoter, the p11 promoter, and the coding sequence of the influenza A virus NA antigen protein are connected in series; The sequence after tandem connection is shown in SEQ ID NO:

2.

7. The method for constructing a recombinant vaccinia virus according to any one of claims 1 to 6, wherein: The method comprises: The coding sequences of influenza A virus NP, M2, HA and NA antigenic proteins were integrated into the genome of vaccinia virus Tiantan strain by homologous recombination; Preferably, the coding sequences of influenza A virus NP and M2 antigen proteins are integrated into the F4L gene position of the vaccinia virus Tiantan strain genome by homologous recombination, and the coding sequences of influenza A virus HA and NA antigen proteins are integrated into the TK gene position of the vaccinia virus Tiantan strain genome by homologous recombination.

8. An influenza vaccine based on a vaccinia virus vector expressing influenza A virus antigen, wherein: The influenza vaccine comprises the recombinant vaccinia virus according to any one of claims 1 to 6.

9. The influenza vaccine according to claim 8, wherein The influenza vaccine is administered through one or more delivery methods including respiratory tract aerosol inhalation, nasal drops, oral administration, direct injection, and mucosal administration.

10. Use of the recombinant vaccinia virus according to any one of claims 1 to 6 in the preparation of an influenza vaccine.

11. Use of the recombinant vaccinia virus according to any one of claims 1 to 6 or the influenza vaccine according to claim 8 or 9 in the preparation of a product for preventing and / or treating influenza.

12. Use of the recombinant vaccinia virus according to any one of claims 1 to 6 in the preparation of a detection reagent or kit for diagnosing influenza.

13. Use of the recombinant vaccinia virus according to any one of claims 1 to 6 as an immunogen in the preparation of influenza A virus antibodies.

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