Broad-spectrum influenza a vaccine immunogen composition and use thereof

Abstract

A broad-spectrum influenza A vaccine immunogen composition and the use thereof. Provided is a nucleic acid molecule encoding an immunogenic peptide, the immunogenic peptide comprising: tandem M2e peptide fragments, an HA2 peptide fragment derived from influenza virus H7N9 subtype; a multimerized motif peptide fragment, and optionally, one or more linker peptide fragments independently located between the peptide fragments, the nucleic acid molecule comprising encoding nucleic acids of the peptide fragments. The broad-spectrum influenza A vaccine can induce organisms to generate antigen-specific immune responses, and can generate a high-titer antibody just in 14 days after the last vaccination, which can completely prevent attacks from various influenza A virus subtypes.

Description

Broad-spectrum influenza A vaccine immunogen composition and application Technical Field

[0001] This application belongs to the field of vaccines and biotechnology. Specifically, this application relates to a broad-spectrum influenza A vaccine immunogen composition and its application. Background Art

[0002] Influenza is an acute respiratory infectious disease caused by influenza virus infection. Influenza virus is a negative-sense, single-stranded RNA virus of the Orthomyxoviridae family, with a genome consisting of eight gene segments. Based on their nucleoprotein (NP) and matrix protein (M), influenza viruses can be divided into four types: A, B, C, and D. Type A influenza virus can infect humans, poultry, pigs, and other organisms, causing disease and often seasonally circulating in humans with the potential for pandemics. Type B influenza virus infects only humans, generally causing seasonal and regional epidemics. Type C influenza virus infection generally causes mild or no symptoms. Type D influenza virus generally does not infect humans. Type A influenza virus can be further divided into multiple subtypes based on its surface proteins, hemagglutinin (HA) and neuraminidase (NA). Recombination between subtypes can occur, giving rise to new subtypes or strains. Furthermore, due to the low fidelity of the replication enzyme, influenza virus replication is prone to mutations, which can also lead to the emergence of new strains. Influenza B is mainly divided into two lineages based on its HA protein, Victoria and Yamagata.

[0003] Globally, seasonal influenza infection in humans causes 3 to 5 million severe cases and 290,000 to 650,000 deaths annually. Influenza viruses are transmitted through the respiratory tract and can cause symptoms such as high fever, body aches, fatigue, and cough, which can be life-threatening in severe cases. Currently, the strains that cause seasonal influenza in humans are primarily H1N1, H3N2, and influenza B viruses.

[0004] Vaccination is the primary way to prevent influenza virus infection. Currently available influenza vaccines, in terms of vaccine strain composition, include trivalent and quadrivalent vaccines. The trivalent vaccine includes one strain from H1N1, H3N2, and type B, while the quadrivalent vaccine includes two strains from H1N1, H3N2, and type B. Vaccine manufacturing processes primarily include split vaccines, recombinant protein vaccines, and attenuated vaccines. The WHO currently predicts and publishes the strains prevalent in the northern and southern hemispheres each year for the flu season. All influenza vaccines must be produced using the WHO-recommended vaccine strains or sequences. Due to inaccuracies in predictions, when the predicted vaccine strain differs from the prevalent strain that year, the protective efficacy of the influenza vaccine can be less than 20%. Due to the rapid changes in prevalent strains, existing influenza vaccines require annual administration, increasing costs and reducing patient compliance. Overcoming the high mutation rate of influenza viruses is a core issue in influenza vaccine development.

[0005] The potency of commercially available influenza vaccines is typically tested using the hemagglutination inhibition (HI) assay, which evaluates the ability of post-immunization serum to inhibit hemagglutination of the influenza virus HA protein. The titer of HI antibodies reflects the protective effect of the vaccine. However, most HI antibodies target the HA protein head, which has a high mutation rate. Once a mutation occurs, the protective antibodies become ineffective, rendering the vaccine ineffective.

[0006] In order to overcome the problem of influenza high mutation causing vaccine failure, it is necessary to be able to induce immunity against the conserved parts of the influenza virus in order to provide a vaccine with broad-spectrum protection. Compared with the highly mutated head region, the stem region of the HA protein is more conservative and is a good candidate for an immunogen. In addition, the extracellular domain (M2e) of the influenza virus M2 protein is highly conserved among multiple subtypes of influenza viruses and is also a good candidate for an immunogen. However, the immunogenicity of the HA stem region and the M2e protein is low, and it is difficult to induce a high-titer immune response through existing vaccination or natural infection. To develop a broad-spectrum influenza vaccine targeting these two regions, it is necessary to improve the immunogenicity of the HA stem region and the M2e protein.

[0007] mRNA (messenger ribonucleic acid) vaccines are an emerging vaccine technology that has achieved promising results in the development of novel coronavirus vaccines, with several companies successfully marketing their COVID-19 mRNA vaccines. The production of mRNA vaccines does not rely on cells, but rather is produced through in vitro transcription and synthesis. Due to its design and process advantages, mRNA vaccines can be designed and produced simply by obtaining the sequence. This allows mRNA vaccine production to skip the traditional strain / cell line culture and expansion process, simplifying the production process and enabling large-scale production in a short period of time. Furthermore, because the entire production process is free of live virus and the in vitro transcription and purification process is cell-free, there are no biosafety risks such as virus leakage, and there is no risk of residual host cell nucleic acid or protein in the finished vaccine. The mRNA production process is easy to scale up quickly, and the vaccine is safe and effective, with high application value.

[0008] T cell immune responses play a key role in clearing viral infections. T cells are known to respond very quickly to secondary exposure to an immunogen. When the body has pre-existing T cell immunity, upon encountering a pathogen, T cells can rapidly respond and proliferate, thereby rapidly controlling the infection. Furthermore, T cell immune responses play a crucial role in clearing long-standing pathogens.

[0009] In summary, the technical problem of how to develop a broad-spectrum and highly effective influenza vaccine based on the conserved immunogen part of influenza, which can induce a high-intensity immune response and has the advantages of rapid production, needs to be solved urgently.

[0010] Summary of the Invention

[0011] In the present invention, in response to the problems existing in the prior art, an mRNA molecule is provided, which encodes the stem region (HA2) of the influenza hemagglutinin protein and the extracellular region (M2e) of the M2 protein, and is connected to a multimerization motif. The mRNA can efficiently express the stem region (HA2) and extracellular region (M2e) of the H7N9 subtype hemagglutinin protein and form a multimer, which can be used for broad-spectrum prevention of influenza A after immunization. On the basis of the mRNA, the present application further provides a composite mRNA comprising the mRNA and connected to other components (such as T cell antigens, interleukin IL-21, etc.) using a connecting element.

[0012] In some aspects of the present application, a nucleic acid molecule encoding an immunogenic peptide is provided, wherein the immunogenic peptide comprises: (a) a tandem M2 protein extracellular region (M2e) peptide segment; (b) a hemagglutinin protein stem region extracellular domain (HA2) peptide segment derived from the H7N9 influenza virus subtype; (c) a multimerization motif peptide segment; (d) optionally, one or more linker peptide segments independently located between each peptide segment; (e) optionally, one or more other peptide segments, such as T cell antigen peptide segments; wherein the nucleic acid molecule comprises the encoding nucleic acid of each peptide segment.

[0013] In some aspects of the present application, an immunogenic peptide is provided, comprising: (a) a tandem M2 protein extracellular region (M2e) peptide segment; (b) a hemagglutinin protein stem region extracellular domain (HA2) peptide segment derived from an H7N9 influenza virus subtype; (c) a multimerization motif peptide segment; (d) one or more linker peptide segments optionally independently located between the peptide segments; and (e) one or more other peptide segments, such as T cell antigen peptide segments. In some embodiments, the immunogenic peptide is encoded by a nucleic acid molecule of the present application.

[0014] In some aspects of the present application, a vector or host cell is provided, which comprises the nucleic acid molecule of the present application.

[0015] In some aspects of the present application, an influenza vaccine is provided, comprising: (i) one or more nucleic acid molecules, immunogenic peptides and / or vectors of the present application; and (ii) an immunologically acceptable carrier, delivery system or adjuvant.

[0016] In some aspects of the present application, provided is the use of the nucleic acid molecule, immunogenic peptide, vector and / or influenza vaccine of the present application in preparing a product for preventing and / or treating influenza.

[0017] In some aspects of the present application, a method for preventing and / or treating influenza A is provided, comprising administering to a subject in need thereof a product comprising the nucleic acid molecule, immunogenic peptide, vector and / or influenza vaccine of the present application.

[0018] In some aspects of the present application, provided are the nucleic acid molecules, immunogenic peptides, vectors and / or influenza vaccines of the present application for use in preventing and / or treating influenza A.

[0019] Those skilled in the art may arbitrarily combine the aforementioned technical solutions and technical features without departing from the inventive concept and protection scope of the present disclosure. Other aspects of the present disclosure will be apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present disclosure will be further described below with reference to the accompanying drawings, which are only for illustrating the embodiments of the present disclosure and are not intended to limit the scope of the present disclosure.

[0021] Figure 1 shows a schematic diagram of the construction of the immunogen 8 tandem repeats M2e (8M2e) or 8M2e-HA (M8H) or 8M2e-HA-collagen (M8HC) (A); and the expression verification of in vitro transcribed mRNA transfected into HEK293 cells (B).

[0022] Figure 2 shows the mouse immunization schedule (A); the levels of humoral immune responses against M2e protein (B) and H7N9 HA protein (C) in mice after priming (i.e., "first dose") and boosting immunizations with mRNA expressing 8M2e, M8H, or M8HC; and the body weight (D, E) and survival (F, G) of mice after infection with lethal influenza A H1N1 (pdmH1N1: A / California / 04 / 2009) and H3N2.

[0023] Figure 3 shows a schematic diagram of the construction of the immunogen 8M2e-HA-collagen (M8HC) or 4M2e-HA-collagen (M4HC) (A); expression verification of mRNA-M4HC in HEK293 cells transfected (B); the levels of humoral immune responses against M2e protein (C) and H7N9 HA protein (D) in mice after prime and boost immunization with mRNA expressing M8HC or M4HC, as well as the body weight (E, F) and survival (G, H) of mice after infection with lethal influenza H1N1 (A / California / 04 / 2009) and H3N2.

[0024] Figure 4 shows a schematic diagram of the construction of the immunogen 4M2e-HA-collagen (M4HC) or 4M2e-HA-foldon (M4HF) (A); expression verification of mRNA-M4HF transfected HEK293 cells (B); the level of humoral immune response against M2e protein (C) and H7N9 HA protein (D) generated in mice after prime and boost immunization with mRNA expressing M4HC or M4HF; body weight (E, F) and survival (G, H) of mice after infection with lethal doses of influenza H1N1 (A / California / 04 / 2009) and H3N2; and the T cell responses to M2e and HA2 peptide libraries 10 days after boost immunization of mice (I, J).

[0025] Figure 5 shows a schematic diagram of the construction of the immunogen 4M2e-HA-foldon-IRES-T (M4HF-T) and T (A).

[0026] Figure 6 shows a schematic diagram of the construction methods of foldon (A) containing only 4M2e and H7N9 HA2 (M4H), containing only H7N9 HA2 and a multimeric sequence (HF), 2M2e-HA-foldon (M2HF) in which the four repeated M2e in M4HF are reduced to two, and 4M2e-H1N1 HA-foldon (M4H1F) and 4M2e-H3N2 HA-foldon (M4H3F) formed by replacing the H7N9-derived HA2 in the designed M4HF with HA2 derived from H1N1 or N3N2, respectively. Specific implementation plan

[0027] The present disclosure relates to the field of vaccines, and in particular to an mRNA vaccine capable of inducing a broad-spectrum anti-influenza A virus immune response and its application.

[0028] All numerical ranges provided herein are intended to expressly include all values ​​falling between the endpoints of the ranges and the ranges therebetween. Features disclosed herein or in the embodiments may be combined. All features disclosed herein may be used in any combination, and each feature disclosed herein may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed herein are merely general examples of equivalent or similar features.

[0029] As used herein, "about" in the context of a numerical value or a range means ±10% of the recited or claimed numerical value or range.

[0030] It should be understood that when a parameter range is provided, the present invention also provides all integers and decimals thereof within the range. For example, "0.1-2.5 mg / day" includes 0.1 mg / day, 0.2 mg / day, 0.3 mg / day, etc. up to 2.5 mg / day.

[0031] As used herein, “containing,” “having,” or “including” encompasses “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of”; “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0032] To address the aforementioned technical problems in the art, the present invention employs a technical solution that provides a nucleic acid molecule encoding a hemagglutinin protein stem region (HA2) of an H7N9 subtype (e.g., strain A / Shanghai / 02 / 2013) and multiple tandem M2 protein extracellular regions (M2e), linked to different multimerization motifs (e.g., collagen / foldon / honeybee melittin / tetrabrachion helical peptide / GCN4). The nucleic acid molecule can be used to efficiently express a corresponding chimeric immunogenic peptide. For example, the nucleic acid molecule can be loaded into an mRNA vector, such that the mRNA efficiently expresses the H7N9 subtype hemagglutinin protein stem region (HA2) and multiple tandem M2 protein extracellular regions (M2e) to form a multimer. This mRNA can be delivered using an appropriate delivery vehicle (e.g., liposome nanoparticle encapsulation) and then used for immunization to prevent or treat infection and pathogenesis caused by various influenza A virus subtypes. The chimeric immunogenic peptides of the present application can also be administered directly to subjects in need.

[0033] Based on the aforementioned technical solutions, the present application further provides an improved solution: providing an mRNA encoding the stem region (HA2) of the hemagglutinin protein and the extracellular region (M2e) of the H7N9 subtype (A / Shanghai / 02 / 2013 strain), linked to different multimerization motifs (collagen / foldon / honeybee melittin / tetrabrachion helical peptide / GCN4), and then linked to a T cell antigen or cytokine. Finally, the mRNA is loaded into an mRNA vector, or the M2e-HA multimerization sequence and T cell antigen are loaded separately. Upon entry into cells, the mRNA efficiently expresses the stem region (HA2) and extracellular region (M2e) of the H7N9 subtype hemagglutinin protein, forming multimers and simultaneously expressing T cell antigens or cytokines. This mRNA can be delivered using an appropriate delivery vehicle (e.g., liposome nanoparticle encapsulation) and then used for immunization to prevent or treat infection and pathogenesis caused by various subtypes of influenza A virus. The chimeric immunogenic peptides of the present application can also be administered directly to a subject in need thereof.

[0034] The broad-spectrum influenza A vaccine designed by the present invention can induce high-titer antibodies with protective effects 7 days after completing the full vaccination (e.g., intramuscular injection). Three weeks after completing the full vaccination, mice are challenged with a lethal dose. The immune response induced by the vaccine can completely resist the attack of lethal doses of H1N1 and H3N2 viruses. The broad-spectrum influenza A vaccine of the present invention (e.g., RNA vaccine) can induce effective protective antibodies after two injections. The broad-spectrum influenza A vaccine of the present invention has good immunogenicity and forms a strong immune protection after immunization. It has low production cost and simple preparation technology. It is suitable for rapid, efficient, and large-scale vaccine preparation and has great development potential. In addition, the introduction of T cell antigens in the improved scheme can induce mice to produce specific T cells that can respond to multiple influenza viruses and secrete cytokines. Three weeks after completing the full vaccination, mice are challenged with a lethal dose. The humoral and cellular immune responses induced by the vaccine enable the mice to survive the attack of lethal doses of H1N1 and H3N2 viruses. The RNA vaccine of the present invention can induce effective humoral and cellular immunity after two injections, which is conducive to the rapid elimination of influenza A virus.

[0035] Immunogenic peptides and their encoding molecules

[0036] As used herein, the term "immunogenic peptide" refers to a peptide capable of inducing an immune response against multiple influenza A viruses, which comprises the following parts: (a) a tandem M2 protein extracellular region (M2e) peptide segment; (b) a hemagglutinin protein stem region extracellular domain (HA2) peptide segment derived from the H7N9 influenza virus subtype; (c) a multimerization motif peptide segment; (d) optionally, one or more linker peptide segments independently located between each peptide segment; (e) optionally, one or more other peptide segments, such as a T cell antigen peptide segment.

[0037] As used herein, the term "linked" has a broad meaning and may include various means such as fusion, covalent linkage, coupling, etc.

[0038] When referring to sequence identity or homology to describe or qualify an amino acid sequence or a nucleotide sequence, the amino acid sequence or nucleotide sequence has a homology or sequence identity of greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, or greater than or equal to 99%, respectively, compared to the reference sequence.

[0039] Immunogenic peptides may also include variant forms thereof, such as deletions, insertions, and / or substitutions of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids, and additions of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the art, substitutions with amino acids having similar or similar properties generally do not alter the function of the protein or polypeptide. For another example, additions of one or more amino acids to the C-terminus and / or N-terminus generally do not alter the function of the protein or polypeptide.

[0040] Immunogenic peptides can be produced by recombinant expression under appropriate circumstances and conditions, for example, by the encoding nucleic acid molecules, vectors, and host cells disclosed herein; they can also be obtained by chemical synthesis, etc., as long as they have the desired amino acid sequence, immunogenicity, and reactivity.

[0041] As used herein, the terms "immunogenic peptide encoding molecule," "coding sequence," and the like are used interchangeably to refer to nucleic acid molecules encoding the immunogenic peptides described herein.

[0042] In some embodiments, the nucleic acid molecule can be selected from, for example: (i) a nucleotide molecule having a nucleotide sequence as shown in SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, or SEQ ID NO: 42; (ii) a molecule that hybridizes with (i) under stringent conditions; (iii) a nucleotide molecule that is homologous or similar to the sequence in (i) and can express a functional immunogenic peptide, for example, each segment thereof is homologous to SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, or SEQ ID NO: 42. The nucleotide sequence shown in NO:42 has a homology or sequence identity of higher than or equal to 80%, higher than or equal to 85%, higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98%, or higher than or equal to 99% with the sequence in (i) or (ii) respectively, or a nucleic acid molecule that has a homology or sequence identity of higher than or equal to 80%, higher than or equal to 85%, higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98%, or higher than or equal to 99% with the sequence in (i) or (ii) and is capable of expressing a functional immunogenic peptide; (iv) a nucleic acid molecule that has one or more nucleotides replaced, deleted or added in the nucleotide sequence defined in (i) or (ii) and is capable of expressing a functional immunogenic peptide.

[0043] As used herein, the term "stringent conditions" refers to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 50%, preferably greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85% or greater than 90%, and more preferably greater than 95%.

[0044] The full-length nucleotide sequences disclosed herein or fragments thereof can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed herein, and amplified using a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template to obtain the relevant sequence. When the sequence is long, two or more PCR amplifications are often required, followed by splicing the fragments amplified in the correct order.

[0045] In some embodiments, the M2e peptide segment in the immunogenic peptide is derived from one or more influenza virus strains selected from the group consisting of H1N1, H2N2, H3N2, H3N8, H5N1, H5N2, H5N6, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2, H10N8, and H11N2 subtype strains. In some embodiments, the M2e peptide segment is derived from an influenza virus strain whose host is an animal selected from the group consisting of mammalian (e.g., human, non-human primate, pig, horse, cattle, sheep, bat, sea lion, seal, rat, mouse, ferret), avian (e.g., poultry or wild bird), or commensal (e.g., zoonotic, avian, or porcine) influenza virus strains. In some embodiments, each M2e region in the M2e peptide segment is derived from the same or different sources.

[0046] In some embodiments, the M2e peptide segment comprises 2 to 12 tandem M2e regions, preferably 4 to 8 tandem Me2 regions, such as 4, 5, 6, 7, 8, 9, 10, 11 or 12 tandem Me2 regions. In some embodiments, the M2e regions in the M2e peptide segment are directly connected or connected through a linker, such as the linker independently selected from: a flexible linker, a rigid linker and a cleavable linker, such as AAA, (G4S) n (e.g. (G4S)3), GSAGSAAGSGEF, (Gly) n 、(EAAAK) n , EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, wherein n is an integer between 2 and 10 (eg, n=3). In some embodiments, the linker comprises one or more repeating segments selected from the group consisting of SEQ ID NOs: 45-50.

[0047] In some embodiments, the M2e region comprises the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleic acid encoding the M2e region comprises the nucleotide sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, or a nucleotide sequence having at least 80% sequence identity thereto (e.g., a sequence obtained by codon optimization or substitution of one or more nucleotides in the aforementioned nucleotide sequences).

[0048] In some embodiments, the M2e peptide segment has an amino acid sequence as set forth in SEQ ID NO: 21 or SEQ ID NO: 23, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleic acid encoding the M2e peptide segment has a nucleotide sequence as set forth in SEQ ID NO: 22 or SEQ ID NO: 24, or a nucleotide sequence having at least 80% sequence identity thereto (e.g., a sequence obtained by codon optimization or one or more nucleotide substitutions in the aforementioned nucleotide sequence).

[0049] In some embodiments, the HA2 peptide segment is derived from an H7N9 subtype strain, such as the A / Shanghai / 02 / 2013 strain or the A / Anhui / 01 / 2013 strain. In some embodiments, the HA2 peptide segment comprises one or more mutations compared to a native HA2 peptide segment, such as an N82 mutation at amino acid position 82, preferably an N82R, N82K, N82D, or N82E mutation; and an N154 mutation at amino acid position 154, preferably an N154H mutation. In some embodiments, the HA2 peptide segment is linked to a partial fragment of HA1, such as an HA2 peptide segment that anchors the HA protein to the stem of the viral lipid envelope and a partial HA1 peptide segment. In some embodiments, the HA2 peptide segment has the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence that has at least 80% sequence identity thereto. In some embodiments, the nucleic acid encoding the HA2 peptide segment has the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence (e.g., a sequence obtained by codon optimization or one or more nucleotide substitutions of the aforementioned nucleotide sequence).

[0050] In some embodiments, the multimerization motif peptide segment is selected from the group consisting of: collagen, the trimerization motif Foldon of T4 phage fibrin, tetrabrachion helical peptide, and GCN. In some embodiments, the multimerization motif peptide segment has an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence. In some embodiments, the nucleic acid encoding the multimerization motif peptide segment has a nucleotide sequence as shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence (e.g., a sequence obtained by codon optimization or substitution of one or more nucleotides in the aforementioned nucleotide sequence).

[0051] In some embodiments, the immunogenic peptide further comprises: (e) one or more other peptide segments. In some embodiments, the other peptide segments include one or more peptide segments selected from the group consisting of: T cell antigen peptide segments, transferrin, FF peptide, cyclic peptide nanotubes, and IL-21.

[0052] In some embodiments, the immunogenic peptide may include other moieties that are further linked, for example, to enhance the stability of the immunogenic stem or fusion protein, improve neutralizing antibody responses, form multimers, increase cellular responses, etc. In some embodiments, the other moieties may include, but are not limited to, proteins of viral or host origin, T cell antigens, transferrin (Fn), IL-2, IL-7, IL-12, IL-18, IL-21, GM-CSF, CD40L, CD40 stimulating antibodies, PD-1 and PD-L1 antibodies, CTLA4 antibodies, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera toxin and its subunits, bacterial flagellin, FimH, etc.

[0053] In some embodiments, the T cell antigen comprises an amino acid sequence as shown in SEQ ID NO: 11, or is encoded by a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 12. In some embodiments, the T cell antigen is as disclosed in Chinese patent application 201880001963.X (application publication number CN 111315407A), for example, the T cell antigen has a sequence as shown in SEQ ID NO: 1 or 2 in the application (i.e., a sequence as shown in SEQ ID NO: 51 and 52 in the present application). In some embodiments, the T cell antigen is obtained using the method disclosed in Chinese patent application 201880001963.X (application publication number CN 111315407A). In some embodiments, the obtained T cell antigen is sequence optimized.

[0054] In some embodiments, the immunogenic peptide may also include elements such as signal peptides, linkers, and molecular tags. For example, a signal peptide element may refer to an amino acid sequence that has the function of guiding protein secretion, localization, and / or delivery, and its length is generally 5-30 amino acids. In some embodiments, the signal peptide element may be selected from: protein self-signal peptide, CD33 protein signal peptide, CD8 protein signal peptide, CD16 protein signal peptide, mouse IgG1 antibody signal peptide, influenza HA protein signal peptide. For example, a connecting peptide sequence (or linker) may refer to a short peptide that plays a role in connecting different elements in the fusion protein herein, and its length is generally 1 to 50 (such as 5 to 50, 5 to 40, 10 to 40) amino acids. Generally, the connecting peptide does not affect or seriously affects the formation of the correct folding and spatial conformation of the peptide of the present invention. In some embodiments, the connecting peptide element can be selected from: (G4S)3 linker, (G4S)n, GSAGSAAGSGEF, (Gly)6, EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, (Gly)8, EGKSSGSGSESKST.

[0055] In some embodiments, the other peptide segments include a T cell antigen peptide segment having an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence.

[0056] In some embodiments, the nucleic acid encoding the T cell antigen peptide comprises the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 80% sequence identity thereto (e.g., a sequence obtained by codon optimization or substitution of one or more nucleotides in the aforementioned nucleotide sequence). In some embodiments, the T cell antigen peptide is linked to a linker, e.g., the linker is selected from the group consisting of IRES, T2A, and P2A. In some embodiments, the T cell antigen peptide is further linked to a degron, e.g., the degron DHFR.

[0057] In some embodiments, the peptide segments of the immunogenic peptides are independently connected directly or through a linker, for example, the linker is independently selected from: a flexible linker, a rigid linker and a cleavable linker, such as AAA, (G4S) n (e.g. (G4S)3), GSAGSAAGSGEF, (Gly) n 、(EAAAK) n , EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, where n is an integer between 2 and 10 (for example, n=3).

[0058] In some embodiments, the immunogenic peptide has an amino acid sequence as shown in SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39, or an amino acid sequence having at least 80% sequence identity thereto.

[0059] In some embodiments, the nucleic acid encoding the immunogenic peptide has a nucleotide sequence as shown in SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 42, or a nucleotide sequence having at least 80% sequence identity thereto (e.g., a sequence obtained by codon optimization or substitution of one or more nucleotides in the aforementioned nucleotide sequence).

[0060] In some embodiments, the nucleic acid molecule is a DNA molecule or an RNA molecule. In some embodiments, the nucleic acid molecule is an mRNA molecule, such as a self-amplifying mRNA (SAM) molecule or a non-replicating mRNA molecule, preferably a SAM molecule; wherein, when the mRNA molecule is a non-replicating mRNA molecule, it further includes a 5'-cap structure and a 3'-poly(A) tail.

[0061] Vectors and host cells

[0062] The present disclosure also relates to vectors containing nucleic acid molecules encoding the immunogenic peptides of the present application, and / or host cells generated by genetic engineering using the vectors. When the constructed nucleic acid molecule is mRNA, the mRNA can be directly used without the need for host cells.

[0063] When host cells are used to produce recombinant immunogenic peptides, the coding sequences disclosed herein can be used to express or produce the recombinant immunogenic peptides using conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally, the following steps are involved:

[0064] (1) transferring the encoding nucleotide molecule of the present disclosure, or a recombinant expression vector containing the nucleotide molecule, into a suitable host cell;

[0065] (2) host cells cultured in a suitable culture medium;

[0066] (3) Isolate and purify proteins or peptides from culture medium or cells.

[0067] In the present disclosure, the term "vector" refers to bacterial plasmids, phages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors well known in the art. The vector can be selected from: mRNA vectors, DNA plasmid vectors, recombinant viral vectors, recombinant bacterial vectors; wherein the mRNA vector is selected from linear, circular and self-replicating vectors; the recombinant viral vector is selected from poxvirus (e.g., Tiantan strain, North American vaccine strain, Wyeth-derived strain, Listeria strain, Ankara-derived strain, Copenhagen strain and New York strain), adenovirus (e.g., adenovirus types 5, 11, 26, 35, 63, 68), adeno-associated virus, herpes simplex virus, measles virus, enterovirus, reovirus, rhabdovirus, flavivirus, influenza virus, parainfluenza virus, respiratory syncytial virus, poliovirus vectors.

[0068] Conventional methods in the art can be used to construct expression vectors containing immunogenic peptide coding sequences and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Expression systems such as pcDNA3.1 vectors, pIRES2-EGFP vectors, AdMax™, and AdC68 can be used in the present disclosure. In addition, the expression vector can contain one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0069] Vectors containing the appropriate DNA sequences described above and appropriate promoters or control sequences can be used to transform appropriate host cells to express proteins or polypeptides. Host cells can be prokaryotes, such as bacteria; lower eukaryotic cells, such as yeast; or higher eukaryotic cells, such as animal cells. Representative examples include Escherichia coli, Streptomyces, and Agrobacterium; fungal cells, such as yeast; and animal cells. In the present disclosure, host cells selected from the group consisting of HEK293, HeLa, CHO, K562, NS0, SP2 / 0, PER.C6, Vero, RD, BHK, HT1080, A549, Cos-7, ARPE-19 and MRC-5 cells; High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5 and Ao38 can be used.

[0070] If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out), centrifugation, osmotic shock, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques, and combinations of these methods.

[0071] Vaccines and immunoconjugates

[0072] Also provided herein is a vaccine comprising the immunogenic peptides, nucleic acid molecules, vectors and / or host cells of the present disclosure, or an immunogenic composition. The vaccine comprises a formulation of the immunogenic peptides and / or nucleic acid molecules of the present disclosure in a form capable of being administered to a vertebrate (preferably a mammal) and induces an immunity-enhancing protective immune response to prevent and / or alleviate influenza A virus infection.

[0073] The term "protective immune response" or "protective response" refers to an immunogen-mediated immune response against an infectious agent or disease, exhibited by a vertebrate (eg, a human) that prevents or alleviates infection with influenza A or reduces at least one disease symptom thereof.

[0074] The term "vertebrate" or "subject" or "patient" refers to any member of the subphylum Chordata, including, but not limited to: humans and other primates, including non-human primates such as chimpanzees and other apes and monkeys; livestock such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats, and guinea pigs; birds, including domesticated, wild, and game birds such as chickens, turkeys, and other gallinaceous birds, ducks, and geese. The terms "mammal" and "animal" are included in this definition and are intended to cover adult, juvenile, and newborn individuals.

[0075] The vaccine herein can be an mRNA vaccine, a recombinant protein vaccine, a recombinant DNA vaccine, a recombinant viral vector vaccine (e.g., an adenovirus vector, a poxvirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a cytomegalovirus vector), a recombinant bacterial vector vaccine, a recombinant yeast vector vaccine, or a recombinant virus-like particle vaccine. In some embodiments, the vaccine herein is preferably an mRNA vaccine.

[0076] The vaccine compositions herein comprise an effective amount of the immunogen or its encoding molecule herein. The term "effective amount" generally refers to an amount of the immunogen or its encoding molecule that can induce a protective immune response sufficient to induce immunity to prevent and / or alleviate infection or disease and / or to reduce influenza A infection or at least one symptom thereof.

[0077] The vaccines herein may also include an adjuvant. Adjuvants known to those of ordinary skill in the art may be used, such as those described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients" (2nd ed.) (incorporated herein by reference in its entirety). Examples of known adjuvants include, but are not limited to, aluminum adjuvants, cholera toxin and its subunits, oligodeoxynucleotides, manganese ion adjuvants, colloidal manganese adjuvants, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I: C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21, and the like.

[0078] The vaccine composition herein may also include pharmaceutically acceptable carriers, diluents, preservatives, solubilizers, emulsifiers and other excipients. For example, pharmaceutically acceptable carriers are known and include, but are not limited to, water for injection, saline solution, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer and combinations thereof. Pharmaceutically acceptable carriers, diluents and other excipients can be found, for example, in Remington's Pharmaceutical Sciences.

[0079] In some embodiments, nucleic acid expression vector (such as mRNA) is contained in packaging material separately, or is combined with the carrier in delivery system.For example, described delivery system is selected from: lipid delivery system, lipid delivery system, polymer delivery system or its combination delivery system, for example, is loaded on the polymer micelle of lipid nanoparticle, cationic liposome, polyurethane (PAA), poly-β amino ester (PBAE), polyethyleneimine (PEI), lipid package.In some embodiments, nucleic acid expression vector is combined with liposome nanoparticle.In some embodiments, liposome nanoparticle comprises: the combination of cationic lipid, structural lipid, helper lipid and stable lipid.

[0080] In some embodiments, the liposome nanoparticles comprise, calculated by molar percentage, 20-50% cationic lipids: 20-50% structural lipids: 5-20% auxiliary lipids: 1-5% stabilizing lipids, more preferably 50% cationic lipids: 38% structural lipids: 10% auxiliary lipids: 2% stabilizing lipids.

[0081] In some embodiments, the cationic lipid in the liposome nanoparticles is one or more selected from the group consisting of (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), didecyl adipate (DDA), 3β-[N-(N′, N′-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), N,N-dimethyl-4-pyridinamine (DMAP), 1,2-triethanolamine-3-trimethylpropane (DOTMA), N-[1-(2,3-dioleoyl)propyl]-N-( Arginine amide) ethyl-N, N-dimethylammonium trifluoroacetate (DOSPA), 4-(N, N-dimethylamino) butyric acid (dilinoleyl) methyl ester (Dlin-MC3-DMA), KC2, N, N-dimethyl-2,2-di-(9Z, 12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethylamine (Dlin-KC2-DMA), preferably 4-(N, N-dimethylamino) butyric acid (dilinoleyl) methyl ester (Dlin-MC3-DMA).

[0082] In some embodiments, the structural lipid in the liposome nanoparticles comprises cholesterol, cholesterol ester, steroid hormone, steroid vitamin or bile acid, preferably cholesterol.

[0083] In some embodiments, the auxiliary lipids in the liposome nanoparticles include dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1,2-distearoyl-3-phosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), distearoylphosphatidylcholine (DSPC), preferably distearoylphosphatidylcholine (DSPC).

[0084] In some embodiments, the stabilizing lipids in the liposome nanoparticles include polyethylene glycol (PEG)-lipids, such as PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified diacylglycerol, preferably PEG-modified diacylglycerol, more preferably long-circulating liposomal 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (PEG 2000-DMG).

[0085] In some embodiments, the mass ratio of cationic liposomes to mRNA in the liposome nanoparticles may be 10 to 30:1, preferably 15 to 20:1.

[0086] In some embodiments, cationic lipid nanoparticles are prepared by mixing cationic liposomes Dlin-MC3-DMA, cholesterol, phospholipid DSPC, and pegylated lipid DMG-PEG2000 in anhydrous ethanol to obtain a mixed organic phase, which is then mixed with an appropriate buffer system (e.g., citrate buffer) to obtain a lipid mixture. The lipid mixture is then added to an extruder and squeezed and filtered to obtain cationic liposome nanoparticles. The prepared mRNA is dissolved in an appropriate buffer system (e.g., citrate buffer), added to the cationic lipid nanoparticle mixture, thoroughly mixed and incubated, and then dialyzed (e.g., overnight dialysis against sterile PBS) to obtain cationic lipid nanoparticles encapsulating the mRNA. In some embodiments, the ratio of the organic phase to the aqueous phase is 1:2 to 1:5, preferably 3:7.

[0087] The vaccine composition herein may be in a form suitable for systemic or local (particularly respiratory) administration. Methods of administering the vaccine composition include, but are not limited to, intramuscular inoculation, intradermal inoculation, subcutaneous inoculation, nasal drops, aerosol inhalation, genital tract, rectal administration, oral administration, or any combination thereof, such as intramuscular injection followed by nasal drops.

[0088] In some embodiments, the vaccine herein prevents, eliminates, or alleviates influenza A virus and at least one symptom thereof in a subject, such as respiratory symptoms (e.g., nasal congestion, sore throat, hoarseness), headache, cough, sputum, fever, rales, wheezing, dyspnea, pneumonia caused by infection, severe acute respiratory syndrome, renal failure, and the like.

[0089] The present invention also relates to an immunoconjugate (also referred to as an immunoconjugate), which comprises the immunogen described herein and other substances conjugated thereto. The other substances may be targeting substances (e.g., moieties that specifically recognize a particular target), therapeutic substances (e.g., drugs, toxins, cytotoxic agents), or labeled substances (e.g., fluorescent markers, radioisotope labels).

[0090] The present disclosure also provides a combination product comprising the immunogenic peptides, nucleotide molecules, vectors, host cells, and / or vaccines of the present disclosure, and may further comprise one or more other substances that contribute to better prevention and / or treatment of influenza virus infection or its symptoms or enhance the stability of the aforementioned substances. For example, the other substances may include other vaccines against influenza viruses, such as other vaccines against influenza virus HA or HA2, wherein the HA or HA2 is derived from, including but not limited to, H1 to H18; other active substances for diseases or conditions that benefit from T cell activation and / or memory immune responses with T cells, such as T cell antigens.

[0091] Immunization methods

[0092] Also provided herein is a method for preventing and / or treating influenza A virus infection and / or its symptoms, comprising administering at least one preventatively and / or therapeutically effective amount of one or more vaccines disclosed herein. Possible vaccination methods include, but are not limited to, systemic immunization methods, such as intramuscular, subcutaneous, and intradermal injections; and intrarespiratory immunization methods, such as nebulization and intranasal drops. In some embodiments, the primary immunization is administered systemically or intrarespirator-directed, with systemic administration being preferred.

[0093] In some embodiments of the present disclosure, the interval between each two vaccinations is at least 1 week, such as 2 weeks, 4 weeks, 2 months, 3 months, 6 months or longer.

[0094] In some embodiments, the immunization method of the present disclosure can adopt a "prime-boost" or "prime-boost-re-boost" approach, can adopt a single systemic immunization or respiratory local immunization approach, or can adopt a combination of the two immunization approaches.

[0095] The combination products herein can be provided in the form of a pharmaceutical pack or kit, for example, one or more vaccine compositions herein or one or more components thereof can be packaged in one or more containers, for example, in sealed containers such as ampoules or sachets indicating the amount of the composition. The vaccine composition can be provided in the form of a liquid, a sterile lyophilized powder or anhydrous concentrate, etc., and can be diluted, reconstituted and / or formulated with an appropriate liquid (e.g., water, saline, etc.) before use to obtain an appropriate concentration and form for administration to a subject.

[0096] Example

[0097] To further illustrate the technical means and effects of the present invention, the present invention will be further described below with reference to specific embodiments. It will be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make appropriate modifications and variations to the present disclosure, and such modifications and variations are within the scope of the present disclosure.

[0098] For experimental methods in the following examples where specific techniques and / or conditions are not specified, conventional methods in the art may be used, for example, as described in Molecular Cloning: A Laboratory Manual (3rd edition, Cold Spring Harbor Laboratory Press, New York, 1989) or according to the conditions recommended by the supplier. All reagents and instruments used were conventional products commercially available through regular channels. Unless otherwise indicated, test methods may be conventional methods in the art or provided by commercial companies.

[0099] Unless otherwise indicated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the disclosed methods. The preferred embodiments and materials described herein are for illustrative purposes only.

[0100] Materials, methods, and animals

[0101] The mRNA preparation, cationic liposome nanoparticle preparation, animal immunization scheme, and detection method involved in the experiments in the examples are summarized as follows:

[0102] I. mRNA Preparation

[0103] The DNA sequence containing the target gene was used as the basic construction unit and optimized according to the codon preference of eukaryotic organisms as a template for subsequent in vitro transcription experiments (all sequences containing HA2 fragments in the examples contain N82R and N154H mutations) (the sequences used can be found in the appendix of the specification and the attached sequence listing). The method for obtaining the basic sequence of the T cell antigen is as described in Chinese application 201880001963.X, and the sequence was optimized according to the specific needs of this application. The amino acid and nucleotide sequences of the T cell antigen used in the examples can be found in SEQ ID NOs: 11 and 12.

[0104] The basic process of in vitro transcription is as follows: a plasmid containing the target gene is constructed using Xba I (Thermo Fisher, FD0685) single enzyme digestion laboratory and recovered and purified as a template, the transcription materials such as NTP required for transcription are added, and the corresponding mRNA is transcribed and purified using T7 polymerase, and then the capping step is completed by the enzymatic capping method. After purification with lithium chloride, it is stored in a -80°C refrigerator until use.

[0105] II. Preparation of Cationic Liposome Nanoparticles (LNPs) for Encapsulating mRNA

[0106] Cationic lipid nanoparticles were prepared by mixing the cationic liposome Dlin-MC3-DMA, cholesterol, the phospholipid DSPC, and the PEGylated lipid DMG-PEG2000 in anhydrous ethanol at a molar ratio of 50:38:10:2 to form a mixed organic phase. The organic phase was then mixed with 50 mM citrate buffer (pH 4.0) at a mass ratio of 20:1 to form a lipid mixture. This lipid mixture was then extruded and filtered using a syringe to produce cationic liposome nanoparticles. The prepared mRNA was dissolved in citrate buffer and added to the cationic lipid nanoparticle mixture at a volume ratio of 1:1. After thorough mixing, the mixture was incubated at 42°C for 1 hour and then dialyzed against sterile PBS overnight. The mixture was then sterile-filtered at 0.22 μm to yield the mRNA-encapsulated cationic lipid nanoparticles.

[0107] III. Animal Immunization Program

[0108] Female BALB / c mice, 6-8 weeks old at the time of primary immunization, were selected. Each experimental and control group consisted of 14 mice (further divided into two subgroups for each influenza virus subtype challenge, with 7 mice for each virus). Each group received an intramuscular injection, with the primary immunization on day 0 and a booster immunization on day 28 (i.e., 4 weeks). Each immunization dose was the same: 5 μg of the corresponding mRNA per mouse, and the single immunization volume was 50 μL.

[0109] IV. Animal Challenge Protocol

[0110] Three weeks after booster immunization, mice in each immunization group were randomly divided into two subgroups:

[0111] The first subgroup: 1000 TCID 50 Intranasal challenge with the pdmH1N1 virus (i.e., A / California / 04 / 2009): Mice were anesthetized with 2.5% tert-amyl alcohol and 1.25% tribromoethanol at a dose of 15 mL / kg body weight via intraperitoneal injection. Once deeply anesthetized, 25 μL of the diluted virus solution was instilled into each nostril. Symptoms of infection were observed, and weight changes and survival rates were recorded over 14 days.

[0112] The second subgroup: 500 TCID 50 The mice were challenged with H3N2 using the same method as the first subgroup. The infection symptoms of the mice after challenge were observed and the weight changes and survival rates within 14 days were recorded.

[0113] V. Detection Methods

[0114] Blood collection

[0115] Blood was collected from the orbital venous plexus of mice at weeks 4, 5, and 6 after the initial immunization. Mice were challenged with the virus under anesthesia three weeks after the final immunization. Whole blood was collected into a 1.5 mL sterile EP tube and incubated at 37°C for 30 minutes to accelerate coagulation. Serum was then centrifuged at 7000g for 15 minutes to isolate the serum. The serum was then inactivated at 56°C for 30 minutes to remove complement activity.

[0116] Serum IgG antibody titer detection using enzyme-linked immunosorbent assay (ELISA)

[0117] H7N9 strain HA protein or M2e protein was diluted in coating buffer (50 mM carbonate buffer, pH 9.6) to a final concentration of 1 μg / mL. 100 μL of the antigen diluent was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. Following coating, PBST containing 5% skim milk was used as a blocking buffer and incubated at 37°C for 2 hours to block any unadsorbed protein areas on the plate. After blocking, mouse serum was diluted 100-fold in the first well, followed by a 2-fold serial dilution, and added to the blocked ELISA plate for antibody incubation. After incubation at room temperature for 3 hours, the antibody was washed away, and the plate was incubated with a secondary antibody (HRP-conjugated goat anti-mouse IgG antibody (1:5000)). After incubation, the secondary antibody was washed away, and color development was initiated. After color development was complete, the plate was read on an end-point ELISA reader at 490 nm. The results were recorded.

[0118] Detection of T cells that specifically recognize M2e or HA2 and secrete IFNγ using enzyme-linked immunospot (ELISpot) assay

[0119] First, remove the plate and dilute the coating antibody with sterile PBS (1:200, 100 μL / well) and coat overnight at 4°C. Wash once with RPMI 1640 medium containing 10% fetal bovine serum (FBS) and then block with the same medium at room temperature for 2 hours. Discard the medium, add the stimulator to the plate, and then add lymphocytes (2.0×10 5Cells / well (stimuli can also be pre-mixed with cells before adding to the plate). In this experiment, the stimuli were either the M2e peptide library or the HA2 peptide library (Qiangyao Biotechnology), diluted to a final concentration of 5 μg / mL. Duplicate wells were prepared for each mouse. Background and blank controls were also set up on the plate. Background controls consisted of culture medium alone, without cells, to exclude nonspecific color development caused by the culture medium. Blank controls consisted of cells added without stimuli, to exclude IFN-γ secretion due to interference from external factors other than experimental factors. The plate was incubated in a 37°C, 5% CO2 incubator for 12-48 hours. The plate was then drained and washed twice with pre-cooled ddH2004 for 3 minutes each time, followed by three washes with PBST (200 μL / well). The detection antibody was diluted in PBS containing 10% FBS (PBS-10% FBS) and added to the plate (1:250, 100 μL / well). Incubated at room temperature for 2 hours, and the plate was then washed. Dilute Streptavidin-HRP (1:100, 100 μL / well) in PBS-10% FBS and add 100 μL per well. Incubate at room temperature for 1 hour, then wash the plate. Add 100 μL / well of colorimetric solution. Stop the reaction when visible spots appear. Wash the plate to terminate the reaction. Allow the plate to dry, read the plate, and analyze the results.

[0120] Example 1. Expression verification of mRNA transfected HEK293T cells

[0121] HEK293T cells were transfected with the prepared mRNA for expression verification. On day 0, HEK293 cells were seeded at a density of 800,000 cells / well in a 6-well plate using complete DMEM medium supplemented with 10% fetal bovine serum. On day 1, transfection was performed using the transfection reagent Lipofectamine 3000, with 3 μg of mRNA per well. The cells were incubated at 37°C for 45-48 hours and then removed. Cells were harvested by pipetting and lysed with 100 μL of RIPA lysis buffer on ice for 1 hour. The cells were centrifuged at 12,000 g for 10 minutes at 4°C, and the supernatant was collected. After adding the appropriate loading buffer, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed, the membrane was transferred, and the cells were blocked in 5% nonfat dry milk for 2 hours at room temperature. The primary antibody (mouse anti-M2e monoclonal antibody, Thermo Fisher, Cat. MA1-082, 1:3000) was incubated overnight at 4°C. The membrane was washed with PBST (PBS buffer containing 0.05% Tween-20) and incubated with goat anti-mouse secondary antibody (Yisheng Bio, Cat. 33201ES60, 1:5000) at room temperature for 1 hour, then washed with PBST (containing 0.05% Tween-20). The membrane was developed by chemiluminescence and photographed.

[0122] The results are shown in Figure 1B , showing that 8 tandem repeats of M2e (8M2e) or 8M2e-HA (M8H) or 8M2e-HA-collagen (M8HC) were all significantly expressed and recognized by specific antibodies.

[0123] Example 2. Humoral immune responses induced by mRNA vaccines 8M2e, M8H, and M8HC in mice

[0124] The normal expression of mRNA (8M2e, M8H, M8HC) in this example has been verified in Example 1. Cationic liposome nanoparticles encapsulating these three mRNAs were prepared using Experimental Method II.

[0125] BALB / c mice were immunized with lipid nanoparticle vaccines encapsulating three types of mRNA to evaluate their immunogenicity. Twelve mice were injected intramuscularly into the hind limbs. Each injection volume was 50 μL liposomes encapsulating 5 μg of the corresponding mRNA. The negative control was an equal volume of cationic lipid nanoparticles without nucleic acid encapsulation (liposome empty). As shown in Figure 2A, the immunization schedule was two immunizations at an interval of 4 weeks. After the first immunization of the three different mRNA vaccines and the liposome empty control group at week 0, a booster immunization was performed at week 4. Peripheral blood was collected from the experimental group mice at weeks 4, 5, and 6, and serum was separated. The titer of antibodies specific to HA protein and M2e protein was detected by enzyme-linked immunosorbent assay.

[0126] The results are shown in Figures 2B and 2C. Four weeks after the first immunization, the geometric mean titers (GMTs) of serum M2e antibodies in mice immunized in the 8M2e, M8H, and M8HC groups were 1076, 328, and 328, respectively, and the GMTs of serum HA antibodies in mice immunized in the M8H and M8HC groups were 164 and 148, respectively. After blood was collected at week 4, booster immunization was performed. At week 5 (one week after the booster), the GMTs of serum M2e antibodies in mice immunized in the 8M2e, M8H, and M8HC groups were 76082, 21000, and 102400, respectively, and the GMTs of serum HA antibodies in mice immunized in the M8H and M8HC groups were 148 and 362, respectively. At week 6 (two weeks after the booster), the GMTs of serum M2e antibodies in mice immunized in the 8M2e, M8H, and M8HC groups were 92746, 25600, and 204800, respectively, and the GMTs of serum HA antibodies in mice immunized in the M8H and M8HC groups were 297 and 362, respectively.

[0127] At week 7 (3 weeks after the boost), mice were anesthetized and challenged intranasally with H1N1 and H3N2. Mice in each immunization group were randomly divided into two subgroups: one challenged with H1N1 and the other with H3N2. The challenge date was designated as day 0, and mouse body weight and survival were recorded continuously for 14 days after challenge.

[0128] Figures D and F show body weight and survival after H1N1 challenge. All groups experienced weight loss after challenge. Weight began to recover after days 7 and 8 in all groups except the empty-vessel control group. However, all mice in the M8HC group survived, while 83% of the M8H group and 67% of the 8M2e group survived. Figures E and G show body weight and survival after H3N2 challenge. All groups experienced a significant weight loss after challenge. Survival revealed that 50% of the mice in the M8HC group and 17% of the mice in the M8H group survived, while all mice in the 8M2e group died. All control groups died.

[0129] The results of this experiment showed that after the initial immunization, serum antibody titers against M2e and HA proteins in all groups were only 100 to 1000. However, after booster immunization, serum antibody titers in all groups increased significantly, with M2e binding antibody titers rising to 100,000 to 100,000 one week after the booster, and antibody titers continued to increase over time. After challenge with H1N1, although all mice in each group experienced weight loss, only the M8HC group survived, while the M2e group survived 67%, suggesting that relying solely on M2e antibodies does not provide complete protection. After challenge with the H3N2 subtype, 50% of the mice in the M8HC group survived, while 17% of the mice in the M8H group survived.

[0130] Results indicate that mRNA formed by linking M2e and HA2 effectively induces an immune response against both M2e and HA2, resulting in superior protection against infection with different subtypes of influenza A virus. Furthermore, the inventors unexpectedly discovered that further linking the low-immunogenic multimerization motif C (collagen) to M2e and HA can even more effectively induce an immune response and achieve even superior protection.

[0131] Example 3. Humoral immune responses induced by mRNA vaccines M8HC and M4HC in mice

[0132] Figure 3A shows the construction of M8HC and M4HC. Normal expression of the mRNA (M8HC) in this example was verified in Example 1. The mRNA (M4HC) was transfected using the method described in Example 1 to verify expression. As shown in Figure 3B , M4HC also showed normal expression. Cationic liposome nanoparticles encapsulating these two mRNAs were prepared using Experimental Method II.

[0133] BALB / c mice were immunized with lipid nanoparticle vaccines containing these two mRNAs to evaluate their immunogenicity. Fourteen mice per group were injected intramuscularly into the hind limbs. Each injection consisted of 50 μL of liposomes encapsulating 5 μg of the corresponding mRNA. Empty liposomes were used as a negative control. The immunization procedure was the same as in Example 2. Peripheral blood was collected from the experimental group mice at weeks 4, 5, and 6, and serum was isolated. Enzyme-linked immunosorbent assays were used to determine antibody titers specific for HA and M2e proteins.

[0134] The results are shown in Figures 3C and 3D. Four weeks after the first immunization, the GMTs of serum M2e antibodies in mice immunized in the M8HC and M4HC groups were 328 and 121, respectively, and the GMTs of serum HA antibodies in mice immunized in the M8HC and M4HC groups were 148 and 134, respectively. After blood was collected on the fourth week, booster immunization was performed. At the fifth week (one week after the booster), the GMTs of serum M2e antibodies in mice immunized in the M8HC and M4HC groups were 102400 and 1766, respectively, and the GMTs of serum HA antibodies in mice immunized in the M8HC and M4HC groups were 362 and 975, respectively. At the sixth week (two weeks after the booster), the GMTs of serum M2e antibodies in mice immunized in the M8HC and M4HC groups were 204800 and 8613, respectively, and the GMTs of serum HA antibodies in mice immunized in the M8HC and M4HC groups were 362 and 883, respectively.

[0135] At week 7 (3 weeks after the boost), mice were anesthetized and challenged intranasally with H1N1 and H3N2. Each immunization group was randomly divided into two subgroups: one challenged with H1N1 and the other with H3N2. The challenge date was designated as day 0, and mouse weight and survival were recorded continuously for 14 days after challenge.

[0136] The results of body weight and survival after H1N1 challenge are shown in Figures 3E and 3G. The weight of mice in the M8HC and M4HC groups gradually decreased after challenge, reaching a minimum average weight on day 7 and then gradually recovering. However, the weight of mice in the empty liposome control group decreased rapidly after challenge and eventually died. Survival results showed that all mice in the M8HC and M4HC groups survived, while all mice in the control group died. The results of body weight and survival after H3N2 challenge are shown in Figures 3F and 3H. All groups experienced rapid weight loss after challenge, but the average weight loss rate of mice in the M4HC group was significantly slower than that in the M8HC group. Survival results showed that 50% of mice in the M8HC group survived, while 83% of mice in the M4HC group survived. All mice in the control group died.

[0137] The experimental results showed that after the number of M2e repeats in the immunogen was reduced from 8 to 4, the antibody titers for M2e and HA binding in all groups of sera after the initial immunization were only 100 to 1000, which was not significantly different from the results in Example 2. However, after the booster immunization, the M4HC group had significantly lower antibody titers for M2e binding than the M8HC group, but the average titer for HA binding increased by approximately 2.7-fold. This suggests that the reduction in the number of M2e repeats reduced M2e-induced antibodies while increasing HA antibodies, resulting in a more balanced antibody response to the two immunogens. After challenge with H1N1, all mice in the M8HC and M4HC groups survived, indicating that the reduction in the number of M2e repeats did not reduce vaccine protection. Results from mice challenged with H3N2 indicated that the M4HC group had better protection against the H3N2 subtype than the M8HC group.

[0138] These results demonstrate that mRNA formed by linking M2e, HA, and a multimerization motif effectively induces immune responses against both M2e and HA, resulting in superior protection against different subtypes of influenza A virus. Furthermore, by adjusting the number of M2e repeats, the response balance between the two immunogens, M2e and HA, can be adjusted, enhancing protection against more viral subtypes and broadening the spectrum of protection.

[0139] Example 4. Humoral and cellular immune responses induced by mRNA vaccines M4HC and M4HF in mice

[0140] Figure 4A shows the construction of M4HC and M4HF. The newly constructed mRNA (M4HF) in this example was transfected and expressed using the method described in Example 1. As shown in Figure 4B, M4HF was expressed normally. M4HC or M4HF mRNA-encapsulated cationic liposome nanoparticles were prepared using Experimental Method II.

[0141] BALB / c mice were immunized with lipid nanoparticle vaccines containing these two mRNAs to evaluate their immunogenicity. Fourteen mice per group were injected intramuscularly into the hind limbs. Each injection consisted of 50 μL of liposomes encapsulating 5 μg of the corresponding mRNA. Empty liposomes were used as a negative control. The immunization procedure was the same as in Example 2. Peripheral blood was collected from the experimental group mice at weeks 4, 5, and 6, and serum was isolated. Enzyme-linked immunosorbent assays were used to determine antibody titers specific for HA and M2e proteins.

[0142] The results are shown in Figures 4C and 4D. Four weeks after the first immunization, the GMTs of serum M2e antibodies in mice in the M4HC and M4HF groups were 121 and 121, respectively, and the GMTs of serum HA antibodies in mice in the immunized mice were 134 and 297, respectively. After blood was collected on the 4th week, booster immunization was performed. At the 5th week (1 week after the booster), the GMTs of serum M2e antibodies in mice in the M4HC and M4HF groups were 1766 and 56529, respectively, and the GMTs of serum HA antibodies in mice were 1188 and 2377, respectively. At the 6th week (2 weeks after the booster), the GMTs of serum M2e antibodies in mice in the M4HC and M4HF groups were 8613 and 11593, respectively, and the GMTs of serum HA antibodies in mice in the M4HC and M4HF groups were 883 and 3533, respectively.

[0143] At week 7 (3 weeks after the boost), mice were anesthetized and challenged intranasally with H1N1 and H3N2. Each immunization group was randomly divided into two subgroups: one challenged with H1N1 and the other with H3N2. The challenge date was designated as day 0, and mouse weight and survival were recorded continuously for 14 days after challenge.

[0144] The results of body weight and survival after H1N1 challenge are shown in Figures 4E and 4G. The weight of mice in the M4HC and M4HF groups gradually decreased after challenge, but gradually recovered after day 7. Survival results showed that all mice in the M4HC and M4HF groups survived, while all mice in the control group died. The results of body weight and survival after H3N2 challenge are shown in Figures 4F and 4H. All groups of mice experienced a significant decrease in body weight after challenge, with an average weight loss of more than 10%. Starting on day 8, their weight gradually recovered. Survival results showed that all mice in the M4HF group survived, while 83% of the mice in the M4HC group survived, while all mice in the control group died.

[0145] The reactivity of T lymphocytes to immunogens after vaccination was detected. The results are shown in Figures 4I and 4J. Ten days after booster immunization, both the M4HC and M4HF groups produced T cells that could specifically recognize M2e and HA2. Among them, the T cells that responded to the M2e peptide library could reach 10 / 10. 6 There are about 1100 (M4HC) or 1900 (M4HF) cells on average, and the number of T cells responding to the HA2 peptide library is about 10 6 There were about 58 (M4HC) or 105 (M4HF) cells on average, which were significantly higher than those in the liposome empty control group.

[0146] The experimental results show that the use of different polymer sequences can change or improve the immunogenicity of the composite immunogen. In this example, the use of Foldon in place of collagen can further enhance the immunogenicity of the immunogen. Specifically, after booster immunization, the serum antibody titers against M2e and HA in the M4HF group were higher than those in the M4HC group. One week after the booster, the serum antibody titers against M2e in the M4HF group were 32 times that of the M4HC group, and the antibody titers against HA were twice that of the M4HC group. The number of T cells in the mice that specifically recognized M2e and HA2 was also significantly higher than that in the control group. After H1N1 challenge, all mice in the M4HC and M4HF groups survived. After H3N2 challenge, only all mice in the M4HF group survived. These results suggest that the M4HF group not only significantly increased antibody titers compared to the M4HC group, but also provided better protection against more subtypes.

[0147] These results demonstrate that mRNA formed by linking M2e, HA, and a multimerization motif effectively induces immune responses against M2e and HA, resulting in excellent protection against different influenza virus infections. Furthermore, by adjusting the multimerization motif used, the immune response can be altered or improved (even significantly), providing broad-spectrum, highly effective protection.

[0148] Example 5. Cellular and humoral immune responses induced by mRNA vaccines M4HF-T and M4HF+T in mice

[0149] Figure 5A shows the construction of M4HF-T and T. The newly constructed mRNA (M4HF-T, T) in this example was transfected and expressed using the method described in Example 1. M4HF-T consists of an IRES-linked M4HF (SEQ ID NO: 27) peptide and a T cell antigen (SEQ ID NO: 11) peptide linked to DHFR (SEQ ID NO: 43). Results show that both M4HF-T and the T cell antigen peptide were expressed normally. Experimental Method II was used to prepare: a. mRNA cationic liposome nanoparticles encapsulating M4HF-T, or b. a mixture of M4HF+T mRNA cationic liposome nanoparticles encapsulating M4HF and T in separate liposomes, followed by mixing the two nanoparticles at a mRNA mass ratio of 1:3.

[0150] BALB / c mice were immunized with lipid nanoparticle vaccines encapsulated with either ①M4HF-T or ②M4HF+T mRNA to evaluate their immunogenicity. Each group of 14 mice received intramuscular injections of both hind limbs. Each injection consisted of 150 μL of liposomes encapsulating 20 μg of the corresponding mRNA (M4HF:T = 1:3). The negative control consisted of an equal volume of empty liposomes. The immunization procedure was the same as in Example 2. Peripheral blood was collected from the experimental group mice at weeks 5 and 6, and serum was isolated. Enzyme-linked immunosorbent assays were used to determine the titers of antibodies specifically binding to HA protein and M2e protein.

[0151] At week 7 (3 weeks after the boost), mice were anesthetized and challenged intranasally with H1N1 and H3N2. Each immunization group was randomly divided into two subgroups: one challenged with H1N1 and the other with H3N2. The challenge date was designated as day 0, and mouse weight and survival were recorded continuously for 14 days after challenge.

[0152] Results of weight and survival data after H1N1 challenge showed that the weight of mice in the M4HF-T and M4HF+T groups gradually decreased after challenge. Survival results showed that both groups had high survival rates, while all mice in the control group died. Results of weight and survival data after H3N2 challenge showed that the average weight of mice in the M4HF-T and M4HF+T groups decreased after challenge, and then gradually recovered. Survival data showed that both groups had high survival rates, while all mice in the control group died.

[0153] At week 6 (two weeks after the booster immunization), five mice were taken from each group, their spleens removed, and lymphocytes isolated. ELISpot analysis was used to measure the number of specific T cells secreting IFN-γ. The results showed that mice immunized with M4HF-T and M4HF+T produced a significant number of specific T cells, significantly higher than those in control mice.

[0154] The experimental results showed that adding T cell antigens to the composite immunogen resulted in a favorable immune response in mice. Specifically, after challenge with either H1N1 or H3N2, the mice maintained a high survival rate and produced a large number of specific T cells. These results suggest that the addition of the immunogen component enhanced the vaccine's protective effect.

[0155] Example 6. Protection of mice by mRNA vaccines M4H1F, M4H3F, M2HF, M4H, and HF

[0156] Figure 6A shows the construction of M4H1F, M4H3F, M2HF, M4H, and HF. The amino acid sequences of M4H1F, M4H3F, M2HF, M4H, and HF are shown in SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37, or are encoded by nucleotide molecules having the sequences shown in SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, and SEQ ID NO: 38, respectively. The newly constructed mRNAs (M4H1F, M4H3F, M2HF, M4H, and HF) in this example were transfected and expressed using the method described in Example 1. The results showed that M4H1F, M4H3F, M2HF, M4H, and HF were all expressed normally. Cationic liposome nanoparticles encapsulating the five mRNAs were prepared using Experimental Method II.

[0157] BALB / c mice were immunized with lipid nanoparticle vaccines encapsulated with M4H1F, M4H3F, M2HF, M4H, and HF, respectively, to evaluate their immunogenicity. Fourteen mice per group were injected intramuscularly into the hind limbs. Each injection consisted of 50 μL of liposomes encapsulating 5 μg of the corresponding mRNA. Negative controls consisted of an equal volume of empty liposomes. The immunization procedure was the same as in Example 2.

[0158] At week 7 (3 weeks after the boost), mice in the M4H, HF, M4H1F, M4H3F, and M2HF groups were challenged intranasally with H1N1 and H3N2 vaccines after anesthesia. Mice in each immunization group were randomly divided into two subgroups: one challenged with H1N1 and the other with H3N2. The challenge date was designated as day 0, and mouse body weight and survival were recorded continuously for 14 days after challenge.

[0159] Experimental results showed that reducing the number of M2e repeats to two significantly decreased M2e antibody titers. Both HF containing only HA2 and multimers and MH containing both M2e and HA2 but without multimers were significantly less protective than M4HF. Substituting the HA2 portion of the vaccine with previously reported H1N1 or H3N2 sources decreased the broad-spectrum protective antibodies of the vaccine. These results suggest that composite immunogens containing the HA2 portion of H7N9 can induce a broader immune response.

[0160] All documents mentioned in this disclosure are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of this disclosure, those skilled in the art may make various changes or modifications to this disclosure, and that such equivalents also fall within the scope of the claims appended hereto.

[0161] Appendix: Sequence Information

[0162] Note: * indicates that the sequence is the sequence used in the examples

[0163] specific sequence

[0164] SEQ ID NO: 1 (H7N9 HA2 extracellular domain amino acid sequence)

[0165] SEQ ID NO: 2 (H7N9 HA2 extracellular domain nucleotide sequence)

[0166] SEQ ID NO: 3 (Collagen amino acid sequence)

[0167] SEQ ID NO: 4 (Collagen nucleotide sequence)

[0168] SEQ ID NO: 5 (Foldon amino acid sequence)

[0169] SEQ ID NO: 6 (Foldon nucleotide sequence)

[0170] SEQ ID NO: 7 (tetrabrachion helical peptide amino acid sequence)

[0171] SEQ ID NO: 8 (quadruple-arm helix peptide nucleotide sequence)

[0172] SEQ ID NO: 9 (GCN4 amino acid sequence)

[0173] SEQ ID NO: 10 (GCN4 nucleotide sequence)

[0174] SEQ ID NO: 11 (T cell antigen amino acid sequence)*

[0175] SEQ ID NO: 12 (T cell antigen nucleotide sequence)

[0176] SEQ ID NO: 13 (amino acid sequence of M2e from a partial H1N1 / H3N2 subtype infecting humans)

[0177] SEQ ID NO: 14 (M2e nucleotide sequence from a partial H1N1 / H3N2 subtype infecting humans)

[0178] SEQ ID NO: 15 (amino acid sequence of M2e from another partial H1N1 / H3N2 subtype infecting humans)

[0179] SEQ ID NO: 16 (M2e nucleotide sequence from another partial H1N1 / H3N2 subtype infecting humans)

[0180] SEQ ID NO: 17 (amino acid sequence of M2e from the H7N9 subtype whose natural host is avian)

[0181] SEQ ID NO: 18 (M2e nucleotide sequence from the H7N9 subtype whose natural host is avian)

[0182] SEQ ID NO: 19 (amino acid sequence of M2e from H5N1 / H9N2 and other subtypes whose natural hosts are birds)

[0183] SEQ ID NO: 20 (M2e nucleotide sequence from H5N1 / H9N2 and other subtypes whose natural hosts are birds)

[0184] SEQ ID NO: 21 (4M2e amino acid sequence)

[0185] SEQ ID NO: 22 (4M2e nucleotide sequence)

[0186] SEQ ID NO: 23 (8M2e amino acid sequence)*

[0187] SEQ ID NO: 24 (8M2e nucleotide sequence)

[0188] SEQ ID NO: 25 (8M2e-HA2-collagen amino acid sequence)*

[0189] SEQ ID NO: 26 (8M2e-HA2-collagen nucleotide sequence)

[0190] SEQ ID NO: 27 (4M2e-HA2-foldon amino acid sequence)*

[0191] SEQ ID NO: 28 (4M2e-HA2-foldon nucleotide sequence)

[0192] SEQ ID NO: 29 (4M2e-H1N1 HA2-foldon amino acid sequence)*

[0193] SEQ ID NO: 30 (4M2e-H1N1 HA2-foldon nucleotide sequence)

[0194] SEQ ID NO: 31 (4M2e-H3N2 HA2-foldon amino acid sequence)*

[0195] SEQ ID NO: 32 (4M2e-H3N2 HA2-foldon nucleotide sequence)

[0196] SEQ ID NO: 33 (2M2e-HA2-foldon amino acid sequence)*

[0197] SEQ ID NO: 34 (2M2e-HA2-foldon nucleotide sequence)

[0198] SEQ ID NO: 35 (4M2e-HA2 amino acid sequence)*

[0199] SEQ ID NO: 36 (4M2e-HA2 nucleotide sequence)

[0200] SEQ ID NO: 37 (HA2-foldon amino acid sequence)*

[0201] SEQ ID NO: 38 (HA2-foldon nucleotide sequence)

[0202] SEQ ID NO: 39 (4M2e-HA2-collagen (M4HC) amino acid sequence)*

[0203] SEQ ID NO: 40 (4M2e-HA2-collagen (M4HC) nucleotide sequence)

[0204] SEQ ID NO: 41 (IRES nucleotide sequence)

[0205] SEQ ID NO: 42 (4M2e-HA2-foldon-IRES-T-DHFR nucleotide sequence)*

[0206] SEQ ID NO: 43 (DHFR amino acid sequence)

[0207] SEQ ID NO: 44 (DHFR nucleotide sequence)

[0208] SEQ ID NO: 45 (peptide linker)

[0209] SEQ ID NO: 46 (peptide linker)

[0210] SEQ ID NO: 47 (peptide linker)

[0211] SEQ ID NO: 48 (peptide linker)

[0212] SEQ ID NO: 49 (peptide linker)

[0213] SEQ ID NO: 50 (peptide linker)

[0214] SEQ ID NO: 51 (T cell antigen 1)

[0215] SEQ ID NO: 52 (T cell antigen 2)

Claims

1. A nucleic acid molecule encoding an immunogenic peptide, wherein the immunogenic peptide comprises: (a) Concatenated peptide fragments of the extracellular region (M2e) of the M2 protein; (b) a peptide fragment of the extracellular domain (HA2) of the stem region of the hemagglutinin protein of the H7N9 influenza virus subtype; (c) multimerization motif peptide; (d) optionally, one or more linker peptide segments independently located between the peptide segments; The nucleic acid molecule comprises the encoding nucleic acid of each peptide segment.

2. The nucleic acid molecule of claim 1, wherein: The M2e peptide segment is derived from one or more influenza virus strains selected from the group consisting of H1N1, H2N2, H3N2, H3N8, H5N1, H5N2, H5N6, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2, H10N8, H11N2 subtypes; and / or The M2e peptide segment is derived from an influenza virus strain whose host is an animal selected from the following group: mammals (e.g., humans, non-human primates, pigs, horses, cattle, sheep, bats, sea lions, seals, rats, mice, ferrets), birds (e.g., poultry or wild birds), or co-occurring (e.g., zoonotic, human-poultry, porcine-poultry) influenza virus strains; and / or Each M2e region in the M2e peptide segment is derived from the same or different sources; and / or The M2e peptide segment comprises 2 to 12 tandem M2e regions, preferably 4 to 8 tandem Me2 regions, for example 4, 5, 6, 7, 8, 9, 10, 11 or 12 tandem Me2 regions; and / or Each M2e region in the M2e peptide segment is directly connected or connected through a linker, for example, the linker is independently selected from: a flexible linker, a rigid linker and a cleavable linker, such as AAA, (G4S) n (e.g. (G4S)3), GSAGSAAGSGEF, (Gly) n 、(EAAAK) n , EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, wherein n is an integer between 2 and 10 (eg, n=3); and / or The M2e region has an amino acid sequence as shown in SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or a sequence having at least 80% sequence identity with the amino acid sequence. Amino acid sequence; and / or The nucleic acid encoding the M2e region has a nucleotide sequence as shown in SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence (e.g., a sequence obtained by codon optimization or one or more nucleotide substitutions of the aforementioned nucleotide sequence); and / or The M2e peptide segment has an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 23, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence; and / or The nucleic acid encoding the M2e peptide segment has a nucleotide sequence as shown in SEQ ID NO: 22 or SEQ ID NO: 24, or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence (e.g., a sequence obtained by codon optimization or one or more nucleotide substitutions of the aforementioned nucleotide sequence).

3. The nucleic acid molecule according to claim 1, wherein The HA2 peptide segment is derived from an H7N9 subtype strain, for example, from an A / Shanghai / 02 / 2013 strain or an A / Anhui / 01 / 2013 strain; and / or The HA2 peptide segment comprises one or more mutations compared to the natural HA2 peptide segment, for example, a mutation of amino acid N82 at position 82, preferably a mutation of N82R, N82K, N82D or N82E; a mutation of amino acid N154 at position 154, preferably a mutation of N154H; and / or The HA2 peptide segment is connected to a partial fragment of HA1, for example, comprising a HA2 peptide segment and a partial peptide segment of HA1 that anchor the HA protein to the stem in the viral lipid envelope; and / or The HA2 peptide segment has the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence; and / or The nucleic acid encoding the HA2 peptide segment has the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence (eg, a sequence obtained by codon optimization or one or more nucleotide substitutions of the aforementioned nucleotide sequence).

4. The nucleic acid molecule according to claim 1, wherein The multimerization motif peptide segment is selected from: collagen, the trimerization motif Foldon of T4 bacteriophage fibrin, tetrabrachion helical peptide, GCN4; and / or The multimerization motif peptide segment has SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, The amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 80% sequence identity thereto; and / or The encoding nucleic acid of the polymerized motif peptide segment has a nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence (e.g., a sequence obtained by codon optimization or one or more nucleotide substitutions of the aforementioned nucleotide sequence).

5. The nucleic acid molecule according to claim 1, wherein The immunogenic peptide further comprises: (e) one or more other peptide segments, For example, the other peptide segments include one or more peptide segments selected from the following group: T cell antigen peptide segment, transferrin, FF peptide, cyclic peptide nanotube, IL-21; For example, the other peptide segments include a T cell antigen peptide segment, wherein the T cell antigen peptide segment has an amino acid sequence as shown in SEQ ID NO: 11, 51 or 52, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence; and / or The nucleic acid encoding the T cell antigen peptide has the nucleotide sequence shown in SEQ ID NO: 12 or is a nucleic acid encoding the polypeptide shown in SEQ ID NO: 51 or 52, or a nucleotide sequence having at least 80% sequence identity with the encoding nucleic acid (e.g., a sequence obtained by codon optimization or one or more nucleotide substitutions of the aforementioned nucleotide sequence); and / or The T cell antigen peptide segment is connected to a connecting element, for example, the connecting element is selected from: IRES, T2A, P2A; and / or The T cell antigen peptide segment is further connected to a degradation factor, such as a degradation factor DHFR.

6. The nucleic acid molecule according to claim 1, wherein The peptide segments of the immunogenic peptides are independently connected directly or through a linker, for example, the linker is independently selected from: a flexible linker, a rigid linker and a cleavable linker, such as AAA, (G4S) n (e.g. (G4S)3), GSAGSAAGSGEF, (Gly) n 、(EAAAK) n , EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, wherein n is an integer between 2 and 10 (eg, n=3); and / or The immunogenic peptides have SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, The amino acid sequence shown in SEQ ID NO: 39, or an amino acid sequence having at least 80% sequence identity thereto; and / or The nucleic acid encoding the immunogenic peptide has a nucleotide sequence as shown in SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 42, or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence (e.g., a sequence obtained by codon optimization or one or more nucleotide substitutions in the aforementioned nucleotide sequence).

7. The nucleic acid molecule according to claim 1, which is a DNA molecule or an RNA molecule, For example, the nucleic acid molecule is an mRNA molecule, such as a self-amplifying mRNA (SAM) molecule or a non-replicating mRNA molecule, preferably a SAM molecule; wherein, When the mRNA molecule is a non-replicative mRNA molecule, it further comprises a 5'-cap structure and a 3'-poly (A) tail.

8. An immunogenic peptide characterized by The method of claim 1 , or the method of claim 2 , wherein the nucleic acid molecule is encoded by the nucleic acid molecule of claim 1 .

9. A vector or host cell comprising the nucleic acid molecule according to any one of claims 1 to 7, For example, the vector is selected from: mRNA vector, DNA plasmid vector, recombinant virus vector, recombinant bacterial vector; wherein the mRNA vector is selected from linear, circular and self-replicating vectors; the recombinant virus vector is selected from poxvirus (e.g., Tiantan strain, North American vaccine strain, Wyeth-derived strain, Listeria strain, Ankara-derived strain, Copenhagen strain and New York strain), adenovirus (e.g., adenovirus 5, 11, 26, 35, 63, 68), adeno-associated virus, herpes simplex virus, measles virus, enterovirus, reovirus, rhabdovirus, flavivirus, influenza virus, parainfluenza virus, respiratory syncytial virus, poliovirus vector; For example, the host cell is a mammalian cell or an insect cell, such as HEK293, HeLa, K562, CHO, NS0, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19 and MRC-5 cells; High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5 and Ao38.

10. An influenza vaccine comprising: (i) one or more nucleic acid molecules according to any one of claims 1 to 7, the immunogenic peptide according to claim 8, or the vector according to claim 9; (ii) an immunologically acceptable carrier, delivery system or adjuvant.

11. The influenza vaccine of claim 10, wherein: The influenza vaccine is an mRNA vaccine, a DNA vaccine, a viral vector vaccine, or a recombinant protein vaccine; and / or The carrier or delivery system is selected from: a lipid delivery system, a lipid delivery system, a polymer delivery system or a combination thereof, such as loaded on lipid nanoparticles (e.g., a combination of cationic lipids, structural lipids, auxiliary lipids and stabilizing lipids), polyurethane (PAA), poly-β-amino ester (PBAE), polyethyleneimine (PEI), lipid-encapsulated polymer micelles; and / or The vaccine further comprises an adjuvant or is used in combination with an adjuvant, for example, the adjuvant is selected from: aluminum adjuvant, cholera toxin and its subunits, oligodeoxynucleotides, manganese ion adjuvants, colloidal manganese adjuvants, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I: C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21; and / or The vaccine is in a form suitable for one or more administration or delivery methods selected from the following group: respiratory tract aerosol inhalation, nasal drops, oral administration, direct injection (e.g., intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection), mucosal administration; and / or The vaccine may be in a form suitable for combined administration of two or more drugs or vaccines, such as co-administration or sequential vaccination.

12. Use of the nucleic acid molecule according to any one of claims 1 to 7, the immunogenic peptide according to claim 8, the vector according to claim 9 and / or the influenza vaccine according to any one of claims 10 to 11 in the preparation of a product for the prevention and / or treatment of influenza.

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