Trivalent influenza nanoparticle vaccine with broad-spectrum epitope and application thereof

Abstract

The invention belongs to the field of biomedicine, and particularly discloses an influenza nanoparticle vaccine as well as a preparation method and application thereof. The fusion protein containing the influenza virus strain HA protein or the fragment thereof, the influenza virus protein epitope and the ferritin can be used as an antigen to induce an immune response effect aiming at influenza viruses, and a vaccine formed by matching the fusion protein with an MF59 adjuvant can be used for inducing to generate a relatively strong challenge protection effect and has a relatively good cross protection effect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a novel fusion protein antigen, a composition comprising the fusion protein antigen, and an application thereof in preparing an influenza vaccine composition, especially a vaccine composition for intramuscular injection. Background Art

[0002] Influenza virus (IV) is a highly contagious respiratory pathogen and one of the major infectious diseases causing morbidity and mortality worldwide, posing a serious threat to global public health. Preventing and controlling seasonal influenza outbreaks and influenza pandemics is crucial, and vaccination is one of the most effective prevention methods. However, current influenza vaccines have some limitations, such as the induced immunity is strain-specific, has poor effects against mismatched strains, and cannot cope with new pandemic strains.

[0003] Based on the current research on T cell immunity of influenza virus in humans and mice, T cells play an important role in clearing the virus. Unlike antibodies, T cells can recognize conserved protein sequences inside the virus and provide broad-spectrum and long-lasting protection. In order to develop a universal T cell influenza vaccine, antigenic regions that are highly conserved in different influenza virus strains are usually selected as immunogens to induce the body to produce cross-protective T cell immune responses to cope with influenza virus mutations.

[0004] In recent years, nanoparticle vaccine platforms have been applied to vaccine development, but most nanoparticle vaccines have problems such as low antigen loading capacity, lengthy and complex preparation processes, and complex structures. Advances in nanotechnology have made synergistic epitope vaccination and cross-protective T cell immune responses possible.

[0005] Therefore, developing a safe and effective multi-epitope influenza vaccine has important research significance and application prospects. Summary of the invention

[0006] To solve the above problems, the present invention discloses a novel recombinant trivalent influenza vaccine and its preparation and application. By using the fusion protein disclosed in the present invention as an antigen and MF59 as an adjuvant, a recombinant influenza nanoparticle vaccine with high safety, effectiveness and broad spectrum is provided.

[0007] The fusion protein provided by the present invention preferably contains influenza virus surface glycoprotein or fragment thereof, lymphocyte B cell, lymphocyte T cell antigen epitope and nanoparticle core at the same time. For example, the surface glycoprotein is hemagglutinin (HA) or fragment thereof; the antigen epitope is derived from other structural proteins of influenza virus except HA, preferably, the other structural proteins are selected from one or more of matrix protein 2 (M2), matrix protein 1 (M1) and ceramidase (NA); the nanoparticle core is derived from a class of iron storage proteins widely present in organisms, and is composed of 24 subunits with an inner diameter of about 8-10nm and an outer diameter of about 12-14nm.

[0008] In one aspect, the present invention provides a fusion protein comprising: (1) an influenza virus HA protein or a fragment thereof; and (2) an antigenic epitope, wherein the antigenic epitope is an antigenic epitope derived from an influenza virus protein. The influenza virus HA protein may be derived from an H1N1 strain, an H3N2 strain, or a BV strain. Preferably, the influenza virus protein is selected from M2, M1, and NA proteins. In some embodiments, the lymphatic B or T cell epitope is derived from an antigenic epitope of M2, M1, and NA, for example, one or more fragments thereof are respectively extracted from M2, M1, and NA proteins. Specifically, the lymphatic B or T cell epitope may be as shown in SEQ ID NO: 4, 5, 6.

[0009] In some embodiments, the HA protein or its fragment is selected from: (a) an HA protein or its fragment derived from an H1N1 strain, which comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 95% identity thereto; (b) an HA protein or its fragment derived from an H3N2 strain, which comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 95% identity thereto; and (c) an HA protein or its fragment derived from a BV strain, which comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 95% identity thereto.

[0010] In some embodiments, the fusion protein comprises an HA protein or a fragment thereof derived from an H1N1 strain, and an epitope derived from any one or more of M2, M1, and NA. In some embodiments, the fusion protein comprises an HA protein or a fragment thereof derived from an H3N2 strain, and an epitope derived from any one or more of M2, M1, and NA. In some embodiments, the fusion protein comprises an HA protein or a fragment thereof derived from a BV strain, and an epitope derived from any one or more of M2, M1, and NA.

[0011] In some embodiments, the fusion protein comprises an HA protein or a fragment thereof derived from an H1N1 strain and an antigenic epitope derived from M2; wherein the HA protein or a fragment thereof derived from an H1N1 strain comprises the amino acid sequence shown in SEQ ID NO:1, and the antigenic epitope derived from M2 comprises the amino acid sequence shown in SEQ ID NO:4.

[0012] In some embodiments, the fusion protein comprises an HA protein or a fragment thereof derived from an H3N2 strain and an antigenic epitope derived from M1; wherein the HA protein or a fragment thereof derived from an H3N2 strain comprises the amino acid sequence shown in SEQ ID NO:2, and the antigenic epitope derived from M1 comprises the amino acid sequence shown in SEQ ID NO:5.

[0013] In some embodiments, the fusion protein comprises an HA protein or a fragment thereof derived from a BV strain and an antigenic epitope derived from NA; wherein the HA protein or a fragment thereof derived from a BV strain comprises the amino acid sequence shown in SEQ ID NO:3, and the antigenic epitope derived from NA comprises the amino acid sequence shown in SEQ ID NO:6.

[0014] In some embodiments, the fusion protein further comprises ferritin, preferably the ferritin is located at the C-terminus of the fusion protein; preferably, the fusion protein is in the form of nanoparticles; optionally, the fusion protein further comprises a signal peptide at the N-terminus. In some embodiments, the ferritin is full-length ferritin or truncated ferritin (e.g., N-terminal truncated M residues, as shown in SEQ ID NO: 7).

[0015] In some embodiments, the ferritin is selected from bacterial ferritin, plant ferritin, algae ferritin, fungal ferritin, insect ferritin or mammalian ferritin, preferably mammalian ferritin or bacterial ferritin. In some embodiments, the bacterial ferritin is Helicobacter pylori ferritin. In some embodiments, the Helicobacter pylori ferritin has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 7.

[0016] In some embodiments, the HA protein or fragment thereof, the antigenic epitope and the ferritin are directly connected or connected via a linker sequence, and the linker sequence is selected from SGS, SGG, EAAAK, GGGGS, GPGPG, AAY and RVRR linkers, preferably, the linker sequence is SGG, GGGGS, and EAAAK.

[0017] In some embodiments, the fusion protein comprises: (a) the amino acid sequence shown in SEQ ID NO: 1, 2 or 3 or a fragment thereof; (b) the amino acid sequence shown in SEQ ID NO: 4, 5 or 6; and (c) the amino acid sequence shown in SEQ ID NO: 7 or a fragment thereof.

[0018] In some embodiments, the fusion protein comprises: (a) the amino acid sequence shown in SEQ ID NO:8, 9 or 10; (b) the amino acid sequence shown in SEQ ID NO:11, 12 or 13; or (c) an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to (a) or (b).

[0019] In one aspect, the present invention provides a fusion protein combination, which comprises a combination of three fusion proteins (referred to as a first fusion protein, a second fusion protein, and a third fusion protein) as disclosed herein, wherein each of the first fusion protein, the second fusion protein, and the third fusion protein comprises the following two parts:

[0020] (1) influenza virus HA protein or a fragment thereof; and

[0021] (2) An antigenic epitope, wherein the antigenic epitope is an antigenic epitope derived from an influenza virus protein, and the influenza virus protein is selected from the group consisting of M2, M1 and NA proteins.

[0022] In some embodiments, the first fusion protein comprises:

[0023] An HA protein or a fragment thereof derived from an H1N1 strain, comprising the amino acid sequence shown in SEQ ID NO: 1; and an antigenic epitope derived from an M2 protein, comprising the amino acid sequence shown in SEQ ID NO: 4.

[0024] In some embodiments, the second fusion protein comprises:

[0025] An HA protein or a fragment thereof derived from an H3N2 strain, comprising the amino acid sequence shown in SEQ ID NO:2; and an antigenic epitope derived from an M1 protein, comprising the amino acid sequence shown in SEQ ID NO:5.

[0026] In some embodiments, the third fusion protein comprises:

[0027] An HA protein or a fragment thereof derived from a BV strain, comprising the amino acid sequence shown in SEQ ID NO:3; and an antigenic epitope derived from a NA protein, comprising the amino acid sequence shown in SEQ ID NO:6.

[0028] Preferably, the first, second and third fusion proteins are all fused with ferritin at the C-terminus and are in the form of nanoparticles. Optionally, the first, second and third fusion proteins are all fused with a signal peptide at the N-terminus.

[0029] In some embodiments, the first, second and third fusion proteins respectively comprise the amino acid sequence shown in SEQ ID NO:8, 9 or 10, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:8, 9 or 10.

[0030] In one aspect, the present invention provides a vaccine composition comprising the antigen composition disclosed herein and a pharmaceutically acceptable carrier and / or adjuvant. In some embodiments, the adjuvant comprises MF59 adjuvant.

[0031] In some embodiments, in a unit dose of the vaccine composition, the antigen composition comprises 2-45 μg of total HA, preferably 5-15 μg of total HA. In some embodiments, the ratio between the volume of the antigen composition and the volume of the MF59 adjuvant is 1:1.

[0032] In one aspect, the present invention provides a nucleic acid molecule encoding any one of the fusion proteins disclosed herein.

[0033] In one aspect, the invention provides a vector comprising a nucleic acid molecule disclosed herein, for example, the vector is a baculovirus vector.

[0034] In one aspect, the present invention provides a host cell comprising a nucleic acid molecule disclosed herein or a vector disclosed herein, optionally wherein the host cell is an E. coli cell or an insect cell.

[0035] In one aspect, the present invention provides a method for preparing the fusion proteins (first, second and / or third fusion proteins) disclosed herein, comprising the following steps: culturing host cells transfected with the vector disclosed herein under suitable conditions; and isolating the fusion protein from the supernatant of the host cells.

[0036] In one aspect, the present invention provides a method for preparing an influenza virus vaccine, comprising the step of mixing the antigen composition of the present invention with an adjuvant, optionally wherein the adjuvant is MF59 adjuvant.

[0037] In one aspect, the present invention relates to the use of the fusion protein disclosed herein or a combination thereof or an antigenic composition in the preparation of a vaccine composition for preventing influenza virus infection in a subject, preferably, the vaccine composition is administered to the subject by intramuscular injection. In some embodiments, the subject is a human or non-human animal, preferably a human. In some embodiments, the influenza virus infection is selected from H1N1 virus infection, H3N2 virus infection and BV virus infection.

[0038] In one aspect, the present invention relates to use of the fusion protein disclosed herein in the preparation of a medicament for inducing an immune response against influenza virus in an organism, wherein the immune response is humoral immunity and / or cellular immunity.

[0039] In one aspect, the present invention relates to the use of the fusion protein disclosed herein in producing antibodies against influenza virus. In some embodiments, the antibody comprises a polyclonal antibody, a monoclonal antibody or a recombinant antibody.

[0040] The present invention also relates to the following embodiments:

[0041] In some aspects, the present invention provides an influenza vaccine, characterized in that:

[0042] The vaccine is composed of a combination of trivalent recombinant influenza ferritin nanoparticle antigen and MF59 adjuvant, and is administered by intramuscular injection.

[0043] The trivalent recombinant influenza ferritin nanoparticle antigen comprises fusion proteins formed by ferritin and HA proteins of H1N1, H3N2, and BV influenza strains, respectively:

[0044] In some aspects, the present invention provides a new influenza vaccine antigen design scheme, characterized in that: the antigen design scheme is any combination of M2, M1, and NA influenza broad-spectrum immune epitopes on the HA protein of H1N1, H3N2, and BV influenza strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The schematic diagram of the composition of three nanoparticles fused with ferritin is shown. The HA proteins of H1N1, H3N2, and BV strains form three fusion proteins with the broad-spectrum epitopes of M2, M1, and NA, as well as ferritin (Ftn). The yellow lines represent peptide linkers, and the black dotted boxes represent the optional presence of broad-spectrum epitopes.

[0046] Figure 2 The SDS-PAGE test results of HA ferritin nanoparticle antigens of H1N1, H3N2, and BV strains are shown, and Mark is a reference for molecular weight.

[0047] Figure 3Shown is the expression of H1N1 hemagglutination-inhibiting antibodies induced by the trivalent influenza nanoparticle vaccine in Balb / c mice.

[0048] Figure 4 Shown is the expression of H3N2 hemagglutination-inhibiting antibodies induced by the trivalent influenza nanoparticle vaccine in Balb / c mice.

[0049] Figure 5 Shown is the expression of BV hemagglutination-inhibitory antibodies induced by the trivalent influenza nanoparticle vaccine in Balb / c mice.

[0050] Figure 6 The trivalent influenza nanoparticle vaccine combined with MF59 adjuvant induced the expression of H1N1, H3N2, and BV antigen-specific IgG in Balb / c mice.

[0051] Figure 7 The figure shows the body weight change curve of Balb / c mice immunized with MF59 adjuvanted trivalent influenza nanoparticle vaccine and positive control vaccine after H1N1 challenge.

[0052] Figure 8 The figure shows the body weight change curve of Balb / c mice immunized with trivalent influenza nanoparticle vaccine without adjuvant and positive control vaccine after H1N1 infection.

[0053] Fig. 9 Shown are the viral loads in the lungs of Balb / c mice immunized with different vaccines after homologous H3N2 challenge.

[0054] Fig.10 Shown are the viral loads in the nasal turbinates of Balb / c mice immunized with different vaccines after homologous H3N2 challenge.

[0055] Fig.11 Shown are the viral loads in the lungs of Balb / c mice immunized with different vaccines after BV challenge.

[0056] Fig.12 The viral load of the nasal turbinates of Balb / c mice immunized with different vaccines after BV challenge is shown.

[0057] Fig.13 The figure shows the body weight change curve of Balb / c mice immunized with MF59 adjuvanted trivalent influenza nanoparticle vaccine and positive control vaccine after heterologous H3N2 challenge.

[0058] Fig.14 The figure shows the body weight change curve of Balb / c mice immunized with unadjuvanted trivalent influenza nanoparticle vaccine and positive control vaccine after heterologous H3N2 challenge.

[0059] Fig.15 Shown are the survival curves of Balb / c mice immunized with MF59-adjuvanted trivalent influenza nanoparticle vaccine and positive control vaccine after heterologous H3N2 challenge. DETAILED DESCRIPTION

[0060] In the present invention, unless otherwise stated, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art. All patents, patent applications and other publications cited herein are incorporated by reference in their entirety. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. If the definition proposed herein conflicts with the definition proposed in the patents, patent applications and other publications incorporated herein by reference, the definition proposed in this article shall prevail.

[0061] Unless the context requires otherwise, terms in the singular shall include the plural, and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a protein" includes multiple proteins; reference to "a cell" includes a mixture of cells, etc. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include the endpoints and all values ​​between the endpoints.

[0062] As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector can be transformed, transduced or transfected into a host cell to express the genetic material elements carried in the host cell. Vectors are well known to those skilled in the art, including but not limited to plasmid phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phages or M13 phages and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). The vector may include multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may include an origin of replication.

[0063] "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of the amino acid residues in the candidate sequence and the identical amino acid residues in the reference polypeptide sequence after the sequences are aligned (and gaps are introduced if necessary) to obtain the maximum percentage sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or DNASTAR software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment for the full length of the compared sequences. When the percentage of sequence identity is mentioned in this application, if not otherwise specifically stated, these percentages are calculated relative to the full length of the longer sequence. Calculations relative to the full length of the longer sequence are applicable to both nucleic acid sequences and polypeptide sequences.

[0064] As used herein, the term "operably linked" (or represented by "-") refers to the juxtaposition of two or more biological sequences of interest (with or without a spacer or linker or intervening sequence) so that they are in a relationship that allows them to function in the intended manner. When used in relation to polypeptides, it refers to the connection of polypeptide sequences in a manner that allows the connected product to have the intended biological function. The operably linked can be a direct connection between the two parts, or a connection through a linker such as a peptide linker.

[0065] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked.

[0066] As used herein, the term "fusion protein" refers to a polypeptide having two (or more) parts operably linked together, wherein each part is a polypeptide having different properties. The various parts can be directly linked by a single peptide bond, or connected by a peptide linker containing one or more amino acid residues. In some embodiments, the fusion protein is a fusion protein of influenza virus HA protein or a fragment thereof, an antigenic epitope and ferritin, wherein the antigenic epitope is an antigenic epitope derived from an influenza virus protein, preferably selected from an antigenic epitope derived from influenza virus M2, M1, and NA protein.

[0067] As used herein, the term "humoral immunity" refers to an immune mechanism that achieves protection by producing antibodies through plasma cells (i.e., effector B cells). After the receptor molecules on the surface of B cells come into contact with antigens, plasma cells and memory B cells are activated and produced, and plasma cells produce specific antibodies that can bind to antigens. Methods for detecting humoral immunity include, but are not limited to, serum immunoglobulin (Ig) determination and secretory IgA (sIgA) determination. sIgA is mainly distributed in exocrine fluids such as saliva, tears, gastrointestinal fluid, milk, and respiratory secretions, and is the main antibody in human mucosal immunity.

[0068] As used herein, the term "cellular immunity" refers to the effect of immune cells to clear foreign bodies. Natural killer cells (NK) and antibody-dependent cell-mediated cytotoxic cells (ADCC) such as macrophages (Mφ) and killer cells (K) and cellular immunity mediated by T cells are all cellular immunity, among which T cell-mediated cellular immunity is specific cellular immunity, mainly including CD4 + T cell responses and CD8 + T cell response. Cellular immunity can be detected by detecting the production of cytokines or cytotoxic substances.

[0069] As used herein, the terms "subject" and "patient" are used interchangeably and refer to any living organism, including humans and animals.

[0070] As used herein, the term "immunogenicity" refers to the ability of an antigen to stimulate the body to form specific antibodies or sensitized lymphocytes. For influenza vaccines, the evaluation indicators are mainly virus strain-specific hemagglutination inhibition (HAI) antibody titer and serum antibody positive conversion rate. Those skilled in the art are familiar with the determination methods of hemagglutination inhibition (HAI) antibody titer and serum antibody positive conversion rate.

[0071] As used herein, "viral load" refers to the total amount of virus in a sample, usually expressed in units of genomes or antigens. Viral load is a commonly used indicator when analyzing the degree of viral infection or the distribution of viral particles.

[0072] As used herein, "virus titer" refers to the activity of the virus, that is, the unit of how much infectivity the virus can produce under certain conditions. Commonly used virus titer units include TCID50 (50% tissue culture infectious dose), PFU (unit containing infectious units), etc.

[0073] As used herein, "viral RNA copy number" refers to the amount of viral RNA in a sample, which is usually measured using techniques such as RT-qPCR. The viral RNA copy number can be used to assess the extent of viral infection and can also be used to detect viral load and viral titer.

[0074] As used herein, the term "valent" when used in relation to a vaccine refers to the type or number of serotypes of the same pathogen targeted by the vaccine. For example, a vaccine containing three influenza virus antigen components, namely, influenza A(H1N1), influenza A(H3N2) and influenza B(Victoria) is a trivalent influenza vaccine.

[0075] Nanoparticle vaccines and ferritin

[0076] Nano vaccines are a general term for new vaccines that use nanoparticles (NP) as carriers or adjuvants. They are one of the key directions of current vaccine research and development, including liposome nanoparticle (LNP) vaccines, metallic nanoparticle (metallic NP) vaccines, and protein nanoparticle (protein NP) vaccines. Nanoparticles are artificially manufactured or naturally formed microparticles with a particle size of 1 to 100 nm. Nanoparticle vaccines have advantages in antigen presentation, antigen assembly, and lymph node accumulation.

[0077] Several types of protein nanoparticles made from proteins from natural sources have been used to deliver antigens. Ferritin (Ftn) is a class of soluble proteins widely present in organisms and plays a key role in iron metabolism. Ferritin is a hollow protein shell (outer diameter 12-14 nm, inner 8-10 nm), its secondary and tertiary structures are highly conserved, and it consists of 24 subunits. Ferritin is present in almost all organisms, including bacteria, fungi, plants, and animals. Ferritin in vertebrates and mammals is composed of two subunits (H and L), while ferritin molecules isolated from plants and bacteria contain only H-type chains. Ferritin can self-assemble into 24-mers with a diameter of about 12-14 nm in vitro. The self-assembled nanoparticle material has good biocompatibility and uniform and stable particle size. Nano-scale macromolecules have very strong antigenicity. Ferritin is used as a carrier for carrying antigen epitopes. Through its self-assembly, antigens can be displayed on the surface of ferritin nanoparticles to enhance their immune activity. In addition, ferritin can be modified by chemical methods or genetic engineering modification methods, for example, a viral peptide is fused and expressed at the N-terminus of a ferritin subunit, and the resulting protein has a ferritin nanostructure.

[0078] In some embodiments, ferritin from Helicobacter pylori (H. pylori) is used to prepare nanoparticles of influenza vaccine compositions. Helicobacter pylori ferritin is a natural and safe self-assembling nanocarrier, and the inventors have found that it has superior immunological activity when used to display and deliver the fusion antigen of the present invention and is suitable for intramuscular administration.

[0079] Influenza virus and antigen

[0080] Influenza virus (IV) belongs to the family Orthomyxoviridae. It is a single-stranded, negative-stranded, segmented RNA virus that can cause seasonal or pandemic influenza in humans and other animals. According to its nucleoprotein and matrix protein, it is divided into four types: A, B, C, and D (or A, B, C, and D). Among them, influenza A, B, and C viruses (IAV, IBV, and ICV) can be transmitted in humans. Compared with influenza C virus, influenza A virus can cross-transmit between different species, causing large-scale outbreaks and epidemics; influenza B virus mainly infects humans and causes local epidemics. Hemagglutinin (HA) and neuraminidase (NA) distributed on the surface of the virus envelope play a vital role in the process of viral infection and replication. According to the genetic and antigenic properties of HA and NA, influenza A virus can be divided into 18 H subtypes (H1-H18) and 11 N subtypes (N1-N11), and influenza B virus can be divided into two antigenic lineages, Victoria lineage (V lineage, BV) and Yamagata lineage (Y lineage, BY). For example, influenza A virus subtypes include, for example, H1N1 and H3N2.

[0081] The genetic material of influenza A and B viruses consists of 8 segments, while the genetic material of influenza C virus consists of 7 segments. Segments 1, 2, and 3 encode RNA polymerase, including PA, PB1 (polymerase basic 1), and PB2, which are responsible for the transcription and replication of viral genetic material RNA; segment 4 encodes HA, which is responsible for virus invasion; segment 5 encodes nucleoprotein (NP), which is responsible for assembly with genetic material RNA; segment 6 encodes NA, which is responsible for virus release; segment 7 encodes matrix protein 1 (M1) and matrix protein 2 (M2), which are responsible for the formation of the matrix protein layer of the virus; segment 8 encodes non-structural proteins (including NS1 and NS2), whose functions are related to RNA nuclear export and antagonism of host antiviral response. Influenza C virus lacks segment 6, and its segment 4 encodes hemagglutinin esterase fusion protein (HEF), which has both the functions of virus invasion and release.

[0082] Since the influenza virus replicates without the involvement of RNA proofreading enzymes, RNA polymerase will make errors for every approximately 10,000 nucleotides it replicates, causing it to mutate more frequently than other viruses. In addition, the segmented nature of the influenza virus genome means that when different types and subtypes of viruses infect a cell at the same time, gene reassortment may occur, leading to larger mutations in the viral genome. In addition, in response to antigenic drift and shifts of influenza viruses, seasonal influenza vaccines need to be updated annually to match prevalent strains. Therefore, it is necessary to develop a broad-spectrum universal influenza vaccine, especially a safe and effective universal influenza vaccine for intramuscular injection, to achieve cross-protection against different influenza viruses.

[0083] In order to develop a universal influenza vaccine, the strategy of the present invention uses the hemagglutinin HA with a high content and the antigenic regions such as M2, M1, and NA with high conservation in the influenza virus strain as immunogens to induce the body to produce a cross-protective T cell immune response to cope with the variation of the influenza virus. Among them, matrix protein 2 (M2) is a protein unique to influenza A virus. The extracellular functional region (M2 ectodomain, M2e) of influenza A virus M2 includes 24 amino acids. M2e is almost completely conserved in all influenza A virus subtypes and can be used as an antigen for a broad-spectrum influenza A vaccine, that is, the development of a universal vaccine using M2e can prevent all subtypes of influenza A virus infection. Neuraminidase (NA) is another major glycoprotein on the surface of influenza virus. It promotes the release of viral particles and prevents the accumulation of progeny viruses on the cell surface by cutting the viral sialic acid receptors on the host cell membrane, and plays an important role in virus transmission. NA protein induces the body to produce NA-specific antibodies, which are cleared by antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), playing an important role in inhibiting virus transmission and controlling influenza infection. NA has a lower frequency of antigenic drift than HA and can provide a wider range of protection, making it an ideal antigen for universal influenza vaccines. Part of the amino acid sequence of the M1 antigen is highly conserved among influenza A viruses, mainly inducing host cellular immunity, especially cytotoxic T lymphocyte (CTL) response. The extracellular functional region M2e of influenza A virus M2 mainly induces humoral immunity. HA protein is a homotrimeric transmembrane glycoprotein on the surface of the viral envelope. Each monomer consists of two disulfide-linked glycosylated polypeptides HA1 and HA2. These polypeptides constitute two structurally different domains: a globular head domain composed of part of HA1, and a stem domain composed of part of HA1 and all of HA2. The head domain contains many dominant epitopes and has strong immunogenicity, but it varies greatly among influenza virus strains, while the stem domain is highly conserved among influenza virus strains.

[0084] In some embodiments, a combination of one or more of M1 protein, M2 protein, HA protein, NA protein, and fragments or portions thereof (e.g., M2e, HA2) is used to prepare antigens for influenza vaccine compositions. Preferably, a combination of two, more preferably three, and more preferably four of the above proteins and fragments or portions thereof is used to prepare antigens. The proteins may be derived from various influenza A virus subtypes such as H1N1 and H3N2, and influenza B viruses such as BV.

[0085] Recombinant influenza fusion protein antigen

[0086] The present invention provides one or more fusion proteins that can be used as influenza virus antigens for preparing vaccine compositions. In some embodiments, the fusion protein comprises two or more parts of highly conserved proteins derived from influenza viruses that are operably linked. Preferably, the fusion protein is further fused to or operably linked to ferritin, and ferritin can be used as a nanocarrier to display antigenic epitopes on the surface of ferritin nanoparticles.

[0087] In some embodiments, the fusion protein comprises: (1) part or all of the HA protein derived from influenza virus, and (2) an epitope derived from a protein selected from influenza virus NA, M2 and M1. For example, the epitope can be derived from M2 protein, M1 protein or NA protein. Parts (1) and (2) can be directly connected or operably connected through a linker. The fusion protein can contain multiple identical or different epitope parts. Optionally, the fusion protein also contains a signal peptide sequence or other tag sequence that facilitates expression or purification at the N-terminus. Preferably, the fusion protein is further fused to ferritin at the C-terminus. This type of ferritin-containing fusion protein is also referred to herein as HA ferritin nanoparticle antigen.

[0088] The influenza virus protein may be a human influenza virus protein, an avian influenza virus protein or a swine influenza virus protein. It may be a human, avian, swine influenza virus protein and its mutant amino acid sequence obtained by artificial synthesis or genetic engineering technology. On the basis of not affecting the immunogenicity of the protein, the present invention also includes the corresponding influenza virus protein mutated by at least one amino acid. The epitope may be derived from proteins with higher conservatism such as M2 protein, M1 protein or NA protein of different influenza virus strains such as influenza A virus and influenza B virus. In some embodiments, the HA protein portion of the influenza virus is respectively from influenza viruses selected from the following: H1N1, H3N2 and BV. More specifically, the HA proteins of H1N1 strains, H3N2 strains and BV strains are shown in SEQ ID NOs: 14, 15 and 16, respectively, and therefore, the HA protein portion in the fusion protein may include or consist of fragments of SEQ ID NOs: 14, 15 and 16.

[0089] In some embodiments, the fusion protein comprises (1) a portion of an influenza virus HA protein derived from influenza virus H1N1, and (2) an epitope derived from an influenza virus M2 protein. In some embodiments, the influenza virus HA protein is an H1N1 strain HA protein (denoted as H1N1-HA), which has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the epitope has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 4. The fusion protein may have, for example, a sequence as shown in SEQ ID NO: 11.

[0090] In some embodiments, the fusion protein comprises (1) a portion of an influenza virus HA protein derived from influenza virus H3N2, and (2) an epitope derived from an influenza virus M1 protein. In some embodiments, the influenza virus HA protein is an H3N2 strain HA protein (denoted as H3N2-HA), which has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the epitope has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 5. The fusion protein may have, for example, a sequence as shown in SEQ ID NO: 12.

[0091] In some embodiments, the fusion protein comprises (1) a portion of an influenza virus HA protein derived from an influenza virus BV, and (2) an epitope derived from an influenza virus NA protein. In some embodiments, the influenza virus HA protein is a BV strain HA protein (denoted as BV-HA), which has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the epitope has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 6. The fusion protein may have, for example, a sequence as shown in SEQ ID NO: 13.

[0092] As described above, the fusion protein may further comprise (3) ferritin, preferably located at the C-terminus of the fusion protein. Ferritin may be full-length ferritin or truncated ferritin, such as a monomeric subunit of full-length ferritin or truncated ferritin. In some embodiments, ferritin may include bacterial-derived ferritin, plant-derived ferritin, algae-derived ferritin, fungal-derived ferritin, insect-derived ferritin or mammal-derived ferritin, etc., preferably mammal-derived ferritin or bacterial-derived ferritin. Preferably, the mammal-derived ferritin includes human-derived ferritin, mouse-derived ferritin or horse spleen ferritin. Preferably, the bacterial-derived ferritin includes Helicobacter pylori ferritin or Escherichia coli ferritin. In some embodiments, ferritin is Helicobacter pylori ferritin, which has an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the amino acid sequence shown in SEQ ID NO:7.

[0093] In the fusion protein, each part or fragment can be directly connected or operably connected through a joint. The joint can be a flexible or non-flexible joint conventionally used in the art, or a joint for epitope connection. In some embodiments, the joint is a peptide joint having any of the following sequences: SGG, SGS, EAAAK, GGGGS, AAY, GPGPG, KK and RVRR joints. In some embodiments, the joint sequence is SGG, GGGGS or EAAAK.

[0094] In one embodiment, the fusion protein comprises H1N1-HA or a fragment thereof, an epitope derived from the M2 protein and ferritin operably linked from the N-terminus to the C-terminus, which is represented as HA(H1N1)-M2e-Ftn, and has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:8.

[0095] In one embodiment, the fusion protein comprises H3N2-HA or a fragment thereof, an epitope derived from the M1 protein, and ferritin operably linked from the N-terminus to the C-terminus, which is represented as HA(H3N2)-M1-Ftn, and has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:9.

[0096] In one embodiment, the fusion protein comprises BV-HA or a fragment thereof, an epitope derived from a NA protein and ferritin operably linked from the N-terminus to the C-terminus, which is represented as HA(BV)-NA-Ftn, and has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:10.

[0097] Compositions containing fusion proteins

[0098] The present invention also provides a composition (eg, a vaccine composition or an antigen composition) comprising any one or a combination of the fusion proteins described above. Preferably, the composition comprises multiple fusion proteins to achieve cross-protection against different influenza viruses.

[0099] In some embodiments, the composition comprises three fusion proteins, wherein the first fusion protein comprises a HA protein portion derived from H1N1 and an epitope derived from M2 protein, the second fusion protein comprises a HA protein portion derived from H3N2 and an epitope derived from M1 protein, and the third fusion protein comprises a HA protein portion derived from BV and an epitope derived from NA protein. The three fusion proteins can be mixed in the composition in different proportions.

[0100] In some embodiments, the first fusion protein has the form of HA (H1N1) -M2e-Ftn, the second fusion protein has the form of HA (H3N2) -M1-Ftn, and the third fusion protein has the form of HA (BV) -NA-Ftn. In some embodiments, the composition comprises the first, second and third fusion proteins in equal or similar proportions. In other embodiments, the proportion of one fusion protein may be the sum of the proportions of the other two fusion proteins. In some embodiments, the proportion of the third fusion protein contained in the composition is higher than the proportion of the first fusion protein, and the proportion of the first fusion protein is higher than the proportion of the second fusion protein. In this article, unless otherwise specified, the proportion of the first, second and third fusion proteins or nanoparticle antigens refers to the weight ratio of the content of the HA antigen components contained therein. For example, the ratio of the first, second and third fusion proteins is 2: 1: 3, which means that the weight ratio of the HA antigen components contained in the first, second and third fusion proteins is 2: 1: 3, for example, the first, second and third fusion proteins contain 10 μg, 5 μg and 15 μg of biologically active hemagglutinin HA antigens, respectively.

[0101] In some embodiments, the composition comprises the first, second and third fusion proteins in a ratio of about (2±1):(2±1):(2±1), i.e., the ratio of each fusion protein can be varied by 50% on the basis of 2:2:2. For example, the ratio between the first, second and third fusion proteins can be adjusted in the range of about (2±1):1:3 to (2±1):3:1, or in the range of about 1:(2±1):3 to 3:(2±1):1, or in the range of about 1:3:(2±1): to 3:1:(2±1).

[0102] In some specific embodiments, the antigen components contained in the antigen composition are fusion proteins H1N1-HA-M2e-Ftn, H3N2-HA-M1-Ftn, and BV-HA-NA-Ftn, wherein H1N1-HA-M2e-Ftn, H3N2-HA-M1-Ftn, and BV-HA-NA-Ftn (also expressed as H1:H3:BV) are combined in a ratio of about 2:2:2. In some specific embodiments, the antigen components contained in the antigen composition are fusion proteins H1N1-HA-M2e-Ftn, H3N2-HA-M1-Ftn, and BV-HA-NA-Ftn, wherein H1N1-HA-M2e-Ftn, H3N2-HA-M1-Ftn, and BV-HA-NA-Ftn are combined in a ratio of about 2:1:3.

[0103] Methods for measuring the content of the HA antigen component in the fusion protein are known to those skilled in the art, such as measurement by single radial immunodiffusion (SRD) (JM Wood et al.: J. Biol. Stand. 5 (1977) 237-247; JM Wood et al., J. Biol. Stand. 9 (1981) 317-330). Alternatively, the content of the HA antigen component can also be determined by methods such as reverse HPLC, enzyme-multiplied immunoassay technique (EMIT), SDS-PAGE and (surface plasmon resonance, SRP).

[0104] Preparation of vaccine compositions

[0105] In some aspects, the present invention provides a vaccine composition comprising the fusion protein disclosed herein and a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier is an adjuvant.

[0106] The vaccine composition of the present invention can be prepared by mixing the fusion protein or antigen composition disclosed herein with various adjuvants known in the art in a certain proportion. The adjuvants include, but are not limited to, TLR (Toll-like receptor) agonists, NOD agonists, CLR (C-type lectin receptor) agonists, RLR agonists, aluminum adjuvants (e.g., aluminum hydroxide, aluminum phosphate), squalene-containing oil-in-water emulsions (e.g., MF59), AS adjuvant systems (e.g., AS01, AS03, AS04), saponin adjuvants, α-galactosylceramide-derived adjuvants, and polysaccharide adjuvants.

[0107] MF59 or MF59 analogs are one of the earliest clinical adjuvants approved for use. It is a water-oil emulsion adjuvant, mainly composed of squalene, Tween-80 and Span85. Its water-in-oil design significantly reduces viscosity and enhances human tolerance. Squalene is an oil phase component, widely distributed in animals, plants and the human body, and has the characteristics of biocompatibility and degradability. Tween-80 and Span85 are surfactants that enhance the stability of the emulsion. MF59 is an ideal adjuvant for viral vaccines. When used in combination with influenza, HIV and other vaccines, it can significantly enhance antibody titers and has good tolerance in different populations. It can promote antigen uptake, recruit a large number of immune cells into the injection site, and recruit monocytes and neutrophils to present antigens and transport them into lymph nodes.

[0108] In one aspect, the present invention provides MF59 adjuvant for preparing influenza virus vaccine compositions, in particular for preparing vaccine compositions for intramuscular injection. The inventors have found that the vaccine composition produced by combining MF59 adjuvant with the fusion protein herein has a strong ability to induce specific humoral immunity and cellular immunity.

[0109] Suitably, the vaccine composition of the present invention can be prepared as a vaccine dose volume of 0.1mL to 1mL, preferably a vaccine dose volume of 0.2mL or 0.25mL or 0.5mL. The dose volume can be slightly adjusted according to the HA concentration in the original bulk sample and according to the delivery route. For example, the vaccine composition of the present invention contains 1 to 15μg HA for each influenza virus strain. The small amount of HA can be as low as practical, provided that it allows the preparation of vaccines that meet Chinese or international (e.g., EU or FDA) efficacy standards. For example, a low amount of HA can be 1 to 7.5μg HA for each influenza virus strain, suitably 1.25 to 7.5μg HA for each influenza virus strain, such as 2.5, 5 or 7.5μg HA for each influenza virus strain.

[0110] Mode of administration of vaccine composition

[0111] The vaccine compositions of the present invention can be administered by any suitable delivery route, such as intradermal, mucosal (eg, intranasal), oral, intramuscular (IM), or subcutaneous. Other delivery routes are well known in the art.

[0112] Preferably, the vaccine composition of the present invention is administered by intramuscular injection. The intramuscular delivery route is particularly suitable for immunogenic compositions, particularly for immunogenic compositions with adjuvants. The composition may be present in a single-dose container, or alternatively, in a multi-dose container. In this case, an antimicrobial preservative, such as thimerosal, may be present to prevent contamination during use. Suitable thimerosal concentrations are 5 μg / 0.5mL doses (i.e., 10 μg / mL) or 10 μg / 0.5mL doses (i.e., 20 μg / mL). Suitable IM delivery devices may be used, such as needle-free liquid jet injection devices, such as Biojector 2000 (Bioject, Portland, Oregon, USA). Alternatively, pen injection devices (e.g., devices for delivering epinephrine at home) can be used for self-administration of vaccines. The use of such delivery devices may be particularly suitable for large-scale immunization activities.

[0113] Methods for establishing strong and lasting immunity generally include repeated immunization, i.e., strengthening the immune response by administering one or more further doses. The vaccine composition of the present invention can be used in a multiple dose administration regimen, such as a two-dose or three-dose administration regimen. The doses administered at different times in the multiple dose administration regimen can be the same or different. The interval between two or three administrations can be two weeks to six months, such as three weeks to three months. Regular long-term booster doses can also be provided, such as once every 2 to 10 years.

[0114] Polynucleotides, vectors and host cells

[0115] In some aspects, the present invention also provides polynucleotides encoding the fusion proteins disclosed herein. The polynucleotides can be used to express the fusion proteins. The polynucleotides encoding the fusion proteins are easy to separate and sequence using conventional procedures known in the art. The polynucleotides can also be obtained by synthetic methods. Preferably, the polynucleotides are codon optimized for expression in eukaryotic host cells, such as insect cells and mammalian cells.

[0116] Using known recombinant techniques, the polynucleotide encoding the fusion protein can be inserted into a vector for further cloning (amplification of the DNA) or expression. The vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0117] In some embodiments, the present invention provides a vector (e.g., an expression vector) comprising a polynucleotide encoding a fusion protein provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the polynucleotide, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), λ phages, and M13 phages, liposomes, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEM EX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM, pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0118] The vector containing the polynucleotide sequence encoding the fusion protein can be introduced into the host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vector herein can be prokaryotes, yeast or higher eukaryotic cells. Suitable prokaryotes for this purpose include true bacteria, such as Gram-negative or Gram-positive bacteria, such as Escherichia coli. In some preferred embodiments, the fusion protein of the present invention is expressed by Escherichia coli DH10Bac-ΔCP-VN, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee (CGMCC, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), with accession number 23911, so as to express it with high yield.

[0119] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for the vectors provided. Saccharomyces cerevisiae or common baker's yeast are the most commonly used in lower eukaryotic host microorganisms. Suitable host cells for expressing the fusion proteins provided herein can also be derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In some other embodiments, the host cell is other mammalian cell lines, such as human cell lines. In some other embodiments, the host cell is an insect cell. In some preferred embodiments, the fusion protein of the present invention is expressed by sf9-RF insect cells deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee (CGMCC, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), accession number 45028, on December 27, 2021, so as to express it in high yield. In some preferred embodiments, the fusion protein of the present invention is expressed by High Five-NV free insect cells with accession number 22356, which were deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​June 21, 2021, so as to express it at a high yield. The fusion protein of the present invention can be obtained by the insect baculovirus expression vector system (BEVS). BEVS is an expression system with insect baculovirus as an exogenous gene vector and insects and insect cells as receptors. The patterns and abilities of translation and post-translational protein modification of insect cells and mammals are similar, including glycosylation, phosphorylation, acylation, signal peptide excision, and cleavage and decomposition of peptide segments, etc. The obtained recombinant protein has similar biological activities such as antigenicity, immunogenicity and function as natural proteins; it can accommodate macromolecular inserts; it is easy to achieve large-scale low-cost production. Currently commonly used BEVS include, for example, Bac-to-Bac system, BaculoDirect system, BacPAK6 and BaculoGOLD system, FlashBAC system, etc. The above BEVS can all be used to express the fusion protein of the present invention.

[0120] In some embodiments, the fusion protein of the present invention is expressed by the Bac-to-Bac system, which mainly includes the following steps: cloning the target gene into the pFastBac vector; transforming the above plasmid into a bacterial strain (e.g., a DH10Bac strain) to prepare a recombinant baculovirus shuttle plasmid (Bacmid); transforming the recombinant Bacmid into insect cells to prepare a recombinant baculovirus; amplifying the baculovirus, infecting insect cells, and expressing the recombinant protein. Examples of pFastBac vectors include, but are not limited to, pFastBac1, pFastBac Dual, pFastBac HT A, pFastBac HT B, and pFastBac HT C vectors. In some embodiments, the pFastBac vector is a pFastBac1 vector. Examples of insect cells include, but are not limited to, Sf9, Sf21, and High Five (HF) cells. In some embodiments, the insect cell is an Sf9 cell. In some embodiments, the insect cell is an Sf9 cell. In some embodiments, the insect cell is an Sf9 and HF cell, wherein Sf9 is used to amplify the baculovirus, and then the HF cell is infected with the obtained baculovirus to express the recombinant protein.

[0121] Host cells are transformed with the above-described expression or cloning vectors for production of fusion proteins and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants or amplifying genes encoding the desired sequences.

[0122] When a recombinant expression vector encoding a target protein is introduced into a host cell, the target protein is produced by culturing the host cell for a period of time to allow the target protein to be expressed in the host cell or secreted into the culture medium in which the host cell is cultured. The target protein can be recovered from the culture medium using standard protein purification methods.

[0123] Illnesses or symptoms caused by influenza virus infection

[0124] In one aspect, the present invention also provides the use of the composition in preparing a medicament for preventing or treating influenza virus infection. The medicament may be a vaccine.

[0125] Influenza A, B and C virus infections can cause acute respiratory infections, namely influenza (flu). Influenza patients and asymptomatic infected persons are the main sources of seasonal influenza. Influenza viruses are mainly transmitted through respiratory droplets produced by sneezing and coughing of infected people, and can also be infected through direct or indirect contact with mucous membranes such as the mouth, nose, and eyes. In certain places, such as crowded, closed or poorly ventilated rooms, it may also be transmitted in the form of aerosols. Infection with influenza viruses can cause symptoms including acute fever, muscle pain, headache, sore throat, cough, runny nose, etc. Different strains of influenza A and B viruses cause regular seasonal outbreaks of influenza.

[0126] The viruses that cause seasonal influenza epidemics are mainly the H1N1 and H3N2 subtypes of influenza A virus and the Victoria strain of influenza B virus. Influenza C virus can infect humans, dogs and pigs, and only causes sporadic cases of upper respiratory tract infection. Since influenza viruses are prone to mutation, the influenza strains that are prevalent each year may change, making it difficult for antibodies previously formed through natural infection or immunization to effectively neutralize them, so the general population is susceptible. Children are at higher risk of contracting influenza because their immune systems are not yet fully developed.

[0127] Vaccination is the best way to prevent the disease, and it can significantly reduce the risk of influenza and serious complications. The influenza vaccines available on the market worldwide are divided into inactivated influenza virus vaccines, live attenuated influenza virus vaccines, and recombinant influenza virus vaccines. According to the components contained in the vaccine, it is divided into trivalent and quadrivalent influenza vaccines. The trivalent influenza vaccine contains A (H3N2) subtype, A (H1N1) pdm09 subtype and a strain of B influenza virus strains, and the quadrivalent influenza vaccine contains A (H3N2) subtype, A (H1N1) pdm09 subtype and B (Victoria) strain and B (Yamagata) strain. According to the production process, it can be divided into chicken embryo culture-based, cell culture-based and recombinant influenza vaccines. Specific vaccine types include standard dose influenza split vaccine and subunit inactivated vaccine based on chicken embryo culture, cell culture-based inactivated influenza vaccine, recombinant influenza vaccine, live attenuated influenza vaccine, high dose inactivated influenza vaccine based on chicken embryo culture, and adjuvanted influenza inactivated vaccine. For influenza vaccines other than live attenuated vaccines, the antigen content is usually expressed as the content of hemagglutinin (HA) in each antigen component, for example, containing 15.0 μg of HA in each antigen component.

[0128] In another aspect, the present invention provides a method for preventing and / or treating influenza disease, comprising administering the vaccine composition described herein to a subject in need thereof, such as a subject at risk of influenza infection. The subject may be a bird, a mammal, and particularly a human. The human subject may be of various ages, such as an elderly person or a young child.

[0129] The present invention has the following beneficial effects:

[0130] 1. The present invention fuses the broad-spectrum immune antigen epitopes of influenza virus protein with HA protein and ferritin, thereby enhancing the broad-spectrum immune activation effect of the antigen.

[0131] 2. The present invention provides a method for preparing and applying a trivalent influenza nanoparticle vaccine, while improving humoral immunity and cellular immunity of the antigen.

[0132] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0133] Example

[0134] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0135] Example 1: Preparation of H1N1, H3N2 and BV recombinant HA ferritin nanoparticles

[0136] 1. Construction of HA ferritin nanoparticle gene plasmids of H1N1, H3N2 and BV strains

[0137] The gene fragments H1N1-HA, H3N2-HA and BV-HA fragments, Ftn fragments and M2 epitopes, M1 epitopes, and NA epitope fragments were synthesized by GenScript Biotechnology Co., Ltd., and the recombinant HA ferritin nanoparticle antigen (composed of multiple fragment sequences, respectively, see Table 1 for details) was obtained by multiple rounds of PCR amplification (primers were synthesized by Qingke Biotechnology Co., Ltd.). The pFastBac1-syn21-HBM empty vector (provided by GenScript Biotechnology Co., Ltd.) was double-digested with EcoR I and Kpn I to recover the large vector fragment. Then, the target fragment was connected to the vector fragment to form a recombinant plasmid by multi-fragment homologous recombination technology. Subsequently, the recombinant plasmid was transformed into Escherichia coli DH5a competent cells. The transformed bacteria were screened with LB solid medium containing ampicillin resistance and expanded. After shaking culture at 37°C overnight, the plasmid was extracted with an endotoxin-free plasmid mini-extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the sequence was confirmed to be correct by sequencing, and then used for the next step of fusion protein expression.

[0138] The prepared fusion proteins were in the form of HA(H1N1)-M2e-Ftn, HA(H3N2)-M1-Ftn, and HA(BV)-NA-Ftn.

[0139] Table 1: Amino acid sequences of three recombinant influenza ferritin nanoparticle fusion proteins, their components and related proteins

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147] 2. Expression of Fusion Protein

[0148] 1) The plasmids containing H1N1-HA-M2e-Ftn, H3N2-HA-M1-Ftn, and BV-HA-NA-Ftn fusion protein genes were transformed into DH10Bac competent cells, and the bacterial solution was resuspended and spread on a culture medium containing Kan+, tetracycline, gentamicin, IPTG, and X-gal. The culture medium was then placed in a 37°C incubator and cultured continuously for 48 hours to observe its growth. After screening with blue-white spots, the white spots were picked and shaken for culture. The recombinant Bacmid was extracted using a baculovirus shuttle plasmid Bacmid small-scale extraction kit (Biyuntian) and sequenced for verification. It was identified that the target gene had been successfully transferred;

[0149] 2) Insect cells Sf9 (our company) in the logarithmic growth phase (cell viability > 90%) were selected and inoculated into six-well plates in 908 medium (Yishengke, catalog number ZJ04F0963), with 1.6×10 5 After transfection, the six-well plate was placed in a 27°C incubator for 5 to 6 hours; then 5 μL of each plasmid was taken, and 5 μL of -Insect Transfection Reagent (Mirus) was added to 250 μL of fresh culture medium, mixed and allowed to stand at room temperature for 20 min, then 260 μL of the mixture was added to the adhered Sf9 cells, and allowed to stand in a 27°C incubator for 5-6 days, and the supernatant was harvested to obtain the P1 virus;

[0150] The above Sf9 cells in the logarithmic growth phase (cell viability> 90%) were selected and inoculated into T25 square flasks and cultured in 908 medium (0.8×10 5 cells / mL, culture volume is 5 mL), incubate in a 27°C incubator for 5-8 h, then add 1 mL of P1 virus, incubate in a 27°C incubator for 5-6 d, harvest the supernatant, and obtain P2 virus;

[0151] Select the above Sf9 cells in the logarithmic growth phase (cell viability> 90%) and dilute the viable cell concentration to 2-3×10 6After 100 cells / mL, inoculate into 125mL cell shake flask (culture volume 100mL) for subculture, add 1mL P2 virus, culture in an incubator at 27°C and 120rpm for 4-5 days, harvest the supernatant, and obtain P3 virus;

[0152] 3) Select HF-NF cells (Thermo Fisher, catalog number B85502) in the logarithmic growth phase with a cell density of 2.0-2.5×10 6 The cells / mL were inoculated into IB907 medium (Yi Shengke, catalog number ZJ04F0963), and the P3 virus was inoculated at 0.1 MOI for fusion protein expression (cultured at 27°C, 120 rpm for 48 h), and samples were taken for virus detection (the target gene was sequenced and analyzed in HF-NF cells inoculated with the P3 virus, and no base insertion, deletion, or mutation was found. After DNA sequence comparison with the full-length sequence of the target gene, it was confirmed that the DNA sequence of the target gene in the cells was completely correct).

[0153] 3. Protein sample purification and preparation process

[0154] 3.1 Clarification process

[0155] 1) Remove solids by low-speed centrifugation, harvest cell supernatant, and fill the obtained cell culture medium into 85% of the maximum capacity of the centrifuge bottle, then balance it, set the centrifuge parameters, centrifuge speed to 3000 rpm, centrifuge time to 20 min, centrifuge temperature to 4°C, harvest cell supernatant, take samples to determine expression level and purity, and weigh the mass of solids.

[0156] 2) High-speed centrifugation to remove trace impurities and harvest the cell supernatant: Fill the refrigerated cell supernatant into 85% of the maximum capacity of the centrifuge tube, then balance it and set the centrifuge parameters to 8000 rpm, 30 min, and 4°C. Harvest the cell supernatant and take samples to test the content, turbidity, purity, and endotoxin, calculate the recovery rate, and measure the pH and conductivity values.

[0157] 3.2 Rough and pure process

[0158] Diamond Layer 400 (Borgron (Zhejiang) Biotechnology Co., Ltd.) filler was used for composite mode purification, and the flow-through mode was adopted to effectively remove some impurities in the product.

[0159] First, the loaded chromatography column is disinfected with 1M NaOH, 30% isopropanol solution, and then the chromatography column is regenerated with 25mM PB, 2M NaCl, pH 7.20±0.05 solution, and then the chromatography column is balanced with 25mM PB, 100mM NaCl, pH 7.20±0.05 solution, and then the sample is loaded. After the loading is completed, 25mM PB, 100mM NaCl, pH 7.20±0.05 solution is used as the elution buffer, and the flow-through peak is collected. The ultraviolet absorption value of the flow-through peak ranges from 20 to 80mAU. The chromatography column is regenerated with 25mM PB, 2M NaClpH7.20±0.05 solution, and the chromatography column is disinfected again with 1M NaOH, 30% isopropanol solution, and then the chromatography column is preserved with 20% ethanol; the collected flow-through sample is the next chromatography sample.

[0160] 3.3 Refined process

[0161] 1) Anion exchange chromatography was performed using Nmacro 50Q filler (Hangzhou Nanomicrobiology Chemical Co., Ltd.) in a binder elution mode to effectively remove HCP, HCD, and some product analog impurities.

[0162] 2) Disinfect the loaded chromatography column with 0.5M NaOH, regenerate the chromatography column with 25mM PB, 2M NaCl, pH 7.20±0.05, and balance the chromatography column with 25mM PB, 100mM NaCl, pH 8.50±0.05 solution. Measure the sample conductivity before loading. If the conductivity is ≥13mS / cm, adjust the sample conductivity to 12±0.5mS / cm with 25mM PB, pH 8.50±0.05 solution, and then load the sample. After loading, first rinse three column volumes with 25mM PB, 100mM NaCl, pH 8.50±0.05 solution, and elute with 25mM PB, 0.24M NaCl, pH 7.20±0.05 solution. Then, the chromatography column was regenerated with 25 mM PB, 2 M NaCl, pH 7.20 ± 0.05, and then disinfected again with 0.5 M NaOH and preserved with 20% ethanol; the eluted sample collected was used as the chromatography sample for the next step.

[0163] 3.4 Preparation of stock solution

[0164] The hollow fiber membrane column (Hangzhou Kebet Filter Equipment Co., Ltd.) is made of PES, with a pore size of 300 kD and a membrane area of ​​56 cm 2Concentrate and replace the solution. First rinse the membrane column with water for injection, then disinfect it with 0.5M NaOH, and then rinse the membrane column with water for injection until the pH is neutral. After the water flux test is completed, balance the membrane column with 25mM PB, 150mM NaCl, pH 7.20±0.05 solution. Then use a hollow fiber membrane column to concentrate to one-fourth, add replacement solution 25mM PB, 130mM NaCl, pH 7.20±0.05 to the original volume, and cycle three times until the conductivity of the filtrate end is 17.0mS / cm and the concentrated protein is about 1mg / mL. Finally, use 25mM PB, 150mM NaCl, pH 7.20±0.05 solution to elute, and rinse 1 column volume into the concentrate until the concentration is between 0.5 and 0.7mg / mL. After the concentration is completed, flush the system with water for injection, disinfect with 0.5M NaOH, and then flush the membrane column with water for injection. After the integrity test and water flux test are completed, use 0.01M NaOH to preserve the membrane column;

[0165] 3.5QC Testing

[0166] Use SDS-PAGE and SEC-HPLC to test sample size and purity. Test HCD residue, HCP residue and endotoxin to ensure that they meet quality requirements. Figure 2 As shown, the sizes of the three fusion proteins were consistent with the antigen design.

[0167] Example 2: Vaccine immunogenicity and challenge protection study

[0168] 1. Vaccine Preparation

[0169] 1.1 MF59 adjuvant vaccine preparation

[0170] Purified fusion proteins H1N1-HA-M2e-Ftn, H3N2-HA-M1-Ftn, and BV-HA-NA-Ftn were mixed at an antigen ratio of 2:1:3 under sterile conditions at room temperature, and then diluted with antigen diluent (25mM PB, 150mM NaCl, 0.005% Tween-80, 100mM arginine hydrochloride, 5% trehalose, pH 7.2), and an appropriate amount of MF59 adjuvant was added. Two intramuscular vaccine compositions containing MF59 adjuvant were prepared, in which the total HA antigen content was 15 or 30μg / mL, and MF59 adjuvant was added at a volume ratio of 1:1, and were numbered HA-np (2:1:3) 2.5μg HA+MF59 and HA-np (2:1:3) 5μg HA+MF59, respectively.

[0171] 1.2 Preparation of non-adjuvanted vaccines

[0172] Purified fusion proteins H1N1-HA-M2e-Ftn, H3N2-HA-M1-Ftn, and BV-HA-NA-Ftn were mixed at an antigen ratio of 2:2:2 under aseptic conditions at room temperature, and then diluted with an antigen diluent (25 mM PB, 150 mM NaCl, 0.005% Tween-80, 100 mM arginine hydrochloride, 5% trehalose, pH 7.2). Two vaccine compositions without MF59 adjuvant were prepared, wherein the total HA antigen content was 90 or 180 μg / mL, and were numbered HA-np (2:2:2) 15 μg HA and HA-np (2:2:2) 30 μg HA, respectively.

[0173] 1.3 Experimental animal grouping and immunization and challenge tests

[0174] Female Balb / c mice aged 4 to 5 weeks (purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd.) were randomly divided into 40 groups, with 6 or 9 mice in each group. The experiment was divided into two stages: the immunization period and the challenge period. During the virus challenge period, four influenza strains, H1N1 (A / Victoria / 2570 / 2019), homologous H3N2 (A / Darwin / 9 / 2021), heterologous H3N2 (A / Aichi / 2 / 1968 Mouse-adapted) and BV (B / Austria / 1359417 / 2021), were used to challenge the immunized mice. The information on animal immunization and challenge grouping is shown in Table 2.

[0175] Table 2 Animal immunization and challenge grouping information

[0176]

[0177]

[0178]

[0179] During the immunization period, each mouse in the H1N1 model group, H3N2 model group, and BV model group was immunized by intramuscular injection with 167 μL of antigen dilution.

[0180] There were 2 positive control groups: one group in which each mouse was intramuscularly injected with a commercially available trivalent inactivated influenza vaccine (15 μg total HA), and the other group in which each mouse was intramuscularly injected with a commercially available quadrivalent inactivated influenza vaccine (20 μg total HA).

[0181] Groups with MF59 adjuvant vaccine: Each animal in Groups 4, 19, 27, and 35 was immunized by intramuscular injection of 2.5 μg total HA in HA-np (2:1:3) 2.5 μg HA + MF59 vaccine, and each animal in Groups 5, 20, 28, and 36 was immunized by intramuscular injection of 5 μg total HA in HA-np (2:1:3) 5 μg HA + MF59 vaccine.

[0182] Vaccine groups without MF59 adjuvant: Each animal in groups 6, 21, 29, and 37 was immunized by intramuscular injection of 15 μg of total HA in HA-np (2:2:2) 15 μg HA vaccine, and each animal in groups 7, 22, 30, and 38 was immunized by intramuscular injection of 30 μg of total HA in HA-np (2:2:2) 30 μg HA vaccine.

[0183] MF59 adjuvant control group: Each mouse was immunized once with 167 μL of MF59 adjuvant by intramuscular injection.

[0184] Blank control group: Each mouse was immunized once intramuscularly with 167 μL of a 1:1 mixture of antigen diluent and MF59 adjuvant.

[0185] The H1N1 challenge group, the homologous H3N2 challenge group, and the BV challenge group were immunized once on day 0, and the animal serum samples were collected 28 days after immunization for hemagglutination inhibition antibody detection and antigen-specific IgG detection to evaluate the immunogenicity of the vaccine. The heterologous H3N2 challenge group was immunized for the first time on day 0 and the second time on day 28, and the animal serum samples were collected 28 days after the first and second immunizations for hemagglutination inhibition antibody detection and antigen-specific IgG detection to evaluate the immunogenicity of the vaccine. Each mouse in the blank control group was immunized once intramuscularly with 167 μL of antigen diluent.

[0186] The H1N1 challenge group was challenged 35 days after the mice were immunized to evaluate the protective effect of the vaccine on the mice. The challenge strain was A / Victoria / 2570 / 2019 (H1N1), and the challenge dose was 5 times LD50, i.e. 8.58lgTCID50 / mouse. The weight of the mice was monitored for 14 consecutive days after the challenge. If the weight loss of the mice exceeded 10% of the weight before the challenge for three consecutive days or more after the challenge, the mice were judged to be ill, and the incidence rate was counted. The protection rate was calculated based on the incidence rate, protection rate = (incidence rate of H1N1 model group-incidence rate of vaccine group) / incidence rate of H1N1 model group * 100%.

[0187] The homologous H3N2 challenge group was challenged 35 days after mice were immunized to evaluate the protective effect of the vaccine on mice. The challenge strain was A / Darwin / 9 / 2021 (H3N2), and the challenge dose was the stock solution, i.e., 10^6.52EID50 / mouse. After the challenge, the weight of the 6 animals in each group was monitored for 14 consecutive days, and the remaining 3 mice were euthanized 3 days after the challenge. The lungs and nasal conchae of the mice were collected, and the viral load was detected using the qPCR method. The virus inhibition rate was calculated, and the virus inhibition rate = (the number of virus copies in the H3N2 model group-the number of virus copies in the vaccine group) / the number of virus copies in the H3N2 model group * 100%.

[0188] The BV challenge group was challenged 35 days after the mice were immunized to evaluate the protective effect of the vaccine on the mice. The challenge strain was B / Austria / 1359417 / 2021 (BV), and the challenge dose was 5*LD50, i.e. 7.39lgTCID50 / mouse. Body weight was monitored continuously after the challenge. On the 6th day, 3 mice in each group were euthanized, and the lungs and nasal conchae of the mice were collected, and the viral load was detected using the qPCR method. The virus inhibition rate was calculated, and the virus inhibition rate = (the number of virus copies in the BV model group-the number of virus copies in the vaccine group) / the number of virus copies in the BV model group * 100%. The remaining 3 animals in each group continued to monitor their weight until 14 days after immunization.

[0189] The heterologous H3N2 challenge group was challenged 33 days after the second immunization of mice to evaluate the protective effect of the vaccine on the mice. The challenge strain was A / Aichi / 2 / 1968 Mouse-adapted (H3N2), and the challenge dose was the stock solution. The weight of the mice was monitored for 14 consecutive days after the challenge. The weight loss of the mice after the challenge exceeded 25% of the weight before the challenge, and the mice were judged to be dead, and the mortality rate was counted. The vaccine protection rate was calculated based on the mortality rate, and the vaccine protection rate = (heterologous H3N2 model group mortality rate-vaccine group mortality rate) / heterologous H3N2 model group mortality rate * 100%.

[0190] 1.4 Hemagglutination inhibition test

[0191] The hemagglutination inhibition antibody assay in the above section 1.3 was performed as follows.

[0192] 1) Removal of nonspecific inhibins (which inhibit erythrocyte agglutination) in serum.

[0193] Take 3 volumes of dissolved Vibrio cholerae receptor destroying enzyme (RDE) and 1 volume of serum (150μL RDE + 50μL serum), and place in a 37℃ water bath for 18h. Then place in a 56℃ water bath for 30min to inactivate the remaining RDE. Add 200μL of normal saline, mix well, and use 250μL for H1N1 and BV antigen hemagglutination inhibition test, and 150μL for H3N2 antigen hemagglutination inhibition test.

[0194] 2) Removal of nonspecific agglutinins in serum

[0195] Preparation of dense red blood cells: according to the test amount, take an appropriate amount of 1% red blood cell suspension (see Table 3 for the amount of 1% red blood cell suspension), centrifuge at 360×g for 10 minutes, remove the upper solution, use a sterile pipette to remove the wall reflux solution, and leave a layer of dense red blood cells for subsequent experiments. Turkey blood dense red blood cells and guinea pig blood dense red blood cells were prepared separately.

[0196] Table 3 1% red blood cell suspension dosage ratio

[0197] Total serum volume treated with RDE 1% red blood cell suspension volume 200μL 1000μL

[0198] In a centrifuge tube, add 1 volume of concentrated red blood cells after centrifugation to 20 volumes of RDE-treated serum (different types of samples should be mixed with corresponding red blood cells, such as H1N1 and BV mixed with concentrated turkey red blood cells; H3N2 mixed with concentrated guinea pig red blood cells). Use a pipette to draw the required volume of red blood cells and add them to the serum. Repeat pipetting several times and use a pipette to mix thoroughly.

[0199] Place at 2-8℃ for 1 hour, during which time, turn the centrifuge tube upside down every 15-20 minutes to resuspend and mix the deposited red blood cells (note: the operation time for resuspending the deposited red blood cells should be controlled within 5 minutes). Then centrifuge at 4℃, 410×g for 5 minutes; aspirate the serum supernatant into a new centrifuge tube for later use (be careful not to aspirate the deposited red blood cells).

[0200] Take out the 96-well micro-hemagglutination plate, and determine the number of wells to add PBS buffer according to the number of serum to be tested. Generally, add 50μL of PBS buffer starting from well A1, add 50μL of serum treated with RDE, make a 2-fold dilution, aspirate 50μL of the 2-fold diluted serum to well A2, make double wells, and add 50μL of 1% red blood cell suspension to each well (select according to the antigen required for detection: 1% turkey red blood cell suspension for H1N1 and BV; 1% guinea pig red blood cell suspension for H3N2), place the 96-well micro-hemagglutination plate on a micro-oscillator and oscillate for 15 to 30 seconds, let it stand at room temperature for 25 to 60 minutes, and observe the test results. If the red blood cells are deposited, it proves that the nonspecific agglutinins in the serum have been completely removed. If not, repeat the above steps.

[0201] 3) Hemagglutination titer test

[0202] Add 50 μL of PBS buffer to each well of the 2nd to 12th columns of the 96-well microhemagglutination plate, add 100 μL of standard antigen to the first column A1 to G1, and add 100 μL of PBS to the H1 well as a negative control for red blood cells. Aspirate 50 μL of standard antigen from each well in the first column, make a 2-fold serial dilution from the 2nd to the 12th column, and discard 50 μL from each well in the last column. If the antigen hemagglutination titer is high, it is recommended to continue to increase the dilution.

[0203] Add 50 μL of 1% red blood cell suspension (selected according to the antigen to be detected: 1% turkey red blood cell suspension for H1N1 and BV; 1% guinea pig red blood cell suspension for H3N2) to each well, place the 96-well micro-hemagglutination plate on a micro-oscillator and oscillate for 15 to 30 seconds to mix thoroughly. Incubate at room temperature, observe the results and take photos to record (observe the 96-well V-shaped micro-hemagglutination plate at an angle of 45°, the negative control should be teardrops flowing downwards; observe the results of the 96-well U-shaped micro-hemagglutination plate from the front, the negative control should be ring-shaped).

[0204] Result judgment: Complete agglutination is when all red blood cells are agglutinated, and is recorded as "+"; only partial agglutination is recorded as "+ / -"; no agglutination is recorded as "-". The determination of the hemagglutination titer is based on the highest dilution at which complete agglutination occurs as the endpoint, and the reciprocal of the dilution is the hemagglutination titer of the antigen.

[0205] 4) Preparation of antigens for 4 hemagglutination units

[0206] One agglutination unit refers to the amount of antigen that can cause an equal amount of standardized hemagglutination of red blood cells. The HAI titer is determined based on this. When performing the hemagglutination inhibition test, 4 hemagglutination units of antigen are generally used.

[0207] When preparing 4 hemagglutination units of antigen, first calculate the total amount of 4 hemagglutination units of antigen required for the hemagglutination inhibition test. If each serum is diluted into 8 wells and 25 μL of antigen is used in each well, 0.2 mL of 4 hemagglutination units of antigen is required to measure one serum. Calculate the total amount of 4 hemagglutination units of antigen required for the experiment based on the number of serum samples to be tested.

[0208] Secondly, calculate the dilution of the antigen. Divide the hemagglutination titer of the antigen by 8, and the quotient obtained is the antigen dilution required to prepare 4 hemagglutination units (the 4 hemagglutination units used in the hemagglutination inhibition test means that every 25 μL of antigen contains 4 hemagglutination units, which is equivalent to 8 hemagglutination units in 50 μL of antigen). For example: the hemagglutination titer of an antigen is 64, which is equal to 8 when divided by 8. Then dilute the antigen at 1:8 (1 mL of antigen plus 7 mL of PBS buffer).

[0209] 5) Calibration of 4 hemagglutination unit antigens

[0210] In order to ensure that the amount of antigen used in the hemagglutination inhibition test is consistent and accurate, the newly prepared 4 hemagglutination units of antigen need to be rechecked and titrated. The operation method is the same as the hemagglutination titer test steps, and the titration results are photographed and recorded.

[0211] If agglutination occurs in the first 4 wells but not in the 5th well, it indicates that each 50 μL of antigen contains 8 hemagglutination units. The antigen dilution is accurate and can be used for the hemagglutination inhibition test. If agglutination also occurs in the 5th well, it indicates that each 50 μL of antigen contains 16 hemagglutination units, and the antigen must be diluted in equal amounts. If only the first 3 wells agglutinate, it indicates that each 50 μL of antigen contains only 4 hemagglutination units, and the amount of antigen needs to be doubled. In addition, the 4 hemagglutination units of antigen must be prepared fresh each time it is used.

[0212] 6) Hemagglutination inhibition titer test

[0213] Take a 96-well microplate and use a pipette to add 25 μL of PBS buffer to each of the 2nd to 11th wells and 50 μL of PBS buffer to the 12th well.

[0214] Add 50 μL of the test serum to the first well, mix thoroughly and remove 25 μL to the second well, and so on, dilute to the 11th well in multiple proportions, and discard 25 μL from the 11th well. A12-D12 4 hemagglutination units are diluted 1:2 to 1:16; E12 to H12 are red blood cell controls. Add 25 μL of 4 hemagglutination units of antigen to each well A1 to A11, place the 96-well micro-hemagglutination plate on a micro-oscillator and oscillate for 15 to 30 seconds to mix the reactants evenly, and let stand at room temperature for 30 minutes.

[0215] Add 50 μL of 1% turkey red blood cell suspension (for H1N1 and BV) or 50 μL of 1% guinea pig red blood cell suspension (for H3N2) to each well from right to left. Place the 96-well micro-hemagglutination plate on a micro-oscillator for 15 to 30 seconds, mix thoroughly, and observe the hemagglutination inhibition results after standing at room temperature for 25 to 60 minutes.

[0216] Result judgment:

[0217] (1) The 96-well V-shaped microplate was tilted at 45°. When the negative control flowed downward in the form of teardrops, a photo was taken from the back of the 96-well V-shaped microplate. The reciprocal of the highest dilution factor that completely inhibited red blood cell aggregation was used as the hemagglutination inhibition titer of the serum.

[0218] (2) When the negative control of the 96-well U-shaped microhemagglutination plate is circular, take a photo from the back of the 96-well U-shaped microhemagglutination plate and determine the hemagglutination inhibition titer of the serum by the reciprocal of the highest dilution factor that completely inhibits red blood cell aggregation.

[0219] 1.5 Antigen-specific IgG ELISA test

[0220] The antigen-specific IgG detection in the above 1.3 was performed as follows.

[0221] 1.5.1 Solution preparation

[0222] Coating solution: carbonate buffer (pH 9.6) (1 L as an example): weigh 1.59 g sodium carbonate and 2.93 g sodium bicarbonate, add ultrapure water to make up to 1 L, filter through a 0.22 μm membrane, and store at 4°C for later use.

[0223] Washing solution: PBST (pH 7.4) (1 L as an example): weigh 8.0 g sodium chloride, 0.2 g potassium chloride, 3.58 g disodium hydrogen phosphate dodecahydrate, 0.27 g potassium dihydrogen phosphate, measure 0.5 mL Tween-20 and add ultrapure water to make up to 1 L, filter through a 0.22 μm membrane, and store at 4°C for later use.

[0224] Blocking solution and antibody diluent (100 mL as an example): Weigh 5 g skim milk powder and dissolve it in 100 mL PBST to make the blocking solution. Weigh 3 g skim milk powder and dissolve it in 100 mL PBST to make the antibody diluent.

[0225] Stop solution: 1M hydrochloric acid solution (120 mL as an example): Measure 10 mL of concentrated hydrochloric acid, add it to 110 mL of water for injection, and mix well.

[0226] 1.5.2 Experimental procedures

[0227] Coating: Dilute the HA subunit antigens of H1N1, H3N2, and BV to 1 μg / mL with coating solution, add 100 μL / well to the ELISA plate, cut the sealing film of appropriate size, tighten it to tightly seal the wells of the ELISA plate, and incubate overnight at 4°C; shake off the liquid in the ELISA plate, wash it 3 times with 200 μL / well of washing solution, 5 minutes each time, and pat dry.

[0228] Blocking: Add 200 μL / well of prepared blocking solution, seal the plate, and incubate at 37°C for 1 hour; shake off the liquid in the ELISA plate, wash 3 times with 200 μL / well of washing solution, each time for 5 minutes, and pat dry.

[0229] Incubate the primary antibody: dilute the serum to be tested to 1:100 with antibody diluent. Add 100 μL / well to the ELISA plate, seal the plate, and incubate at 37°C for 1 hour; shake off the liquid in the ELISA plate, wash it 5 times with 200 μL / well of washing solution, 5 minutes each time, and pat dry.

[0230] Incubate with secondary antibody: dilute the corresponding secondary antibody 1:5000 with antibody diluent, add 100 μL / well to the ELISA plate, seal the plate, and incubate at 37°C for 1 hour; shake off the liquid in the ELISA plate, wash 5 times with 200 μL / well of washing solution, and pat dry.

[0231] Color development: Add 100 μL / well of TMB color development solution and develop at 37°C for 10-20 min (determine by color change).

[0232] Stop: Add 50 μL / well of stop solution to stop the reaction; read the OD on the microplate reader 450 nm (reference wavelength 620nm) value.

[0233] 1.6qPCR detection

[0234] The qPCR assay in 1.3 above was performed as follows.

[0235] (1) After the animal is euthanized, the lungs and nasal conchae are collected and weighed. An appropriate volume of PBS is added and the collected lungs and nasal conchae are homogenized in a tissue disruptor. The homogenate is centrifuged at 10,000 rpm and 4°C for 10 min, and the supernatant is collected. RNA in the supernatant is extracted using the QIAGEN RNeasy Mini Kit.

[0236] (2) Add 5 μL of β-mercaptoethanol to each tube, mix well, then add 600 μL of 70% ethanol and mix thoroughly.

[0237] (3) Take out the 2 mL collection tube with the filter column from the kit, open the package and mark it. Take 600 μL of the mixed solution in step (2) and add it to the filter column. Centrifuge at 12,000 rpm for 15 seconds and discard the centrifuge in the collection tube. Put the filter column back on the collection tube, add all the remaining mixed solution in step (2) to the filter column, centrifuge at 12,000 rpm for 15 seconds and discard the centrifuge.

[0238] (4) Add 700 μL Wash Buffer RW1 to the filter column and centrifuge at 12,000 rpm for 15 seconds. Take a clean 2 mL collection tube from the QIAGEN RNeasy Mini Kit, transfer the centrifuged filter column to a new collection tube, add 500 μL Wash Buffer RPE to the filter column, centrifuge at 12,000 rpm for 15 seconds. Discard the centrifuge in the collection tube, add 500 μL Wash Buffer RPE to the filter column, centrifuge at 13,000-14,000 rpm for 2 minutes.

[0239] (5) Transfer the filter column to a clean 1.5 mL eppendorf tube, add 30-50 μL of nuclease-free water to the filter column, and let it stand at room temperature for 1-3 minutes. Then centrifuge at 12,000 rpm for 1 minute, collect the centrifuge liquid, which is the extracted viral RNA, and use it immediately in experiments or store it below -20°C.

[0240] (6) After calculating the total number of reactions based on the following reagents and the required volumes for each reaction, add the total number of reactions + 1 into a 1.5 mL Eppendorf tube in sequence, and prepare the universal A, H5, H7, H9, and RNP primers and probes in separate tubes.

[0241]

[0242] Reaction solution distribution: Dispense the reaction solution into 0.2mL PCR tubes at 20μL / tube and mark them. Transfer the PCR tubes with reaction solution to the nucleic acid extraction area and add negative control, test specimen and positive control reaction template RNA at 5μL / tube.

[0243] Transfer the PCR tube with the above reaction system to the nucleic acid amplification area workroom, turn on the fluorescent quantitative PCR instrument and the online computer in turn, place the PCR tube on the fluorescent quantitative PCR instrument for amplification reaction, act at 60℃ for 5 min, 50℃ for 30 min, 95℃ for 15 min, denature at 95℃ for 15 s, anneal at 55℃ for 30 s, extend at 72℃ for 30 s, for 45 cycles, and act at 72℃ for 5 min.

[0244] Result observation: judge the results when the quality control standard is valid.

[0245] 1.7 Test results

[0246] 28 days after the first immunization of mice, low-dose (2.5 μg HA) and high-dose (5 μg) trivalent influenza nanoparticle antigens (H1:H3:BV=2:1:3) combined with MF59 adjuvant induced strong H1N1, H3N2, BV hemagglutination inhibition antibodies and antigen-specific IgG, such as Figure 3-Figure 6 As shown in Figure 2, the MF59 adjuvanted vaccine with HA antigen content of 1 / 3 of the positive control dose is sufficient to achieve an effect comparable to or better than the commercially available trivalent influenza vaccine and the commercially available quadrivalent influenza vaccine.

[0247] Figure 7 and Figure 8 , Fig.13 and Fig.14 The results showed that the weight changes were milder with the vaccine composition adjuvanted with MF59.

[0248] Low-dose and high-dose trivalent influenza nanoparticle vaccines with MF59 adjuvant induced strong protection against H1N1, H3N2, and BV in mice, and had good cross-protection against heterologous H3N2 strains ( Fig.15). As shown in Table 4, the incidence of mice immunized with MF59 adjuvant vaccine at a dose of 5 μg HA was 0% and the protection rate was 100% when challenged with H1N1. When challenged with homologous H3N2, the virus inhibition rate in the nasal turbinates of mice was 81.5%, and the virus inhibition rate in the lungs was 92.3%. Similarly, when challenged with BV, there were high virus inhibition rates in the lungs and nasal turbinates, which were 89.8% and 100%, respectively. For the 1968 H3N2 strain with lower homology, the MF59 adjuvant vaccine at a dose of 5 μg HA also had a significant cross-protection effect, with a protection rate of 100%.

[0249] Table 4 Statistical table of the protective effect of trivalent influenza nanoparticle vaccine

[0250]

[0251]

[0252] The above results show that the trivalent influenza nanoparticle vaccine with MF59 adjuvant prepared by the present invention can induce strong hemagglutination inhibition antibodies and antigen-specific IgG, and induce strong protection against H1N1, H3N2, and BV in mice, and has good cross-protection against heterologous H3N2 strains. Compared with the commercially available trivalent influenza vaccine and the commercially available quadrivalent influenza vaccine, it achieves a better immune effect.

[0253] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fusion protein combination, comprising a first fusion protein, a second fusion protein and a third fusion protein, wherein each of the first fusion protein, the second fusion protein and the third fusion protein comprises the following two parts: (1) influenza virus HA protein or a fragment thereof; and (2) An antigenic epitope, wherein the antigenic epitope is an antigenic epitope derived from an influenza virus protein, and the influenza virus protein is selected from the group consisting of M2, M1 and NA proteins.

2. The fusion protein combination of claim 1, wherein the antigenic epitope is selected from the following: (a) an antigenic epitope derived from the M2 protein, comprising the amino acid sequence shown in SEQ ID NO: 4; (b) an antigenic epitope derived from the M1 protein, comprising the amino acid sequence shown in SEQ ID NO: 5; (c) An antigenic epitope derived from NA protein, which comprises the amino acid sequence shown in SEQ ID NO:

6.

3. The fusion protein combination of claim 1 or 2, wherein the HA protein or its fragment is selected from the following: (a) an HA protein or a fragment thereof derived from an H1N1 strain, comprising the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 95% identity thereto; (b) an HA protein derived from an H3N2 strain or a fragment thereof, comprising the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 95% identity thereto; and (c) an HA protein or a fragment thereof derived from a BV strain, comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 95% identity thereto.

4. The fusion protein combination according to any one of claims 1 to 3, wherein the first fusion protein comprises: an HA protein or a fragment thereof derived from an H1N1 strain, comprising an amino acid sequence as shown in SEQ ID NO: 1; and The antigenic epitope derived from the M2 protein comprises the amino acid sequence shown in SEQ ID NO:

4.

5. The fusion protein combination according to any one of claims 1 to 4, wherein the second fusion protein comprises: an HA protein or a fragment thereof derived from an H3N2 strain, comprising an amino acid sequence as shown in SEQ ID NO: 2; and The antigenic epitope derived from the M1 protein comprises the amino acid sequence shown in SEQ ID NO:

5.

6. The fusion protein combination according to any one of claims 1 to 5, wherein the third fusion protein comprises: an HA protein or a fragment thereof derived from a BV strain, comprising an amino acid sequence as shown in SEQ ID NO: 3; and The antigenic epitope derived from the NA protein comprises the amino acid sequence shown in SEQ ID NO:

6.

7. The fusion protein combination of any one of claims 1 to 6, wherein each of the first fusion protein, the second fusion protein and the third fusion protein further comprises ferritin, preferably the ferritin is located at the C-terminus of the fusion protein, Preferably, the fusion protein is in the form of nanoparticles. Optionally, the fusion protein further comprises a signal peptide at the N-terminus.

8. The fusion protein combination of any one of claims 1 to 7, wherein the ferritin is a full-length ferritin or a truncated ferritin.

9. The fusion protein combination according to any one of claims 1 to 8, wherein the ferritin is selected from bacterial ferritin, plant ferritin, algae ferritin, fungus ferritin, insect ferritin or mammalian ferritin, preferably mammalian ferritin or bacterial ferritin.

10. The fusion protein combination of claim 9, wherein the bacterial-derived ferritin is Helicobacter pylori ferritin.

11. The fusion protein combination of claim 10, wherein the Helicobacter pylori ferritin comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

7.

12. The fusion protein combination of any one of claims 1-11, wherein the HA protein or fragment thereof and the antigenic epitope, and in the presence of ferritin, the ferritin and the antigenic epitope are directly connected or connected via a linker sequence, and the linker sequence is selected from SGS, SGG, EAAAK, GGGGS, GPGPG and RVRR linkers, preferably, the linker sequence is SGG, GGGGS or EAAAK.

13. The fusion protein combination according to any one of claims 1 to 12, wherein: The first fusion protein comprises the amino acid sequence shown in SEQ ID NO: 8 or 11, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 8 or 11; The second fusion protein comprises the amino acid sequence shown in SEQ ID NO: 9 or 12, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 9 or 12; The third fusion protein comprises the amino acid sequence shown in SEQ ID NO:10 or 13 or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:10 or 13.

14. A vaccine composition comprising the fusion protein combination of any one of claims 1 to 13 and a pharmaceutically acceptable carrier and / or adjuvant, Preferably, the adjuvant is MF59 adjuvant.

15. The vaccine composition of claim 14, wherein in a unit dose, the vaccine composition comprises a total HA content of 2 to 30 μg, preferably a total HA content of 2.5-15 μg.

16. Nucleic acid molecules encoding the first, second and third fusion proteins of the fusion protein combination of any one of claims 1 to 13.

17. A vector comprising the nucleic acid molecule of claim 16, for example, said vector is a baculovirus vector.

18. A host cell comprising the nucleic acid molecule of claim 16 or the vector of claim 17, optionally wherein the host cell is an E. coli cell or an insect cell.

19. A method for preparing an influenza virus vaccine, comprising the step of mixing the fusion protein combination according to any one of claims 1 to 13 with an adjuvant, wherein the adjuvant is optionally MF59 adjuvant.

20. Use of the fusion protein combination according to any one of claims 1 to 13 in the preparation of a vaccine composition for preventing influenza virus infection in a subject, Preferably, the vaccine composition is administered to the subject by intramuscular injection.

21. The use according to claim 20, wherein the subject is a human or a non-human animal, preferably a human.

22. The use of claim 21, wherein the influenza virus infection is selected from the group consisting of H1N1 virus infection, H3N2 virus infection and BV virus infection.

23. Use of the fusion protein combination according to any one of claims 1 to 13 in the preparation of a medicament for inducing an immune response against influenza virus in an organism, wherein the immune response is humoral immunity and / or cellular immunity.

24. Use of the fusion protein combination according to any one of claims 1 to 13 in producing antibodies against influenza virus.

25. The use of claim 24, wherein the antibody comprises a polyclonal antibody, a monoclonal antibody or a recombinant antibody.

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