MRNA vaccine and application thereof

Abstract

The invention relates to an mRNA vaccine and application thereof. The mRNA vaccine comprises nucleic acids encoding a fusion F protein of a respiratory syncytial virus and at least one matrix protein (M) of a metapneumovirus. The mRNA vaccine disclosed by the invention can induce animals to generate specific humoral immune response (neutralizing antibody) aiming at RSV (Respiratory Syncytial Virus), can protect the animals from death caused by infection of the RSV, and can reduce virus load after challenge.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an mRNA vaccine and its application. Background Art

[0002] Respiratory syncytial virus (RSV) is a common cause of acute respiratory illness. RSV infection occurs in people of all ages and can be life-threatening in infants, the elderly, and immunocompromised adults. Epidemiological studies worldwide have shown that 2-5% of children with RSV infection require hospitalization. RSV disease causes 100,000-200,000 deaths worldwide each year. A key contributing factor to this huge disease burden is the limited prevention and treatment options available.

[0003] Respiratory syncytial virus is a single-stranded, negative-sense, non-segmented enveloped RNA virus classified as a member of the genus Pneumovirus in the family Paramyxoviridae. There are two glycoproteins on the surface of RSV - attachment protein (RSV G) and fusion protein (RSV F). RSV G enhances pathogenicity in vivo and promotes attachment to host cells. RSV F is a class I fusion protein that mediates the fusion of the viral membrane with the host cell membrane and is the main target for inducing the production of neutralizing antibodies by the human immune system.

[0004] The F protein exists in two conformations: before infecting human cells, it is in the prefusion conformation, in which the F protein exists as a trimer and contains the major antigenic site It is the main target of neutralizing antibodies in the body. After binding to the receptor on the surface of the host cell, the F protein undergoes an irreversible conformational change, namely the post-fusion conformation. This change causes it to insert into the host cell membrane and leads to the fusion of the virus and host cell membranes, but at this time the main neutralizing antibody target disappears. If the immune system is able to encounter the F protein in the pre-fusion conformation, it can produce strong neutralizing antibodies, but if the F protein is in the post-fusion conformation, it will hardly stimulate the production of neutralizing antibodies with high neutralizing titers. Although about 50% of the surface is shared before and after fusion, the antigenic sites most sensitive to neutralization are only located on the pre-fusion conformation. Therefore, the design of antigens targeting the pre-fusion conformation of the F protein is critical.

[0005] Studies have shown that RSV F protein polymer precursor is activated in Golgi by the cutting post-translation of furin protease on site I and II. This cutting process produces two subunits, F1 and F2, which are connected by two disulfide bond cysteine-cysteine ​​bridges, and release the short glycoprotein of 27 amino acids that is called as p27. Therefore, the N-terminal of F1 subunit exposes hydrophobic fusogenic peptide (FP), which causes virion-cell fusion after inserting in target membrane. The C-terminal of RSV F1 comprises cytosolic tail (CT) domain, which interacts with matrix protein (M) during virion assembly.

[0006] Currently, several antibody drugs have been developed, such as palivizumab, but their use is limited to prophylactic use in high-risk newborns. This limitation is a direct result of the low neutralizing potency of the developed RSV antibodies, which requires multiple, high-dose administration.

[0007] Therefore, it is necessary to develop an efficient and safe RSV vaccine. Summary of the invention

[0008] In view of this, the purpose of the present invention is to propose an mRNA vaccine and its application, which can induce animals to produce specific humoral immune response (neutralizing antibodies) against RSV, protect animals from death caused by respiratory syncytial virus (RSV) infection, and reduce the viral load after infection.

[0009] Based on the above objectives, the first aspect of the present invention provides an mRNA vaccine comprising a nucleic acid encoding a fusion F protein of a respiratory syncytial virus and a matrix protein (M) of at least one metapneumovirus.

[0010] In a preferred embodiment of the present invention, the fusion F protein of the respiratory syncytial virus (RSV F) is the pre-fusion F protein of the respiratory syncytial virus (RSV pre F), and the at least one matrix protein (M) of the metapneumovirus is one and / or two matrix proteins (M) of the metapneumovirus;

[0011] Preferably, the prefusion F protein of respiratory syncytial virus (RSV pre F) is the prefusion F protein of human respiratory syncytial virus, and the matrix protein (M) of metapneumovirus (MPV) is the matrix protein (M) of human metapneumovirus (hMPV).

[0012] In a preferred embodiment of the present invention, the pre-fusion F protein of the respiratory syncytial virus has the following amino acid sequence:

[0013] (1) the amino acid sequence shown in SEQ ID NO.1; or

[0014] (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0015] (3) An amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2).

[0016] In a preferred embodiment of the present invention, the matrix protein (M) of the metapneumovirus has the following amino acid sequence:

[0017] (1) the amino acid sequence shown in SEQ ID NO.2; or

[0018] (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0019] (3) An amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2).

[0020] In a preferred embodiment of the present invention, a fusion F protein of a respiratory syncytial virus is linked to at least one matrix protein (M) of a metapneumovirus via a tandem gene expression element;

[0021] Preferably, a nucleic acid encoding a pre-fusion F protein of a respiratory syncytial virus and a nucleic acid encoding a matrix protein (M) of two metapneumoviruses are operably linked to a tandem gene expression element, which is an internal ribosome entry site (IRES);

[0022] More preferably, the nucleic acid sequence of the IRES is SEQ ID NO:3.

[0023] In a preferred embodiment of the present invention, the fusion F protein of the respiratory syncytial virus comprises modifications to one or more amino acid residues;

[0024] Preferably, the wild-type amino acid residue at one or more residues in positions 112, 155, 190, 207, 290, 379 and / or 447 of the pre-fusion F protein of the respiratory syncytial virus is replaced by another amino acid residue relative to the native F protein of the respiratory syncytial virus.

[0025] In a preferred embodiment of the present invention, the transmembrane domain of the pre-fusion F protein of the respiratory syncytial virus (RSV pre F) is replaced with the transmembrane domain of the matrix protein (M) of the metapneumovirus (MPV); and / or the cytosolic tail domain (CT) of the pre-fusion F protein of the respiratory syncytial virus (RSV pre F) is replaced with the cytosolic tail domain (CT) of the matrix protein (M) of the metapneumovirus (MPV);

[0026] Preferably, the transmembrane domain of the prefusion F protein of the respiratory syncytial virus (RSV pre F) is replaced by the transmembrane domain of the matrix protein (M) of the human metapneumovirus (hMPV); and the cytosolic tail domain (CT) of the prefusion F protein of the respiratory syncytial virus (RSV preF) is replaced by the cytosolic tail domain (CT) of the matrix protein (M) of the human metapneumovirus (hMPV);

[0027] More preferably, amino acid residues 525-574 of the prefusion F protein of respiratory syncytial virus (RSV pre F) are replaced with amino acid residues 489-539 of the matrix protein (M) of human metapneumovirus (hMPV).

[0028] In a preferred embodiment of the present invention, the nucleic acid of the mRNA vaccine has:

[0029] (4) the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 and / or SEQ ID NO.6; or

[0030] (5) a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as (4); or

[0031] (6) A nucleotide sequence having at least 70% homology to the nucleotide sequence shown in (4) or (5).

[0032] The second aspect of the present invention provides the use of the above-mentioned mRNA vaccine in the preparation of respiratory syncytial virus vaccine.

[0033] A third aspect of the present invention provides a DNA construct comprising nucleic acid sequences encoding the fusion F protein of respiratory syncytial virus (RSV F) and the matrix protein (M) of metapneumovirus (MPV);

[0034] Preferably, the fusion F protein of respiratory syncytial virus (RSV F) is the pre-fusion F protein of respiratory syncytial virus (RSV pre F);

[0035] More preferably, the respiratory syncytial virus prefusion F protein (RSV pre F) is the human respiratory syncytial virus prefusion F protein, and the metapneumovirus matrix protein (M) is the matrix protein (M) of human metapneumovirus (hMPV).

[0036] In a preferred embodiment of the present invention, the transmembrane domain of the pre-fusion F protein of the respiratory syncytial virus (RSV pre F) is replaced with the transmembrane domain of the matrix protein (M) of the metapneumovirus (MPV); and / or the cytosolic tail domain (CT) of the pre-fusion F protein of the respiratory syncytial virus (RSV pre F) is replaced with the cytosolic tail domain (CT) of the matrix protein (M) of the metapneumovirus (MPV);

[0037] Preferably, the transmembrane domain of the prefusion F protein of the respiratory syncytial virus (RSV pre F) is replaced by the transmembrane domain of the matrix protein (M) of the human metapneumovirus (hMPV); and the cytosolic tail structure (CT) domain of the prefusion F protein of the respiratory syncytial virus (RSV preF) is replaced by the cytosolic tail domain (CT) of the matrix protein (M) of the human metapneumovirus (hMPV);

[0038] More preferably, amino acid residues 525-574 of the prefusion F protein of respiratory syncytial virus (RSV pre F) are replaced with amino acid residues 489-539 of the matrix protein (M) of human metapneumovirus (hMPV).

[0039] The beneficial effects of the present invention are:

[0040] The mRNA vaccine of the present invention can induce animals to produce specific humoral immune responses (neutralizing antibodies) against RSV, can protect animals from death caused by respiratory syncytial virus (RSV) infection, and reduce the viral load after infection, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The ELISA method was used to detect the binding activity of serum from mice immunized with mRNA vaccines (FM-mRNA-1, FM-mRNA-2, FM-mRNA-3 and DS-Cav1 protein);

[0042] Figure 2 Immunofluorescence assay was used to detect the neutralizing activity of serum from mice immunized with mRNA vaccine (FM-mRNA-1, FM-mRNA-2, FM-mRNA-3 and DS-Cav1 protein);

[0043] Figure 3Shows the detection of lung virus load (FM-mRNA-1, FM-mRNA-2, FM-mRNA-3 and DS-Cav1protein) in mice immunized with mRNA vaccine after RSV (LONG strain) virus challenge;

[0044] Figure 4 The ELISA method was used to detect the binding activity of serum from mice immunized with mRNA vaccines (FM-mRNA-2, DS-Cav1mRNA, DS2 mRNA, and DS-Cav1 protein);

[0045] Figure 5 Immunofluorescence assay was used to detect the neutralizing activity of serum from mice immunized with mRNA vaccine (FM-mRNA-2, DS-Cav1mRNA, DS2 mRNA and DS-Cav1 protein);

[0046] Figure 6 The results show the detection of lung viral load (FM-mRNA-2, DS-Cav1 mRNA, DS2 mRNA and DS-Cav1 protein) in mice immunized with mRNA vaccine after RSV (LONG strain) virus infection. DETAILED DESCRIPTION

[0047] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in this application should have the common meanings understood by technicians in the relevant field.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0049] As used herein and in the appended claims, the singular forms "a," "an," "another," and "the" include plural referents unless the context clearly dictates otherwise.

[0050] Amino acid substitution: An amino acid in a polypeptide is replaced / substituted with a different amino acid or with no amino acid (ie, deleted).

[0051] Cytosolic tail domain (CT): A continuous region in a transmembrane protein that includes the terminal end (either N or C terminus) of the protein and extends from the cytoplasmic surface of the cell membrane or viral envelope into the cytoplasm of the cell or enveloped virus. In the case of type I transmembrane proteins, the CT includes the C terminus of the protein. In the case of type II transmembrane proteins, the CT includes the N terminus of the protein.

[0052] Transmembrane domain (TM): An amino acid sequence that spans a membrane lipid bilayer, such as that of a cell or virus. A transmembrane domain can be used to anchor an antigen to the membrane.

[0053] In the present invention, the native respiratory syncytial virus F protein refers to the respiratory syncytial virus F protein before and / or after fusion.

[0054] "Sequence identity" between two polypeptide or nucleic acid sequences refers to the percentage of the number of identical residues between the sequences to the total number of residues. In calculating percent identity, the sequences being compared are aligned in a manner that produces the largest match between the sequences, and gaps in the alignment, if any, are resolved by a particular algorithm. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package, including GAP, BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410). The above programs are publicly available from the International Center for Biotechnology Information (NCBI) and other sources. The well-known Smith Waterman algorithm can also be used to determine identity.

[0055] The present invention provides an mRNA vaccine comprising a nucleic acid encoding a fusion F protein of a respiratory syncytial virus and a matrix protein (M) of at least one metapneumovirus.

[0056] In the present invention, the mRNA vaccine includes at least one of the following structural modifications:

[0057] 1. Replace / substitute the amino acid residues at certain positions of RSV F protein to keep the RSV F protein in a pre-fusion conformation; for example, the wild-type amino acid residue at one or more residues in positions 112, 155, 190, 207, 290, 379 and / or 447 of the pre-fusion F protein of the respiratory syncytial virus is replaced by another amino acid residue relative to the native respiratory syncytial virus F protein;

[0058] 2. Replace the transmembrane domain of RSV F protein with the corresponding transmembrane domain of matrix protein (M) of metapneumovirus (MPV);

[0059] 3. The cytosolic tail domain of the prefusion F protein of respiratory syncytial virus (RSV pre F) was replaced with the corresponding cytosolic tail domain of the matrix protein (M) of metapneumovirus (MPV).

[0060] Preferably, the mRNA vaccine includes the first structural modification and the second structural modification, or the first structural modification and the third structural modification, or includes the first structural modification, the second structural modification and the third structural modification at the same time.

[0061] In the present invention, the nucleic acid vaccine designed according to this method can stimulate the ability to produce humoral and cellular immune responses in vivo.

[0062] The mRNA vaccine of the present invention is co-expressed by recombinant genetic engineering of the matrix protein (M protein) of metapneumovirus (MPV) and the RSV glycoprotein (F protein). When the mRNA vaccine is administered to a subject, it can induce an immune response, including inducing neutralizing antibodies. The mRNA vaccine can induce higher immune protection and prevent the occurrence of viral mutants that can evade the immune system.

[0063] In a preferred embodiment of the present invention, the pre-fusion F protein of the respiratory syncytial virus has the following amino acid sequence:

[0064] (1) the amino acid sequence shown in SEQ ID NO.1; or

[0065] (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0066] (3) An amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2).

[0067] In a preferred embodiment of the present invention, the matrix protein (M) of the metapneumovirus has the following amino acid sequence:

[0068] (1) the amino acid sequence shown in SEQ ID NO.2; or

[0069] (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0070] (3) An amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2).

[0071] In the mRNA vaccine of the present invention, it simultaneously comprises the amino acid sequence of the fusion F protein of the respiratory syncytial virus and the amino acid sequence of the matrix protein (M) of at least one metapneumovirus, and the nucleic acid sequence of the tandem gene expression element;

[0072] Preferably, a nucleic acid encoding a pre-fusion F protein of a respiratory syncytial virus and a nucleic acid encoding a matrix protein (M) of two metapneumoviruses are operably linked to a tandem gene expression element, which is an internal ribosome entry site (IRES);

[0073] More preferably, the nucleic acid sequence of the IRES is SEQ ID NO:3.

[0074] In a preferred embodiment of the present invention, the nucleic acid of the mRNA vaccine has:

[0075] (4) the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 and / or SEQ ID NO.6; or

[0076] (5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as (4); herein, the same or similar function as (4) means that the nucleotide sequence of (4) can achieve the same or similar function as the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 and / or SEQ ID NO.6 in inducing animals to produce RSV-specific humoral immunity (neutralizing antibodies); or

[0077] (6) A nucleotide sequence having at least 70% homology to the nucleotide sequence shown in (4) or (5).

[0078] The present invention also provides a DNA construct comprising a nucleic acid sequence encoding a fusion F protein of respiratory syncytial virus (RSVF) and a matrix protein (M) of metapneumovirus (MPV);

[0079] Preferably, the fusion F protein of respiratory syncytial virus (RSV F) is the pre-fusion F protein of respiratory syncytial virus (RSV pre F);

[0080] More preferably, the respiratory syncytial virus prefusion F protein (RSV pre F) is the human respiratory syncytial virus prefusion F protein, and the metapneumovirus matrix protein (M) is the matrix protein (M) of human metapneumovirus (hMPV).

[0081] The DNA construct of the present invention is introduced into cells to form a matrix protein (M) expressing the fusion F protein of respiratory syncytial virus (RSVF) and metapneumovirus (MPV), thereby forming the mRNA vaccine of the present invention.

[0082] In a preferred embodiment of the present invention, the transmembrane domain of the pre-fusion F protein of the respiratory syncytial virus (RSV pre F) is replaced with the transmembrane domain of the matrix protein (M) of the metapneumovirus (MPV); and / or the cytosolic tail domain (CT) of the pre-fusion F protein of the respiratory syncytial virus (RSV pre F) is replaced with the cytosolic tail domain (CT) of the matrix protein (M) of the metapneumovirus (MPV);

[0083] Preferably, the transmembrane domain of the prefusion F protein of the respiratory syncytial virus (RSV pre F) is replaced by the transmembrane domain of the matrix protein (M) of the human metapneumovirus (hMPV); and the cytosolic tail structure (CT) domain of the prefusion F protein of the respiratory syncytial virus (RSV preF) is replaced by the cytosolic tail domain (CT) of the matrix protein (M) of the human metapneumovirus (hMPV);

[0084] More preferably, amino acid residues 525-574 of the prefusion F protein of respiratory syncytial virus (RSV pre F) are replaced with amino acid residues 489-539 of the matrix protein (M) of human metapneumovirus (hMPV).

[0085] The present invention is further described below by way of examples. The examples of the present invention are intended to illustrate the present invention rather than to limit the present invention. Simple modifications to the present invention made according to the essence of the present invention all fall within the scope of protection claimed in the present invention. The present invention is further described below in conjunction with the examples:

[0086] Example 1: mRNA vaccine preparation

[0087] The amino acid sequence and nucleic acid sequence corresponding to the mRNA vaccine are shown in SEQ ID NO.1-6. The sequences are all prepared by Wuhan Hanhai Xinzyme Biotechnology Co., Ltd. For specific operation steps, refer to Cimica V, Hélène Boigard, Bhatia B, et al. Novel Respiratory Syncytial Virus-Like Particle Vaccine Composed of the Postfusion and Prefusion Conformations of the F Glycoprotein. [J]. Clinical & Vaccine Immunology Cvi, 2016, 23 (6): 451-459.

[0088] Example 2: mRNA vaccine immunization program

[0089] Set up the following experimental and control groups:

[0090] Experimental group: 6-8 weeks old female BALB / c mice, 5 mice / group, 1 μg / mouse, 5 μg / mouse, 10 μg / mouse mRNA vaccine (FM-mRNA-1, FM-mRNA-2 and FM-mRNA-3, whose nucleic acid sequences are shown in SEQ ID NO.4-6, respectively, all prepared by Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.) and 1 μg / mouse, 5 μg / mouse, 10 μg / mouse DS-Cav1protein (according to reference: Structure-Based Design of a Fusion Glycoprotein Vaccine for Respiratory Syncytial Virus [J]. Science, 2013, 342 (6158): 592-598.) were used to immunize mice. The mice were immunized twice, on the 1st day and the 22nd day, and the serum was collected on the 32nd day for detection. On the 33rd day, RSV (LONG strain, purchased from ATCC) virus (titer 2*10 6 FFU / each), after the mice were anesthetized (estimated 2-5 min), 25 μL of poison was administered intranasally, and lung tissue was obtained 3 days after the challenge (D36), and the supernatant was obtained after grinding for evaluation of the protection against the challenge.

[0091] Example 3: Detection of serum binding activity of mice immunized with mRNA vaccine (ELISA method)

[0092] RSV pre F protein was coated in the coated plate in advance, 2μg / mL, 100μL per well, and coated overnight at 2-8℃. The plate was washed 3 times with PBS-T, 2min / time, and the blocking solution was added, incubated at 37℃ for 1h, and the plate was washed 3 times with PBS-T, 2min / time; serum in the first well: PBS was 1:100, 5-fold serial gradient dilution, and only antibody diluent was added to the blank well. 100μL / well of the sample was added to the coated plate and incubated at 37℃ for 1h; the plate was washed 3 times with PBS-T, and HRP-labeled goat anti-mouse IgG (H+L) secondary antibody was added, and incubated at 37℃ for 1h; TMB colorimetric solution (A and B solutions were mixed in equal amounts) was added, color was developed at room temperature in the dark for 15min, and 2MH 2 SO 4 Stop color development and measure the absorbance with an enzyme-labeled instrument.

[0093] The experimental results are as follows Figure 1 Different doses of FM-mRNA-1, FM-mRNA-2 and FM-mRNA-3 all had significant immune activity in a dose-dependent manner. Among them, the immune effect of 10μg / mL FM-mRNA-2 was significantly higher than that of 10μg / mL DS-Cav1 protein.

[0094] Example 4: Detection of serum neutralization activity of mice immunized with mRNA vaccine (indirect immunofluorescence method)

[0095] The neutralizing activity of BALB / c mouse serum against mRNA vaccine after immunization was detected by indirect immunofluorescence. Synagis (purchased from Immunity Pharma) was diluted to 50 μg / mL in DMEM / F12 medium as a primary antibody positive control. The 10-fold diluted serum sample to be tested and the positive control antibody Synagis were incubated with RSV (LONG strain) virus (titer 2*10 8 FFU / mL, 1.0 mL / tube, purchased from ATCC) and incubated for 1 h. HEp-2 cells (density 1.2*10 6 / mL) and incubate at 37℃ for 24h. No virus is added to cells that are negative. After 24h, discard the supernatant, add 80% cold acetone, and fix the cells at 4℃. After 30min, discard the acetone, dry the acetone, dilute the primary antibody Synagis (1mg / mL) 1:1000, 50μL / well, and incubate at 37℃ for 1h. Then, discard the supernatant, wash 3 times with PBS, add Goat anti-human IgG-FITC (1:400 dilution, 50μL / well, purchased from Southernbiotech), and incubate at 37℃ for 1h. Wash 3 times with PBS, and observe the proportion of fluorescent foci under a fluorescence microscope.

[0096] The experimental results are as follows Figure 2 :Negative cells without fluorescence, RSV (LONG strain): Virus positive fluorescent foci 80%-95%. 5μg / mL, 10μg / mL FM-mRNA-1, FM-mRNA-2 and FM-mRNA-3 all had significant immune activity, and were dose-dependent. Among them, the immune effects of 10μg / mL FM-mRNA-1 and 10μg / mL FM-mRNA-3 were comparable to those of 10μg / mL DS-Cav1protein, but the immune effect of 10μg / mL FM-mRNA-2 was significantly higher than that of 10μg / mL DS-Cav1protein.

[0097] Example 5: Evaluation of the protection of mice immunized with mRNA vaccines

[0098] After grinding the lung tissue, the supernatant was taken for lung viral load detection (qPCR). During the experiment, the hair loss of the immunized mice was observed and recorded. RNA was extracted according to the instructions of Viral RNA Kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) and then tested.

[0099] The experimental results are as follows Figure 3Different doses of FM-mRNA-1, FM-mRNA-2 and FM-mRNA-3 can protect animals from death caused by lethal RSV infection and reduce lung virus load after challenge. Among them, 10μg / mL FM-mRNA-2 has the best effect.

[0100] The above examples show that different doses of FM-mRNA-1, FM-mRNA-2, and FM-mRNA-3 all have significant binding activity and can induce RSV-specific humoral immunity (neutralizing antibodies) in animals. Each mRNA vaccine can protect animals from death caused by lethal RSV virus infection and reduce lung virus load after challenge. The relationship between immunogenicity and protection between the same dose groups is: FM-mRNA-2≥DS-Cav1 protein>FM-mRNA-1=FM-mRNA-3.

[0101] Example 6: mRNA vaccine immunization program

[0102] Set up the following experimental and control groups:

[0103] Experimental groups: 6-8 week old female BALB / c mice, 5 mice / group, were respectively given 1 μg / mouse, 5 μg / mouse, and 10 μg / mouse mRNA vaccine (FM-mRNA-2 was produced by Wuhan Hanhai Xinzyme Biotechnology Co., Ltd.), DS-Cav1 mRNA, DS2 mRNA, and DS-Cav1 protein (DS-Cav1 mRNA: according to reference: Structure-Based Design of a Fusion Glycoprotein Vaccine for Respiratory Syncytial Virus[J]. Science, 2013, 342(6158):592-598. DS2 mRNA: according to reference: Joyce MG, Zhang B, Ou L, et al. Iterative structure-based improvement of a respiratory syncytial virus fusion glycoprotein vaccine[J]. Nature Structural & Molecular Biology, 2016, 23(9):811-820. and DS-Cav1 Mice were immunized with RSV (LONG strain) at day 39 (titer 2*10 6 FFU / each), after the mice were anesthetized (estimated 2-5 min), 25 μL of virus was injected into each nostril, and lung tissue was obtained 3 days after challenge (D42), and the supernatant was obtained after grinding for challenge protection evaluation.

[0104] Example 7: Detection of serum binding activity of mice immunized with mRNA vaccine (ELISA method)

[0105] RSV pre F protein was coated in the coated plate in advance, 2μg / mL, 100μL per well, and coated overnight at 2-8℃. The plate was washed 3 times with PBS-T, 2min / time, and the blocking solution was added, incubated at 37℃ for 1h, and the plate was washed 3 times with PBS-T, 2min / time; serum in the first well: PBS was 1:500, 5-fold serial gradient dilution, and only antibody diluent was added to the blank well, and 100μL / well of the sample was added to the coated plate and incubated at 37℃ for 1h; the plate was washed 3 times with PBS-T, and HRP-labeled goat anti-mouse IgG (H+L) secondary antibody was added, and incubated at 37℃ for 1h; TMB colorimetric solution (A and B solutions were mixed in equal amounts) was added, and color was developed at room temperature in the dark for 15min, 2MH2SO4 was added to stop color development, and the absorbance value was measured with an enzyme marker.

[0106] The experimental results are as follows Figure 4 : 1μg / mouse, 5μg / mouse and 10μg / mouse FM-mRNA-2, DS-Cav1mRNA, DS2 mRNA and DS-Cav1 protein all had significant immune activity. Among them, the immune effect of 10μg / mL FM-mRNA-2 was equivalent to that of 10μg / mL DS-Cav1 protein.

[0107] Example 8: Detection of serum neutralization activity of mice immunized with mRNA vaccine (indirect immunofluorescence method)

[0108] The neutralizing activity of BALB / c mouse serum against mRNA vaccine after immunization was detected by indirect immunofluorescence. Synagis (purchased from Immunity Pharma) was diluted to 50 μg / mL in DMEM / F12 medium as a primary antibody positive control. The 10-fold diluted serum sample to be tested and the positive control antibody Synagis were incubated with RSV (LONG strain) virus (titer 2*10 8 FFU / mL, 1.0 mL / tube, purchased from ATCC) and incubated for 1 h before adding HEp-2 cells (density 1.2*10 6 / mL) and incubate at 37℃ for 24h. No virus is added to cells that are negative. After 24h, discard the supernatant, add 80% cold acetone, and fix the cells at 4℃. After 30min, discard the acetone, dry the acetone, dilute the primary antibody Synagis (1mg / mL) 1:1000, 50μL / well, and incubate at 37℃ for 1h. Then, discard the supernatant, wash 3 times with PBS, add Goat anti-human IgG-FITC (1:400 dilution, 50μL / well, purchased from Southernbiotech), and incubate at 37℃ for 1h. Wash 3 times with PBS, and observe the proportion of fluorescent foci under a fluorescence microscope.

[0109] The experimental results are as follows Figure 5: Cells negative and no fluorescence, RSV (LONG strain): virus positive fluorescent foci 80%-95%. After the second immunization with 10μg / mL FM-mRNA-2, a significantly higher level of neutralizing antibodies was produced than with 10μg / mL DS-Cav1protein.

[0110] Example 9: Evaluation of the protection of mice immunized with mRNA vaccines against virus

[0111] After grinding the lung tissue, the supernatant was taken for lung viral load detection (qPCR). During the experiment, the hair loss of the immunized mice was observed and recorded. RNA was extracted according to the instructions of Viral RNA Kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) and then tested.

[0112] The experimental results are as follows Figure 6 Different doses of FM-mRNA-2, DS-Cav1 mRNA, DS2mRNA and DS-Cav1 protein can protect animals from death caused by lethal RSV virus infection and reduce lung viral load after challenge. 10μg / mL FM-mRNA-2 can reduce lung viral load after challenge more than 10μg / mL DS-Cav1 protein.

[0113] The results of the above examples show that different doses of FM-mRNA-2, DS-Cav1 mRNA, DS2 mRNA and DS-Cav1 protein all have obvious binding activity and can induce animals to produce specific humoral immunity (neutralizing antibodies) against RSV. Each vaccine can protect animals from death caused by lethal RSV virus infection and reduce lung virus load after challenge. The strength relationship between immunogenicity and protection between the same dose groups is: FM-mRNA-2>DS-Cav1 protein>DS2mRNA>DS-Cav1mRNA.

[0114] Although only specific implementation examples of the present invention are described above, those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these implementations without departing from the principles and essence of the present invention, but these changes or modifications all fall within the protection scope of the present invention.

Claims

1. An mRNA vaccine comprising a nucleic acid encoding a fusion F protein of respiratory syncytial virus and at least one matrix protein (M) of metapneumovirus.

2. The mRNA vaccine according to claim 1, in, The fusion F protein of the respiratory syncytial virus is the pre-fusion F protein of the respiratory syncytial virus, and the at least one matrix protein (M) of the metapneumovirus is one and / or two matrix proteins (M) of the metapneumovirus; Preferably, the pre-fusion F protein of the respiratory syncytial virus is the pre-fusion F protein of the human respiratory syncytial virus, and the matrix protein (M) of the metapneumovirus is the matrix protein (M) of the human metapneumovirus.

3. The mRNA vaccine according to claim 1 or 2, in, The pre-fusion F protein of the respiratory syncytial virus has the following amino acid sequence: (1) the amino acid sequence shown in SEQ ID NO.1; or (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or (3) An amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2).

4. The mRNA vaccine according to claim 1 or 2, in, The matrix protein (M) of the metapneumovirus has the following amino acid sequence: (1) the amino acid sequence shown in SEQ ID NO.2; or (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or (3) An amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2).

5. The mRNA vaccine according to claims 1-4, in, A fusion F protein of respiratory syncytial virus and at least one matrix protein (M) of metapneumovirus are linked via tandem gene expression elements; Preferably, a nucleic acid encoding a pre-fusion F protein of a respiratory syncytial virus and a nucleic acid encoding a matrix protein (M) of two metapneumoviruses are operably linked to a tandem gene expression element, which is an internal ribosome entry site (IRES); More preferably, the nucleic acid sequence of the IRES is SEQ ID NO:

3.

6. The mRNA vaccine according to claims 1-5, in, The fusion F protein of the respiratory syncytial virus comprises modifications to one or more amino acid residues; Preferably, the wild-type amino acid residue at one or more residues in positions 112, 155, 190, 207, 290, 379 and / or 447 of the pre-fusion F protein of the respiratory syncytial virus is replaced by another amino acid residue relative to the native F protein of the respiratory syncytial virus.

7. The mRNA vaccine according to claim 6, in, The transmembrane domain of the prefusion F protein of the respiratory syncytial virus is replaced by the transmembrane domain of the matrix protein (M) of the metapneumovirus; and / or the cytosolic tail domain of the prefusion F protein of the respiratory syncytial virus is replaced by the cytosolic tail domain of the matrix protein (M) of the metapneumovirus; Preferably, the transmembrane domain of the prefusion F protein of the respiratory syncytial virus is replaced with the transmembrane domain of the matrix protein (M) of the human metapneumovirus; and the cytosolic tail domain of the prefusion F protein of the respiratory syncytial virus is replaced with the cytosolic tail domain of the matrix protein (M) of the human metapneumovirus; More preferably, amino acid residues 525-574 of the pre-fusion F protein of respiratory syncytial virus are replaced with amino acid residues 489-539 of the matrix protein (M) of human metapneumovirus.

8. The mRNA vaccine according to any one of claims 1 to 7, in, The nucleic acid of the mRNA vaccine has: (4) the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 and / or SEQ ID NO.6; or (5) a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as (4); or (6) A nucleotide sequence having at least 70% homology to the nucleotide sequence shown in (4) or (5).

9. Use of the mRNA vaccine according to any one of claims 1-8 in the preparation of respiratory syncytial virus vaccine.

10. A DNA construct comprising a nucleic acid sequence encoding a fusion F protein of respiratory syncytial virus and a matrix protein (M) of metapneumovirus; Preferably, the fusion F protein of respiratory syncytial virus is the pre-fusion F protein of respiratory syncytial virus; More preferably, the pre-fusion F protein of respiratory syncytial virus is the pre-fusion F protein of human respiratory syncytial virus, and the matrix protein (M) of metapneumovirus is the matrix protein (M) of human metapneumovirus.

11. The DNA construct according to claims 1-10, in, The transmembrane domain of the prefusion F protein of the respiratory syncytial virus is replaced by the transmembrane domain of the matrix protein (M) of the metapneumovirus; and / or the cytosolic tail domain of the prefusion F protein of the respiratory syncytial virus is replaced by the cytosolic tail domain of the matrix protein (M) of the metapneumovirus; Preferably, the transmembrane domain of the prefusion F protein of the respiratory syncytial virus is replaced with the transmembrane domain of the matrix protein (M) of the human metapneumovirus; and the cytosolic tail domain of the prefusion F protein of the respiratory syncytial virus is replaced with the cytosolic tail domain of the matrix protein (M) of the human metapneumovirus; More preferably, amino acid residues 525-574 of the pre-fusion F protein of respiratory syncytial virus are replaced with amino acid residues 489-539 of the matrix protein (M) of human metapneumovirus.

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