mRNA vaccine and use thereof

Abstract

Provided are an mRNA vaccine and use thereof. The mRNA vaccine comprises nucleic acids encoding a fusion F protein of respiratory syncytial virus and at least one matrix protein (M) of metapneumovirus. The mRNA vaccine can induce animals to generate specific humoral immune responses (neutralizing antibodies) against respiratory syncytial virus (RSV), can protect the animals from death caused by RSV infection, and reduces the viral load after challenge.

Description

An mRNA vaccine and its application 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. Global epidemiological studies indicate that 2–5% of children with RSV infection require hospitalization. RSV disease causes 100,000–200,000 deaths worldwide annually. A key contributing factor to this enormous disease burden is the limited availability of prevention and treatment options.

[0003] Respiratory syncytial virus (RSV) is a single-stranded, negative-sense, non-segmented enveloped RNA virus classified as a member of the genus Pneumovirus in the family Paramyxoviridae. RSV has two surface glycoproteins: 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 fusion of the viral membrane with the host cell membrane and is the primary target for inducing neutralizing antibodies in 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 host cell surface, the F protein undergoes an irreversible conformational change, namely the post-fusion conformation. This change allows 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 target of neutralizing antibodies 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 between the pre-fusion and post-fusion phases, the antigenic sites most sensitive to neutralization are only located in the pre-fusion conformation. Therefore, the design of antigens targeting the pre-fusion conformation of the F protein is very critical.

[0005] Research shows that RSV F protein polymer precursor is activated after translation by the cutting of furin on site I and II in Golgi apparatus.This cutting process produces two subunits, F1 and F2, and they are connected by two disulfide bond cysteine-cysteine ​​bridges, and discharge the 27 amino acid whose short glycoprotein 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, and this domain interacts with matrix protein (M) during virion assembly.

[0006] Currently, several antibody drugs, such as palivizumab, have been developed, 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, necessitating multiple, high-dose administration.

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

[0008] Summary of the Invention

[0009] 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 responses (neutralizing antibodies) against RSV, protect animals from death caused by respiratory syncytial virus (RSV) infection, and reduce the viral load after infection.

[0010] 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 respiratory syncytial virus and at least one matrix protein (M) of metapneumovirus.

[0011] In a preferred embodiment of the present invention, the fusion F protein of respiratory syncytial virus (RSV F) is the prefusion F protein of respiratory syncytial virus (RSV pre F), and the at least one matrix protein (M) of metapneumovirus is one and / or two matrix proteins (M) of metapneumovirus;

[0012] Preferably, the respiratory syncytial virus prefusion F protein (RSV pre F) is the human respiratory syncytial virus prefusion F protein, and the metapneumovirus (MPV) matrix protein (M) is the human metapneumovirus (hMPV) matrix protein (M).

[0013] In a preferred embodiment of the present invention, the pre-fusion F protein of the respiratory syncytial virus has the following amino acid sequence:

[0014] (1) the amino acid sequence shown in SEQ ID NO. 1; or

[0015] (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

[0016] (3) An amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2).

[0017] In a preferred embodiment of the present invention, the matrix protein (M) of the metapneumovirus has the following amino acid sequence:

[0018] (1) the amino acid sequence shown in SEQ ID NO. 2; or

[0019] (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

[0020] (3) An amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2).

[0021] In a preferred embodiment of the present invention, a fusion F protein of respiratory syncytial virus is linked to at least one matrix protein (M) of metapneumovirus via tandem gene expression elements;

[0022] Preferably, a nucleic acid encoding a prefusion F protein of respiratory syncytial virus and a nucleic acid encoding two matrix proteins (M) of metapneumovirus are operably linked to a tandem gene expression element, which is an internal ribosome entry site (IRES);

[0023] More preferably, the nucleic acid sequence of the IRES is SEQ ID NO: 3.

[0024] 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;

[0025] Preferably, the wild-type amino acid residue at one or more residues among positions 112, 155, 190, 207, 290, 379 and / or 447 of the prefusion F protein of the respiratory syncytial virus is substituted by another amino acid residue relative to the native F protein of the respiratory syncytial virus.

[0026] In a preferred embodiment of the present invention, the transmembrane domain of the prefusion F protein of respiratory syncytial virus (RSV pre F) is replaced by the transmembrane domain of the matrix protein (M) of metapneumovirus (MPV); and / or the cytosolic tail domain (CT) of the prefusion F protein of respiratory syncytial virus (RSV pre F) is replaced by the cytosolic tail domain (CT) of the matrix protein (M) of metapneumovirus (MPV);

[0027] Preferably, the transmembrane domain of the prefusion F protein of respiratory syncytial virus (RSV pre F) is replaced by the transmembrane domain of the matrix protein (M) of human metapneumovirus (hMPV); and the cytosolic tail domain (CT) of the prefusion F protein of respiratory syncytial virus (RSV pre F) is replaced by the cytosolic tail domain (CT) of the matrix protein (M) of human metapneumovirus (hMPV);

[0028] 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).

[0029] In a preferred embodiment of the present invention, the nucleic acid of the mRNA vaccine has:

[0030] (4) the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 and / or SEQ ID NO.6; or

[0031] (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

[0032] (6) A nucleotide sequence having at least 70% homology to the nucleotide sequence shown in (4) or (5).

[0033] The second aspect of the present invention provides the use of the above-mentioned mRNA vaccine in the preparation of respiratory syncytial virus vaccine.

[0034] 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);

[0035] Preferably, the fusion F protein of respiratory syncytial virus (RSV F) is the pre-fusion F protein of respiratory syncytial virus (RSV pre F);

[0036] 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 human metapneumovirus (hMPV) matrix protein (M).

[0037] In a preferred embodiment of the present invention, the transmembrane domain of the prefusion F protein of respiratory syncytial virus (RSV pre F) is replaced by the transmembrane domain of the matrix protein (M) of metapneumovirus (MPV); and / or the cytosolic tail domain (CT) of the prefusion F protein of respiratory syncytial virus (RSV pre F) is replaced by the cytosolic tail domain (CT) of the matrix protein (M) of metapneumovirus (MPV);

[0038] Preferably, the transmembrane domain of the prefusion F protein of respiratory syncytial virus (RSV pre F) is replaced by the transmembrane domain of the matrix protein (M) of human metapneumovirus (hMPV); and the cytosolic tail structure (CT) domain of the prefusion F protein of respiratory syncytial virus (RSV pre F) is replaced by the cytosolic tail structure domain (CT) of the matrix protein (M) of human metapneumovirus (hMPV);

[0039] 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).

[0040] The beneficial effects of the present invention are:

[0041] The mRNA vaccine of the present invention can induce animals to produce specific humoral immune responses (neutralizing antibodies) against RSV, 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

[0042] Figure 1 shows the ELISA assay for the binding activity (FM-mRNA-1, FM-mRNA-2, FM-mRNA-3, and DS-Cav1 protein) of serum from mice immunized with mRNA vaccines;

[0043] Figure 2 shows the neutralizing activity (FM-mRNA-1, FM-mRNA-2, FM-mRNA-3 and DS-Cav1 protein) of serum from mice immunized with mRNA vaccines detected by immunofluorescence assay;

[0044] FIG3 shows the detection of lung viral load (FM-mRNA-1, FM-mRNA-2, FM-mRNA-3 and DS-Cav1 protein) in mice immunized with mRNA vaccine after RSV (LONG strain) virus challenge;

[0045] Figure 4 shows the ELISA assay for the binding activity of serum from mice immunized with the mRNA vaccine (FM-mRNA-2, DS-Cav1 mRNA, DS2 mRNA, and DS-Cav1 protein);

[0046] Figure 5 shows the neutralizing activity (FM-mRNA-2, DS-Cav1 mRNA, DS2 mRNA and DS-Cav1 protein) of serum from mice immunized with mRNA vaccines detected by immunofluorescence assay;

[0047] FIG6 shows 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 challenge. DETAILED DESCRIPTION

[0048] It should be noted that, unless otherwise defined, the technical or scientific terms used in this application should have the common meanings understood by those skilled in the art.

[0049] 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.

[0050] 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.

[0051] Amino acid substitution: An amino acid in a polypeptide is replaced / substituted with a different amino acid or no amino acid (ie, deleted).

[0052] Cytosolic tail domain (CT): A continuous region of a transmembrane protein that includes the terminal end (either the 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.

[0053] 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.

[0054] In the present invention, the native respiratory syncytial virus F protein refers to the respiratory syncytial virus F protein before and / or after fusion.

[0055] "Sequence identity" between two polypeptide or nucleic acid sequences refers to the percentage of residues that are identical between the sequences to the total number of residues. In calculating percent identity, the sequences being compared are aligned to produce 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 aforementioned programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources. The well-known Smith-Waterman algorithm can also be used to determine identity.

[0056] The present invention provides an mRNA vaccine comprising nucleic acid encoding a fusion F protein of respiratory syncytial virus and at least one matrix protein (M) of metapneumovirus.

[0057] In the present invention, the mRNA vaccine includes at least one of the following structural modifications:

[0058] 1. Substitution / replacement of amino acid residues at certain positions of the RSV F protein to maintain 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 substituted with another amino acid residue relative to the native respiratory syncytial virus F protein;

[0059] 2. The transmembrane domain of the RSV F protein was replaced with the corresponding transmembrane domain of the matrix protein (M) of the metapneumovirus (MPV);

[0060] 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).

[0061] 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.

[0062] 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.

[0063] 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.

[0064] In a preferred embodiment of the present invention, the pre-fusion F protein of the respiratory syncytial virus has the following amino acid sequence:

[0065] (1) the amino acid sequence shown in SEQ ID NO. 1; or

[0066] (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

[0067] (3) An amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2).

[0068] In a preferred embodiment of the present invention, the matrix protein (M) of the metapneumovirus has the following amino acid sequence:

[0069] (1) the amino acid sequence shown in SEQ ID NO. 2; or

[0070] (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

[0071] (3) An amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2).

[0072] In the mRNA vaccine of the present invention, it comprises simultaneously the amino acid sequence of the fusion F protein of 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;

[0073] Preferably, a nucleic acid encoding a prefusion F protein of respiratory syncytial virus and a nucleic acid encoding two matrix proteins (M) of metapneumovirus are operably linked to a tandem gene expression element, which is an internal ribosome entry site (IRES);

[0074] More preferably, the nucleic acid sequence of the IRES is SEQ ID NO: 3.

[0075] In a preferred embodiment of the present invention, the nucleic acid of the mRNA vaccine has:

[0076] (4) the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 and / or SEQ ID NO.6; or

[0077] (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 function of the nucleotide sequence of (4) being the same or similar to that of (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

[0078] (6) A nucleotide sequence having at least 70% homology to the nucleotide sequence shown in (4) or (5).

[0079] The present invention also 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);

[0080] Preferably, the fusion F protein of respiratory syncytial virus (RSV F) is the pre-fusion F protein of respiratory syncytial virus (RSV pre F);

[0081] 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 human metapneumovirus (hMPV) matrix protein (M).

[0082] The DNA construct of the present invention is introduced into cells to form the expression of the fusion F protein of respiratory syncytial virus (RSV F) and the matrix protein (M) of metapneumovirus (MPV), thereby forming the mRNA vaccine of the present invention.

[0083] In a preferred embodiment of the present invention, the transmembrane domain of the prefusion F protein of respiratory syncytial virus (RSV pre F) is replaced by the transmembrane domain of the matrix protein (M) of metapneumovirus (MPV); and / or the cytosolic tail domain (CT) of the prefusion F protein of respiratory syncytial virus (RSV pre F) is replaced by the cytosolic tail domain (CT) of the matrix protein (M) of metapneumovirus (MPV);

[0084] Preferably, the transmembrane domain of the prefusion F protein of respiratory syncytial virus (RSV pre F) is replaced by the transmembrane domain of the matrix protein (M) of human metapneumovirus (hMPV); and the cytosolic tail structure (CT) domain of the prefusion F protein of respiratory syncytial virus (RSV pre F) is replaced by the cytosolic tail structure domain (CT) of the matrix protein (M) of human metapneumovirus (hMPV);

[0085] 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).

[0086] The present invention is further illustrated by the following examples. The examples of the present invention are intended to illustrate the present invention rather than to limit the present invention. Simple modifications made to the present invention based on the essence of the present invention fall within the scope of protection claimed in the present invention. The present invention is further illustrated by the following examples:

[0087] Example 1: mRNA vaccine preparation

[0088] 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.

[0089] Example 2: mRNA vaccine immunization program

[0090] Set up the following experimental and control groups:

[0091] Experimental group: 6-8 week old female BALB / c mice, 5 mice / group, were immunized with 1 μg / mouse, 5 μg / mouse, and 10 μg / mouse of 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, and 10 μg / mouse of DS-Cav1 protein (according to the reference: Structure-Based Design of a Fusion Glycoprotein Vaccine for Respiratory Syncytial Virus [J]. Science, 2013, 342(6158): 592-598.) for a total of 2 immunizations, on day 1 and day 22, respectively. Serum was collected on day 32 for testing. On day 33, 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 challenge protection evaluation.

[0092] Example 3: Detection of serum binding activity of mice immunized with mRNA vaccine (ELISA method)

[0093] Pre-coat the plate with RSV pre F protein at 2 μg / mL, 100 μL per well, and coat overnight at 2-8°C. Wash the plate three times with PBS-T (2 min / time), add blocking buffer, and incubate at 37°C for 1 hour. Wash the plate three times with PBS-T (2 min / time). Serum in the first well is diluted 1:100 in PBS, and a five-fold serial dilution series is performed. For blank wells, add only antibody diluent. Add 100 μL / well of sample to the coated plate and incubate at 37°C for 1 hour. Wash the plate three times with PBS-T, add HRP-labeled goat anti-mouse IgG (H+L) secondary antibody, and incubate at 37°C for 1 hour. Add TMB colorimetric solution (equal volumes of solution A and solution B) and develop at room temperature in the dark for 15 minutes. Stop development with 2 M H2SO4, and measure absorbance with a microplate reader.

[0094] The experimental results are shown in Figure 1: Different doses of FM-mRNA-1, FM-mRNA-2, and FM-mRNA-3 all showed significant immune activity in a dose-dependent manner. Among them, the immune effect of 10 μg / mL FM-mRNA-2 was significantly higher than that of 10 μg / mL DS-Cav1 protein.

[0095] Example 4: Detection of serum neutralization activity in mice immunized with mRNA vaccine (indirect immunofluorescence assay)

[0096] The neutralizing activity of BALB / c mouse serum against mRNA vaccine 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 serum sample to be tested and the positive control antibody Synagis were diluted 10 times and then 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 was added to cells that were negative. After 24h, the supernatant was discarded and 80% cold acetone was added to fix the cells at 4℃. After 30min, the acetone was discarded and dried in the air. The primary antibody Synagis (1mg / mL) was diluted 1:1000, 50μL / well, and incubated at 37℃ for 1h. Subsequently, the supernatant was discarded, the cells were washed 3 times with PBS, and Goat anti-human IgG-FITC (1:400 dilution, 50μL / well, purchased from Southern biotech) was added and incubated at 37℃ for 1h. The cells were washed 3 times with PBS and the proportion of fluorescent foci was observed under a fluorescence microscope.

[0097] The experimental results are shown in Figure 2: cells were negative for fluorescence, while RSV (LONG strain) showed 80%-95% virus-positive fluorescent foci. FM-mRNA-1, FM-mRNA-2, and FM-mRNA-3 at 5 μg / mL and 10 μg / mL all showed significant immune activity in a dose-dependent manner. The immune effects of 10 μg / mL FM-mRNA-1 and 10 μg / mL FM-mRNA-3 were comparable to those of 10 μg / mL DS-Cav1 protein, but the immune effect of 10 μg / mL FM-mRNA-2 was significantly higher than that of 10 μg / mL DS-Cav1 protein.

[0098] Example 5: Evaluation of protection against infection in mice immunized with mRNA vaccines

[0099] After grinding lung tissue, the supernatant was collected for lung viral load analysis (qPCR). Hair loss in immunized mice was observed and recorded during the experiment. RNA was extracted according to the instructions of the Viral RNA Kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) and subsequently tested.

[0100] The experimental results are shown in Figure 3: Different doses of FM-mRNA-1, FM-mRNA-2, and FM-mRNA-3 were able to protect animals from lethal RSV infection and reduce lung viral load after challenge. 10 μg / mL FM-mRNA-2 was the most effective.

[0101] The above examples demonstrate that FM-mRNA-1, FM-mRNA-2, and FM-mRNA-3 at varying doses all exhibit significant binding activity and can induce RSV-specific humoral immunity (neutralizing antibodies) in animals. Each mRNA vaccine can protect animals from lethal RSV infection and reduce lung viral loads after challenge. The correlation between immunogenicity and protective efficacy across dose groups is: FM-mRNA-2 ≥ DS-Cav1 protein > FM-mRNA-1 = FM-mRNA-3.

[0102] Example 6: mRNA vaccine immunization program

[0103] Set up the following experimental and control groups:

[0104] 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 of 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) protein (produced by GenScript Biotech Co., Ltd.) for a total of two immunizations, one on day 1 and the other on day 22. Serum was collected on day 36 for testing. Blood was collected from the cheeks and placed in a 1.5 ml EP tube. Serum was separated by centrifugation at 13,000 rpm for 10 min. On day 39, RSV (LONG strain) virus (titer 2*10 6FFU / 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 the challenge (D42), and the supernatant was obtained after grinding for challenge protection evaluation.

[0105] Example 7: Detection of serum binding activity of mice immunized with mRNA vaccine (ELISA method)

[0106] Pre-coat the plate with RSV pre F protein at 2 μg / mL, 100 μL per well, and coat overnight at 2-8°C. Wash the plate three times with PBS-T for 2 minutes each time, add blocking buffer, and incubate at 37°C for 1 hour. Wash the plate three times with PBS-T for 2 minutes each time. Serum in the first well was diluted 1:500 in PBS, and a five-fold serial dilution series was performed. For the blank wells, only antibody diluent was added. 100 μL / well of the sample was added to the coated plate and incubated at 37°C for 1 hour. Wash the plate three times with PBS-T, add HRP-labeled goat anti-mouse IgG (H+L) secondary antibody, and incubate at 37°C for 1 hour. Add TMB colorimetric solution (equal volumes of solution A and solution B) and develop at room temperature in the dark for 15 minutes. 2M H2SO4 was added to terminate the color development, and the absorbance was measured using a microplate reader.

[0107] The experimental results are shown in Figure 4: FM-mRNA-2, DS-Cav1 mRNA, DS2 mRNA, and DS-Cav1 protein at 1 μg / mouse, 5 μg / mouse, and 10 μg / mouse all produced significant immune activity. The immune effect of 10 μg / mL FM-mRNA-2 was comparable to that of 10 μg / mL DS-Cav1 protein.

[0108] Example 8: Detection of neutralizing activity of serum from mice immunized with mRNA vaccine (indirect immunofluorescence assay)

[0109] The neutralizing activity of BALB / c mouse serum against mRNA vaccine 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 serum sample to be tested and the positive control antibody Synagis were diluted 10 times and then 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 was added to cells that were negative. After 24h, the supernatant was discarded and 80% cold acetone was added to fix the cells at 4℃. After 30min, the acetone was discarded and dried in the air. The primary antibody Synagis (1mg / mL) was diluted 1:1000, 50μL / well, and incubated at 37℃ for 1h. Subsequently, the supernatant was discarded, the cells were washed 3 times with PBS, and Goat anti-human IgG-FITC (1:400 dilution, 50μL / well, purchased from Southern biotech) was added and incubated at 37℃ for 1h. The cells were washed 3 times with PBS and the proportion of fluorescent foci was observed under a fluorescence microscope.

[0110] The experimental results are shown in Figure 5: cells were negative and had no fluorescence, RSV (LONG strain): virus-positive fluorescent foci were 80%-95%. After the second immunization with 10 μg / mL FM-mRNA-2, the level of neutralizing antibodies was significantly higher than that with 10 μg / mL DS-Cav1 protein.

[0111] Example 9: Evaluation of protection against infection in mice immunized with mRNA vaccines

[0112] After grinding lung tissue, the supernatant was collected for lung viral load analysis (qPCR). Hair loss in immunized mice was observed and recorded during the experiment. RNA was extracted according to the instructions of the Viral RNA Kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) and subsequently tested.

[0113] The experimental results are shown in Figure 6. Different doses of FM-mRNA-2, DS-Cav1 mRNA, DS2 mRNA, and DS-Cav1 protein all protected animals from lethal RSV infection and reduced lung viral load after challenge. 10 μg / mL FM-mRNA-2 reduced lung viral load more than 10 μg / mL DS-Cav1 protein after challenge.

[0114] The results of the above examples indicate that different doses of FM-mRNA-2, DS-Cav1 mRNA, DS2 mRNA, and DS-Cav1 protein all exhibited significant binding activity and induced specific humoral immunity (neutralizing antibodies) against RSV in animals. Each vaccine protected animals from lethal RSV infection and reduced lung viral load after challenge. The correlation between immunogenicity and protective efficacy across dose groups was: FM-mRNA-2 > DS-Cav1 protein > DS2 mRNA > DS-Cav1 mRNA.

[0115] Although only specific embodiments of the present invention have been described above, those skilled in the art will appreciate that these are merely illustrative and that the scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, and such changes or modifications are intended to fall within the 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, wherein 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, wherein 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, wherein 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, wherein 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, wherein 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, wherein 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, wherein 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, wherein: 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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