Respiratory syncytial virus vaccine and its preparation method and application

Abstract

The present disclosure relates to the technical field of gene drugs, and particularly relates to a nucleic acid molecule or a mixture of nucleic acid molecules encoding a human respiratory syncytial virus (hRSV) antigen, wherein the human respiratory syncytial virus (hRSV) antigen is a prefusion hRSV F protein.

Description

[0001] This application claims the priority of a Chinese patent application with an application date of May 19, 2023 (Application No.: 202310570847.2, Invention Title: Respiratory Syncytial Virus Vaccine and Its Preparation Method and Application), and part of the content of this Chinese patent application is incorporated into this application by reference in its entirety. Technical Field

[0002] The present disclosure relates to the field of gene drug technology, and in particular to a respiratory syncytial virus vaccine and its preparation method and application. Background Art

[0003] The statements herein only provide background information related to the present disclosure and do not necessarily constitute prior art.

[0004] Respiratory syncytial virus (RSV) is the most common pathogen of bronchiolitis and pneumonia in infants and young children, seriously endangering the health of infants and young children. After RSV infection, lasting immunity cannot be obtained, and therefore, repeated infections are very common. The incidence of RSV infection is global, and local outbreaks can occur, which has become a worldwide public health problem. However, there is currently no approved RSV vaccine.

[0005] The RSV genome is about 15 - 16 kb in size and encodes 11 proteins, including 8 structural proteins and 3 non - structural proteins (NS1, NS2, and M2 - 2). The structural proteins include 3 transmembrane surface proteins (G, F, SH), 2 matrix proteins (M and M2 - 1), and 3 nucleocapsid proteins (L, N, and P). The G protein mediates the binding of the virus to host cells, and the F protein mediates the fusion of the virus with the host cell membrane, enabling the virus to enter the cell. Both are crucial for virus replication and contain B - cell and T - cell epitopes, making them the most important viral antigen proteins that stimulate the body to produce humoral and cellular immunity. The coding region of the G protein has high variability and can be divided into A and B subtypes based on this variability. The neutralizing antibodies induced by the G protein are subtype - specific; the coding region of the F protein is highly conserved, and the amino acid sequences of the F proteins of A and B subtypes are at least 90% identical. Therefore, the neutralizing antibodies induced by the F protein can inhibit the infection of both A and B subtype RSVs. The structure of the F protein shows dynamic changes. First, it is transcribed and translated into a single inactive polypeptide (F0) in host cells; then it is first cleaved by host cell furin protease to generate a partially cleaved pre - fusogenic protein; subsequently, a second furin protease cleavage occurs, generating F2 and F1 subunits. The two subunits are linked by two disulfide covalent bonds to form a monomer, and then three monomers form a metastable functional pre - fusion F protein trimer; thereafter, it can undergo conformational rearrangement to form a thermodynamically stable post - fusion F protein without further processing. The time and cellular location of the two furin protease cleavages and conformational rearrangement are not fully understood, and the conditions inducing conformational rearrangement are also unclear. On the surface of the F protein, the antigenic epitopes related to neutralizing activity are mainly of types, among which epitopes I, II, III, and IV exist in both pre - fusion and post - fusion F proteins; V and are specific antigenic sites of pre - fusion F protein, and the post - fusion F protein does not have these two epitopes.

[0006] Epidemiological studies have shown that RSV neutralizing antibodies can prevent severe RSV - ALRI. The neutralizing activity of the monoclonal antibody against epitope is 10 - 100 times that of the monoclonal antibody against epitope II, and the neutralizing activity of the monoclonal antibody against epitope VIII is also very high. Therefore, most of the RSV neutralizing activity in serum is only against the antigenic sites of pre - fusion F protein. The inventors have recognized that inducing high - titer neutralizing antibodies is the main goal in developing RSV vaccines, and the pre - fusion F protein with specific antigenic epitopes has become the most popular RSV vaccine target. However, the pre - fusion F protein is essentially an unstable protein, and making various stability modifications to it without losing important antigenic epitopes is one of the difficulties in RSV vaccine research and development. SUMMARY OF THE INVENTION

[0007] The present disclosure provides a nucleic acid molecule or a mixture of nucleic acid molecules encoding a human respiratory syncytial virus (hRSV) antigen, wherein the human respiratory syncytial virus (hRSV) antigen is a prefusion hRSV F protein.

[0008] In some embodiments, the amino acid sequence of the prefusion hRSV F protein is as shown in Seq ID NO.30, Seq ID NO.44, Seq ID NO.46, Seq ID NO.48, or comprises an amino acid sequence having at least 80% identity with Seq ID NO.30, Seq ID NO.44, Seq ID NO.46, Seq ID NO.48, and can be, for example but not limited to, an amino acid sequence having at least 80%, 85%, 90%, 95% or 98% identity with Seq ID NO.30, Seq ID NO.44, Seq ID NO.46, Seq ID NO.48.

[0009] In some embodiments, the prefusion hRSV F protein comprises at least one of the S46G substitution, E92D substitution, P102A substitution, A149C substitution, L373R substitution, I379V substitution, M447V substitution, Y458C substitution, K465Q substitution, D486C substitution, D489C substitution.

[0010] In some embodiments, the prefusion hRSV F protein comprises the D486C substitution and the D489C substitution.

[0011] In some embodiments, the prefusion hRSV F protein comprises the S46G substitution and the E92D substitution.

[0012] In some embodiments, the prefusion hRSV F protein comprises the A149C substitution, the Y458C substitution and the K465Q substitution.

[0013] In some embodiments, the F2 sequence of the prefusion hRSV F protein is selected from amino acid sequences in which at least 1, 2, 3, 4, 5 or 6 amino acids are continuously or intermittently deleted from positions 104 to 109, or the RR1 sequence is selected from amino acid sequences in which at least 1, 2, 3, 4, 5, 6, 7, 8 amino acids are continuously or intermittently deleted from positions 137 to 144, or the RR2 sequence is selected from amino acid sequences in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 amino acids are continuously or intermittently deleted from positions 504 to 524.

[0014] In some embodiments, the F2 sequence of the pre-fusion hRSV F protein is selected from the amino acid sequences with 6 amino acids deleted at positions 104-109.

[0015] In some embodiments, the RR1 sequence of the pre-fusion hRSV F protein is selected from the amino acid sequences with 8 amino acids deleted at positions 137-144.

[0016] In some embodiments, the RR2 sequence of the pre-fusion hRSV F protein is selected from the amino acid sequences with consecutive deletions at positions 514-524, 504-524, and 510-519 in the amino acids at positions 504-524.

[0017] In some embodiments, the GS sequence of the pre-fusion hRSV F protein is selected from one of (GnS)m, (GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q; where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; o is an integer from 1 to 5; p is an integer from 1 to 40; q is an integer from 1 to 5.

[0018] In some embodiments, the GS sequence of the pre-fusion hRSV F protein is GS.

[0019] In some embodiments, the sequence of the multimerization element of the pre-fusion hRSV F protein can also be selected from one of SeqID NO.1-11, Seq ID NO.50.

[0020] In some embodiments, the sequence of the multimerization element of the pre-fusion hRSV F protein can also be selected from one of SeqID NO.3, Seq ID NO.50.

[0021] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein is as shown in Seq ID NO.28, SeqID NO.32, Seq ID NO.26 or Seq ID NO.27, or, comprises an amino acid sequence having at least 80% identity with Seq ID NO.28, Seq ID NO.32, Seq ID NO.26 or Seq ID NO.27, for example, can be but is not limited to an amino acid sequence having at least 80%, 85%, 90%, 95% or 98% identity with Seq ID NO.28, Seq ID NO.32, Seq ID NO.26 or Seq ID NO.27.

[0022] In some embodiments, the amino acid sequence of the prefusion hRSV F protein is as shown in Seq ID NO.28 or Seq ID NO.32, or comprises an amino acid sequence having at least 80% identity with Seq ID NO.28 or Seq ID NO.32, and can be, for example but not limited to, an amino acid sequence having at least 80%, 85%, 90%, 95% or 98% identity with Seq ID NO.28 or Seq ID NO.32.

[0023] In some embodiments, the amino acid sequence of the prefusion hRSV F protein is as shown in Seq ID NO.32, or comprises an amino acid sequence having at least 80% identity with Seq ID NO.32, and can be, for example but not limited to, an amino acid sequence having at least 80%, 85%, 90%, 95% or 98% identity with Seq ID NO.32.

[0024] In some embodiments, the nucleic acid molecule or nucleic acid molecule mixture encoding a human respiratory syncytial virus (hRSV) antigen comprises a DNA molecule and / or an RNA molecule.

[0025] In some embodiments, the DNA molecule comprises a linear DNA molecule and / or a circular DNA molecule. In some embodiments, the RNA molecule comprises mRNA or circular RNA.

[0026] In some embodiments, the mRNA molecule comprises an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) is as shown in Seq ID NO.31, Seq ID NO.36, Seq ID NO.38, Seq ID NO.40-43, Seq ID NO.45, Seq ID NO.47, Seq ID NO.49, or comprises a nucleotide sequence having at least 80% identity with Seq ID NO.31, Seq ID NO.36, Seq ID NO.38, Seq ID NO.40-43, Seq ID NO.45, Seq ID NO.47, Seq ID NO.49, and can be, for example but not limited to, a nucleotide sequence having at least 80%, 85%, 90%, 95% or 98% identity with Seq ID NO.31, Seq ID NO.36, Seq ID NO.38, Seq ID NO.40-43, Seq ID NO.45, Seq ID NO.47, Seq ID NO.49.

[0027] In some embodiments, the nucleotide sequence of the open reading frame (ORF) is as shown in Seq ID NO.25, Seq ID NO.29, Seq ID NO.31, Seq ID NO.23, Seq ID NO.24, Seq ID NO.33, or comprises a nucleotide sequence having at least 80% identity to Seq ID NO.25, Seq ID NO.29, Seq ID NO.31, Seq ID NO.23, Seq ID NO.24, Seq ID NO.33, and can be, for example but not limited to, a nucleotide sequence having at least 80%, 85%, 90%, 95% or 98% identity to Seq ID NO.25, Seq ID NO.29, Seq ID NO.31, Seq ID NO.23, Seq ID NO.24, Seq ID NO.33.

[0028] In some embodiments, the nucleic acid molecule can optimize the mRNA sequence by sequence optimization means to improve the characteristics related to the expression efficacy after in vivo administration: for example, to increase mRNA stability, increase the translation efficacy in the target tissue, reduce the number of truncated proteins expressed, improve the folding of the expressed protein or prevent its misfolding, reduce the toxicity of the expression product, reduce the cell death caused by the expression product, increase and / or reduce protein aggregation, and obtain an mRNA with improved characteristics. The purposes of sequence optimization also include: optimizing the formulation and delivery characteristics of nucleic acid-based therapeutic agents while maintaining structural and functional integrity; overcoming the expression threshold; increasing the expression rate; half-life and / or protein concentration; optimizing protein localization; and avoiding adverse biological responses such as immune responses and / or degradation pathways. Sequence optimization means include: (1) codon optimization according to the codon frequency in a specific organ and / or host organism to ensure proper folding and appropriate expression; (2) adjusting the G / C content to increase mRNA stability or reduce secondary structure; (3) minimizing tandem repeat codons or base runs that may damage gene construction or expression; (4) customizing transcription and translation control regions; (5) reducing or eliminating problematic secondary structures within the polynucleotide.

[0029] "Sequence identity" between two nucleotide sequences indicates the percentage of identical nucleotides between the sequences. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0030] The term "percent identity" or similar terms refers to the percentage of identical nucleotides or amino acids between the sequences to be compared under optimal alignment. This percentage is purely statistical, and the differences between the two sequences may (but not necessarily) be randomly distributed throughout the length of the sequences to be compared. The comparison of two sequences is usually carried out by comparing these sequences relative to a fragment or "comparison window" after optimal alignment to identify local regions of the corresponding sequences.

[0031] In some embodiments, the 5' end and / or 3' end of the nucleic acid molecule or nucleic acid molecule mixture has a protective modification group.

[0032] In some embodiments, the nucleic acid molecule or nucleic acid molecule mixture is an mRNA molecule, and the 5' end modification group of the mRNA molecule is selected from ARCA, m7G(5'')ppp(5'')(2''OMeA)pG, m7G(5'')ppp(5'')(2''OMeG)pG, m7(3''OMeG)(5'')ppp(5'')(2''OMeG)pG, m7(3''OMeG)(5'')ppp(5'')(2''OMeA)pG, mCAP, dmCAP, tmCAP or dmCAP.

[0033] In some embodiments, the 3' end protective modification group of the mRNA molecule is poly(A), and the length of the poly(A) is 50 - 200, preferably 80 - 200.

[0034] In some embodiments, the mRNA molecule further contains a 5'UTR; in some embodiments, the length of the 5'UTR is preferably 10 - 200 nucleotides, more preferably 15 - 100 nucleotides; in some embodiments, the nucleotide sequence of the 5'UTR is as shown in Seq ID NO.12 - 14.

[0035] In some embodiments, the mRNA fragment further contains a 3'UTR; in some embodiments, the 3'UTR sequence is as shown in Seq ID NO.15 - 17.

[0036] In some embodiments, the mRNA comprises, in order from the 5' end to the 3' end, a 5' cap, a 5'UTR, an ORF, a 3'UTR and a 3' poly(A) tail.

[0037] In some embodiments, based on the provided RNA sequence, one of ordinary skill in the art will be able to obtain the corresponding DNA sequence (e.g., uracil is converted to thymine). Similarly, based on the provided DNA sequence, one of ordinary skill in the art will obtain the corresponding RNA sequence (e.g., thymine is converted to uracil). In some embodiments, based on the provided RNA or DNA sequence, one of ordinary skill in the art will be able to obtain the corresponding amino acid sequence.

[0038] In some embodiments, one or more uridines in the mRNA are replaced with modified nucleosides. In some embodiments, the modified nucleoside that replaces uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0039] In some embodiments, the present disclosure provides a nucleic acid molecule vaccine, which includes the above nucleic acid molecule and / or nucleic acid molecule mixture, and is complexed with an adapted carrier to obtain a vaccine that can be used clinically; preferably, the carrier includes liposome nanoparticles.

[0040] In some embodiments, the mass ratio of mRNA to liposome nanoparticles in the nucleic acid molecule vaccine is selected from 5:1 to 40:1, 8:1 to 40:1, 10:1 to 30:1, 15:1 to 30:1, 10:1 to 25:1, 5:1 to 25:1, 12:1 to 18:1, 14:1 to 17:1, 15:1 to 16:1.

[0041] The present disclosure also provides a preparation method of the above nucleic acid molecule vaccine. By mixing the aqueous phase containing the above nucleic acid molecule with the organic phase containing the carrier components, a nucleic acid molecule vaccine is obtained. This method is simple and easy to operate and is suitable for industrial promotion.

[0042] In some embodiments, the carrier includes liposome nanoparticles.

[0043] In some embodiments, the liposome nanoparticles include 20% - 50% of cationic lipid by mole percentage, for example, but not limited to 20%, 25%, 30%, 35%, 40%, 45% or 50%; 20% - 50% of DOPG, for example, but not limited to 20%, 25%, 30%, 35%, 40%, 45% or 50%; 5% - 20% of cholesterol, for example, but not limited to 5%, 10%, 15% or 20%; and 1% - 5% of PEG-DMG, for example, but not limited to 1%, 2%, 3%, 4% or 5%.

[0044] In some embodiments, the liposome nanoparticles comprise 20% to 50% of cationic lipid by mole percentage, for example, but not limited to, 20%, 25%, 30%, 35%, 40%, 45% or 50%; 20% to 50% of DSCP, for example, but not limited to, 20%, 25%, 30%, 35%, 40%, 45% or 50%; 5% to 20% of cholesterol, for example, but not limited to, 5%, 10%, 15% or 20%; and 1% to 5% of PEG-DMG, for example, but not limited to, 1%, 2%, 3%, 4% or 5%.

[0045] In some embodiments, the liposome nanoparticles comprise 50% of Dlin-MC3-DMA, 10% of DOPG, 38.5% of cholesterol and 1.5% of PEG-DMG by mole percentage.

[0046] In some embodiments, the liposome nanoparticles comprise 50% of Dlin-MC3-DMA, 10% of DSCP, 38.5% of cholesterol and 1.5% of PEG-DMG by mole percentage.

[0047] The present disclosure provides a method for preparing the nucleic acid molecule vaccine according to any one of the foregoing embodiments. The nucleic acid molecule is dissolved in a buffer to obtain an aqueous phase, and each lipid component of the liposome nanoparticles is measured and dissolved in an organic solvent to obtain an organic phase. After mixing the aqueous phase and the organic phase, the organic phase is removed to obtain the nucleic acid molecule vaccine.

[0048] In some embodiments, the volume ratio of the aqueous phase to the organic phase is 1:2 to 4, preferably 1:3. In some embodiments, the buffer comprises a citrate buffer or sodium acetate, preferably a citrate buffer. In some embodiments, the pH of the buffer is 3 to 7, preferably 4. In some embodiments, the concentration of the nucleic acid molecule in the aqueous phase is 0.05 mg / mL to 0.5 mg / mL, preferably 0.1 mg / mL. In some embodiments, the organic solvent is selected from C1-C4 lower alcohols, preferably absolute ethanol. In some embodiments, the concentration of the lipid component in the organic phase is 5 mg / mL to 7 mg / mL, preferably 6 mg / mL. In some embodiments, a microfluidic device is used to mix the aqueous phase and the organic phase, and the organic solvent is filtered by tangential flow filtration. Preferably, the flow rate of the microfluidic device is > 3 ml / min, more preferably 12 mL / min.

[0049] In some embodiments, a concentration step is further included after mixing. The concentration step makes the final concentration of the nucleic acid molecule 50 μg / mL to 200 μg / mL, preferably 100 μg / mL.

[0050] In some embodiments, the diameter of the lipid nanoparticles is less than about 200 nm. In some embodiments, the diameter of the lipid nanoparticles is less than about 150 nm. In some embodiments, the diameter of the lipid nanoparticles is less than 100 nm. In some embodiments, the diameter of the lipid nanoparticles is from about 55 nm to about 90 nm. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present disclosure. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0052] Figure 1 A schematic diagram showing the wild-type hRSVF protein is shown.

[0053] Figure 2 A schematic diagram showing the variant hRSVF protein is shown.

[0054] Figure 3 Showing the RSVF protein-specific IgG antibody in the mouse serum detected by ELISA in Example 5.

[0055] Figure 4 Showing the TNF-α+CD4+ and TNF-α+CD8+ T cells detected by flow cytometry in Example 5.

[0056] Figure 5 Showing the IFN-γ and IL-4 in the cell culture supernatant detected by ELISA in Example 5.

[0057] Figure 6 Showing the RSV neutralizing antibody titer in the mouse serum in Example 5.

[0058] Figure 7 Showing the RSVF protein-specific IgG antibody in the mouse serum detected by ELISA in Example 6.

[0059] Figure 8 Showing the IFN-γ and IL-4 in the cell culture supernatant detected by ELISA in Example 6.

[0060] Figure 9 Showing the TNF-α+CD4+ and TNF-α+CD8+ T cells detected by flow cytometry in Example 6.

[0061] Figure 10 Showing the RSV neutralizing antibody titer in the mouse serum in Example 6.

[0062] Figure 11 Shows the RSV neutralizing antibody titer in the mouse serum of Example 7.

[0063] Figure 12 Shows the RSV neutralizing antibody titer in the mouse serum of Example 8.

[0064] Figure 13 Shows the RSV neutralizing antibody titer in the mouse serum of Example 9.

[0065] Figure 14 Shows the RSV neutralizing antibody titer in the mouse serum of Example 10. Detailed implementation mode

[0066] Figure 1 Shows a schematic diagram of the wild-type hRSVF protein; Figure 2 Shows a schematic diagram of the variant hRSVF protein.

[0067] For furin cleavage; SP is the signal peptide sequence; p27 is the 27aa peptide sequence removed after cleavage; RR1 is the repeat folding region 1, spanning amino acid residues 137 to 216 of the hRSV F protein (wild type), including the fusion peptide and heptapeptide repeat sequence A (HRA); RR2 is the repeat folding region 2, which forms the C-terminal stem in the pre-fusion hRSV F protein spike, including the heptapeptide repeat sequence B (HRB) repositioned to the other side of the head of the RSV F glycoprotein; TM is the transmembrane region sequence.

[0068] F2 usually contains amino acid residues 26 to 109 of the F0 precursor; F1 usually contains amino acid residues 137 to 574 of the F0 precursor; F1 and F2 are linked by disulfide bonds to form a heterodimer, which is called the RSV F "protomer".

[0069] The C-terminus can be deleted or selected from at least one of the TM transmembrane sequence and the multimerization element sequence. In some embodiments, the C-terminus of the hRSVF protein variant is deleted. In some embodiments, the C-terminus of the hRSVF protein variant is the TM transmembrane sequence. In some embodiments, the C-terminus of the hRSVF protein variant is the TM transmembrane sequence and the multimerization element sequence connected in sequence, where the C-terminus of the TM transmembrane sequence is connected to the N-terminus of the multimerization element sequence. In some embodiments, the C-terminus of the hRSVF protein variant is the multimerization element sequence.

[0070] The hRSV F protein variant described in the present disclosure is an artificially mutated and modified F protein. The mutations and modifications (including but not limited to amino acid residue substitution, amino acid residue insertion and / or addition, amino acid residue deletion, and covalent modification, etc.) are obtained by mutating and modifying the wild type to conform to the amino acid sequence of the hRSV F protein. In the present disclosure, "hRSV F protein variant", "hRSV F protein in the present disclosure" or "hRSV F protein" all refer to "hRSV F protein variant", while "hRSV F protein wild type" or "hRSV F protein (wild type)" all refer to the wild type hRSV F protein without artificial modification or mutation. Additionally, unless otherwise specified, in the present disclosure, except for the background art section, "hRSV F protein" or "F protein" in other parts all refer to "hRSV F protein variant".

[0071] The positions of the amino acid sequences described in the present disclosure, such as S46G substitution, E92D substitution, P102A substitution, A149C substitution, L373R substitution, I379V substitution, M447V substitution, Y458C substitution, K465Q substitution, D486C substitution, D489C substitution are located based on the full-length amino acid sequence of the hRSV F protein wild type shown in Figure 1 For example, the S46G substitution is based on the hRSV F protein wild type, and the Figure 1 S amino acid residue at the 46th position of the full-length amino acid of the hRSV F protein wild type shown is replaced with a G amino acid residue, and this mutation occurs in the F2 sequence.

[0072] The multimerization elements described in the present disclosure include dimerization elements, trimerization elements, tetramerization elements, and oligomerization elements. When used in combination with the hRSV F protein in the present disclosure, they can lead to the formation of multimeric hRSV F protein complexes. The multimerization elements can be located at the N-terminus or C-terminus of the hRSV F protein, and at the nucleic acid level, their coding sequences are usually placed in the 5' or 3' frame of the coding sequence.

[0073] Among them, the dimerization element can be selected from, for example, the dimerization element / domain of heat shock protein, the immunoglobulin Fc domain, and leucine zipper (the dimerization domain of basic region leucine zipper-like transcription factors). The specific amino acid sequence is shown in Seq ID NO.1 for reference. The trimerization and tetramerization elements can be selected from, for example, engineered leucine zipper (engineered -helical coiled peptides in a parallel trimer state), the fibritin folding domain of bacteriophage T4 of Escherichia coli, GCN4PLL, CCN4-PLI, p53, GCN4. The specific amino acid sequences are shown in Seq ID NO.2 - 9, Seq ID NO.50 (for trimerization) and Seq ID NO.10 - 11 (for tetramerization)) for reference.

[0074] In some embodiments, the nucleic acid molecule or nucleic acid molecule mixture described in the present disclosure is selected from mRNA. The mRNA consists of a sequence including a 5' cap, 5' UTR, ORF, 3' UTR, and 3' poly(A) tail in sequence from the 5' end to the 3' end.

[0075] The 5′ untranslated region (UTR) described in the present disclosure refers to the sequence of mRNA that does not encode a polypeptide and is located immediately upstream of the start codon. When an RNA transcript is produced, the 5′ UTR may contain a promoter sequence. Such promoter sequences are known in the art. The RNA sequence of the 5′ UTR is shown as one of Seq ID NO.12, Seq ID NO.13, or Seq ID NO.14, or alternatively, the 5' UTR sequence disclosed prior to the filing date of the present application may be introduced into the present disclosure.

[0076] The 3′ untranslated region (UTR) described in the present disclosure refers to the sequence of mRNA that does not encode a polypeptide and is located downstream of the stop codon. The RNA sequence of the 3′ UTR is shown as one of Seq ID NO.15, Seq ID NO.16, or Seq ID NO.17, or alternatively, the 3' UTR sequence disclosed prior to the filing date of the present application may be introduced into the present disclosure.

[0077] The poly(A) tail described in the present disclosure is the sequence of mRNA located downstream of the 3′ UTR containing multiple consecutive adenosine monophosphates. The poly(A) tail may contain 10 to 300 adenosine monophosphates. In cells and / or in vivo, the poly(A) tail is used to protect mRNA from enzymatic degradation, and helps with transcription termination, and / or mRNA export from the nucleus and translation.

[0078] In an alternative embodiment, the nucleic acid molecule vaccine described in the present disclosure may further comprise at least one cryoprotectant. In an alternative embodiment, the cryoprotectant is selected from at least one of sucrose and glycerol. In an alternative embodiment, the cryoprotectant of the nucleic acid molecule vaccine is selected from one of 5% (w / v) - 18% (w / v) sucrose, 6% (w / v) - 16% (w / v) sucrose, 7% (w / v) - 14% (w / v) sucrose, 7% (w / v) - 12% (w / v) sucrose, 8% (w / v) - 11% (w / v) sucrose. In an alternative embodiment, the cryoprotectant of the nucleic acid molecule vaccine is selected from one of 1% (w / v) - 9% (w / v) glycerol, 1.5% (w / v) - 7% (w / v) glycerol, 1.75% (w / v) - 6% (w / v) glycerol, 1% (w / v) - 6% (w / v) glycerol, 3% (w / v) - 6% (w / v) glycerol. In an alternative embodiment, the cryoprotectant of the nucleic acid molecule vaccine is selected from one of the combinations of 5% (w / v) - 18% (w / v) sucrose and 1% (w / v) - 9% (w / v) glycerol, 6% (w / v) - 16% (w / v) sucrose and 1.5% (w / v) - 7% glycerol, 7% (w / v) - 14% (w / v) sucrose and 1.75% (w / v) - 6% (w / v) glycerol, 7% (w / v) - 12% (w / v) sucrose and 1% (w / v) - 6% (w / v) glycerol, 8% (w / v) - 11% (w / v) sucrose and 3% (w / v) - 6% (w / v) glycerol.

[0079] The nucleic acid molecule vaccine described in the present disclosure can be administered by the following, but not limited to the following routes: intramuscular injection, subcutaneous injection, oral inhalation, intranasal drip, nasal spray, oral-nasal inhalation.

[0080] The nucleic acid molecule vaccine described herein can be formulated into the following, but not limited to the following dosage forms: injection, aerosol, dry powder inhaler / powder aerosol, spray, and / or nebulized inhalation solution.

[0081] The aerosol, dry powder inhaler / powder aerosol, spray, and / or nebulized inhalation solution of the present disclosure generally contains the drug to be delivered, optionally formulated together with a surfactant, such as a nonionic surfactant (e.g., polysorbate - 80), and one or more buffering agents, provided that the inclusion of the surfactant does not disrupt the structure of the lipid formulation. In an alternative embodiment, the aerosol, dry powder inhaler / powder aerosol, spray, and / or nebulized inhalation solution further contains a propellant. The pH value of the aerosol, spray, and / or nebulized inhalation solution is 6.8 - 7.2. The drug solvent used can also be a slightly acidic aqueous buffer with a pH value of 4 - 6. Other components can be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases.

[0082] In order to formulate the nucleic acid molecule vaccine of the present disclosure for oral inhalation administration, intranasal administration, nasal spray administration, and oral-nasal inhalation administration, the nucleic acid molecule vaccine comprising mRNA and LNP can be combined with various pharmaceutically acceptable additives. The additives are selected from one or more of pH control agents, local anesthetics, adsorption inhibitors, isosmotic agents, solubility enhancers, stabilizers, reducing agents, etc. The pH control agent is selected from one or more of arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid. The local anesthetic is selected from benzyl alcohol). The isosmotic agent is selected from one or more of sodium chloride, mannitol, sorbitol. The adsorption inhibitor is selected from Tween 80. The solubility enhancer is selected from one or more of cyclodextrin and its derivatives. The stabilizer is selected from serum albumin. The reducing agent is selected from glutathione.

[0083] The nucleic acid molecule vaccine comprising mRNA and LNP can be dispersed in an alkali or a vehicle, and the alkali or vehicle may contain a hydrophilic compound having the ability to disperse the nucleic acid molecule vaccine and any desired additives. The alkali can be selected from a wide range of suitable carriers, including but not limited to polycarboxylic acids or their salts, copolymers of carboxylic anhydrides (selected from maleic anhydride) and other monomers selected from (meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid and their non-toxic metal salts. Generally, biodegradable polymers are selected as the alkali or vehicle, for example, polylactic acid, poly(lactic-glycolic acid) copolymer, polyhydroxybutyrate, poly(hydroxybutyrate-glycolic acid) copolymer, and mixtures thereof. Optionally or additionally, synthetic fatty acid esters such as polyglycerol fatty acid esters, sucrose fatty acid esters, etc. can be used as the vehicle. The hydrophilic polymer and other carriers can be used alone or in combination, and the carrier can be given enhanced structural integrity by partial crystallization, ionic bonding, crosslinking, etc. The vehicle can be provided in various forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and membranes for direct application to the nasal mucosa. The use of the selected vehicle in this case can result in the promotion of the absorption of the nucleic acid molecule vaccine.

[0084] The nucleic acid molecule vaccine of the present disclosure can alternatively contain pharmaceutically acceptable carrier substances required for near-physiological conditions, such as pH regulators and buffers, tonicity regulators and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, dehydrated sorbitol monolaurate, triethanolamine oleate, and mixtures thereof. For solid nucleic acid molecule vaccines, conventional non-toxic pharmaceutically acceptable carriers can be used, which include, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc.

[0085] In an alternative embodiment, the nucleic acid molecule vaccine of the present disclosure can be atomized or otherwise delivered as a particulate liquid or solid before or after administration to a subject. The solid or liquid particulate nucleic acid molecule vaccine for administration is an atomized aqueous solution or suspension loaded in at least one suitable device that is prone to generating particles that can be breathed or inhaled by the subject. In an alternative embodiment, the device is selected from a metered-dose inhaler, a jet nebulizer, an ultrasonic nebulizer, a dry powder inhaler, a propellant-based inhaler, or a blow-in device.

[0086] Example 1

[0087] A lipid nanoparticle comprising RNA encoding a respiratory syncytial virus antigen, wherein the lipid nanoparticle comprises 50% Dlin-MC3-DMA, 20% DOPG, 29% cholesterol, and 1% PEG-DMG by mole percentage.

[0088] The preparation method is as follows:

[0089] (a) Dissolve the RNA in a citrate buffer at pH 4 and adjust the concentration to 0.1 mg / ml to obtain an aqueous phase.

[0090] (b) Dissolve Dlin-MC3-DMA, DOPG, cholesterol, and PEG-DMG in anhydrous ethanol according to the formula amount and adjust the concentration of the lipid components in the organic phase to 6 mg / mL to obtain an organic phase.

[0091] (c) Mix the aqueous phase of step (a) and the organic phase of step (b) at a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 mL / min. Immediately dilute the mixture 100-fold with a PBS solution at pH 7.4 and use tangential flow filtration (TFF) to remove the ethanol component in the solution, and then concentrate to a concentration of 55 μg / ml of mRNA in the system to obtain a lipid nanoparticle comprising RNA encoding a respiratory syncytial virus antigen.

[0092] Example 2

[0093] Using luciferase as a reporter gene, the efficiency of delivering mRNA encoding the luciferase gene in mice was studied by in vivo fluorescence imaging technology for different vaccine carrier formulations (as shown in Table 1 below, "MC3" refers to Dlin-MC3-DMA, "+": luciferase expression in mice was detected by a small animal in vivo fluorescence imaging system after administration), and the physicochemical indexes of different composite preparations (the preparation method refers to Example 1) were detected.

[0094] Table 1

[0095]

[0096] The results are shown in Table 1. Through research, it was found that increasing the lipid-to-mRNA mass ratio is beneficial to increasing the encapsulation efficiency of mRNA in lipid nanoparticles, thereby making it more stable. In addition, moderately increasing the content of polyethylene glycol (PEG) in the formulation is beneficial to improving the in vivo expression efficiency of mRNA. Therefore, considering factors such as the mRNA encapsulation efficiency and the in vivo delivery efficiency of mRNA, Formulations 3 and 4 were selected for the subsequent research on mRNA vaccines.

[0097] Example 3

[0098] The ability of cationic lipid nanoparticles with different formulations to encapsulate mRNA encoding the full-length S protein and the particle size data of the formed nanoparticles are shown in Table 2. All formulations can compress the S protein mRNA into nanoparticles with a particle size below 100 nm and a net neutral surface potential, and can encapsulate at least 50% of the mRNA, so they can all have a certain in vivo delivery effect. "MC3" refers to Dlin-MC3-DMA.

[0099] Table 2

[0100]

[0101] Example 4

[0102] BALB / c mice (female, 5-6 weeks old, average body weight 20-25 g, purchased from Zhuhai Besttone Biotechnology Co., Ltd.) were selected for the evaluation of vaccine immunogenicity. The experimental animals were randomly divided into groups of 5 mice each. The immunization dose was 15 μg / mouse. A booster immunization with the same dose was given 2 weeks after the vaccine inoculation. Twelve days after the second immunization, the cellular immune response and humoral immune response indexes were detected.

[0103] The lymphocytes of the mice were isolated according to the following method: The mice were sacrificed by cervical dislocation and soaked in 75% ethanol; the spleens of the mice were taken out in a biosafety cabinet; 4 mL - 5 mL of mouse lymphocyte separation medium (restored to room temperature and shaken well before use) was placed in a 35 mm culture dish; ground, and the separation medium containing spleen cells was immediately transferred to a 15 mL centrifuge tube, and centrifuged at 800 g for 30 min at room temperature with a horizontal rotor. After centrifugation, the lymphocyte layer was aspirated, and then 10 mL of RPMI 1640 medium was added and washed by inversion. The cells were collected by centrifugation at room temperature.

[0104] 1. Detection of RSV F protein-specific IgG antibodies in mouse serum by ELISA

[0105] The hRSVF protein (His Tag) (2 mg / mL) diluted to 2 μg / mL was added to the multi-well plate (100 μL per well) with washing solution and coated overnight. Washed, and then a 2% BSA solution was added for blocking.

[0106] Dilute all serum samples to be tested with washing solution starting from 1:100 in 10-fold gradients until 1:1,000,000. The negative control sample is diluted in the same multiples as the serum samples to be tested, resulting in a total of 6 gradient samples.

[0107] Add the prepared serum sample solution to the enzyme-labeled multi-well plate, incubate and then wash. Add the diluted enzyme-labeled antibody solution, incubate and then wash. Perform color development, and measure the OD value at 450nm / 630nm on an enzyme-labeled instrument.

[0108] Take 2.1 times the arithmetic mean of the signals of the negative serum samples as the Cutoff value.

[0109] 2. Flow cytometry detection of TNF-α+CD4+ and TNF-α+CD8+ T cells

[0110] Take the isolated mouse lymphocytes, add peptide library stimulation, and place them in a 37°C, 5% CO2 incubator for 72h. Stimulate overnight with BD GolgiPlμgTM (containing brefeldin A) protein transport inhibitor (containing BSA) solution. Centrifuge the mouse lymphocyte suspension to obtain a precipitate.

[0111] Resuspend the mouse lymphocyte precipitate, add specific monoclonal fluorescent antibodies such as CD4, CD8, etc., incubate and centrifuge to obtain a mouse lymphocyte precipitate.

[0112] Add fixation / permeabilization solution, incubate and centrifuge to obtain a mouse lymphocyte precipitate. Add buffer to the mouse lymphocyte precipitate, mix well and centrifuge to obtain a mouse lymphocyte precipitate.

[0113] Resuspend the mouse lymphocyte precipitate with staining buffer, add cytokine fluorescent antibodies such as IFN-γ, TNF-α, etc., incubate in the dark and centrifuge to obtain a mouse lymphocyte precipitate. Then resuspend the mouse lymphocyte precipitate with staining buffer and detect it on a flow cytometer.

[0114] The expression of TNF-α in CD3+ / CD4+ and CD3+ / CD8+ T lymphocytes was detected by flow cytometry.

[0115] 3. ELISA detection of IFN-γ and IL-4 in the supernatant of cell culture medium

[0116] Take the isolated mouse lymphocytes, add peptide library stimulation, and place them in a 37°C, 5% CO2 incubator for 72h. Centrifuge the mouse lymphocyte suspension to obtain the supernatant.

[0117] The contents of IFN-γ and IL-4 in the supernatant were detected using a mouse IL-4 ELISA kit (purchased from Xinbosheng Biotechnology Co., Ltd.) and a mouse IFN-γ ELISA kit (purchased from Xinbosheng Biotechnology Co., Ltd.).

[0118] 4. Neutralizing activity of serum

[0119] The neutralizing activity of the serum of the immunized group was detected as follows: (1) Digest HEp-2 cells, adjust the cell density, inoculate into a 96-well plate, and culture overnight. (2) Add each mouse serum dilution and virus dilution to the positive wells of the multi-well plate, add the virus dilution to the negative wells, and then add the diluted virus to the positive and negative wells of the multi-well plate for neutralization culture. (3) Add the neutralization products in the positive and negative wells of the multi-well plate to the multi-well plate containing digested HEp-2 cells respectively, and culture; aspirate the neutralization products, and add DMEM + 2% FBS medium to each well for continued culture. (4) Discard the supernatant of the cells, add 4% paraformaldehyde to each well to fix the cells, wash with PBS washing solution, and block the cells with a 1:1 mixed solution of 4% BSA and 0.2% triton. Discard the blocking solution, add F6-6-88 antibody dilution, incubate and wash; spin dry the multi-well plate and use the CTL device to read the values.

[0120] Example 5

[0121] The nucleotide sequences described in Table 3 are mRNA sequences. The mRNA sequences described in Table 3 were respectively prepared into lipid nanoparticles containing RNA encoding respiratory syncytial virus antigen according to the method described in Example 1.

[0122] Table 3

[0123]

[0124] In addition to the reading frame sequences in the above table, the RNA vaccine encoding respiratory syncytial virus antigen also includes a 5' cap, 5' UTR (as shown in Seq ID NO.12), 3' UTR (as shown in Seq ID NO.15), and a 3' tail of 100 polyA.

[0125] Sample Y1 is a lipid nanoparticle containing RNA encoding the wild-type amino acid sequence (Wt) of RSV F glycoprotein; the amino acid sequence encoded by sample Y2 is based on the wild-type of RSV F glycoprotein, with the TM sequence removed and Fibritin Dominain added at the C-terminus (the amino acid sequence is shown in Seq ID NO.3); the amino acid sequence encoded by Y3 has mutations at the S155C / S190F / V207L / S290C sites, with the TM sequence removed and Fibritin Dominain added at the C-terminus (the amino acid sequence is shown in Seq ID NO.3); the amino acid sequence encoded by Y4 has mutations at the N67I / S215P sites, the P27 sequence is deleted, a GS linker sequence is added in front of the terminal amino acid Arg of the F2 sequence, the TM sequence is removed, and Fibritin Dominain is added at the C-terminus (the amino acid sequence is shown in Seq ID NO.3); the amino acid sequence encoded by Y5 has mutations at the S46G / E92D / A149C / S155C / S190F / V207L / S215P / S290C / Y458C / K465Q sites, the P27 sequence is deleted, GS linker sequences are added in the F2 sequence and the RR1 sequence, the TM sequence is removed, and Fibritin Dominain is added at the C-terminus (the amino acid sequence is shown in Seq ID NO.3).

[0126] The vaccine immunogenicity of the prepared lipid nanoparticles encoding the RNA of respiratory syncytial virus antigen was evaluated according to the method of Example 4.

[0127] 1. Detection of RSV F protein-specific IgG antibodies in mouse serum by ELISA

[0128] The experimental results are shown in Fig. 3A - 3E. Fig. 3A - 3E are the OD450 values measured by indirect ELISA after the serum was diluted from 100 - fold to 1,000,000 - fold. If the sample OD450 ≥ 2 times the average OD450 of the negative control at the minimum dilution factor (1:100), it is determined to be positive. The dotted lines in Fig. 3A - 3E are the cutoff lines. Among them, in the Y1 immunized group, positive values did not appear until the serum was diluted to 1,000,000 - fold. Fig. 3F is the determination of the serum IgG antibody titer. The results show that the IgG antibody titers in the RSV F glycoprotein variant mRNA vaccine immunized groups are all significantly higher than those in the PBS control group, and the Y1 immunized group (the mRNA vaccine immunized group encoding the wild - type RSV F glycoprotein) is significantly higher than other immunized groups.

[0129] 2. Detection of TNF - α+CD4+ and TNF - α+CD8+ T cells by flow cytometry

[0130] Except for the Y5 immune group, the other immune groups were significantly increased compared with the PBS control group. The results showed that the RSVF glycoprotein variant mRNA vaccine could stimulate the Th1-type cellular immune response in mice.

[0131] 3. Detection of IFN-γ and IL-4 in the supernatant of cell culture medium by ELISA

[0132] IFN-γ in the RSV F glycoprotein variant mRNA vaccine immune group was significantly increased compared with the PBS control group (P<0.01) (Th1-type cellular immune response), while IL-4 in the RSV F mutant mRNA vaccine immune group was also significantly increased compared with the PBS control group (P<0.01).

[0133] 4. Neutralizing activity of serum

[0134] The results of RSV neutralizing antibody titers in mouse sera are shown in the figure. Compared with the PBS control group, the RSV neutralizing antibody titers in all RSVF glycoprotein variant mRNA immune groups were significantly increased. The neutralizing antibody titers in the Y2, Y4, and Y6 mRNA immune groups encoding the RSV F glycoprotein variant were relatively high.

[0135] Example 6

[0136] The nucleotide sequences described in Table 4 are mRNA sequences. The mRNA sequences described in Table 4 were respectively prepared into lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens according to the method described in Example 2.

[0137] In addition to the reading frame sequences in the above table, the RNA vaccine encoding respiratory syncytial virus antigens also includes a 5' cap, 5' UTR (as shown in Seq ID NO.12), 3' UTR (as shown in Seq ID NO.15), and a 3' tail of 100 polyA.

[0138] Table 4

[0139]

[0140] The vaccine immunogenicity of the prepared lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens was evaluated according to the method of Example 4.

[0141] (1) Detection of specific IgG of RSV F protein in serum by ELISA

[0142] Figs. 7A - 7D show the OD450 values measured by indirect ELISA after diluting the serum from 100 - fold to 1,000,000 - fold. If the sample OD450 ≥ 2 times the average OD450 of the negative control at the minimum dilution (1:100), it is determined to be positive. The dotted lines in Figs. 7A - 7D are the cutoff lines. Among them, in the Y6 immunized group, no positive value appeared in the sample until it was diluted to 10,000 - fold. Fig. 7E shows the determination of the serum IgG antibody titer.

[0143] The results showed that the IgG antibody titers in the RSV F glycoprotein variant mRNA vaccine immunized groups were all significantly higher than those in the PBS control group.

[0144] (2) ELISA detection of IFN - γ and IL - 4 in the cell culture supernatant

[0145] The IFN - γ in the RSV F glycoprotein variant mRNA vaccine immunized group was significantly increased compared with the PBS control group (P < 0.01) (Th1 - type cellular immune response); the IL - 4 in the RSV F glycoprotein variant mRNA vaccine immunized group was also significantly increased compared with the PBS control group (P < 0.01).

[0146] (3) Flow cytometry detection of TNF - α+CD4+ and TNF - α+CD8+ T cells

[0147] After immunization for 12 days, mouse spleen lymphocytes were isolated, and the expression of TNF - α in CD3+ / CD4+ and CD3+ / CD8+ T lymphocytes was detected by flow cytometry. The RSV F glycoprotein variant immunized group had a significant increase compared with the PBS control group. The results showed that the RSV F glycoprotein variant mRNA vaccine could stimulate the Th1 - type cellular immune response in mice.

[0148] (4) RSV neutralizing antibody titer in serum

[0149] After the second immunization for 12 days, the RSV neutralizing antibody titer in mouse serum was detected by the RSV live virus plaque reduction assay. As shown in the figure, compared with the PBS control group, the RSV neutralizing antibody titers in all RSV F glycoprotein variant mRNA immunized groups were significantly increased.

[0150] Example 7

[0151] The C-terminal propeptide of the respiratory syncytial virus antigen hRSVF protein variant was replaced with the sequences shown in amino acid sequences such as SeqID NO.4 and Seq ID NO.6 respectively, to obtain respiratory syncytial virus antigen hRSVF protein variants with amino acid sequences such as Seq ID NO.35 and Seq IDNO.37. The C-terminal propeptide sequence of the respiratory syncytial virus antigen hRSVF protein variant encoded by RNA in sample Y25 is the wild-type TM sequence.

[0152] The nucleotide sequences described in Table 5 are mRNA sequences, and the mRNA sequences described in Table 5 were respectively prepared into lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen according to the method described in Example 2.

[0153] Table 5

[0154]

[0155] In addition to the reading frame sequences in the above table, the RNA vaccine encoding the respiratory syncytial virus antigen also includes a 5' cap, 5' UTR (shown in Seq ID NO.12), 3' UTR (shown in Seq ID NO.15), and a 3' tail of 100 polyA.

[0156] The prepared lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen were evaluated for vaccine immunogenicity according to the method described in Example 4. The test results are shown in the figure.

[0157] In addition to the respiratory syncytial virus antigen hRSV F protein variants shown in Table 5 (amino acid sequences such as Seq IDNO.35, Seq ID NO.37, and Seq ID NO.39), the C-terminal propeptide of the present application was also replaced with the sequences shown in amino acid sequences such as Seq ID NO.7, Seq ID NO.8, Seq ID NO.9, and Seq ID NO.5 respectively, to obtain the amino acid sequences of new respiratory syncytial virus antigen hRSVF protein variants. Corresponding lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen were designed and prepared according to the new amino acid sequences, and the vaccine immunogenicity was evaluated according to the method of Example 4. The neutralization activity NT50 of the serum of the experimental mice was about 3000 - 3200.

[0158] For the respiratory syncytial virus antigen hRSVF protein variant with the sequence shown in Seq ID NO.32, the propeptide before its C-terminus was replaced with the sequences shown in Seq ID NO.4, Seq ID NO.5-9, and Seq ID NO.50 respectively, to obtain the amino acid sequences of the new respiratory syncytial virus antigen hRSVF protein variants. According to the new amino acid sequences, the corresponding RNA lipid nanoparticles encoding the respiratory syncytial virus antigen were designed and prepared, and the vaccine immunogenicity was evaluated according to the method of Example 4. The neutralizing activity NT50 of the sera of the experimental mice was about 4400-4900.

[0159] Example 8

[0160] The amino acid sequences of the RR2 region of the respiratory syncytial virus antigen hRSV F protein variant were mutated to obtain the sequences shown in Seq ID NO.44, Seq ID NO.46, and Seq ID NO.48. According to the new amino acid sequences, the corresponding RNA sequences encoding the respiratory syncytial virus antigen were designed. The sequence information is as follows: Sample Y6, with the amino acid sequence shown in SeqID NO.30 and the ORF sequence shown in Seq ID NO.31; Sample Y11, with the amino acid sequence shown in Seq ID NO.30 and the ORF sequence shown in Seq ID NO.41; Sample Y12, with the amino acid sequence shown in Seq ID NO.30 and the ORF sequence shown in SeqID NO.42; Sample Y13, with the amino acid sequence shown in Seq ID NO.30 and the ORF sequence shown in Seq ID NO.43; Sample Y14, with the amino acid sequence shown in Seq ID NO.44 and the ORF sequence shown in Seq ID NO.45; Sample Y15, with the amino acid sequence shown in Seq ID NO.46 and the ORF sequence shown in Seq ID NO.47; Sample Y16, with the amino acid sequence shown in Seq IDNO.48 and the ORF sequence shown in Seq ID NO.49.

[0161] According to the method described in Example 1, the mRNAs shown in Table 6 were prepared into lipid nanoparticles encoding the respiratory syncytial virus RNA vaccine. The sequences of these RNA vaccines also include a 5' cap, a 5' UTR (shown in Seq ID NO.12), a 3' UTR (shown in Seq ID NO.15), and a 3' tail with 100 polyAs.

[0162] The prepared lipid nanoparticles were evaluated for vaccine immunogenicity according to the method of Example 4. The experimental results are shown in the figure.

[0163] In addition to the respiratory syncytial virus antigen hRSVF protein variants shown in this example (amino acid sequences are shown in SeqID NO.30, Seq ID NO.44, Seq ID NO.46, and Seq ID NO.48), the amino acid sequence of the RR2 region in this application was mutated to obtain a new respiratory syncytial virus antigen hRSVF protein variant. According to the amino acid sequence, the corresponding RNA lipid nanoparticles encoding the respiratory syncytial virus antigen were designed and prepared, and the vaccine immunogenicity was evaluated according to the method of Example 4. The neutralization activity NT50 of the experimental mouse serum was about 3000-3200.

[0164] For the respiratory syncytial virus antigen hRSVF protein variant with the sequence shown in Seq ID NO.32, the amino acid sequence of the RR2 region in this application was also mutated (substituted with RR2 sequences similar to those shown in Seq ID NO.44, Seq ID NO.46, and SeqID NO.48), to obtain a new respiratory syncytial virus antigen hRSVF protein variant. According to the new amino acid sequence, the corresponding RNA lipid nanoparticles encoding the respiratory syncytial virus antigen were designed and prepared, and the vaccine immunogenicity was evaluated according to the method of Example 4. The neutralization activity NT50 of the experimental mouse serum was about 4600-4900.

[0165] Example 9

[0166] The nucleotide sequences described in Table 6 are mRNA sequences. The mRNA sequences described in Table 4 were respectively prepared into lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen according to the method described in Example 2. The prepared lipid nanoparticles were evaluated for vaccine immunogenicity according to the method of Example 4. The experimental results are shown in the figure.

[0167] Table 6

[0168] Serial number Name ORF nucleotide sequence 3’UTR 5’UTR 1 Y17 Seq ID NO.31 Seq ID NO.16 Seq ID NO.13 2 Y18 Seq ID NO.31 Seq ID NO.17 Seq ID NO.13 3 Y19 Seq ID NO.31 Seq ID NO.15 Seq ID NO.13 4 Y20 Seq ID NO.31 Seq ID NO.16 Seq ID NO.14 5 Y21 Seq ID NO.31 Seq ID NO.17 Seq ID NO.14 6 Y22 Seq ID NO.31 Seq ID NO.15 Seq ID NO.14 7 Y23 Seq ID NO.31 Seq ID NO.16 Seq ID NO.12 8 Y24 Seq ID NO.31 Seq ID NO.17 Seq ID NO.12 9 Y6 Seq ID NO.31 Seq ID NO.15 Seq ID NO.12

[0169] Example 10

[0170] Take the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen prepared from Y6, Y7, and Y10 in Example 5, and the syncytial virus F glycoprotein (purchased from ATCC), and evaluate the vaccine immunogenicity according to the method described in Example 4. The results are shown in the figure.

[0171] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A nucleic acid molecule or mixture of nucleic acid molecules encoding a human respiratory syncytial virus (hRSV) antigen, characterized in that, The human respiratory syncytial virus (hRSV) antigen is the prefusion hRSV F protein; the amino acid sequence of the prefusion hRSV F protein is as shown in Seq ID NO.30; the nucleic acid molecule is an RNA molecule; the RNA molecule is an mRNA.

2. The nucleic acid molecule or nucleic acid molecule mixture according to claim 1, characterized in that, The mRNA molecule includes an open reading frame, and the nucleotide sequence of the open reading frame is as shown in any one of Seq ID NO.31, Seq ID NO.41-43.

3. The nucleic acid molecule or mixture of nucleic acid molecules according to claim 2, wherein, The 5'-end and / or 3'-end of the nucleic acid molecule or nucleic acid molecule mixture has a protective modification group; the nucleic acid molecule or nucleic acid molecule mixture is an mRNA molecule, and the 5'-end modification group of the mRNA molecule is selected from ARCA, m7G(5'')ppp(5'')(2''OMeA)pG, m7G(5'')ppp(5'')(2''OMeG)pG, m7(3''OMeG)(5'')ppp(5'')(2''OMeG)pG, m7(3''OMeG)(5'')ppp(5'')(2''OMeA)pG, mCAP, dmCAP, tmCAP or dmCAP.

4. The nucleic acid molecule or nucleic acid molecule mixture according to claim 1, wherein The 3'-end protective modification group of the mRNA molecule is poly(A), and the length of the poly(A) is 50-200.

5. The nucleic acid molecule or mixture of nucleic acid molecules according to claim 1, characterized in that, The length of the poly(A) is 80-200.

6. The nucleic acid molecule or nucleic acid molecule mixture according to claim 1, wherein The mRNA molecule further contains a 5'UTR; the nucleotide sequence of the 5'UTR is as shown in any one of Seq ID NO.12-14.

7. The nucleic acid molecule or mixture of nucleic acid molecules according to claim 6, wherein, The mRNA fragment further contains a 3'UTR; the 3'UTR sequence is as shown in any one of Seq ID NO.15-17.

8. The nucleic acid molecule or mixture of nucleic acid molecules according to claim 6, characterized in that, One or more uridines in the mRNA molecule are replaced with modified nucleosides; the modified nucleosides for replacing the uridines are pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U).

9. A nucleic acid molecule vaccine, characterized in that, The nucleic acid molecule vaccine includes the nucleic acid molecule and / or nucleic acid molecule mixture as described in any one of claims 1-8, and is complexed with a suitable carrier to obtain a vaccine that can be used clinically.

10. The nucleic acid molecule vaccine according to claim 9, wherein, The carrier includes liposome nanoparticles.

11. The nucleic acid molecule vaccine according to claim 10, wherein The mass ratio of mRNA to liposome nanoparticles in the nucleic acid molecule vaccine is selected from 5:1 to 40:

1.

12. The method for preparing the nucleic acid molecule vaccine according to claim 11, wherein, The nucleic acid molecule vaccine is obtained by mixing an aqueous phase containing the nucleic acid molecule as described in any one of claims 1-8 with an organic phase containing carrier components.

13. The method for preparing the nucleic acid molecule vaccine according to claim 12, wherein The carrier includes liposome nanoparticles; The liposome nanoparticles include 20%-50% of cationic lipid, 20%-50% of DOPG, 5%-20% of cholesterol, and 1%-5% of PEG-DMG in terms of molar percentage.

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