Respiratory syncytial virus vaccine, and its manufacturing method and use

Abstract

This disclosure relates to an immunocomposition comprising or encoding a human respiratory syncytial virus antigen, selected from the group consisting of nucleic acid immunocompositions, polypeptide immunocompositions, or viral immunocompositions.

Description

Technical Field

[0001] This application claims priority to a Chinese patent application filed on May 19, 2023 (Application No.: 202310570847.2, Title of Invention: Respiratory Syncytial Virus Vaccine, and Its Manufacturing Method and Use), which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to the field of gene medicine technology, and particularly to respiratory syncytial virus vaccines, and their manufacturing methods and uses.

Background Art

[0003] The descriptions herein only provide background information related to this 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 seriously harms the health of infants. Since durable immunity cannot be obtained after RSV infection, reinfection is common. The incidence of RSV infection is global and can spread explosively locally, and it has already become a global public health problem. However, there is currently no approved RSV vaccine.

[0005] The RSV genome is approximately 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 core-shell proteins (L, N, and P). The G protein mediates the binding of the virus to the host cell, and the F protein mediates the fusion of the virus to the host cell membrane, allowing the virus to enter the cell. Both are extremely important for viral replication and all contain B cell and T cell epitopes, making them the most important viral antigen proteins that stimulate the body and bring about humoral and cellular immunity. The coding region of the G protein is highly variable, allowing for its classification into A and B subtypes. The neutralizing antibodies induced by the G protein exhibit subtype specificity. The coding region of the F protein is highly conserved, and at least 90% of the amino acid sequences of the F proteins of subtypes A and B are identical. Therefore, the neutralizing antibodies induced by the F protein can simultaneously suppress infection with both A and B subtype RSV. The structure of the F protein undergoes dynamic changes. First, it is transcribed and translated into a single inactive polypeptide (F0) in the host cell. Next, it undergoes a first cleavage by furin protease in the host cell, generating a partially cleaved prefusogenic protein. Subsequently, a second cleavage by furin protease occurs, generating F2 and F1 subunits. The two subunits are then bonded to a single monomer by two disulfide covalent bonds. The three monomers then form a metastable functional prefusion protein trimer. After that, it can undergo conformational rearrangement without requiring further processing to form a thermodynamically stable postfusion protein. The timing and cellular location of the two furin protease cleavages and conformational rearrangement are not yet fully understood, nor are the conditions that induce conformational rearrangement clear.On the surface of the F protein, there are mainly six types of antigenic epitopes associated with neutralizing activity: I, II, III, IV, V, V, and Φ. Of these, epitopes I, II, III, and IV are present in the F protein both before and after fusion, while V and Φ are specific antigenic sites on the pre-fusion F protein, and these two types of epitopes are absent on the post-fusion F protein. Epidemiological studies have shown that RSV neutralizing antibodies can prevent severe RSV-ALRI, and that the neutralizing activity of epitope Φ monoclonal is 10 to 100 times greater than that of epitope II monoclonal, and that the neutralizing activity of epitope VIII monoclonal is also high. Therefore, it has become clear that in serum, most RSV neutralizing activity targets only the antigenic sites of the pre-fusion F protein. The inventors of this invention recognized that inducing high-titer neutralizing antibodies is a major goal in RSV vaccine development, and that pre-fusion F proteins with specific antigen epitopes are the most popular RSV vaccine targets. However, pre-fusion F proteins are inherently unstable proteins, and performing multiple stability modifications on them without losing important antigen epitopes is one of the challenges in RSV vaccine development. [Overview of the project]

[0006] In some embodiments, the present disclosure provides an immunocomposition comprising or encoding a human respiratory syncytial virus (hRSV) antigen that can induce an effective neutralizing antibody response against human respiratory syncytial virus (hRSV).

[0007] Some aspects of the present disclosure provide an immunocomposition comprising or encoding a human respiratory syncytial virus (hRSV) antigen, selected from the group consisting of nucleic acid immunocompositions, polypeptide immunocompositions, or viral immunocompositions.

[0008] In some embodiments, the antigen is a pre-fusion hRSV F protein whose amino acid sequence includes a TM sequence and a polymerizing element.

[0009] In some embodiments, the amino acid sequences of the polymerizing elements are shown in SEQ ID NOs: 1-12 and 14-15.

[0010] In some embodiments, the amino acid sequence of the polymerizing element is one shown in SEQ ID NO: 7 and SEQ ID NO: 12.

[0011] In some embodiments, the P27 sequence of the pre-fusion hRSV F protein mutant is replaced with a GS sequence.

[0012] In some embodiments, the GS sequence includes (GnS)m, (GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q, for example, sequence numbers 48-61, sequence number 74 (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, and q is an integer from 1 to 5).

[0013] In some embodiments, the amino acid sequence of the GS sequence is shown in SEQ ID NO: 74.

[0014] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein variant includes at least one of the following substitutions: S46G substitution, N67I substitution, E92D substitution, P102A substitution, A149C substitution, S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, L373R substitution, S215P substitution, I379V substitution, M447V substitution, Y458C substitution, K465Q substitution, D486C substitution, and D489C substitution.

[0015] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes at least one of the following substitutions: S155C, S190F, V207L, or S290C, and / or the P27 sequence of the pre-fusion hRSV F protein is replaced with a GS sequence.

[0016] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes an S155C substitution, an S190F substitution, a V207L ​​substitution, and an S290C substitution, the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74, and the pre-fusion hRSV F protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO: 12.

[0017] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes an S155C substitution, an S190F substitution, a V207L ​​substitution, and an S290C substitution; the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74; the pre-fusion hRSV F protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO: 12; and the amino acid sequence of the pre-fusion hRSV F protein further includes at least one of the following substitutions: an S46G substitution, an N67I substitution, an E92D substitution, a P102A substitution, an A149C substitution, an L373R substitution, an S215P substitution, an I379V substitution, an M447V substitution, a Y458C substitution, a K465Q substitution, a D486C substitution, and a D489C substitution.

[0018] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes at least one of the following substitutions: S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, D486C substitution, and D489C substitution.

[0019] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes at least one of the following substitutions: S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, D486C substitution, and D489C substitution, and / or the P27 sequence of the pre-fusion hRSV F protein is substituted with a GS sequence.

[0020] In some embodiments, the amino acid sequence of the pre-fusion hRSV protein includes an S155C substitution, an S190F substitution, a V207L ​​substitution, an S290C substitution, a D486C substitution, and a D489C substitution, and / or the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74, and / or the pre-fusion hRSV F protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO: 12.

[0021] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, D486C substitution, and D489C substitution, the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74, the pre-fusion hRSV protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO: 12, and the amino acid sequence of the pre-fusion hRSV F protein further includes at least one of S46G substitution, N67I substitution, E92D substitution, P102A substitution, A149C substitution, L373R substitution, S215P substitution, I379V substitution, M447V substitution, Y458C substitution, and K465Q substitution.

[0022] In some embodiments, the pre-fusion hRSV F protein mutant includes an amino acid sequence in which at least one amino acid in the F2 sequence, RR1 sequence, RR2 sequence, 525-529 sequence, or 551-574 sequence is deleted consecutively or intermittently.

[0023] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein is shown in SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 76, and SEQ ID NO: 78.

[0024] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein is shown in SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 76, and SEQ ID NO: 78.

[0025] In some embodiments, the amino acid sequences of the pre-fusion hRSV F protein are shown in SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 76, and SEQ ID NO: 78.

[0026] In some embodiments, the polymerizing element of the pre-fusion hRSV F protein may be selected from the amino acid sequences shown in SEQ ID NOs: 1-12 and SEQ ID NOs: 14-15.

[0027] In some embodiments, the immune composition is a nucleic acid immune composition.

[0028] In some embodiments, the nucleic acid immunocomposition comprises nucleic acid molecules.

[0029] In some embodiments, the nucleic acid molecule includes a DNA molecule and / or an RNA molecule.

[0030] In some embodiments, the DNA molecule includes a chain-like DNA molecule and / or a circular DNA molecule.

[0031] In some embodiments, the RNA molecule includes mRNA or circular RNA.

[0032] In some embodiments, the mRNA molecule includes an open reading frame (ORF) whose nucleotide sequence is shown in SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 84-103.

[0033] In some embodiments, the mRNA molecule includes an open reading frame (ORF) whose nucleotide sequence is shown in SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NOs: 84-103.

[0034] In some embodiments, the mRNA molecule includes an open reading frame (ORF) whose nucleotide sequence is shown in SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, and SEQ ID NO: 92-103.

[0035] In some embodiments, the mRNA molecule includes an open reading frame (ORF) whose nucleotide sequence is shown in SEQ ID NO: 63, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, and SEQ ID NO: 96-103.

[0036] In some embodiments, the mRNA molecule includes an open reading frame (ORF) whose nucleotide sequence is shown in SEQ ID NO: 67.

[0037] In some embodiments, the mRNA molecule includes an open reading frame (ORF) whose nucleotide sequence is shown in SEQ ID NO: 71.

[0038] In some embodiments, the nucleotide sequences of the polymerized elements in the open reading frame (ORF) are also shown in SEQ ID NOs: 36-47 and SEQ ID NO: 13.

[0039] In some embodiments, the immunotherapy composition comprises a delivery formulation. In some embodiments, the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0040] In some embodiments, the mRNA is mixed with a delivery formulation containing lipid nanoparticles or cationic liposomes to form an immune composition.

[0041] In some embodiments, the Disclosure provides an isolated mRNA wherein the coding region nucleic acid sequence of the mRNA encodes a pre-fusion hRSV F protein whose amino acid sequence comprises a TM sequence and a multimerizing element, preferably one in which the amino acid sequence of the multimerizing element is shown in SEQ ID NOs: 1-12 and SEQ ID NOs: 14-15, and preferably one in which the amino acid sequence of the multimerizing element is shown in SEQ ID NOs: 7 and SEQ ID NOs: 12.

[0042] In some embodiments, the mRNA further comprises at least one of a 5' terminal cap, a 5' UTR, a 3' UTR, and a 3' terminal poly-A tail.

[0043] In some embodiments, the 5' end cap is selected from the group consisting of ARCA, mCAP, dmCAP, tmCAP, m7G(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, dmCAP, or m7G(5')ppp(5')(2'OMeG)pG.

[0044] In some embodiments, the 5' end cap is m7Gppp(5')(2'-OMeA)pG.

[0045] In some embodiments, the length of the 3' end poly-A tail is 50 to 200.

[0046] In some embodiments, the length of the 3' end poly-A tail is 80 to 200.

[0047] In some embodiments, the length of the 3' end poly A tail is 80 to 120. In some embodiments, the length of the 3' end poly A tail is 120.

[0048] In some embodiments, the length of the 5'UTR is preferably 10 to 200 nucleotides.

[0049] In some embodiments, the length of the 5'UTR is 15 to 100 nucleotides.

[0050] In some embodiments, the nucleotide sequences of the 5'UTR are shown in SEQ ID NOs: 16-18.

[0051] In some embodiments, the nucleotide sequence of the 5'UTR is shown in SEQ ID NO: 16.

[0052] In some embodiments, the sequence of the 3'UTR is shown in sequence numbers 19-21.

[0053] In some embodiments, the sequence of the 3'UTR is shown in sequence number 19.

[0054] In some embodiments, one or more uridines in the mRNA are replaced with modified nucleosides.

[0055] In some embodiments, the modified nucleoside is pseudouridine (Ψ), N 1-methyl-psoidouridine (m1Ψ), or 5-methyl-uridine (m5U).

[0056] In some embodiments, the modified nucleoside is N1-methylpsoiduridine (m1Ψ).

[0057] In some embodiments, the disclosure provides isolated DNA which is obtained by reverse transcription of isolated mRNA nucleic acid or which is a transcriptionable sequence of isolated mRNA.

[0058] In some embodiments, the present disclosure provides a biomaterial comprising an expression cassette, a vector, a process bacterium, or a cell line, which contains or expresses the isolated mRNA or the isolated DNA. [Brief explanation of the drawing]

[0059] To more clearly illustrate specific embodiments of the present disclosure or technical aspects of the prior art, the following briefly describes drawings that may be used in the description of specific embodiments or the prior art. Clearly, the drawings in the following description are some embodiments of the present disclosure, and those skilled in the art can obtain other drawings based on these without any creative effort. [Figure 1] A schematic diagram of the hRSV F protein wild type is shown. [Figure 2] A schematic diagram of the hRSV F protein mutant is shown. [Figure 3] A schematic diagram of the experimental evaluation flow for mice is shown. [Figure 4] The TNFα+CD4+ and TNF-α+CD8+ T cells detected by flow cytometry in Example 5 are shown. [Figure 5] The IFN-γ and L4 in the cell culture supernatant detected by ELISA in Example 5 are shown. [Figure 6] The titer of RSV-neutralizing antibodies in mouse serum in Example 5 is shown. [Figure 7] The statistical graph of RSV-F protein-specific IgG antibody titers in mouse serum detected by ELISA in Example 6 is shown. [Figure 8] The TNFα+CD4+ and TNF-α+CD8+ T cells detected by flow cytometry in Example 6 are shown. [Figure 9] The IFN-γ and IL2 in the cell culture supernatant detected by ELISA in Example 6 are shown. [Figure 10] The IL4 and IL10 detected in the cell culture supernatant by ELISA in Example 6 are shown. [Figure 11] The titer of RSV neutralizing antibody in mouse serum in Example 6 is shown. [Figure 12] The TNFα+CD4+ and TNF-α+CD8+ T cells detected by flow cytometry in Example 7 are shown. [Figure 13]The IFN-γ+CD4+ and IFN-γ+CD8+ T cells detected by flow cytometry in Example 7 are shown. [Figure 14] The IFN-γ and IL2 in the cell culture supernatant detected by ELISA in Example 7 are shown. [Figure 15] The IL4 and IL10 detected in the cell culture supernatant by ELISA in Example 7 are shown. [Figure 16] The titer of RSV neutralizing antibody in mouse serum in Example 7 is shown. [Figure 17] The TNFα+CD4+ and TNF-α+CD8+ T cells detected by flow cytometry in Example 8 are shown. [Figure 18] The IFN-γ+CD4+ and IFN-γ+CD8+ T cells detected by flow cytometry in Example 8 are shown. [Figure 19] The IFN-γ and IL2 in the cell culture supernatant detected by ELISA in Example 8 are shown. [Figure 20] The IL4 and IL10 detected in the cell culture supernatant by ELISA in Example 8 are shown. [Figure 21] The titer of RSV neutralizing antibody in mouse serum in Example 8 is shown. [Figure 22] The titer of RSV neutralizing antibody in mouse serum in Example 9 is shown. [Figure 23] The titer of RSV neutralizing antibody in mouse serum in Example 11 is shown. [Figure 24] The titer of RSV neutralizing antibody in mouse serum in Example 12 is shown. [Figure 25] The titer of RSV neutralizing antibody in mouse serum in Example 12 is shown. [Figure 26] The titer of RSV neutralizing antibody in mouse serum in Example 12 is shown. [Figure 27] The trend diagram of mouse body weight changes in Example 13 is shown. [Figure 28] The trend diagram of the rate of change in mouse body weight in Example 13 is shown. [Figure 29] The statistical graph of RSV-F protein-specific IgG antibody titers in mouse serum detected by ELISA in Example 13 is shown. [Figure 30] The results of measuring neutralizing antibody titers against RSV-A / B venom strains in mouse serum in Example 13 are shown. [Figure 31] The results of measuring the viral titer in mouse lung tissue after RSV infection in Example 13 are shown. [Figure 32] The pathological examination results of mouse lung tissue after RSV infection in Example 13 are shown. [Modes for carrying out the invention]

[0060] Figure 1 shows a schematic diagram of the pre-fusion hRSV F protein wild type. Figure 2 shows a schematic diagram of one pre-fusion hRSV F protein (pre-fusion hRSV F protein variant) of the immunocomposition antigen according to this disclosure.

[0061] [ka] is cleavage by furin protease. SP is the signal peptide sequence. P27 is the sequence that removes the 27aa peptide after cleavage. RR1 is repeat folding region 1, which spans 137-216 amino acid residues of the pre-fusion hRSV F protein wild type and contains the fusion peptide and heptapeptide repeat sequence A (HRA). RR2 is repeat folding region 2, which contains the heptapeptide repeat sequence B (HRB), which is rearranged on the opposite side of the pre-fusion RSV F protein head and forms the C-terminal stalk of the pre-fusion hRSV F protein spike. Sequences 525-529 are segments of the sequence that binds RR2 and TM. TM is the transmembrane region sequence. Sequences 551-574 are sequences ligated to the C-terminus of the TM sequence.

[0062] F2 typically contains amino acid residues 26-109 of the F0 precursor. F1 typically contains amino acid residues 137-574 of the F0 precursor. F1 and F2 are linked by a disulfide bond to form a heterodimer called the RSV F "protemer".

[0063] In this disclosure, "pre-fusion hRSV F protein wild type" or "pre-fusion hRSV F protein (wild type)" both refer to the wild-type pre-fusion hRSV F protein, which has not undergone artificial modification or mutation.

[0064] The C-terminus shown in Figure 2 may be deleted or selected from at least one of the group consisting of TM sequences, sequences 525-529, sequences 551-574, and multimerization elements. In some embodiments, the C-terminus of the pre-fusion hRSV F protein variant according to this disclosure is deleted. In some embodiments, the C-terminus of the pre-fusion hRSV F protein variant is a TM sequence. In some embodiments, the C-terminus of the pre-fusion hRSV F protein variant is a sequentially linked TM sequence and a multimerization element, where the C-terminus of the TM sequence is linked to the N-terminus of the multimerization element. In some embodiments, the C-terminus of the pre-fusion hRSV F protein variant is a TM sequence. In some embodiments, the C-terminus of the pre-fusion hRSV F protein variant is a sequentially linked TM sequence, sequences 551-574, and a multimerization element, where the C-terminus of the TM sequence is linked to sequences 551-574, and the C-terminus of sequences 551-574 is linked to the N-terminus of the multimerization element. In some embodiments, the C-terminus of the pre-fusion hRSV F protein mutant is a multimerizing element.

[0065] The multimerizing elements relating to this disclosure include dimerizing elements, trimerizing elements, tetramerizing elements, and oligomerizing elements, and when used in combination with the hRSV F protein relating to this disclosure, can result in the formation of a multimerized hRSV F protein complex. The multimerizing elements may be located at the N-terminus or C-terminus of the hRSV F protein, and at the nucleic acid level, their coding sequences are typically located within the 5' or 3' frame of the coding sequence.

[0066] In some embodiments, the dimerization element can be selected from, for example, the dimerization element / domain of heat shock proteins, immunoglobulin Fc domains, and leucine zippers (dimerization domains of leucine zipper-system transcription factors in the basic region), with specific amino acid sequences referring, for example, to SEQ ID NOs: 1-2. The trimerization and tetramerization elements can be selected from, for example, the processed leucine zipper (using processed helical peptides in a parallel trimer state), the fibrin folding domain of E. coli phage T4, GCN4PLL, CCN4-PLI, p53, and GCN4, with specific amino acid sequences referring to SEQ ID NOs: 3-12 (trimerization) and SEQ ID NOs: 14-15 (tetramerization).

[0067] The pre-fusion hRSV F protein variants relating to this disclosure are F proteins that have undergone artificial mutation and modification, and such mutations and modifications (including, but not limited to, amino acid residue substitutions, insertions and / or additions, deletions and covalent modifications of amino acid residues to the amino acid sequence) obtain an amino acid sequence that matches the pre-fusion hRSV F protein through mutation and modification of the pre-fusion hRSV F protein wild type. In some embodiments of this disclosure, unless otherwise stated as "pre-fusion hRSV F protein wild type" or "pre-fusion hRSV F protein (wild type)", "pre-fusion hRSV F protein" is a pre-fusion hRSV F protein variant.

[0068] In some embodiments, the GS sequence includes, for example, (GnS)m, (GGGGS), GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q, for example, SEQ ID NOs: 48-61, SEQ ID NO: 74 (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, and q is an integer from 1 to 5). Furthermore, in some embodiments, the amino acid sequence of the GS sequence is shown in SEQ ID NO: 74, or the sequence-optimized coding amino acid sequence is the nucleotide sequence shown in SEQ ID NO: 74.

[0069] In some embodiments, the present disclosure provides an immunocomposition comprising or encoding a human respiratory syncytial virus (hRSV) antigen, the antigen capable of inducing an effective neutralizing antibody response against human respiratory syncytial virus (hRSV).

[0070] In some aspects of this disclosure, an immunocomposition comprising or encoding a human respiratory syncytial virus antigen is provided, selected from the group consisting of nucleic acid immunocompositions, polypeptide immunocompositions, or viral immunocompositions.

[0071] In some embodiments, the antigen is a pre-fusion hRSV F protein whose amino acid sequence includes a TM sequence and a polymerizing element.

[0072] In some embodiments, the amino acid sequences of the polymerizing elements are shown in SEQ ID NOs: 1-12 and 14-15.

[0073] In some embodiments, the amino acid sequence of the polymerizing element is one shown in SEQ ID NO: 7 and SEQ ID NO: 12.

[0074] In some embodiments, the P27 sequence of the pre-fusion hRSV F protein mutant is replaced with a GS sequence.

[0075] In some embodiments, the GS sequence includes (GnS)m, (GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q, for example, sequence numbers 48-61, sequence number 74 (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, and q is an integer from 1 to 5).

[0076] In some embodiments, the amino acid sequence of the GS sequence is shown in SEQ ID NO: 74.

[0077] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein variant includes at least one of the following substitutions: S46G substitution, N67I substitution, E92D substitution, P102A substitution, A149C substitution, S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, L373R substitution, S215P substitution, I379V substitution, M447V substitution, Y458C substitution, K465Q substitution, D486C substitution, and D489C substitution.

[0078] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes at least one of the following substitutions: S155C, S190F, V207L, or S290C, and / or the P27 sequence of the pre-fusion hRSV F protein is replaced with a GS sequence.

[0079] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes an S155C substitution, an S190F substitution, a V207L ​​substitution, and an S290C substitution, the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74, and the pre-fusion hRSV F protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO: 12.

[0080] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes an S155C substitution, an S190F substitution, a V207L ​​substitution, and an S290C substitution; the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74; the pre-fusion hRSV F protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO: 12; and the amino acid sequence of the pre-fusion hRSV F protein further includes at least one of the following substitutions: an S46G substitution, an N67I substitution, an E92D substitution, a P102A substitution, an A149C substitution, an L373R substitution, an S215P substitution, an I379V substitution, an M447V substitution, a Y458C substitution, a K465Q substitution, a D486C substitution, and a D489C substitution.

[0081] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes an S155C substitution, an S190F substitution, a V207L ​​substitution, and an S290C substitution; the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74; the pre-fusion hRSV F protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO: 12; and the pre-fusion hRSV F protein variant includes an amino acid sequence in which at least one amino acid in the F2 sequence, RR1 sequence, RR2 sequence, 525-529 sequence, or 551-574 sequence is deleted consecutively or intermittently.

[0082] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes at least one of the following substitutions: S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, D486C substitution, and D489C substitution, and / or the P27 sequence of the pre-fusion hRSV F protein is substituted with a GS sequence.

[0083] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, D486C substitution, and D489C substitution, the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74, and the pre-fusion hRSV F protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO: 12.

[0084] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, D486C substitution, and D489C substitution; the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74; the pre-fusion hRSV protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO: 12; and the amino acid sequence of the pre-fusion hRSV F protein includes at least one of S46G substitution, N67I substitution, E92D substitution, P102A substitution, A149C substitution, L373R substitution, S215P substitution, I379V substitution, M447V substitution, Y458C substitution, and K465Q substitution.

[0085] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein includes an S155C substitution, an S190F substitution, a V207L ​​substitution, an S290C substitution, a D486C substitution, and a D489C substitution; the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74; the pre-fusion hRSV protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO: 12; and the pre-fusion hRSV F protein variant includes an amino acid sequence in which at least one amino acid in the F2 sequence, RR1 sequence, RR2 sequence, 525-529 sequence, or 551-574 sequence is deleted consecutively or intermittently.

[0086] In some embodiments, the pre-fusion hRSV F protein mutant includes an amino acid sequence in which at least one amino acid in the F2 sequence, RR1 sequence, RR2 sequence, 525-529 sequence, or 551-574 sequence is deleted consecutively or intermittently.

[0087] In some embodiments, the 98th to 109th amino acids in the F2 sequence of the pre-fusion hRSV F protein mutant have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid sequences deleted consecutively or intermittently.

[0088] In some embodiments, the RR1 sequence of the pre-fusion hRSV F protein mutant has at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acid sequences deleted consecutively or intermittently.

[0089] In some embodiments, the 504-524th amino acids in the RR2 sequence of the pre-fusion hRSV F protein mutant have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 amino acid sequences deleted consecutively or intermittently.

[0090] In some embodiments, the amino acids in sequences 525-529 of the pre-fusion hRSV F protein variant have at least 1, 2, 3, 4, or 5 amino acid sequences deleted consecutively or at intervals.

[0091] In some embodiments, the amino acids in sequences 551-574 of the pre-fusion hRSV F protein variant have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid sequences deleted consecutively or intermittently.

[0092] The amino acid sequence positions relating to this disclosure, such as S46G substitution, E92D substitution, P102A substitution, A149C substitution, L373R substitution, I379V substitution, M447V substitution, Y458C substitution, K465Q substitution, D486C substitution, and D489C substitution, are positioned in the full-length amino acid sequence of the pre-fusion hRSV F protein wild type shown in Figure 1. For example, the S46G substitution is based on the pre-fusion hRSV F protein wild type, substituting the 46th S amino acid residue with a G amino acid residue in the full-length amino acid sequence of the pre-fusion hRSV F protein wild type shown in Figure 1, and this mutation occurs in the F2 sequence. For example, the 525-529 sequence is based on the pre-fusion hRSV F protein wild type, with the RR2 sequence and TM sequence being joined to the pre-fusion hRSV F protein wild type shown in Figure 1. For example, sequences 551-574 are based on the pre-fusion hRSV F protein wild type and are bound to the C-terminus of the TM sequence in the pre-fusion hRSV F protein wild type shown in Figure 1.

[0093] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein is shown in SEQ ID NO: 62, SEQ ID NO: 76, SEQ ID NO: 78, or comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 62, SEQ ID NO: 76, SEQ ID NO: 78, and may, for example, comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 62, SEQ ID NO: 76, SEQ ID NO: 78, but is not limited to these.

[0094] In some embodiments, the amino acid sequence of the stabilized pre-fusion hRSV F protein mutant is shown in SEQ ID NO: 66, or comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 66, and may, for example, comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 66.

[0095] In some embodiments, the amino acid sequence of the stabilized pre-fusion hRSV F protein mutant is shown in SEQ ID NO: 70, or comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 70, and may, for example, comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 70.

[0096] In some embodiments, the amino acid sequence of the stabilized pre-fusion hRSV F protein mutant is shown in SEQ ID NO: 64, or comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 64, and may, for example, comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 64.

[0097] In some embodiments, the amino acid sequence of the stabilized pre-fusion hRSV F protein mutant is shown in SEQ ID NO: 68, or comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 68, and may, for example, comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 68.

[0098] In some embodiments, the amino acid sequence of the stabilized pre-fusion hRSV F protein mutant is shown in SEQ ID NO: 72, or comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 72, and may, for example, comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 72.

[0099] In some embodiments, the pre-fusion hRSV F protein mutant includes an amino acid sequence in which at least one amino acid in the F2 sequence, RR1 sequence, RR2 sequence, 525-529 sequence, or 551-574 sequence is deleted consecutively or intermittently.

[0100] In some embodiments, the 98th to 109th amino acids in the F2 sequence of the pre-fusion hRSV F protein mutant have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid sequences deleted consecutively or intermittently.

[0101] In some embodiments, the RR1 sequence of the pre-fusion hRSV F protein mutant has at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acid sequences deleted consecutively or intermittently.

[0102] In some embodiments, the 504-524th amino acids in the RR2 sequence of the pre-fusion hRSV F protein mutant have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 amino acid sequences deleted consecutively or intermittently.

[0103] In some embodiments, the amino acids in sequences 525-529 of the pre-fusion hRSV F protein variant have at least 1, 2, 3, 4, or 5 amino acid sequences deleted consecutively or at intervals.

[0104] In some embodiments, the amino acids in sequences 551-574 of the pre-fusion hRSV F protein variant have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid sequences deleted consecutively or intermittently.

[0105] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein is shown in SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 78, and / or an amino acid sequence obtained by sequentially or intermittently deleting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acids from positions 98 to 109 in the F2 sequence, and / or an amino acid sequence obtained by sequentially or intermittently deleting at least 1, 2, 3, 4, 5, 6, 7, 8 amino acids from positions 137 to 144 in the RR1 sequence, and / or the RR2 sequence The amino acid sequences include those in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 amino acids in the 504-524 positions are deleted consecutively or intermittently, and / or amino acid sequences in which at least 1, 2, 3, 4, and 5 amino acids in the 525-529 sequences are deleted consecutively or intermittently, and / or amino acid sequences in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 amino acids in the 551-574 sequences are deleted consecutively or intermittently.

[0106] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein is shown in SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 76, SEQ ID NO: 78, and / or an amino acid sequence obtained by deleting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acids from positions 98 to 109 in the F2 sequence, either consecutively or at intervals, and / or an amino acid sequence obtained by deleting at least 1, 2, 3, 4, 5, 6, 7, 8 amino acids from positions 137 to 144 in the RR1 sequence, and / or an amino acid sequence obtained by deleting at least 504- in the RR2 sequence The amino acid sequence includes an amino acid sequence 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 deleted consecutively or intermittently, and / or an amino acid sequence in which at least 1, 2, 3, 4, 5 amino acids are deleted consecutively or intermittently in sequences 525-529, and / or an amino acid sequence in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 amino acids are deleted consecutively or intermittently in sequences 551-574.

[0107] In some embodiments, the amino acid sequence of the pre-fusion hRSV F protein is shown in SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 76, SEQ ID NO: 78, and / or an amino acid sequence obtained by deleting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acids from positions 98 to 109 in the F2 sequence, and / or an amino acid sequence obtained by deleting at least 1, 2, 3, 4, 5, 6, 7, 8 amino acids from positions 137 to 144 in the RR1 sequence, and / or a sequence obtained by deleting at least 504 to 524 amino acids in the RR2 sequence The amino acid sequence comprises an amino acid sequence 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 deleted consecutively or intermittently, and / or an amino acid sequence in which at least 1, 2, 3, 4, 5 amino acids in sequences 525-529 are deleted consecutively or intermittently, and / or an amino acid sequence in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 amino acids in sequences 551-574 are deleted consecutively or intermittently.

[0108] In some embodiments, the multimerizing element described in the pre-fusion hRSV F protein may be selected from the amino acid sequences shown in SEQ ID NOs: 1-12 and SEQ ID NOs: 14-15.

[0109] In some embodiments, the immunocomposition encoding the human respiratory syncytial virus (hRSV) antigen is a nucleic acid immunocomposition comprising nucleic acid molecules, including DNA molecules and / or RNA molecules.

[0110] In some embodiments, the DNA molecule includes a chain-like DNA molecule and / or a circular DNA molecule.

[0111] In some embodiments, the RNA molecule includes mRNA or circular RNA.

[0112] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-103, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-103, and may, for example, include a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-103.

[0113] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-103, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-103, and may include, for example, a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-103.

[0114] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, SEQ ID NO: 92-103, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, SEQ ID NO: 92-103, and may, for example, include a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, SEQ ID NO: 92-103.

[0115] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 63, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, SEQ ID NO: 96-103, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 63, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, SEQ ID NO: 96-103, and may, for example, include a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 63, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, SEQ ID NO: 96-103.

[0116] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 63, SEQ ID NO: 84-87, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 63, SEQ ID NO: 84-87, and may include, for example, a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 63, SEQ ID NO: 84-87.

[0117] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 63, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 63, and may, for example, include a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 63, but is not limited to these.

[0118] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 77, SEQ ID NO: 96-99, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 77, SEQ ID NO: 96-99, and may, for example, include a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 77, SEQ ID NO: 96-99.

[0119] In some embodiments, the mRNA molecule comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 77, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 77, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 77, but is not limited to these.

[0120] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 79, SEQ ID NO: 100-103, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 79, SEQ ID NO: 100-103, and may, for example, include a nucleotide sequence that is at least 80%, 85%, 95%, or 98% identical to SEQ ID NO: 79, SEQ ID NO: 100-103.

[0121] In some embodiments, the mRNA molecule comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 79, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 79, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 79, but is not limited to these.

[0122] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 65, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 65, and may, for example, include a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 65.

[0123] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 67, SEQ ID NO: 88-91, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 67, SEQ ID NO: 88-91, and may include, for example, a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 67, SEQ ID NO: 88-91.

[0124] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) is shown in SEQ ID NO: 67, or includes a nucleotide sequence that is at least 80% identical to SEQ ID NO: 67, and may, for example, include a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 67.

[0125] In some embodiments, the mRNA molecule includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 69, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 69, and may, for example, include a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 69.

[0126] In some embodiments, the mRNA molecule comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) comprising a nucleotide sequence shown in SEQ ID NO: 71, SEQ ID NOs: 92-95, or a nucleotide sequence that is at least 80% identical to SEQ ID NO: 71, SEQ ID NOs: 92-95, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 71, SEQ ID NOs: 92-95.

[0127] In some embodiments, the mRNA molecule comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 71, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 71, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 71, but is not limited to these.

[0128] In some embodiments, the mRNA molecule comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 73, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 73, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 73, but is not limited to these.

[0129] In some embodiments, the nucleotide sequence of the dimerized element is shown in SEQ ID NO: 36 and SEQ ID NO: 42, or the sequence-optimized coding amino acid sequence is the amino acid sequence shown in SEQ ID NOs: 1-2. The trimerized nucleotide sequence is shown in SEQ ID NOs: 37-40, SEQ ID NOs: 43-46 and SEQ ID NO: 13, or the sequence-optimized coding amino acid sequence is the nucleotide sequence shown in SEQ ID NOs: 3-12. The nucleotide sequence of the tetramerized element is shown in SEQ ID NOs: 41 and SEQ ID NO: 47, and the sequence-optimized coding amino acid sequence is the nucleotide sequence shown in SEQ ID NOs: 14-15.

[0130] Furthermore, in some embodiments, the nucleotide sequences of the polymerized elements in the open reading frame (ORF) are also shown in SEQ ID NOs: 36-47 and SEQ ID NO: 13.

[0131] In some embodiments, nucleic acid molecules can have their mRNA sequences optimized by sequence optimization means to improve properties related to expression effects after in vivo administration, such as improved mRNA stability, increased translational effect in target tissues, reduced number of expressed truncated proteins, improved or prevention of incorrect folding of expressed proteins, reduced toxicity of expression products, reduced cell death by expression products, increased and / or decreased protein aggregation, and acquisition of mRNA with improved properties. The objectives of sequence optimization further include optimizing the characteristics of preparation and delivery of nucleic acid-based therapeutic agents while maintaining structural and functional integrity, overcoming expression thresholds, increasing expression rates, half-lives and / or protein concentrations, optimizing protein localization, and avoiding unfavorable biological responses such as immune responses and / or degradation pathways. Sequence optimization means include (1) codon optimization to ensure proper folding and expression based on the frequency of codon use in a specific organ and / or host organism; (2) adjusting the G / C content to increase mRNA stability or reduce secondary structures; (3) minimizing serial repeat codons or base runs that may impair gene construction or expression; (4) tailoring transcription and translation regulatory regions; and (5) reducing or removing problematic secondary structures in polynucleotides.

[0132] Sequence identity between two nucleotide sequences indicates the proportion of identical nucleotides in those sequences. Sequence identity between two amino acid sequences indicates the proportion of identical amino acids in those sequences.

[0133] The term "% identity" or similar terms refer to the percentage of identical nucleotides or amino acids between sequences being compared, in optimal alignment. This percentage is purely statistical, and the differences between two sequences may, but not necessarily, be randomly distributed across the entire length of the sequences being compared. Comparison between two sequences is typically performed by comparing modified sequences to a fragment or "comparison window" after optimal alignment to identify the local regions of the corresponding sequences.

[0134] In some embodiments, based on the open reading frame sequence of a provided mRNA, a person skilled in the art can obtain the open reading frame sequence of a corresponding circular RNA and, according to the contents of published literature such as CN202180048567.4, can prepare the complete sequence of the circular RNA capable of encoding the same amino acid sequence. In some embodiments, based on the provided mRNA sequence, a person skilled in the art can obtain the corresponding DNA sequence (e.g., converting uracil to thymine). Similarly, based on the provided DNA sequence, a person skilled in the art can obtain the corresponding RNA sequence (e.g., converting thymine to uracil). In some embodiments, based on the provided RNA or DNA sequence, a person skilled in the art can obtain the corresponding amino acid sequence.

[0135] An open reading frame (ORF) as relating to this disclosure is a segment of continuous DNA or RNA that begins with a start codon such as ATG or AUG and ends with a stop codon such as TAA, TAG or TGA, or UAA, UAG or UGA.

[0136] In some embodiments, the mRNA according to this disclosure consists of a sequence comprising a 5' cap, a 5' UTR, an ORF, a 3' UTR, and a 3' poly(A) tail in order from the 5' end to the 3' end.

[0137] The 5' untranslated region (UTR) as relating to this disclosure refers to a sequence located immediately upstream (i.e., 5') of the start codon of a non-polypeptide mRNA (i.e., the first codon of the mRNA transcript being translated by the ribosome). If an RNA transcript is produced, the 5' UTR may include a promoter sequence. Such promoter sequences are known in the art. The RNA sequence of the 5' UTR is shown in one of SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, or a 5' UTR sequence disclosed prior to the filing date of this disclosure may be incorporated into this disclosure.

[0138] The 3' untranslated region (UTR) as relating to this disclosure refers to a sequence located downstream of the stop codon of mRNA that does not encode a polypeptide. The RNA sequence of the 3' UTR is shown in one of SEQ ID NOs: 19, 20, or 21, or a 3' UTR sequence disclosed prior to the filing date of this disclosure may be incorporated into this disclosure.

[0139] The poly(A) tails relating to this disclosure are sequences located downstream of the 3'UTR of mRNA, containing multiple consecutive adenosine monophosphates. The poly(A) tails can contain 10 to 300 adenosine monophosphates. In cells and / or in vivo, poly(A) tails are used to protect mRNA from enzymatic degradation and to assist in transcription termination and / or the export of mRNA from the cell nucleus and translation.

[0140] In some embodiments, the mRNA molecule includes either a self-replicating mRNA molecule or a non-self-replicating mRNA molecule.

[0141] In some embodiments, the mRNA relating to this disclosure is a self-replicating mRNA having a sequence capable of expressing RNA polymerase (RNA-dependent RNA polymerase, RdRP), and specifically, the sequence design of Chinese Patent CN202110424124.2 relating to self-replicating mRNA is incorporated into this disclosure.

[0142] In some embodiments, the immunotherapy composition includes a delivery formulation.

[0143] In some embodiments, the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0144] In some embodiments, the components of the lipid nanoparticles include at least one of a protonable cationic lipid, a structural lipid, an auxiliary lipid, and a surfactant.

[0145] In some embodiments, the components of the lipid nanoparticles include, by weight, 40 to 60 parts of protonable cationic lipids, 20 to 40 parts of structural lipids, 10 to 30 parts of auxiliary lipids, and 0.5 to 5 parts of surfactant.

[0146] In some embodiments, the protonable cationic lipid is selected from at least one of Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA.

[0147] In some embodiments, the structural lipids include cholesterol and / or cholesterol derivatives.

[0148] In some embodiments, the auxiliary lipid includes at least one of DSPC, DOPE, DOPC, DOPG, and DOPS.

[0149] In some embodiments, the surfactant comprises at least one of PEG-DMG, PEG-DSPE, and TPGS.

[0150] In some embodiments, the liposome nanoparticles contain, in mole percent, 20% to 50% (for example, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) cationic lipids, 20% to 50% (for example, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) DOPG, 5% to 20% (for example, 5%, 10%, 15%, or 20%) cholesterol, and 1% to 5% (for example, 1%, 2%, 3%, 4%, or 5%) PEG-DMG.

[0151] In some embodiments, the liposome nanoparticles contain, in mole percent, 20% to 50% (for example, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) cationic lipids, 20% to 50% (for example, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) DSCP, 5% to 20% (for example, 5%, 10%, 15%, or 20%) cholesterol, and 1% to 5% (for example, 1%, 2%, 3%, 4%, or 5%) PEG-DMG.

[0152] In some embodiments, the liposome nanoparticles contain, in mole percent, 50% Dlin-MC3-DMA, 10% DOPG, 38.5% cholesterol, and 1.5% PEG-DMG.

[0153] In some embodiments, the liposome nanoparticles contain, in mole percent, 50% Dlin-MC3-DMA, 10% DSCP, 38.5% cholesterol, and 1.5% PEG-DMG.

[0154] In some embodiments, the present disclosure provides a method for producing an immune composition according to any one of the embodiments, characterized by mixing the mRNA with a delivery formulation comprising lipid nanoparticles or cationic liposomes to form an immune composition.

[0155] In some embodiments, the method for producing the immunocomposition involves dissolving mRNA in a buffer to obtain an aqueous phase, weighing out each lipid component of liposome nanoparticles and dissolving them in an organic solvent to obtain an organic phase, mixing the aqueous phase and the organic phase, and then removing the organic phase to obtain the immunocomposition.

[0156] In some embodiments, the volume ratio of the aqueous phase to the organic phase is 1:2 to 4, preferably 1:3.

[0157] In some embodiments, the buffer solution comprises a citrate buffer or sodium acetate, and is preferably a citrate buffer.

[0158] In some embodiments, the pH of the buffer solution is 3 to 7, preferably 4.

[0159] In some embodiments, the mRNA concentration in the aqueous phase is 0.05 mg / mL to 0.5 mg / mL, preferably 0.1 mg / mL.

[0160] In some embodiments, the organic solvent is selected from C1-C4 low-carbon alcohols, preferably anhydrous ethanol.

[0161] In some embodiments, the lipid component concentration in the organic phase is 5 mg / mL to 7 mg / mL, preferably 6 mg / mL.

[0162] In some embodiments, the aqueous phase and the organic phase are mixed using a microfluidic, and the organic solvent is filtered using tangential flow. Preferably, the flow rate of the microfluidic is >3 ml / min, and more preferably 12 ml / min.

[0163] In some embodiments, the process also includes a concentration step after mixing to bring the final mRNA concentration to 50 μg / mL to 200 μg / mL, preferably 100 μg / mL.

[0164] In some embodiments, the diameter of the lipid nanoparticles is less than approximately 200 nm. In some embodiments, the diameter of the lipid nanoparticles is less than approximately 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 between approximately 55 nm and approximately 90 nm.

[0165] In some embodiments, the immunocomposition containing the human respiratory syncytial virus (hRSV) antigen is a polypeptide immunocomposition. The polypeptide immunocomposition is an immunocomposition prepared by chemical synthesis or genetic engineering techniques according to the amino acid sequence of a known or predicted antigen epitope in the human respiratory syncytial virus antigen gene. In some embodiments, the polypeptide immunocomposition according to the disclosure is an immunocomposition prepared by chemical synthesis techniques after artificial mutations and modifications are added according to the amino acid sequence of the human respiratory syncytial virus pre-fusion hRSV F protein. In some embodiments, the polypeptide immunocomposition according to the disclosure is obtained after artificial mutations and modifications are added according to the amino acid sequence of the human respiratory syncytial virus pre-fusion hRSV F protein, and then fermented by genetic engineering techniques (e.g., construction of genetically engineered bacteria by genetic engineering means).

[0166] In some embodiments, the polypeptide immunocomposition comprises a pre-fusion hRSV F protein, the amino acid sequence of the pre-fusion hRSV F protein is shown in SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 78, and / or an amino acid sequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acids from positions 98 to 109 in the F2 sequence deleted consecutively or at intervals, and / or an amino acid sequence having at least 1, 2, 3, 4, 5, 6, 7, 8 amino acids from positions 137 to 144 in the RR1 sequence deleted consecutively or at intervals, and / or an amino acid sequence having at least 1, 2, 3, 4, 5, 6, 7, 8 amino acids from positions 137 to 144 in the RR2 sequence The amino acid sequence includes an amino acid sequence 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 in the 504-524 position are deleted consecutively or intermittently, and / or an amino acid sequence in which at least 1, 2, 3, 4, 5 amino acids in the 525-529 sequence are deleted consecutively or intermittently, and / or an amino acid sequence in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 amino acids in the 551-574 sequence are deleted consecutively or intermittently.

[0167] In some embodiments, the viral immune compositions according to this disclosure introduce the gene for the human respiratory syncytial virus antigen, hRSV F protein, into humans via harmless microorganisms, thereby inducing an immune response in the host's immune system. Key viruses used in cellular immunity induction studies include rinderpest virus variants and poliovirus.

[0168] In some embodiments, the disclosure provides isolated mRNA characterized in that the coding region nucleic acid sequence of the mRNA encodes a pre-fusion hRSV F protein comprising a TM sequence and a multimerization element. Preferably, the amino acid sequence of the multimerization element is shown in SEQ ID NOs: 1-12 and SEQ ID NOs: 14-15. Preferably, the amino acid sequence of the multimerization element is one shown in SEQ ID NOs: 7 and SEQ ID NOs: 12.

[0169] In some embodiments, the disclosure provides isolated mRNA in which the P27 sequence of the pre-fusion hRSV F protein is replaced with a GS sequence. Preferably, the GS sequence includes (GnS)m, (GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q, e.g., SEQ ID NOs: 48-61, SEQ ID NOs: 74 (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, and q is an integer from 1 to 5). Preferably, the amino acid sequence of the GS sequence is shown in SEQ ID NOs: 74.

[0170] In some embodiments, the disclosure provides isolated mRNA in which the amino acid sequence of the pre-fusion hRSV F protein includes at least one of the following substitutions: S46G substitution, N67I substitution, E92D substitution, P102A substitution, A149C substitution, S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, L373R substitution, S215P substitution, I379V substitution, M447V substitution, Y458C substitution, K465Q substitution, D486C substitution, and D489C substitution.

[0171] In some embodiments, the disclosure provides isolated mRNA in which the amino acid sequence of the pre-fusion hRSV F protein comprises at least one of the following substitutions: S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, D486C substitution, and D489C substitution.

[0172] In some embodiments, the Disclosure provides isolated mRNA in which the amino acid sequence of the pre-fusion hRSV F protein comprises at least one of S155C substitution, S190F substitution, V207L ​​substitution, and S290C substitution, and / or the P27 sequence of the pre-fusion hRSV F protein is substituted with a GS sequence.

[0173] In some embodiments, the disclosure provides isolated mRNA in which the amino acid sequence of the pre-fusion hRSV F protein comprises an S155C substitution, an S190F substitution, a V207L ​​substitution, and an S290C substitution, the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74, and the pre-fusion hRSV F protein comprises one of the multimerization elements shown in SEQ ID NO: 7 and SEQ ID NO: 12.

[0174] In some embodiments, the Disclosure provides isolated mRNA in which the amino acid sequence of the pre-fusion hRSV F protein comprises an S155C substitution, an S190F substitution, a V207L ​​substitution, or an S290C substitution, the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74, the pre-fusion hRSV F protein comprises one of the multimerization elements shown in SEQ ID NO: 7 or SEQ ID NO: 12, and the amino acid sequence of the pre-fusion hRSV F protein further comprises at least one of the following substitutions: S46G substitution, N67I substitution, E92D substitution, P102A substitution, A149C substitution, L373R substitution, S215P substitution, I379V substitution, M447V substitution, Y458C substitution, K465Q substitution, D486C substitution, or D489C substitution.

[0175] In some embodiments, the Disclosure provides isolated mRNA in which the amino acid sequence of the pre-fusion hRSV F protein comprises at least one of S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, D486C substitution, and D489C substitution, and / or the P27 sequence of the pre-fusion hRSV F protein is substituted with a GS sequence.

[0176] In some embodiments, the disclosure provides isolated mRNA in which the amino acid sequence of the pre-fusion hRSV F protein comprises S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, D486C substitution, and D489C substitution, the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74, and the pre-fusion hRSV protein comprises one of the multimerization elements shown in SEQ ID NO: 7 and SEQ ID NO: 12.

[0177] In some embodiments, the Disclosure provides isolated mRNA in which the amino acid sequence of the pre-fusion hRSV F protein comprises an S155C substitution, an S190F substitution, a V207L ​​substitution, an S290C substitution, a D486C substitution, or a D489C substitution, the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74, the pre-fusion hRSV protein comprises one of the multimerization elements shown in SEQ ID NO: 7 or SEQ ID NO: 12, and the amino acid sequence of the pre-fusion hRSV F protein further comprises at least one of the following substitutions: an S46G substitution, an N67I substitution, an E92D substitution, a P102A substitution, an A149C substitution, an L373R substitution, an S215P substitution, an I379V substitution, an M447V substitution, a Y458C substitution, or a K465Q substitution.

[0178] In some embodiments, the disclosure provides isolated mRNA comprising an amino acid sequence in which the pre-fusion hRSV F protein variant has consecutive or intermittent deletions of at least one amino acid in the F2 sequence, RR1 sequence, RR2 sequence, 525-529 sequence, or 551-574 sequence.

[0179] In some embodiments, the present disclosure provides isolated mRNA of the pre-fusion hRSV F protein whose amino acid sequence is shown in SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 76, and SEQ ID NO: 78.

[0180] In some embodiments, the present disclosure provides isolated mRNA of the pre-fusion hRSV F protein whose amino acid sequence is shown in SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 76, and SEQ ID NO: 78.

[0181] In some embodiments, the present disclosure provides isolated mRNA of the pre-fusion hRSV F protein whose amino acid sequence is shown in SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 76, and SEQ ID NO: 78.

[0182] In some embodiments, the present disclosure provides isolated mRNA selected from amino acid sequences shown in SEQ ID NOs: 1-12 and SEQ ID NOs: 14-15, as well as the multimerization elements described in the pre-fusion hRSV F protein.

[0183] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown as SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-103, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 63, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 96-103, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 63, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 96-103.

[0184] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-103, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-103, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-103.

[0185] In some embodiments, the isolated mRNA includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, SEQ ID NO: 92-103, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, SEQ ID NO: 92-103, and may include, for example, a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, SEQ ID NO: 92-103.

[0186] In some embodiments, the isolated mRNA includes an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 63, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, SEQ ID NO: 96-103, or including a nucleotide sequence that is at least 80% identical to SEQ ID NO: 63, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, SEQ ID NO: 96-103, and may include, for example, a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, SEQ ID NO: 96-103.

[0187] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 63, SEQ ID NOs: 84-87, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 63, SEQ ID NOs: 84-87, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 63, SEQ ID NOs: 84-87.

[0188] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 63, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 63, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 63.

[0189] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 77, SEQ ID NOs: 96-99, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 77, SEQ ID NOs: 96-99, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 77, SEQ ID NOs: 96-99.

[0190] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 77, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 77, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 77, but is not limited to these.

[0191] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 79, SEQ ID NO: 100-103, or may, but is not limited to, a nucleotide sequence that is at least 80%, 85%, 95%, or 98% identical to SEQ ID NO: 79, SEQ ID NO: 100-103.

[0192] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) comprises a nucleotide sequence shown in SEQ ID NO: 79, or a nucleotide sequence that is at least 80% identical to SEQ ID NO: 79, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 79, but is not limited to these.

[0193] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 65, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 65, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 65.

[0194] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) comprises a nucleotide sequence shown in SEQ ID NO: 67, SEQ ID NOs: 88-91, or a nucleotide sequence that is at least 80% identical to SEQ ID NO: 67, SEQ ID NOs: 88-91, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 95%, or 98% identical to SEQ ID NO: 67, SEQ ID NOs: 88-91.

[0195] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 67, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 67, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 67, but is not limited to these.

[0196] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 69, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 69, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 69.

[0197] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 71, SEQ ID NOs: 92-95, or a nucleotide sequence that is at least 80% identical to SEQ ID NO: 71, SEQ ID NOs: 92-95, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 95%, or 98% identical to SEQ ID NO: 71, SEQ ID NOs: 92-95.

[0198] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 71, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 71, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 71, but is not limited to these.

[0199] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), the nucleotide sequence of the open reading frame (ORF) being shown in SEQ ID NO: 73, or comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 73, and may, for example, comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 73.

[0200] Furthermore, in some embodiments, the nucleotide sequences of the multimerization elements in the open reading frame (ORF) of the isolated mRNA are also shown in SEQ ID NOs: 36-47 and SEQ ID NO: 13.

[0201] In some embodiments, the present disclosure provides isolated DNA characterized by being obtained by reverse transcription of the isolated mRNA or mRNA molecule described in any one of the preceding paragraphs, or by being a sequence or mRNA molecule capable of transcribing the isolated mRNA described in any one of the preceding paragraphs.

[0202] In some embodiments, the isolated DNA is isolated chain DNA or isolated circular DNA.

[0203] In some embodiments, the present disclosure provides a biomaterial comprising an expression cassette, a vector, a process bacterium, or a cell line, which contains or expresses isolated mRNA or isolated DNA as described in any one of the preceding paragraphs.

[0204] In some embodiments, the Disclosure provides a method of administering to a subject an effective amount of at least one of the immunocompositions described in any one of the above, isolated mRNA described in any one of the above, isolated DNA described in any one of the above, or biomaterial described in any one of the above, in order to induce a neutralizing antibody response to human syncytial virus in the subject's body.

[0205] In some embodiments, the method comprises administering to a subject at least twice an effective amount of at least one of the immunocompositions, isolated mRNA, isolated DNA, or biomaterials described in any one of the above.

[0206] In some embodiments, the time interval between the first and second doses in the at least two doses is 14 days or more.

[0207] In some embodiments, the subjects have a weakened immune system. In some embodiments, the subjects are between 5 years old and 65 years old or older.

[0208] In some embodiments, the effective amount relating to this disclosure is 40 μg, 30 μg, 25 μg, 20 μg, 15 μg, 10 μg, 5 μg, 3 μg, or 1 μg of any one of the above-mentioned immunocompositions, any one of the above-mentioned isolated mRNA, or any one of the above-mentioned isolated DNA, or any one of the above-mentioned biomaterials. [Examples]

[0209] Example 1: RNA-based LNP production process These are lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens, comprising 50% Dlin-MC3-DMA, 20% DOPG, 29% cholesterol, and 1% PEG-DMG in mole percent.

[0210] The preparation method is as follows: (a) RNA was dissolved in citrate buffer at pH 4, and the concentration was adjusted to 0.1 mg / ml to obtain the aqueous phase. (b) Depending on the amount used, Dlin-MC3-DMA, DOPG, cholesterol, and PEG-DMG were dissolved in anhydrous ethanol to adjust the concentration of lipid components in the organic phase to 6 mg / mL, thereby obtaining the organic phase. (c) The aqueous phase of step (a) and the organic phase of step (b) were mixed in a volume ratio of 1:3 using a microfluidic apparatus at a flow rate of 12 mL / min. The mixture was immediately diluted 100-fold with pH 7.4 PBS solution, and the ethanol component in the solution was removed by tangential flow filtration (TFF). Subsequently, the mRNA concentration in the system was concentrated to 55 μg / ml to obtain lipid nanoparticles containing RNA encoding respiratory syncytial virus antigen.

[0211] Example 2 Luciferase was used as the reporter gene, and different vaccine carrier formulations were investigated using biofluorescence imaging technology (shown in Table 1 below).

[0212] [Table 1]

[0213] "MC 3" refers to Dlin-MC3-DMA, and "+" indicates that luciferase expression in mice was detected after administration using a small animal biofluorescence imaging system. The efficiency of delivering mRNA encoding the luciferase gene in mice and the physicochemical indicators of different compound formulations (see Example 1 for preparation methods) were detected, and the results are shown in Table 1. This study revealed that increasing the mass ratio of lipids to mRNA is advantageous in increasing the encapsulation rate of mRNA in lipid nanoparticles and providing higher stability, and that moderately increasing the polyethylene glycol (PEG) content in the formulation is advantageous in increasing the efficiency of mRNA expression in vivo. Therefore, considering factors such as mRNA encapsulation rate and mRNA delivery efficiency into the body, formulations 3 and 4 were selected and used in subsequent mRNA vaccine research.

[0214] Example 3 [Table 2]

[0215] As shown in Table 2, the ability of different formulations of cationic lipid nanoparticles to encapsulate luciferase-encoding mRNA and the particle size data of the nanoparticles formed, several formulations can compress luciferase mRNA into net surface potential neutral nanoparticles with a particle size of 100 nm or less, while simultaneously encapsulating at least 50% of the mRNA, thus all of which can provide a certain level of intracellular delivery effect. "MC3" refers to Dlin-MC3-DMA.

[0216] Example 4 BALB / c mice (female, 5-6 weeks old, average body weight 20-25g, purchased from ZhuHai Bestest Biotechnology Co., Ltd.) were selected, and the immunogenicity of the vaccine was evaluated.

[0217] The experimental animals were randomly divided into groups of 5 mice each. The immunization dose was 15 μg / mice, and the same dose of reinforcement immunization was administered 2 weeks after vaccination. Indicators of cellular and humoral immune responses were measured 12 days after secondary immunization.

[0218] Here, mouse lymphocytes were isolated using the following method: Mice were killed by decapitation and immersed in 75% ethanol. Mouse spleens were removed on a sterile workbench. 4-5 mL of mouse lymphocyte isolate (returned to room temperature and shaken uniformly before collection) was placed in a 35 mm petri dish. The isolated solution, after being polished and suspending the spleen cells, was immediately transferred to a 15 mL centrifuge tube and centrifuged at room temperature with an 800 g horizontal rotor for 30 min. After centrifugation, the lymphocyte layer was aspirated, and 10 mL of RPMI 1640 medium was added and washed upside down. Cells were collected by centrifugation at room temperature. The experimental flow can be seen in Figure 3.

[0219] 1. Detection of RSV-F protein-specific IgG antibodies in mouse serum by ELISA (humoral immunity) hRSV F protein wild-type (His Tag) (2 mg / mL), diluted to 2 μg / mL with washing solution, was added to a porous plate (100 μL per well) and coated overnight. After washing, 2% BSA solution was added for blocking.

[0220] All target serum samples were diluted with a washing solution in a 10-fold gradient from 1:100 to 1:1,000,000. The negative control sample was identical to the target serum sample and was diluted by the same factor, resulting in a total of six gradient samples.

[0221] The prepared serum sample solution was added to an enzyme-labeled porous plate, incubated, washed, and then diluted enzyme-labeled antibody solution was added. After incubation and washing, the plate was colorimetrically treated, and the OD values ​​at 450 nm / 630 nm were measured using a microplate reader.

[0222] The cutoff value was set at 2.1 times the arithmetic mean of the negative serum sample signal.

[0223] 2. TNFα by flow cytometry + CD4 + and TNF-α + CD8 + Detection of T cells (cellular immunity) Isolated mouse lymphocytes were collected, stimulated in a peptide bank, and cultured for 72 hours in a 5% CO2 culture box at 37°C. They were stimulated overnight with a BD GolgiPlμg™ (containing Brefeldin A) protein transport inhibitor (containing BSA) solution. The mouse lymphocyte suspension was centrifuged to retain the precipitate.

[0224] The mouse lymphocyte precipitate was resuspended, specific monoclonal fluorescent antibodies such as CD4 and CD8 were added, incubated, and then centrifuged to retain the mouse lymphocyte precipitate.

[0225] Fixation / permeation solution was added, incubated, and centrifuged to retain a precipitate (pellet) of mouse lymphocytes. Buffer solution was added to the mouse lymphocyte precipitate, mixed uniformly, and centrifuged to retain the mouse lymphocyte precipitate.

[0226] The mouse lymphocyte pellet was resuspended in staining buffer, cytokine fluorescent antibodies such as IFN-γ and TNF-α were added, incubated in the dark, and centrifuged to leave the mouse lymphocyte pellet. Then, the mouse lymphocyte pellet was resuspended in staining buffer and detected by flow cytometry.

[0227] CD3 + / CD4 + and CD3 + / CD8 + The expression of TNF-α in T lymphocytes of CD3 + / CD4 + and CD3 + / CD8 + was detected by flow cytometry.

[0228] 3. Detection of IFN-γ by flow cytometry + CD4 + and IFN-γ + CD8 + Detection of T cells (cellular immunity) Isolated mouse lymphocytes were collected, stimulated with a peptide bank, and cultured in a 37°C, 5% CO2 incubator for 72 h. They were stimulated overnight with a BD GolgiPlμgTM (containing brefeldin A) protein transport inhibitor (containing BSA) solution. The mouse lymphocyte suspension was centrifuged to leave the pellet.

[0229] The mouse lymphocyte pellet was resuspended, specific monoclonal fluorescent antibodies such as CD4 and CD8 were added, incubated, and centrifuged to leave the mouse lymphocyte pellet.

[0230] A fixation / permeabilization solution was added, incubated, and centrifuged to leave the mouse lymphocyte pellet. Buffer was added to the mouse lymphocyte pellet, mixed uniformly, and centrifuged to leave the mouse lymphocyte pellet.

[0231] The mouse lymphocyte pellet was resuspended in staining buffer, cytokine fluorescent antibodies such as IFN-γ and TNF-α were added, incubated in the dark, and centrifuged to leave the mouse lymphocyte pellet. Then, the mouse lymphocyte pellet was resuspended in staining buffer and detected by flow cytometry.

[0232] CD3 + / CD4 + and CD3 + / CD8 + The expression of IFN-γ in T lymphocytes of CD3 / CD4 and CD3 / CD8 was detected by flow cytometry.

[0233] 4. Detection of IFN-γ and IL2 in cell culture supernatant by ELISA (cellular immunity) Isolated mouse lymphocytes were collected, stimulated with a peptide bank, cultured in a 37°C, 5% CO2 incubator for 72 h, and the mouse lymphocyte suspension was centrifuged to collect the supernatant.

[0234] The IFN-γ and IL2 contents in the supernatant were detected using a mouse IL-2 ELISA kit (purchased from Neobioscience Technology Co., Ltd.) and a mouse IFN-γ ELISA kit (purchased from Neobioscience Technology Co., Ltd.).

[0235] 5. Detection of IL4 and IL10 in cell culture supernatant by ELISA (cellular immunity) Isolated mouse lymphocytes were collected, stimulated with a peptide bank, cultured in a 37°C, 5% CO2 incubator for 72 h, and the mouse lymphocyte suspension was centrifuged to collect the supernatant. The IL4 and IL10 contents in the supernatant were detected using a mouse IL4 ELISA kit (purchased from Neobioscience Technology Co., Ltd.) and a mouse IL10 ELISA kit (purchased from Neobioscience Technology Co., Ltd.).

[0236] 6. Neutralizing activity of serum The method for detecting the neutralizing activity of serum in the immunocomposition group is as follows. HEp-2 cells were digested, the cell density was adjusted, inoculated into a 96-well plate, and cultured overnight.

[0237] Each mouse serum diluent and virus diluent were added to the positive wells of the porous plate, and the virus diluent was added to the negative wells. Further, the diluted virus was added to the positive and negative wells of the porous plate, and the mixture was cultured and neutralized.

[0238] The neutralization products in the positive and negative wells of the porous plate were respectively added to a porous plate filled with digested HEp-2 cells and cultured. The neutralization products were aspirated, and DMEM + 2% FBS medium was added to each well to continue the culture.

[0239] The cells discarded the supernatant, 4% paraformaldehyde was added to each well to fix the cells, washed with PBS washing solution, and blocked with a 1:1 mixed solution of 4% BSA and 0.2% Triton. The blocking solution was discarded, F6-6-488 antibody diluent was added, incubated and washed. The porous plate was dehydrated by centrifugal force and read using a CTL device.

[0240] Example 5 The nucleotide sequences described in the following table are mRNA sequences, and the mRNA sequences described in the following table were respectively prepared as lipid nanoparticles containing RNA encoding respiratory syncytial virus antigen according to the method described in Example 1.

[0241]

Table 3

[0242] The mRNA related to the mRNA immunocomposition encoding respiratory syncytial virus antigen, in addition to the reading frame sequences in the above table, contains a 5' cap (m7Gppp(5')), 5'UTR (shown in SEQ ID NO: 16), 3'UTR (shown in SEQ ID NO: 19), and a 3' polyA tail consisting of 100 adenine nucleotides, and the uracil of the mRNA was replaced with 5' pseudouridine.

[0243] In addition to the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen mentioned above, the experiment also included an F-protein experimental group and a PBS blank control group.

[0244] Sample Wt was an RNA lipid nanoparticle containing the amino acid sequence encoding the wild-type RSV F protein. The amino acid sequences of the RSV F protein mutants from samples F7-5 were based on the wild-type RSV F protein, with the S155C / S190F / V207L / S290C substitution occurring and Fibritin Dominain (amino acid sequence shown in SEQ ID NO: 7) added to the C-terminus. The amino acid sequence of the RSV F protein mutant encoded by sample DS-Cav1 was based on the wild-type RSV F protein, with the S155C / S190F / V207L / S290C substitution occurring. The amino acid sequence of the RSV F protein mutant encoded by sample DS-Cav1+Tric was based on the wild-type RSV F protein, with the S155C / S190F / V207L / S290C substitution occurring, the TM sequence being deleted, and Fibritin Dominain (amino acid sequence shown in SEQ ID NO: 7) added to the C-terminus.

[0245] The prepared lipid nanoparticles of RNA encoding respiratory syncytial virus antigen were evaluated for vaccine immunogenicity according to the method of Example 4.

[0246] 1. TNFα by flow cytometry + CD4 + and TNF-α + CD8 + Detection of T cells The results are shown in Figure 4. From these results, the TNF-α group of mRNA immunocompositions + CD8 + It was revealed that there was no significant difference (P>0.01) between the mRNA immunocomposition group and the TNF-α group. + CD4 +There was no significant difference compared to the F-protein immunocomposition group (P>0.01). The mRNA immunocomposition group and the F-protein immunocomposition group were able to significantly stimulate the Th1-type cellular immune response compared to the PBS group (P<0.01).

[0247] 2. Detection of IFN-γ and IL-4 in cell culture supernatant by ELISA The IFN-γ detection results are shown in Figure 5. These results revealed that the IFN-γ levels in the mRNA immunotherapy group were not significantly different from those in the F-protein immunotherapy group (P>0.01). Both the mRNA immunotherapy group and the F-protein immunotherapy group significantly stimulated the Th1-type cellular immune response compared to the PBS group.

[0248] The IL-4 detection results are shown in Figure 5. From these results, it was clear that, with the exception of the DS-Cav1 immunocomposition group, IL-4 levels in the remaining mRNA immunocomposition groups were significantly higher than in the F-protein immunocomposition group (P<0.01). The mRNA immunocomposition group and the F-protein immunocomposition group were able to significantly stimulate the Th2-type cellular immune response compared to the PBS group.

[0249] 3. Serum neutralizing activity Figure 6 shows the results of detecting RSV neutralizing antibody titers in mouse serum by an experiment to reduce RSV live virus bacteriophage plaques 14 days after secondary immunization. Compared to the RSV F protein wild-type mRNA immunization group and other RSV F protein mutant mRNA immunization groups, the geometric mean titer of RSV neutralizing antibodies in the F7-5 immunization group was 10119.3, which was significantly higher than the other mRNA immunization groups, the F-Protein immunization group, and the PBS control group (P<0.01).

[0250] In this example, mRNA immunocompositions of RSV F protein mutants with S155C / S190F / V207L / S290C substitutions, based on the wild-type RSV F protein, showed significantly higher RSV neutralizing antibody titers in mouse serum when they retained the TM sequence and increased the multimerizing elements (as in sample F7-5) compared to those that retained only the TM sequence (as in sample DS-Cav1) or those that increased only the multimerizing elements (as in sample DS-Cav1+Tric).

[0251] Example 6 According to the method described in Example 2, lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens were prepared as samples Wt, F7, F7-3, F7-1, and F7-2, respectively.

[0252] The sequence of the RNA open reading frame in the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in sample Wt is shown in SEQ ID NO: 25, and the encoded respiratory syncytial virus antigen was the RSV F protein wild type (amino acid sequence shown in SEQ ID NO: 24).

[0253] The RNA open reading frame sequence in the lipid nanoparticle containing RNA encoding the respiratory syncytial virus antigen in sample F7 is shown in SEQ ID NO: 23, and the encoded respiratory syncytial virus antigen was an RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 22). The amino acid sequence of the encoded RSV F protein mutant described in sample F7 is based on the wild-type RSV F protein, with the S155C / S190F / V207L / S290C / D486C / D489C substitution, the P27 sequence replaced with the GS sequence shown in SEQ ID NO: 74, the TM sequence deleted, and Fibritin Dominain (amino acid sequence shown in SEQ ID NO: 7) added to the C-terminus.

[0254] The sequence of the RNA open reading frame in the lipid nanoparticle containing the RNA encoding the respiratory syncytial virus antigen according to Sample F7-3 is shown in SEQ ID NO: 29, and the encoded respiratory syncytial virus antigen was an RSV F protein mutant (the amino acid sequence is shown in SEQ ID NO: 28). The amino acid sequence of the encoded RSV F protein mutant described in Sample F7-3 was based on the wild-type RSV F protein, with S155C / S190F / V207L / S290C / D486C / D489C substitutions occurring, the P27 sequence replaced with the GS sequence shown in SEQ ID NO: 28, the TM sequence deleted, and a GCN4 Dominain (the amino acid sequence is shown in SEQ ID NO: 12) added to the C-terminus.

[0255] The sequence of the RNA open reading frame in the lipid nanoparticle containing the RNA encoding the respiratory syncytial virus antigen according to Sample F7-1 is shown in SEQ ID NO: 63, and the encoded respiratory syncytial virus antigen was an RSV F protein mutant (the amino acid sequence is shown in SEQ ID NO: 62). The amino acid sequence of the encoded RSV F protein mutant described in Sample F7-1 was based on the wild-type RSV F protein, with S155C / S190F / V207L / S290C / D486C / D489C substitutions occurring, the P27 sequence replaced with the GS sequence shown in SEQ ID NO: 74, and a Fibritin Dominain (the amino acid sequence is shown in SEQ ID NO: 7) added to the C-terminus.

[0256] The sequence of the RNA open reading frame in the lipid nanoparticle containing the RNA encoding the respiratory syncytial virus antigen according to Sample F7-2 is shown in SEQ ID NO: 65, and the encoded respiratory syncytial virus antigen was an RSV F protein mutant (the amino acid sequence is shown in SEQ ID NO: 64). The amino acid sequence of the encoded RSV F protein mutant described in Sample F7-2 was based on the wild-type RSV F protein, with S155C / S190F / V207L / S290C / D486C / D489C substitutions occurring, the P27 sequence replaced with the GS sequence shown in SEQ ID NO: 74.

[0257] The mRNA described in the mRNA immunocomposition encoding the respiratory syncytial virus antigen included, in addition to the above-mentioned reading frame sequence, a 5' cap (m7Gppp(5')), a 5' UTR (shown in SEQ ID NO: 16), a 3' UTR (shown in SEQ ID NO: 19), and a 3' poly-A tail consisting of 100 adenine nucleotides, and the uracil of the mRNA was substituted with 5' pseudouracil.

[0258] In addition to the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen mentioned above, the experiment also included an F-protein (purchased from Sino Biological, Inc.) experimental group and a PBS blank control group.

[0259] The prepared lipid nanoparticles of RNA encoding respiratory syncytial virus antigen were evaluated for vaccine immunogenicity according to the method of Example 4.

[0260] (1) Detection of RSV-F protein-specific IgG in serum by ELISA Fourteen days after secondary immunization, RSV-F protein-specific IgG antibodies were detected in mouse serum by ELISA. After diluting the serum 100-10,000,000 times, the OD450 was measured by indirect ELISA. Statistical analysis of the specific IgG antibody titers in mice from the RSV F protein mRNA immunization groups (see Figure 7) revealed that the IgG antibody titers of the RSV F protein wild-type mRNA immunization group (Wt) and the RSV F protein mutant mRNA immunization groups (F7, F7-3, F7-1, and F7-2) were not significantly different from those of the F-protein immunization group.

[0261] (2) Flow cytometry of TNFα + CD4 + and TNF-α + CD8 + Detection of T cells 14 days after immunization, mouse splenic lymphocytes were isolated and CD3 + / CD4 + and CD3+ / CD8 + TNF-α expression in T lymphocytes was detected by flow cytometry. The results are shown in Figure 8.

[0262] Based on these results, the TNF-α of the F7-3 immunocomposition group and the F7-2 immunocomposition group + CD8 + It was revealed that the TNF-α group showed significantly higher levels compared to the F-protein immunocomposition group (P<0.01). + CD4 + There was no significant difference compared to the F-protein immunocomposition group (P>0.01). TNF-α in the RSV F protein wild-type mRNA immunocomposition group (Wt), the RSV F protein mutant mRNA immunocomposition groups (F7, F7-3, F7-1 and F7-2), and the F-protein immunocomposition group + CD8 + / CD4 + This group showed significantly higher levels than the PBS group (P<0.01).

[0263] The results above clearly show that the RSV F protein wild-type mRNA immune composition group (Wt), the RSV F protein mutant mRNA immune composition groups (F7, F7-3, F7-1, and F7-2), and the F-protein immune composition group can all significantly stimulate the Th1-type cellular immune response in mice.

[0264] (3) Detection of IFN-γ and IL-2 in the supernatant of splenic T lymphocyte culture medium by ELISA IFN-γ and IL-2 expression in lymphocyte supernatant was detected by ELISA 14 days after immunization. See Figure 9 for results.

[0265] These results revealed that IFN-γ levels in the F7-3, F7-1, and F7-2 immunocomposition groups were significantly higher than in the F-protein immunocomposition group (P<0.01). IL-2 levels in the F7-3 and F7-2 immunocomposition groups were also significantly higher than in the F-protein immunocomposition group (P<0.01). IFN-γ and IL-2 levels in the RSV F protein wild-type mRNA immunocomposition group (Wt), the RSV F protein mutant mRNA immunocomposition groups (F7, F7-3, F7-1, and F7-2), and the F-protein immunocomposition group were significantly higher than in the PBS group (P<0.01).

[0266] (4) Detection of IL-4 and IL-10 in the supernatant of splenic T lymphocyte culture medium by ELISA Fourteen days after immunization, the expression of IL-4 and IL-10 in lymphocyte supernatant was detected by ELISA. The results are shown in Figure 10. From these results, it was clear that IL-4 levels in the F7-3 and F7-2 immunization groups were significantly higher than in the F-protein immunization group (P<0.01). IL-10 levels in the F7-1 immunization group were significantly higher than in the F-protein immunization group (P<0.01). IL-4 and IL-10 levels in the RSV F protein wild-type mRNA immunization group (Wt), the RSV F protein mutant mRNA immunization groups (F7, F7-3, F7-1, and F7-2), and the F-protein immunization group were significantly higher than in the PBS group (P<0.01).

[0267] The results above clearly show that the RSV F protein wild-type mRNA immune composition group (Wt), the RSV F protein mutant mRNA immune composition groups (F7, F7-3, F7-1, and F7-2), and the F-protein immune composition group were all able to significantly stimulate the Th2-type cellular immune response in mice.

[0268] (5) Serum titer of RSV neutralizing antibody Fourteen days after secondary immunization, the titer of RSV neutralizing antibodies in mouse serum was detected by an experiment to reduce the number of live RSV virus bacteriophage plaques. As shown in Figure 11, the geometric mean titer of RSV neutralizing antibodies in the F7-1 immunization group was 36162, which was significantly higher than that of the other mRNA immunization groups, compared to the RSV F protein wild-type mRNA immunization group (Wt), the RSV F protein mutant mRNA immunization groups (F7-3, F7, and F7-2), and the F-protein immunization group (P<0.01).

[0269] In this example, the group of RSV F protein mutant mRNA immunocompositions, based on the wild-type RSV F protein, underwent S155C / S190F / V207L / S290C substitutions, D486C / D489C substitutions, and P27 sequence substitution with the GS sequence shown in SEQ ID NO: 74. When the TM sequence was retained and the multimerization element was increased (as in sample F7-1), the RSV neutralizing antibody titer in mouse serum was significantly higher than when only the TM sequence was retained (as in sample F7-2) or only the multimerization element was increased (as in sample F7).

[0270] Example 7 According to the method described in Example 2, lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens were prepared as samples Wt, F7-1, DS-Cav1, and mVRC-1(v2), respectively.

[0271] The sequence of the RNA open reading frame in the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in sample Wt is shown in SEQ ID NO: 25, and the encoded respiratory syncytial virus antigen was the RSV F protein wild type (amino acid sequence shown in SEQ ID NO: 24).

[0272] The RNA open reading frame sequence in the lipid nanoparticle containing RNA encoding the respiratory syncytial virus antigen in sample F7-1 is shown in SEQ ID NO: 63, and the encoded respiratory syncytial virus antigen was an RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 62). The amino acid sequence of the encoded RSV F protein mutant described in sample F7-1 is based on the wild-type RSV F protein, with the S155C / S190F / V207L / S290C / D486C / D489C substitution, the P27 sequence being replaced with the GS sequence shown in SEQ ID NO: 74, and Fibritin Dominain (amino acid sequence shown in SEQ ID NO: 7) being added to the C-terminus.

[0273] The RNA open reading frame sequence in lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in sample DS-Cav1 is shown in SEQ ID NO: 27, and the encoded respiratory syncytial virus antigen was an RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 26). The amino acid sequence of the RSV F protein mutant encoded by sample DS-Cav1 is based on the wild-type RSV F protein, with the S155C / S190F / V207L / S290C substitution occurring.

[0274] The sequence of the RNA open reading frame in lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen from sample mVRC-1(v2) is shown in SEQ ID NO: 31, and the encoded respiratory syncytial virus antigen was the RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 30).

[0275] The mRNA described in the RNA immunocomposition encoding the respiratory syncytial virus antigen included, in addition to the above-mentioned reading frame sequence, a 5' cap (m7 Gppp(5')), a 5' UTR (shown in SEQ ID NO: 16), a 3' UTR (shown in SEQ ID NO: 19), and a 3' poly-A tail consisting of 100 adenine nucleotides, and the uracil of the mRNA was substituted with 5' pseudouracil.

[0276] In addition to the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen mentioned above, the experiment also included an F-protein experimental group and a PBS blank control group.

[0277] The prepared lipid nanoparticles of RNA encoding respiratory syncytial virus antigen were evaluated for vaccine immunogenicity according to the method of Example 4.

[0278] (1) Flow cytometry of TNFα + CD4 + and TNF-α + CD8 + Detection of T cells 14 days after immunization, mouse splenic lymphocytes were isolated and CD3 + / CD4 + and CD3 + / CD8 + TNF-α expression in T lymphocytes was detected by flow cytometry. The results are shown in Figure 12.

[0279] Based on these results, the TNF-α group of mRNA immunocompositions + CD8 + It was revealed that there was no significant difference (P>0.01) between the mRNA immunocomposition group and the TNF-α group. + CD4 + There was no significant difference compared to the F-protein immunocomposition group (P>0.01). Compared to the PBS group, both the mRNA immunocomposition group and the F-protein immunocomposition group were able to significantly stimulate the Th1-type cellular immune response.

[0280] (2) Flow cytometry of IFN-γ + CD4 + and IFN-γ + CD8 + Detection of T cells 14 days after immunization, mouse splenic lymphocytes were isolated and CD3 + / CD 4 + and CD3 + / CD8+ IFN-γ in T lymphocytes + The expression of [substance name] was detected by flow cytometry. The results are shown in Figure 13.

[0281] Based on these results, the mRNA immunocomposition group IFN-γ + CD8 + It was revealed that IFN-γ was significantly increased compared to the F-protein immunocomposition group (P<0.01). Excluding the mVRC-1(v2) immunocomposition group, the remaining mRNA immunocomposition groups showed a significant increase in IFN-γ. + CD4 + The levels were significantly higher in the mRNA immunocomposition group than in the F-protein immunocomposition group (P<0.01). Compared to the PBS group, both the mRNA immunocomposition group and the F-protein immunocomposition group were able to significantly stimulate the Th1-type cellular immune response.

[0282] (3) Detection of IFN-γ and IL-2 in the supernatant of splenic T lymphocyte culture medium by ELISA Fourteen days after immunization, IFN-γ and IL-2 expression in lymphocyte supernatant was detected by ELISA. The results are shown in Figure 14. From these results, it was clear that IFN-γ levels in the mRNA immunization group were not significantly different from those in the F-protein immunization group (P>0.01). Excluding the Wt immunization group, IL-2 levels in the remaining mRNA immunization groups were significantly elevated compared to the F-protein immunization group (P<0.01). Compared to the PBS group, both the mRNA immunization group and the F-protein immunization group were able to significantly stimulate the Th1-type cellular immune response.

[0283] (4) Detection of IL-4 and IL-10 in the supernatant of splenic T lymphocyte culture medium by ELISA Fourteen days after immunization, the expression of IL-4 and IL-10 in the lymphocyte supernatant was detected by ELISA. The results are shown in Figure 15. From these results, it was clear that, excluding the DS-Cav1 immunization group, IL-4 levels in the remaining mRNA immunization groups were significantly elevated compared to the F-protein immunization group (P<0.01). Excluding the DS-Cav1 immunization group, IL-10 levels in the remaining mRNA immunization groups were significantly elevated compared to the F-protein immunization group (P<0.01). Compared to the PBS group, both the mRNA immunization group and the F-protein immunization group were able to significantly stimulate the Th2-type cellular immune response.

[0284] (5) Serum titer of RSV neutralizing antibody Fourteen days after secondary immunization, the titer of RSV neutralizing antibodies in mouse serum was detected by an experiment to reduce the amount of RSV live virus bacteriophage plaques. As shown in Figure 16, the geometric mean titer of RSV neutralizing antibodies in the F7-1 immunization group was 20821, which was significantly higher than that of the other mRNA immunization groups (P<0.01).

[0285] Example 8 According to the method described in Example 2, lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens were prepared as samples Wt, F7-1, F7-2, F7-3, F7-4, F7-5, and F7-6, respectively.

[0286] The sequence of the RNA open reading frame in the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in sample Wt is shown in SEQ ID NO: 25, and the encoded respiratory syncytial virus antigen was the RSV F protein wild type (amino acid sequence shown in SEQ ID NO: 24).

[0287] The RNA open reading frame sequence in the lipid nanoparticle containing RNA encoding the respiratory syncytial virus antigen in sample F7-1 is shown in SEQ ID NO: 63, and the encoded respiratory syncytial virus antigen was an RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 62). The amino acid sequence of the encoded RSV F protein mutant described in sample F7-1 is based on the wild-type RSV F protein, with the S155C / S190F / V207L / S290C / D486C / D489C substitution, the P27 sequence being replaced with the GS sequence shown in SEQ ID NO: 74, and Fibritin Dominain (amino acid sequence shown in SEQ ID NO: 7) being added to the C-terminus.

[0288] The RNA open reading frame sequence in the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in sample F7-2 is shown in SEQ ID NO: 65, and the encoded respiratory syncytial virus antigen was an RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 64). The amino acid sequence of the encoded RSV F protein mutant described in sample F7-2 is based on the wild-type RSV F protein, with the S155C / S190F / V207L / S290C / D486C / D489C substitution occurring, and the P27 sequence being replaced with the GS sequence shown in SEQ ID NO: 74.

[0289] The RNA open reading frame sequence in the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in sample F7-3 is shown in SEQ ID NO: 67, and the encoded respiratory syncytial virus antigen was an RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 66). The amino acid sequence of the encoded RSV F protein mutant described in sample F7-3 is based on the wild-type RSV F protein, with the S155C / S190F / V207L / S290C / D486C / D489C substitution, the P27 sequence being replaced with the GS sequence shown in SEQ ID NO: 74, and GCN4 Dominain (amino acid sequence shown in SEQ ID NO: 12) being added to the C-terminus.

[0290] The RNA open reading frame sequence in the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in sample F7-4 is shown in SEQ ID NO: 69, and the encoded respiratory syncytial virus antigen was an RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 68). The amino acid sequence of the encoded RSV F protein mutant described in sample F7-4 is based on the wild-type RSV F protein, with the S155C / S190F / V207L / S290C substitution occurring, the P27 sequence being replaced with the GS sequence shown in SEQ ID NO: 74, and Fibritin Dominain (amino acid sequence shown in SEQ ID NO: 7) being added to the C-terminus.

[0291] The RNA open reading frame sequence in the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in sample F7-5 is shown in SEQ ID NO: 71, and the encoded respiratory syncytial virus antigen was an RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 70). The amino acid sequence of the RSV F protein mutant encoded by sample F7-5 is based on the wild-type RSV F protein, with the S155C / S190F / V207L / S290C substitution and the addition of fibritin Dominain (amino acid sequence shown in SEQ ID NO: 7) to the C-terminus.

[0292] The RNA open reading frame sequence in the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in sample F7-6 is shown in SEQ ID NO: 73, and the encoded respiratory syncytial virus antigen was an RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 72). The amino acid sequence of the encoded RSV F protein mutant described in sample F7-6 is based on the wild-type RSV F protein, with the S155C / S190F / V207L / S290C / D486C / D489C substitution and the addition of Fibritin Dominain (amino acid sequence shown in SEQ ID NO: 7) to the C-terminus.

[0293] The mRNA described in the RNA immunocomposition encoding the respiratory syncytial virus antigen included, in addition to the reading frame sequence described above, a 5' cap (m7Gppp(5')), a 5' UTR (shown in SEQ ID NO: 16), a 3' UTR (shown in SEQ ID NO: 19), and a 3' poly-A tail consisting of 100 adenine nucleotides, and the uracil of the mRNA was substituted with 5' pseudouracil.

[0294] In addition to the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen mentioned above, a PBS blank control group was also included in the experiment.

[0295] The prepared lipid nanoparticles of RNA encoding respiratory syncytial virus antigen were evaluated for vaccine immunogenicity according to the method of Example 4.

[0296] (1) Flow cytometry of TNFα + CD4 + and TNF-α + CD8 + Detection of T cells 14 days after immunization, mouse splenic lymphocytes were isolated and CD3 + / CD4 + and CD3 + / CD8 + TNF-α expression in T lymphocytes was detected by flow cytometry. The results are shown in Figure 17.

[0297] Based on these results, the F7-1 immunocomposition group contains TNF-α + CD8 + It was revealed that there was no significant difference (P>0.01) compared to the other mRNA immunocomposition groups. In the mRNA immunocomposition group, TNF-α + CD4 + No significant increase was detected (P>0.01). Compared to the PBS group, all mRNA immunocomposition groups were able to significantly stimulate the Th1-type cellular immune response.

[0298] (2) Flow cytometry of IFN-γ + CD4+ and IFN-γ + CD8 + Detection of T cells 14 days after immunization, mouse splenic lymphocytes were isolated and CD3 + / CD4 + and CD3 + / CD8 + IFN-γ in T lymphocytes + The expression of [substance name] was detected by flow cytometry. The results are shown in Figure 18.

[0299] Based on these results, excluding the F7-2 immunocomposition group, the IFN-γ of the F7-1 immunocomposition group... + CD8 + It was revealed that there was no significant difference compared to the other mRNA immunocomposition groups. In the mRNA immunocomposition group, IFN-γ + CD4 + No significant increase was detected (P>0.01). Compared to the PBS group, all mRNA immunocomposition groups were able to significantly stimulate the Th1-type cellular immune response.

[0300] (3) Detection of IFN-γ and IL-2 in the supernatant of splenic T lymphocyte culture medium by ELISA Fourteen days after immunization, IFN-γ and IL-2 expression in lymphocyte supernatant was detected by ELISA. The results are shown in Figure 19. From these results, it was clear that, with the exception of the F7-4 and F7-6 immunization groups, the IFN-γ expression in the F7-1 immunization group was not significantly different from that of the other mRNA immunization groups. In the F7-1 immunization group, IL-2 was significantly elevated compared to the other mRNA immunization groups (P<0.01). Compared to the PBS group, all mRNA immunization groups were able to significantly stimulate the Th1-type cellular immune response.

[0301] (4) Detection of IL-4 and IL-10 in the supernatant of splenic T lymphocyte culture medium by ELISA Fourteen days after immunization, IL-4 and IL-10 expression in lymphocyte supernatant was detected by ELISA. The results are shown in Figure 20. From these results, it was clear that, with the exception of the Wt and F7-3 immunization groups, IL-4 levels in the F7-1 immunization group were not significantly different from those in the other mRNA immunization groups. With the exception of the Wt, F7-2, and F7-3 immunization groups, IL-10 levels in the F7-1 immunization group were significantly elevated compared to those in the other mRNA immunization groups. Compared to the PBS group, all mRNA immunization groups were able to significantly stimulate the Th2-type cellular immune response.

[0302] (5) Serum titer of RSV neutralizing antibody Fourteen days after secondary immunization, the titer of RSV neutralizing antibodies in mouse serum was detected by an experiment to reduce the amount of RSV live virus bacteriophage plaques. The results are shown in Figure 21. As shown in the figure, the geometric mean titer of RSV neutralizing antibodies in the F7-1 immunization group was 20730, which was significantly higher than the other mRNA immunization groups and the F-Protein immunization groups (P<0.01). The geometric mean titer of RSV neutralizing antibodies in the F7-3 immunization group was 16026, and the geometric mean titer of RSV neutralizing antibodies in the F7-5 immunization group was 10871, which were significantly higher than the Wt, F7-2, F7-4, F7-6 immunization groups and the F-Protein immunization groups (P<0.01).

[0303] Example 9 According to the method described in Example 2, lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens were prepared as samples Wt, F7-1, F7-a, and F7-b, respectively.

[0304] The sequence of the RNA open reading frame in the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in sample Wt is shown in SEQ ID NO: 25, and the encoded respiratory syncytial virus antigen was the RSV F protein wild type (amino acid sequence shown in SEQ ID NO: 24).

[0305] The sequence of the RNA open reading frame in the lipid nanoparticle containing RNA encoding the respiratory syncytial virus antigen in sample F7-1 is shown in SEQ ID NO: 63, and the encoded respiratory syncytial virus antigen was the RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 62).

[0306] The sequence of the RNA open reading frame in the lipid nanoparticle containing RNA encoding the respiratory syncytial virus antigen in sample F7-a is shown in SEQ ID NO: 77, and the encoded respiratory syncytial virus antigen was the RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 76).

[0307] The RNA open reading frame sequence in the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in sample F7-b is shown in SEQ ID NO: 79, and the encoded respiratory syncytial virus antigen was the RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 78).

[0308] In addition to the nucleotide ORF sequences described above, the sequences of these RNA vaccines included a 5' cap (m7Gppp(5')), a 5' UTR (shown in SEQ ID NO: 16), a 3' UTR (shown in SEQ ID NO: 19), and a 3' poly-A tail consisting of 100 adenine nucleotides, with the uracil in the mRNA being replaced with 5' pseudouracil.

[0309] In addition to the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen mentioned above, a PBS blank control group was also included in the experiment.

[0310] The lipid nanoparticles of RNA encoding the prepared respiratory syncytial virus antigen were evaluated in serum for RSV neutralizing antibody titers according to the method of Example 4.

[0311] Fourteen days after secondary immunization, the titer of RSV neutralizing antibodies in mouse serum was detected by an experiment to reduce the amount of RSV live virus bacteriophage plaques. As shown in Figure 22, the geometric mean titers of RSV neutralizing antibodies in the F7-1, F7-a, and F7-b immunization groups all exceeded 18,000 compared to the Wt immunization group, which was significantly higher than the Wt immunization group (P<0.01).

[0312] Example 10 The nucleotide sequences listed in the table below are mRNA sequences. Cells were transfected with the mRNAs shown in the table below, and the expression of full-length RSV F protein in the cells was detected. The results are shown in the table below.

[0313] The detailed method is as follows: HEK 293 cells were transfected with each mRNA for 24 hours, then degraded. A sample of 10 μg of total protein was used to specifically detect the target protein using SDS-PAGE immunoblotting. In this example, anti-RSV F protein antibody was used as the primary antibody and goat anti-mouse-HRP antibody as the secondary antibody. After incubation, color development was performed. When the protein expression levels were analyzed, standardized quantification was performed using β-actin as an internal standard. Simultaneously, cells that were not transfected with mRNA were set as a negative control, and the difference in the amount of expressed protein after transfecting cells with different mRNAs was compared. The results showed that the expression of the full-length RSV F protein could be detected in all cases. The expression level of each sequence was measured using relative OD values, as shown in the table below. Here, the relative OD value was calculated as OD value of sample / OD value of sample 1.

[0314] [Table 4]

[0315] Example 11 The RNA sequences numbered 1 to 9 in Table 4 of Example 10 were each prepared as lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens according to the method described in Example 2, and these were designated as Samples 01 to 09.

[0316] In addition to the nucleotide sequences described above, the sequences of these RNA vaccines included a 5' cap (m7Gppp(5')) and a 3' poly-A tail consisting of 100 adenine nucleotides, and the uracil in the mRNA was replaced with 5' pseudouracil.

[0317] In addition to the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen mentioned above, a PBS blank control group was added to the experiment, and the sample weight (Wt) prepared according to Example 9 was predicted.

[0318] The lipid nanoparticles of RNA encoding the prepared respiratory syncytial virus antigen were evaluated in serum for RSV neutralizing antibody titers according to the method of Example 4.

[0319] Fourteen days after secondary immunization, the titer of RSV neutralizing antibodies in mouse serum was detected by an experiment to reduce the amount of RSV live virus bacteriophage plaques. As shown in Figure 23, compared to the Wt immunization composition group, the geometric mean titers of RSV neutralizing antibodies in samples 01-09 all exceeded 10,000, which was significantly higher than that of the Wt immunization composition group (P<0.01). Furthermore, the geometric mean titer of RSV neutralizing antibodies in sample 09 was slightly higher than that of the other samples 01-08.

[0320] Example 12 Lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens were prepared according to the method described in Example 2, as samples Wt, F124-F138, respectively.

[0321] The sequence of the RNA open reading frame in the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in sample Wt is shown in SEQ ID NO: 25, and the encoded respiratory syncytial virus antigen was the RSV F protein wild type (amino acid sequence shown in SEQ ID NO: 24).

[0322] The sequences of the RNA open reading frames in lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in samples 124 to 128 are shown in SEQ ID NO: 63 and SEQ ID NOs: 84-87, and the encoded respiratory syncytial virus antigen was the RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 62).

[0323] The sequences of the RNA open reading frames in lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in samples 129 to 133 are shown in SEQ ID NO: 67 and SEQ ID NOs: 88-91, and the encoded respiratory syncytial virus antigen was the RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 66).

[0324] The sequences of the RNA open reading frames in lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in samples 134 to 138 are shown in SEQ ID NO: 71 and SEQ ID NOs: 92-95, and the encoded respiratory syncytial virus antigen was the RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 70).

[0325] The sequences of the RNA open reading frames in lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen in samples 139 to 143 are shown in SEQ ID NO: 77 and SEQ ID NOs: 96-99, and the encoded respiratory syncytial virus antigen was the RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 76).

[0326] The RNA open reading frame sequences in lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens in samples 144 to 148 are shown in SEQ ID NO: 79 and SEQ ID NOs: 100-103, and the encoded respiratory syncytial virus antigen was the RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 78).

[0327] The sequence of the RNA open reading frame in the lipid nanoparticles containing the RNA encoding the respiratory syncytial virus antigen, as shown in SEQ ID NO: 103, indicated that the encoded respiratory syncytial virus antigen was the RSV F protein mutant (amino acid sequence shown in SEQ ID NO: 78).

[0328] In addition to the nucleotide ORF sequences described above, the sequences of these RNA vaccines included a 5' cap (m7Gppp(5')), a 5' UTR (shown in SEQ ID NO: 16), a 3' UTR (shown in SEQ ID NO: 19), and a 3' poly-A tail consisting of 100 adenine nucleotides, with the uracil in the mRNA being replaced with 5' pseudouracil.

[0329] In addition to the lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen mentioned above, a PBS blank control group was also included in the experiment.

[0330] The lipid nanoparticles of RNA encoding the prepared respiratory syncytial virus antigen were evaluated in serum for RSV neutralizing antibody titers according to the method of Example 4.

[0331] Fourteen days after secondary immunization, the titer of RSV neutralizing antibodies in mouse serum was detected by an experiment to reduce the amount of RSV live virus bacteriophage plaques. As shown in Figures 24-26, the geometric mean titers of RSV neutralizing antibodies in the F124-F148 immunization groups all exceeded 10,000 compared to the Wt immunization group, which was significantly higher (P<0.01).

[0332] Respiratory syncytial virus antigens that edit the amino acid sequences shown in SEQ ID NO: 62, SEQ ID NO: 76, and SEQ ID NO: 78 are RSV F protein mutants. The RSV neutralizing antibody titers in samples 124-128 and 140-148 were higher than those in the other samples.

[0333] Example 13 We selected BALB / c mice (female, 5 weeks old, average weight approximately 19g, purchased from ZhuHai Bestest Biotechnology Co., Ltd.) and performed vaccine immunogenicity evaluation. The experimental animals were divided into five random groups of six mice each, and the administration methods were as follows: (1) Vegetable group: 50 μg / mouse intramuscularly injected with PBS buffer containing blank lipid nanoparticles; (2) F protein group: 50 μg / mouse intramuscularly injected with PBS buffer containing 0.05 μg of pre-fusion hRSV F protein; (3) F7-1 (5 μg) group: 25 μg / mouse intramuscularly injected with PBS buffer containing 5 μg of lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen, the RNA open reading frame sequence of which is shown in SEQ ID NO: 63; (4) F7-1 (15 μg) group: 75 μg / mouse intramuscularly injected with PBS buffer containing 15 μg of lipid nanoparticles containing RNA encoding the respiratory syncytial virus antigen, the RNA open reading frame sequence of which is shown in SEQ ID NO: 63; (5) Healthy group: No administration. On day 21 after vaccination, the same dose of enhanced immunization was administered. Serum samples were collected on day 35 after vaccination, and the animals were killed and lung tissue collected on day 40 after vaccination. On day 35 after vaccination, RSV / A2 virus was administered intranasally to mice in groups 1-4. Mouse body weight and body weight changes during the experiment, as well as RSV-F protein-specific IgG antibodies, serum neutralizing activity, detection of viral titers in mouse lung tissue after RSV infection, and detection of lung histopathology in mice after RSV infection were monitored. For the method of detecting RSV-F protein-specific IgG antibodies and serum neutralizing activity, refer to Example 4 of this disclosure. Lung tissue lesions, perivascular, peribronchial, alveolar, and interstitial inflammation were evaluated by HE staining. Lung tissue viral titers were measured by plaque assay.

[0334] Lipid nanoparticles containing RNA encoding respiratory syncytial virus antigen were prepared as Sample F7-1 according to the method described in Example 2. In addition to the nucleotide ORF sequences described above, the sequences of these RNA vaccines included a 5' cap (m7Gppp(5')), a 5' UTR (shown in SEQ ID NO: 16), a 3' UTR (shown in SEQ ID NO: 19), and a 3' poly-A tail consisting of 100 adenine nucleotides, and the uracil of the mRNA was substituted with 5' pseudouracil.

[0335] (1) Mouse body weight and changes in body weight Figures 27 and 28 show the body weight and changes in body weight of the mice during the experimental process. Two to five days after the first vaccination, the body weight of the F7-1 (5 μg) and F7-1 (15 μg) immunized groups decreased significantly (Figure 27). The F7-1 (5 μg) immunized group lost about 5% of its body weight two days after the first vaccination, while the F7-1 (15 μg) immunized group lost about 10% of its body weight two days after the first vaccination (Figure 28). One day after the second vaccination (22 days after the first vaccination), the body weight of the F7-1 (5 μg) and F7-1 (15 μg) immunized groups decreased significantly (Figure 27). The F7-1 (5 μg) immunized group lost about 5% of its body weight two days after the second vaccination, while the F7-1 (15 μg) immunized group lost about 10% of its body weight two days after the second vaccination (Figure 28). Compared to before nasal administration of the RSV / A2 virus, on day 5 after attack, the body weight of the vehicle immunization group decreased by approximately 3%, the body weight of the F protein immunization group decreased by approximately 5%, the body weight of the F7-1 (5 μg) immunization group decreased by approximately 4%, but the body weight of the F7-1 (15 μg) immunization group increased by 1%.

[0336] (2) Detection of RSV-F protein-specific IgG in serum by ELISA 35 days after the initial vaccination, RSV-F protein-specific IgG antibodies were detected in mouse serum by ELISA. After diluting the serum 100- to 8,000,000 times, the OD450 was measured by indirect ELISA.

[0337] A sample is considered positive if its OD450 is greater than or equal to twice the mean OD450 of the negative control group at the minimum dilution ratio (1:100) (Bao-Zhong Wang, et al., PLoS ONE. 2010; Teena Mohan, et al., J Control Release. 2016). The final dilution titer was reached when serum samples from the F7-1 (15 μg) immunization group were diluted 8 million times.

[0338] Figure 29 shows the statistical results of RSV-F protein-specific IgG antibody titers in serum. These results clearly show that the F7-1 (15 μg) immunization group had the highest IgG titer GMT value. The IgG antibody titers in the F7-1 (5 μg) and F7-1 (15 μg) immunization groups were significantly higher than those in the F protein immunization group.

[0339] (3) Serum titer of RSV neutralizing antibody 35 days after the initial vaccination, RSV neutralizing antibody titers in mouse serum were detected by a plaque reduction experiment using RSV-A / B venomous strain virus bacteriophage. As shown in the left panel of Figure 30, the neutralizing antibody titers against the RSV-A2 venomous strain were significantly higher in the F7-1 (5 μg) (GMT=20030) and F7-1 (15 μg) (GMT=27335) immunized groups than in the F protein (GMT=54) immunized group (P<0.05). There was no significant difference between the F7-1 (5 μg) immunized group and the F7-1 (15 μg) immunized group, suggesting that there is no significant difference between the high and low dose groups of the F7-1 vaccine. As shown in the right-hand panel of Figure 30, the neutralizing antibody titers against the RSV-18537 strain were significantly higher in the F7-1 (5 μg) (GMT=4177) and F7-1 (15 μg) (GMT=9392) immunization groups than in the F protein (GMT=37) immunization group (P<0.05). However, there was no significant difference in neutralizing antibody titers between the F7-1 (5 μg) immunization group and the F7-1 (15 μg) immunization group (P>0.05), suggesting that there was no significant difference between the high-dose and low-dose F7-1 groups.

[0340] (4) Mouse lung tissue virus titer after RSV infection Five days after RSV infection, RSV virus titers were measured in homogenized right lung tissue of mice after nasal inoculation using a Plaque Reduction Neutralization Test (see Figure 31). The lung tissue virus titers in the immunized group were significantly higher than those in the F protein, F7-1 (5 μg), F7-1 (15 μg), and healthy control groups. Here, F7-1 (5 μg), F7-1 (15 μg), and healthy control groups were all at the lower limit of quantification (LLOQ). These results demonstrate that the F protein (RSV inactivated vaccine), F7-1 (5 μg), and F7-1 (15 μg) mRNA vaccines can significantly reduce the lung tissue virus titers of mice in the immunized group.

[0341] (5) Lung histopathology of mice after RSV infection Five days after RSV infection, lung tissue lesions (perivascular, peribronchial, alveolar, and interstitial inflammation) were evaluated by HE staining. As shown in Figure 32, the scores for perivascular, peribronchial, alveolar, and interstitial inflammation were significantly higher in the F protein immunization group than in the media immunization group. Mild pathological reactions were observed in the media, 0.07-5μg, and 0.07-15μg immunization groups after RSV infection. These results indicate that the F7-1(5μg) and F7-1(15μg) immunization groups can induce high-titer IgG-specific and neutralizing antibodies to protect animals from RSV infection without causing vaccine-enhanced respiratory disease (VERD).

[0342] Finally, while the above embodiments are used solely to illustrate the technical ideas of the Disclosure and are not limitations thereof, those skilled in the art can modify the technical ideas described in the above embodiments or replace some or all of their technical features with equivalent ones, and such modifications or replacements will not cause the essence of the corresponding technical ideas to deviate from the scope of the technical ideas of the embodiments of the Disclosure.

Claims

1. An immunocomposition comprising or encoding human respiratory syncytial virus antigen, selected from the group consisting of nucleic acid immunocompositions, polypeptide immunocompositions, or viral immunocompositions, Preferably, the antigen is a pre-fusion hRSV F protein whose amino acid sequence includes a TM sequence and a multimerizing element, preferably the amino acid sequence of the multimerizing element is shown in SEQ ID NOs: 1-12 and SEQ ID NOs: 14-15, and preferably the amino acid sequence of the multimerizing element is one of those shown in SEQ ID NOs: 7 and SEQ ID NOs: 12, characterized in that the immunocomposition is characterized in that.

2. The P27 sequence of the pre-fusion hRSV F protein is replaced with a GS sequence, preferably the GS sequence comprises (GnS)m, (GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q, SEQ ID NOs: 48-61, SEQ ID NOs: 74 (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, and q is an integer from 1 to 5), preferably the amino acid sequence of the GS sequence is shown in SEQ ID NOs:

74. Preferably, the pre-fusion hRSV F protein contains at least one of the following substitutions: S46G substitution, N67I substitution, E92D substitution, P102A substitution, A149C substitution, S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, L373R substitution, S215P substitution, I379V substitution, M447V substitution, Y458C substitution, K465Q substitution, D486C substitution, and D489C substitution, as described in claim 1.

3. The amino acid sequence of the pre-fusion hRSV F protein includes at least one of the following substitutions: S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, D486C substitution, and D489C substitution. Preferably, the amino acid sequence of the pre-fusion hRSV F protein includes at least one of the following: S155C substitution, S190F substitution, V207L ​​substitution, and / or the P27 sequence of the pre-fusion hRSV F protein is substituted with a GS sequence. Preferably, the amino acid sequence of the pre-fusion hRSV F protein includes an S155C substitution, an S190F substitution, a V207L ​​substitution, and an S290C substitution, the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74, and the pre-fusion hRSV F protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO:

12. Preferably, the amino acid sequence of the pre-fusion hRSV F protein includes an S155C substitution, an S190F substitution, a V207L ​​substitution, and an S290C substitution, the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74, the pre-fusion hRSV F protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO: 12, and the amino acid sequence of the pre-fusion hRSV F protein further includes at least one of the following substitutions: S46G substitution, N67I substitution, E92D substitution, P102A substitution, A149C substitution, L373R substitution, S215P substitution, I379V substitution, M447V substitution, Y458C substitution, K465Q substitution, D486C substitution, and D489C substitution. Preferably, the amino acid sequence of the pre-fusion hRSV F protein includes at least one of the following substitutions: S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, D486C substitution, and / or the P27 sequence of the pre-fusion hRSV F protein is substituted with a GS sequence. Preferably, the amino acid sequence of the pre-fusion hRSV F protein includes S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, D486C substitution, and D489C substitution, the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74, and the pre-fusion hRSV F protein includes one of the polymerizing elements shown in SEQ ID NO: 7 and SEQ ID NO:

12. Preferably, the amino acid sequence of the pre-fusion hRSV F protein includes an S155C substitution, an S190F substitution, a V207L ​​substitution, an S290C substitution, a D486C substitution, or a D489C substitution; the P27 sequence of the pre-fusion hRSV F protein is substituted with the GS sequence shown in SEQ ID NO: 74; the pre-fusion hRSV F protein includes one of the polymerizing elements shown in SEQ ID NO: 7 or SEQ ID NO: 12; and the amino acid sequence of the pre-fusion hRSV F protein includes at least one of the following substitutions: an S46G substitution, an N67I substitution, an E92D substitution, a P102A substitution, an A149C substitution, an L373R substitution, an S215P substitution, an I379V substitution, an M447V substitution, a Y458C substitution, or a K465Q substitution, as described in claim 1 or 2.

4. The immunocomposition according to claim 1 or 2, characterized in that the pre-fusion hRSV F protein contains an amino acid sequence in which at least one amino acid in the F2 sequence, RR1 sequence, RR2 sequence, 525-529 sequence, or 551-574 sequence is deleted consecutively or intermittently.

5. The amino acid sequence of the pre-fusion hRSV F protein is shown in SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 76, and SEQ ID NO:

78. Preferably, the amino acid sequence of the pre-fusion hRSV F protein is shown in SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 76, SEQ ID NO: 78, Preferably, the amino acid sequence of the pre-fusion hRSV F protein is shown in SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 76, and SEQ ID NO: 78, the immunocomposition according to claim 1 or 2.

6. The immunocomposition according to claim 5, wherein the polymerizing element of the pre-fusion hRSV F protein is further selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 1-12 and SEQ ID NOs: 14-15.

7. The aforementioned immune composition is a nucleic acid immune composition, the nucleic acid immune composition comprises nucleic acid molecules, the nucleic acid molecules comprising DNA molecules and / or RNA molecules, preferably the DNA molecules comprising chain DNA molecules and / or circular DNA molecules, preferably the RNA molecules comprising mRNA or circular RNA, preferably the mRNA molecules comprising open reading frames (ORFs) whose nucleotide sequences are shown in SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-103. Preferably, the mRNA molecule includes an open reading frame (ORF) whose nucleotide sequence is shown in SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-103. Preferably, the mRNA molecule includes an open reading frame (ORF) whose nucleotide sequence is shown in SEQ ID NO: 63, SEQ ID NO: 71, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, and SEQ ID NO: 92-103. Preferably, the mRNA molecule includes an open reading frame (ORF) whose nucleotide sequence is shown in SEQ ID NO: 63, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84-87, and SEQ ID NO: 96-103. Preferably, the mRNA molecule includes an open reading frame (ORF) whose nucleotide sequence is shown in SEQ ID NO:

67. Preferably, the mRNA molecule includes an open reading frame (ORF) whose nucleotide sequence is shown in SEQ ID NO:

71. Preferably, the nucleotide sequences of the polymerizing elements in the open reading frame (ORF) are also shown in SEQ ID NOs: 36-47 and SEQ ID NO: 13, as described in claim 1 or 2.

8. The immunocomposition according to claim 1 or 2, wherein the immunocomposition comprises a delivery formulation, preferably the delivery formulation comprises lipid nanoparticles or cationic liposomes.

9. A method for producing an immune composition according to claim 7, characterized by mixing the mRNA with a delivery formulation containing lipid nanoparticles or cationic liposomes to form an immune composition.

10. The coding region nucleic acid sequence of the mRNA encodes a pre-fusion hRSV F protein whose amino acid sequence includes a TM sequence and a multimerizing element, preferably the amino acid sequence of the multimerizing element is shown in SEQ ID NOs: 1-12 and SEQ ID NOs: 14-15, and preferably the amino acid sequence of the multimerizing element is one of those shown in SEQ ID NOs: 7 and SEQ ID NOs:

12. Preferably, the mRNA further comprises at least one of a 5' terminal cap, a 5' UTR, a 3' UTR, and a 3' terminal poly-A tail, and preferably, the 5' terminal cap is selected from the group consisting of ARCA, mCAP, dmCAP, tmCAP, m7G(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, dmCAP, or m7G(5')ppp(5')(2'OMeG)pG. Preferably, the length of the 3' end poly A tail is 50 to 200, preferably, the length of the 3' end poly A tail is 80 to 200, preferably, the length of the 3' end poly A tail is 80 to 120, preferably, the length of the 3' end poly A tail is 120. Preferably, the length of the 5'UTR is 10 to 200 nucleotides, preferably 15 to 100 nucleotides, and preferably the nucleotide sequence of the 5'UTR is shown in SEQ ID NOs: 16-18, and preferably the nucleotide sequence of the 5'UTR is shown in SEQ ID NO:

16. Preferably, the sequence of the 3'UTR is shown in sequence numbers 19 to 21, and preferably, the sequence of the 3'UTR is shown in sequence number 19. Isolated mRNA, preferably characterized in that one or more uridines in the mRNA are replaced with modified nucleosides, preferably the modified nucleosides being pseudouridine (Ψ), N1-methyl-psoidouridine (m1Ψ), or 5-methyluridine (m5U) (m5U), and preferably the modified nucleosides being N1-methyl-psoidouridine (m1Ψ).

11. Isolated DNA, characterized by being obtained by reverse transcription of the isolated mRNA nucleic acid described in claim 10, or by being a sequence capable of transcribing the isolated mRNA described in claim 10.

12. A biomaterial comprising an expression cassette, a vector, a process bacterium, or a cell line, wherein the biomaterial contains or expresses isolated mRNA according to claim 10 or isolated DNA according to claim 11.

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