Immune compositions comprising human respiratory syncytial virus antigens, methods of making and using the same

Abstract

The present disclosure relates to the technical field of gene drugs, and particularly relates to an immunological composition containing a human respiratory syncytial virus antigen, wherein the human respiratory syncytial virus (hRSV) antigen is a pre-fusion hRSV F protein.

Description

[0001] This application claims priority to Chinese Patent Application (Application No. 202310570847.2, Invention Title: Respiratory Syncytial Virus Vaccine and Preparation Method and Application Thereof) with a filing date of May 19, 2023, the contents of which are incorporated by reference in their entirety into this application. TECHNICAL FIELD

[0002] The present disclosure relates to the technical field of gene drugs, and in particular to an immunological composition comprising human respiratory syncytial virus (hRSV) antigens, wherein the hRSV antigens are pre-fusion hRSV F proteins. BACKGROUND

[0003] Respiratory syncytial virus (RSV) is the most common pathogen of bronchiolitis and pneumonia in infants and young children, and seriously endangers the health of infants and young children. After RSV infection, no long-term immunity can be obtained, and therefore, repeated infection is very common. RSV infection occurs globally and can cause local outbreaks, and has become a worldwide public health problem. However, there is currently no approved RSV vaccine.

[0004] The RSV genome is about 15-16 kb in size, encoding 11 proteins, including 8 structural proteins and 3 non-structural proteins (NS1, NS2 and M2-2), the structural proteins including 3 transmembrane surface proteins (G, F, SH), 2 matrix proteins (M and M2-1), 3 nucleocapsid 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 with the host cell membrane, allowing the virus to enter the cell, both of which are essential for viral replication, and both contain B cell and T cell epitopes, and are the most important viral antigen proteins for stimulating the body to produce humoral and cellular immunity. The G protein coding region varies greatly, and can be divided into A and B subtypes based on its variation, and the neutralizing antibodies induced by the G protein have subtype specificity; the F protein coding region is highly conserved, and the F protein amino acid sequences of A and B subtypes are at least 90% identical, so the neutralizing antibodies induced by the F protein can simultaneously inhibit A and B subtypes of RSV infection. The structure of the F protein is dynamic, first being transcribed and translated into a single inactive polypeptide (F0) in the host cell; then the first furin protease cleavage generates a partially cleaved prefusogenic protein; then the second furin protease cleavage generates F2 and F1 subunits, which are connected into a monomer by two disulfide covalent bonds, and then three monomers form a metastable functional prefusion protein trimer; thereafter, without further processing, conformational rearrangement occurs to form a thermodynamically stable postfusion protein; the time and cellular location of the two furin protease cleavages and conformational rearrangement are not completely clear, and the conditions that induce conformational rearrangement are not well understood. There are six antigen epitopes related to neutralizing activity on the surface of the F protein, including I, II, III, IV, V, and Ø, of which epitopes I, II, III, and IV are present in both the prefusion and postfusion F proteins; V and Ø are specific antigen sites of the prefusion F protein, and the postfusion F protein does not have these two epitopes. Epidemiological studies have shown that RSV neutralizing antibodies can prevent severe RSV-ALRI, and the neutralizing activity of epitope VIII monoclonal antibodies is 10-100 times higher than that of epitope II monoclonal antibodies, and the neutralizing activity of epitope VIII monoclonal antibodies is also very high, therefore, most of the RSV neutralizing activity in serum is only directed against the prefusion F protein antigen sites. Inducing high-titer neutralizing antibodies is the main goal of RSV vaccine development, and the prefusion F protein with specific antigen epitopes has become the most popular target for RSV vaccine development, but the prefusion F protein is essentially an unstable protein, and various stability modifications without losing important antigen epitopes are one of the difficulties in RSV vaccine development. SUMMARY

[0005] In some embodiments, the present disclosure provides an immunological composition comprising a human respiratory syncytial virus antigen, which is capable of eliciting an effective neutralizing antibody response against a human respiratory syncytial virus (hRSV) antigen.

[0006] Some aspects of the present disclosure provide an immunological composition comprising a human respiratory syncytial virus antigen, which is selected from a nucleic acid immunological composition, a polypeptide immunological composition, or a viral immunological composition.

[0007] In some embodiments, the human respiratory syncytial virus antigen is a pre-fusion hRSV F protein, the F1 sequence of which is set forth in Seq ID NO. 10-11, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 10, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 10; the F2 sequence of which is set forth in Seq ID NO. 2-5, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 2-5, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 2-5; the RR1 sequence of which is set forth in Seq ID NO. 6-9, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 6-9, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 6-9; the RR2 sequence of which is set forth in Seq ID NO. 12-20, Seq ID NO. 88-93, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 12-20, Seq ID NO. 88-93, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 12-20, Seq ID NO. 88-93; the C-terminal sequence of which is set forth in Seq ID NO. 21-57, Seq ID NO. 85-87, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 21-57, Seq ID NO. 85-87, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 21-57, Seq ID NO. 85-87.

[0008] In some embodiments, the P27 sequence of the pre-fusion hRSV F protein is set forth in Seq ID NO. 76; the F2 sequence is set forth in Seq ID NO. 3, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 3, for example, can be, but is not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 3; the RR1 sequence is set forth in Seq ID NO. 7, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 7, for example, can be, but is not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 7; the Fl sequence is set forth in Seq ID NO. 10, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 10, for example, can be, but is not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 10; the RR2 sequence is set forth in Seq ID NO. 91, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 91, for example, can be, but is not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 91; and the C-terminal sequence is set forth in Seq ID NO. 85-87, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 85-87, for example, can be, but is not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 85-87.

[0009] In some embodiments, the F2 sequence of the pre-fusion hRSV F protein is connected to the RR1 sequence by a GS-linker sequence, the F2 sequence is set forth in Seq ID NO. 3, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 3, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 3; the RR1 sequence is set forth in Seq ID NO. 7, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 7, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 7; the Fl sequence is set forth in Seq ID NO. 10, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 10, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 10; the RR2 sequence is set forth in Seq ID NO. 91, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 91, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 91; and the C-terminal sequence is set forth in Seq ID NO. 85-87, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 85-87, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 85-87.

[0010] In some embodiments, the F2 sequence of the pre-fusion hRSV F protein is connected to the RR1 sequence by a GS-linker sequence, the F2 sequence is set forth in Seq ID NO. 3, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 3, for example, can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 3; the RR1 sequence is set forth in Seq ID NO. 7, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 7, for example, can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 7; the Fl sequence is set forth in Seq ID NO. 10, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 10, for example, can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 10; the RR2 sequence is set forth in Seq ID NO. 91, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 91, for example, can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 91; the C-terminal sequence is set forth in Seq ID NO. 21-57, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 21-57, for example, can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 21-57; in some embodiments, the C-terminal sequence is set forth in Seq ID NO. 41, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 41, for example, can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 41.

[0011] In some embodiments, the F2 sequence of the pre-fusion hRSV F protein is connected to the RR1 sequence by a GS-linker sequence, the F2 sequence is set forth in Seq ID NO. 4, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 4, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 4; the RR1 sequence is set forth in Seq ID NO. 7, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 7, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 7; the Fl sequence is set forth in Seq ID NO. 10, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 10, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 10; the RR2 sequence is set forth in Seq ID NO. 91, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 91, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 91; the C-terminal sequence is set forth in Seq ID NO. 21-57, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 21-57, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 21-57; in some embodiments, the C-terminal sequence is set forth in Seq ID NO. 41, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 41, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 41.

[0012] In some embodiments, the F2 sequence of the pre-fusion hRSV F protein is linked to the RR1 sequence by a GS-linker sequence, the F2 sequence is set forth in Seq ID NO. 4, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 4, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 4; the RR1 sequence is set forth in Seq ID NO. 8, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 8, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 8; the Fl sequence is set forth in Seq ID NO. 11, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 11, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 11; the RR2 sequence is set forth in Seq ID NO. 17, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 17, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 17; the C-terminal sequence is set forth in Seq ID NO. 21-57, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 21-57, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 21-57. In some embodiments, the C-terminal sequence is set forth in Seq ID NO. 41, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 41, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 41.

[0013] In some embodiments, the pre-fusion hRSV F protein has an amino acid sequence set forth in Seq ID NO. 64, Seq ID NO. 78, Seq ID NO. 95-97, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 64, Seq ID NO. 78, Seq ID NO. 95-97, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 64, Seq ID NO. 78, Seq ID NO. 95-97.

[0014] In some embodiments, the pre-fusion hRSV F protein has an amino acid sequence as set forth in Seq ID NO. 64, Seq ID NO. 97, or an amino acid sequence at least 80% identical to Seq ID NO. 64, Seq ID NO. 97, for example, but not limited to, an amino acid sequence at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 64, Seq ID NO. 97.

[0015] In some embodiments, the pre-fusion hRSV F protein has a contiguous or intermittent deletion of at least 1, 2, 3, 4, 5, or 6 amino acid residues in the F2 sequence from amino acid 104 to 109, or a contiguous or intermittent deletion of at least 1 amino acid residue in the RR1 sequence from amino acid 137 to 144, or a contiguous or intermittent deletion of at least 1 amino acid residue in the RR2 sequence from amino acid 504 to 524.

[0016] In some embodiments, the P27 sequence of the pre-fusion hRSV F protein is replaced by a GS-linker sequence selected from one of (GnS)m, ((GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q; wherein n is 1-10, 15, or 20; m is 1-10, 15, or 20; o is an integer from 1-5; p is an integer from 1-40; q is an integer from 1-5.

[0017] In some embodiments, the immunogenic composition is a nucleic acid immunogenic composition comprising a DNA molecule and / or an RNA molecule encoding a pre-fusion hRSV F protein.

[0018] In some embodiments, the DNA molecule comprises a linear DNA molecule and / or a circular DNA molecule. In some embodiments, the RNA molecule comprises an mRNA molecule or a circular RNA molecule. In some embodiments, the RNA molecule is an mRNA.

[0019] In some embodiments, the mRNA molecule comprises an open reading frame (ORF) having a nucleotide sequence as set forth in Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 80-81, Seq ID NO. 83-84, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112, or a nucleotide sequence at least 80% identical to Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 80-81, Seq ID NO. 83-84, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112, for example, but not limited to, a nucleotide sequence at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 80-81, Seq ID NO. 83-84, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112.

[0020] In some embodiments, the mRNA molecule comprises an open reading frame (ORF) having a nucleotide sequence as set forth in Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112, or a nucleotide sequence at least 80% identical to Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112, for example, but not limited to, a nucleotide sequence at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112.

[0021] In some embodiments, the mRNA molecule comprises an open reading frame (ORF) having a nucleotide sequence as set forth in Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112, or a nucleotide sequence at least 80% identical to Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112, for example, but not limited to, a nucleotide sequence at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112.

[0022] In some embodiments, the 3' end protective modification group of the mRNA molecule is poly(A), which is 50-200 in length, preferably 80-200.

[0023] In some embodiments, the mRNA molecule further comprises a 5' UTR. In some embodiments, the 5' UTR is preferably 10-200 nucleotides in length, preferably 15-100 nucleotides in length. In some embodiments, the 5' UTR nucleotide sequence is set forth in SEQ ID. NO. 58-60.

[0024] In some embodiments, the mRNA molecule further comprises a 3' UTR. In some embodiments, the 3' UTR sequence is set forth in SEQ ID. NO. 61-63.

[0025] In some embodiments, one or more uridines in the mRNA molecule are replaced with modified nucleosides; in some embodiments, the modified nucleoside replacing uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0026] In some embodiments, the nucleic acid immunization composition comprises a carrier adapted to nucleic acid.

[0027] In some embodiments, the carrier is selected from the group consisting of a liposomal nanoparticle, a cationic nanoemulsion, a LLP.

[0028] Some aspects of the present disclosure provide a method of preparing the immunization composition of any one of the above, characterized in that the nucleic acid immunization composition is obtained by mixing an aqueous phase comprising the mRNA molecule described above with an organic phase comprising a carrier component. In some embodiments, the carrier comprises a liposomal nanoparticle;

[0029] In some embodiments, the liposomal nanoparticle comprises 20-50% cationic lipid by mole percent, for example, but not limited to, 20%, 25%, 30%, 35%, 40%, 45%, or 50%; 20-50% DSCP, for example, but not limited to, 20%, 25%, 30%, 35%, 40%, 45%, or 50%; 5-20% cholesterol, for example, but not limited to, 5%, 10%, 15%, or 20%; and 1-5% PEG-DMG, for example, but not limited to, 1%, 2%, 3%, 4%, or 5%.

[0030] In some embodiments, the liposomal nanoparticle comprises 50% Dlin-MC3-DMA, 10% DOPG, 38.5% cholesterol, and 1.5% PEG-DMG by mole percent.

[0031] In some embodiments, the mass ratio of mRNA to total lipid in the nucleic acid immunization composition is selected from the group consisting of 5:1-40:1, 8:1-40:1, 10:1-30:1, 15:1-30:1, 10:1-25:1, 5:1-25:1, 12:1-18:1, 14:1-17:1, 15:1-16:1.

[0032] In some embodiments, the diameter of the lipid nanoparticle is less than about 200 nm; in some embodiments, the diameter of the lipid nanoparticle is less than about 150 nm; in some embodiments, the diameter of the lipid nanoparticle is less than 100 nm; in some embodiments, the diameter of the lipid nanoparticle is about 55 nm to about 90 nm.

[0033] In some embodiments, the mRNA molecule is dissolved in a buffer to obtain an aqueous phase, and each lipid component of the liposomal nanoparticle is dissolved in an organic solvent to obtain an organic phase, and the aqueous phase and the organic phase are mixed to remove the organic phase to obtain the nucleic acid immunization composition.

[0034] In some embodiments, the volume ratio of the aqueous phase to the organic phase is 1:2-4, preferably 1:3.

[0035] In some embodiments, the buffer comprises a citrate buffer or sodium acetate, preferably a citrate buffer.

[0036] In some embodiments, the pH of the buffer is 3-7, preferably 4.

[0037] In some embodiments, the concentration of the nucleic acid molecule in the aqueous phase is 0.05 mg / mL-0.5 mg / mL, preferably 0.1 mg / mL.

[0038] In some embodiments, the organic solvent is selected from the group consisting of C1-C4 low-carbon alcohol, preferably anhydrous ethanol.

[0039] In some embodiments, the concentration of the lipid component in the organic phase is 5 mg / mL-7 mg / mL, preferably 6 mg / mL.

[0040] In some embodiments, the aqueous phase and the organic phase are mixed using microfluidics, and the organic solvent is filtered using tangential flow.

[0041] Preferably, the flow rate of the microfluidics is >3 ml / min, further preferably 12 mL / min.

[0042] In some embodiments, the mixing is followed by a concentration step that results in a final concentration of the mRNA molecules of 50 μg / mL to 200 μg / mL, preferably 100 μg / mL.

[0043] Some aspects of the present disclosure provide a nucleic acid molecule, characterized in that the nucleic acid molecule comprises a nucleic acid encoding a human respiratory syncytial virus antigen, the human respiratory syncytial virus antigen being a pre-fusion hRSV F protein.

[0044] In some embodiments, the human respiratory syncytial virus antigen is a pre-fusion hRSV F protein, the F1 sequence of which is set forth in Seq ID NO. 10-11, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 10-11, for example, but not limited to, an amino acid sequence that comprises at least 80%, 85%, 90%, 95%, or 98% identity to Seq ID NO. 10-11; the F2 sequence of which is set forth in Seq ID NO. 2-5, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 2-5, for example, but not limited to, an amino acid sequence that comprises at least 80%, 85%, 90%, 95%, or 98% identity to Seq ID NO. 2-5; the RR1 sequence of which is set forth in Seq ID NO. 6-9, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 6-9, for example, but not limited to, an amino acid sequence that comprises at least 80%, 85%, 90%, 95%, or 98% identity to Seq ID NO. 6-9; the RR2 sequence of which is set forth in Seq ID NO. 12-20, Seq ID NO. 88-93, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 12-20, Seq ID NO. 88-93, for example, but not limited to, an amino acid sequence that comprises at least 80%, 85%, 90%, 95%, or 98% identity to Seq ID NO. 12-20, Seq ID NO. 88-93; the C-terminal sequence of which is set forth in Seq ID NO. 21-57, Seq ID NO. 85-87, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 21-57, Seq ID NO. 85-87, for example, but not limited to, an amino acid sequence that comprises at least 80%, 85%, 90%, 95%, or 98% identity to Seq ID NO. 21-57, Seq ID NO. 85-87.

[0045] In some embodiments, the P27 sequence of the pre-fusion hRSV F protein is set forth in Seq ID NO. 76, or comprises an amino acid sequence at least 80% identical to Seq ID NO. 76, for example, but not limited to, an amino acid sequence comprising at least 80%, 85%, 90%, 95%, or 98% identity to Seq ID NO. 76; the F2 sequence is set forth in Seq ID NO. 3, or comprises an amino acid sequence at least 80% identical to Seq ID NO. 3, for example, but not limited to, an amino acid sequence comprising at least 80%, 85%, 90%, 95%, or 98% identity to Seq ID NO. 3; the RR1 sequence is set forth in Seq ID NO. 7, or comprises an amino acid sequence at least 80% identical to Seq ID NO. 7, for example, but not limited to, an amino acid sequence comprising at least 80%, 85%, 90%, 95%, or 98% identity to Seq ID NO. 7; the Fl sequence is set forth in Seq ID NO. 10, or comprises an amino acid sequence at least 80% identical to Seq ID NO. 10, for example, but not limited to, an amino acid sequence comprising at least 80%, 85%, 90%, 95%, or 98% identity to Seq ID NO. 10; the RR2 sequence is set forth in Seq ID NO. 91, or comprises an amino acid sequence at least 80% identical to Seq ID NO. 91, for example, but not limited to, an amino acid sequence comprising at least 80%, 85%, 90%, 95%, or 98% identity to Seq ID NO. 91; the C-terminal sequence is set forth in Seq ID NO. 85-87, or comprises an amino acid sequence at least 80% identical to Seq ID NO. 85-87, for example, but not limited to, an amino acid sequence comprising at least 80%, 85%, 90%, 95%, or 98% identity to Seq ID NO. 85-87.

[0046] In some embodiments, the F2 sequence of the pre-fusion hRSV F protein is connected to the RR1 sequence by a GS-linker sequence, the F2 sequence is set forth in Seq ID NO. 3, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 3, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 3; the RR1 sequence is set forth in Seq ID NO. 7, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 7, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 7; the Fl sequence is set forth in Seq ID NO. 10, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 10, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 10; the RR2 sequence is set forth in Seq ID NO. 91, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 91, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 91; and the C-terminal sequence is set forth in Seq ID NO. 85-87, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 85-87, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 85-87.

[0047] In some embodiments, the F2 sequence of the pre-fusion hRSV F protein is connected to the RR1 sequence by a GS-linker sequence, the F2 sequence is set forth in Seq ID NO. 3, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 3, for example, can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 3; the RR1 sequence is set forth in Seq ID NO. 7, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 7, for example, can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 7; the Fl sequence is set forth in Seq ID NO. 10, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 10, for example, can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 10; the RR2 sequence is set forth in Seq ID NO. 91, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 91, for example, can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 91; the C-terminal sequence is set forth in Seq ID NO. 21-57, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 21-57, for example, can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 21-57; in some embodiments, the C-terminal sequence is set forth in Seq ID NO. 41, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 41, for example, can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 41.

[0048] In some embodiments, the F2 sequence of the pre-fusion hRSV F protein is connected to the RR1 sequence by a GS-linker sequence, the F2 sequence is set forth in Seq ID NO. 4, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 4, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 4; the RR1 sequence is set forth in Seq ID NO. 7, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 7, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 7; the Fl sequence is set forth in Seq ID NO. 10, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 10, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 10; the RR2 sequence is set forth in Seq ID NO. 91, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 91, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 91; the C-terminal sequence is set forth in Seq ID NO. 21-57, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 21-57, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 21-57; in some embodiments, the C-terminal sequence is set forth in Seq ID NO. 41, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 41, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 41.

[0049] In some embodiments, the F2 sequence of the pre-fusion hRSV F protein is linked to the RR1 sequence by a GS-linker sequence, the F2 sequence is set forth in Seq ID NO. 4, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 4, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 4; the RR1 sequence is set forth in Seq ID NO. 8, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 8, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 8; the Fl sequence is set forth in Seq ID NO. 11, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 11, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 11; the RR2 sequence is set forth in Seq ID NO. 17, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 17, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 17; the C-terminal sequence is set forth in Seq ID NO. 21-57, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 21-57, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 21-57. In some embodiments, the C-terminal sequence is set forth in Seq ID NO. 41, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 41, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 41.

[0050] In some embodiments, the pre-fusion hRSV F protein has an amino acid sequence set forth in Seq ID NO. 64, Seq ID NO. 97, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO. 64, Seq ID NO. 97, for example, but not limited to, an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 64, Seq ID NO. 97.

[0051] In some embodiments, the pre-fusion hRSV F protein has at least a 1 amino acid residue contiguous or intermittent deletion in the F2 sequence from amino acid 104 to 109, or, at least a 1 amino acid residue contiguous or intermittent deletion in the RR1 sequence from amino acid 137 to 144, or, at least a 1 amino acid residue contiguous or intermittent deletion in the RR2 sequence from amino acid 504 to 524.

[0052] In some embodiments, the P27 sequence of the pre-fusion hRSV F protein is replaced by a GS-linker sequence selected from one of (GnS)m, ((GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q; wherein n is 1-10, 15, or 20; m is 1-10, 15, or 20; o is an integer from 1 to 5; p is an integer from 1 to 40; q is an integer from 1 to 5.

[0053] In some embodiments, the nucleic acid encoding a human respiratory syncytial virus antigen comprises a DNA molecule and / or an RNA molecule. In some embodiments, the DNA molecule comprises a linear DNA molecule and / or a circular DNA molecule. In some embodiments, the RNA molecule comprises an mRNA or a circular RNA.

[0054] In some embodiments, the mRNA molecule comprises an open reading frame (ORF) having a nucleotide sequence as set forth in Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 80-81, Seq ID NO. 83-84, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112, or a nucleotide sequence at least 80% identical to Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 80-81, Seq ID NO. 83-84, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112, for example, but not limited to, a nucleotide sequence at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 80-81, Seq ID NO. 83-84, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112.

[0055] In some embodiments, the mRNA molecule comprises an open reading frame (ORF) having a nucleotide sequence as set forth in Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112, or a nucleotide sequence at least 80% identical to Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112, for example, but not limited to, a nucleotide sequence at least 80%, 85%, 90%, 95%, or 98% identical to Seq ID NO. 65, Seq ID NO. 79, Seq ID NO. 94, Seq ID NO. 98, Seq ID NO. 111-112.

[0056] In some embodiments, nucleic acid molecules can be optimized by sequence optimization means to improve properties related to expression efficacy upon in vivo administration: for example, to improve mRNA stability, to increase translation efficacy in target tissues, to reduce the amount of truncated proteins expressed, to improve folding or prevent misfolding of expressed proteins, to reduce toxicity of expression products, to reduce cell death caused by expression products, to increase and / or reduce protein aggregation, resulting in mRNA with improved properties. The purpose of sequence optimization also includes: optimizing the characteristics of formulation and delivery of nucleic acid-based therapeutics while maintaining structural and functional integrity; overcoming thresholds of expression; increasing expression rates; half-life and / or protein concentration; optimizing protein localization; and avoiding adverse biological responses such as immune responses and / or degradation pathways. Sequence optimization means include: (1) codon optimization according to codon frequencies in a particular organ and / or host organism to ensure proper folding and proper expression; (2) modulation of G / C content to increase mRNA stability or reduce secondary structures; (3) minimization of tandem repeated codons or base runs that can impair gene construction or expression; (4) customization of transcription and translation control regions; (5) reduction or elimination of problematic secondary structures within the polynucleotide.

[0057] “Sequence identity” between two nucleotide sequences indicates the percent of nucleotides that are the same between the sequences. “Sequence identity” between two amino acid sequences indicates the percent of amino acids that are the same between the sequences.

[0058] The term "% identity" or like terms refers to the percentage of nucleotides or amino acids that are the same between the sequences being compared, after optimal alignment. The percentage is purely statistical, and the differences between the two sequences can be, but are not necessarily, randomly distributed throughout the length of the sequences being compared. Comparison of two sequences is typically done by comparing the sequences against each other, after optimal alignment, over a segment or "comparison window" to identify local regions of the sequences that correspond.

[0059] In some embodiments, the 5' end and / or the 3' end of the mRNA molecule has a protective modification group. In some embodiments, the 5' end modification group of the mRNA molecule is selected from ARCA, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, mCAP, dmCAP, tmCAP, or dmCAP.

[0060] In some embodiments, the 3' end protective modification group of the mRNA molecule is poly(A) with a length of 50-200, preferably 80-200.

[0061] In some embodiments, the mRNA molecule further comprises a 5' UTR. In some embodiments, the 5' UTR has a length of 10-200 nucleotides, preferably 15-100 nucleotides. In some embodiments, the 5' UTR nucleotide sequence is set forth in SEQ ID. NO. 58-60.

[0062] In some embodiments, the mRNA fragment further comprises a 3' UTR. In some embodiments, the 3' UTR sequence is set forth in SEQ ID. NO. 61-63.

[0063] In some embodiments, based on the provided RNA sequence, one of ordinary skill in the art would be able to derive the corresponding DNA sequence (e.g., uracil to thymine conversion). Likewise, based on the provided DNA sequence, one of ordinary skill in the art would be able to derive the corresponding RNA sequence (e.g., thymine to uracil conversion). In some embodiments, based on the provided RNA or DNA sequence, one of ordinary skill in the art would be able to derive the corresponding amino acid sequence.

[0064] In some embodiments, one or more uridines in the mRNA molecule are replaced with a modified nucleoside; in some embodiments, the modified nucleoside replacing the uridine is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U). BRIEF DESCRIPTION OF DRAWINGS

[0065] In order to more clearly illustrate the technical solutions in the specific embodiments of the present disclosure or the prior art, the drawings required to be used in the specific embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present disclosure, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0066] Figure 1 A schematic diagram of the wild-type hRSV F protein is shown.

[0067] Figure 2 A schematic diagram of the hRSV F protein variant is shown.

[0068] Figure 3 ELISA detection of RSV F protein-specific IgG antibodies in mouse sera in Example 5 is shown.

[0069] Figure 4 Flow cytometric detection of TNF-a+ CD4+ and TNF-a+ CD8+ T cells in Example 5 is shown.

[0070] Figure 5 ELISA detection of IFN-g and IL-4 in cell culture supernatants in Example 5 is shown.

[0071] Figure 6 RSV neutralizing antibody titers in mouse sera in Example 5 are shown.

[0072] Figure 7 ELISA detection of RSV F protein-specific IgG antibodies in mouse sera in Example 6 is shown.

[0073] Figure 8 ELISA detection of IFN-g and IL-4 in cell culture supernatants in Example 6 is shown.

[0074] Figure 9 Flow cytometric detection of TNF-a+ CD4+ and TNF-a+ CD8+ T cells in Example 6 is shown.

[0075] Figure 10 RSV neutralizing antibody titers in mouse sera in Example 6 are shown.

[0076] Figure 11 RSV neutralizing antibody titers in mouse sera in Example 7 are shown.

[0077] Figure 12 RSV neutralizing antibody titers in mouse sera in Example 8 are shown.

[0078] Figure 13 RSV neutralizing antibody titers in mouse sera from Example 9 are shown.

[0079] Figure 14 RSV neutralizing antibody titers in mouse sera from Example 10 are shown.

[0080] Figure 15 RSV neutralizing antibody titers in mouse sera from Example 11 are shown.

[0081] Figure 16 RSV neutralizing antibody titers in mouse sera from Example 12 are shown.

[0082] Figure 17 RSV neutralizing antibody titers in mouse sera from Example 13 are shown.

[0083] Figure 18 RSV neutralizing antibody titers in mouse sera from Example 14 are shown. DETAILED DESCRIPTION

[0084] Figure 1 A schematic of the hRSV F protein wild type is shown; Figure 2 A schematic of the hRSV F protein variant is shown.

[0085] Fl is a fusion protein that is cleaved by furin; SP is a signal peptide sequence; p27 is a 27 aa peptide sequence removed after cleavage; RR1 is a repeat folding region 1 that spans amino acid residues 137 to 216 of the hRSV F protein (wild type), including a fusion peptide and a heptad repeat sequence A (HRA); RR2 is a repeat folding region 2 that forms a C-terminal stalk in the prefusion hRSV F protein spike, including a heptad repeat sequence B (HRB) that is relocated to the other side of the head of the RSV F protein; TM is a transmembrane region sequence.

[0086] F2 generally comprises amino acid residues 26 to 109 of the F0 precursor; Fl generally comprises amino acid residues 137 to 574 of the F0 precursor; Fl and F2 are linked by disulfide bonds to form a heterodimer, which is referred to as the RSV F "protomer".

[0087] The C-terminus can be deleted, or selected from at least one of a TM transmembrane sequence, a multimerization element sequence. In some embodiments, the C-terminus of the hRSV F protein variant is deleted. In some embodiments, the C-terminus of the hRSV F protein variant is a TM transmembrane sequence. In some embodiments, the C-terminus of the hRSV F protein variant is a TM transmembrane sequence and a multimerization element sequence connected in sequence, wherein the C-terminus of the TM transmembrane sequence is connected to the N-terminus of the multimerization element sequence. In some embodiments, the C-terminus of the hRSV F protein variant is a multimerization element sequence.

[0088] The hRSV F protein variant described in the present disclosure is an F protein artificially mutated and engineered, the mutations and engineering, including but not limited to amino acid residue substitution, amino acid residue insertion and / or addition, amino acid residue deletion, and covalent modification, etc. to the amino acid sequence of the wild type hRSV F protein; in the present disclosure, "hRSV F protein variant", "hRSV F protein in the present disclosure" or "hRSV F protein" all refer to "hRSV F protein variant", and "hRSV F protein wild type" or "hRSV F protein (wild type)" all refer to wild type hRSV F protein without artificial modification or variation. In addition, unless otherwise specified, "hRSV F protein" or "F protein" in other parts of the present disclosure, except the background section, all refer to "hRSV F protein variant".

[0089] The position of the amino acid sequence described in the present disclosure, such as S46G substitution, E92D substitution, P102A substitution, A149C substitution, L373R substitution, I379V substitution, M447V substitution, Y458C substitution, K465Q substitution, D486C substitution, and D489C substitution, is based on the full-length amino acid sequence of the wild type hRSV F protein shown below. Figure 1 The S46G substitution is based on the wild type hRSV F protein, and the S amino acid residue at the 46th position of the wild type hRSV F protein full-length amino acid sequence is replaced by a G amino acid residue. Figure 1 The S46G substitution is based on the wild type hRSV F protein, and the S amino acid residue at the 46th position of the wild type hRSV F protein full-length amino acid sequence is replaced by a G amino acid residue.

[0090] The multimerization element described in the present disclosure includes a dimerization element, a trimerization element, a tetramerization element, and an oligomerization element, which can cause the formation of a multimeric hRSV F protein complex when used in combination with the hRSV F protein in the present disclosure; the multimerization element can be located at the N-terminus or C-terminus of the hRSV F protein, and its coding sequence is usually placed in the 5' or 3' frame of the coding sequence at the nucleic acid level.

[0091] Among them, the dimerization element can be selected from, for example, the dimerization element / domain of heat shock protein, immunoglobulin Fc domain and leucine zipper (dimerization domain of basic region leucine zipper transcription factor), and the specific amino acid sequence is shown in SEQ ID NO. 21~24. The trimerization and tetramerization element can be selected from, for example, engineered leucine zipper (engineered-helix helix coiled peptide in parallel trimer state), fibritin folding domain of colibacteriophage T4, GCN4PLL, CCN4-PLI, p53, GCN4, and the specific amino acid sequence is shown in SEQ ID NO. 25~53 and SEQ ID NO. 54~57 (tetramerization)).

[0092] In some embodiments, the polypeptide immunization composition described in the present disclosure is prepared by chemical synthesis technology or genetic engineering technology according to the amino acid sequence of a known or predicted antigen epitope of the human respiratory syncytial virus antigen gene. In some embodiments, the polypeptide immunization composition described in the present disclosure is prepared by chemical synthesis technology according to the amino acid sequence of the human respiratory syncytial virus pre-fusion hRSV F protein, combined with artificial mutations and modifications. In some embodiments, the polypeptide immunization composition described in the present disclosure is prepared by fermentation after genetic engineering technology (such as construction of genetically engineered bacteria by genetic engineering technology) according to the amino acid sequence of the human respiratory syncytial virus pre-fusion hRSV F protein, combined with artificial mutations and modifications.

[0093] In some embodiments, the virus immunization composition described in the present disclosure carries the human respiratory syncytial virus antigen gene into the human body through harmless microorganisms, and induces the immune system of the body to make an immune response. Important viruses used in the cell immunity test include variants of bovine disease virus, poliovirus, etc.

[0094] In some embodiments, the mRNA described in the present disclosure comprises, from 5' end to 3' end, the sequence of 5' cap, 5' UTR, ORF, 3' UTR and 3' poly(A) tail.

[0095] In some embodiments, the mRNA described in the present disclosure also carries a sequence capable of expressing RNA polymerase (RNA-dependent RNA polymerase, RdRP), and the sequence design content of Chinese patent CN202110424124.2 for self-replicating mRNA is introduced into the present disclosure.

[0096] The 5' untranslated region (UTR) as described in the present disclosure refers to the sequence of the mRNA that is located directly upstream of the start codon that does not encode a polypeptide. The 5' UTR can comprise a promoter sequence when the RNA transcript is produced. Such promoter sequences are known in the art. The RNA sequence of the 5' UTR is shown in one of Seq ID NO. 58, Seq ID NO. 59, or Seq ID NO. 60, or a 5' UTR sequence disclosed prior to the filing date of the present disclosure can also be incorporated into the present disclosure.

[0097] The 3' untranslated region (UTR) as described in the present disclosure refers to the sequence of the mRNA that is located downstream of the stop codon that does not encode a polypeptide. The RNA sequence of the 3' UTR is shown in one of Seq ID NO. 61, Seq ID NO. 62, or Seq ID NO. 63, or a 3' UTR sequence disclosed prior to the filing date of the present disclosure can also be incorporated into the present disclosure.

[0098] The poly(A) tail as described in the present disclosure is a sequence of the mRNA that is located downstream of the 3' UTR containing multiple consecutive adenosine monophosphates. The poly(A) tail can contain 10 to 300 adenosine monophosphates. In cells and / or in vivo, the poly(A) tail serves to protect the mRNA from enzymatic degradation, and aids in transcription termination, and / or mRNA export from the nucleus and translation.

[0099] In alternative embodiments, the immunological composition comprising human respiratory syncytial virus antigen described in the present disclosure can further comprise at least one cryoprotectant. In alternative embodiments, the cryoprotectant is selected from at least one of sucrose, glycerol. In alternative embodiments, the cryoprotectant of the immunological composition comprising human respiratory syncytial virus antigen is selected from one of 5% (w / v) to 18% (w / v) sucrose, 6% (w / v) to 16% (w / v) sucrose, 7% (w / v) to 14% (w / v) sucrose, 7% (w / v) to 12% (w / v) sucrose, 8% (w / v) to 11% (w / v) sucrose. In alternative embodiments, the cryoprotectant of the immunological composition comprising human respiratory syncytial virus antigen is selected from one of 1% (w / v) to 9% (w / v) glycerol, 1.5% (w / v) to 7% (w / v) glycerol, 1.75% (w / v) to 6% (w / v) glycerol, 1% (w / v) to 6% (w / v) glycerol, 3% (w / v) to 6% (w / v) glycerol. In alternative embodiments, the cryoprotectant of the immunological composition comprising human respiratory syncytial virus antigen is selected from one of 5% (w / v) to 18% (w / v) sucrose and 1% (w / v) to 9% (w / v) glycerol combination, 6% (w / v) to 16% (w / v) sucrose and 1.5% (w / v) to 7% glycerol combination, 7% (w / v) to 14% (w / v) sucrose and 1.75% (w / v) to 6% (w / v) glycerol combination, 7% (w / v) to 12% (w / v) sucrose and 1% (w / v) to 6% (w / v) glycerol combination, 8% (w / v) to 11% (w / v) sucrose and 3% (w / v) to 6% (w / v) glycerol combination.

[0100] The immunological composition comprising human respiratory syncytial virus antigen described in the present disclosure can be administered by, but not limited to, intramuscular injection, subcutaneous injection, oral inhalation administration, nasal instillation administration, nasal spray administration, oral-nasal inhalation administration.

[0101] The immunological composition comprising human respiratory syncytial virus antigen described herein can be formulated into, but not limited to, the following dosage forms: injection, aerosol, dry powder inhalation / powder mist, spray, and / or atomized inhalation solution.

[0102] The aerosol, dry powder inhaler / powder mist, spray, and / or nebulized inhalation solution of the present disclosure generally comprises a drug to be delivered, optionally formulated with a surfactant, such as a non-ionic surfactant (e.g., polysorbate 80), and one or more buffers, provided that the inclusion of the surfactant does not disrupt the structure of the lipid formulation. In alternative embodiments, the aerosol, dry powder inhaler / powder mist, spray, and / or nebulized inhalation solution further comprises a propellant. The pH of the aerosol, spray, and / or nebulized inhalation solution is 6.8-7.2. The drug solvent used can also be a slightly acidic aqueous buffer with a pH of 4-6. Other components can be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases.

[0103] For formulating the immunocomposition comprising human respiratory syncytial virus antigen of the present disclosure for oral inhalation administration, drop administration, nasal spray administration, or oronasal inhalation administration, the immunocomposition comprising human respiratory syncytial virus antigen comprising mRNA and LNP can be combined with various pharmaceutically acceptable additives. The additives are selected from one or more of pH controlling agents, local anesthetics, adsorption inhibitors, isotonic agents, solubility enhancers, stabilizing agents, reducing agents. The pH controlling agents are selected from one or more of arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid. The local anesthetics are selected from benzyl alcohol. The isotonic agents are selected from one or more of sodium chloride, mannitol, sorbitol. The adsorption inhibitors are selected from Tween 80. The solubility enhancers are selected from one or more of cyclodextrin and its derivatives. The stabilizing agents are selected from serum albumin. The reducing agents are selected from glutathione.

[0104] The immunological compositions comprising human respiratory syncytial virus antigens comprising mRNA and LNPs can be dispersed in a base or vehicle, which can comprise hydrophilic compounds having the ability to disperse the immunological compositions comprising human respiratory syncytial virus antigens and any desired additives. The base can be selected from a wide range of suitable carriers, including but not limited to polycarboxylic acids or their salts, copolymers of carboxylic anhydride (selected from maleic anhydride) with other monomers selected from (meth)acrylic acid methyl ester, acrylic acid, etc., hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid and its nontoxic metal salts. Typically, biodegradable polymers are chosen as the base or vehicle, for example, polylactic acid, poly(lactic-co-glycolic acid), copolymers, polyhydroxybutyric acid, poly(hydroxybutyric acid-co-glycolic acid), and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglyceryl fatty acid esters, sucrose fatty acid esters, etc. can be used as vehicles. Hydrophilic polymers and other carriers can be used alone or in combination, and can be endowed with enhanced structural integrity of the carrier by partial crystallization, ionic bonding, cross-linking, etc. The vehicles can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and films for direct administration to the nasal mucosa. The use of a selected vehicle in this context can result in the promotion of absorption of the immunological compositions comprising human respiratory syncytial virus antigens.

[0105] The immunological compositions comprising human respiratory syncytial virus antigens of the present disclosure can alternatively contain agents that enhance the effectiveness of the immunological compositions comprising human respiratory syncytial virus antigens, including, for example, penetration enhancers. The penetration enhancers can be selected from a wide range of suitable agents, including but not limited to surfactants, fatty acids, bile salts, and the like. The immunological compositions comprising human respiratory syncytial virus antigens can also contain minor amounts of wetting or lubricating agents, pH adjusting agents and buffers, such as sodium acetate, lactate, citrate, and phosphate, and agents for the adjustment of tonicity such as sodium chloride, dextrose, mannitol, and sorbitol.

[0106] In alternative embodiments, the immunological compositions comprising human respiratory syncytial virus antigens of the present disclosure can be nebulized or otherwise delivered as particulate liquids or solids, either before or after administration to a subject. The means for administering such solid or liquid particulate immunological compositions comprising human respiratory syncytial virus antigens are aqueous solutions or suspensions that are nebulized, loaded into at least one suitable device that readily produces particles that are breathed or inhaled by the subject. In alternative embodiments, the device is selected from a metered dose inhaler, a jet nebulizer, an ultrasonic nebulizer, a dry powder inhaler, a propellant-based inhaler, or an insufflator.

[0107] Example 1: Process for RNA manufacture of LNPs

[0108] A lipid nanoparticle comprising RNA encoding a respiratory syncytial virus antigen, wherein the lipid nanoparticle comprises, by mole percent, Dlin-MC3-DMA 50%, DOPG 20%, cholesterol 29%, and PEG-DMG 1%.

[0109] The preparation method is as follows: (a) dissolve the RNA in a citrate buffer at pH 4, adjust the concentration to 0.1 mg / ml to obtain an aqueous phase.

[0110] (b) dissolve Dlin-MC3-DMA, DOPG, cholesterol and PEG-DMG in anhydrous ethanol according to the formula amount, adjust the concentration of the lipid components in the organic phase to 6 mg / mL to obtain an organic phase.

[0111] (c) mix the aqueous phase of step (a) and the organic phase of step (b) according to a volume ratio of 1:3, using a microfluidic device, at a flow rate of 12 mL / min, immediately dilute the mixture with a PBS solution at pH 7.4 by 100 times, and remove the ethanol component in the solution using tangential flow filtration (TFF), and then concentrate to a concentration of 55 ug / ml of mRNA in the system to obtain a lipid nanoparticle comprising RNA encoding a respiratory syncytial virus antigen.

[0112] Example 2

[0113] Using luciferase as a reporter gene, the efficiency of different vaccine carrier formulations (as shown in Table 1 below, "MC3" refers to Dlin-MC3-DMA, "+" indicates that luciferase expression in mice was detected by a small animal live fluorescence imaging system after administration) in delivering luciferase gene-encoding mRNA in mice was studied by live fluorescence imaging technology, and the physicochemical indexes of different complex formulations (preparation method, see Example 1) were detected, and the results are shown in Table 1. Through research, it is found that increasing the mass ratio of lipid to mRNA is beneficial to increasing the encapsulation rate of mRNA in the lipid nanoparticle, thereby making it have higher stability, in addition, moderately increasing the content of polyethylene glycol (PEG) in the formula is beneficial to increasing the expression efficiency of mRNA in vivo. Therefore, considering factors such as mRNA encapsulation rate and mRNA in vivo delivery efficiency, Formulations No. 3 and No. 4 are selected for subsequent mRNA vaccine research.

[0114] Table 1

[0115] .

[0116] Example 3

[0117] The ability of different formulations of cationic lipid nanoparticles to encapsulate luciferase-encoding mRNA and the particle size data for the nanoparticles formed are shown in Table 2. Several formulations were able to compress luciferase mRNA into nanoparticles with a particle size of less than 100 nm and a net neutral surface potential, while also being able to encapsulate at least 50% of the mRNA, and thus can have some in vivo delivery effect. "MC3" refers to Dlin-MC3-DMA.

[0118] Table 2

[0119]

[0120] Example 4

[0121] BALB / c mice (female, 5-6 weeks old, average weight 20-25 g, purchased from Zhuhai Baitishun Biotechnology Co., Ltd.) were selected for vaccine immunogenicity evaluation.

[0122] The experimental animals were randomly grouped, with 5 mice in each group. The immunization dose was 15 pg per mouse, and the same dose was given for booster immunization 2 weeks after vaccination. Twelve days after the second immunization, the cellular immune response and humoral immune response indicators were detected.

[0123] The mouse lymphocytes were isolated according to the following method: the mouse was sacrificed by breaking the neck and immersed in 75% ethanol; the mouse spleen was taken out in a clean bench; 4-5 mL of mouse lymphocyte separation medium (recovered to room temperature and shaken before use) was added to a 35 mm culture dish; grinded, and the separation medium with the spleen cells was immediately transferred to a 15 mL centrifuge tube, centrifuged at room temperature at 800g with a horizontal rotor for 30 min. After centrifugation, the lymphocyte layer was aspirated, and 10 mL of RPMI 1640 medium was added for washing. The cells were collected by centrifugation at room temperature.

[0124] 1. ELISA detection of RSV-F protein specific IgG antibodies in mouse serum

[0125] hRSV F protein (His Tag) (2 mg / mL) diluted to 2 pg / mL was added to the multi-well plate (100 pL per well) with washing solution and coated overnight. After washing, 2% BSA solution was added for blocking. All serum samples to be tested were diluted by 10 times gradient from 1:100 to 1:1,000,000, and the negative control sample was consistent with the serum sample to be tested, and was diluted by the same multiple. There were 6 gradient samples in total. The prepared serum sample solution was added to the enzyme-labeled multi-well plate, incubated, washed, and then enzyme-labeled antibody solution was added, incubated, washed, and developed. The OD value was measured on the enzyme-labeled instrument at 450 nm / 630 nm. The arithmetic mean of the negative serum sample signal was 2.1 times the Cutoff value.

[0126] 2. Flow cytometry to detect TNF-α+ CD4+ and TNF-α+ CD8+ T cells

[0127] Isolated mouse lymphocytes were stimulated with peptide pools and incubated at 37°C in a 5% CO2 incubator for 72 h. BD GolgiPlug™ (with brefeldin A) protein transport inhibitor (with BSA) solution was used to stimulate overnight. The mouse lymphocyte suspension was centrifuged to obtain a pellet. The mouse lymphocyte pellet was resuspended and specific monoclonal fluorescent antibodies, such as CD4, CD8, etc., were added. The mouse lymphocyte pellet was incubated and centrifuged. A fixation / permeabilization solution was added and the mouse lymphocyte pellet was incubated and centrifuged. Buffer was added to the mouse lymphocyte pellet, mixed and centrifuged. The mouse lymphocyte pellet was resuspended with staining buffer and cytokine fluorescent antibodies, such as IFN-γ, TNF-α, etc., were added. The mouse lymphocyte pellet was incubated in the dark and centrifuged. The mouse lymphocyte pellet was then resuspended and analyzed by flow cytometry. The expression of TNF-α in CD3+ / CD4+ and CD3+ / CD8+ T lymphocytes was detected by flow cytometry.

[0128] 3. ELISA to detect IFN-γ and IL4 in cell culture supernatant

[0129] Isolated mouse lymphocytes were stimulated with peptide pools and incubated at 37°C in a 5% CO2 incubator for 72 h. The mouse lymphocyte suspension was centrifuged to obtain supernatant. Mouse IL-4 ELISA kit (purchased from Xinbosheng Biotechnology Co., Ltd.) and mouse IFN-γ ELISA kit (purchased from Xinbosheng Biotechnology Co., Ltd.) were used to detect the content of IFN-γ and IL4 in the supernatant.

[0130] 4. Neutralizing activity of serum

[0131] The method for detecting the neutralizing activity of immune group serum is as follows:

[0132] HEp-2 cells were digested and adjusted to a certain cell density. The cells were inoculated into a 96-well plate and cultured overnight. Mouse serum diluents and virus diluents were added to positive wells of the multi-well plate, and virus diluents were added to negative wells. Diluted virus was added to positive and negative wells of the multi-well plate for neutralization. The neutralization products in the positive and negative wells of the multi-well plate were added to the multi-well plate containing the digested HEp-2 cells, respectively, and cultured. The neutralization products were aspirated, and DMEM+2% FBS medium was added to each well for further culture. The supernatant was discarded, and 4% paraformaldehyde was added to each well to fix the cells. PBS washing solution was used for washing, and a 1:1 mixed solution of 4% BSA and 0.2% triton was used to block the cells. The blocking solution was discarded, and F6-6-488 antibody diluent was added for incubation and washing. The multi-well plate was dried, and the reading was taken using CTL equipment.

[0133] Example 5

[0134] The nucleotide sequences described in Table 3 are mRNA sequences, and the mRNA sequences described in Table 3 were each prepared into a lipid nanoparticle containing RNA encoding a respiratory syncytial virus antigen according to the method described in Example 1.

[0135] The RNA vaccine encoding a respiratory syncytial virus antigen includes, in addition to the reading frame sequence in the table, a 5' cap, a 5' UTR (as shown in Seq ID NO. 58), a 3' UTR (as shown in Seq ID NO. 61), and a 3' tail of 100 polyA.

[0136] Table 3

[0137]

[0138] Sample Y1 is a lipid nanoparticle comprising RNA encoding the wild type amino acid sequence of RSV F protein (Wt, the amino acid sequence is shown as Seq ID NO. 66); sample Y2 encodes an amino acid sequence based on the wild type of RSV F protein, removes the TM sequence, and adds Fibritin Dominain (the amino acid sequence of Fibritin Dominain is shown as Seq ID NO. 41) at the C-terminal; sample Y3 encodes an amino acid sequence based on the wild type of RSV F protein, removes the P27 sequence, and connects with a GS-linker-linker between the F2 sequence and the RR1 sequence; Y4 encodes an amino acid sequence with S155C / S190F / V207L / S290C site mutations, removes the TM sequence, and adds Fibritin Dominain (the amino acid sequence of Fibritin Dominain is shown as Seq ID NO. 41) at the C-terminal; Y5 encodes an amino acid sequence with N67I / S215P site mutations, deletes the P27 sequence, adds a GS-linker connecting sequence before the terminal amino acid Arg of the F2 sequence, removes the TM sequence, and adds Fibritin Dominain (the amino acid sequence of Fibritin Dominain is shown as Seq ID NO. 41) at the C-terminal; Y6 encodes an amino acid sequence with N67I / S155C / S190F / V207L / S215P / S290C / D486C / D489C site mutations, the amino acid sequence encoded by Y6 is shown as Seq ID NO. 78, which includes the SP sequence shown as Seq ID NO. 1, the F2 sequence shown as Seq ID NO. 3, the P27 sequence shown as Seq ID NO. 76, the RR1 sequence shown as Seq ID NO. 7, the F1 sequence shown as Seq ID NO. 10, the RR2 sequence shown as Seq ID NO. 91, and the terminal sequence shown as Seq ID NO. 85-87.

[0139] The prepared lipid nanoparticles of RNA encoding respiratory syncytial virus antigens were subjected to vaccine immunogenicity evaluation according to the method of Example 4.

[0140] 1. ELISA detection of RSV-F protein specific IgG antibodies in mouse serum

[0141] The experimental results are shown in Fig. 3A-3E. Fig. 3A-3E are the OD450 determined by indirect ELISA after the serum is diluted from 100 times to 1 million times. The sample is determined to be positive when the OD450 is greater than or equal to 2 times the average value of the OD450 of the negative control at the minimum dilution (1:100). The dotted line in Fig. 3A-3E is the cutoff line. Among them, the Y1 immunization group did not have a positive value until it was diluted to 1 million times. Fig. 3F is the determination of serum IgG antibody titer.

[0142] The results show that the IgG antibody titers of the RSV F protein variant mRNA vaccine immunization groups are significantly higher than those of the PBS control group, and the Y1 immunization group (mRNA vaccine immunization group encoding wild-type RSV F protein) is significantly higher than the other immunization groups.

[0143] 2. Flow cytometry detection of TNF-α+ CD4+ and TNF-α+ CD8+ T cells

[0144] Except for the Y5 immunization group, the rest of the immunization groups were significantly higher than the PBS control group. The results show that the RSV F protein variant mRNA vaccine can stimulate Th1 type cellular immune response in mice.

[0145] 3. ELISA detection of IFN-γ and IL4 in cell culture medium supernatant

[0146] The IFN-γ of the RSV F protein variant mRNA vaccine immunization group was significantly higher than that of the PBS control group (P<0.01) (Th1 type cellular immune response), and the IL-4 of the RSV-F mutant mRNA vaccine immunization group was also significantly higher than that of the PBS control group (P<0.01).

[0147] 4. Neutralizing activity of serum

[0148] The results of RSV neutralizing antibody titers in mouse serum are shown in the figure. Compared with the PBS control group, the RSV neutralizing antibody titers of all RSV F protein variant mRNA immunization groups were significantly increased. The neutralizing antibody titers of the Y2, Y3, Y4 and Y6 mRNA immunization groups encoding RSV F protein variants were higher.

[0149] Example 6

[0150] The amino acid sequence encoded by sample Y7 is based on the RSV F protein mutant encoded by sample Y6, P27 is removed, and a GS-linker-linker sequence is added between the F2 sequence and the RR1 sequence. The amino acid sequence of the RSV F protein mutant encoded by sample Y7 includes the SP sequence shown as Seq ID NO. 1, the F2 sequence shown as Seq ID NO. 3, the GS-linker-linker sequence shown as Seq ID NO. 77, the RR1 sequence shown as Seq ID NO. 7, the F1 sequence shown as Seq ID NO. 10, the RR2 sequence shown as Seq ID NO. 91, and the terminal sequence shown as Seq ID NO. 85-87. The mRNA sequence of the ORF is shown as Seq ID NO. 80.

[0151] The amino acid sequence encoded by sample Y8 is based on the RSV F protein mutant encoded by sample Y6, the TM sequence is removed, and a Fibritin Dominain sequence is added to the terminal sequence. The amino acid sequence of the RSV F protein mutant encoded by sample Y8 includes the SP sequence shown as Seq ID NO. 1, the F2 sequence shown as Seq ID NO. 3, the P27 sequence shown as Seq ID NO. 76, the RR1 sequence shown as Seq ID NO. 7, the F1 sequence shown as Seq ID NO. 10, the RR2 sequence shown as Seq ID NO. 91, and the terminal sequence Fibrintin Dominain shown as Seq ID NO. 41. The mRNA sequence of the ORF is shown as Seq ID NO. 81.

[0152] The amino acid sequence encoded by sample Y9 is based on the RSV F protein mutant encoded by sample Y6, the P27 sequence is removed, the GS-linker-linker sequence is added between the F2 sequence and the RR1 sequence, the TM sequence is removed, and a Fibritin Dominain sequence is added to the C-terminal end (the amino acid sequence is shown as Seq ID NO. 41). The mRNA sequence of the ORF is shown as Seq ID NO. 82. (DS2 mutant)

[0153] The amino acid sequence encoded by Y10 is based on the RSV F protein mutant encoded by sample Y7, with the TM sequence removed, the Fibritin Dominain added at the terminal sequence, with S46G / E92D site mutations, the amino acid sequence of the RSV F protein mutant encoded thereby is shown as Seq ID NO. 95, comprising the SP sequence shown as Seq ID NO. 1, the F2 sequence shown as Seq ID NO. 4, the GS-linker-linker sequence shown as Seq ID NO. 77, the RR1 sequence shown as Seq ID NO. 7, the F1 sequence shown as Seq ID NO. 10, the RR2 sequence shown as Seq ID NO. 91, the terminal sequence Fibrintin Dominain shown as Seq ID NO. 41, the mRNA sequence of the ORF thereof is shown as Seq ID NO. 83;

[0154] The amino acid sequence encoded by Y11 is based on the RSV F protein mutant encoded by sample Y7, with Y458C / K465Q / A149C site mutations, the amino acid sequence of the RSV F protein mutant encoded thereby is shown as Seq ID NO. 96, comprising the SP sequence shown as Seq ID NO. 1, the F2 sequence shown as Seq ID NO. 3, the GS-linker-linker sequence shown as Seq ID NO. 77, the RR1 sequence shown as Seq ID NO. 8, the F1 sequence shown as Seq ID NO. 11, the RR2 sequence shown as Seq ID NO. 17, the terminal sequence Fibrintin Dominain shown as Seq ID NO. 41, the mRNA sequence of the ORF thereof is shown as Seq ID NO. 84.

[0155] The amino acid sequence encoded by Y12 is based on the RSV F protein mutant encoded by sample Y10, with S46G / E92D site mutations, the amino acid sequence of the RSV F protein mutant encoded thereby is shown as Seq ID NO. 97, comprising the SP sequence shown as Seq ID NO. 1, the F2 sequence shown as Seq ID NO. 4, the GS-linker-linker sequence shown as Seq ID NO. 77, the RR1 sequence shown as Seq ID NO. 8, the F1 sequence shown as Seq ID NO. 11, the RR2 sequence shown as Seq ID NO. 17, the terminal sequence Fibrintin Dominain shown as Seq ID NO. 41, the mRNA sequence of the ORF thereof is shown as Seq ID NO. 94.

[0156] Lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens were prepared according to the above description and the method described in Example 2, respectively, as samples Y7-Y12.

[0157] The RNA vaccine encoding respiratory syncytial virus antigens includes, in addition to the above-described reading frame sequence, a 5' cap, a 5' UTR (as shown in Seq ID NO. 58), a 3' UTR (as shown in Seq ID NO. 61), and a 3' tail of 100 polyA.

[0158] The prepared lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens were subjected to vaccine immunogenicity evaluation according to the method of Example 4.

[0159] (1) ELISA detection of specific IgG of RSV-F protein in serum

[0160] Fig. 7A-7D are the OD450 determined by indirect ELISA after the serum is diluted from 100 times to 1 million times. The sample OD450≥2 times the average value of the negative control OD450 of the minimum dilution multiple (1:100), then it is determined to be positive, and the dotted line on Fig. 7A-7D is the cutoff line. Among them, the Y6 immunization group did not appear positive value until diluted to 10,000 times. Fig. 7E is the determination of serum IgG antibody titer.

[0161] The results show that the IgG antibody titers of the RSV F protein variant mRNA vaccine immunization group are significantly higher than those of the PBS control group.

[0162] (2) ELISA detection of IFN-γ and IL4 in cell culture supernatant

[0163] The IFN-γ of the RSV F protein variant mRNA vaccine immunization group is significantly higher than that of the PBS control group (P<0.01) (Th1 type cellular immune response); the IL-4 of the RSV F protein variant mRNA vaccine immunization group is also significantly higher than that of the PBS control group (P<0.01).

[0164] (3) Flow detection of TNFα+ CD4+ and TNF-α+ CD8+ T cells

[0165] After 12 days of immunization, the mouse spleen lymphocytes were isolated, and the expression of TNF-α in CD3+ / CD4+ and CD3+ / CD8+ T lymphocytes was detected by flow cytometry. The RSV F protein variant immunization group was significantly higher than the PBS control group.

[0166] The results show that the RSV F protein variant mRNA vaccine can stimulate Th1 type cellular immune response in mice.

[0167] (4) Serum RSV neutralizing antibody titers

[0168] RSV live virus reduction assay was performed to test the serum RSV neutralizing antibody titers of the mice at 12 days post the second immunization. As shown in the figure, the RSV neutralizing antibody titers of all the RSV F protein variant mRNA immunization groups were significantly increased compared with the PBS control group.

[0169] Example 7

[0170] Based on the RSV F protein mutant encoded by sample Y6 (the amino acid sequence is shown as Seq ID NO. 78), the F2 sequence, RR1 sequence and RR2 sequence were mutated at partial sites or sequences, and based on the nucleotide ORF sequence of the RSV F protein mutant encoded by sample Y6 shown as Seq ID NO. 79, the mutation sites were replaced with commonly used codons of human, and the lipid nanoparticles of the RSV F protein mutant encoded by sample Y6-1, sample Y6-2, sample Y6-3, sample Y6-4, sample Y6-5, sample Y6-6, sample Y6-7 and sample Y6-8 were prepared according to the method described in Example 2.

[0171] Specifically, sample Y6-1 was replaced with the F2 sequence shown as Seq ID NO. 2; sample Y6-2 was replaced with the RR1 sequence shown as Seq ID NO. 6; sample Y6-3 was replaced with the RR2 sequence shown as Seq ID NO. 88; sample Y6-4 was replaced with the RR2 sequence shown as Seq ID NO. 89; sample Y6-5 was replaced with the RR2 sequence shown as Seq ID NO. 90; sample Y6-6 was replaced with the RR2 sequence shown as Seq ID NO. 92; sample Y6-7 was replaced with the RR2 sequence shown as Seq ID NO. 93; and sample Y6-8 was replaced with the F2 sequence shown as Seq ID NO. 2, the RR1 sequence shown as Seq ID NO. 6 and the RR2 sequence shown as Seq ID NO. 88.

[0172] In addition to the above nucleotide ORF sequence, the sequences of these RNA vaccines also include 5' cap, 5' UTR (shown as Seq ID NO. 58), 3' UTR (shown as Seq ID NO. 61) and 100 polyA 3' tail.

[0173] The obtained lipid nanoparticle samples Y6-1, Y6-2, Y6-3, Y6-4, Y6-5, Y6-6, Y6-7 and Y6-8 were subjected to vaccine immunogenicity evaluation according to the method of Example 4. The experimental results are shown in Table 6. Figure 11 .

[0174] Example 8

[0175] The RSV F protein mutant RR2 sequence encoded by sample 7 was replaced with the amino acid sequence of Seq ID NO. 90, and the nucleotide ORF sequence of the RSV F protein mutant encoded by sample Y7 was replaced with the nucleotide sequence of Seq ID NO. 80. The amino acid mutation sites were replaced with the commonly used codons of human beings. Sample Y7-1 was prepared according to the method described in Example 2.

[0176] The RSV F protein mutant RR2 sequence encoded by sample 7 was replaced with the amino acid sequence of Seq ID NO. 89, and the nucleotide ORF sequence of the RSV F protein mutant encoded by sample Y11 was replaced with the nucleotide sequence of Seq ID NO. 80. The RNA sequence of the amino acid mutation sites was obtained with the commonly used codons of human beings. Sample Y7-2 was prepared according to the method described in Example 2.

[0177] The RSV F protein mutant RR2 sequence encoded by sample 8 was replaced with the amino acid sequence of Seq ID NO. 92, and the nucleotide ORF sequence of the RSV F protein mutant encoded by sample Y8 was replaced with the nucleotide sequence of Seq ID NO. 81. The amino acid mutation sites were replaced with the commonly used codons of human beings. Sample Y8-1 was prepared according to the method described in Example 2.

[0178] The F2 sequence of the RSV F protein mutant encoded by sample 8 was replaced with the amino acid sequence of Seq ID NO. 2, the RR1 sequence of Seq ID NO. 7 and the RR2 sequence of Seq ID NO. 88, and the nucleotide ORF sequence of the RSV F protein mutant encoded by sample Y8 was replaced with the nucleotide sequence of Seq ID NO. 81. The amino acid mutation sites were replaced with the commonly used codons of human beings. Sample Y8-2 was prepared according to the method described in Example 2.

[0179] The RR2 sequence of the RSV F protein mutant encoded by sample 10 was replaced with the amino acid sequence of Seq ID NO. 88, and the nucleotide ORF sequence of the RSV F protein mutant encoded by sample Y10 was replaced with the nucleotide sequence of Seq ID NO. 83. The amino acid mutation sites were replaced with the commonly used codons of human beings. Sample Y10-1 was prepared according to the method described in Example 2.

[0180] The RR2 sequence of the RSV F protein mutant encoded by sample 10 was replaced with the RR2 sequence of amino acid sequence Seq ID NO. 93 to obtain sample Y10-2, which was prepared according to the method described in Example 2 based on the nucleotide ORF sequence of the RSV F protein mutant encoded by sample Y10 as shown in Seq ID NO. 83, and the mutation site was replaced with the commonly used codon of human.

[0181] In addition to the nucleotide ORF sequence described above, the sequence of these RNA vaccines also includes a 5' cap, a 5' UTR (as shown in Seq ID NO. 58), a 3' UTR (as shown in Seq ID NO. 61), and a 3' tail of 100 polyA.

[0182] The lipid nanoparticle samples Y7-1, Y7-2, sample Y8-1, sample Y8-2, sample Y10-1, and sample Y10-2 prepared were subjected to vaccine immunogenicity evaluation according to the method of Example 4. The experimental results are shown in Table 5. Figure 12 .

[0183] Example 9

[0184] The amino acid sequence encoded by sample Y12-1 is based on the RSV F protein mutant encoded by sample Y12, which encodes a RSV F protein mutant with an amino acid sequence as shown in Seq ID NO. 64, including an SP sequence as shown in Seq ID NO. 1, an F2 sequence as shown in Seq ID NO. 5, a GS-linker-linker sequence as shown in Seq ID NO. 77, an RR1 sequence as shown in Seq ID NO. 9, an F1 sequence as shown in Seq ID NO. 11, an RR2 sequence as shown in Seq ID NO. 12, and a terminal sequence as shown in Seq ID NO. 41, and the ORF mRNA sequence thereof is as shown in Seq ID NO. 98.

[0185] The RR2 sequence of the RSV F protein mutant encoded by sample 12-1 was replaced with the RR2 sequence of amino acid sequence Seq ID NO. 13-Seq ID NO. 20 to obtain samples Y12-2, Y12-3, Y12-4, Y12-5, Y12-6, Y12-7, Y12-8, and Y12-9, which were prepared according to the method described in Example 2 based on the nucleotide ORF sequence of the RSV F protein mutant encoded by sample Y12-1 as shown in Seq ID NO. 98.

[0186] In addition to the nucleotide ORF sequence described above, the sequence of these RNA vaccines also includes a 5' cap, a 5' UTR (as shown in Seq ID NO. 58), a 3' UTR (as shown in Seq ID NO. 61), and a 3' tail of 100 polyA.

[0187] The obtained lipid nanoparticle samples Y12-2, Y12-3, Y12-4, Y12-5, Y12-6, Y12-7, Y12-8 and Y12-9 were prepared, and the vaccine immunogenicity evaluation was carried out according to the method of Example 4. The experimental results are shown in Table 6. Figure 13 .

[0188] Example 10

[0189] Sample Y6.1, sample Y6.2, sample Y6.3, sample Y6.4, sample Y6.5, sample Y6.6 are mRNA lipid nanoparticles encoding respiratory syncytial virus antigen hRSV F protein variants prepared according to the method described in Example 2.

[0190] The mRNA lipid nanoparticles encoding respiratory syncytial virus antigen hRSV F protein variants in sample Y6.1, the amino acid sequence of the hRSV F protein includes SP sequence as shown in Seq ID NO. 1, F2 sequence as shown in Seq ID NO. 3, P27 sequence as shown in Seq ID NO. 76, RR1 sequence as shown in Seq ID NO. 7, F1 sequence as shown in Seq ID NO. 10, RR2 sequence as shown in Seq ID NO. 91, dimerization element sequence as shown in Seq ID NO. 21; the RNA sequence of the dimerization element sequence is shown in Seq ID NO. 99.

[0191] The mRNA lipid nanoparticles encoding respiratory syncytial virus antigen hRSV F protein variants in sample Y6.2, the amino acid sequence of the hRSV F protein includes SP sequence as shown in Seq ID NO. 1, F2 sequence as shown in Seq ID NO. 3, P27 sequence as shown in Seq ID NO. 76, RR1 sequence as shown in Seq ID NO. 7, F1 sequence as shown in Seq ID NO. 10, RR2 sequence as shown in Seq ID NO. 91, trimerization element sequence as shown in Seq ID NO. 25; the RNA sequence of the trimerization element sequence is shown in Seq ID NO. 100.

[0192] The mRNA lipid nanoparticles in sample Y6.3 encoding a variant of the hRSV F protein of the respiratory syncytial virus antigen, the amino acid sequence of the original hRSV F protein comprising a SP sequence as shown in Seq ID NO. 1, a F2 sequence as shown in Seq ID NO. 3, a P27 sequence as shown in Seq ID NO. 76, a RR1 sequence as shown in Seq ID NO. 7, a F1 sequence as shown in Seq ID NO. 10, a RR2 sequence as shown in Seq ID NO. 91, a trimerization element sequence as shown in Seq ID NO. 31; the RNA sequence of the trimerization element sequence is shown in Seq ID NO. 101.

[0193] The mRNA lipid nanoparticles in sample Y6.4 encoding a variant of the hRSV F protein of the respiratory syncytial virus antigen, the amino acid sequence of the original hRSV F protein comprising a SP sequence as shown in Seq ID NO. 1, a F2 sequence as shown in Seq ID NO. 3, a P27 sequence as shown in Seq ID NO. 76, a RR1 sequence as shown in Seq ID NO. 7, a F1 sequence as shown in Seq ID NO. 10, a RR2 sequence as shown in Seq ID NO. 91, a trimerization element sequence as shown in Seq ID NO. 42; the RNA sequence of the trimerization element sequence is shown in Seq ID NO. 102.

[0194] The mRNA lipid nanoparticles in sample Y6.5 encoding a variant of the hRSV F protein of the respiratory syncytial virus antigen, the amino acid sequence of the original hRSV F protein comprising a SP sequence as shown in Seq ID NO. 1, a F2 sequence as shown in Seq ID NO. 3, a P27 sequence as shown in Seq ID NO. 76, a RR1 sequence as shown in Seq ID NO. 7, a F1 sequence as shown in Seq ID NO. 10, a RR2 sequence as shown in Seq ID NO. 91, a trimerization element sequence as shown in Seq ID NO. 48; the RNA sequence of the trimerization element sequence is shown in Seq ID NO. 103.

[0195] The mRNA lipid nanoparticles in sample Y6.6 encoding the hRSV F protein variant of the respiratory syncytial virus antigen, the original hRSV F protein amino acid sequence of which comprises the SP sequence as shown in Seq ID NO. 1, the F2 sequence as shown in Seq ID NO. 3, the P27 sequence as shown in Seq ID NO. 76, the RR1 sequence as shown in Seq ID NO. 7, the F1 sequence as shown in Seq ID NO. 10, the RR2 sequence as shown in Seq ID NO. 91, and the tetramerization element sequence as shown in Seq ID NO. 54; the RNA sequence of the tetramerization element sequence is shown in Seq ID NO. 104.

[0196] The RNA vaccine encoding the respiratory syncytial virus antigen further comprises a 5' cap, a 5' UTR (as shown in Seq ID NO. 58), a 3' UTR (as shown in Seq ID NO. 61), and a 3' tail of 100 polyA in addition to the reading frame sequence in the above table.

[0197] The prepared RNA encoding the respiratory syncytial virus antigen lipid nanoparticles were subjected to vaccine immunogenicity evaluation according to the method described in Example 4. The detection results are shown in the figure.

[0198] The application also replaces the multimerization element with the sequence as shown in amino acid sequences Seq ID NO. 21-Seq ID NO. 57 to obtain a new amino acid sequence of the hRSV F protein variant of the respiratory syncytial virus antigen, and designs and prepares the corresponding RNA lipid nanoparticles containing the RNA encoding the respiratory syncytial virus antigen according to the new amino acid sequence, and performs vaccine immunogenicity evaluation according to the method of Example 4. The neutralization activity NT50 of the serum of the experimental mouse of the dimerization element is about 3000; the neutralization activity NT50 of the serum of the experimental mouse of the trimerization element is about 3300; and the neutralization activity NT50 of the serum of the experimental mouse of the tetramerization element is about 3200.

[0199] Example 11

[0200] Sample Y12.1, sample Y12.2, sample Y12.3, sample Y12.4, sample Y12.5, and sample Y12.6 are mRNA lipid nanoparticles encoding the hRSV F protein variant of the respiratory syncytial virus antigen prepared according to the method described in Example 2.

[0201] mRNA Lipid Nanoparticles in sample Y12.1 encoding a respiratory syncytial virus antigen hRSV F protein variant, the prot hRSV F protein amino acid sequence comprising a SP sequence as shown in Seq ID NO. 1, a F2 sequence as shown in Seq ID NO. 4, a GS-linker-linker sequence as shown in Seq ID NO. 77, a RR1 sequence as shown in Seq ID NO. 8, a Fl sequence as shown in Seq ID NO. 11, a RR2 sequence as shown in Seq ID NO. 17, a dimerization element sequence as shown in Seq ID NO. 22; the RNA sequence of the dimerization element sequence is shown in Seq ID NO. 105.

[0202] mRNA Lipid Nanoparticles in sample Y12.2 encoding a respiratory syncytial virus antigen hRSV F protein variant, the prot hRSV F protein amino acid sequence comprising a SP sequence as shown in Seq ID NO. 1, a F2 sequence as shown in Seq ID NO. 4, a GS-linker-linker sequence as shown in Seq ID NO. 77, a RR1 sequence as shown in Seq ID NO. 8, a Fl sequence as shown in Seq ID NO. 11, a RR2 sequence as shown in Seq ID NO. 17, a trimerization element sequence as shown in Seq ID NO. 26; the RNA sequence of the trimerization element sequence is shown in Seq ID NO. 106.

[0203] mRNA Lipid Nanoparticles in sample Y12.3 encoding a respiratory syncytial virus antigen hRSV F protein variant, the prot hRSV F protein amino acid sequence comprising a SP sequence as shown in Seq ID NO. 1, a F2 sequence as shown in Seq ID NO. 4, a GS-linker-linker sequence as shown in Seq ID NO. 77, a RR1 sequence as shown in Seq ID NO. 8, a Fl sequence as shown in Seq ID NO. 11, a RR2 sequence as shown in Seq ID NO. 17, a trimerization element sequence as shown in Seq ID NO. 37; the RNA sequence of the trimerization element sequence is shown in Seq ID NO. 107.

[0204] The mRNA lipid nanoparticle in sample Y12.4 encoding a respiratory syncytial virus antigen hRSV F protein variant, the original hRSV F protein amino acid sequence of which comprises an SP sequence as shown in Seq ID NO. 1, a F2 sequence as shown in Seq ID NO. 4, a GS-linker-linker sequence as shown in Seq ID NO. 77, a RR1 sequence as shown in Seq ID NO. 8, a F1 sequence as shown in Seq ID NO. 11, a RR2 sequence as shown in Seq ID NO. 17, a trimerization element sequence as shown in Seq ID NO. 50; the RNA sequence of the trimerization element sequence is shown in Seq ID NO. 108.

[0205] The mRNA lipid nanoparticle in sample Y12.5 encoding a respiratory syncytial virus antigen hRSV F protein variant, the original hRSV F protein amino acid sequence of which comprises an SP sequence as shown in Seq ID NO. 1, a F2 sequence as shown in Seq ID NO. 4, a GS-linker-linker sequence as shown in Seq ID NO. 77, a RR1 sequence as shown in Seq ID NO. 8, a F1 sequence as shown in Seq ID NO. 11, a RR2 sequence as shown in Seq ID NO. 17, a trimerization element sequence as shown in Seq ID NO. 53; the RNA sequence of the trimerization element sequence is shown in Seq ID NO. 109.

[0206] The mRNA lipid nanoparticle in sample Y12.6 encoding a respiratory syncytial virus antigen hRSV F protein variant, the original hRSV F protein amino acid sequence of which comprises an SP sequence as shown in Seq ID NO. 1, a F2 sequence as shown in Seq ID NO. 4, a GS-linker-linker sequence as shown in Seq ID NO. 77, a RR1 sequence as shown in Seq ID NO. 8, a F1 sequence as shown in Seq ID NO. 11, a RR2 sequence as shown in Seq ID NO. 17, a tetramerization element sequence as shown in Seq ID NO. 57; the RNA sequence of the tetramerization element sequence is shown in Seq ID NO. 110.

[0207] The RNA vaccine encoding a respiratory syncytial virus antigen further comprises a 5’ cap, a 5’ UTR (as shown in Seq ID NO. 58), a 3’ UTR (as shown in Seq ID NO. 61), and a 3’ tail of 100 polyA in addition to the reading frame sequences in the above table.

[0208] The prepared lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens were subjected to vaccine immunogenicity evaluation according to the method described in Example 4. The test results are shown in the figure.

[0209] The application also replaces the multimerization element with the sequence shown in amino acid sequences such as Seq ID NO. 21-Seq ID NO. 57, respectively, to obtain the amino acid sequence of the new respiratory syncytial virus antigen hRSV F protein variant. According to the new amino acid sequence, the corresponding lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens are designed and prepared, and subjected to vaccine immunogenicity evaluation according to the method of Example 4. The neutralizing activity NT50 of the serum of the experimental mouse with the dimerization element is about 3800; the neutralizing activity NT50 of the serum of the experimental mouse with the trimerization element is about 4000; and the neutralizing activity NT50 of the serum of the experimental mouse with the tetramerization element is about 3700.

[0210] Example 12

[0211] The nucleotide sequences described in Table 7 are mRNA sequences. The mRNA sequences described in Table 7 are prepared into lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens according to the method described in Example 2.

[0212] The prepared lipid nanoparticles are subjected to vaccine immunogenicity evaluation according to the method of Example 4. The experimental results are shown in the figure.

[0213] Table 7

[0214]

[0215] Example 13

[0216] The lipid nanoparticles containing RNA encoding respiratory syncytial virus antigens prepared from Y6, Y12, Y12-1 in Example 5, and syncytial virus F glycoprotein (purchased from ATCC) are subjected to vaccine immunogenicity evaluation according to the method described in Example 4. The results are shown in the figure.

[0217] Example 14

[0218] Heterologous protein expression requires codon optimization, choosing synonymous codons corresponding to high-abundance tRNA to improve protein translation efficiency. In 2006, Grzegorz Kudla et al. published the article High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells, and found that genes with high GC content have several to one hundred times higher expression efficiency than genes with low GC content in mammalian cells. This phenomenon is due to the more efficient mRNA transcription or processing of genes with high GC content, resulting in more stable mRNA.

[0219] Codon Adaptation Index (CAI) refers to the degree of consistency between the codons in the heterologous mRNA sequence and the optimal codon usage frequency of the host cell. The closer this value is to 1, theoretically, the higher the protein expression of the exogenous mRNA in the host cell. Therefore, the most basic principle of codon optimization is to replace the codons in the exogenous mRNA sequence with synonymous codons with high usage frequency in the host cell, to ensure that the codon usage bias of the exogenous mRNA sequence and the host cell is more consistent, and to avoid the use of rare codons. However, we need to know that codon is not the only factor affecting protein expression, there are other factors, such as rare codons, GC content, secondary structure (free energy), etc. When all the codons in the exogenous mRNA sequence are replaced with the optimal codons of the host cell, it may actually lead to the failure of protein expression, because the presence of rare codons is required for the expression of some proteins, to slow down the speed of ribosome advancement, and to provide enough time for the correct folding of proteins.

[0220] In October 2021, Zhang He et al. published the article Linear Design: Efficient Algorithms for Optimized mRNA Sequence Design, developed a new algorithm to optimize mRNA sequence more efficiently, website http: / / rna.baidu.com / , the algorithm takes into account the Codon Adaptation Index (CAI) and the folding free energy (MFE) of mRNA, can obtain more stable mRNA structure, prolong the half-life of mRNA and protein expression time, thus improving the final yield of mRNA in cells.

[0221] Sample Y21, sample Y22, sample Y23 are respectively RNA vaccine LNP encoding respiratory syncytial virus antigen comprising RNA sequence of reading frame sequence as shown in Seq ID NO. 65, Seq ID NO. 111, Seq ID NO. 112, in addition to the reading frame sequence, the mRNA further comprises 5' cap, 5' UTR (as shown in Seq ID NO. 58), 3' UTR (as shown in Seq ID NO. 61) and 100 polyA 3' tail. The reading frame sequence contained in the RNA vaccine LNP of sample Y21, sample Y22, sample Y23 and Y12-1 is obtained by using different optimization methods, and the ΔG, GC content and CAI of the four reading frame sequences are measured by using online tools, and the results are as follows: ΔG of Seq ID NO. 65 is -879.60 kcal / mol, GC is 52.24%, CAI is 0.73; ΔG of Seq ID NO. 98 is -493.20 kcal / mol, GC is 53.57%, CAI is 0.93; ΔG of Seq ID NO. 111 is -413.30 kcal / mol, GC is 46.92%, CAI is 0.81; ΔG of Seq ID NO. 112 is -483.70 kcal / mol, GC is 53.84%, CAI is 0.93.

[0222] According to the method of Example 4, sample Y21, sample Y22, sample Y23, sample Y12-1 were subjected to vaccine immunogenicity evaluation. The experimental results are shown in Table 1. Figure 18 .

[0223] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features thereof; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An immunological composition comprising an antigen of human respiratory syncytial virus, characterized in that, The immunological composition is selected from a nucleic acid immunological composition or a viral immunological composition; the human respiratory syncytial virus antigen is a pre-fusion hRSV F protein; the amino acid sequence of the pre-fusion hRSV F protein is shown as SEQ ID NO.

64.

2. The immunological composition of claim 1, wherein The immunological composition is a nucleic acid immunological composition, which comprises a nucleic acid encoding a pre-fusion hRSV F protein, and the nucleic acid is a DNA molecule or an RNA molecule.

3. The immunological composition of claim 2, wherein The nucleic acid is a DNA molecule, which is a linear DNA molecule or a circular DNA molecule.

4. The immunological composition of claim 2, wherein The nucleic acid is an RNA molecule, which is an mRNA molecule or a circular RNA molecule.

5. The immunological composition of claim 4, wherein the polypeptide is a polypeptide of SEQ ID NO: 1 or a polypeptide of SEQ ID NO:

2. The RNA molecule is an mRNA, which comprises an open reading frame, and the nucleotide sequence of the open reading frame is shown as SEQ ID NO.

98.

6. The immunological composition of claim 5, wherein The 5' end of the mRNA molecule has a protective modification group, which is selected from ARCA, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, mCAP, dmCAP, tmCAP or dmCAP.

7. The immune composition according to claim 5, characterized in that, The 3' end of the mRNA molecule has a protective modification group, which is poly(A) with a length of 50-200.

8. The immunological composition of claim 7, wherein the polypeptide is a polypeptide of SEQ ID NO: 1 or a polypeptide of SEQ ID NO:

2. The poly(A) has a length of 80-200.

9. The immunological composition of claim 5, wherein The mRNA molecule further comprises a 5'UTR and a 3'UTR, the nucleotide sequence of the 5'UTR is shown as one of SEQ ID NO. 58-60, and the sequence of the 3'UTR is shown as one of SEQ ID NO. 61-63.

10. The immunological composition of claim 5, wherein One or more uridines in the mRNA molecule are replaced by modified nucleosides, and the modified nucleosides replacing the uridines are pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U).

11. The immunological composition of claim 5, wherein The nucleic acid immunological composition comprises a carrier adapted to the nucleic acid, and the carrier is selected from a liposome nanoparticle, a cationic nanoemulsion or LLP.

12. The method of preparing an immunological composition according to claim 5, wherein The nucleic acid immunological composition is obtained by mixing an aqueous phase containing the above mRNA molecule with an organic phase containing a carrier component; the carrier is a liposome nanoparticle; the liposome nanoparticle comprises 20-50% of a cationic lipid, 20-50% of DOPG, 5-20% of cholesterol and 1-5% of PEG-DMG in terms of molar percentage.

13. The method of preparing an immunological composition according to claim 12, wherein the step of mixing the antigen and the adjuvant is performed at a temperature of 4°C to 37°C. The mRNA molecule is dissolved in a buffer to obtain an aqueous phase, and each lipid component of the liposome nanoparticle is dissolved in an organic solvent to obtain an organic phase, and the nucleic acid immunological composition is obtained by mixing the aqueous phase and the organic phase and removing the organic phase.

14. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleic acid encoding a human respiratory syncytial virus antigen, and the human respiratory syncytial virus antigen is a pre-fusion hRSV F protein; the amino acid sequence of the pre-fusion hRSV F protein is shown as SEQ ID NO.

64.

15. The nucleic acid molecule of claim 14, wherein The nucleic acid encoding the human respiratory syncytial virus antigen is a DNA molecule or an RNA molecule.

16. The nucleic acid molecule of claim 15, wherein The nucleic acid is a DNA molecule, which is a linear DNA molecule or a circular DNA molecule.

17. The nucleic acid molecule of claim 16, wherein The nucleic acid is an RNA molecule, which is mRNA or circular RNA.

18. The nucleic acid molecule of claim 17, wherein The RNA molecule is mRNA, which comprises an open reading frame, and the nucleotide sequence of the open reading frame is shown as SEQ ID NO. 98.

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