Polynucleotide for coding RSV (Respiratory Syndrome Virus) pre-fusion F protein and application thereof in mRNA (Messenger Ribonucleic Acid) vaccine

Abstract

The invention provides polynucleotide for coding an RSV (Respiratory Syndrome Virus) pre-fusion F protein and application of the polynucleotide in an mRNA (Messenger Ribonucleic Acid) vaccine. The sequence of the polynucleotide is as shown in SEQ ID No: 1. Experiments prove that the antigen-specific IgG titer of the polynucleotide, the vaccine and the pharmaceutical composition provided by the invention is 2-4 times that of an unoptimized sequence, so that more efficient expression of target protein in vivo is prompted, and meanwhile, the geometric mean titer value of a neutralizing antibody is also higher than that of the unoptimized sequence, so that good immunogenicity is prompted. The GMT value and the antigen specificity IFN-gamma, IL-5 and IL-17A levels of the neutralizing antibody generated by induction of the RSV mRNA vaccine provided by the invention are obviously higher than those of a control group in the prior art, so that the RSV mRNA vaccine has excellent application prospects.

Description

Technical Field

[0001] This disclosure relates to the field of biomedicine, and in particular, to a polynucleotide encoding an antigenic polypeptide and its applications. Background Technology

[0002] Respiratory syncytial virus (RSV) is one of the leading pathogens causing lower respiratory tract infections in infants, the elderly, and immunocompromised individuals worldwide. The associated disease burden is severe, posing a significant challenge to global public health. The RSV fusion protein (F protein) plays a crucial role in viral invasion of host cells and is a primary target for neutralizing antibodies, thus becoming the core antigen in current RSV vaccine development. To date, three RSV vaccines have been approved for marketing globally, including two recombinant protein vaccines and one mRNA vaccine. All of these vaccines use the prefusion conformation F protein, which has undergone stable mutation modification, as the immunogen.

[0003] With the successful development and widespread application of mRNA vaccines, mRNA technology has shown great promise in infectious disease control and tumor immunotherapy. The antigen structure and sequence design employed in existing vaccines (including RSV protein vaccines and the first RSV mRNA vaccine) have laid an important foundation for research and development, but their immunogenicity and protective efficacy still have room for improvement. By rationally designing novel mRNA sequences, it is possible to optimize the translation efficiency and stability of antigens, inducing higher levels and more durable neutralizing antibodies as well as a more balanced cellular immune response, potentially providing more comprehensive and robust immune protection, especially in combating viral mutations and preventing severe lower respiratory tract infections. Different codon optimization methods (such as those based on human codon bias, mRNA secondary structure content, and tRNA abundance matching) will produce drastically different sequences. Through novel computational design and experimental screening, it is hoped that novel RSV mRNA vaccine sequences with better immunogenicity can be obtained. Therefore, designing more stable RSV mRNA sequences with higher translation efficiency and immunogenicity is an urgent priority. Summary of the Invention

[0004] Technical problems to be solved:

[0005] One aspect of this disclosure is to address the problems of low translation efficiency and poor immunogenicity in existing respiratory syncytial virus mRNA vaccines by providing a polynucleotide encoding an antigenic polypeptide and its application.

[0006] Specifically, the technical solution disclosed herein creatively employs a genetic algorithm to design the codon sequence encoding the pre-fusion F protein by optimizing multiple empirical indicators (such as codon preference index, GC content, system free energy, secondary structure length and content, etc.). This method synergistically considers multiple key indicators during the optimization process, achieving optimized design of the pre-fusion F protein mRNA sequence and thus solving the aforementioned problems.

[0007] Technical solution:

[0008] A polynucleotide encoding an antigenic polypeptide, the sequence of which is shown in SEQ ID No:1.

[0009] In some embodiments, the polynucleotide is mRNA without introns.

[0010] In some embodiments, the polynucleotide sequence described above may be unmodified mRNA. In other embodiments, it may be nucleoside-modified mRNA.

[0011] In some embodiments, the above-mentioned nucleoside modification is selected from at least one of the following modifications: pseudouridine, N1-methylpseuuridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazo-pseuuridine, 2-thio-1-methyl-pseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methyl-pseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, 5-methoxyuridine, or 2'-O-methyluridine.

[0012] In some implementations, the open reading frame (ORF) formed by the polynucleotides described above is codon-optimized.

[0013] In some embodiments, the two ends of the above sequence further include:

[0014] (1) The untranslated region and cap structure located at the end of the 5' sequence; and

[0015] (2) The untranslated region and the stabilized tail structure located at the end of the sequence 3'.

[0016] In some embodiments, the stabilized tail structure is a 3' end stabilizing element comprising a polyadenylated sequence (PolyA) or a functional analogue thereof, wherein the number of adenosine nucleotides in the polyadenylated sequence is 100 to 150.

[0017] In some embodiments, the sequence of the untranslated region at the 3' end is as shown in SEQ ID No:4, or a sequence that has more than 80% identity with the sequence and has the same or similar function.

[0018] In some embodiments, the cap structure (5'Cap) described above is a cap structure containing 7-methylguanosine. The 7-methylguanosine cap structure can be m... 7 The cap structure represented by G(5')ppp(5')N or its pharmaceutically acceptable salt or derivative.

[0019] In some embodiments, the sequence of the untranslated region at the 5' end is as shown in SEQ ID No:5, or a sequence that has more than 80% identity with the sequence and has the same or similar function.

[0020] In this disclosure, having more than 80% identity means a sequence that has at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the target sequence.

[0021] In some embodiments, the polynucleotide is a liquid formulation. In other embodiments, the polynucleotide is a lyophilized agent. In still other embodiments, the polynucleotide is a cryogenic formulation. In yet another embodiment, the polynucleotide is an injectable formulation.

[0022] Another aspect of this disclosure is to provide a nucleic acid vaccine comprising the aforementioned polynucleotides.

[0023] In some embodiments, the polynucleotides are configured in carriers, such as lipid nanoparticles, polymer nanoparticles, peptide carriers, or exosomes, in the nucleic acid vaccine. Further, in other embodiments, the polynucleotides are configured for delivery / transport within lipid nanoparticles (LNPs). The lipid nanoparticles may include: (1) ionizable oxygen-ionized lipids; (2) neutral lipids; (3) steroids; and (4) polymer-coupled lipids. In some embodiments, the lipid nanoparticles may be modified.

[0024] In some embodiments, the cationic ionizable lipids include ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate). In some embodiments, the steroids include cholesterol. In some embodiments, the neutral lipids include phospholipids. In some embodiments, the phospholipids include distearate phosphatidylcholine (DSPC). In some embodiments, the polymer-coupled lipids include polyethylene glycol (PEG)-lipids. In some embodiments, the PEG lipids include 2-[(polyethylene glycol)-2000]- / V z / V-bistetradecylacetamide.

[0025] In some implementations, the aforementioned nucleic acid vaccine can also be multivalent.

[0026] Another aspect of this disclosure is to provide a pharmaceutical composition comprising the aforementioned polynucleotide or nucleic acid vaccine.

[0027] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, delivery medium, excipient, stabilizer, diluent, or combination thereof. The pharmaceutical composition may be a vaccine composition.

[0028] Another aspect of this disclosure is the use of the aforementioned polynucleotide, the aforementioned nucleic acid vaccine, or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention or treatment of respiratory syncytial virus infection.

[0029] Another aspect of this disclosure is a method for generating a specific immune response against respiratory syncytial virus in a subject, the method being the administration of an effective dose of the aforementioned polynucleotide, the aforementioned nucleic acid vaccine, or the aforementioned pharmaceutical composition to the subject. In some embodiments, this may be a first administration. In other embodiments, it may be a booster administration.

[0030] Another aspect of this disclosure is to provide a method for preventing or treating respiratory syncytial virus infection, the method being to administer to a subject an effective dose of the above-mentioned polynucleotide, the above-mentioned nucleic acid vaccine, or the above-mentioned pharmaceutical composition.

[0031] Beneficial effects:

[0032] Experimental verification showed that the antigen-specific IgG titers of the polynucleotides, vaccines, and pharmaceutical compositions provided in this disclosure were 2-4 times higher than those of the unoptimized sequences, indicating more efficient expression of the target protein in vivo. Simultaneously, the geometric mean titer of neutralizing antibodies was also higher than that of the unoptimized sequences, indicating good immunogenicity. The neutralizing antibody GMT values, antigen-specific IFN-γ, IL-5, and IL-17A levels induced by the RSV mRNA vaccine provided in this disclosure were significantly higher than those of the prior art control group, thus demonstrating excellent application prospects. Attached Figure Description

[0033] Figure 1A This is the result of antigen-specific IgG antibody detection after immunization in one embodiment of this disclosure. Figure 1B The results of antigen-specific IgG antibody detection after the second immunization in this embodiment of the present disclosure;

[0034] Figure 2 The results of neutralizing antibody detection after the second immunization in this embodiment of the present disclosure;

[0035] Figure 3 The results of neutralizing antibody detection are shown in the embodiments of this disclosure;

[0036] Figure 4 The results of cell-mediated immunity detection in the embodiments of this disclosure;

[0037] Figure 5 The results of mouse weight changes in the embodiments of this disclosure;

[0038] Figure 6 The results of viral load detection in mouse lung tissue in this embodiment of the present disclosure;

[0039] Figure 7 The results of mouse lung tissue pathology (HE staining) in the embodiments of this disclosure are shown.

[0040] Sequence description.

[0041] SEQ ID No:1 is the TriM5 mRNA sequence in the embodiments of this disclosure;

[0042] SEQ ID No:2 is the mRNA sequence of the wild-type sequence in the embodiments of this disclosure;

[0043] SEQ ID No:3 is the mRNA sequence of the Moderna control in the embodiments of this disclosure;

[0044] SEQ ID No:4 is the nucleic acid sequence of the untranslated region at the 3' end in the embodiments of this disclosure;

[0045] SEQ ID No:5 is the nucleic acid sequence of the untranslated region at the 5' end in this embodiment. Detailed Implementation

[0046] This document discloses a polynucleotide encoding an antigenic polypeptide and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately alter and combine the content described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0047] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., shall be understood to include the stated elements or components without excluding other elements or other components. The term "a," "an," and "the" includes plural indicators. The term "a plurality of" means two or more. The terms "such as," "for example," etc., are intended to refer to exemplary embodiments and are not intended to limit the scope of this disclosure.

[0048] In this disclosure, when a range of values ​​is provided, it should be understood that, unless the context otherwise explicitly indicates otherwise, the range includes endpoints and each intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range and any value within a smaller range between specified values.

[0049] In this disclosure, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0050] In this disclosure, terms such as "one embodiment," "an example," "some embodiments," "a particular embodiment," "related embodiment," "a certain embodiment," "some embodiments," "additional embodiment," or "further embodiment," "further implementation," or "another embodiment," "some other embodiments," mean that at least one feature or characteristic description is included in relation to the embodiment. Therefore, throughout this disclosure, the above phrases do not necessarily refer to the same embodiment. Furthermore, specific features may be combined in any suitable manner in one or more embodiments.

[0051] The use of any and all examples or exemplary language (e.g., "for example") provided in this disclosure is intended only to better illustrate this disclosure and does not limit the scope of the claims of this disclosure. No language in the specification should be construed as indicating any unclaimed elements essential to the practice of this disclosure.

[0052] As used in this disclosure, “and / or” should be understood as a specific disclosure of either of the two specified features or components, whether or not the other is included. For example, “X and / or Y” should be understood as a specific disclosure of (i) X, (ii) Y, and (iii) X and Y, as if each were listed separately herein.

[0053] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions of common molecular biology terms can be found in Lewin's *GENES*, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Jones & Bartlett Learning. Definitions of common biochemistry terms can be found in Lehninger's *Principles of Biochemistry*, Eighth Edition, David L. Nelson, Michael M. Cox, WHFreeman. Definitions of common cell biology terms can be found in *Molecular Biology of the Cell*, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Garland Science. Definitions of common genetics terms can be found in *Genetics: Analysis of Genes and Genomes*, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Jones & Bartlett Learning.

[0054] Unless otherwise specified, the experimental techniques used in this paper employ standard techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard books such as *Molecular Cloning: A Laboratory Manual* and *Cell Biology: A Laboratory Handbook*.

[0055] definition:

[0056] The term "respiratory syncytial virus" (RSV) refers to a common virus that causes lung and respiratory infections. RSV is an enveloped, negative-sense, single-stranded RNA virus belonging to the genus *Orthopneumovirus* in the family Pneumoviridae. Its genome encodes 11 proteins, including 9 structural proteins and 2 non-structural proteins. Two transmembrane glycoproteins on the surface of the viral particle, attachment protein G and fusion protein F, play important roles in the initiation of viral infection and are the main targets of neutralizing antibodies. RSV has two subtypes: subtype A and subtype B. The G protein differs significantly between the two subtypes, while the F protein is relatively conserved, with sequence homology exceeding 90% (Ref: Harshbarger W, et al. PLoS Pathog., 2020, 16:e1008943.).

[0057] The term "polynucleotide" refers to a chain of nucleotides of any length, including DNA or RNA. It can include any known nucleotide analogs or modified nucleotides or bases.

[0058] The term "cap structure," also known as "5' cap" or "mRNA 5' modification," refers to 7-methylguanosine (mRNA) linked to the transcription initiation nucleotide of eukaryotic mRNA molecules via a 5'-5' triphosphate bridge. 7 G) or its derivatives. This structure is the initiation signal necessary for mRNA molecules to be recognized by ribosomes and initiate protein translation, while effectively protecting mRNA from degradation by exonucleases in the 5'→3' direction, thereby significantly improving the intracellular stability of mRNA and protein expression levels. In some embodiments, this term encompasses all modified guanosine structures that are achieved through chemical synthesis, enzymatic transformation, or co-transcriptional incorporation and can equivalently perform the aforementioned core functions of "translation initiation" and "stabilization," for example, including but not limited to m 7 GpppN (where N is any nucleoside), anti-reverse cap analogs, and CleanCap-conjugated analogs ® The hat form produced by the technology.

[0059] The term "stabilizing tail" refers to a nucleotide sequence or modified structure attached to the 3' end of an mRNA molecule that significantly enhances the intracellular stability of the mRNA and its protein expression levels. Its core function is to resist degradation by exonucleases along the 3'→5' direction, prolong the mRNA half-life, and synergistically promote efficient cycling of the translation complex with the 5' cap structure. In some embodiments, the term primarily includes the typical polyadenylated tail with a length of approximately 70 to approximately 200 adenosine nucleotides, but also encompasses other sequence forms that achieve equivalent stabilization and enhanced expression function, such as mixed tails containing non-adenosine nucleotides, chemically modified nucleotide tails, or 3' end structures that provide stability through other molecular mechanisms (such as forming specific secondary structures or binding to stabilizing proteins).

[0060] The term "identity" or "sequence identity" refers to the complete similarity of two sequences at the same nucleotide or amino acid residue site. It is a fundamental and important concept in bioinformatics, commonly used to compare the degree of similarity between two nucleic acid or protein sequences. Sequence identity is usually expressed as a percentage, reflecting the degree of consistency between sequences.

[0061] Example:

[0062] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0063] Example 1: Preparation of nucleic acid vaccine.

[0064] 1. Synthesize the following mRNA sequence:

[0065] (1) The optimized mRNA sequence of this disclosure (as shown in SEQ ID No:1, denoted as “TriM-5 mRNA sequence”);

[0066] (2) Control group: RSV F protein mRNA sequence before optimization (as shown in SEQ ID No:2, referred to as "wild sequence");

[0067] (3) Control group: Moderna's marketed RSV mRNA vaccine sequence (mRESVIA) ® As shown in SEQ ID No:3, it is described in patent WO2022 / 221336A1 and referred to as "Moderna Comparison").

[0068] 2. Encapsulate the above sequences respectively.

[0069] In this embodiment, the cationic lipid nanoparticles used to encapsulate mRNA consist of cationic lipids (SM102 or BM028), phospholipids (DSPC), cholesterol (Cholesterol), and polyethylene glycol-modified lipids (DMG-PEG2000). The cationic lipid SM102 was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number: 06040008800. The BM028 lipid was synthesized according to the invention patent with patent number ZL202411204254.5. Cholesterol was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number: 06040010300. The phospholipid (DSPC, 1,2-distearyl-sn-glycerol-3-phosphocholine, Distearoylphosphatidylcholine) was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number: 06030001100. Polyethylene glycol-modified lipids (DMG-PEG2000, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) were purchased from Xiamen Sinobond Biotechnology Co., Ltd., product number: 06020112402. The molar ratio of the four components of SM102 lipid nanoparticles was cationic lipid SM102: DSPC: Cholesterol: DMG-PEG2000 = 50: 10: 38.5:1.5. The ratio of cationic lipids to mRNA was N / P = 2.5. The molar ratio of the four components of BM028 lipid nanoparticles was cationic lipid BM028: DSPC: Cholesterol: DMG-PEG2000 = 48.5: 10: 40: 1.5. The ratio of cationic lipids to mRNA was N / P = 5.0.

[0070] Both methods for preparing mRNA-encapsulated lipid nanoparticles employed a microfluidic mixing system. Specifically, cationic lipid compounds, cholesterol, phospholipids, and polyethylene glycol-modified lipids were dissolved separately in anhydrous ethanol to prepare solutions with a concentration of 1.0 mg / mL. The corresponding volumes of the four solutions were then mixed according to the aforementioned molar ratio to prepare an organic phase solution. The mRNA was dissolved in 20 mM citrate-sodium citrate buffer solution (pH = 4.5) to prepare an aqueous mRNA solution with a concentration of 33 µg / mL. The organic phase containing the dissolved lipid mixture and the aqueous phase containing the dissolved mRNA were then mixed using a microfluidic system at a flow rate of 1:3, with a total mixing flow rate of 20 mL / min. Finally, the prepared lipid nanoparticles encapsulated with mRNA were diluted with 20 times their volume of phosphate buffer solution (135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2.0 mM NaH2PO4, pH 7.3 ± 0.1), and the solution was ultrafiltered to the desired volume using an ultrafiltration tube with a molecular weight cutoff of 30 kDa (Millipore). The obtained lipid nanoparticle-mRNA composition was further analyzed using dynamic light scattering to measure the particle size and polydispersity index (PDI). The encapsulation efficiency of the composition was characterized using RiboGreen reagent (Thermo Fisher Scientific, Invitrogen™, R11491). The final two lipid nanoparticle-mRNA compositions had particle sizes in the range of 77 nm–118 nm, PDIs in the range of 0.06–0.15, and encapsulation efficiencies of over 90%.

[0071] Example 2: Immunogenicity evaluation of RSV-mRNA vaccine.

[0072] Sequences (1) and (2) from Example 1 were encapsulated using SM102 lipid nanoparticles to prepare mRNA vaccines. The RSV mRNA vaccine prepared using the SEQ No:1 sequence is referred to as "TriM5-mRNA vaccine", and the RSV mRNA vaccine prepared using the SEQ No:2 sequence is referred to as "wild-type sequence control vaccine". BALB / c mice (female, 42-62 days old) were immunized by intramuscular injection at week 0 and week 2, with PBS as a negative control (100 μL / dose / mouse, mRNA content 2 μg / dose / mouse). Serum was collected 10 days after the first and second immunizations. The level of IgG antibody specific to the pre-fusion F protein was detected by ELISA. In addition, the level of neutralizing antibody against RSV A2 strain was detected in the serum 10 days after the second immunization using a live virus micro-neutralization assay.

[0073] The results of specific IgG antibody detection are shown in Table 1 and Figure 1A , Figure 1B As shown, 10 days after one injection ( Figure 1A ) and 10 days after 2 doses of immunization ( Figure 1B The results showed that the antigen-specific IgG antibody titer of the TriM5-mRNA vaccine was significantly higher than that of the wild-type sequence control vaccine (P < 0.001), indicating that the mRNA sequence optimized and designed using this embodiment can be effectively expressed in vivo and stimulate the production of high levels of antibodies. The serum neutralizing antibody results 10 days after two doses of immunization are shown in Table 1 and... Figure 2 As shown, 10 days after two doses of immunization, the neutralizing antibody level of the TriM5-mRNA vaccine was significantly higher than that of the wild-type sequence control vaccine (P < 0.001). These results demonstrate that the mRNA vaccine prepared using the optimized nucleotide sequence shown in SEQ No:1 of this embodiment can effectively stimulate the body to produce high levels of neutralizing antibodies, exhibits good immunogenicity, and is an ideal candidate RSV mRNA vaccine.

[0074] Table 1. GMT values ​​of F protein-specific IgG antibodies and neutralizing antibodies before fusion.

[0075] Example 3: Immunogenicity comparison with Moderna control vaccine

[0076] Sequences (1) and (3) from Example 1 were encapsulated with BM028 lipid nanoparticles to prepare mRNA vaccines. The RSV mRNA vaccine prepared using the SEQ No:1 sequence is referred to as "TriM5-mRNA vaccine", and the RSV mRNA vaccine prepared using the SEQ No:4 sequence is referred to as "Moderna control vaccine". BALB / c mice (female, 42-62 days old) were immunized with saline as a negative control. Intramuscular injections were administered at week 0 and week 3 (100 μL / dose / mouse, mRNA content 10 μg / dose / mouse). Serum was collected at week 4, and the neutralizing antibody level of RSVA2 strain was detected using a live virus micro-neutralization assay. In addition, spleen lymphocytes were collected from 5 mice to detect antigen-specific cellular immunity levels.

[0077] The results of neutralizing antibody detection are shown in Table 2 and Figure 3 As shown, the TriM5-mRNA vaccine induced a neutralizing antibody GMT value of 17560 against the RSV A2 strain, which is 3.03 times that of the Moderna control vaccine (GMT=5793), and the difference is statistically significant. Meanwhile, the results of cellular immune assays are shown in Table 3 and... Figure 4As shown, the TriM5-mRNA vaccine induced 7368, 425, and 200 spot-forming cells (SFCs) / 10 of IFN-γ, IL-5, and IL-17A, respectively. 6 The number of spleen lymphocytes was higher than that of the Moderna control vaccine. These results demonstrate that the mRNA vaccine prepared using the optimized nucleotide sequence shown in SEQ No. 1 of this invention possesses both humoral and cellular immune response capabilities, and both are superior to the Moderna control vaccine.

[0078] Table 2. GMT values ​​of neutralizing antibodies

[0079] Table 3. Mean values ​​of antigen-specific cytokines (ELISPOT multicolor fluorescence)

[0080] Example 4: Comparison of in vivo protective efficacy against Moderna control vaccine

[0081] Sequences (1) and (3) from Example 1 were encapsulated using BM028 lipid nanoparticles to prepare mRNA vaccines. The RSV mRNA vaccine prepared using the SEQ No:1 sequence is referred to as "TriM5-mRNA vaccine", and the RSV mRNA vaccine prepared using the SEQ No:4 sequence is referred to as "Moderna control vaccine". BALB / c mice (female, 42-62 days old) were immunized with saline as a negative control. Immunization was performed via intramuscular injection at week 0 and week 3 (100 μL / dose / mouse, mRNA content 10 μg / dose / mouse). At week 5, RSV A2 live virus was challenged via nasal instillation at a dose of 1 x 10⁻⁶ mc². 6 TCID 50 / animal, weight and survival status were monitored for 5 consecutive days after challenge. Lungs were collected on the 5th day after challenge for viral load in lung tissue (detected by PCR) and pathological examination (HE staining) to evaluate the in vivo protective effect of the vaccine against challenge.

[0082] The changes in body weight of mice after challenge are shown in Table 4 and Figure 5 The TriM5-mRNA vaccine and the Moderna control vaccine showed similar patterns of weight change. After challenge, both groups of immunized mice experienced a slight decrease in body weight, but recovered largely by day 4-5, with no significant difference between the two groups. The viral load results in lung tissue are shown in Table 5. Figure 6The viral load in lung tissue of both the TriM-5 mRNA vaccine and the Moderna control vaccine was significantly lower than that in the virus control group, with no significant difference between the two groups; lung tissue pathology results were as follows. Figure 7 Compared to the virus control group, no significant pathological changes were observed in the lung tissue of mice treated with the TriM-5 mRNA vaccine and the Moderna control vaccine, consistent with the negative control. No immunopathological phenomena were also observed. These results demonstrate that the mRNA vaccine prepared using the optimized nucleotide sequence shown in SEQ No. 1 of this invention exhibits good in vivo challenge protection, comparable to the Moderna control vaccine, making it an ideal candidate RSV mRNA vaccine.

[0083] Table 4. Changes in mouse body weight

[0084] Table 5. Results of viral load in lung tissue

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polynucleotide encoding an antigenic polypeptide, characterized in that, The sequence of the polynucleotide is shown as SEQ ID No:

1.

2. The polynucleotide of claim 1, wherein, The sequence is modified by nucleosides.

3. The polynucleotide of claim 2, wherein, The nucleoside modification is at least one selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2'-O-methyluridine.

4. The polynucleotide of claim 1, 2, or 3, wherein, The sequence further comprises at both ends thereof: (1) an untranslated region and a cap structure at the 5' end of the sequence; and (2) an untranslated region and a stabilization tail structure at the 3' end of the sequence.

5. The polynucleotide of claim 4, wherein, The stabilization tail structure is a 3' end stabilization element comprising a poly(A) sequence or a functional analog thereof, wherein the number of adenylate in the poly(A) sequence is 100-150.

6. The polynucleotide of claim 4, wherein The sequence of the untranslated region at the 3' end is shown as SEQ ID No:

4.

7. The polynucleotide of claim 4, wherein The cap structure is a cap structure comprising 7-methylguanosine.

8. The polynucleotide of claim 4, wherein The sequence of the untranslated region at the 5' end is shown as SEQ ID No:

5.

9. A nucleic acid vaccine, characterized in that, The nucleic acid vaccine comprises the polynucleotide according to any one of claims 1-8.

10. The nucleic acid vaccine of claim 9, wherein The nucleic acid vaccine further comprises a lipid nanoparticle, a polymeric nanoparticle, a polypeptide carrier, or an exosome.

11. The nucleic acid vaccine according to claim 9 or 10, characterized in that, The nucleic acid vaccine is multivalent.

12. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the polynucleotide according to any one of claims 1-8, or the nucleic acid vaccine according to claim 9, 10, or 11.

13. Use of the polynucleotide according to any one of claims 1-8, the nucleic acid vaccine according to claim 9, 10, or 11, or the pharmaceutical composition according to claim 12 in the preparation of a medicament for preventing or treating respiratory syncytial virus infection.

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