Respiratory syncytial virus mRNA vaccine and its preparation method and application

Abstract

This invention belongs to the field of nucleic acid vaccine technology and provides a respiratory syncytial virus (RSV) mRNA vaccine and its preparation method. The mRNA molecule of the invention contains antigenic sequences encoding RSV ON1 genotype F protein and partial fragments of the BA9 genotype F protein. Through structural modification, the mRNA encodes the preF protein or a protein containing its major antigenic epitopes. A specific plasmid was selected during vector construction to optimize the mRNA antigen sequence. After successful vaccine preparation, antigen mRNA expression was assayed and mouse immunization experiments were conducted. The results showed that the R01 vaccine had the best humoral immune effect, with no significant differences in cellular immune response among the groups, confirming that the vaccine of the invention can effectively induce both humoral and cellular immunity against RSV in mice.

Description

Technical Field

[0001] The present invention relates to the fields of biopharmaceuticals and nucleic acid vaccines, and more specifically, to RSV mRNA vaccines, preparation methods, and applications thereof. Background Art

[0002] Respiratory syncytial virus (RSV) belongs to the genus Pneumovirus in the family Paramyxoviridae, subfamily Pneumovirinae. Its genome is a single-stranded negative-sense RNA, 15.2 kb in length, encoding 11 proteins. Adhesion protein (G) and fusion protein (F) are the primary glycoproteins on the viral surface and are the most critical proteins mediating viral infection of human cells. RSV has only one serotype, divided into subtypes A and B. Internationally, RSV is further classified into different genotypes based on genetic variation in the G gene. Currently, the ON1 genotype of subtype A and the BA9 genotype of subtype B are the dominant genotypes prevalent globally and have also been the predominant genotypes prevalent in my country in recent years.

[0003] RSV can cause respiratory infections in people of all ages, with children, the elderly, and adults with immunodeficiency or underlying medical conditions being particularly susceptible. The clinical manifestations of RSV infection vary widely, ranging from mild respiratory infections to acute lower respiratory tract infections. Severe infections can also affect organs beyond the respiratory system. Furthermore, children infected with RSV early in life are at risk for recurrent wheezing and airway hyperresponsiveness. Therefore, the development of vaccines or treatments against RSV is crucial.

[0004] The search for an RSV vaccine began in the 1860s. For a long time, both inactivated and recombinant RSV vaccines had limited success. It wasn't until researchers deciphered the structural variations of RSV during infection that they realized the prefusion conformation (preF) of the F protein was the primary factor in initiating the immune response against RSV. Since then, RSV vaccines have used the preF protein as an antigen, achieving promising results.

[0005] Nucleic acid vaccines consisting of in vitro transcribed mRNAs encapsulated in lipid nanoparticles (LNPs) can elicit strong humoral and cellular immune responses. Delivery of mRNA antigens via LNPs can enhance immune responses to vaccines, as LNPs protect mRNA from enzymatic degradation and promote efficient mRNA uptake and intracellular release by target cells. However, no mRNA vaccines have been reported to be effective for RSV prevention and treatment. Summary of the Invention

[0006] The object of the present invention is to provide a respiratory syncytial virus mRNA vaccine, which comprises mRNA antigen molecules encoding partial fragments of respiratory syncytial virus ON1 genotype F protein and BA9 genotype F protein, which can effectively prevent and treat RSV infection.

[0007] The present invention provides an mRNA molecule encoding a respiratory syncytial virus (RSV) protein, comprising an antigen sequence encoding a partial fragment of RSV ON1 genotype F protein and a partial fragment of BA9 genotype F protein.

[0008] In one embodiment, the ends of the sequences encoding the partial fragment of RSV ON1 genotype F protein and the partial fragment of BA9 genotype F protein are respectively spliced ​​with sequences encoding T4 foldon structure, or the ends of the sequences encoding the partial fragment of RSV ON1 genotype F protein and the partial fragment of BA9 genotype F protein are respectively spliced ​​with Fc sequences encoding human IgG1.

[0009] In one embodiment, the antigen sequence encoding a partial fragment of the F protein of the respiratory syncytial virus ON1 genotype is encoded by the nucleotide sequence shown in SEQ ID NO.3, 5 or 7, and the antigen sequence encoding a partial fragment of the F protein of the respiratory syncytial virus BA9 genotype is encoded by the nucleotide sequence shown in SEQ ID NO.4, 6 or 8.

[0010] In one embodiment, the mRNA molecule further comprises a cap structure, a 5'UTR, a 3'UTR, and a poly A; the 5'UTR is selected from CYBA, DEN2, hHBB, RpL38, RpS25, TCV, or TOP; the 3'UTR is selected from hHBA1, hHBB, BYDV, hHBBx2, CYBA_1.5x, BMV, TSV, or TCV; and the poly A is selected from 60A, 100A, 30A-10X-70A, 30A-10X-70A-10C-A, or 40A-10X-60A-10C-A. Preferably, the 5'UTR is DEN2, the 3'UTR is hHBBx2, and the poly A is 100A.

[0011] The present invention also provides an expression vector for the mRNA molecule, which comprises the following elements from the 5' end to the 3' end: a 5'UTR, a coding sequence comprising a partial fragment of the RSV F protein, a 3'UTR and poly A; optionally, the coding sequence comprising a partial fragment of the RSV F protein is ligated into a pKL plasmid, preferably, the pKL plasmid is obtained by enzyme digestion of the pKL-Luci plasmid of SEQ ID NO.2.

[0012] In one embodiment, the 5'UTR of the expression vector of the mRNA molecule is selected from CYBA, DEN2, hHBB, RpL38, RpS25, TCV or TOP; the 3'UTR is selected from hHBA1, hHBB, BYDV, hHBBx2, CYBA_1.5x, BMV, TSV or TCV; the poly A is selected from 60A, 100A, 30A-10X-70A, 30A-10X-70A-10C-A or 40A-10X-60A-10C-A.

[0013] In one embodiment, the respiratory syncytial virus mRNA vaccine comprises the above-mentioned mRNA molecule.

[0014] In one embodiment, the respiratory syncytial virus mRNA vaccine further comprises lipid nanoparticles (LNPs), which encapsulate the mRNA molecules. Preferably, the LNP comprises D-Lin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 in a molar ratio of 50:10:38.5:1.5. Preferably, the nitrogen-phosphorus ratio of the lipid nanoparticles to the mRNA is (4-8):1 (the nitrogen-phosphorus ratio of the lipid nanoparticles to the mRNA refers to the molar ratio of the nitrogen element of the cationic lipid in the lipid nanoparticles to the phosphorus element of the mRNA molecule).

[0015] In one embodiment, the LNP-encapsulated mRNA molecule is a mixture of mRNA molecules encoding partial fragments of RSV ON1 genotype F protein and partial fragments of BA9 genotype F protein, respectively, wherein the molar ratio of the mRNA molecules encoding partial fragments of RSV ON1 genotype F protein and partial fragments of BA9 genotype F protein is (0.5-4):1.

[0016] The present invention also provides the use of the mRNA molecule, the expression vector, and the RSV mRNA vaccine in the preparation of a medicament for preventing and / or treating RSV infection. Preferably, the diseases include, but are not limited to, mild respiratory tract infections, acute lower respiratory tract infections, severe infections involving organs other than the respiratory system, and early-life RSV infection leading to subsequent recurrent wheezing and airway hyperresponsiveness in children.

[0017] Beneficial effects of the present invention:

[0018] This study designed and prepared a respiratory syncytial virus (RSV) mRNA vaccine containing antigenic mRNA molecules encoding fragments of the F protein of the RSV ON1 and BA9 genotypes. Through structural modification, both mRNAs encode preF proteins or proteins containing the major antigenic epitopes of the preF protein. Mouse immunization studies confirmed that the RSV mRNA vaccine effectively induced humoral and cellular immunity against RSV in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of pKL-EGFP plasmid.

[0020] Figure 2A This is the expression effect of pKL-EGFP in 293FT cells.

[0021] Figure 2B Expression effect of mRNA prepared from pKL-EGFP in 293FT cells.

[0022] Figure 3 The effect of different 5'UTR and 3'UTR on luciferase expression.

[0023] Figure 4 The effects of different 5'UTR and 3'UTR combinations on luciferase expression.

[0024] Figure 5 The effect of different poly A on luciferase mRNA expression.

[0025] Figure 6 The effects of different 5'UTR, 3'UTR and poly A combinations on luciferase mRNA expression.

[0026] Figure 7 Schematic diagram of the pKL-DEN2-Luci-hHBB1x2-poly A (100A) plasmid.

[0027] Figure 8 Western blot was used to detect the expression of antigen mRNA.

[0028] Figure 9 Western blot experiments were used to detect the expression of antigen epitopes. Figures A, B, C, and D show the expression of Φ, V, II, and III epitopes, respectively.

[0029] Figure 10 This is a recombinant real virus neutralizing antibody test to detect the humoral immunity level in mice after vaccination with RSV mRNA vaccine. Figures A and B show the neutralizing antibody levels against RSV type A and type B viruses, respectively.

[0030] Figure 11 This is an ELISpot experiment to detect the cellular immunity level in mice after vaccination with RSV mRNA vaccine. Figures A and B show the expression levels of IFN-γ and IL-2, respectively. DETAILED DESCRIPTION

[0031] The following examples are only used to further illustrate the present invention, but are not intended to limit the present invention in any way.

[0032] Unless otherwise specified, the reagents, materials, and instruments used in the following examples and test examples can be obtained from commercial sources; operations not otherwise specified are routine operations in the art.

[0033] Example 1. Construction of antigen carrier

[0034] The pKL plasmid serves as an mRNA expression vector, wherein the 5'UTR is selected from CYBA, DEN2, hHBB, RpL38, RpS25, TCV, or TOP; the 3'UTR is selected from hHBA1, hHBB, BYDV, hHBBx2, CYBA_1.5x, BMV, TSV, or TCV; and the poly A sequence is selected from 60A, 100A, 30A-10X-70A, 30A-10X-70A-10C-A, or 40A-10X-60A-10C-A. The vector also includes a promoter, specifically a CMV promoter or a T7 promoter, or a tandem CMV promoter and a T7 promoter; and carries a resistance gene, such as a kanamycin resistance gene, an ampicillin resistance gene, or a tetracycline resistance gene. The mRNA expression vector backbone is a pKL vector backbone. Furthermore, the vector further comprises any one of the following elements: a capping site, an enzyme cleavage site, a signal peptide expression element, and a tag protein element.

[0035] Preferably, the 5'UTR is DEN2, the 3'UTR is hHBB1x2, and the ploy A is 100 consecutive adenylate residues, ie, DEN2-hHBBx2-100A.

[0036] First, the pKL-EGFP plasmid sequence encoding enhanced green fluorescent protein (EGFP) was designed (SEQ ID NO. Figure 1). The pKL-EGFP plasmid uses the pUC replicon and carries the kanamycin resistance gene. The promoter includes a tandem CMV promoter and a T7 promoter, wherein the CMV promoter can initiate the transcription of the target gene in eukaryotic cells, and the T7 promoter can be recognized by the T7 transcriptase in the in vitro transcription system and synthesize mRNA; the sequences of the 5'UTR, 3'UTR and poly A of the plasmid are consistent with the corresponding element sequences of BNT162b2 (DOI: 10.3390 / vaccines9070734) (as shown in Table 1); in addition, the plasmid also contains a Bsa I restriction site at the 3' end of the poly A for linearizing the circular plasmid. The pKL-EGFP plasmid was transfected into 293FT cells using a cell transfection reagent (Invtrogen). The control group only added the cell transfection reagent. After 24 hours, the cells were observed to show strong green fluorescence ( Figure 2A After the pKL-EGFP circular plasmid was cut into a linearized DNA fragment using Bsa I endonuclease (Vazyme), the linearized fragment was used as a template to synthesize mRNA in vitro under the catalysis of T7 transcriptase (Vazyme). Then, under the catalysis of vaccinia capping enzyme (Vazyme) and 2-oxomethyltransferase (Vazyme), EGFP mRNA containing Cap1 structure was synthesized. Finally, pure EGFP mRNA was purified using magnetic beads and stored in RNA storage solution (Invtrogen). The mRNA concentration was measured using a Qubit4 fluorometer. 1 μg of EGFP mRNA was transfected into 1x 10 cells using cell transfection reagent. 6 293FT (single well of 6-well plate), the control group only added cell transfection reagent, and after 24 hours, the cells showed strong green fluorescence ( Figure 2B ), indicating that pKL can effectively express the target gene it carries and can serve as a template for in vitro transcription (IVT) of mRNA to synthesize the mRNA of the target gene.

[0037] Table 1. Partial sequence information of pKL-EGFP plasmid

[0038]

[0039] After confirming that pKL-EGFP could effectively synthesize mRNA via IVT, we generated the firefly luciferase-carrying plasmid pKL-Luci01 (the plasmid's 5'UTR, 3'UTR, and poly A sequences were identical to those of pKL-EGFP and BNT162b2) to screen for effective 5'UTR, 3'UTR, and poly A elements. We commissioned a gene synthesis company to synthesize the luciferase gene sequence with SpeI and XhoI restriction sites at the 5' and 3' ends, respectively. The synthesized luciferase gene sequence and the pKL-EGFP plasmid were then digested with SpeI (NEB) and XhoI (NEB) endonucleases, respectively, at 37°C for 30 minutes. Finally, the digested luciferase gene sequence was mixed with the pKL plasmid and ligated with T4 ligase (NEB) at room temperature for 30 minutes. The luciferase gene was then subcloned into the pKL plasmid to generate the pKL-Luci01 plasmid. To screen for effective 5'UTRs, pKL-R1-Luci plasmids containing a series of different 5'UTRs (Table 2) were prepared (the 3'UTRs and poly A of these plasmids were still consistent with BNT162b2). The operation method is as follows: First, a gene synthesis company was commissioned to synthesize a series of primers carrying 5'UTR cloning adapters (the primers include two parts: 5'UTR sequence and seamless cloning homology arms, Table 3). Then, DNA polymerase (NEB) and these primers were used to perform PCR reactions using pKL-Luci01 as a template. The resulting products were then incubated at 37°C for 30 minutes using a seamless cloning kit (Vyzame) to obtain pKL-R1-Luci plasmids in which the 5'UTR of the pKL-Luci01 plasmid was replaced with another 5'UTR. R1 was one of CYBA, DEN2, hHBB, RpL38, RpS25, TCV, or TOP.

[0040] Table 2. Sequences of different 5'UTRs

[0041]

[0042] Table 3. 5'UTR PCR primers

[0043]

[0044]

[0045] Similarly, the present invention replaced the 3'UTR of the pKL-Luci01 plasmid with other 3'UTRs (Tables 4 and 5) to obtain the pKL-Luci-R2 plasmid (the 5'UTR and poly A of these plasmids were still consistent with BNT162b2), and R2 was one of hHBA1, hHBB, BYDV, hHBBx2, CYBA_1.5x, BMV, TSV, or TCV. Next, the expression efficiency of the plasmids containing different 5'UTRs or 3'UTRs was tested using a reporter gene assay. 293FT cells were co-transfected with pKL-R1-Luci plasmid, pKL-Luci-R2 plasmid, or pKL-Luci01 plasmid (control group) expressing firefly luciferase (Fluc) and pRL-SV40 plasmid expressing Renilla luciferase (Rluc). After 48 hours, the cells were collected and the Fluc and Rluc levels were measured using a dual-luciferase reporter gene assay kit (Vyzame). The Fluc / Rluc ratio was calculated as an indicator of luciferase expression. The results of two repeated experiments showed that in the 5'UTR, the effects of CYBA, DEN2 and hHBB on the expression efficiency of luciferase were close to or better than those of the 5'UTR of pKL-Luci01; in the 3'UTR, the effects of hHBA1, hHBB and hHBBx2 on the expression efficiency of luciferase were close to or better than those of the 3'UTR of pKL-Luci01 ( Figure 3 , unpaired two-tailed t test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0046] Table 4. Sequences of different 3'UTRs

[0047]

[0048]

[0049] Table 5. 3'UTR PCR primers

[0050]

[0051] Based on the above results, in order to test the effects of different 5'UTR and 3'UTR combinations on gene expression, a series of pKL-R3-Luci-R4 plasmids were prepared (the poly A of these plasmids is still consistent with BNT162b2), wherein R3 is the 5'UTR, which is one of CYBA, DEN2 or hHBB, and R4 is the 3'UTR, which is one of hHBA1, hHBB or hHBBx2. The present application utilizes PCR primers carrying R3 5'UTR (Table 2) and DNA polymerase, and uses pKL-Luci-R2 plasmid as a template (R2 is hHBA1, hHBB or hHBBx2) for PCR reaction. The obtained product is then incubated with a seamless cloning kit at 37°C for 30 minutes to obtain a pKL-R3-Luci-R4 plasmid in which the 5'UTR is replaced with the R3 5'UTR. Similarly, the present application used a reporter gene assay to test the expression efficiency of the pKL-R3-Luci-R4 plasmid containing different 5'UTR and 3'UTR combinations. The control group (Control) was transfected with the pKL-Luci01 plasmid. The results showed that the gene expression efficiency was highest when the 5'UTR was one of CYBA, DEN2 or hHBB and the 3'UTR was hHBBx2 ( Figure 4 , unpaired two-tailed t test, ***P<0.001, ****P<0.0001).

[0052] Next, the effects of different poly A residues on gene expression efficiency were screened. To make poly A screening plasmids, different poly A sequences carrying 5'-end NheI and 3'-end AvrII restriction sites were synthesized (Table 6). The synthesized poly A sequence and pKL-Luci01 plasmid were then digested with NheI (NEB) and AvrII (NEB) endonucleases at 37°C for 30 minutes. Finally, the digested poly A sequence was mixed with the pKL-Luci01 plasmid and ligated with T4 ligase at room temperature for 30 minutes to obtain pKL-Luci01-R5 plasmids containing different poly A sequences (the 5'UTR and 3'UTR of these plasmids were still consistent with BNT162b2). R5 was 60A, 100A, 30A-10X-70A, 30A-10X-70A-10C-A, or 40A-10X-60A-10C-A. The screening of poly A also used a reporter gene test. Since poly A mainly affects the stability of mRNA, this application used the aforementioned method to linearize the pKL-Luci-R5 plasmid, and then used in vitro transcription and capping reagents to produce mRNA containing different poly A. These mRNAs were co-transfected with pRL-SV40 plasmids into 293FT cells, and the cells were collected after 24, 48, 72 and 96 hours. The control group (Control) was transfected with pKL-Luci01 plasmid, and the gene expression efficiency was detected using a dual luciferase reporter gene assay kit. The results showed that 100A and 30A-10X-70A had better mRNA stability ( Figure 5 ), so these two poly A structures were selected as candidate poly A.

[0053] Table 6. Poly A sequence

[0054]

[0055] Based on the above data, in order to screen the most effective 5'UTR, 3'UTR and poly A combination, pKL-R6-Luci-hHBBx2-R7 plasmids were prepared (the 3'UTR of these plasmids is hHBBx2), R6 is the 5'UTR, which is CYBA, DEN2 or hHBB, and R7 is poly A, which is 100A or 30A-10X-70A. In this application, the aforementioned synthesized R7 poly A sequence carrying NheI and AvrII restriction sites was digested with the aforementioned pKL-R3-Luci-hHBBx2 plasmid (R3 is CYBA, DEN2 or hHBB) at 37°C for 30 minutes, and then T4 ligase was added to ligate at room temperature for 30 minutes to prepare a series of pKL-R6-Luci-hHBBx2-R7 plasmids. Then, the corresponding mRNA was produced using in vitro transcription and capping reagents. These mRNAs were co-transfected with pRL-SV40 plasmids into 293FT cells. Cells were collected after 24, 48, 72, and 96 hours. The control group (Control) was transfected with pKL-Luci01 plasmid, and the gene expression efficiency was detected using a dual-luciferase reporter gene assay kit. The results showed that except for pKL-CYBA-Luci-hHBB1x2-poly A (30A-10X-70A), the effects of the other combinations on luciferase expression were close to or better than BNT162b2, especially pKL-DEN2-Luci-hHBB1x2-poly A (100A) had the best effect ( Figure 6 ).

[0056] In summary, the present application selected pKL-R8-Luci-hHBB1x2-R9 as an efficient mRNA synthesis vector. The 3'UTR of these plasmids is hHBBx2, wherein R8 and R9 are 5'UTR and poly A, respectively, specifically CYBA, 100A; or DEN2, 100A; or hHBB, 100A; or DEN2, 30A-10X-70A; or hHBB, 30A-10X-70A. The best combination is pKL-DEN2-Luci-hHBB1x2-poly A (100A) (abbreviated as pKL-Luci, SEQ ID NO. 2, Figure 7 ), wherein the 5'UTR is DEN2, the 3'UTR is hHBB1x2, and the poly A is 100 consecutive A's. Finally, this plasmid is used to synthesize the expression vector of the following mRNA vaccine.

[0057] Next, we used the pKL-Luci plasmid screened above to make an mRNA expression vector. This application designs a bivalent mRNA vaccine for RSV. The protein encoded by the antigen mRNA contains partial fragments of RSV ON1 genotype and BA9 genotype F protein. The F protein contains several domains, among which the furin cleavage sites at both ends of the p27 domain cause the F protein to be cut by the furin enzyme into three segments: F1, p27, and F2, promoting the F protein to transform from a pre-fusion conformation (preF) to a post-fusion conformation (postF). In addition, it also includes two hydrophobic domains, HR1 and HR2, a transmembrane domain (TM), and an intracellular region (CT). RA01 of the present application is the extracellular segment coding sequence of the F protein of the ON1 genotype excluding the p27 domain and the furin cleavage site. The front and back segments of the p27 domain are connected with a "GSGSG" sequence (SEQ ID NO.3). RB01 is similar to RA01, but the viral genotype is BA9 (SEQ ID NO.4); RA02 removes the TM domain on the basis of RA01 and introduces the T4 foldon structure sequence. The two are connected with a "GSGSGS" sequence (SEQ ID NO.5). RB02 is similar to RA02, but the viral genotype is BA9 (SEQ ID NO.6); RA03 is similar to RA01, and the p27 domain and the furin cleavage site are also removed, and are connected with a "GSGSG" sequence, but the length of the F1 and F2 structures is shortened, and the Fc segment protein sequence of human IgG1 is introduced at the end (SEQ ID NO.7). RB03 is similar to RA03, but the viral genotype is BA9 (SEQ ID NO.8). All the above antigen sequences have been nucleotide sequence optimized, and the specific sequences are shown in Table 7.

[0058] Table 7 Antigen sequences

[0059]

[0060] Next, the present applicant commissioned a gene synthesis company to synthesize the RA01, RA02, RA03, RB01, RB02 and RB03 antigen nucleotide sequences containing the 5'-terminal extension sequence (5'-TTTTAATTAGAGAGCAGATCTCTGGCCACC-3') and the 3'-terminal extension sequence (5'-GGACAGCAAGAAAGCGAGC-3'), and then used BgIII and SacI endonucleases to digest the pKL-Luci plasmid at 37°C for 30 minutes. Finally, the digested plasmid was mixed with the synthesized antigen sequence, and the reaction was carried out at room temperature for 30 minutes using a seamless cloning kit to obtain the antigen vectors pKL-RA01, pKL-RA02, pKL-RA03, pKL-RB01, pKL-RB02 and pKL-RB03.

[0061] Example 2. mRNA vaccine preparation

[0062] The above-mentioned pKL-RA01, pKL-RA02, pKL-RA03, pKL-RB01, pKL-RB02 and pKL-RB03 plasmids were cut into linearized DNA fragments with Bsa I endonuclease, and the linearized fragments were used as templates to synthesize RA01, RA02, RA03, RB01, RB02 and RB03 antigen mRNAs in vitro, respectively, using in vitro transcription and capping reagents, in which 1-methylpseudouracil was used instead of uracil.

[0063] D-Lin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to produce a lipid mixture. RA01 and RB01 mRNA stock solutions were then mixed at a molar ratio of 1:1 and designated R01. R01 was then diluted in 50 mM sodium citrate solution at pH 4.0. Finally, the lipid mixture was mixed with the antigen mRNA using a lipid nanoparticle preparation device (Maianna Instrument Technology Co., Ltd.) to produce lipid nanoparticles containing the mRNA antigen (mRNA-LNPs). The mRNA-LNP solution was then diluted 30-fold with PBS, replaced and concentrated using an ultrafiltration concentrator (Millipore), and finally sterilized by filtration through a 0.22 μm sterile filter to produce the R01 vaccine containing RA01 and RB01 mRNA molecules. Similarly, the R02 vaccine containing RA02 and RB02 mRNA molecules and the R03 vaccine containing RA03 and RB03 mRNA molecules were prepared.

[0064] Using RediPlate TM 96 RiboGreen TM RNA Quantitation Kit (Invtrogen) was used to measure the LNP encapsulation efficiency and the mRNA molecule encapsulation concentration. In addition, the lipid mixture and 50 mM sodium citrate solution at pH 4.0 were prepared using a lipid nanoparticle preparation device using the above method to prepare empty LNPs as a control vaccine.

[0065] Example 3. Detection of antigen mRNA expression

[0066] RA01, RA02, RA03, RB01, RB02, and RB03 antigen mRNAs were prepared according to the method of Example 2. Antibodies against RSV F protein were purchased from Santa Cruz Biotechnology, and antibodies against F protein Φ, II, III, and V epitopes were purchased from Suzhou Jinan Protein Co., Ltd. RSV A2 genotype preF and postF proteins were purchased from Suzhou Jinan Protein Co., Ltd. HEK293FT cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% streptavidin.

[0067] HEK293FT cells were transfected with 1 μg of RA01, RA02, RA03, RB01, RB02, and RB03 antigen mRNA using a transfection reagent. A control group received only the transfection reagent. Cells were harvested 48 hours later, and protein expression was assessed by western blot.

[0068] The results showed that all antigen mRNAs could express the correct antigen protein ( Figure 8 In addition, it was found that RA01 is consistent with the preF protein and has a Φ epitope unique to the pre-fusion conformation of the F protein ( Figure 9 A) and V( Figure 9 B) epitopes, while the postF protein does not have these two epitopes, indicating that the antigen mRNA designed by the R01 structure of the present application expresses the pre-fusion conformation of the F protein.

[0069] Example 4. RSV mRNA vaccine efficacy test

[0070] With reference to Example 2, R01, R02, and R03 vaccines were prepared. Twenty 6-8 week old female BALB / c mice were weighed and randomly divided into groups of 5 mice each; the experimental group was vaccinated with mRNA vaccine, and the control group was vaccinated with control vaccine (LNP without mRNA encapsulation). Each mouse was vaccinated with one dose of vaccine at week 1 and week 3, each dose including 100 μl control vaccine or RSV mRNA vaccine containing 1 μg mRNA. Blood was collected one week after the second immunization to detect neutralizing antibodies against RSV type A and type B recombinant true viruses; 9 weeks after the second immunization, the mice were killed, spleen lymphocytes were isolated, and the lymphocytes were stimulated with a mixture of polypeptides of RSV ON1 genotype and BA9 genotype F protein, and the levels of INF-γ and IL-2 expressed by mouse spleen lymphocytes were detected by ELISpot test.

[0071] The results showed that the neutralizing antibody levels of group R01 and group R02 for subtype A and subtype B viruses were significantly higher than those of group R03, and the neutralizing antibody level of group R01 for subtype B virus was significantly higher than that of group R02 ( Figure 10, *P>0.05, ***P>0.001, ****P>0.0001). This shows that the humoral immune effect of the R01 regimen is the best, followed by the R02 regimen. In terms of cellular immune effect, Figure 11 As shown in the figure, there was no significant difference between the groups. The above results indicate that the R01 antigen vaccine has the best immunogenicity.

[0072] Finally, it should be noted that the foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will be able to modify the technical solutions described in the foregoing embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An mRNA molecule encoding a respiratory syncytial virus protein, characterized in that Comprising an antigen sequence encoding a partial fragment of RSV ON1 genotype F protein or a partial fragment of BA9 genotype F protein; The antigen sequence encoding a partial fragment of RSV ON1 genotype F protein is encoded by the nucleotide sequence shown in SEQ ID NO.3, 5 or 7, and the antigen sequence encoding a partial fragment of RSV BA9 genotype F protein is encoded by the nucleotide sequence shown in SEQ ID NO.4, 6 or 8.

2. The mRNA molecule encoding respiratory syncytial virus protein according to claim 1, characterized in that The mRNA molecule further comprises a cap structure, a 5'UTR element, a 3'UTR element and poly A; the 5'UTR is selected from CYBA, DEN2, or hHBB; the 3'UTR is selected from hHBBx2; and the poly A is selected from 100A, 30A-10X-70A.

3. The mRNA molecule encoding respiratory syncytial virus protein according to claim 2, characterized in that The 5'UTR is DEN2; the 3'UTR is hHBBx2, and the poly A is 100A.

4. An expression vector comprising the mRNA molecule encoding the respiratory syncytial virus protein according to any one of claims 1 to 3.

5. The expression vector according to claim 4, wherein The antigen sequence encoding the RSV ON1 genotype F protein partial fragment or the BA9 genotype F protein partial fragment is connected to the pKL plasmid, and the pKL plasmid is obtained by enzyme digestion of the pKL-Luci plasmid of SEQ ID NO.

2.

6. A respiratory syncytial virus mRNA vaccine, characterized in that: The mRNA molecule encoding a partial fragment of RSV ON1 genotype F protein and the mRNA molecule encoding a partial fragment of RSV BA9 genotype F protein are included. Wherein, the mRNA molecule encoding the partial fragment of RSV ON1 genotype F protein is encoded by the nucleotide sequence shown in SEQ ID NO.3, and the mRNA molecule encoding the partial fragment of RSV BA9 genotype F protein is encoded by the nucleotide sequence shown in SEQ ID NO.4; The mRNA molecule encoding the partial fragment of RSV ON1 genotype F protein is encoded by the nucleotide sequence shown in SEQ ID NO.5, and the mRNA molecule encoding the partial fragment of RSV BA9 genotype F protein is encoded by the nucleotide sequence shown in SEQ ID NO.6; or The mRNA molecule encoding the partial fragment of RSV ON1 genotype F protein is encoded by the nucleotide sequence shown in SEQ ID NO.7, and the mRNA molecule encoding the partial fragment of RSV BA9 genotype F protein is encoded by the nucleotide sequence shown in SEQ ID NO.

8.

7. The respiratory syncytial virus mRNA vaccine according to claim 6, characterized in that The mRNA vaccine also contains lipid nanoparticles, which encapsulate the mRNA molecules.

8. The respiratory syncytial virus mRNA vaccine according to claim 7, characterized in that The mRNA molecules encapsulated by the lipid nanoparticles are a mixture of mRNA molecules encoding partial fragments of RSV ON1 genotype F protein and partial fragments of BA9 genotype F protein, respectively, wherein the molar ratio of the mRNA molecules encoding partial fragments of RSV ON1 genotype F protein and partial fragments of BA9 genotype F protein is (0.5-4):

1.

9. Use of the mRNA molecule according to any one of claims 1-3, the expression vector according to claim 4 or 5, or the mRNA vaccine according to any one of claims 6-8 in the preparation of a medicament for treating diseases related to respiratory syncytial virus infection, wherein the disease is selected from respiratory tract infections with mild symptoms, acute lower respiratory tract infections, and recurrent wheezing or airway hyperresponsiveness in children caused by RSV infection in early life.

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