Respiratory syncytial virus mRNA vaccine as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nucleic acid vaccines, and provides an RSV (respiratory syncytial virus) mRNA (messenger ribonucleic acid) vaccine and a preparation method thereof, an mRNA molecule comprises an antigen sequence for encoding partial fragments of RSV ON1 genotype F protein and BA9 genotype F protein, and the mRNA encodes preF protein or protein containing main antigen epitopes of the preF protein through structural modification. In vector construction, a specific plasmid is selected, and an mRNA antigen sequence is optimized. After the vaccine is successfully prepared, antigen mRNA expression detection and a mouse immune experiment are carried out, and the result shows that the vaccine of the R01 scheme has the optimal humoral immune effect, the cellular immune effect is not obviously different from each other, and the vaccine is proved to be capable of effectively inducing humoral immune and cellular immune aiming at RSV in a mouse body.

Description

Technical Field

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

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

[0003] RSV can cause respiratory infections in people of all ages, among whom children, the elderly, and adults with immunodeficiency or underlying diseases are particularly susceptible. The clinical manifestations of RSV infection vary greatly, ranging from mild respiratory infections to acute lower respiratory tract infections. Severe infections can also affect organs other than the respiratory system. In addition, children infected with RSV early in life are at risk of recurrent wheezing and airway hyperresponsiveness. Therefore, it is of great significance to develop vaccines or drugs against RSV.

[0004] People began to explore RSV vaccines in the 1860s. For a long time, both inactivated RSV vaccines and recombinant protein RSV vaccines had unsatisfactory results. It was not until people deciphered the structural variation of RSV when it infects human cells and realized that the prefusion conformation (preF) of the F protein was the main factor in inducing the human immune response to RSV that RSV vaccines thereafter used preF protein as antigen and achieved good 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 the immune response to the vaccine, as LNPs can protect mRNA from enzymatic degradation and promote effective uptake and intracellular release of mRNA by target cells. However, there are no reports of mRNA vaccines that are effective in preventing and treating RSV. 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 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 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; 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 of the mRNA molecule, which comprises the following elements from the 5′ end to the 3′ end: 5′UTR, a coding sequence comprising a partial fragment of RSV F protein, 3′UTR and poly A; optionally, the coding sequence comprising a partial fragment of RSV F protein is connected to 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 (LNP), 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 respiratory syncytial virus mRNA vaccine in the preparation of a drug for preventing and / or treating respiratory syncytial virus infection. Preferably, the disease includes but is not limited to respiratory tract infection with mild symptoms, acute lower respiratory tract infection, severe infection involving organs other than the respiratory system, and children infected with RSV in early life who subsequently develop recurrent wheezing and airway hyperresponsiveness.

[0017] Beneficial effects of the present invention:

[0018] The present invention designs and prepares a respiratory syncytial virus mRNA vaccine, which contains antigen mRNA molecules of partial fragments of F protein of RSV ON1 genotype and BA9 genotype, and through structural modification, both mRNAs encode preF protein or protein containing the main antigenic epitope of preF protein. Mouse immunization experiments have confirmed that the respiratory syncytial virus mRNA vaccine of the present invention can effectively induce humoral immunity and cellular immunity against RSV in mice. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0021] Figure 2B The expression effect of mRNA prepared for 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 the expression of luciferase mRNA.

[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 pKL-DEN2-Luci-hHBB1x2-poly A (100A) plasmid.

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

[0028] Fig. 9 Western blot experiment was 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] Fig.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] Fig.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 do not constitute any limitation to the present invention.

[0032] Unless otherwise specified, the reagents, materials and instruments used in the following embodiments 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 is used 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; poly A 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 also carries a resistance gene, such as a kanamycin resistance gene, an ampicillin resistance gene, and a tetracycline resistance gene. The backbone of the mRNA expression vector 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 nucleotides, ie, DEN2-hHBBx2-100A.

[0036] First, the pKL-EGFP plasmid sequence encoding enhanced green fluorescent protein (EGFP) was designed (SEQ ID NO.1, 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 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 poly A for linearizing the circular plasmid. The pKL-EGFP plasmid was transfected into 293FT cells using a cell transfection reagent (Invtrogen), and only the cell transfection reagent was added to the control group. 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), and then EGFP mRNA containing Cap1 structure was synthesized under the catalysis of vaccinia capping enzyme (Vazyme) and 2-oxomethyltransferase (Vazyme). Finally, pure EGFP mRNA was purified by magnetic beads and stored in RNA storage solution (Invtrogen). The mRNA concentration was measured using 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 can effectively synthesize mRNA through IVT, the pKL-Luci01 plasmid carrying firefly luciferase was produced (the 5'UTR, 3'UTR and poly A of this plasmid are the same as pKL-EGFP and consistent with BNT162b2) for screening effective 5'UTR, 3'UTR and poly A elements. First, a gene synthesis company was commissioned to synthesize a luciferase gene sequence with 5'-end SpeI and 3'-end XhoI restriction sites, and then the synthesized luciferase gene sequence and pKL-EGFP plasmid were digested with SpeI (NEB) and XhoI (NEB) endonucleases at 37°C for 30 minutes, respectively. Finally, the digested luciferase gene sequence was mixed with the pKL plasmid, and T4 ligase (NEB) was added to connect at room temperature for 30 minutes, and the luciferase gene was subcloned into the pKL plasmid to obtain the pKL-Luci01 plasmid. In order to screen effective 5'UTR, pKL-R1-Luci plasmids containing a series of different 5'UTRs (Table 2) were prepared (the 3'UTR and poly A of these plasmids are 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 (primers include 5'UTR sequence and seamless cloning homology arms, Table 3), and then DNA polymerase (NEB) and these primers were used to perform PCR reaction with pKL-Luci01 as a template. The obtained product was then incubated at 37°C for 30 minutes with a seamless cloning kit (Vyzame) to obtain a pKL-R1-Luci plasmid in which the 5'UTR of the pKL-Luci01 plasmid was replaced with other 5'UTRs, and R1 was one of CYBA, DEN2, hHBB, RpL38, RpS25, TCV, and TOP.

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

[0041]

[0042] Table 3. 5'UTR PCR Primers

[0043]

[0044]

[0045] Similarly, the present application replaces 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 are still consistent with BNT162b2), and R2 is one of hHBA1, hHBB, BYDV, hHBBx2, CYBA_1.5x, BMV, TSV, and TCV. Next, the expression efficiency of the plasmids containing different 5'UTRs or 3'UTRs was tested using a reporter gene assay. The pKL-R1-Luci plasmid, pKL-Luci-R2 plasmid or pKL-Luci01 plasmid (control group) expressing firefly luciferase (Fluc) and containing different 5'UTR, 3'UTR or poly A were co-transfected with the pRL-SV40 plasmid expressing Renilla luciferase (Rluc) into 293FT cells. After 48 hours, the cells were collected and the Fluc and Rluc values ​​were measured using the dual-luciferase reporter gene assay kit (Vyzame). The Fluc / Rluc value was calculated as an indicator of the luciferase expression effect. 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 (Table 2) carrying R3 5'UTR and DNA polymerase, and uses pKL-Luci-R2 plasmid as a template (R2 is hHBA1, hHBB or hHBBx2) for PCR reaction, and the obtained product is then incubated at 37°C for 30 minutes using a seamless cloning kit 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, and the control group (Control) was transfected with the pKL-Luci01 plasmid. The results showed that when the 5'UTR was one of CYBA, DEN2 or hHBB and the 3'UTR was hHBBx2, the gene expression efficiency was the highest ( Figure 4 , unpaired two-tailed t test, ***P<0.001, ****P<0.0001).

[0052] Next, the effects of different poly A on gene expression efficiency were screened. In order to make poly A screening plasmids, different poly A sequences carrying 5'-end NheI and 3'-end AvrII restriction sites were synthesized (Table 6), and then the synthesized poly A sequence and pKL-Luci01 plasmid were 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 T4 ligase was added to connect 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 are still consistent with BNT162b2), R5 is 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, the present application linearized the pKL-Luci-R5 plasmid using the aforementioned method, 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 detection kit. The results showed that 100A and 30A-10X-70A had better stability for mRNA ( Figure 5 ), so these two poly A structures were selected as candidate poly A.

[0053] Table 6. Poly A sequence

[0054]

[0055] Combined with the above data, in order to screen the most effective 5'UTR, 3'UTR and poly A combination, pKL-R6-Luci-hHBBx2-R7 plasmids (the 3'UTR of these plasmids is hHBBx2) were prepared, R6 is 5'UTR, which is CYBA, DEN2 or hHBB, and R7 is poly A, which is 100A or 30A-10X-70A. The present application digested the aforementioned synthesized R7 poly A sequence carrying NheI and AvrII restriction sites with the aforementioned prepared pKL-R3-Luci-hHBBx2 plasmid (R3 is CYBA, DEN2 or hHBB) at 37°C for 30 minutes, and then added T4 ligase to connect at room temperature for 30 minutes to prepare a series of pKL-R6-Luci-hHBBx2-R7 plasmids. Then, the corresponding mRNA was produced by in vitro transcription and capping reagents, and these mRNAs were co-transfected with pRL-SV40 plasmids into 293FT cells. The cells were collected after 24, 48, 72 and 96 hours, and the control group (Control) was transfected with pKL-Luci01 plasmid. 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 the expression of luciferase 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 5'UTR is DEN2, 3'UTR is hHBB1x2, and 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, and 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 from the pre-fusion conformation (preF) to the 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 coding sequence of the extracellular segment of the F protein of the ON1 genotype except the p27 domain and the furin restriction site. The two segments before and after the p27 domain are connected by the "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, and the two are connected by the "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 restriction site are also removed, and they are connected by the "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 optimized for nucleotide sequence, 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. Preparation of mRNA vaccine

[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 obtain a lipid mixture; then the RA01 and RB01 mRNA stock solutions were mixed at a molar ratio of 1:1 and named R01, and then R01 was diluted in a 50mM sodium citrate solution at pH 4.0; finally, the lipid mixture and antigen mRNA were mixed using a lipid nanoparticle preparation device (Maianna Instrument Technology Co., Ltd.) to prepare lipid nanoparticles (mRNA-LNP) containing mRNA antigens, and then the mRNA-LNP solution was diluted 30 times with PBS, and then replaced and concentrated with an ultrafiltration concentration device (Millipore), and finally filtered and sterilized with a 0.22μm sterile filter to obtain R01 vaccine containing RA01 and RB01 mRNA molecules. Similarly, R02 vaccine containing RA02 and RB02 mRNA molecules and 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 encapsulation efficiency of LNP and the encapsulation concentration of mRNA molecules. In addition, the lipid mixture and 50 mM sodium citrate solution at pH 4.0 were used as the control group vaccine by using the lipid nanoparticle preparation equipment.

[0065] Example 3. Detection of antigen mRNA expression

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

[0067] HEK293FT cells were transfected with 1 μg of RA01, RA02, RA03, RB01, RB02 and RB03 antigen mRNA using transfection reagents, and only transfection reagents were added to the control group. Cells were harvested 48 hours later, and protein expression was detected 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 ( Fig. 9 A) and V( Fig. 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, 5 mice in each group; the test 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 in the first and third weeks, each dose including 100 μl control vaccine or RSV mRNA vaccine containing 1 μg mRNA. Blood was collected in the first week after the second immunization to detect neutralizing antibodies against RSV type A strain and type B strain recombinant true virus; 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 R01 and R02 groups for subtype A and B viruses were significantly higher than those of R03 group, and the neutralizing antibody level of R01 group for subtype B virus was significantly higher than that of R02 group ( Fig.10, *P>0.05, ***P>0.001, ****P>0.0001). This indicates that the humoral immune effect of the vaccine of R01 scheme is the best, followed by R02 scheme. In terms of cellular immune effect, Fig.11 As shown, there was no significant difference between the groups. The above results show that the vaccine with R01 antigen scheme has the best immunogenicity.

[0072] Finally, it should be noted that the above is only 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 aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned various embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An mRNA molecule encoding a respiratory syncytial virus (RSV) protein, characterized in that: It contains antigen sequences encoding partial fragments of RSV ON1 genotype F protein and partial fragments of BA9 genotype F protein.

2. The mRNA molecule encoding respiratory syncytial virus protein according to claim 1, characterized in that The ends of the sequences encoding the RSV ON1 genotype F protein partial fragment and the BA9 genotype F protein partial fragment are respectively spliced ​​with sequences encoding the T4 foldon structure, or the ends of the sequences encoding the RSV ON1 genotype F protein partial fragment and the BA9 genotype F protein partial fragment are respectively spliced ​​with Fc sequences encoding human IgG1.

3. The mRNA molecule encoding respiratory syncytial virus protein according to claim 2, characterized in that 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.

4. The mRNA molecule encoding respiratory syncytial virus protein according to any one of claims 1 to 3, 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, 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; preferably, the 5'UTR is DEN2; the 3'UTR is hHBBx2, and the poly A is 100A.

5. An expression vector comprising the mRNA molecule according to any one of claims 1 to 4, characterized in that: It comprises the following elements from 5' to 3' end: 5'UTR, a coding sequence comprising a partial fragment of RSV F protein, 3'UTR and poly A; optionally, the coding sequence comprising a partial fragment of RSV F protein is connected to a pKL plasmid, preferably, the pKL plasmid is obtained by enzyme digestion of the pKL-Luci plasmid of SEQ ID NO.

2.

6. The expression vector according to claim 5, characterized in that 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; the poly A is selected from 60A, 100A, 30A-10X-70A, 30A-10X-70A-10C-A or 40A-10X-60A-10C-A.

7. A respiratory syncytial virus mRNA vaccine, characterized in that: Comprising the mRNA molecule described in any one of claims 1-4.

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

9. The respiratory syncytial virus mRNA vaccine according to claims 7-8, 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.

10. Use of the mRNA molecule described in any one of claims 1-4, the expression vector described in claim 5 or 6, and the mRNA vaccine described in any one of claims 7-9 in the preparation of a medicament for treating diseases related to respiratory syncytial virus infection, wherein the diseases include but are not limited to respiratory tract infections with mild symptoms, acute lower respiratory tract infections, severe infections involving organs other than the respiratory system, and RSV infection in early life resulting in subsequent recurrent wheezing and airway hyperresponsiveness in children.

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