Novel human syncytial virus RSV B mRNA vaccine

Abstract

The present invention provides a recombinant human syncytial virus RSV B pre F mRNA, and a human syncytial virus vaccine prepared by using the mRNA and its application. The present invention obtains a recombinant human syncytial virus mRNA by screening and modifying the virus strain and optimizing the mRNA sequence, and further successfully obtains a human syncytial virus mRNA / LNP nanoparticle vaccine. The prepared vaccine can produce specific antibodies and neutralizing antibodies after immunizing mice, and the protection time detection of the vaccine shows that the immune protection period of the human syncytial virus vaccine can reach more than one year. After the virus infection, the booster vaccine is given again, which greatly increases the antibody content, indicating that the human syncytial virus vaccine has a very good virus protection effect. Cotton rat lung experiments have shown that the vaccine can effectively control the amplification of RSV virus in the lungs, and the immune effect is significantly better than similar syncytial virus vaccine products on the market.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically to the design and application of a novel human syncytial virus RSV B mRNA vaccine, belonging to the field of immune drug technology. Background Art

[0002] Intracellular delivery technologies based on biological macromolecules such as nucleic acids have received widespread attention in the field of innovative drug research and development in recent years. Among them, new vaccines based on mRNA technology have been rapidly promoted in research and development, clinical trials and marketing approval under urgent needs, demonstrating to the world for the first time the clinical effectiveness, safety and commercial value of the mRNA technology platform.

[0003] Compared with traditional drugs that directly use proteins or peptides as active ingredients, mRNA technology has many advantages: 1) Higher biological activity and safety: For drugs using mRNA technology, the complex protein modification processes such as protein expression, glycosylation of related structures, terminal modification, and demethylation are all completed in the human body's own cells according to natural pathways. Whether from the molecular structure of the synthetic raw materials or the synthetic environment, they are highly homologous to the human body. Therefore, compared with the in vitro bacterial system or cell system of traditional protein preparations, endogenous protein products based on mRNA sequences have more accurate action specificity, higher affinity and more expected biological activity. 2) Longer biological half-life: Existing experimental results have confirmed that proteins generated by mRNA, including antibodies, especially those using self-replicating nucleic acids or circular nucleic acids, can maintain effective blood concentrations for a longer period of time, thereby reducing the frequency and dosage of medication, reducing drug side effects, and improving patient compliance. 3) More application scenarios: Through the development of dosage forms based on delivery technology and diversified administration methods, mRNA preparations can reach a variety of clinical targets, improve the local concentration and efficacy of active drug ingredients, and enrich the scope of new drug development while also enriching the clinical treatment options for patients. 4) Rapid response: The technologies used in the product development process of mRNA vaccines are mostly basic molecular biology methods, and technology transfer is relatively easy. A mature mRNA platform only takes a few weeks from gene sequencing, design to production for different pathogens. It can respond quickly to epidemics with urgent time requirements and play an important role in vaccines in a timely manner.

[0004] Human Respiratory Syncytial Virus (hRSV) is an enveloped, non-segmented, negative-strand RNA virus of the genus Pneumovirus in the family Paramyxoviridae. It is a highly contagious respiratory virus that has been prevalent worldwide for a long time. Its infectivity and pathogenicity are second only to influenza, causing bronchiolitis in infants, the common cold in adults, and more serious respiratory diseases such as pneumonia in the elderly and immunocompromised people. It affects more than 64 million people worldwide each year. Since there is no preventive vaccine or effective antiviral drug before 2023, RSV infection poses a huge health risk and economic burden. RSV infection causes more than 3 million hospitalizations and nearly 60,000 deaths worldwide each year, nearly half of which occur in infants under 6 months old. In the United States alone, RSV infection in the elderly leads to 177,000 hospitalizations and 14,000 deaths each year, with hospitalization costs exceeding $1 billion.

[0005] The RSV genome is approximately 15,000 nucleotides in total length, consisting of 10 genes encoding 11 proteins. The interior of the virus particle is composed of nucleoprotein (N), tetrameric phosphoprotein (P) and large polymerase protein (L) combined with viral RNA. It is surrounded by matrix protein (M) and wrapped in a lipid bilayer, in which fusion protein F, glycoprotein G and small hydrophobic protein SH are distributed. According to the response of F and G proteins to monoclonal antibodies, RSV virus is divided into two antigenic subtypes: type A and type B, which are transmitted simultaneously or alternately in local epidemics. Because the F protein plays an important role in the process of RSV entering host cells, and it is also conserved across strains, protein stability, and less glycosylation, it eventually became a popular antigen for vaccines.

[0006] The F protein sequence is highly conserved between strains and exists in two conformations. Before fusion, it is in the Pre-F state, where the protein exists as a trimer and contains the main antigenic site. After binding to the target on the host cell surface, Pre-F undergoes conformational changes to close the distance between the viral membrane and the cell membrane. It will be lost due to encapsulation inside and transformed into a more stable post-fusion Post-F conformation. In the cell, the F protein is initially expressed as a single polypeptide precursor F0, which is cleaved by furin to form F1, F2 and Pep27 polypeptides. Among them, F1 and F2 are connected by two disulfide bonds to form a mature F protein, which is anchored in the viral membrane as a trimer structure. Because the RSV F protein plays an important role in the process of RSV entry into cells, it is the target of neutralizing antibodies and the main body of vaccine development. However, like other RSV antigens, previous vaccine development efforts based on RSV F protein have proven to be unsuccessful.

[0007] Since RSV was first discovered in humans in 1957, attempts to develop an RSV vaccine have never stopped for decades. However, due to the lag in technology, from inactivated vaccines causing more serious infections to later protein vaccines and viral vector vaccines, researchers have been struggling with many issues, such as titer, G protein, F protein, or N protein as antigen, and whether to mainly induce T cell cellular immunity or B cell humoral immunity. Despite accumulating a lot of experience, they have never achieved a successful RSV vaccine.

[0008] In recent years, with the intervention of structural molecular biology, people have had a breakthrough understanding of how vaccines work, which has enabled the first two RSV protein vaccines (GSK and Pfizer) to be launched. Our design combines the latest discoveries in antigen protein structural biology with a deep understanding of viral epidemiology and strains to design and synthesize our unique RSV B mRNA / LNP vaccine. Summary of the invention

[0009] The purpose of the present invention is to provide a RSV B mRNA / LNP vaccine to address the problem that RSV vaccines in the prior art are insufficient or have unsatisfactory effects.

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0011] The present invention discloses a recombinant human syncytial virus mRNA vaccine, wherein the DNA coding sequence corresponding to the vaccine RSV BF is shown as SEQ ID NO: 1, the mRNA sequence is shown as SEQ ID NO: 2, and the protein sequence is shown as SEQ ID NO: 3.

[0012] The invention discloses an expression vector and a DNA molecule described in the expression vector.

[0013] Preferably, the sequence of the expression vector is as shown in SEQ ID NO:4.

[0014] The invention discloses a cell, which comprises the expression vector.

[0015] The invention discloses a recombinant protein, which is obtained by translating the mRNA.

[0016] Preferably, the sequence of the recombinant protein is as shown in SEQ ID NO:3.

[0017] The invention discloses nucleotides, which encode the recombinant protein.

[0018] The invention discloses a plasmid, which comprises the nucleotide.

[0019] The invention discloses an mRNA / LNP complex, comprising the mRNA molecule and lipid nanoparticles.

[0020] The present invention discloses a method for preparing an mRNA / LNP complex, which is characterized by comprising the following steps:

[0021] 1) dissolving the mRNA in acetate buffer;

[0022] 2) preparing a mixed solution of ionizable cations, DSPC, cholesterol and PEG-2000 in proportion;

[0023] 3) The solution in step 2) is mixed with the solution in step 1) at high speed in a microfluidic system, and diluted and the pH value is increased to obtain an mRNA / LNP complex.

[0024] Preferably, the mRNA is obtained by in vitro transcription and capping.

[0025] Preferably, the pH value of the acetate buffer is 4.0 and the concentration is 50 mM.

[0026] The invention discloses an mRNA / LNP complex, and the complex is prepared according to the method.

[0027] The present invention discloses a vaccine composition, which comprises the mRNA and / or the expression vector and / or the recombinant protein and / or the nucleotide and / or the plasmid and / or the mRNA / LNP complex.

[0028] The present invention discloses the use of the mRNA and / or the expression vector and / or the recombinant protein and / or the nucleotide and / or the plasmid and / or the mRNA / LNP complex in preparing a human syncytial virus vaccine composition.

[0029] The present invention discloses use of the mRNA and / or the expression vector and / or the recombinant protein and / or the nucleotide and / or the plasmid and / or the mRNA / LNP complex in preparing a medicine for treating a disease caused by syncytial virus infection.

[0030] Preferably, the disease is a respiratory disease, and the respiratory disease is pneumonia or bronchiolitis or a cold.

[0031] The present invention discloses use of the mRNA and / or the expression vector and / or the recombinant protein and / or the nucleotide and / or the plasmid and / or the mRNA / LNP complex in preparing a medicine for treating patients with diseases caused by syncytial virus infection.

[0032] Preferably, the disease is a respiratory disease, and the respiratory disease is pneumonia or bronchiolitis or a cold.

[0033] Preferably, the patient has been previously infected with syncytial virus.

[0034] Preferably, the infection is performed once or multiple times.

[0035] The present invention obtains a recombinant human syncytial virus mRNA by screening and modifying virus strains and optimizing mRNA sequences. Further, the coding gene of the mRNA is constructed into a plasmid to achieve efficient transcription and capping in vitro, and lipid nanoparticles in which mRNA is wrapped are formed by increasing pH, and the encapsulation rate reaches more than 91%, and the mRNA / LNP nanoparticle vaccine is successfully obtained. The expression of RSV F mRNA / LNP nanoparticles in cells is western identified. After immunizing mice, specific antibodies and neutralizing antibodies are produced. The protection time detection of the vaccine shows that the antibody level only shows a decrease after six months of immunization, but the amplitude is very limited, which proves that the prepared vaccine has long-term protection against the virus. After the virus is infected, the booster vaccine is given again, which greatly increases the antibody content, indicating that it has a very good virus protection effect. The cotton rat lung experiment proves that the vaccine can effectively control the amplification of RSV virus in the lungs, and the immune effect is significantly better than the similar syncytial virus vaccine products on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 .JY-B-009-V02-2 vaccine sequence. A) RSV-F antigen encoding DNA sequence (SEQ ID NO: 1); B) RSV-F antigen encoding corresponding RNA sequence (SEQ ID NO: 2) C) RSV-F antigen protein amino acid sequence (SEQ ID NO: 3); D) plasmid DNA complete sequence (SEQ ID NO: 4).

[0037] Figure 2 .Plasmid JY-B-009-V02-2 map.

[0038] Figure 3 . Restriction endonuclease detection of plasmid JY-B-009-V02-2.

[0039] Figure 4 .Analysis of the characteristics of mRNA transcribed from the linearized plasmid JY-B-009-V02-2. Figure 4 A is the purity of mRNA analyzed by capillary electrophoresis; Figure 4 B is the purity of mRNA analyzed by SEC-HPLC aggregate content; Figure 4 C is the analysis of mRNA capping rate.

[0040] Figure 5 .Western Blot was used to detect the expression of JY-B-009-V02-2 mRNA / LNP nanoparticles in 293T cells after transfection.

[0041] Figure 6 .Antibody response induced by JY-B-009-V02-2 mRNA / LNP vaccine in mice.

[0042] Figure 7 .In vivo dose effect of antibody response to JY-B-009-V02-2 mRNA / LNP vaccine, Figure 7 A is the antibody titer after intramuscular injection of different doses, showing the relationship between different doses and time after immunization; Figure 7 B is Figure 7 A corresponds to the mean value of each group (n=5); Figure 7 C takes the 21st and 28th days after immunization as an example. Compared with 15μg and 200μg, there was no significant difference in antibody titer on day 21, and the difference on day 28 was p<0.05.

[0043] Figure 8 .The humoral immunity induced by JY-B-009-V02-2 mRNA / LNP vaccine in mice is long-lasting.

[0044] Fig. 9 .Identification of neutralizing antibodies induced by JY-B-009-V02-2 mRNA / LNP vaccine in mice. Fig. 9 A is for neutralization of RSV A2 virus; Fig. 9 B is for neutralization of RSV B18537 virus.

[0045] Fig.10 .JY-B-009-V02-2 mRNA / LNP vaccine can be used as a booster shot to induce high titer humoral immunity in animals that have been infected with RSV virus. Fig.10 A is the experimental design; Fig.10 B is the serum antibody titer after the second immunization or the first infection; Fig.10 C is the serum antibody titer after the second immunization, after infection / immunization, or after infection only.

[0046] Fig.11 .JY-B-009-V02-2 mRNA / LNP vaccine has the effect of reducing RSV infection in cotton rat lungs. Fig.11 A and B are experimental designs; Fig.11 C is the final quantitative detection of RSV virus content in cotton rat lung tissue.

[0047] Fig.12 .Compared with the RSV vaccines that have been on the market, the virus-specific antibodies induced by the JY-B-009-V02-2 mRNA / LNP vaccine have advantages. Fig.12 A is the experimental design; Fig.12 B is the antibody titer in the serum after one immunization or one infection; Fig.12 C is the antibody titer in the serum after the second immunization, or infection / immunization. DETAILED DESCRIPTION

[0048] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0049] Example 1 Screening, modification and mRNA sequence optimization of virus strains

[0050] 1.1 Initial virus strain screening

[0051] According to traditional serological classification, RSV virus can be divided into subtype A and subtype B. In epidemiology, they are almost equal in weight, showing randomness in epidemic years and regions, and comparable pathogenicity. Therefore, no matter which subtype is focused on in the design, the significance is the same. This patent mainly focuses on RSV B subtype, and the listed design sequences all start from RSV B subtype.

[0052] The general design of the current virus vaccine is based on the F fusion protein gene of the traditional RSV's most primitive strain RSV-A2. The immune response induced by such a vaccine, especially the antiviral antibodies, has the same "key and lock" matching relationship as RSV-A2, and can better neutralize the RSV-A2 virus strain to achieve the effect of immunity. However, the viral gene sequence will undergo genetic variation as the virus repeatedly infects the host and continuously propagates, which will inevitably cause the immune response induced by the original strain of RSV-A2 isolated in 1962 to be difficult to completely control the modern strain sixty years later. For this reason, after searching for the F gene sequences of hundreds of RSV B serotypes, we selected a clinical strain sequence as the starting point of our design according to the geographical location and time of the virus epidemic: RSV B, BE / 5150 / 08, 2008, from Belgium, GenBank: JX576730.1.

[0053] 1.2 Sequence Optimization:

[0054] mRNA sequence design is one of the most core and critical issues in the entire mRNA pharmaceutical process. It is closely related to the drugability of mRNA. It affects the yield of mRNA in vitro transcription, the immunogenicity of mRNA molecules, the translation efficiency and the stability of mRNA molecules. Currently, the common mRNA is composed of five elements: 5'cap (cap structure), 5'UTR (non-coding region), ORF (open reading frame encoding protein), 3'UTR and 3'poly A tail (polyadenylic acid tail).

[0055] The 5′Cap structure protects mRNA from degradation by the cytoplasmic exonuclease Xrn1 and the nuclear Xrn2 enzyme. Capped mRNA has a longer half-life and higher stability than uncapped mRNA, which significantly increases the yield of intracellular protein synthesis. The chemical nature of the cap structure is a special structure located at the 5' end of mRNA formed by modification during mRNA transcription, namely the m7GPPPN structure, also known as the methylguanosine cap. It is formed under the catalytic action of RNA triphosphatase, guanylyltransferase, mRNA (guanine-N7) methyltransferase and mRNA (nucleoside-2') methyltransferase. Depending on the degree of methylation modification, the cap can be divided into three structures: Cap 0, Cap 1 and Cap 2. Since uncapped mRNA or Cap 0 structure will be recognized by the natural immune receptor RIG-1, while Cap 1 and Cap 2 can protect themselves from being recognized by innate immune sensors, our design is the commonly used Cap1 structure.

[0056] The T7 promoter sequence can be divided into two domains: the binding domain and the transcription initiation domain. For the transcription initiation domain, any base substitution will seriously affect the strength of the promoter. Our design is TAATACGACTCACTATA, followed by agg. Kozak is GCCGCCACC.

[0057] The regulation of eukaryotic mRNA translation initiation is mainly determined by the characteristics of the 5′UTR, including secondary structure, sequence elements and 5′UTR length, which will affect the stability of mRNA. Our design uses the human cytochrome B-245α polypeptide (CYBA) UTR with relatively high protein expression. Of course, the performance of the UTR also depends on the cell type.

[0058] For the protein sequence of RSV, in order for the vaccine-induced antibodies to block the virus more effectively, the selection of a metastable pre-fusion form is key, which can achieve structural stability through point mutations. We arranged four point mutations on the original sequence: S155C and S290C mutations of DS, introducing non-native disulfide bonds; S190F and V207L ​​mutations of Cav1, filling the cavity with amino acids. At the same time, in order to induce a uniform and more significant immune response, the transmembrane segment (TM) and intracellular segment at the C-terminus were removed and replaced with a Fibritin sequence, so that the F protein can be presented in a more stable trimer form. For the DNA sequence of RSV-F, choosing appropriate codons can optimize the overall translation efficiency of mRNA. First, the overall approach to the host cell, that is, the high-frequency synonymous codons in human cells to replace the codons in the viral sequence, ensure that the codon usage bias is more consistent and avoid too many rare codons. However, it is also noted that retaining a small number of viral codons slows down the speed of ribosome advancement and provides sufficient time for the correct folding of proteins, thereby more accurately restoring the antigenic protein structure of the virus. Another point of attention is the GC content. A slightly higher GC content is believed to increase the stability of mRNA and increase protein expression in the body. Finally, the secondary structure of the mRNA sequence is also under consideration. It is reported that the hairpin structure of the secondary structure can affect the degradation of mRNA and contribute to the stability of mRNA.

[0059] Similar to 5′UTR, 3′UTR also contains many regulatory elements, which play an important role in mRNA stability, subcellular localization and translation efficiency. The 3'UTR we used was from human AES / TLE5 gene.

[0060] The poly A tail plays an indispensable role in the protein translation process. It binds to the poly A tail binding protein (PAPB), which then interacts with the translation initiation factor eIF4G to form a "closed loop" structure, recruiting the 40S translation initiation complex to the mRNA, and synergizing with the 5' end cap structure to stimulate translation initiation. During bacterial amplification, plasmids carrying long poly A nucleotide sequences will produce unpredictable recombination events. The poly A tail on the plasmid shortens as the bacteria continue to amplify, causing trouble for plasmid cloning and amplification. Studies have shown that spacing the poly A sequence can significantly reduce recombination events during plasmid DNA amplification, maintain the tail length, and do not affect the translation efficiency and half-life of the mRNA generated by in vitro transcription. Following this principle, our poly A is 40-spacer-80 bases.

[0061] The optimized RSV-F coding sequence obtained is as follows Figure 1 shown. Figure 1A shows the RSV-F antigen encoding DNA sequence of the JY-B-009-V02-2 vaccine (SEQ ID NO: 1); Figure 1 B is the mRNA sequence after transcription (SEQ ID NO: 2); Figure 1 C is the amino acid sequence of the translated protein (SEQ ID NO: 3); Figure 1 D is to add the DNA sequence of RSV-F antigen to the N-terminal T7 transcriptase binding site, 5'UTR, Kozak, C-terminal termination code, 3'UTR, poly (A), and then insert it into the vector through the HindIII and SapI sites.

[0062] Example 2 Plasmid synthesis and DNA extraction of mRNA vaccine

[0063] 2.1 Plasmid synthesis

[0064] After the plasmid sequence design was completed, it was synthesized by CRO company ( Figure 2 ).

[0065] 2.2 Plasmid DNA extraction

[0066] 2.2.1 Reagent preparation

[0067] Plates and liquid LB medium containing peptone, sodium chloride, yeast extract and corresponding antibiotics.

[0068] 2.2.2 Plasmid transformation

[0069] Plasmid DNA was added to competent cells and placed in an ice box for 30 minutes; heat-shocked in a 42°C water bath for 45 seconds, quickly placed in an ice box for 2 minutes, 500 μl of LB culture medium without antibiotics was added, and cultured in a shaking incubator at 37°C for 1 hour, then plated and cultured at 37°C overnight. The next day, clones were picked from the plate.

[0070] 2.2.3 Plasmid DNA extraction

[0071] The overnight cultured bacterial solution is extracted using a small extraction or large extraction kit.

[0072] 2.3 Plasmid DNA restriction enzyme digestion verification

[0073] Use restriction endonucleases for single, double and triple digestion verification. Qualified plasmids enter the next experiment. According to the sequence design, three groups of enzyme double digestion verification ( Figure 3). The first group of enzymes is EcoR I and Hind III, and the theoretical size after enzyme digestion is 1723bp+3279bp; the second group of enzymes is Pml I and SnaB I, and the theoretical size after enzyme digestion is 1179bp+3823bp; the third group of enzymes is Pme I and Bbs I, and the theoretical size after enzyme digestion is 211bp+4791bp.

[0074] Example 3 In vitro transcription capping, purification and quality testing of RSV B

[0075] 3.1 Plasmid DNA linearization

[0076] This design uses a reverse BspQI at the 3' end of poly A to linearize the plasmid. After reacting at 50°C for 3 hours, SDS-PAGE gel electrophoresis is used to determine whether the enzyme digestion is complete. The 3M sodium acetate precipitation method is used for purification; the DNA concentration is then determined by the Nanodrop method.

[0077] 3.2 In vitro transcription, capping and purification

[0078] The JY-B-009-V02-2 plasmid is designed with agg between the T7 polymerase binding site and the 5'UTR, so in vitro transcription is suitable for co-transcription. The co-transcription IVT system contains template DNA, NTP (UTP is pseudouracil), Capanalogs, T7 transcriptase, 10X transcription buffer solution, pyrophosphatase, RNase inhibitor, and DEPC water. The amount of DNA template and the volume of the fixed volume depend on the specific experiment. After reacting at 37°C for 3 hours, DNase I was added to remove the DNA template, and SDS-PAGE gel was used to determine the mRNA synthesis and DNA template digestion. The purification method is lithium chloride precipitation.

[0079] 3.3 mRNA integrity and capping rate detection

[0080] The quality of mRNA largely determines the quality of the final product. Due to the instability of mRNA itself, it is easily degraded or hydrolyzed by ubiquitous nucleases, which makes mRNA vaccines challenging in production, processing, and long-term storage. Therefore, a simple and efficient method for purity analysis and molecular size characterization of mRNA vaccines is crucial to its quality, efficacy, safety, and optimized manufacturing process.

[0081] 3.3.1. Capillary gel electrophoresis (CE)

[0082] Capillary gel electrophoresis was used to determine the proportion of incomplete or fragmented mRNA in DS. The method parameters were as follows:

[0083] Agilent 5200 fragment analyzer was used for analysis with Agilent DNF-471 RNA analysis kit. After the test sample was diluted to 20 μg / mL, it was heated and denatured at 70°C with ladder for 2 minutes, immediately cooled to 4°C and kept on ice, 22 μL RNA Diluent Marker (15nt) solution was added to the 96-well PCR plate, and then 2 μL of the test sample or ladder solution was added. After mixing, the 96-well PCR plate was placed in the 5200 fragment analyzer. Separation was performed at 8.0 kV for 40 minutes. Figure 4 As shown in A, the purity of our mRNA sample reached 97.1%.

[0084] 3.3.2SEC-HPLC analysis

[0085] The purity of mRNA was analyzed from the perspective of aggregate content by SEC-HPLC. The method parameters were as follows: the mobile phase was 150 mM PB, pH 7.0, the analysis was performed on the chromatographic column under the conditions of an isocratic elution of 20 min and a flow rate of 1.0 mL / min, the sample load was 2000 ng, the detection wavelength was 260 nm, and the column temperature was 30°C. The results are shown in Figure 4 B shows that the mRNA purity is 98.42%.

[0086] 3.3.3 Cap Rate Analysis

[0087] The 5' cap structure of mRNA plays a role in preventing mRNA degradation, prolonging the half-life and providing mRNA translation ability, which is a key quality attribute of mRNA drugs. The 5' capped nucleic acid fragments are specifically cleaved by combining RNaseH enzyme and specific probe, and then the capped fragments are purified by streptavidin-coupled magnetic beads. The capped and non-capped nucleic acid fragments are separated by ion pair reversed phase chromatography (IP-RP-HPLC), and the capping rate of mRNA is calculated by area normalization method.

[0088] RNaseH enzyme-specific digestion: Add digestion probe, buffer and enzyme-free water to the sample, mix well and place on a PCR instrument, slowly cool down from 95°C to 22°C for annealing. Then add magnetic beads, incubate at room temperature for 30 minutes, then add RNase H (50μnits) and incubate at 37°C for 3h. After the digestion is completed, the supernatant is removed by magnetic rack enrichment, and then rinsed with enzyme-free water 3 times. Next, add 75% methanol and incubate at 80°C for 3 minutes, and collect the eluate by magnetic rack enrichment. The eluate is placed in a vacuum centrifugal concentrator to dry, and finally a resuspension solution (100μM EDTA / 1% methanol) is added, which can be used for subsequent IP-RP-HPLC analysis.

[0089] IP-RP-HPLC analysis: Mobile phase A was 200 mM hexafluoroisopropanol + 8.15 mM triethylamine, mobile phase B was MeOH, and the proportion of mobile phase B was increased from 5% to 75% at a flow rate of 0.3 mL / min within 20 min. The sample load was 100 ng, the detection wavelength was 260 nm, and the column temperature was 75°C. The results are as follows Figure 4 C shows that the mRNA capping rate is 90.45%.

[0090] Example 4 Preparation of mRNA / LNP Nanoparticles

[0091] This study adopted the preparation method of nanoparticles LNP commonly used in the industry.

[0092] LNP is composed of ionizable cations (ionizable lipid): DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine): cholesterol (cholesterol): DMG-PEG-2000 in a ratio of 50:10:38.5:1.5, mixed with mRNA dissolved in pH 4 acetate buffer in a high-impact nanoparticle preparation device, and formed lipid nanoparticles (mRNA / LNP) in which mRNA is encapsulated with subsequent dilution and pH increase. The sample was concentrated and purified by ultrafiltration centrifugation. The final product was tested for particle size, PDI, ZP, encapsulation efficiency and effective mRNA concentration.

[0093] 4.1. Buffer preparation

[0094] All reagents were sterilized by autoclave at 121°C for 20 min and prepared using DEPC water.

[0095] 1) 50 mM acetate buffer, pH 4.0

[0096] 2) 25 mM Tris-HCl buffer, pH 7.5

[0097] 3) 0.6 g / mL sucrose solution

[0098] 4.2 Preparation of lipid solution and mRNA solution

[0099] Table 1: Preparation of lipid solutions

[0100]

[0101] 1) Before weighing lipids, they need to be restored to room temperature;

[0102] 2) All ingredients were dissolved in anhydrous ethanol, and an ultrasonic device was used to ensure complete dissolution.

[0103] 3) The prepared lipid solutions were mixed in a ratio of 1:1:1:1 to obtain a 12 mg / mL lipid mixed solution that met the formula.

[0104] Table 2: Preparation of mRNA solution

[0105]

[0106] According to the calculation method and buffer type shown in the above table, the in vitro transcribed and capped mRNA stock solution prepared in Example 3 was diluted to the required concentration of 0.2 mg / mL.

[0107] 4.3 Preparation of mRNA / LNP (taking a total volume of 4 mL as an example)

[0108] 4.3.1 INano LTM, the rapid nanomedicine preparation system of Maianna. Set the parameters as needed.

[0109] 4.3.2 Rinse the chip before use

[0110] Prepare three 5mL syringes, one for absolute ethanol, one for acetate buffer, and one for air. On the left, 5mL of acetate buffer → air 3 times; on the right, 5mL of ethanol → 5mL of ethanol → air 3 times, and clean the chip by hand. After cleaning, install the chip into the card slot.

[0111] 4.3.3 Operation of microfluidic device to prepare LNP

[0112] Use a 3ml syringe to draw 3ml of the above-configured mRNA solution, remove the bubbles, and load it into the syringe slot on the left side of the machine. Use a 1ml syringe to draw 1ml of the above-configured lipid mixture solution, remove the bubbles, and load it into the syringe slot on the right side of the machine. Insert a 15ml centrifuge tube into the waste liquid collection area and sample collection area of ​​the machine for waste liquid and sample collection. After confirming that the parameters are correct, click "Start", and the Maianna Rapid Nanodrug Preparation System INano LTM will start immediately, that is, LNP preparation will be carried out. After the preparation is completed, take out the centrifuge tube at the sample collection area.

[0113] 4.3.4 Cleaning the chip after use

[0114] Immediately after the LNP preparation, use 5 mL of DEPC water on the left, 5 mL of ethanol on the right → 5 mL of ethanol → 3x of air in this order, load the syringe into the chip injection port, clean the chip by hand push, and then let it dry naturally.

[0115] 4.3.5 Processing of collected mRNA / LNP samples

[0116] The collected mRNA / LNP suspension was immediately diluted with 25mM Tris-Hcl pH7.5 buffer to about 25 times the volume of mRNA / LNP, and then ultrafiltration centrifuged at 3000rpm, 4°C, 1h. This process was repeated until the solution volume was less than or equal to the volume before dilution (3.6mL, i.e., the total volume of 4mL minus 0.4mL of waste liquid). Then, it was sterilized by 0.22μm filter membrane, and 0.6g / mL sucrose solution of 0.2 times the volume of the solution was added to finally obtain the mRNA / LNP preparation product.

[0117] 4.3.6 mRNA / LNP physical property detection

[0118] The prepared mRNA / LNP should be tested for physical properties as soon as possible to determine the quality of the product and facilitate the next step of biological activity research. The test uses a Malvern Zetasizer to obtain particle size, PDI, and ZP data.

[0119] Encapsulation efficiency analysis:

[0120] Use 1X TE Bμffer and 2% Triton TE Bμffer to dilute the mRNA control (100μg / ml) from 2000ng / mL to 7 standard curve points (2000, 1600, 1200, 800, 400, 200ng / mL). When measuring the total RNA content, use 2% Triton TE Buffer to dilute the test LNP 50 times, and when measuring the free RNA content, use 1X TEBuffer to dilute the test sample 20 times. Transfer 100μL of the above standard curve solution and test solution to a black ELISA plate, then add 100μL 0.005×Ribogreen (200-fold dilution) respectively, react at room temperature in the dark for 2-5min, read the fluorescence response value with the ELISA plate (excitation wavelength 480nm, emission wavelength 520nm), and calculate the encapsulation efficiency according to the following formula:

[0121] Encapsulation efficiency EE (%) = (C total RNA - C free RNA) / C total RNA * 100%.

[0122] The encapsulation efficiency of the test sample obtained according to the above method is: 91.56%.

[0123] Example 5 Expression of RSV F mRNA / LNP Nanoparticles Transfected into 293T Cells

[0124] To ensure that the mRNA in the lipid nanoparticles can express the correct protein, the first step is to transfect the specific mRNA / LNP into appropriate cells, and then Elisa, Western Blot, flow cytometry and other methods can be used to detect the correctness of protein expression.

[0125] 5.1 Cell transfection

[0126] One day before transfection, 293T (CRL-3216, ATCC) cells were plated in 24-well plates at 2x10^5 / well. The complete culture medium was DMEM plus 10% fetal bovine serum and double antibody. The culture conditions were 37°C, 5% CO2. The next day, the cell culture medium in each well was aspirated and gently rinsed with PBS; after adding 500μl of complete culture medium to each well, gradient RSV F mRNA / LNP nanoparticles ranging from 0.5 to 3μgRNA / well were added to the cells. After gently shaking to disperse the nanoparticles, the cells were placed at 37°C, 5% CO2 for 48-72 hours.

[0127] 5.2 Western Blot (WB) method to detect the expression of antigen protein

[0128] The WB method can be used to qualitatively detect whether mRNA / LNP is translated into the correct antigen target protein after entering the cell. Since the designed antigen protein contains a signal peptide, the extracellular protein is measured here.

[0129] 5.2.1 Electrophoresis sample preparation

[0130] Collect the cell supernatant 48-72 hours after transfection, centrifuge at 2500rpm, 5min, 4℃, and take the supernatant into a new 1.5mL tube. If the expression level is too low, choose a Millipore ultrafiltration tube of appropriate specifications to do moderate concentration first. Then determine the protein content, adjust each sample to the same loading volume, and add 6×SDS loading buffer to a final concentration of 1×. Heat the sample at 92℃ for 5min to denature the protein before loading.

[0131] 5.2.2 SDS-PAGE electrophoresis

[0132] The electrophoresis used 10% separation gel and 4% concentration gel, and the sample was immediately loaded after heating. During electrophoresis, the voltage of the concentration gel section was 110V, and the voltage of the separation gel section was 150V.

[0133] 5.2.3 Transfer

[0134] Depending on the molecular weight, nitrocellulose membrane or PVDF membrane is used. Generally, transfer is performed at 80V for 1 hour, or at 60V for 2 hours, with ice packs to control the heat. After transfer, stain with 1× Ponceau red solution for 5 minutes to check the transfer effect.

[0135] 5.2.4 Blockade and immune response

[0136] First, the membrane was blocked with 5% no fat milk at 4℃ overnight. The next day, after washing 3 times with PBST solution, an appropriately diluted primary antibody (mouse anti-RSV-F) was added for hybridization and shaken at room temperature for 90 minutes. Then, the membrane was washed 3 times with PBST solution on a shaker, each time for 5 to 10 minutes. The secondary antibody was anti-mouse-HRP, and the membrane was shaken at room temperature for 60 minutes. Then, the membrane was washed 3 times with PBST solution on a shaker, each time for 5 to 10 minutes.

[0137] 5.2.5 Development and Exposure

[0138] According to the instructions of the ECL kit, mix the test solution A and the test solution B in a ratio of 1:1 and put them into the membrane. Shake the reaction in a dark room and then expose it to the chemiluminescence image analyzer for detection. The correctness of the target protein size can be determined based on the marker band.

[0139] The results are as follows Figure 5 The results showed that after JY-B-009-V02-2 was transfected into 293T cells, mRNA / LNP was internalized into the cells, released into the cytoplasm in the acidic environment of the lysosome, completed protein translation and modification in the endoplasmic reticulum and Golgi apparatus, and released into the extracellular space under the guidance of the signal peptide. The encoded RSV-F antigen protein can bind to the F antibody, and the protein specificity and protein molecular weight also meet the design requirements.

[0140] Example 6 Immunization of mice with JY-B-009-V02-2 mRNA / LNP vaccine and expression and identification of virus-specific antibodies

[0141] In order to detect the expression and performance of vaccine antigens in an in vivo environment, we used mice as an animal model for testing.

[0142] 6.1 Animal immunization

[0143] Balb / c mice, female, 6 weeks old, were acclimated to the animal room for one week after arrival. The vaccine was the JY-B-009-V02-2 mRNA / LNP vaccine prepared in Example 4. The intramuscular injection was 10 μg / mouse, and the injection was divided into two injections at an interval of 14 days. Blood was collected one day before the first immunization and 7 days after the second immunization, and the serum was used for subsequent antibody Elisa detection.

[0144] 6.2 Elisa for detection of anti-RSV antibodies

[0145] One of the key indicators of vaccines is to induce specific antiviral antibodies in animals. We used Elisa to detect the expression of anti-RSV-F antibodies.

[0146] 6.2.1 Antigen Coating

[0147] The antigen (RSV-A2, F) was diluted to 62.5 ng / mL with PBS and coated in a 96-well ELISA plate, 100 μl per well, at 4° C. for 16 h.

[0148] 6.2.2 Washing and blocking

[0149] Take out the ELISA plate coated with antigen, wash the plate once with 300 μL / well PBST, then add 100 μL blocking solution (5% milk) at room temperature for 30 min, and then wash the plate 3 times with 300 μL / well PBST.

[0150] 6.2.3 Sample dilution and loading

[0151] After the serum samples of immunized mice were appropriately diluted in a 4-fold decreasing gradient, 100 μl was added to the ELISA plate in triplicate, incubated at room temperature for 1.5 h, and then washed three times with 300 μL / well PBST.

[0152] 6.2.4 Add detection antibodies

[0153] According to the number of wells to be loaded with samples, take an appropriate amount of matching detection antibody (Goat Anti-Mouse IgG, Peroxidase-Conjugated, H+L), dilute it to 0.8 ng / mL, add 80 μL to each well, incubate at room temperature for 1 hour, and wash the plate 5 times with 300 μL / well PBST.

[0154] 6.2.5 Color development and termination

[0155] Mix color development solution A (containing H202) and color development solution B (containing TMB) in a ratio of 1:1, add 100 μL to each well, leave at room temperature for 15-30 minutes, add 30 μL 1M HCL stop solution to each well, and then read at 450 nm using an enzyme reader.

[0156] 6.2.6 Standard curve

[0157] If absolute quantification is required, the purchased anti-RSV-F antibody can be formulated into a suitable concentration range from low to high according to the amount of antibody protein, and Elisa can be performed simultaneously with the sample. The results can be plotted with the amount of antibody protein as the horizontal axis and OD450 as the vertical axis.

[0158] from Figure 6 The results show that the JY-B-009-V02-2 mRNA / LNP vaccine can induce a strong antibody response in mice, with the median serum dilution value above 32K.

[0159] Example 7 Dose exploration of JY-B-009-V02-2 mRNA / LNP vaccine

[0160] As a vaccine, dosage is a key factor to consider. We first studied the dose effect of JY-B-009-V02-2 mRNA / LNP vaccine and the time frame of IgG antibody production.

[0161] 7.1 Animal immunization

[0162] Balb / c mice, female, 6 weeks old, were acclimated for one week after arrival in the animal room. The vaccine was in the form of mRNA / LNP, and was administered intramuscularly in a single dose of 15, 30, 45, 60, 75, 90, 120, 200 μg / mouse.

[0163] 7.2 Elisa for detection of anti-RSV antibodies

[0164] 7.2.1 Antigen Coating

[0165] The antigen (RSV-A2, F) was diluted to 62.5 ng / mL with PBS and coated in a 96-well ELISA plate, 100 μl per well, at 4° C. for 16 h.

[0166] 7.2.2 Washing and sealing

[0167] Take out the ELISA plate coated with antigen, wash the plate once with 300 μL / well PBST, then add 100 μL blocking solution (5% milk) at room temperature for 30 min, and then wash the plate 3 times with 300 μL / well PBST.

[0168] 7.2.3 Sample dilution and loading

[0169] After the serum samples of immunized mice were appropriately diluted in a 4-fold decreasing gradient, 100 μl was added to the ELISA plate in triplicate, incubated at room temperature for 1.5 h, and then washed three times with 300 μL / well PBST.

[0170] 7.2.4 Add detection antibodies

[0171] According to the number of wells to be loaded with samples, take an appropriate amount of matching detection antibody (Goat Anti-Mouse IgG, Peroxidase-Conjugated, H+L), dilute it to 0.8 ng / mL, add 80 μL to each well, incubate at room temperature for 1 hour, and wash the plate 5 times with 300 μL / well PBST.

[0172] 7.2.5 Color development and termination

[0173] Mix colorimetric solution A (containing H202) and colorimetric solution B (containing TMB) in a 1:1 ratio, add 100 μL to each well, leave at room temperature for 15-30 minutes, add 30 μL 1M HCL stop solution to each well, and then read at 450 nm using an ELISA reader.

[0174] 7.2.6. Standard curve

[0175] For absolute quantification of antibodies, anti-RSV-F antibodies were purchased and prepared into appropriate concentrations from low to high according to the amount of antibody protein. Elisa was performed simultaneously with the samples, and the results were plotted with the amount of antibody protein as the horizontal axis and OD450 as the vertical axis. The absolute amount of anti-RSV F antibodies in serum was calculated based on the standard curve.

[0176] The dose finding results are as follows Figure 7 The results showed that the mRNA / LNP vaccine of JY-B-009-V02-2 can induce high titer virus-specific antibodies in animals, with no significant difference between the doses of 15 and 200 μg. The antibody concentration tended to stabilize 28-35 days after immunization, but still showed a small increase, especially in the high-dose group, which may be related to the stability of the RSV F protein antigen after point mutation modification. This result laid the foundation for subsequent experimental design and vaccine development.

[0177] Example 8 Detection of the long-term protective effect of JY-B-009-V02-2 mRNA / LNP vaccine

[0178] The protective effect of vaccines is an important indicator for evaluating vaccines. An effective vaccine that can provide protection during the infection season should be effective for at least four months. To this end, we investigated the protective effect of the JY-B-009-V02-2 mRNA / LNP vaccine.

[0179] 8.1 Animal immunization

[0180] Balb / c mice, female, 6 weeks old, will be acclimated for one week after arriving at the animal house. The vaccine is in the form of mRNA / LNP, 15 μg / mouse is administered intramuscularly, one immunization and two boosters, three doses of the same amount, 14 days apart. Blood samples were collected before immunization, 7 days, 21 days, 6 months, and 12 months after the last injection.

[0181] 8.2 Elisa for detecting anti-RSV antibodies

[0182] 8.2.1 Antigen Coating

[0183] The antigen (RSV-A2, F) was diluted to 62.5 ng / mL with PBS and coated in a 96-well ELISA plate, 100 μl per well, at 4° C. for 16 h.

[0184] 8.2.2 Washing and blocking

[0185] Take out the ELISA plate coated with antigen, wash the plate once with 300 μL / well PBST, then add 100 μL blocking solution (5% milk) at room temperature for 30 min, and then wash the plate 3 times with 300 μL / well PBST.

[0186] 8.2.3 Sample dilution and loading

[0187] After the serum samples of immunized mice were appropriately diluted in a 4-fold decreasing gradient, 100 μl was added to the ELISA plate in triplicate, incubated at room temperature for 1.5 h, and then washed three times with 300 μL / well PBST.

[0188] 8.2.4 Add detection antibodies

[0189] According to the number of wells to be loaded with samples, take an appropriate amount of matching detection antibody (Goat Anti-Mouse IgG, Peroxidase-Conjugated, H+L), dilute it to 0.8 ng / mL, add 80 μL to each well, incubate at room temperature for 1 hour, and wash the plate 5 times with 300 μL / well PBST.

[0190] 8.2.5 Color development and termination

[0191] Mix colorimetric solution A (containing H2O2) and colorimetric solution B (containing TMB) in a 1:1 ratio, add 100 μL to each well, leave at room temperature for 15-30 minutes, add 30 μL 1M HCL stop solution to each well, and read at 450 nm using an ELISA reader.

[0192] The results are as follows Figure 8 As shown, on the 7th day after the vaccine booster, the virus-specific antibodies reached a very high titer; the antibody titer on day 21 remained at the same level, and the median serum dilution was above 32k. At six months, it showed a decline, but the magnitude was very limited. At one year, it declined significantly, but the median serum dilution was still around 6-7K. The data show that the JY-B-009-V02-2mRNA / LNP vaccine has long-term protection against the virus.

[0193] Example 9 Detection of Antibody Neutralization Ability to Viruses

[0194] One of the golden indicators of vaccine testing is to detect whether the specific antiviral antibodies induced in animals have the ability to neutralize the virus and prevent viral infection. Neutralization virus testing is designed for this purpose. Since RSV is a biosafety level 2 virus, all the following steps must be completed in a biosafety level 2 laboratory in accordance with the corresponding requirements and specifications.

[0195] 9.1 Preparation of cells

[0196] HEp-2 (ATCC, CCL-23) human laryngeal epidermoid carcinoma cells are the most suitable cells for RSV growth. The day before the neutralization experiment, the cells were digested and resuspended and washed, then inoculated into a 24-well plate, 2x10^5 / well, MEM 10% FBS complete medium, and cultured overnight at 37°C 5% CO2.

[0197] 9.2 Antibodies and Virus Neutralization

[0198] 9.2.1 Serum Serial Dilution

[0199] The serum dilution gradient and dilution method commonly used in this laboratory are shown in Table 6 below. For RSV, the culture medium used for serum dilution is Opti-MEM without fetal bovine serum.

[0200] Table 3. Serum dilution and dilution method

[0201]

[0202] 9.2.2 Serum Antibodies and Virus Neutralization

[0203] The dosage of RSV virus is preferably 30-40PFμ / well (24-well plate), and the dilution medium is Opti-MEM without fetal bovine serum. Take 120μl of virus dilution and add it to the diluted serum well, and mix gently. It is necessary to set up a virus control group (that is, virus solution without diluted serum). Incubate in a 37℃ incubator for 1 hour.

[0204] 9.2.3 Inoculation of cells

[0205] Take 70 μl of the above virus-antibody mixture and add it to the 24-well cell culture plate where the cells have grown into a monolayer. Inoculate 3 wells of cells for each dilution. At the same time, set up 4 wells of normal cell control and 4 wells of virus control. Place the culture plate in a 37°C, 5% CO2 incubator for 1 hour, and shake it gently every 15 minutes. After the incubation, discard the mixture in the well and add 500 μl / well of the pre-prepared warm culture medium, which is MEM, 3% FBS, 0.75% methylcellulose. Culture at 37°C, 5% CO2 for 4 days.

[0206] 9.3 Result color display

[0207] 9.3.1 Virus and cell fixation

[0208] On the fourth day of culture, discard the culture solution in the wells, wash again with PBS, then add 150 μl / well of pre-cooled 85% methanol and fix at 4°C for 1 hour. Then discard the solution in the wells and wash again with PBS.

[0209] 9.3.2 Blocking and plate washing

[0210] Add 300 μL of blocking solution (5% milk) and shake gently on a shaker at room temperature for 30 min. Then wash the plate 3 times with 500 μL / well PBST.

[0211] 9.3.3 Adding detection antibodies

[0212] According to the number of wells to be loaded, take the matching detection antibody (Anti-RSV-F IgG, H+L, HRP Conjugated), dilute it by an appropriate multiple, add 150 μL to each well, incubate at room temperature for 90 min, and wash the plate three times with 500 μL / well PBST.

[0213] 9.3.4 Adding secondary antibody

[0214] Take the matching secondary antibody, dilute it at an appropriate multiple of 1:1000 to 1:5000, add 150 μL to each well, incubate at room temperature for 60 min, and wash the plate 5 times with 500 μL / well PBST.

[0215] 9.3.5 Color development and termination

[0216] Add 150 μL of TrueBlue to each well. TM Peroxidase Substrate, shake gently, virus-positive plaques will appear quickly. Then wash away the reaction solution with water. Dry in the dark. Virus plaques can be counted the next day.

[0217] It can be seen that for RSV A2 virus ( Fig. 9 A), whether the B-009-V02-2 mRNA / LNP vaccine of RSV B type is used alone or in combination with the A type vaccine (#7), the vaccine-induced antibodies have the same ability to neutralize the virus, with an EC50 of 10K serum dilution. When neutralizing RSV B 18537 virus ( Fig. 9 B), EC50 is similar or slightly higher, and B-009-V02-2 mRNA / LNP vaccine of RSV B type is better. The results show that JY-B-009-V02-2 mRNA / LNP vaccine has good cross-protection effect.

[0218] Example 10 Simulating the real world, immune boosting of animals that have been infected with RSV virus

[0219] RSV virus respiratory infection is a very common respiratory infection. Except for newborn babies who are infected for the first time within one year old, almost everyone faces repeated infection from viruses brought by the environment. For most people with a complete immune system, reinfection is not a very serious problem, but for young children whose immune systems are still developing, the elderly with declining immune systems, and patients with defective immune systems, RSV infection is often a serious threat. This experimental design is to understand whether the vaccine has a protective effect on animals that have been infected with RSV, and the extent of the protective effect.

[0220] 10.1 Animal Immunization

[0221] 10.1.1 Animals

[0222] The animals used were Balb / c female mice, 6 weeks old. After the animals arrived at the CRO biosafety level 2 animal room, they were acclimated there for one week.

[0223] 10.1.2 Immunization and sampling

[0224] The time points of immunization and sampling were as follows Fig.10 As shown in A.

[0225] On the first day, blood samples were collected from the eye socket before immunization. After each blood collection, the samples were placed at 4°C for 2 hours, then centrifuged at 2500 rpm, 5 min, 4°C, and the supernatant was collected and aliquoted. The serum was stored in a -80°C low-temperature refrigerator.

[0226] On the second day, the first group was injected intramuscularly with 10 μg / animal JY-B-009-V02-2 mRNA / LNP vaccine; the second and third groups were nasally infected for 10 5 PFU / RSV A2; the fourth group was PBS control.

[0227] On the fourteenth day after the first immunization, the first group of mice received a second immunization with the same species, dose and immunization route as the first one.

[0228] On the fifteenth day after the second immunization, blood was collected from the eye sockets of all animals. This was the blood after the first immunization or infection. On the second day after the blood was collected, that is, on the 30th day after the mice were infected with RSV virus, the second group of mice were intramuscularly injected with 10μg / mouse JY-B-009-V02-2mRNA / LNP vaccine as a booster shot.

[0229] Fourteen days later, all animals were bled from their orbits, which was the blood after the second immunization or infection.

[0230] On the second day after blood collection, all mice were infected with RSV A2,10 5 PFU / nasal challenge.

[0231] The experiment was terminated on the fourth day after infection. After the animals were euthanized by inhalation anesthesia, the complete lungs were removed and weighed according to the SOP autopsy process.

[0232] 10.2 Elisa for detection of anti-RSV antibodies

[0233] The serum after the first and second immunization or infection was used to detect the specific antibody content by Elisa.

[0234] 10.2.1 Antigen Coating

[0235] The coating antigen (RSV-A2, F) was diluted to 62.5 ng / mL with PBS and coated in a 96-well ELISA plate, 100 μl per well, at 4° C. for 16 h.

[0236] 10.2.2 Washing and sealing

[0237] Take out the ELISA plate coated with antigen, wash the plate once with 300 μL / well PBST, then add 100 μL blocking solution (5% milk) at room temperature for 30 min, and then wash the plate 3 times with 300 μL / well PBST.

[0238] 10.2.3 Sample dilution and loading

[0239] The mouse serum samples were appropriately diluted in a 4-fold gradient, and 100 μl was added to the ELISA plate. After incubation at room temperature for 1.5 h, the plate was washed three times with 300 μL / well PBST.

[0240] 10.2.4 Add detection antibodies

[0241] According to the number of sample wells to be added, take an appropriate amount of matching detection antibody (Goat Anti-Mouse IgG, Peroxidase-Conjugated, H+L), dilute it to 0.8 ng / mL, add 60 μL to each well, incubate at room temperature for 1 hour, and wash the plate 5 times with 300 μL / well PBST.

[0242] 10.2.5 Color development and termination

[0243] Mix colorimetric solution A (containing H202) and colorimetric solution B (containing TMB) in a 1:1 ratio, add 100 μL to each well, leave at room temperature for 15-30 minutes, add 30 μL 1M HCL stop solution to each well, and then read at 450 nm using an ELISA reader.

[0244] 10.2.6 Standard Curve

[0245] If absolute quantification is required, the purchased anti-RSV-F antibody can be formulated into a suitable concentration range from low to high according to the amount of antibody protein, and Elisa can be performed simultaneously with the sample. The results can be plotted with the antibody protein amount as the horizontal axis and OD450 as the vertical axis.

[0246] Results Fig.10 The amount of antibodies induced by the second dose of immunization in mice was equivalent to the amount of antibodies induced by the first viral infection ( Fig.10 B). However, giving a booster vaccine after viral infection greatly increased the antibody content ( Fig.10 C), indicating that it has a very good virus protection effect. In other words, giving booster immunization in the population can greatly control RSV virus infection.

[0247] Example 11 JY-B-009-V02-2 mRNA / LNP vaccine inhibits RSV infection in cotton rat lungs

[0248] The ultimate testing indicator of a vaccine is to detect whether the specific immune response induced by the vaccine in the body is effective in protecting the host and reducing viral infection.

[0249] 11.1 Animal Models and Immunization

[0250] 11.1.1 Animal Models

[0251] The relatively ideal animal model for RSV is the cotton rat, because after RSV infects the cotton rat, the virus can replicate and proliferate in its lungs, and the immune response and lung structure are also closer to humans. The animal model used in this embodiment is the cotton rat.

[0252] 11.1.2 Immunization and sampling

[0253] Female cotton rats aged 8 weeks were kept in a biosafety animal room. Fig.11 Experiments shown in A and 11B. For each operation, cotton rats were anesthetized with isoflurane.

[0254] On the first day, blood samples were collected from the eye sockets before immunization, placed at 4°C for 2 hours, and then centrifuged at 2500 rpm for 5 min at 4°C. The supernatant was collected and aliquoted. Serum was stored in a -80°C freezer.

[0255] On the second day, the third group of animals were nasally infected with RSV A2, 10^5 / rat. Two weeks later, the first group was immunized with JY-B-009-V02-2 mRNA / LNP vaccine, intramuscular injection, 60μg / rat; the second group was injected intramuscularly with JY-B-009-V02-2 mRNA / LNP vaccine and RSV A2-#7B vaccine, 30μg / rat each. The fourth group was the control group, injected with PBS.

[0256] Three weeks later, blood was collected from the eye sockets. On the second day after the blood was collected, the first and second groups were given the same vaccine as the first immunization. The third group, which had been previously infected with RSV, was given a booster injection of 60μg / rat of JY-B-009-V02-2mRNA / LNP vaccine intramuscularly to simulate the common situation in the real world where people are infected first and then boosted.

[0257] Two weeks later, another round of blood draws was conducted. On the second day after the blood draw, the rats were challenged with RSV B18537, 5x10^5 / rat.

[0258] The experiment was terminated on the fourth day after the challenge. After the animals were euthanized by inhalation anesthesia, the complete lungs of cotton rats were removed and weighed according to the SOP autopsy process.

[0259] 11.2 Lung Viral Load Detection

[0260] 11.2.1 Preparation of lung homogenate

[0261] Prepare cryotubes and add 2 mL of 4°C precooled virus protection solution; place fresh lung tissue in the cryotube containing virus protection solution and homogenize on the same day to avoid freeze-thaw affecting the experimental results. Use a high-throughput tissue grinder to homogenize the lung tissue at low temperature until there are no obvious particles, draw tissue fluid into 1.5 mL EP tubes, and freeze at -80°C in separate tubes.

[0262] 11.2.2 Preparing cell plates

[0263] The cells used for virus titration were Hep-2 laryngeal epithelial cancer cells. The day before virus titration, the cells were digested and resuspended, washed, and then seeded in 24-well plates at 2x10^5 / well in MEM 10% FBS complete medium at 37°C and 5% CO2 overnight.

[0264] 11.2.3 Viral Infection

[0265] The lung was homogenized at a ratio of 1:10, 1:10 2 ,1:10 3 ,1:10 4 ,1:10 5 ,1:10 6Dilution, the dilution medium is Opti-MEM, without fetal bovine serum. Then gently wash the 24-well plate laid out the day before with PBS, add 100μl of diluted virus solution to each well, and set up 3 replicates for each concentration. At the same time, set up 4 wells of normal cell control and 4 wells of virus control. Place the culture plate in a 37℃, 5% CO2 incubator and incubate for 1h, gently shaking every 15min. After the incubation, discard the mixed solution in the well and add 500μl / well of pre-prepared warm culture medium, the culture medium is MEM, 3% FBS, 0.75% methylcellulose. Culture at 37℃, 5% CO2 for 4 days.

[0266] 11.2.4 Virus and cell fixation

[0267] On the fourth day of culture, discard the culture solution in the wells, wash again with PBS, then add 100 μl / well of pre-cooled 85% methanol and fix at 4°C for 1 hour. Then discard the solution in the wells and wash again with PBS.

[0268] 11.2.5 Blocking and plate washing

[0269] Add 300 μL of blocking solution (5% milk) and shake gently on a shaker at room temperature for 30 min. Then wash the plate 3 times with 500 μL / well PBST.

[0270] 11.2.6 Adding detection antibodies

[0271] According to the number of wells to be loaded, take the matching detection antibody (Anti-RSV-F IgG, H+L, HRP Conjugated), dilute it by an appropriate multiple, add 150 μL to each well, incubate at room temperature for 90 min, and wash the plate three times with 500 μL / well PBST.

[0272] 11.2.7 Adding secondary antibodies

[0273] Take the matching secondary antibody, dilute it at an appropriate multiple of 1:1000 to 1:5000, add 150 μL to each well, incubate at room temperature for 60 min, and wash the plate 5 times with 500 μL / well PBST.

[0274] 11.2.8 Color development and termination

[0275] Add 150 μL of TrueBlue to each well. TM Peroxidase Substrate, shake gently, virus-positive plaques will appear quickly. Then wash away the reaction solution with water. Dry in the dark. Virus plaques can be counted the next day.

[0276] The results are as follows Fig.11The results showed that the JY-B-009-V02-2 mRNA / LNP vaccine can effectively control the proliferation of RSV virus in the lungs. Although there were differences in the induced antibody titers in the three cases of 60μg / animal immunization, 30+30μg / animal bivalent vaccine, and post-infection booster injection, no difference was seen in the amount of lung virus. Whether this is related to the virus detection method needs further study. Because the PCR method is based on viral nucleic acid, it cannot distinguish between infectious live viruses and neutralized and non-infectious viruses; and the immune plaque method only detects infectious live viruses.

[0277] Example 12 Comparison of the immune effects of JY-B-009-V02-2 mRNA / LNP vaccine and Pfizer vaccine

[0278] ABRYSVO (Pfizer) is a protein bivalent vaccine designed to provide broad protection against all RSV virus subgroups, not only for the general population, but also for the elderly and infants. Using ABRYSVO, the world's leading vaccine, as a control, we compared the immune effects of the JY-B-009-V02-2 mRNA / LNP vaccine with the Pfizer vaccine.

[0279] 12.1 Animal Immunization

[0280] 12.1.1 Animals

[0281] The animals used were Balb / c female mice, 6 weeks old. After the animals arrived at the CRO biosafety level 2 animal room, they were acclimated there for one week.

[0282] 12.1.2 Immunization and sampling

[0283] The time points of immunization and sampling were as follows Fig.12 As shown in A.

[0284] 12.2 Immunization and Sampling

[0285] The animals used were Balb / c female mice, 6 weeks old. After the animals arrived at the CRO biosafety level 2 animal room, they were acclimated there for one week.

[0286] On the first day, blood samples were collected from the eye sockets before immunization, placed at 4°C for 2 hours, and then centrifuged at 2500 rpm for 5 min at 4°C. The supernatant was collected and aliquoted. Serum was stored in a -80°C freezer.

[0287] On the second day, the third group of animals were nasally infected with RSV A2, 5.5x10^4 / mouse

[0288] Two weeks later, the first group was immunized with JY-B-009-V02-2 mRNA / LNP vaccine, intramuscular injection, 20μg / mouse; the second group was immunized with JY-B-009-V02-2 mRNA / LNP vaccine and RSV A2-#7B vaccine, 10μg / mouse each, intramuscular injection; the fourth group was injected with 10μg / mouse ABRYSVO intramuscularly; the fifth group was injected with 20μg / mouse ABRYSVO intramuscularly; the sixth group was the control group, injected with PBS.

[0289] Two weeks later, blood was collected from the eye sockets. This is the blood sample after the first immunization.

[0290] On the second day after blood collection, groups 1, 2, 4, and 5 were given the same vaccine as the first immunization. Group 3, which had been previously infected with RSV, was given a booster injection of 20 μg / rat of JY-B-009-V02-2 mRNA / LNP vaccine intramuscularly to simulate the common situation of infection first and then boosting in the real world.

[0291] Two weeks later, another round of blood draws. This is the blood sample after the first immunization.

[0292] On the second day after blood collection, the mice were challenged with RSV B-18537, 6.8x10^4 / mouse.

[0293] The experiment was terminated on the fourth day after the challenge. After the animals were euthanized by inhalation anesthesia, the complete lungs were removed and weighed according to the SOP autopsy process.

[0294] 12.3 Elisa for detection of anti-RSV antibodies

[0295] The serum after the first and second immunization or infection was used to detect the specific antibody content by Elisa.

[0296] 12.3.1 Antigen Coating

[0297] The coating antigen RSV-A2, F was diluted to 62.5 ng / mL with PBS and coated in a 96-well ELISA plate, 100 μl per well, at 4° C. for 16 h.

[0298] 12.3.2 Washing and blocking

[0299] Take out the ELISA plate coated with antigen, wash the plate once with 300 μL / well PBST, then add 100 μL blocking solution (5% milk) at room temperature for 30 min, and then wash the plate 3 times with 300 μL / well PBST.

[0300] 12.3.3 Sample dilution and loading

[0301] The mouse serum samples were appropriately diluted in a 4-fold gradient, and 100 μl was added to the ELISA plate. After incubation at room temperature for 1.5 h, the plate was washed three times with 300 μL / well PBST.

[0302] 12.3.4 Adding detection antibodies

[0303] According to the number of sample wells to be added, take an appropriate amount of matching detection antibody (Goat Anti-Mouse IgG, Peroxidase-Conjugated, H+L), dilute it to 0.8 ng / mL, add 60 μL to each well, incubate at room temperature for 1 hour, and wash the plate 5 times with 300 μL / well PBST.

[0304] 12.3.5 Color development and termination

[0305] Mix colorimetric solution A (containing H202) and colorimetric solution B (containing TMB) in a 1:1 ratio, add 100 μL to each well, leave at room temperature for 15-30 minutes, add 30 μL 1M HCL stop solution to each well, and then read at 450 nm using an ELISA reader.

[0306] 12.3.6. Standard Curve

[0307] If absolute quantification is required, the purchased anti-RSV-F antibody can be formulated into a suitable concentration range from low to high according to the amount of antibody protein, and Elisa can be performed simultaneously with the sample. The results can be plotted with the antibody protein amount as the horizontal axis and OD450 as the vertical axis.

[0308] The results are as follows Fig.12 As shown in B, the JY-B-009-V02-2 mRNA / LNP vaccine, whether used alone or in the form of a bivalent vaccine with the same total amount, induced F protein-specific antibodies that were comparable, higher than the protein vaccine ABRYSVO at 10 or 20 μg / mouse, and showed good cross-protection characteristics because the coating protein of Elisa is RSV-A2F protein. It is very meaningful that for mice previously infected with RSV A2, the antibody titer increased greatly when the JY-B-009-V02-2 mRNA / LNP vaccine was given as a booster shot five weeks after infection, which may indicate that for the general population who have been previously infected with RSV virus, the JY-B-009-V02-2 mRNA / LNP vaccine as a booster shot will have a very good protective effect.

[0309] The present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An expression vector, characterized in that The sequence of the expression vector is shown in SEQ ID NO:

4.

2. A cell, characterized in that The cell comprises the expression vector of claim 1.

3. A vaccine composition, characterized in that The vaccine composition comprises the expression vector according to claim 1.

4. Use of the expression vector according to claim 1 in preparing a human syncytial virus vaccine composition.

5. Use of the expression vector according to claim 1 in the preparation of a vaccine drug for preventing diseases caused by syncytial virus infection.

6. The use according to claim 5, characterized in that The disease is a respiratory disease, and the respiratory disease is pneumonia or bronchiolitis.

7. Use of the expression vector according to claim 1 in the preparation of a medicament for treating a patient suffering from a disease caused by syncytial virus infection.

8. The use according to claim 7, characterized in that The disease is a respiratory disease, and the respiratory disease is pneumonia or bronchiolitis.

9. The use according to any one of claims 7-8, characterized in that: The patient had previously been infected with syncytial virus.

10. The use according to claim 9, characterized in that The infection times are one or more times.

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