Novel human syncytial virus RSV b mRNA vaccine

Abstract

Provided are a recombinant human syncytial virus RSV B pre F mRNA, a human syncytial virus vaccine prepared from the mRNA, and a use thereof. A recombinant human syncytial virus mRNA is obtained by screening and modifying a virus strain and performing mRNA sequence optimization and a human syncytial virus mRNA / LNP nanoparticle vaccine is further successfully obtained. Mice immunized with the prepared vaccine can generate a specific antibody and a neutralizing antibody, and protection duration testing of the vaccine indicated that the human syncytial virus vaccine can provide an immune protection period of one year or more. After virus infection, a booster vaccine is given, so that the content of the antibody is greatly increased, suggesting that the human syncytial virus vaccine has a very good virus protection effect. A cotton rat lung experiment had demonstrated that the vaccine can effectively control the amplification of RSV viruses in lung, and has an immune effect significantly superior to that of the same class of commercially available similar syncytial virus vaccine products.

Description

Novel human syncytial virus RSV B mRNA vaccine TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine technology. Specifically, it relates to the design and application of a novel human syncytial virus RSV B mRNA vaccine, and belongs to the field of immunological drugs. BACKGROUND

[0002] In recent years, intracellular delivery technology based on biological macromolecules such as nucleic acids has attracted widespread attention in the field of innovative drug research and development. Among them, new vaccines based on mRNA technology have been rapidly developed, clinically tested, and approved for marketing under the urgent demand of the COVID-19 pandemic. This is the first time that the mRNA technology platform has demonstrated its clinical effectiveness, safety, and commercial value to the world.

[0003] Compared with traditional drugs directly using proteins or polypeptides as active ingredients, mRNA technology has many advantages: 1) higher biological activity and safety: the protein expression, related structural glycosylation, end modification, demethylation, and other complex protein modification processes of mRNA technology drugs are all completed in human cells according to the natural path. From the molecular structure of the synthetic raw material to the synthetic environment, it is highly homologous to the human body. Therefore, compared with traditional protein preparations in bacterial or cell systems, 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 using self-replicating nucleic acids or circular nucleic acids, can maintain effective blood concentrations for a long time, thereby reducing the frequency and amount of medication, reducing drug side effects, and improving patient compliance. 3) More application scenarios: through the development of delivery technology-based dosage forms and diverse administration methods, mRNA preparations can access a variety of clinical action targets, improve the local area concentration and efficacy of the drug active ingredient, and enrich the range of new drug development while also enriching the selection of patient treatment methods in the clinic. 4) Rapid response: mRNA vaccines use basic molecular biology methods in the product development process, and technology transfer is relatively easy. A mature mRNA platform, from gene sequencing for different pathogens to design and production, only takes a few weeks, and can quickly respond to time requirements in an emergency, such as the COVID-19 pandemic, 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 family Paramyxoviridae, subfamily Pneumovirinae, and is a highly contagious and globally highly prevalent respiratory virus. Its infectivity and pathogenicity are second only to influenza, causing bronchiolitis in infants, common cold in adults, and more severe respiratory diseases such as pneumonia in the elderly and immunocompromised individuals. It affects more than 64 million people worldwide each year. Due to the lack of a preventive vaccine or effective antiviral drugs before 2023, RSV infection poses a huge health risk and economic burden. Each year, more than 3 million people worldwide are hospitalized due to RSV infection, and nearly 60,000 people die, half of whom are infants under 6 months old. In the United States alone, RSV infection in the elderly results in 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 length and consists of 10 genes, encoding 11 proteins. The interior of the viral particle is composed of nucleoprotein (N), tetrameric phosphoprotein (P), and large polymerase protein (L) bound to viral RNA, which is surrounded by matrix protein (M) and encapsulated in a lipid bilayer containing fusion protein F, glycoprotein G, and small hydrophobic protein SH. Based on the reaction of F and G proteins to monoclonal antibodies, RSV viruses are divided into two antigenic subtypes: type A and type B, which are transmitted simultaneously or alternately in local epidemics. Since the F protein plays an important role in the entry of RSV into host cells, it is a popular antigen for vaccines due to its cross-strain type conservation, protein stability, and less glycosylation.

[0006] The F protein sequence is highly conserved among strains and exists in two conformational forms. Pre-F state is the pre-fusion state, and the protein exists in a trimeric form and contains the main antigenic site Ø. After binding to the target on the host cell surface, Pre-F undergoes a conformational change to convert to the more stable post-fusion Post-F conformation, and site Ø is lost due to being wrapped inside. In cells, the initial F protein is expressed as a single polypeptide precursor F0, which is cleaved by furin protease to form F1, F2, and Pep27 polypeptides. F1 and F2 are connected by two disulfide bonds to form mature F protein and anchor in the viral membrane in a trimeric structure. Since the RSV F protein plays an important role in the entry of RSV into cells, it is the target of neutralizing antibodies and the main subject of vaccine development. However, like other RSV antigens, previous vaccine development efforts based on RSV F protein have proven unsuccessful.

[0007] Since RSV was first discovered in human population in 1957, attempts for RSV vaccine have never stopped for decades. However, due to the lag of technology, from inactivated vaccine causing more serious infection, to later protein vaccine, virus carrier vaccine, researchers have been entangled in many problems such as valence, G protein, F protein, or N protein as antigen, mainly inducing T cell cellular immunity or B cell humoral immunity, etc., accumulated a lot of experience, but still failed to obtain a successful RSV vaccine.

[0008] In recent years, with the intervention of structural molecular biology, people have a breakthrough understanding of the working principle of the vaccine, which enables the initial two RSV protein vaccines (GSK and Pfizer) to be put on the market. Our design is just combined with the latest antigen protein structure biology discovery and in-depth understanding of virus epidemiology and strain, and designed and synthesized our unique RSV B mRNA / LNP vaccine. SUMMARY

[0009] The purpose of the present application is to solve the problems of the prior art RSV vaccine, and provide a RSV B mRNA / LNP vaccine.

[0010] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

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

[0012] The present application discloses an expression vector, wherein the DNA molecule of the expression vector is shown in SEQ ID NO: 4.

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

[0014] The present application discloses a cell, wherein the cell comprises the expression vector.

[0015] The present application discloses a recombinant protein, wherein the recombinant protein is obtained by translation of the mRNA.

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

[0017] The present application discloses a nucleotide, wherein the nucleotide encodes the recombinant protein.

[0018] The present application discloses a plasmid, wherein the plasmid comprises the nucleotide.

[0019] The application discloses an mRNA / LNP complex, comprising the mRNA molecule and a lipid nanoparticle.

[0020] The application discloses a preparation method of an mRNA / LNP complex, characterized by comprising the following steps:

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

[0022] 2) proportionally configuring an ionizable cation, DSPC, cholesterol and PEG-2000 into a mixed solution;

[0023] 3) high-speed cross-collision mixing the solution in step 2) with the solution in step 1) in a microfluidic device, and performing dilution and pH value lifting to obtain the 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 application discloses an mRNA / LNP complex, which is prepared according to the method.

[0027] The application 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 application discloses application 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 preparation of a human syncytial virus vaccine composition.

[0029] The application discloses application 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 preparation of a drug for treating diseases 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 application discloses application 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 preparation of a drug for treating patients suffering from diseases caused by syncytial virus infection.

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

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

[0034] Preferably, the number of infections is one or more.

[0035] The present application obtains a recombinant human syncytial virus mRNA through screening, modification of virus strains, and optimization of mRNA sequences. Further, the coding gene of the mRNA is constructed into a plasmid to realize efficient transcription and capping in vitro, and the mRNA is encapsulated in a lipid nanoparticle through pH promotion, with an encapsulation rate of more than 91%, and an mRNA / LNP nanoparticle vaccine is successfully obtained. Western blotting is performed on the expression of the RSV F mRNA / LNP nanoparticle in cells. After immunization of mice, specific antibodies and neutralizing antibodies are produced, and the protection duration of the vaccine is detected, which shows that the antibody level decreases very limitedly at six months after immunization, proving that the prepared vaccine has long-term protection against viruses. After viral infection, a booster vaccine is given, which greatly increases the antibody content, indicating that it has very good virus protection effect. A cotton rat lung experiment proves that the vaccine can effectively control the amplification of the RSV virus in the lung, and the immunization effect is obviously better than that of a commercially available syncytial virus vaccine product. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0038] Figure 3. Plasmid JY-B-009-V02-2 restriction enzyme detection.

[0039] Figure 4. mRNA synthesized by linearization of plasmid JY-B-009-V02-2 property analysis. Among them, Figure 4A is capillary electrophoresis analysis of the purity of mRNA; Figure 4B is SEC-HPLC aggregate content analysis of the purity of mRNA; Figure 4C is mRNA capping rate analysis.

[0040] Figure 5. Western Blot detection of expression of JY-B-009-V02-2 mRNA / LNP nanoparticle in cells after transfection into 293T cells.

[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 JY-B-009-V02-2 mRNA / LNP vaccine antibody response, wherein Figure 7A is antibody titers after immunization with different doses of intramuscular injection, showing the relationship between different doses and time after immunization; Figure 7B is the average value of each group (n=5) corresponding to Figure 7A; Figure 7C is an example of 21 days and 28 days after immunization, 15 μg and 200 μg, there is no significant difference in antibody titers at 21 days, and the difference at 28 days is p<0.05.

[0043] Figure 8. Humoral immunity induced by JY-B-009-V02-2 mRNA / LNP vaccine in mice has long-term effect.

[0044] Figure 9. Identification of neutralizing antibodies induced by JY-B-009-V02-2 mRNA / LNP vaccine in mice. Wherein, Figure 9A is neutralizing RSV A2 virus; Figure 9B is neutralizing RSV B 18537 virus.

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

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

[0047] Figure 12. Virus-specific antibodies induced by JY-B-009-V02-2 mRNA / LNP vaccine have advantages compared with marketed RSV vaccines. Wherein, Figure 12A is the experimental design; Figure 12B is the antibody titer in serum after one immunization or one infection; Figure 12C is the antibody titer in serum after two immunizations or infection / immunization. DETAILED DESCRIPTION

[0048] The specific embodiments of the present application are described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the present application.

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

[0050] 1.1 Screening of starting virus strains

[0051] According to the traditional serological classification, RSV viruses can be divided into A and B subtypes. In epidemiology, they are almost equal in weight, which is reflected in the randomness of the epidemic year and region, and the equivalent degree of pathogenicity. Therefore, it is the same regardless of which subtype the design focuses on. This patent mainly focuses on RSV B subtype, and the design sequences listed are all started from RSV B subtype.

[0052] The general design of the current virus vaccine is to take the F fusion protein gene of the most original strain of RSV, RSV-A2, as the starting point. The immune response induced by such a vaccine, especially the anti-virus antibody, has a complete "key and lock" matching relationship with RSV-A2, which can better neutralize the RSV-A2 strain and achieve the effect of immunity. However, the virus gene sequence will undergo genetic variation as the virus repeatedly infects the host and continuously passes on, which will inevitably result in the difficulty of the immune response induced by the original strain RSV-A2 isolated in 1962 to completely control modern strains after sixty years. Therefore, after searching for several hundred F gene sequences 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 manufacturing process, and it is inseparable from the mRNA manufacturing process. It affects the yield of mRNA in vitro transcription, the immunogenicity of mRNA molecules, the translation efficiency and the stability of mRNA molecules. The current general mRNA is composed of 5' cap (cap structure), 5' UTR (non-coding region), ORF (protein-coding open reading frame), 3' UTR and 3' poly A tail (poly A tail).

[0055] 5' Cap structure protects mRNA from degradation by cytosolic exonuclease Xrn1 and nuclear Xrn2 enzyme. Capped mRNA has a longer half-life and higher stability than uncapped mRNA, significantly improving the yield of protein synthesis in cells. The chemical nature of the cap structure is a special structure at the 5' end of mRNA formed during mRNA transcription, namely the m7GPPPN structure, also known as the methyl guanosine cap. It is formed under the catalysis of RNA triphosphatase, guanylyltransferase, mRNA (guanine-N7) methyltransferase and mRNA (nucleoside-2') methyltransferase. According to 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 can be recognized by natural immune receptor RIG-1, and Cap 1 and Cap 2 can protect themselves from being recognized by innate immune sensors, our design is to use 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 translation initiation of eukaryotic mRNA is mainly determined by the characteristics of 5' UTR, including secondary structure, sequence elements and 5' UTR length, which will affect the stability of mRNA. Our design is to use the human cytochrome B-245 alpha polypeptide (CYBA) UTR with relatively high protein expression. Of course, the performance of UTR also depends on the cell type.

[0058] For the protein sequence of RSV, in order to make the antibodies induced by the vaccine more effectively block the virus, it is crucial to select the metastable pre-fusion form, which can achieve structural stability through point mutations. We arranged four point mutations on the original sequence: S155C and S290C mutations of DS to introduce non-native disulfide bonds; S190F and V207L mutations of Cav1 to fill the cavity with amino acids. At the same time, in order to induce uniform and more significant immune response, the transmembrane segment (TM) and intracellular segment at the C-terminal were removed and replaced with a Fibritin sequence, so that the F protein can be presented in the form of a relatively stable trimer.

[0059] For the DNA sequence of RSV-F, the overall translation efficiency of mRNA can be optimized by choosing appropriate codons. One is to replace the codons in the viral sequence with synonymous codons that are frequently used in host cells, i.e. human cells, to ensure that the codon usage bias is more compatible and to avoid excessive rare codons. However, it is also noted that a small number of viral codons are retained to slow down the speed of ribosome advancement and provide sufficient time for the correct folding of the protein, thereby more accurately restoring the antigenic protein structure of the virus. Another point to note is the GC content. A slightly higher GC content is believed to increase the stability of mRNA and improve protein expression in vivo. Finally, the secondary structure of the mRNA sequence is also taken into consideration. It has been reported that the hairpin structure of the secondary structure can affect the degradation of mRNA and contribute to the stability of mRNA.

[0060] Similar to the 5' UTR, the 3' UTR also contains many regulatory elements that play an important role in the stability, subcellular localization, and translation efficiency of mRNA. The 3' UTR we used is from the human AES / TLE5 gene.

[0061] The poly A tail plays an indispensable role in protein translation. It binds to poly A tail binding protein (PAPB), which in turn interacts with translation initiation factor eIF4G to form a "closed loop" structure, recruiting the 40S translation initiation complex to the mRNA, working in conjunction with the 5' terminal cap structure to stimulate translation initiation. During bacterial amplification, plasmids carrying long poly A nucleotide sequences can produce unpredictable recombination events, and the poly A tail on the plasmid can be shortened with the continuous amplification of bacteria, 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, maintaining the length of the tail, while not affecting the translation efficiency and half-life of mRNA generated by in vitro transcription. Following this principle, our poly A is 40-spaced-80 bases.

[0062] The optimized RSV-F coding sequence obtained is shown in Figure 1. Figure 1A shows the DNA sequence of the RSV-F antigen coding sequence of JY-B-009-V02-2 vaccine (SEQ ID NO: 1); Figure 1B is the mRNA sequence after transcription (SEQ ID NO: 2); Figure 1C is the protein amino acid sequence after translation (SEQ ID NO: 3); and Figure 1D is the DNA sequence of the RSV-F antigen with the addition of the N-terminal T7 transcription enzyme binding site, 5' UTR, Kozak, C-terminal stop code, 3' UTR, poly(A), and then inserted into the vector through HindIII and Sap I sites.

[0063] Example 2 Plasmid synthesis and DNA extraction of mRNA vaccine

[0064] 2.1 Plasmid synthesis

[0065] After the plasmid sequence design was completed, it was synthesized by a CRO company (Figure 2).

[0066] 2.2 Plasmid DNA extraction

[0067] 2.2.1 Reagent preparation

[0068] LB plates and liquid media containing peptone, sodium chloride, yeast extract and corresponding antibiotics.

[0069] 2.2.2 Plasmid transformation

[0070] Plasmid DNA was added to competent cells, which were placed in an ice box for 30 min; 42°C water bath heat shock for 45 seconds, quickly placed in an ice box for 2 min, added 500 μl of LB culture solution without resistance, 37°C shaking bed culture for 1 h, then plated, and cultured at 37°C overnight. The next day, the colonies were picked.

[0071] 2.2.3 Plasmid DNA extraction

[0072] The overnight culture was extracted by the method of small or large extraction kit.

[0073] 2.3 Plasmid DNA restriction enzyme verification

[0074] Single, double and triple enzyme digestion was performed using restriction enzymes. Qualified plasmids were used for the next step. According to the sequence design, three groups of enzymes were used for double enzyme digestion verification (Figure 3). The first group of enzymes was EcoR I and Hind III, and the theoretical size after digestion was 1723 bp + 3279 bp; the second group of enzymes was Pml I and SnaB I, and the theoretical size after digestion was 1179 bp + 3823 bp; the third group of enzymes was Pme I and Bbs I, and the theoretical size after digestion was 211 bp + 4791 bp.

[0075] Example 3 In vitro transcription capping, purification and quality detection of RSV B

[0076] 3.1 Plasmid DNA linearization

[0077] This design uses a reverse BspQI at the 3' end of polyA to linearize the plasmid. After 3 hours of reaction at 50°C, SDS-PAGE gel electrophoresis was used to determine whether the digestion was complete. Purification was performed using 3M sodium acetate precipitation method; then the DNA concentration was determined by Nanodrop method.

[0078] 3.2 In vitro transcription capping and purification

[0079] The JY-B-009-V02-2 plasmid design is added agg between the T7 polymerase binding site and the 5' UTR, so the in vitro transcription is suitable for using co-transcription method. The co-transcription IVT system contains template DNA, NTP (UTP is pseudo-uracil), Cap analogs, T7 transcriptase, 10 X transcription buffer solution, pyrophosphatase, RNase inhibitor enzyme, and DEPC water. The amount of DNA template and the volume of constant volume are determined according to the specific experiment. After 37°C reaction for 3 hours, DNase I is added to remove DNA template, and SDS-PAGE gel is used to judge mRNA synthesis and DNA template digestion. The purification method is lithium chloride precipitation method.

[0080] 3.3 mRNA integrity and capping rate detection

[0081] The quality of mRNA determines the quality of the final product to a large extent. Due to the instability of mRNA itself, it is easily degraded or hydrolyzed by ubiquitous nucleases, which makes mRNA vaccine face challenges in production, process, long-term storage, etc. Therefore, a simple and efficient mRNA vaccine purity analysis and molecular size characterization method is crucial for its quality, efficacy, safety and optimized manufacturing process.

[0082] 3.3.1. Capillary gel electrophoresis (CE)

[0083] Capillary gel electrophoresis is used to determine the proportion of incomplete or fragmented mRNA in DS, and the method parameters are as follows:

[0084] Agilent 5200 fragment analyzer is used for analysis using Agilent DNF-471 RNA analysis kit. After the test sample is diluted to 20 μg / mL and the Ladder is heated and denatured at 70°C for 2 minutes, it is immediately cooled to 4°C and kept on ice. Add 22 μL of RNA Diluent Marker (15 nt) solution to the 96-well PCR plate, then add 2 μL of test sample or Ladder solution, mix well, and then put the 96-well PCR plate into the 5200 fragment analyzer. Separate at 8.0 kV for 40 min. As shown in Figure 4A, the purity of our mRNA sample reaches 97.1%.

[0085] 3.3.2 SEC-HPLC analysis

[0086] The purity of mRNA was analyzed by SEC-HPLC in terms of aggregate content. The method parameters were as follows: analysis was performed using a mobile phase of 150 mM PB, pH 7.0, isocratic elution on a column at a flow rate of 1.0 mL / min for 20 min, a sample load of 2000 ng, a detection wavelength of 260 nm, and a column temperature of 30°C. As shown in FIG. 4B, the purity of mRNA was 98.42%.

[0087] 3.3.3 Analysis of capping rate

[0088] The 5' end cap structure of mRNA plays a role in preventing mRNA degradation, prolonging the half-life, and providing mRNA translation ability, and is a key quality attribute of mRNA drugs. The 5' end capped nucleic acid fragment was specifically cleaved by RNaseH enzyme and specific probe combination, and then purified by streptavidin-coupled magnetic beads. The capped and uncapped nucleic acid fragments were separated by ion pair reversed phase chromatography (IP-RP-HPLC), and the capping rate of mRNA was calculated by area normalization method.

[0089] RNaseH enzyme specific cleavage: enzyme cleavage probe, buffer and enzyme-free water were added to the sample, mixed well and placed in a PCR instrument, and slowly annealed from 95°C to 22°C. Then magnetic beads were added, incubated at room temperature for 30 min, and then RNase H (50 units) was added and incubated at 37°C for 3 h. After enzyme cleavage, the supernatant was removed by magnetic stand enrichment, and then rinsed with enzyme-free water for 3 times. Then, 75% methanol was added and incubated at 80°C for 3 min, and the eluate was collected by magnetic stand enrichment. The eluate was placed in a vacuum centrifugal concentrator for drying, and finally resuspended in resuspension solution (100 μM EDTA / 1% methanol) for subsequent IP-RP-HPLC analysis.

[0090] IP-RP-HPLC analysis: using a mobile phase A of 200 mM hexafluoroisopropanol + 8.15 mM triethylamine and a mobile phase B of MeOH, eluting the mobile phase B from 5% to 75% in a gradient at a flow rate of 0.3 mL / min for 20 min on a column, a sample load of 100 ng, a detection wavelength of 260 nm, and a column temperature of 75°C. As shown in FIG. 4C, the capping rate of mRNA was 90.45%.

[0091] Example 4 mRNA / LNP nanoparticle preparation

[0092] In this study, the commonly used method for preparing nanoparticles LNP in the industry was used.

[0093] LNP was prepared by mixing ionizable lipid: DSPC (1, 2-distearoyl-sn-glycero-3- phosphocholine): cholesterol: DMG-PEG-2000 at a ratio of 50:10:38.5:1.5 with mRNA dissolved in pH 4 acetate buffer in a high shear nanoparticle preparation device and forming lipid nanoparticles (mRNA / LNP) with mRNA encapsulated within as a result of the 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.

[0094] 4.1. Buffer preparation

[0095] All reagents were autoclaved at 121 °C for 20 min and prepared using DEPC water.

[0096] 1) 50 mM acetate buffer, pH 4.0

[0097] 2) 25 mM Tris-HCL buffer, pH 7.5

[0098] 3) 0.6 g / mL sucrose solution

[0099] 4.2. Lipid solution and mRNA solution preparation

[0100] Table 1: Lipid solution preparation

[0101]

[0102] 1) The lipid was allowed to return to room temperature before weighing;

[0103] 2) All components were dissolved in anhydrous ethanol and sonicated to ensure complete dissolution.

[0104] 3) The prepared lipid solution was mixed at a ratio of 1:1:1:1 to obtain a 12 mg / mL lipid mixture solution according to the formula.

[0105] Table 2: mRNA solution preparation

[0106]

[0107] The in vitro transcribed and capped mRNA stock solution prepared in Example 3 was diluted to the desired concentration of 0.2 mg / mL according to the calculation method and buffer type shown in the above table.

[0108] 4.3. Preparation of mRNA / LNP (4 mL total volume as an example)

[0109] 4.3.1 Use of the rapid nanomedicine preparation system INano LTM. Set the parameters as needed.

[0110] 4.3.2 Pre-use cleaning of the chip

[0111] Prepare 5 mL syringes, 3 of which are used to draw anhydrous ethanol, 1 to draw acetate buffer, and 1 to draw air. On the left side, draw 5 mL of acetate buffer -> 3 times of air; on the right side, draw 5 mL of ethanol -> 5 mL of ethanol -> 3 times of air, and push the chip to clean it. After cleaning, load the chip into the card slot.

[0112] 4.3.3 Running the microfluidic device to prepare LNP

[0113] Use a 3 mL syringe to draw 3 mL of the above mRNA solution, and remove the air bubbles, and load it into the left syringe card slot of the machine. Use a 1 mL syringe to draw 1 mL of the above lipid mixture solution, and remove the air bubbles, and load it into the right syringe card slot of the machine. Insert a 15 mL centrifuge tube at the waste liquid collection and sample collection of the machine for waste liquid and sample collection. After confirming that the parameters are correct, click “Start”, and the rapid nanomedicine preparation system INano LTM will immediately run, i.e., to prepare LNP. After the preparation is completed, take out the centrifuge tube at the sample collection.

[0114] 4.3.4 Post-use cleaning of the chip

[0115] Immediately after the LNP preparation is completed, use the left 5 mL DEPC water and the right 5 mL ethanol -> 5 mL ethanol -> 3 times of air to load the syringes into the chip sample inlet, and push the chip to clean it, and then naturally dry it.

[0116] 4.3.5 Processing of the collected mRNA / LNP sample

[0117] Immediately dilute the collected mRNA / LNP suspension with 25 mM Tris-Hcl pH7.5 buffer to about 25 times the volume of mRNA / LNP, and then centrifuge for ultrafiltration at 3000 rpm, 4°C, for 1 h. Repeat this process until the solution volume is less than or equal to the pre-dilution volume (3.6 mL, i.e., the total volume of 4 mL minus the waste liquid of 0.4 mL). Then, sterilize it through a 0.22 μm filter membrane, and add 0.2 times the solution volume of 0.6 g / mL sucrose solution, to finally obtain the mRNA / LNP preparation product.

[0118] 4.3.6 Detection of the physical properties of mRNA / LNP

[0119] The finished 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 Malvern Zetasizer, and the particle size, PDI, ZP data can be obtained.

[0120] Encapsulation efficiency analysis:

[0121] The mRNA control (100 μg / ml) was diluted from 2000 ng / mL to 7 standard curve points (2000, 1600, 1200, 800, 400, 200 ng / mL) using 1X TE Buffer and 2% Triton TE Buffer, respectively. When measuring the total RNA content, the test sample LNP was diluted 50 times using 2% Triton TE Buffer, and when measuring the free RNA content, the test sample was diluted 20 times using 1X TE Buffer. 100 μL of the above standard curve solution and test sample solution were transferred into a black microplate, and then 100 μL of 0.005x Ribogreen (200-fold dilution) was added, respectively, and reacted at room temperature for 2-5 min in the dark. The fluorescence response value (excitation wavelength 480 nm, emission wavelength 520 nm) was read on the microplate, and the encapsulation efficiency was calculated according to the following formula:

[0122] Encapsulation efficiency EE (%) = (Ctotal RNA - Cfree RNA) / Ctotal RNA * 100%.

[0123] The encapsulation efficiency of the test sample obtained according to the above method was 91.56%.

[0124] Example 5 Expression of RSV F mRNA / LNP Nanoparticle Transfection into 293T Cells

[0125] To determine that the mRNA in the lipid nanoparticle can express the correct protein, the first step is to transfect the specific mRNA / LNP into the appropriate cells, and then the protein expression can be detected by Elisa, Western Blot, flow cytometry, etc.

[0126] 5.1 Cell transfection

[0127] One day before transfection, 293T (CRL-3216, ATCC) cells were plated at 2x10^5 per well in a 24-well plate in complete medium, DMEM plus 10% fetal bovine serum and antibiotics, in a 37°C, 5% CO2 incubator. The next day, the cell culture medium was aspirated from each well and the cells were gently washed with PBS. After adding 500 μl of complete medium to each well, 0.5 to 3 μg of RSV F mRNA / LNP nanoparticles were added to the cells. After gently shaking to disperse the nanoparticles, the cells were incubated at 37°C, 5% CO2 for 48-72 hours.

[0128] 5.2 Western Blot (WB) method for detecting expression of antigen protein

[0129] By WB method, the mRNA / LNP translated into the correct antigen protein after entering the cells can be qualitatively detected. Since the designed antigen protein has a signal peptide, the extracellular protein is determined here.

[0130] 5.2.1 Preparation of electrophoresis sample

[0131] The supernatant of the cells after transfection for 48-72 hours was collected, centrifuged at 2500 rpm, 5 min, 4°C, and the supernatant was taken to a new 1.5 mL tube. If the expression is too low, a suitable size Millipore ultrafiltration tube can be selected for moderate concentration. Then the protein content was determined, and each sample was adjusted to the same sample amount, and 6x SDS loading buffer was added to a final concentration of 1x. The sample was heated at 92°C for 5 min to denature the protein before loading.

[0132] 5.2.2 SDS-PAGE electrophoresis

[0133] The electrophoresis used 10% separation gel and 4% concentration gel, and the sample was loaded immediately after heating. During electrophoresis, the voltage of the concentration gel segment was 110V, and the voltage of the separation gel segment was 150V.

[0134] 5.2.3 Transferring membrane

[0135] According to the size of the molecular weight, nitrocellulose membrane or PVDF membrane was selected. Generally, 80V was used for 1h, or 60V was used for 2h, and ice bag was used for heat control. After transferring, 1x rose red staining solution was used for 5min to detect the transfer effect.

[0136] 5.2.4 Blocking and immunoreaction

[0137] Firstly, the membrane was blocked with 5% no fat milk at 4℃ overnight. The next day, after washing with PBST solution for 3 times, the diluted primary antibody (mouse anti-RSV-F) was hybridized at room temperature for 90 min. Then, the membrane was washed with PBST solution for 3 times, each for 5-10 min. The secondary antibody was anti-mouse-HRP, which was hybridized at room temperature for 60 min. Subsequently, the membrane was also washed with PBST solution for 3 times, each for 5-10 min

[0138] 5.2.5 Development and exposure

[0139] According to the ECL kit instructions, the test solution A and B were mixed at a ratio of 1:1 and placed in the membrane. After shaking in the dark room, the membrane was exposed to the chemiluminescence image analyzer for detection. According to the marker band, the size of the target protein was determined to be correct.

[0140] The results are shown in Figure 5. After transfection of 293T cells with JY-B-009-V02-2, mRNA / LNP was endocytosed into cells, released into the cytoplasm in the lysosomal acidic environment, and translated and modified in the endoplasmic reticulum and Golgi apparatus. Under the guidance of the signal peptide, it was released into the extracellular space. The encoded RSV-F antigen protein can bind to the F antibody, and the protein specificity and molecular weight meet the design requirements.

[0141] Example 6 Immunization of mice with JY-B-009-V02-2 mRNA / LNP vaccine and identification of virus-specific antibody expression

[0142] To detect the expression and performance of the vaccine in vivo, we used mice as animal models for detection.

[0143] 6.1 Animal immunization

[0144] Balb / c mice, female, 6 weeks old, will be adapted for a week after arriving at the animal house. The vaccine is the JY-B-009-V02-2 mRNA / LNP vaccine prepared in Example 4. Intramuscular injection, the dose is 10 μg / mouse, divided into two injections with an interval of 14 days. Blood was taken one day before the first immunization and 7 days after the second immunization, and the serum was used for subsequent antibody Elisa detection.

[0145] 6.2 Elisa detection of anti-RSV antibody

[0146] One of the key indicators of the vaccine is to induce specific anti-virus antibodies in animals. We used Elisa to detect the expression of anti-RSV-F antibodies.

[0147] 6.2.1 Antigen coating

[0148] Dilute antigen (RSV-A2, F) to 62.5 ng / mL with PBS, coat antigen in 96-well enzyme-labeled plate, 100 μl per well, 4℃, 16h.

[0149] 6.2.2 Wash and block

[0150] Take out the enzyme-labeled plate coated with antigen, wash the plate once with 300 μL / well PBST. Then add 100 μL blocking solution (5% milk), room temperature, 30 min. Then wash the plate 3 times with 300 μL / well PBST.

[0151] 6.2.3 Sample dilution and sample addition

[0152] After the serum sample of the immunized mouse is diluted by 4 times in a suitable gradient, 100 μl is added to the enzyme-labeled plate, three replicates, incubated at room temperature for 1.5 h, and then washed 3 times with 300 μL / well PBST.

[0153] 6.2.4 Add detection antibody

[0154] According to the number of wells to be added, take the appropriate amount of detection antibody (Goat Anti-Mouse IgG, Peroxidase-Conjugated, H+L) matched, dilute to 0.8 ng / mL, add 80 μL per well, incubate at room temperature for 1 h, and then wash the plate 5 times with 300 μL / well PBST.

[0155] 6.2.5 Color development and termination

[0156] Mix color developing solution A (containing H202) and color developing solution B (containing TMB) at a ratio of 1:1, add 100 μL per well, and incubate at room temperature for 15-30 min. Then add 30 μL 1 M HCL termination solution per well, and then read at 450 nm using an enzyme-labeled instrument.

[0157] 6.2.6 Standard curve

[0158] If absolute quantification is required, purchase anti-RSV-F antibodies, prepare a suitable low-to-high concentration range according to the antibody protein amount, and perform Elisa at the same time as the sample. The results are plotted with antibody protein amount as the abscissa and OD450 as the ordinate.

[0159] As can be seen from the results of Figure 6, JY-B-009-V02-2 mRNA / LNP vaccine can induce a strong antibody response in mice, with a median serum dilution value of more than 32K.

[0160] Example 7 Dose exploration of JY-B-009-V02-2 mRNA / LNP vaccine

[0161] As a vaccine, dosage is a key consideration factor. We first investigated the dose effect and time frame of IgG antibody production of JY-B-009-V02-2 mRNA / LNP vaccine.

[0162] 7.1 Animal immunization

[0163] Balb / c mice, female, 6 weeks old, will be acclimated for one week after arrival in the animal room. The vaccine is mRNA / LNP dosage form, respectively, single dose of intramuscular administration of 15, 30, 45, 60, 75, 90, 120, 200 μg / each.

[0164] 7.2 Elisa detection of anti-RSV antibody

[0165] 7.2.1 Antigen coating

[0166] Dilute the antigen (RSV-A2, F) to 62.5 ng / mL with PBS, and coat the antigen in a 96-well enzyme-labeled plate, 100 μl per well, 4°C, 16h.

[0167] 7.2.2 Washing and blocking

[0168] Take out the enzyme-labeled plate with coated antigen, wash the plate once with 300 μL / well PBST. Then add 100 μL blocking solution (5% milk), room temperature, 30 min. Then wash the plate 3 times with 300 μL / well PBST.

[0169] 7.2.3 Sample dilution and sample addition

[0170] After the serum samples of immunized mice are diluted by 4 times in a suitable gradient, 100 μl is added to the enzyme-labeled plate, three replicates, incubated at room temperature for 1.5 h, and then washed 3 times with 300 μL / well PBST.

[0171] 7.2.4 Add detection antibody

[0172] According to the number of wells to be added, take the appropriate amount of detection antibody (Goat Anti-Mouse IgG, Peroxidase-Conjugated, H+L) matched, dilute to 0.8 ng / mL, add 80 μL per well, incubate at room temperature for 1 h, and then wash the plate 5 times with 300 μL / well PBST.

[0173] 7.2.5 Color development and termination

[0174] Mix color developing solution A (containing H202) and color developing solution B (containing TMB) at a ratio of 1:1, add 100 μL per well, and let stand at room temperature for 15-30 minutes. Then add 30 μL of 1 M HCL per well to stop the reaction, and then read the OD at 450 nm using an enzyme-labeled instrument.

[0175] 7.2.6. Standard curve

[0176] For antibody absolute quantification, anti-RSV-F antibodies were purchased, and a suitable low-to-high concentration range was prepared according to the amount of antibody protein. Elisa was performed simultaneously with the sample, and the results were plotted with the amount of antibody protein as the abscissa and OD450 as the ordinate. The absolute amount of anti-RSV F antibody in the serum was calculated according to the standard curve.

[0177] The dose exploration results are shown in FIG. 7. The mRNA / LNP vaccine of JY-B-009-V02-2 can induce high-titer virus-specific antibodies in animals, and there is no significant difference between the doses of 15 to 200 μg. The antibody concentration tends to be stable 28-35 days after immunization, but still shows 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 lays a foundation for subsequent experimental design and vaccine development.

[0178] Example 8. Detection of long-term protection effect of JY-B-009-V02-2 mRNA / LNP vaccine

[0179] The protection time of the vaccine is an important indicator for investigating the vaccine. An effective vaccine that can protect against infection during the season should have an effective period of at least four months. Therefore, we investigated the protection time of the JY-B-009-V02-2 mRNA / LNP vaccine.

[0180] 8.1 Animal immunization

[0181] Balb / c mice, female, 6 weeks old, will be adapted for one week after arriving at the animal house. The vaccine is an mRNA / LNP dosage form, and the administration is intramuscular injection of 15 μg per mouse, one immunization and two boosts, three doses of the same amount, with an interval of 14 days. Blood samples are taken before immunization, 7 days, 21 days, 6 months, and 12 months after the last injection.

[0182] 8.2 Elisa detection of anti-RSV antibodies

[0183] 8.2.1 Antigen coating

[0184] Dilute the antigen (RSV-A2, F) to 62.5 ng / mL with PBS, and coat the antigen in a 96-well enzyme-labeled plate at 100 μl per well, 4°C, 16h.

[0185] 8.2.2 Wash plate and seal

[0186] Take out the enzyme-labeled plate coated with antigen, wash the plate once with 300 μL / well PBST. Then add 100 μL blocking solution (5% milk), room temperature, 30 min. Then wash the plate with 300 μL / well PBST for 3 times.

[0187] 8.2.3 Sample dilution and sample addition

[0188] After the serum samples of immunized mice are diluted by a suitable 4-fold gradient, 100 μl is added to the enzyme-labeled plate, three replicates, incubated at room temperature for 1.5 h, and then washed with 300 μL / well PBST for 3 times.

[0189] 8.2.4 Add detection antibody

[0190] According to the number of sample wells, take the appropriate amount of detection antibody (Goat Anti-Mouse IgG, Peroxidase - Conjugated, H+L) matched, dilute to 0.8 ng / mL, add 80 μL per well, incubate at room temperature for 1 h, and then wash the plate with 300 μL / well PBST for 5 times.

[0191] 8.2.5 Color development and termination

[0192] Mix color developing solution A (containing H2O2) and color developing solution B (containing TMB) at a ratio of 1:1, add 100 μL per well, and incubate at room temperature for 15-30 min. Then add 30 μL of 1 M HCL termination solution per well, and then read at 450 nm using an enzyme-labeled instrument.

[0193] The results are shown in Figure 8. On the 7th day after the booster injection of the vaccine, the virus-specific antibody reached a very high titer; the antibody titer remained at the same level on the 21st day, and the median serum dilution was above 32k. At six months, there was a very limited decrease, but at one year, the decrease was obvious, but there was still a titer with a median serum dilution of 6-7K. The data show that the JY-B-009-V02-2 mRNA / LNP vaccine has long-term protection against the virus.

[0194] Example 9 Detection of the neutralization ability of antibodies against viruses

[0195] One of the gold standard assays for a vaccine is to determine if the specific anti-virus antibodies induced in the animal have the ability to neutralize the virus and prevent infection. The neutralization assay is designed to do just that. Since RSV is a biosafety level 2 virus, all the following steps must be done in a biosafety level 2 laboratory following the appropriate requirements and regulations.

[0196] 9.1 Preparation of cells

[0197] HEp-2 (ATCC, CCL-23) human laryngeal epidermoid carcinoma cells are the most suitable cells for RSV growth. The day before the neutralization assay, cells are trypsinized and resuspended and washed, then seeded into 24-well plates at 2 x 105 / well in MEM 10% FBS complete medium and incubated overnight at 37°C in 5% CO2.

[0198] 9.2 Antibody and virus neutralization

[0199] 9.2.1 Serum dilution gradient

[0200] The serum dilution gradient and dilution method used in this laboratory are shown in Table 6. For RSV, the dilution medium is Opti-MEM without fetal bovine serum.

[0201] Table 3, Serum dilution and dilution method

[0202]

[0203] 9.2.2 Serum antibody and virus neutralization

[0204] The amount of RSV virus used is 30-40 PFU / well (24-well plate), and the dilution medium is Opti-MEM without fetal bovine serum. Take 120 μl of the virus dilution and add it to the diluted serum well, and mix gently. Set up a virus control group (i.e., virus solution without diluted serum). Incubate in a 37°C incubator for 1 h.

[0205] 9.2.3 Seeding of cells

[0206] Take 70 μl of the virus-antibody mixture described above and add it to the 24-well cell culture plate in which the cells have grown into a monolayer, 3 wells for each dilution. At the same time, set up 4 wells of normal cells as a control and 4 wells of virus as a control. Incubate the plate in a 37°C, 5% CO2 incubator for 1 h, and gently shake it every 15 min. After incubation, discard the mixture in the wells, and add 500 μl / well of pre-prepared warm medium, which is MEM, 3% FBS, and 0.75% methylcellulose. Incubate at 37°C in 5% CO2 for 4 days.

[0207] 9.3 Results coloration

[0208] 9.3.1 Virus and cell fixation

[0209] On the fourth day of culture, discard the culture solution in the well, and then wash it once with PBS, and then add 150 μl of pre-cooled 85% methanol per well, and fix it at 4°C for 1 hour. Then discard the solution in the well, and wash it once with PBS.

[0210] 9.3.2 Blocking and washing the plate

[0211] Add 300 μL of blocking solution (5% milk), and incubate at room temperature with gentle shaking for 30 min. Then wash the plate 3 times with 500 μL / well of PBST.

[0212] 9.3.3 Add detection antibody

[0213] According to the number of sample wells, take the matching detection antibody (Anti-RSV-F IgG, H+L, HRP Conjugated), dilute it by an appropriate multiple, and then add 150 μL per well, and incubate at room temperature for 90 min. Then wash the plate 3 times with 500 μL / well of PBST.

[0214] 9.3.4 Add secondary antibody

[0215] Take the matching secondary antibody, dilute it by an appropriate multiple of 1:1000 to 1:5000, and then add 150 μL per well, and incubate at room temperature for 60 min. Then wash the plate 5 times with 500 μL / well of PBST.

[0216] 9.3.5 Coloration and termination

[0217] Add 150 μL of TrueBlue™ Peroxidase Substrate per well, and gently shake. Virus-positive plaques will appear quickly. Then wash away the reaction solution with water. Dry in the dark. The next day, count the virus plaques.

[0218] As can be seen, in terms of RSV A2 virus (Figure 9A), whether it is RSV B type B-009-V02-2 mRNA / LNP vaccine alone or combined with A type vaccine (#7), the neutralization ability of the antibodies induced by the vaccine to the virus is similar, with an EC50 of 10K serum dilution. When neutralizing RSV B 18537 virus (Figure 9B), the EC50 is similar or slightly higher, and the RSV B type B-009-V02-2 mRNA / LNP vaccine is better. The results show good cross-protection effect of JY-B-009-V02-2 mRNA / LNP vaccine.

[0219] Example 10 Immunization boost in animals that have been infected with RSV virus

[0220] RSV virus respiratory infection is a very common respiratory infection disease, almost all people are faced with the virus infection repeatedly by the environment, except for the first infection within one year of newborn infants. For most people with a perfect immune system, re-infection is not a very serious problem, but for young children whose immune system is still developing, elderly people whose immune system is declining, and patients with immune system defects, RSV infection is often a serious threat. The purpose of this experiment is to understand whether the vaccine has a protective effect on animals that have been infected with RSV, and the degree of protection.

[0221] 10.1 Animal immunization

[0222] 10.1.1 Animals

[0223] The animals used are Balb / c mice, female, 6 weeks old. After the animals arrive at the CRO biosafety level II animal house, they will be adapted there for one week.

[0224] 10.1.2 Immunization and sampling

[0225] The time points of immunization and sampling are shown in Figure 10A.

[0226] On the first day, the pre-immune blood sample was taken from the orbit. After each blood collection, the sample was placed at 4°C for 2 hours, then centrifuged at 2500 rpm for 5 min at 4°C, the supernatant was taken and aliquoted. The serum was stored in a -80°C refrigerator.

[0227] 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 infected with 10 5 PFU / animal RSV A2; the fourth group was the PBS control.

[0228] Fourteen days after the first immunization, the first group of mice was given a second immunization, with the same type, dose and route of immunization as the first time.

[0229] Fifteen days after the second immunization, all animals were bled from the orbit, which was the blood after the first immunization or infection.

[0230] The second day after blood collection, that is, the 30th day after the mice were infected with RSV virus, the second group of mice was injected intramuscularly with 10 μg / animal JY-B-009-V02-2 mRNA / LNP vaccine as a booster.

[0231] Fourteen days later, all animals were bled from the orbit, which was the blood after the second immunization or infection.

[0232] The day after blood collection, all mice were challenged intranasally with RSV A2, 10 5 PFU / mouse. Nasal challenge.

[0233] The experiment was terminated on the fourth day post-infection. Animals were euthanized by inhalation anesthesia and the entire lung was removed and weighed according to the SOP dissection procedure.

[0234] 10.2 Elisa detection of anti-RSV antibodies

[0235] The sera collected after the first and second immunization or infection were used to detect the specific antibody content by Elisa.

[0236] 10.2.1 Coating of the antigen

[0237] The coating antigen (RSV-A2, F) was diluted in PBS to 62.5 ng / mL and used to coat the 96-well ELISA plates, 100 μl / well, 4°C, 16h.

[0238] 10.2.2 Washing and blocking

[0239] The ELISA plates with the coated antigen were removed and washed once with 300 μL / well PBST. Then, 100 μL of blocking solution (5% milk) was added and incubated at room temperature for 30 min. Subsequently, the plates were washed three times with 300 μL / well PBST.

[0240] 10.2.3 Sample dilution and addition

[0241] After the appropriate 4-fold dilution of the mouse serum samples, 100 μl was added to the ELISA plates and incubated at room temperature for 1.5 h. Then, the plates were washed three times with 300 μL / well PBST.

[0242] 10.2.4 Addition of detection antibody

[0243] The appropriate amount of detection antibody (Goat Anti-Mouse IgG, Peroxidase-Conjugated, H+L) was added to the wells according to the number of wells, diluted to 0.8 ng / mL and incubated at room temperature for 1 h. Then, the plates were washed five times with 300 μL / well PBST.

[0244] 10.2.5 Color development and termination

[0245] Mix color developing solution A (containing H202) and color developing solution B (containing TMB) at a ratio of 1:1, add 100 μL per well, and place at room temperature for 15-30 minutes. Then add 30 μL of 1 M HCL per well to stop the reaction, and then read the OD at 450 nm using an enzyme-labeled instrument.

[0246] 10.2.6 Standard curve

[0247] If absolute quantification is required, purchase anti-RSV-F antibodies, and prepare a suitable range of concentrations from low to high according to the amount of antibody protein. Perform Elisa on the sample at the same time, and plot the results with the amount of antibody protein as the abscissa and the OD450 as the ordinate.

[0248] The results are shown in Figure 10. The amount of antibody induced by two doses of immunization and the amount of antibody after one viral infection were comparable (Figure 10B). However, the administration of a booster vaccine after viral infection greatly increased the antibody content (Figure 10C), indicating a very good viral protection effect. That is, the administration of a booster immunization in the human population can greatly control RSV viral infection.

[0249] Example 11 Inhibition of RSV viral infection in the lungs of cotton rats by JY-B-009-V02-2 mRNA / LNP vaccine

[0250] The ultimate detection indicator of the vaccine is whether the specific immune response induced by the vaccine in vivo has the efficiency of protecting the host from viral infection.

[0251] 11.1 Animal model and immunization

[0252] 11.1.1 Animal model

[0253] The relatively ideal animal model for RSV is the cotton rat, because after RSV viral infection in the cotton rat, viral replication and proliferation can occur in the lungs, and the immune response and lung structure are also more similar to humans. The animal model used in this embodiment is the cotton rat.

[0254] 11.1.2 Immunization and sampling

[0255] 8-week-old female cotton rats were used to perform the experiments shown in Figures 11A and 11B in a biosafety animal room. Each operation was performed after the cotton rats were anesthetized with isoflurane.

[0256] On the first day, an orbital blood sample was taken before immunization, and the sample was placed at 4°C for 2 hours, then centrifuged at 2500 rpm for 5 min at 4°C, and the supernatant was taken and aliquoted. The serum was stored in a -80°C refrigerator.

[0257] The next day, the third group of animals were infected with RSV A2 in the nose, 10^5 / rat. Two weeks later, the first group was immunized with JY-B-009-V02-2 mRNA / LNP vaccine, intramuscularly, 60 μg / rat; the second group was immunized with JY-B-009-V02-2 mRNA / LNP vaccine and RSV A2 - #7B vaccine, each 30 μg / rat, intramuscularly. The fourth group was the control group, injected with PBS.

[0258] Three weeks later, the orbital blood was taken. The next day after blood collection, the first and second groups were each given the same vaccine as the first immunization. The third group, which had been previously infected with RSV, was given a booster injection of JY-B-009-V02-2 mRNA / LNP vaccine, 60 μg / rat, intramuscularly, to simulate the real-world scenario of universal pre-infection and post-boost.

[0259] Two weeks later, another round of blood was drawn. The next day after blood collection, the animals were challenged with RSV B18537, 5x10^5 / rat, intranasally.

[0260] Four days after the challenge, the experiment ended. After the animals were euthanized by inhalation anesthesia, the complete lungs of the cotton rats were taken and weighed according to the SOP dissection procedure.

[0261] 11.2 Lung viral load detection

[0262] 11.2.1 Preparation of lung homogenate

[0263] Prepare a cryotube and add 2 mL of 4°C pre-cooled virus protection solution; place the fresh lung tissue in the cryotube containing the virus protection solution, homogenize on the same day to avoid the impact of freeze-thaw on the experimental results. Homogenize the lung tissue under low temperature using a high-throughput tissue grinder until there are no obvious particles, and then transfer the tissue liquid to a 1.5 mL EP tube and store at -80°C.

[0264] 11.2.2 Preparation of cell plates

[0265] The cells used for virus titer determination are Hep-2 laryngeal epithelial carcinoma cells. The day before virus titration, the cells were digested and resuspended after washing, inoculated into 24-well plates at 2x10^5 / well, MEM 10% FBS complete medium, 37°C 5% CO2 incubation overnight.

[0266] 11.2.3 Virus infection

[0267] The lung homogenate was diluted according to 1:10, 1:10 2 , 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6Dilution, dilution medium is Opti-MEM, no fetal bovine serum. Then the day before the 24-well plate gently washed with PBS, each hole 100 μl diluted virus, each concentration of 3 wells. At the same time set 4 normal cell control and set 4 hole virus control group. Incubate the plate in 37℃, 5% CO2 incubator for 1 h, gently shake once every 15 min. Incubation after the hole mixed solution, add pre-prepared warm medium 500 μl / well, medium is MEM, 3% FBS, 0.75% methylcellulose. 37℃, 5% CO2 culture for 4 days.

[0268] 11.2.4 virus and cell fixation

[0269] On the fourth day of culture, discard the hole culture solution, and then wash with PBS, and then add pre-cooled 85% methanol 100 μl / well, 4℃ fixation for 1 hour. After that, discard the hole solution, and then wash with PBS.

[0270] 11.2.5 blocking and washing plate

[0271] Add 300 μL blocking solution (5% milk), room temperature, gently shake, 30 min. Then wash the plate with 500 μl / well PBST 3 times.

[0272] 11.2.6 add detection antibody

[0273] According to the number of wells, take the matching detection antibody (Anti-RSV-F IgG, H+L, HRP Conjugated), appropriate dilution, each hole 150 μL, room temperature incubation for 90 min, and then wash the plate with 500 μl / well PBST 3 times.

[0274] 11.2.7 add secondary antibody

[0275] Take the matching secondary antibody, 1:1000 to 1:5000 appropriate dilution, each hole 150 μL, room temperature incubation for 60 min, and then wash the plate with 500 μl / well PBST 5 times.

[0276] 11.2.8 color and stop

[0277] Add 150 μL of TrueBlue™ Peroxidase Substrate to each well, gently shake, and virus positive plaque will appear soon. Then wash the reaction solution with water. Dry in the dark. The next day, count the virus plaque.

[0278] Results As shown in Figure 11, JY-B-009-V02-2 mRNA / LNP vaccine can effectively control the amplification of RSV virus in the lungs. Although there are differences in the induced antibody titers, there is no difference in lung virus load in the three cases of immunization dose of 60 μg / animal, 30+30 μg / animal of bivalent vaccine, and post-infection booster. 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 virus and virus that has been neutralized and has no infectivity; while the immunoplaque method detects only infectious live virus.

[0279] Example 12 Comparison of the immunization effect of JY-B-009-V02-2 mRNA / LNP vaccine and Pfizer vaccine

[0280] 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. With ABRYSVO, the world's leading vaccine, as a control, we compared the immunization effect of JY-B-009-V02-2 mRNA / LNP vaccine and Pfizer vaccine.

[0281] 12.1 Animal immunization

[0282] 12.1.1 Animals

[0283] The animals used were Balb / c mice, female, 6 weeks old. After the animals arrived at the CRO biosafety level II animal house, they would be adapted there for one week.

[0284] 12.1.2 Immunization and sampling

[0285] The time points of immunization and sampling are shown in Figure 12A.

[0286] 12.2 Immunization and sampling

[0287] The animals used were Balb / c mice, female, 6 weeks old. After the animals arrived at the CRO biosafety level II animal house, they would be adapted there for one week.

[0288] On the first day, the pre-immune blood sample was taken from the orbit, the sample was placed at 4°C for 2 hours, then centrifuged at 2500 rpm, 5 min, 4°C, the supernatant was taken and aliquoted. The serum was stored at -80°C in a low temperature refrigerator.

[0289] On the second day, the third group of animals was infected with RSV A2, 5.5x10^4 / mouse

[0290] Two weeks later, the first group was immunized with JY-B-009-V02-2 mRNA / LNP vaccine, intramuscularly, 20 μg / mouse; the second group was immunized with JY-B-009-V02-2 mRNA / LNP vaccine and RSV A2 - #7B vaccine, 10 μg / mouse each, intramuscularly; the fourth group was injected with ABRYSVO, 10 μg / mouse, intramuscularly; the fifth group was injected with ABRYSVO, 20 μg / mouse, intramuscularly; the sixth group was the control group, injected with PBS.

[0291] Two weeks later, the animals were bled from the orbital plexus. This is the post-bleed sample.

[0292] The next day after the bleed, the first, second, fourth, and fifth groups were each given the same vaccine as the first immunization. The third group, which was previously infected with RSV, was given a booster injection of JY-B-009-V02-2 mRNA / LNP vaccine, 20 μg / mouse, intramuscularly, to mimic the real-world scenario of a common infection followed by a booster.

[0293] Two weeks later, the animals were bled again. This is the post-bleed sample.

[0294] The next day after the bleed, the animals were challenged with RSV B-18537, 6.8x10^4 / mouse, intranasally.

[0295] Four days after the challenge, the experiment was terminated. The animals were euthanized by inhalation anesthesia, and the entire lung was removed and weighed according to the SOP dissection procedure.

[0296] 12.3 Elisa detection of anti-RSV antibodies

[0297] The sera from the first and second immunizations or infections were each tested for specific antibody content using Elisa.

[0298] 12.3.1 Coating of antigen

[0299] The coating antigen, RSV-A2, F, was diluted with PBS to 62.5 ng / mL, and the antigen was coated in a 96-well enzyme-linked immunosorbent assay (ELISA) plate, 100 μl per well, at 4°C for 16 hours.

[0300] 12.3.2 Washing and blocking

[0301] The enzyme-linked immunosorbent assay (ELISA) plate with coated antigen was removed, and the plate was washed once with 300 μL / well PBST. Then, 100 μL of blocking solution (5% milk) was added, and the plate was incubated at room temperature for 30 minutes. Next, the plate was washed three times with 300 μL / well PBST.

[0302] 12.3.3 Sample dilution and addition

[0303] After the serum samples of mice were diluted by a suitable gradient of 4 times in decreasing order, 100 μl was added to the enzyme-labeled plate, and after incubation at room temperature for 1.5 h, the plate was washed with 300 μL / well PBST for 3 times.

[0304] 12.3.4 Detection antibody

[0305] According to the number of sample wells, the appropriate amount of detection antibody (Goat Anti-Mouse IgG, Peroxidase - Conjugated, H+L) was taken and diluted to 0.8 ng / mL, and then 60 μL was added to each well, and after incubation at room temperature for 1 h, the plate was washed with 300 μL / well PBST for 5 times.

[0306] 12.3.5 Color development and termination

[0307] Color developing solution A (containing H202) and color developing solution B (containing TMB) were mixed according to the ratio of 1:1, 100 μL was added to each well, and after standing at room temperature for 15-30 minutes, 30 μL of 1 M HCL termination solution was added to each well, and then the enzyme-labeled instrument was read at 450 nm.

[0308] 12.3.6. Standard curve

[0309] If absolute quantification is required, the purchased anti-RSV-F antibody is prepared into a suitable range of low to high concentrations according to the amount of antibody protein, and Elisa is performed at the same time as the sample, and the results are plotted with the amount of antibody protein as the abscissa and OD450 as the ordinate.

[0310] The results are shown in Figure 12B. 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 to and higher than 10 or 20 μg / mouse of the protein vaccine ABRYSVO, and showed good cross-protection characteristics because the coating protein of Elisa was RSV-A2 F protein. It is very meaningful that for mice previously infected with RSV A2, the antibody titer greatly increased after five weeks of infection when JY-B-009-V02-2 mRNA / LNP vaccine was given as a booster, which is likely to indicate that for the general population who have been previously infected with RSV virus, JY-B-009-V02-2 mRNA / LNP vaccine as a booster will have very good protective effect.

[0311] The present application is illustrated by the above examples, but the present application is not limited to the above process steps, i.e. it is not meant that the present application must rely on the above process steps to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of the raw materials selected by the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the scope of protection and disclosure of the present application.

Claims

1. A recombinant human syncytial virus mRNA, characterized in that The coding sequence of the mRNA is shown in SEQ ID NO:

1.

2. An expression vector, characterized in that The expression vector expresses the mRNA molecule according to claim 1.

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

4. A recombinant protein, characterized in that The recombinant protein is obtained by translating the mRNA according to claim 1.

5. Nucleotide, characterized in that The nucleotide sequence encodes the recombinant protein according to claim 4.

6. A plasmid, characterized in that The plasmid comprises the nucleotide sequence of claim 5.

7. mRNA / LNP complex, characterized in that Comprising the mRNA molecule and lipid nanoparticles according to claim 1.

8. A method for preparing an mRNA / LNP complex, characterized in that: The steps include: 1) dissolving the mRNA according to claim 1 in acetate buffer; 2) Prepare a mixed solution of ionizable cations, DSPC, cholesterol, and PEG-2000 in appropriate proportions; 3) The solution in step 2) is mixed with the buffer in step 1), and diluted to increase the pH value to obtain an mRNA / LNP complex.

9. mRNA / LNP complex, characterized in that The composite is prepared according to the method of claim 8.

10. A vaccine composition, characterized in that The vaccine composition comprises the mRNA of claim 1 and / or the expression vector of claim 2 and / or the recombinant protein of claim 4 and / or the nucleotide of claim 5 and / or the plasmid of claim 6 and / or the mRNA / LNP complex of claim 7.

11. Use of the mRNA according to claim 1 and / or the expression vector according to claim 2 and / or the recombinant protein according to claim 4 and / or the nucleotide according to claim 5 and / or the plasmid according to claim 6 and / or the mRNA / LNP complex according to claim 7 in preparing a human syncytial virus vaccine composition.

12. Use of the mRNA according to claim 1 and / or the expression vector according to claim 2 and / or the recombinant protein according to claim 4 and / or the nucleotide according to claim 5 and / or the plasmid according to claim 6 and / or the mRNA / LNP complex according to claim 7 in the preparation of a medicament for treating diseases caused by syncytial virus infection.

13. The use according to claim 12, characterized in that The disease is a respiratory disease, and the respiratory disease is pneumonia, bronchiolitis or a cold.

14. Use of the mRNA according to claim 1 and / or the expression vector according to claim 2 and / or the recombinant protein according to claim 4 and / or the nucleotide according to claim 5 and / or the plasmid according to claim 6 and / or the mRNA / LNP complex according to claim 7 in the preparation of a medicament for treating a patient suffering from a disease caused by syncytial virus infection.

15. The use according to claim 14, characterized in that The disease is a respiratory disease, and the respiratory disease is pneumonia, bronchiolitis or a cold.

16. The use according to any one of claims 14-15, characterized in that The patient had previously been infected with syncytial virus.

17. The use according to claim 16, characterized in that The infection times are one or more times.

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