Influenza virus vaccine as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an influenza virus vaccine as well as a preparation method and application thereof. According to the influenza virus vaccine, M15-HA is adopted as a target antigen of the influenza mRNA vaccine, nucleic acid modified M15-HAmRNA is prepared, then lipid nanoparticles are used for wrapping, the influenza virus vaccine is prepared, the lipid nanoparticles comprise ionizable lipid, auxiliary phospholipid, cholesterol and PEG lipid, and the molar ratio of the ionizable lipid to the auxiliary phospholipid to the cholesterol to the PEG lipid is 50: (10-15): (38-40): (1.5-2.5). Compared with the prior art, the influenza vaccine provided by the invention has the advantages of high antigen expression, higher long-acting immune protection effect, higher particle size uniformity, higher encapsulation efficiency and better targeted delivery efficiency and tissue permeability.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to an influenza virus vaccine, a preparation method thereof, and an application thereof. Background Art

[0002] A / Michigan / 45 / 2015 (H1N1) is a strain of the H1N1 subtype of influenza A virus, which was isolated in Michigan, USA in 2015. It belongs to the evolutionary branch of the 2009 H1N1 pandemic virus (pdm09). Antigenic drift occurred in its HA (hemagglutinin) protein (such as mutations at sites K163Q, S185T, etc.), resulting in a decrease in the protective efficacy of the previous vaccine strain (A / California / 7 / 2009). The World Health Organization listed A / Michigan / 45 / 2015 (H1N1) as a recommended component of the influenza vaccine in the Northern Hemisphere for 2017 - 2018, replacing the previous A / California / 7 / 2009 strain. This adjustment is based on data from the global influenza surveillance network, indicating that this strain has become the dominant epidemic strain. This strain has been widely used in research on influenza virus evolution, immune escape mechanisms, and broad-spectrum vaccines, and the conserved region of its HA stem has become a target for universal vaccine design.

[0003] The article "Study on the Preparation and Booster Immunization Strategy of mRNA Vaccine Based on Hemagglutinin of Influenza A Virus H1N1 Subtype" by Shen Haiqian et al. disclosed the mRNA vaccine of this strain, and constructed the FlucmRNA-lipid nanoparticle (LNP) vaccine. The mRNA vaccine can directly direct the production of viral antigens in the cells of the vaccinated person, which mimics part of the natural infection process, thereby triggering a stronger and more persistent immune response. mRNA itself is non-infectious and will not integrate into the human genome, theoretically reducing the risk of long-term side effects. Lipid nanoparticles, as carriers, have been proven to be relatively safe and effective drug delivery tools, showing acceptable safety characteristics in clinical trials.

[0004] However, during the R & D process, the applicant found that in the above-mentioned scheme, the antibody neutralization activity of low-dose mRNA-LNP significantly decayed after the secondary immunization, resulting in insufficient antigen expression and affecting long-term immune protection; at the same time, the particle size uniformity was insufficient, leading to a reduction in the targeted delivery efficiency and tissue permeability. Summary of the Invention

[0005] Aiming at the above problems, the purpose of the present invention is to provide an influenza virus vaccine, a preparation method thereof, and an application thereof.

[0006] To achieve the above purpose, the present invention provides the following technical solutions: In the first aspect of the present invention, an influenza virus vaccine is provided. The influenza virus vaccine uses M15-HA as the target antigen of the influenza mRNA vaccine, prepares nucleic acid-modified M15-HA mRNA, and then wraps it with lipid nanoparticles. The lipid nanoparticles include ionizable lipids, helper phospholipids, cholesterol, and PEG lipids.

[0007] Preferably, the molar ratio of the ionizable lipid, helper phospholipid, cholesterol, and PEG lipid is 50:(10 - 15):(38 - 40):(1.5 - 2.5).

[0008] More preferably, the molar ratio of the ionizable lipid, helper phospholipid, cholesterol, and PEG lipid is 50:10:38.5:1.5.

[0009] Preferably, the ionizable lipid is selected from SM-102.

[0010] Preferably, the helper phospholipid is selected from DOPE.

[0011] Preferably, the PEG lipid includes short-chain PEG and long-chain PEG.

[0012] Preferably, the short-chain PEG is DMG-PEG2000.

[0013] Preferably, the long-chain PEG is ALC-0159.

[0014] Preferably, the molar ratio of the short-chain PEG and the long-chain PEG is 1:(1.3 - 1.6).

[0015] More preferably, the molar ratio of the short-chain PEG and the long-chain PEG is 1:1.5.

[0016] Preferably, the preparation method of the mRNA is as follows: Using pUC57-Fluc and pUC57-M15HA plasmids as templates, T7 RNA polymerase is used to synthesize mRNA precursors by in vitro transcription, and a 5' cap structure is added to the mRNA precursors through enzymatic reactions.

[0017] In the second aspect of the present invention, a preparation method of an influenza virus vaccine is provided, including the following steps: Dissolve M15-HA mRNA in sodium citrate buffer to obtain an aqueous phase; dissolve the lipid nanoparticles in ethanol to obtain an ethanol phase; mix the ethanol phase and the aqueous phase to obtain M15-HA-LNP, and replace the mRNA-LNP solvent with phosphate buffer by dialysis method, and store at -80°C.

[0018] Preferably, the concentration of M15-HA mRNA in the sodium citrate buffer is 80 - 100 μg / mL.

[0019] Preferably, the concentration of M15-HAmRNA in sodium citrate buffer is 90 μg / mL.

[0020] Preferably, the concentration of LNP in ethanol is 3 - 5 mg / mL.

[0021] Preferably, the concentration of LNP in ethanol is 4 mg / mL.

[0022] Preferably, the pH of the sodium citrate buffer is 4.

[0023] Preferably, the ethanol phase and the aqueous phase are mixed at a volume ratio of 1:(3.2 - 3.5), the flow rate of the aqueous phase is 12 - 15 mL / min, and the flow rate of the ethanol phase is 3 - 5 mL / min.

[0024] Preferably, the ethanol phase and the aqueous phase are mixed at a volume ratio of 1:3.3, the flow rate of the aqueous phase is 13 mL / min, and the flow rate of the ethanol phase is 4 mL / min.

[0025] The third aspect of the present invention provides an application of an influenza virus vaccine, which is used in the preparation of a drug for preventing and / or treating influenza A virus or its related diseases.

[0026] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: The present invention provides a novel influenza virus vaccine. On the basis of the prior art, the particle distribution uniformity, encapsulation efficiency, immune response intensity and persistence, and particle size are optimized and designed. This not only improves the overall quality and application effect of the vaccine, but also provides solid technical support for the wide application of mRNA vaccines in the clinical and public health fields.

[0027] (1) By optimizing the preparation process of lipid nanoparticles (LNP), the present invention significantly reduces the polydispersity coefficient of the particle size distribution and achieves a more uniform LNP system. A low PDI means a high degree of particle size consistency, which is beneficial to improving the delivery efficiency of mRNA in vivo and the consistency of cell uptake. In practical applications, this highly uniform LNP system can reduce the differences in immune responses among individuals, improve the stability and repeatability of the vaccine, and thus enhance the overall vaccination effect. In addition, the uniform particle distribution helps to improve the physical stability of the preparation, reduce the risk of aggregation or precipitation during storage and transportation, extend the shelf life, and has important practical significance.

[0028] (2) By regulating the component ratio of the LNP, the present invention effectively improves the encapsulation efficiency of mRNA. A high encapsulation rate means that more mRNA is successfully encapsulated inside the LNP, avoiding its degradation or loss in the in vitro environment, thereby ensuring that it can exert its maximum efficacy after entering the body. This improvement can not only significantly enhance the utilization rate of the effective dose of the vaccine, but also reduce raw material loss and production costs, which is particularly important in large-scale vaccine production. At the same time, a higher encapsulation rate also helps to reduce non-specific immune stimulation caused by unencapsulated mRNA, improve the safety and tolerance of the vaccine, and lay a foundation for industrial promotion.

[0029] (3) By optimizing the LNP delivery system, the present invention significantly enhances the humoral and cellular immune responses induced by the vaccine. This vaccine can induce a high-level and stable geometric mean titer (GMT) of serum antibodies and maintain a high antibody level for a long time, indicating its good ability to form immune memory. This strong and persistent immune response is particularly important for coping with the rapid mutation of influenza viruses, can provide longer-term protection for vaccinated individuals, and reduce the need for frequent vaccinations.

[0030] (4) The present invention makes fine adjustments in the control of the LNP particle size, so that this range can not only meet the requirements of efficient cell uptake, but also ensure good physicochemical stability. This makes the immune activation of the vaccine more targeted. In practical applications, this optimized design helps to improve the bioavailability and safety of the vaccine, and provides an important reference for the research and development of future multivalent or multi-target mRNA vaccines. Specific Embodiments

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0032] The preparation of M15-HA mRNA in Examples 1-3 and Comparative Examples 1-5 of the present invention is the same as that in "Research on the Preparation and Booster Immunization Strategy of mRNA Vaccine Based on Hemagglutinin of Influenza A Virus Subtype H1N1", and will not be elaborated here.

[0033] The plasmids pUC57-Fluc and pUC57-M15-HA used in the present invention are both from the Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine.

[0034] The following raw materials of the present invention are all commercially available products: SM-102, purchased from MedChemExpress (MCE).

[0035] DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0036] DMG-PEG2000 was purchased from MedChemExpress (MCE).

[0037] ALC-0159 was purchased from MedChemExpress (MCE).

[0038] Cholesterol, CAS Registry Number: 57-88-5.

[0039] Example 1: This example provides an influenza virus vaccine. The influenza virus vaccine uses M15-HA as the target antigen of the influenza mRNA vaccine, prepares nucleic acid-modified M15-HA mRNA, and then uses lipid nanoparticles for encapsulation. The lipid nanoparticles include ionizable lipids, helper phospholipids, cholesterol, and PEG lipids; the molar ratio of the ionizable lipid, helper phospholipid, cholesterol, and PEG lipid is 50:10:38.5:1.5. The ionizable lipid is SM-102. The helper phospholipid is DOPE. The PEG lipid includes short-chain PEG and long-chain PEG; the short-chain PEG is DMG-PEG2000; the long-chain PEG is ALC-0159; the molar ratio of the short-chain PEG and long-chain PEG is 1:1.5.

[0040] The preparation method of the above influenza virus vaccine includes the following steps: Dissolve M15-HA mRNA in sodium citrate buffer with a pH of 4 and a concentration of 90 μg / mL of M15-HA mRNA in the sodium citrate buffer to obtain an aqueous phase; dissolve the lipid nanoparticles in ethanol with a concentration of 4 mg / mL of LNP in ethanol to obtain an ethanol phase; mix the ethanol phase and the aqueous phase at a volume ratio of 1:3.3, with the flow rate of the aqueous phase being 13 mL / min and the flow rate of the ethanol phase being 4 mL / min to obtain M15-HA-LNP, and replace the mRNA-LNP solvent with phosphate buffer by dialysis and store at -80 °C.

[0041] Example 2: This example provides an influenza virus vaccine. The influenza virus vaccine uses M15-HA as the target antigen of the influenza mRNA vaccine. Modified M15-HA mRNA is prepared and then encapsulated with lipid nanoparticles. The lipid nanoparticles include ionizable lipids, helper phospholipids, cholesterol, and PEG lipids. The molar ratio of the ionizable lipids, helper phospholipids, cholesterol, and PEG lipids is 50:10:40:1.5. The ionizable lipid is SM-102. The helper phospholipid is DOPE. The PEG lipid includes short-chain PEG and long-chain PEG. The short-chain PEG is DMG-PEG2000. The long-chain PEG is ALC-0159. The molar ratio of the short-chain PEG and long-chain PEG is 1:1.5.

[0042] The preparation method of the above influenza virus vaccine includes the following steps: Dissolve M15-HA mRNA in sodium citrate buffer with a pH of 4, and the concentration of M15-HA mRNA in the sodium citrate buffer is 90 μg / mL to obtain an aqueous phase. Dissolve the lipid nanoparticles in ethanol with a concentration of 4 mg / mL of LNP in ethanol to obtain an ethanol phase. Mix the ethanol phase and the aqueous phase at a volume ratio of 1:3.3, with the flow rate of the aqueous phase being 13 mL / min and the flow rate of the ethanol phase being 4 mL / min to obtain M15-HA-LNP. Replace the mRNA-LNP solvent with phosphate buffer by dialysis and store at -80°C.

[0043] Example 3: This example provides an influenza virus vaccine. The influenza virus vaccine uses M15-HA as the target antigen of the influenza mRNA vaccine. Modified M15-HA mRNA is prepared and then encapsulated with lipid nanoparticles. The lipid nanoparticles include ionizable lipids, helper phospholipids, cholesterol, and PEG lipids. The molar ratio of the ionizable lipids, helper phospholipids, cholesterol, and PEG lipids is 50:15:38:2.5. The ionizable lipid is SM-102. The helper phospholipid is DOPE. The PEG lipid includes short-chain PEG and long-chain PEG. The short-chain PEG is DMG-PEG2000. The long-chain PEG is ALC-0159. The molar ratio of the short-chain PEG and long-chain PEG is 1:1.5.

[0044] The preparation method of the above influenza virus vaccine comprises the following steps: dissolving M15-HA mRNA in sodium citrate buffer solution with a pH of 4 and a concentration of 90 μg / mL of M15-HA mRNA in the sodium citrate buffer solution to obtain an aqueous phase; dissolving lipid nanoparticles in ethanol with a concentration of 4 mg / mL of LNP in the ethanol to obtain an ethanol phase; mixing the ethanol phase and the aqueous phase at a volume ratio of 1:3.3, with the flow rate of the aqueous phase being 13 mL / min and the flow rate of the ethanol phase being 4 mL / min to obtain M15-HA-LNP, and replacing the mRNA-LNP solvent with phosphate buffer solution by dialysis method, and storing at -80 °C.

[0045] Comparative Example 1 The difference between this comparative example and Example 1 is: different lipid nanoparticles.

[0046] The lipid nanoparticles used in this comparative example are specifically: the optimal formulation parameters of LNP in the article "Study on the Preparation and Boosting Immunization Strategy of mRNA Vaccine Based on Hemagglutinin of Influenza A Virus Subtype H1N1": molar ratio 46.3% ALC-0315, 1.6% ALC-0159, 9.4% DSPC and 42.7% cholesterol.

[0047] Comparative Example 2 The difference between this comparative example and Example 1 is: the molar ratio of the ionizable lipid, the helper phospholipid, the cholesterol and the PEG lipid is 50:8:42:1.

[0048] Comparative Example 3 The difference between this comparative example and Example 1 is: the lipid nanoparticles comprise an ionizable lipid, a helper phospholipid and cholesterol; the molar ratio of the ionizable lipid, the helper phospholipid and the cholesterol is 50:10:38.5.

[0049] Comparative Example 4 The difference between this comparative example and Example 1 is: replacing the helper phospholipid DOPE with: the helper phospholipid DSPC.

[0050] Comparative Example 5 The difference between this comparative example and Example 1 is: the molar ratio of the short-chain PEG and the long-chain PEG is 1.5:1.

[0051] Performance Test Perform performance tests on M15-HA mRNA-LNP prepared in Examples 1-3 and Comparative Examples 1-5.

[0052] 1. Particle size and PDI: Use dynamic light scattering (DLS).

[0053] 2. Determine the encapsulation efficiency by RiboGreen fluorescence method.

[0054] 3. Immunogenicity: The immunogenicity of M15-HAmRNA-LNP was evaluated in female BALB / c mice. The immunization protocol was the same as that in Figure 4A of "Study on the Preparation and Boosting Immunization Strategy of mRNA Vaccine Based on Hemagglutinin of Influenza A Virus Subtype H1N1". The dosage used was: 1 μg in terms of the amount of mRNA in the influenza virus vaccine M15-HAmRNA-LNP. Two immunizations were carried out on day 0 and day 21. The levels of M15-HA specific IgG in serum were evaluated at days 0, 14, 35, and 49, and the geometric mean titer (GMT) of antibodies in the immunized group was determined.

[0055] The results are shown in Table 1.

[0056] Table 1 Performance Test Results As can be seen from Table 1, the average particle size of Examples 1-3 matches the size of virus particles, enhancing cell uptake. The PDI is less than 0.1, the dispersion is more uniform, and the encapsulation efficiency is greater than 95%. At the same time, with a dosage of 1 μg, continuous antigen stimulation can be achieved after the second injection, and the antigen levels at 35 days and 49 days remain unchanged. This shows that the lipid nanoparticles in the influenza virus vaccine of the present invention can achieve persistent infection, maintain the long-term presence of antigens in the body. After boosting immunization, memory B cells and follicular helper T cells are activated, continuously generating plasma cells through the germinal center reaction, and maintaining antibody levels. The effect is significantly better than that of the prior art in Comparative Example 1.

[0057] In Comparative Example 2, the composition and ratio of the lipid nanoparticles were changed, resulting in a decrease in particle size and poorer particle homogeneity. Under these conditions, a dosage of 1 μg was not sufficient to induce enough long-lasting plasma cells, resulting in insufficient antibody maintenance ability and a decrease at 49 days.

[0058] In Comparative Example 3, PEG was not used, and the lipid particle size increased significantly, larger than the size of virus particles, which was not conducive to cell uptake. The absence of PEG led to particle aggregation and a decrease in stability.

[0059] In Comparative Example 4, DOPE was replaced by DSPC, resulting in an increase in particle size. The high membrane fluidity of DOPE used in the examples can promote the fusion of LNP with the endosome membrane and help mRNA escape into the cytoplasm; while the rigid structure of DSPC will reduce the efficiency of this process.

[0060] In Comparative Example 5, the ratio of short-chain PEG to long-chain PEG was different. Compared with Example 1, the GMT of the plateau period at 35 days decreased.

[0061] In summary, the average particle sizes of Examples 1-3 of the present invention are concentrated in the range of 74.6-76.4 nm, the PDI values are 0.091-0.096, the particle sizes are moderate and the distribution is highly uniform. Examples 1-3 of the present invention achieve a balance between particle size and uniformity, taking into account both delivery efficiency and stability. The higher encapsulation efficiency of the present invention means that more mRNA is effectively encapsulated, reducing the waste and potential toxicity of free mRNA. The GMT values of Examples 1-3 of the present invention are higher than those of Comparative Examples 1-5, indicating stronger initial immune activation ability and stability. The GMT of Comparative Examples 1-4 decreased significantly, and the GMT of Comparative Example 5 remained at 67,963 at 49 days, but was still lower than that of Example 1. In the prior art of Comparative Example 1, the particle size is significantly different from that of virus particles, the PDI is not ideal, the encapsulation efficiency is low, and the GMT decreased significantly at 49 days, indicating that PEG lipids are crucial for stability and long-term immunity.

[0062] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An influenza virus vaccine, characterized in that, The influenza virus vaccine uses M15-HA as the target antigen of the influenza mRNA vaccine. Prepare M15-HA mRNA modified by nucleic acid, and then wrap it with lipid nanoparticles. The lipid nanoparticles include ionizable lipids, helper phospholipids, cholesterol, and PEG lipids; the molar ratio of the ionizable lipids, helper phospholipids, cholesterol, and PEG lipids is 50:(10-15):(38-40):(1.5-2.5); the ionizable lipid is selected from SM-102; the helper phospholipid is selected from DOPE; the PEG lipid includes short-chain PEG and long-chain PEG.

2. The influenza virus vaccine according to claim 1, wherein The short-chain PEG is DMG-PEG2000.

3. The influenza virus vaccine according to claim 1, characterized in that, The long-chain PEG is ALC-0159.

4. The influenza virus vaccine according to claim 1, characterized in that, The molar ratio of the short-chain PEG and the long-chain PEG is 1:(1.3-1.6).

5. A method for preparing the influenza virus vaccine according to any one of claims 1-4, characterized in that, It includes the following steps: dissolve M15-HA mRNA in sodium citrate buffer to obtain an aqueous phase; dissolve the lipid nanoparticles in ethanol to obtain an ethanol phase; mix the ethanol phase and the aqueous phase to obtain M15-HA-LNP, and replace the mRNA-LNP solvent with phosphate buffer by dialysis method and store it.

6. Use of the influenza virus vaccine according to any one of claims 1 to 4, characterized in that, The application of the influenza virus vaccine in the preparation of a drug for preventing and / or treating influenza A virus or its related diseases.

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