MRNA (messenger ribonucleic acid) vaccine composition for preventing enterovirus A71 infection as well as preparation method and application of mRNA vaccine composition

Abstract

The present invention relates to an mRNA vaccine composition for the prevention of enterovirus A71 (EV-A71) infection. The composition comprises an mRNA encoding the VP1 protein of EV-A71 and lipid nanoparticles (LNPs) comprising specific cationic lipids. Compared with the conventional cationic lipid system, the imidazopyridyl cationic lipid A5 with strong membrane fusion capability is adopted, so that obvious neutralizing antibody and cellular immune response can be induced under single-time and low-dose immune conditions, and excellent safety and children applicability are shown.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an mRNA vaccine composition for preventing enterovirus A71 (EV-A71) infection, its preparation method and application. Background Technology

[0002] Enterovirus type A 71 (EV-A71) is a type of human enterovirus. EV-A71 infection can cause a variety of diseases, with hand, foot and mouth disease (HFMD) being the most common. Although HFMD is a syndrome caused by multiple pathogens (coxsackievirus A16 (CV-A16) is also one of the major causative agents), severe HFMD and related deaths are mainly caused by EV-A71 infection. Since 2007, EV-A71-associated HFMD has been prevalent among infants and young children in my country and Southeast Asia, with a high incidence rate and a certain percentage of deaths among affected children.

[0003] Because EV-A71 virus is primarily transmitted through contact, children are generally susceptible and have a high rate of asymptomatic infection, making routine hygiene and preventative measures difficult to implement and less effective. Inactivated vaccines are available in my country, and the Chinese Center for Disease Control and Prevention recommends EV-A71 vaccination for susceptible children aged 6 months and older, with earlier vaccination being better; it encourages completion of the vaccination schedule before 12 months of age to ensure early protection. However, vaccine production is costly and time-consuming, requiring two doses to achieve the desired protective effect. Immunogenicity may weaken after vaccination for a period, leading to insufficient stimulation of cellular immune responses. Furthermore, increasing evidence suggests that intra- and inter-type recombination and mutations in EV-A71 strains drive rapid viral evolution, posing a potential challenge to inactivated vaccines.

[0004] mRNA vaccines have become a hot topic in vaccine development due to their strong immunogenicity, lack of adjuvant requirements, high safety, and rapid development speed. However, no mRNA prophylactic vaccine against EV-A71 for hand-foot-mouth disease has yet been developed. Developing a low-dose, single-dose EV-A71 mRNA vaccine that can induce a protective immune response and has excellent safety is of great significance for preventing hand-foot-mouth disease in young children. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an EV-A71 mRNA vaccine composition. This composition, through an optimized lipid nanoparticle delivery system, achieves a comprehensive effect of highly efficient single-dose immunization and superior safety. The mRNA encodes the EV-A71 VP1 protein, comprising a cap structure, optimized 5' / 3' UTR, poly(A) tail, and N1-methylpseudouridine modification; the mRNA is encapsulated in an LNP composed of cholesterol, DSPC, PEG-lipids, and cationic lipid A5. A single immunization with this composition can induce protective neutralizing antibodies and a significant cellular immune response, and significantly reduce inflammatory responses.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the invention, an mRNA vaccine composition for preventing enterovirus A71 infection is provided, comprising: (a) An mRNA molecule encoding the EV-A71 VP1 protein, the mRNA comprising a cap structure, a 5' untranslated region and a 3' untranslated region, a sequence encoding VP1, and a PolyA tail of 80-150 nucleotides. (b) Lipid nanoparticles encapsulating the mRNA.

[0007] Furthermore, the mRNA is one of the sequences SEQ ID NO.6-7.

[0008] Furthermore, the nucleotide sequence encoding the EV-71 viral VP1 protein further includes: A sequence based on the above nucleotide sequence by substitution, deletion, addition and / or replacement of one or more bases; or a sequence with more than 90% homology to the above nucleotide sequence.

[0009] Furthermore, the vaccine also includes lipid nanoparticles encapsulating the mRNA.

[0010] Furthermore, the lipid nanoparticles comprise: ionizable cationic lipids, distearate phosphatidylcholine, polyethylene glycol lipids, and cholesterol. The molar percentage of the ionizable lipids is 20%-60%, the molar percentage of the auxiliary lipids is 3%-15%, the molar percentage of the sterols is 20%-75%, and the molar percentage of the polymer is 0%-5%; the sum of the molar percentages of the ionizable lipids, auxiliary lipids, sterols, and polymers is 100%.

[0011] Furthermore, the ionizable cationic lipid is an imidazopyridyl lipid compound selected from the following compounds, and the specific preparation method is described in patent CN115232128B:

[0012] (I)

[0013] (II)

[0014] (III)

[0015] (IV) The compound I is abbreviated as cationic lipid molecule A5, the compound II as cationic lipid molecule A10, the compound III as cationic lipid molecule A20, and the compound IV as cationic lipid molecule A23.

[0016] In a second aspect of the invention, a method for preparing the mRNA vaccine is provided, the method comprising: S1. Prepare the mRNA molecule by in vitro transcription, wherein the in vitro transcription includes capping the mRNA and introducing the 5'UTR, 3'UTR and polyA tail, and selectively using modified nucleoside triphosphates to replace the natural nucleoside triphosphates during the transcription process; S2. Prepare a lipid mixture; S3. Mix the aqueous mRNA solution obtained in step S1 with the lipid mixture obtained in step S2 by microfluidic mixing or an equivalent rapid mixing method to form an LNP encapsulating the mRNA; S4. Dilute, concentrate and replace the LNP with buffer to obtain the final formulation.

[0017] As one specific implementation method, the method includes: The nucleotide sequence shown in SEQ ID NO. 6 or SEQ ID NO. 7 is synthesized and then ligated into a plasmid to obtain a recombinant plasmid; The recombinant plasmid was linearized using restriction endonucleases to obtain linearized DNA; mRNA was prepared by transcription using the linearized DNA as a template. A5 lipid mixture solution was obtained; The mRNA is mixed with the lipid mixture, diluted, ultrafiltered, and concentrated to obtain the mRNA vaccine.

[0018] Furthermore, the plasmid is pUC57-Kan.

[0019] Furthermore, the restriction endonucleases are BglI and MluI.

[0020] Furthermore, the lipid mixture solution is a solution obtained by mixing ionizable cationic lipids, neutral lipids, steroidal compounds, polyethylene glycol lipids, and organic solvents.

[0021] Furthermore, the volume ratio of the mRNA to the lipid mixture is (2-4):1, preferably 3:1.

[0022] In a third aspect of the invention, the use of the mRNA vaccine in the preparation of a vaccine for the prevention of EV-71 infection is provided.

[0023] The mRNA vaccine of the present invention can significantly increase the titer of VP1 neutralizing antibodies against EV-71 and the specific cellular immune response produced in vivo.

[0024] The present invention has the following advantages and beneficial effects: 1. The mRNA vaccine of the present invention optimizes the codons by linking the VP1 antigen with a signal peptide sequence that promotes extracellular secretion, and then selects a suitable promoter and 5'... UTR, 3' UTR and 3' The PolyA terminal combines to form complete mRNA nucleic acid.

[0025] 2. This invention improves the composition of LNP by using an imidazopyridyl lipid compound with better membrane fusion ability and lower cytotoxicity to prepare an mRNA vaccine, thereby enhancing its targeting in vivo and activation efficiency of immune cells. After immunizing mice, it produces a strong and stable immune effect, especially in terms of the level of neutralizing antibodies and cellular immunity, without affecting the normal growth of animals.

[0026] 3. The mRNA-LNP vaccine of this invention can induce a neutralizing antibody titer (PRNT) at a single dose of 5 µg. 50 The ratio of IL-6 to alanine aminotransferase (ALT) was ≥1:1000, and the levels of IL-6 and ALT were significantly lower than those of the inactivated vaccine. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 For mRNA integrity analysis (capillary electrophoresis).

[0029] Figure 2 Particle size and PDI analysis of mRNA-LNP.

[0030] Figure 3 The titers of neutralizing antibodies after immunization with mRNA-LNP containing different cationic lipids are given.

[0031] Figure 4 The level of cellular immunity after immunization with mRNA-LNP containing different cationic lipids.

[0032] Figure 5 To assess the in vivo protective effect of neutralizing antibodies against mRNA-LNP containing different cationic lipids after immunization. Detailed Implementation

[0033] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0034] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0035] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0036] The overall concept of this invention is as follows: According to one embodiment of the present invention, an EV-71 mRNA vaccine is provided, comprising mRNA expressing VP1 protein and lipid nanoparticles containing cationic lipid molecule A5: 1. As shown in the data of Example 1 of the present invention, the improvement in expression level of mRNA (without LNP delivery) is limited by optimization alone, and the neutralizing antibody titer after naked mRNA immunization is <1:32 (Table 2), which proves that single optimization cannot solve the fundamental problem.

[0037] 2. The "non-obviousness" of the combination and the synergistic effect The binding of A5 lipids to VP1 mRNA in the EV-A71 vaccine produced a synergistic effect far exceeding expectations: (1) Breakthrough in immune response: Highly effective protection achieved with a single low dose Comparison data: Existing technology (SM-102-LNP): Neutralizing antibody titer after a single 5μg immunization is 682.7±264.4; This invention (A5-LNP): Under the same conditions, the titer was increased to 938.7±209.0 (p<0.05), and the challenge protection rate increased from 80% to 100%. Figure 5 ); Unexpectedly, A5 is structurally similar to A10, A20, and A23, but its immunogenicity is significantly better than all controls (including similar lipids such as A10, A20, and A23 and SM-102), indicating that A5 has a specific enhancing effect on the delivery of EV-A71 antigen and the induced immune reactivity.

[0038] (2) Unexpected activation of cellular immunity Conventional mRNA vaccines based on viral antigens are typically effective primarily by producing high titers of neutralizing antibodies, but A5-LNP also stimulates strong cellular immunity. The number of IFN-γ spot-forming cells reached 800 SFC / 10 6 Spleen cells ( Figure 4 (), far exceeding the SM-102 group (approximately 300 SFC) and the commercially available inactivated vaccine group.

[0039] This effect is rare in EV-A71 vaccines because inactivated vaccines are almost unable to activate cellular immunity, while A5 enhances intracellular antigen expression and antigen presentation through enhanced membrane fusion capabilities, thereby activating T cells and generating a higher level of cellular immunity.

[0040] (3) Significant advantages in safety Although A5 lipids are known, there is a lack of safety data for them in infant vaccines. This invention has found that: The level of the inflammatory factor IL-6 was only 30% of that in the SM-102 group (Table 4), and the ALT liver toxicity index was also significantly reduced.

[0041] This contradicts the common knowledge that "lipid toxicity usually increases with enhanced immune response," demonstrating that A5 achieves an unexpected balance between high efficacy and safety.

[0042] Clinical significance: This characteristic makes the vaccine suitable for infants and young children over 6 months of age, while existing technologies (such as SM-102) have a stronger inflammatory response and may produce more adverse reactions in younger populations.

[0043] (4) Comparison with traditional technologies Table 1

[0044] The present invention provides an EV-A71 mRNA vaccine composition based on cationic lipid A5, which achieves a combination of highly effective protection and excellent safety under a single low-dose immunization by optimizing the mRNA sequence design and lipid nanoparticle (LNP) delivery system.

[0045] In a preferred embodiment of the present invention, the multivalent vaccine comprises mRNA encoding EV-A71 VP1 protein (such as SEQ ID NO: 6 or 7) and mRNA encoding Coxsackievirus A16 VP1 protein. These two mRNAs can be mixed in a certain ratio (e.g., 1:1) and co-encapsulated using the LNP system containing cationic lipid A5 described in this invention to form a bivalent mRNA-LNP vaccine for the simultaneous prevention of hand-foot-mouth disease caused by EV-A71 and CV-A16.

[0046] The present application will now be described in detail with reference to embodiments and experimental data.

[0047] Example 1: Design, preparation, and expression validation of EV-A71 VP1 mRNA The encoded VP1 structural protein sequence was derived from enterovirus A71 C4 subtype (GenBank: KP861243.1), and its amino acid sequence is shown in SEQ NO.1. A human tissue plasminogen activator (tPA) signal peptide is linked at the N-terminus to promote VP1 secretion, and its amino acid sequence is shown in SEQ NO.2. The sequence was codon-optimized, taking into account both expression level (codon adaptation index (CAI) and stability (minimum free-energy change (MFE)). The GC content was 50–70%; the codon adaptation index (CAI) was ≥0.80; and the minimum free-energy change (MFE) of the mRNA secondary structure was below -400 kcal / mol. The sequence and optimized parameters are shown in Table 2.

[0048] The sequence was synthesized by a bioengineering company and recombined into the plasmid pUC57-Kan, which already contained the 5'UTR and 3'UTR of the BNT162b2 vaccine. After amplification culture, the plasmid was extracted and linearized.

[0049] In the in vitro transcription reaction, linearized DNA was used as a template, and a cap analog, NTP, and modified pseudouracil were added. One-step in vitro transcription (IVT) was performed using T7 RNA polymerase in a reaction flask to produce mRNA, resulting in capped, tailed mRNA with modified nucleosides. The IVT product was then digested with DNase I to remove the template DNA from the reactants. After purification, the mRNA sequence length and integrity were assessed by capillary electrophoresis (results are shown in [link to results]). Figure 1 and Figure 2 ).

[0050] Table 2. mRNA sequence design

[0051] Next, HEK293 cells were transfected with the purified mRNA described in Table 2, and then the expression of VP1 in the intracellular and extracellular spaces was analyzed by Western Blot.

[0052] Cell lysates and culture supernatants were mixed with SDS-PAGE loading buffer 24 hours after transfection, boiled, and separated by SDS-polyacrylamide gel electrophoresis at 100 V for 1 hour. The separated cells were then transferred to a polyvinylidene fluoride (PVDF) membrane at a constant current of 1800 mA for 2 hours. After transfer, the membrane was placed in 5% skim milk and blocked at room temperature for 2 hours. The membrane was then incubated with primary and secondary antibodies sequentially: the primary antibody was rabbit anti-EV-A71 VP1 antibody (GTX132338, GeneTex), incubated overnight at 4°C; the secondary antibody was peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (711-035-152, Jackson ImmunoResearch), incubated for 45 minutes at room temperature. GAPDH was used as an internal control for standardized VP1 protein quantification.

[0053] Image J software was used to analyze the protein band intensity, and the VP1 intensity was normalized to the GAPDH band intensity. Then, based on the expression level of VP1 (SEQ ID NO: 3) without codon optimization, the relative expression level of the target protein in each sequence was calculated. The specific quantitative results are shown in Table 3.

[0054] Table 3 Comparison of intracellular and extracellular levels of mRNA-expressed antigens

[0055] The results showed that VP1 protein was correctly expressed in HEK293 cells, with a molecular weight of approximately 35–40 kD, consistent with expectations. Codon optimization significantly improved the expression level of VP1 protein; simultaneously, the introduction of the tPA signal peptide into the mRNA sequence significantly enhanced the content of VP1 protein in the cell supernatant. Based on these results, mRNA expressing tPA-VP1-2 (SEQ ID NO: 7) was selected for subsequent experiments.

[0056] Example 2: Preparation and Characterization of mRNA-LNP In this embodiment, ionizable lipids (A5, A10, A20, A23, and SM-102), auxiliary lipid DSPC, cholesterol, and polymer DMG-PEG2000 were dissolved in ethanol solutions at a concentration of 10 mg / mL. The ionizable lipids, DSPC, cholesterol, and DMG-PEG2000 were mixed in a molar ratio of 50:38:10:2 and anhydrous ethanol was added to control the total lipid concentration at 10 mM. The mRNA molecule (tPA-VP1-2, whose coding nucleotide sequence is shown in SEQ ID NO. 7) obtained by in vitro transcription in step 1 was diluted in citrate buffer (pH 4.0) at a concentration of 90 μg / mL, ensuring an N / P ratio of 4 for both mRNA and lipids. The prepared organic phase (lipids) and aqueous phase (mRNA) were used to prepare LNPs encapsulating the mRNA using a microfluidic instrument, with the flow rate of the aqueous phase at 12 mL / min and the flow rate of the organic phase at 4 mL / min. Immediately after obtaining the LNPs containing mRNA, they were diluted 10-fold with sterile ultrapure water. The diluted LNPs were then ultrafiltered using a 100 kDa ultrafiltration tube at 3000 rpm and 4°C. When the liquid was reduced to 1 mL, sterile ultrapure water was added to bring the volume to 15 mL, and the mixture was centrifuged again, repeating this process three times to remove ethanol from the liquid.

[0057] Table 4. LNP characterization results of different cationic lipids

[0058] The results showed that the encapsulation efficiency of all five cations was around 95% and the PDI was less than 0.2, with the zeta potential within a reasonable range, making them suitable for further evaluation in animal experiments. Among them, A5 and SM-102 both achieved encapsulation efficiencies of over 90%. The LNP prepared by A5 had the smallest particle size and PDI and the most stable potential, indicating that it produced the best particle dispersibility.

[0059] Example 3: Measurement of humoral immunity levels in mice immunized with a single vaccine dose Female BALB / c mice aged 6-8 weeks were randomly divided into 5 groups (n = 6): A5, A10, A20, A23, SM-102, and a naked mRNA LNP-free group. A commercially available inactivated vaccine served as a positive control. Mice in each group received a single intramuscular injection of mRNA-LNP (5 µg / mouse); the commercially available inactivated vaccine was administered at 1 / 5 of the human dose. Blood samples were collected on day 21 post-immunization, serum was separated, and the total specific antibody titer in the serum was detected using enzyme-linked immunosorbent assay (ELISA). The simplified procedure was as follows: A 96-well ELISA plate was coated with 100 µg / mL VP1 protein and incubated overnight at 4°C. After washing four times with PBST, 100 µL of blocking buffer (PBST containing 1% BSA) was added to each well, and the plate was blocked at 37°C for 2 hours. After washing three more times, 100 µL of serially diluted mouse serum was added to each well, and the plate was incubated at 37°C for 2 hours. After four washes, 100 µL of horseradish peroxidase-labeled goat anti-mouse IgG (1:5,000 dilution) was added to each well, and the plate was incubated at 37°C for 1 hour. After four washes, 50 µL of TMB solution (NCM Biotech) was added to each well, and the plate was incubated at room temperature for 10–30 minutes. Finally, 50 µL of 2 mol / L sulfuric acid was added to each well to stop the reaction, and the absorbance was measured at 450 nm.

[0060] Neutralizing antibody titer assay: Vero cells were seeded at a density of 10,000 cells per well in 96-well plates and cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C for 24 hours. The following day, mouse serum was serially diluted 1:32 using DMEM containing 2% FBS. The diluted serum was then mixed with an equal volume of DMEM containing 50 TCID50. 50 The EV-A71 virus mixture was incubated at 37°C for 2 hours. Then, 100 μL of the mixture was seeded into Vero cells and incubated for another 2 hours. The virus-containing supernatant was discarded. Cytopathic effect (CPE) was assessed in each well on day 5 post-infection. Triple-dose wells were used for each sample, and the 50% neutralizing antibody titer (NT50) was calculated.

[0061] Table 5. Comparison of humoral immune levels of vaccines containing different cationic lipids

[0062] As shown in Table 5, the IgG titers in group A5 (12800~6400) were significantly higher than those in other groups (6400~3200). The titers of neutralizing antibodies (see...) Figure 3The A5 group (938.7 ± 209.0) also had the highest value (p<0.05) in Table 2, which was significantly higher than the SM-102 group (682.7 ± 264.4, p<0.05) and the commercially available inactivated vaccine group (170.7 ± 66.1, p<0.001), suggesting that the LNP system containing A5 can induce a significantly enhanced humoral immune response under a single dose.

[0063] Example 4: Measurement of Cellular Immunity Levels Spleens were collected from mice 6 weeks post-immunization (day 42) as described in Example 3. Lymphocytes were aseptically isolated and a single-cell suspension was prepared. The cells were stimulated with an overlapping peptide pool (15 amino acids in length, with adjacent peptides overlapping by 10 amino acids) of the EV-71 VP1 protein for 20 hours. The number of IFN-γ-secreting lymphocytes was detected by enzyme-linked immunospot assay, and the number of speckle-forming cells per million spleen cells (SFC / 102) was used as the criterion. 6 splenocytes represent the level of cellular immune response.

[0064] The results are as follows Figure 4 As shown, there are significant differences in the intensity of cellular immune responses induced by different vaccine groups. Specifically: In the naked mRNA group, used as a negative control, almost no specific IFN-γ secreting cells were detected, while all LNP-encapsulated mRNA vaccine groups induced varying degrees of cellular immune responses. Among them, the A5 LNP group described in this invention induced the highest SFC value of IFN-γ, reaching approximately 800 SFC / 102. 6 Spleen cells showed a significantly stronger immune response than the comparative LNP groups (A10, A20, A23, SM-102, etc.) and far surpassed those of commercially available inactivated vaccines. The response levels of the comparative SM-102 and A20 groups were similar, both significantly lower than those of the A5 group. The response levels of the comparative A23 and A10 groups were between those of the A5 and SM-102 groups. Cellular immunity typically peaks 7-14 days post-immunization. We measured cellular immunity levels 6 weeks post-immunization; the A5 group still showed a very strong cellular level, significantly superior to the other groups.

[0065] Experimental conclusion: The above results demonstrate that the LNP delivery system based on A5 cationic lipids provided by this invention can elicit a more potent specific T-cell immune response in vivo than existing technologies (such as SM-102 and commercially available inactivated vaccines) and other comparative technologies (such as A10, A20, and A23) when delivering mRNA encoding antigens. This indicates that it has an unexpectedly superior effect in inducing cellular immunity.

[0066] Example 5: Passive Immunoprotection Test in Mice After Viral Challenge Considering the difficulty in establishing an active challenge model of EV-A71 in immunocompetent mice, passive immunization-challenge in A129 newborn mice was used to assess serum protective efficacy, consistent with industry practice. One-day-old A129 mice were randomly divided into 5 groups (n=6). Each group of mice received an intraperitoneal injection of 50 μL of mixed serum from the corresponding immunized mice described in Example 3. Twenty-four hours after injection, the mice were challenged via intramuscular injection (im) at a lethal dose of EV-A71 virus (2 × 10⁻⁶). 7 TCID 50 / 50 μL). Weight changes, clinical symptoms, and survival were monitored daily during the experiment until day 21 post-infection.

[0067] The protective effect results are as follows Figure 5 As shown: Mice in the naked mRNA group all died early after infection, with a survival rate of 0%, confirming that their serum did not provide effective protection. The commercially available inactivated vaccine group provided partial protection, a result consistent with its single immunization (its normal immunization schedule requires an additional booster shot) and the fact that antibody levels had not yet reached peak levels. All LNP-mRNA vaccine groups provided significant protection, with the final survival rates of the A10, A20, A23, and SM102 groups ranging from approximately 40% to 80%. The A5 vaccine group exhibited the best protective efficacy (100%), with a significantly higher survival rate than all other groups.

[0068] Experimental Conclusion: The results of the challenge protection experiment clearly show that the LNP-mRNA vaccine based on A5 cationic lipids provided by this invention can provide 100% survival protection for susceptible mice after a single immunization, and its protective efficacy is significantly better than other cationic lipids such as SM-102 and commercially available inactivated vaccines. This proves that the vaccine can rapidly induce the production of high-quality protective antibodies and has outstanding immunoprotective effects.

[0069] Example 6: Safety Evaluation Given that this vaccine is intended for use in infants and young children, its safety is a crucial evaluation parameter. 6-8 week old BALB / c mice were intramuscularly injected according to the grouping method described in Example 2. The vaccine dose was 5 μg / mouse, and the commercially available vaccine dose was 1 / 5 of the human dose, with 3 mice per group. Body temperature and weight changes in the mice were continuously monitored after vaccination. Blood was collected 24 hours post-vaccination, and serum was separated. The levels of the inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the serum were detected by ELISA. The levels of liver injury markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by biochemical analysis. All data were normalized to multiples of the mean values ​​in the SM-102-containing mRNA vaccine (Vaccine 5) group. The results are shown in Table 6.

[0070] Table 6. Cytokine levels and liver function-related enzymes ALT and AST levels after vaccine injection

[0071] As shown in Table 6, this invention has an excellent safety profile. Compared with commercially available inactivated vaccines, the levels of inflammatory factors (IL-6, TNF-α) and liver injury markers (ALT, AST) in all LNP-mRNA vaccine groups (Vaccine 1-5) were significantly reduced, demonstrating the inherent advantages of the mRNA-LNP technology platform in avoiding systemic inflammatory responses and hepatotoxicity, making it more suitable for infants and young children.

[0072] The lipid A5 of this invention exhibits the best safety profile on the LNP platform: Among all LNP formulations, the vaccine (Vaccine 1) containing lipid A5 described in this invention demonstrates the best safety profile. The inflammatory response (IL-6 and TNF-α levels) it induces is significantly lower than that of Vaccine 5 in the comparative ready-to-use cationic lipid SM-102 (only 30% and 56% of the SM-102 group, respectively), and its hepatotoxicity indicators (ALT and AST levels) are also significantly lower than those in the SM-102 group (87% and 65% of the SM-102 group, respectively), indicating that its impact on the liver is also effectively reduced.

[0073] Comparison with negative controls: Although all indicators of the naked mRNA group were the lowest, it could not effectively deliver mRNA into cells and elicit an effective immune response (as shown in Examples 2 and 4), and therefore had no practical application value. In contrast, the A5LNP of this invention achieves a highly efficient immune response while exhibiting significantly better safety indicators than other LNP groups; this balance is its outstanding advantage.

[0074] In summary, the LNP delivery system containing A5 cationic lipids provided by this invention can minimize systemic inflammatory responses and potential hepatotoxicity caused by vaccination when used in mRNA vaccines. Its excellent safety profile makes it of great application potential and clinical value in the development of infant vaccines.

[0075] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0076] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0077] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An mRNA vaccine composition for preventing enterovirus A71 infection, characterized in that, Include: (a) An mRNA molecule encoding the EV-A71 VP1 protein, said mRNA comprising a cap structure, a 5' untranslated region and a 3' untranslated region, encoding the VP1 protein sequence, and a PolyA tail of 80-150 nucleotides; and (b) Lipid nanoparticles encapsulating the mRNA.

2. The composition according to claim 1, characterized in that, The mRNA corresponds to one of the nucleic acid sequences SEQ ID NO. 6-7.

3. The composition according to claim 1 or 2, characterized in that, The LNP comprises an ionizable cationic lipid, cholesterol, 1,2-distearate-sn-glycerol-3-phosphocholine, and a polyethylene glycol-modified lipid; the ionizable cationic lipid is A5 or a structural analogue of A5, the structural formula of which is shown below: （I）。 4. The composition according to claim 3, characterized in that, The LNP is composed of the following components in molar percentage: 45%-55% ionizable cationic lipids, 35%-40% distearate phosphatidylcholine (DSPC), 8%-12% cholesterol, and 1%-3% polyethylene glycol modified lipids, and the sum of the molar percentages of each component is 100%.

5. The mRNA vaccine composition according to claim 1, characterized in that, The mRNA molecule has a signal peptide linked to its N-terminus, with the amino acid sequence shown in SEQ ID NO.2, which is used to promote the secretion of VP1 protein.

6. The composition according to any one of claims 1–5, wherein the LNP has a particle size of 50–150 nm, an encapsulation efficiency of ≥60%, and a zeta potential of 5–20 mV.

7. A method for preparing the mRNA vaccine composition according to any one of claims 1-6, characterized in that, include: S1. Prepare the mRNA molecule by in vitro transcription, wherein the in vitro transcription includes capping the mRNA and introducing the 5'UTR, 3'UTR and PolyA tail, and during the transcription process, modified nucleoside triphosphates may be selectively used to replace the natural nucleoside triphosphates; S2. Prepare a lipid mixture; S3. Mix the aqueous mRNA solution obtained in step S1 with the lipid mixture obtained in step S2 by microfluidic mixing or an equivalent rapid mixing method to form an LNP encapsulating the mRNA; S4. Dilute, concentrate and replace the LNP with buffer to obtain the final formulation.

8. The use of the mRNA vaccine composition according to any one of claims 1-7 in the preparation of a vaccine for the prevention of EV-A71 virus infection.

9. The application according to claim 8, characterized in that, The drug is administered via intramuscular injection.

10. The application according to claim 8, characterized in that, The drug is a multivalent or combination vaccine, comprising the mRNA vaccine composition for preventing enterovirus A71 infection as described in claim 1.

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