Trivalent beta coronavirus s protein trimer mRNA-lnp vaccine and preparation method

Abstract

The present application relates to the technical field of biological medicine, and in particular to a trivalent beta coronavirus S protein trimer mRNA-LNP vaccine and a preparation method. The first nucleic acid of the composition is an mRNA encoding a SARS-CoV-2 S protein, the second nucleic acid is an mRNA encoding a SARS-CoV-1 S protein, and the third nucleic acid is an mRNA encoding a MERS-CoV S protein. Each S protein includes a proline stabilizing mutation, and an exogenous trimerization domain is inserted between the C-terminal end of the extracellular domain and the transmembrane region. The SARS-CoV-2 S protein and the MERS-CoV S protein include a furin cleavage site inactivation mutation. The mass ratio of each nucleic acid is 1:1:1. After co-delivery, the vaccine does not produce immune interference, the neutralizing antibody in each component is better than that of a monovalent strategy, and trimerization significantly enhances the specific IgG level, the neutralizing antibody level, and the cross-neutralization ability to variant strains.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a trivalent β-coronavirus S protein trimer mRNA-LNP vaccine and its preparation method. Background Technology

[0002] Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoV-1), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are three highly pathogenic human beta coronaviruses that all mediate host cell invasion via their spike protein (S protein). Although these viruses belong to different subgenera (Sarbecovirus and Merbecovirus), their S protein exhibits a degree of conservation in structure and function. Epidemiological evidence suggests that cross-species transmission of highly pathogenic coronaviruses is inherently recurrent, and the World Health Organization (WHO) has prioritized broad-spectrum coronavirus vaccines as a key area for pandemic preparedness.

[0003] Currently approved mRNA coronavirus vaccines all target only SARS-CoV-2 (including the original strain and updated versions such as Omicron variants), essentially being single-pathogen vaccines with fundamental limitations: on the one hand, the continuous emergence of new variants necessitates frequent vaccine updates, and the phenomenon of immune imprinting exacerbates the uncertainty of variant updates; on the other hand, these vaccines lack the ability to simultaneously cover other highly pathogenic β-coronaviruses such as SARS-CoV-1 and MERS-CoV, leaving gaps in protection against future emerging epidemics. Therefore, there is an urgent need to develop broad-spectrum vaccines capable of simultaneously covering multiple highly pathogenic human β-coronaviruses. Co-delivering multiple heterologous antigens in the same delivery system is a straightforward strategy for achieving broad-spectrum coverage, but it faces the core technical obstacle of immune interference—that is, when multiple antigens are co-expressed simultaneously, the immune response to some antigen components is suppressed or weakened due to imbalances in immunogenicity, antigen presentation competition, or immunodominance shifts. Immune interference has been reported in studies of multivalent influenza vaccines, multivalent pneumococcal conjugate vaccines, and multivalent coronavirus vaccines based on protein nanoparticles.

[0004] On the mRNA-lipid nanoparticle (LNP) platform, when multiple heterologous coronavirus S protein mRNAs are co-encapsulated in the same LNP, each mRNA needs to compete for ribosomal translation resources. Moreover, the lengths (approximately 3800~4400 nt) and structures of the various S protein coding sequences differ. Whether co-expression will lead to an imbalance in translation efficiency and cause an immune dominance shift remains to be experimentally verified.

[0005] At the level of antigen conformation optimization, strategies such as exogenous trimerization domains (e.g., T4 bacteriophage fibritin foldon (T4-Fd)), proline stabilization mutations (e.g., 2P diproline mutation or 6P / HexaPro hexaproline mutation), and furin protease cleavage site inactivation mutations to maintain the pre-fusion trimer conformation of the S protein have been proven to effectively enhance immunogenicity in monovalent vaccines.

[0006] However, most previous studies on the aforementioned strategies have been limited to single coronavirus antigens. Furthermore, coronaviruses primarily invade the host through the respiratory mucosa. Existing intramuscular mRNA vaccines mainly induce a systemic immunoglobulin G (IgG) response, with relatively insufficient induction of local respiratory mucosal immunoglobulin A (IgA), making it difficult to form an effective first line of defense at the viral entry point.

[0007] In summary, the existing technologies have the following problems that urgently need to be solved: (1) The monovalent strategy has a narrow coverage and cannot prevent multiple highly pathogenic β coronaviruses at the same time; (2) Multivalent co-delivery faces the core technical obstacle of immune interference, and it is difficult to maintain the efficient immune response of each component at the same time when multiple heterologous S protein mRNAs are co-delivered; (3) The multiple antigen conformation optimization strategy (proline stable mutation, furin protease cleavage site mutation, trimerization) has not been systematically integrated and verified on the multivalent heterologous coronavirus mRNA platform; (4) Intramuscular injection cannot effectively induce respiratory mucosal IgA protection.

[0008] Therefore, there is an urgent need for a technical solution that can integrate multiple antigen conformation optimization and multivalent co-delivery on the same mRNA-LNP platform, overcome immune interference, and effectively induce respiratory mucosal immunity. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a trivalent β-coronavirus S protein trimer mRNA-LNP vaccine and its preparation method.

[0010] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides a trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition, the composition comprising a first nucleic acid, a second nucleic acid, and a third nucleic acid, wherein the first nucleic acid comprises mRNA encoding SARS-CoV-2 S protein, the second nucleic acid comprises mRNA encoding SARS-CoV-1 S protein, and the third nucleic acid comprises mRNA encoding MERS-CoV S protein; The SARS-CoV-2 S protein, the SARS-CoV-1 S protein, and the MERS-CoV S protein all contain stable proline mutations, and an exogenous trimerized domain is inserted between the C-terminus of the extracellular domain and the transmembrane region of each S protein. The SARS-CoV-2 S protein and the MERS-CoV S protein also include inactivation mutations at the furin cleavage site.

[0011] Based on the above technical solution, the present invention can be further improved as follows.

[0012] Furthermore, in the amino acid sequence of the SARS-CoV-2 S protein, the furin protease cleavage site inactivation mutation is changed from RRAR to GSAS, the proline stable mutation is changed to 6P proline stable mutation, and the mutation sites are F817P, A892P, A899P, A942P, K986P, and V987P; the exogenous trimerization domain is a T4 phage fibrin fold; and the amino acid sequence of the SARS-CoV-2 S protein has a primitive transmembrane region and a cytoplasmic tail region.

[0013] Furthermore, in the amino acid sequence of the SARS-CoV-1 S protein, the proline is stably mutated to a 6P proline stable mutation, with mutation sites F799P, A874P, A881P, A924P, K968P, and V969P; the exogenous trimerization domain is a T4 phage fibrin fold; and the amino acid sequence of the SARS-CoV-1 S protein has a primitive transmembrane region and a cytoplasmic tail region.

[0014] Furthermore, in the amino acid sequence of the MERS-CoV S protein, the furin protease cleavage site inactivation mutation is changed from RSVR to ASVG, the proline stable mutation is changed to 2P proline stable mutation, and the mutation sites are V1060P and L1061P; the exogenous trimerization domain is a T4 phage fibrin fold; and the amino acid sequence of the MERS-CoV S protein has a primitive transmembrane region and a cytoplasmic tail region.

[0015] Furthermore, the amino acid sequence of the SARS-CoV-2 S protein is shown in SEQ ID NO:1, the amino acid sequence of the SARS-CoV-1 S protein is shown in SEQ ID NO:3, and the amino acid sequence of the MERS-CoV S protein is shown in SEQ ID NO:5.

[0016] Furthermore, the mRNA nucleotide sequence for encoding the SARS-CoV-2 S protein is shown in SEQ ID NO:2, the mRNA nucleotide sequence for encoding the SARS-CoV-1 S protein is shown in SEQ ID NO:4, and the mRNA nucleotide sequence for encoding the MERS-CoV S protein is shown in SEQ ID NO:6.

[0017] Furthermore, the 5'UTR sequences of the first nucleic acid, the second nucleic acid, and the third nucleic acid are identical, as are the 3'UTR sequences of the first nucleic acid, the second nucleic acid, and the third nucleic acid.

[0018] Furthermore, in the composition, the mass ratio of the first nucleic acid, the second nucleic acid, and the third nucleic acid is 1:1:1.

[0019] The present invention also provides a trivalent beta-coronavirus S protein trimer mRNA-LNP vaccine, comprising lipid nanoparticles, wherein the lipid nanoparticles comprise the trivalent beta-coronavirus S protein trimer mRNA composition as described above and liposomes for encapsulating the composition.

[0020] The present invention also provides a method for preparing the trivalent β-coronavirus S protein trimer mRNA-LNP vaccine as described above, wherein an aqueous phase containing the trivalent β-coronavirus S protein trimer mRNA composition and a lipid phase containing the liposomes are mixed and encapsulated under acidic conditions using a microfluidic method to obtain the lipid nanoparticles, and then the lipid nanoparticles are mixed with a pharmaceutically acceptable solvent to prepare the vaccine.

[0021] The beneficial effects of this invention are as follows: (1) The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition of the present invention optimizes each S protein by multiple reconfiguration, effectively retains the natural conformational epitopes to enhance the immunogenicity of each component, so that each antigen can be correctly folded after expression to maintain the pre-fusion conformation for recognition by the immune system, thereby reducing the immune advantage shift caused by changes in the natural conformation of the antigen from the source. (2) The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition of the present invention uses a uniform UTR for each mRNA, which can ensure the balanced translation of each component in the host cell from the translation regulation level and reduce the risk of antigen expression imbalance caused by differences in translation efficiency; (3) The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition of the present invention adopts a strategy of co-delivery of trivalent components in equal mass ratio, which can ensure the uniformity of the proportion of each component mRNA in LNP from the formulation level, and reduce the risk of antigen expression imbalance in synergy with the molecular design of unified UTR. (4) The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine of the present invention did not produce immune interference after co-delivery. The neutralizing antibodies of each component were superior to those of the monovalent strategy. Trimerization significantly enhanced the level of specific IgG, the level of neutralizing antibodies, and the cross-neutralization ability against variants. Attached Figure Description

[0022] Figure 1 These are schematic diagrams of the structures of the protein trimer mRNA constructs and the monomeric mRNA constructs without T4-Fd in Example 1 of the present invention; Figure 1The structures in AC represent, in order, the SARS-CoV-2 S protein trimer mRNA construct, the SARS-CoV-1 S protein trimer mRNA construct, and the MERS-CoV S protein trimer mRNA construct; Figure 1 The DF components in the middle are, in order, the SARS-CoV-2 S protein monomer mRNA construct, the SARS-CoV-1 S protein monomer mRNA construct, and the MERS-CoV S protein monomer mRNA construct; Figure 2 This is an electrophoresis image of recombinant plasmid digestion identification in Example 1 of the present invention; Figure 2 In the middle, A represents the linearization result of single enzyme digestion with EcoRI; Figure 2 The images in the middle (BD) represent the SpeI / EcoRI double enzyme digestion identification results of the pGEM-SARS2-Trimer plasmid, pGEM-SARS1-Trimer plasmid, and pGEM-MERS-Trimer plasmid, respectively. Figure 3 This is a formaldehyde denaturing gel electrophoresis image of the mRNA in vitro transcription product in Example 2 of the present invention; Figure 4 This is a Western blot diagram of the in vitro expression of trimeric mRNA in Example 3 of the present invention; Figure 4 The results of HEK293T cell lysates transfected with SARS-CoV-2, SARS-CoV-1, and MERS-CoV trimeric mRNA are shown in the middle AC column, respectively. Figure 5 This is a characterization diagram of the morphological and physicochemical properties of mRNA-LNP in Example 4 of the present invention; Figure 5 In the image, A is a TEM image of mRNA-LNP, with a scale bar of 100 nm; Figure 5 In the middle section, B represents the average particle size and polydispersity index (PDI) of different vaccine components. Figure 5 The middle section (C) shows a comparison of the zeta potentials of each component. Figure 5 D represents the encapsulation efficiency (EE%) of each component. Figure 6 This is a graph showing the detection of specific IgG antibody titers in the serum of immunized mice in Example 5 of the present invention. Figure 6 The figures in the middle (AC) are, in order, the IgG titer detection results for SARS-CoV-2, SARS-CoV-1, and MERS-CoV S proteins; Figure 7 This is a neutralization inhibition curve of immunized mouse serum against SARS-CoV-2 (original strain, XBB.1.5), SARS-CoV-1 and MERS-CoV pseudovirus in Example 5 of the present invention; Figure 7The AD values ​​in the middle section represent the neutralization and inhibition curves of the original SARS-CoV-2 strain, the SARS-CoV-2 XBB.1.5 variant, and the SARS-CoV-1 and MERS-CoV trimer constructs, respectively. Figure 7 The EH values ​​represent, in order, the neutralization and inhibition curves of the original SARS-CoV-2 strain, the SARS-CoV-2 XBB.1.5 variant, and the SARS-CoV-1 and MERS-CoV monomer constructs. Figure 8 In Example 5 of the present invention, the titer of pseudovirus neutralizing antibody (pVNT) in the serum of immunized mice was... 50 Comparison chart; Figure 9 This is a comparison of specific IgA levels in bronchoalveolar lavage fluid (BALF) induced by different immunization routes of the trimeric mixed trivalent mRNA vaccine in Example 6 of the present invention. Detailed Implementation

[0023] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0024] The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition of the present invention comprises a first nucleic acid, a second nucleic acid, and a third nucleic acid, wherein the first nucleic acid comprises mRNA encoding SARS-CoV-2 S protein antigen, the second nucleic acid comprises mRNA encoding SARS-CoV-1 S protein antigen, and the third nucleic acid comprises mRNA encoding MERS-CoV S protein antigen. The amino acid sequences of the SARS-CoV-2 S protein antigen, the SARS-CoV-1 S protein antigen, and the MERS-CoV S protein antigen all contain stable proline mutations, and exogenous trimerized domains are inserted between the C-terminus of the extracellular domain and the transmembrane region of each S protein antigen. The amino acid sequences of the SARS-CoV-2 S protein antigen and the MERS-CoV S protein antigen also include inactivation mutations at the furin cleavage site.

[0025] The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition of this invention optimizes each S protein through multiple reconfigurations, effectively preserving native conformational epitopes to enhance the immunogenicity of each component. Inactivation mutations at the furin cleavage site maintain pre-fusion conformational integrity, preventing irreversible conformational changes that could alter the neutralizing epitope conformation. Proline stabilization mutations further lock the pre-fusion conformation of the S protein. For all three S proteins, an exogenous trimerization domain is inserted between the C-terminus of the extracellular domain and the transmembrane region to drive uniform trimer formation while preserving the transmembrane and cytoplasmic tail regions, anchoring the antigen to the cell membrane surface and mimicking the display mode of the native viral S protein. This achieves both trimer conformational uniformity and antigen membrane surface display. These multiple modifications ensure that each antigen can correctly fold and maintain its pre-fusion conformation for recognition by the immune system, reducing the immune advantage shift caused by conformational changes in native antigens from the source.

[0026] This invention achieves effective expression and antigen display of each component by structurally engineering the three heterologous S proteins and co-delivering them in equal mass ratios. This reduces or mitigates the risk of immune interference in trivalent co-delivery, ensuring that the trivalent group has a neutralizing antibody response to the three viruses that is no lower than that of the corresponding monovalent group, and exhibits higher neutralizing activity in some viruses or variants.

[0027] Preferably, each S protein of the present invention is a mutation and modification based on the following original strains: SARS-CoV-2 (original strain, GenBank: NC_045512.2), SARS-CoV-1 (GenBank: AAP51227.1) and MERS-CoV (GenBank: YP_009047204.1).

[0028] Preferably, the exogenous trimerization domain is the T4 phage fibrin fold.

[0029] Preferably, in the amino acid sequence of the SARS-CoV-2 S protein antigen, the furin protease cleavage site inactivation mutation is changed from RRAR to GSAS, the proline stable mutation is changed to 6P proline stable mutation, and the mutation sites are F817P, A892P, A899P, A942P, K986P, and V987P; the exogenous trimerization domain is the T4 phage fibrin fold; the amino acid sequence of the SARS-CoV-2 S protein antigen retains the original transmembrane region and cytoplasmic tail region.

[0030] Preferably, the amino acid sequence of the SARS-CoV-2 S protein antigen is shown in SEQ ID NO:1, and the mRNA nucleotide sequence encoding the SARS-CoV-2 S protein antigen is shown in SEQ ID NO:2.

[0031] Preferably, in the amino acid sequence of the SARS-CoV-1 S protein antigen, the proline is stably mutated to a 6P proline stable mutation, with mutation sites at F799P, A874P, A881P, A924P, K968P, and V969P; the exogenous trimerization domain is the T4 phage fibrin fold; and the amino acid sequence of the SARS-CoV-1 S protein antigen retains the original transmembrane region and cytoplasmic tail region. Since the SARS-CoV-1 S protein S1 / S2 junction does not contain a typical furin cleavage site, there is no need for furin cleavage site inactivation mutations.

[0032] Preferably, the amino acid sequence of the SARS-CoV-1 S protein antigen is shown in SEQ ID NO:3, and the mRNA nucleotide sequence encoding the SARS-CoV-1 S protein antigen is shown in SEQ ID NO:4.

[0033] Preferably, in the amino acid sequence of the MERS-CoV S protein antigen, the furin cleavage site inactivation mutation is changed from RSVR to ASVG, the proline stable mutation is changed to 2P proline stable mutation, and the mutation sites are V1060P and L1061P; the exogenous trimerization domain is the T4 phage fibrin fold; the amino acid sequence of the MERS-CoV S protein antigen retains the original transmembrane region and cytoplasmic tail region.

[0034] Preferably, the amino acid sequence of the MERS-CoV S protein antigen is shown in SEQ ID NO:5, and the mRNA nucleotide sequence encoding the MERS-CoV S protein antigen is shown in SEQ ID NO:6.

[0035] Preferably, the first, second, and third nucleic acids of the present invention all have a 5'Cap1 capped structure and a 3'Poly(A) tail, and all are modified nucleosides by completely replacing the natural uridine substrate with N1-methylpseudouridine triphosphate (N1-mΨTP) to improve mRNA stability, translation efficiency, and reduce innate immune activation.

[0036] Preferably, the untranslated regions (UTRs) in the first, second, and third nucleic acids of the present invention all adopt the 5'UTR and 3'UTR combination of Moderna mRNA-1273. The UTR sequence plays a decisive role in the translational efficiency and half-life of the mRNA vaccine in the host. Using a uniform UTR can ensure the balanced translation of each component in the host cell from the perspective of translational regulation, and reduce the risk of antigen expression imbalance caused by differences in translation efficiency. This is a key design element to reduce the risk of immune dominance shift in trivalent co-delivery.

[0037] Further preferred, the 5'UTR nucleotide sequence of Moderna mRNA-1273 is shown in SEQ ID NO: 13, and the 3'UTR nucleotide sequence of Moderna mRNA-1273 is shown in SEQ ID NO: 14.

[0038] In the composition of this invention, the mass ratio of the first nucleic acid, the second nucleic acid, and the third nucleic acid is 1:1:1. Co-delivery of the trivalent nucleic acids in equal mass ratios allows for balanced antigen expression within the same LNP delivery system. The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine of this invention comprises lipid nanoparticles, which include the aforementioned trivalent β-coronavirus S protein trimer mRNA composition and liposomes for encapsulating the composition; the liposomes include ionizable lipids, as well as helper phospholipids, cholesterol, and PEG-modified lipids.

[0039] Preferably, the molar ratio of ionizable lipids, cofactor phospholipids, cholesterol and PEG-modified lipids is 50:10:38.5:1.5.

[0040] Preferably, the ionizable lipid is SM-102, the cofactor phospholipid is 1,2-distearate-sn-glycerol-3-phosphatidylcholine (DSPC), and the PEG-modified lipid is PEG2000-DMG.

[0041] Experimental verification showed that the trivalent β-coronavirus S protein trimer mRNA-LNP vaccine of the present invention did not produce immune interference during co-delivery, and the neutralizing antibodies of each component were superior to those of the monovalent strategy. The introduction of exogenous trimerization domains improved the overall level of specific IgG, neutralizing antibody, and cross-neutralization ability against variants.

[0042] The method for preparing the trivalent β-coronavirus S protein trimer mRNA-LNP vaccine of the present invention involves mixing and encapsulating an aqueous phase containing a trivalent β-coronavirus S protein trimer mRNA composition and a lipid phase containing liposomes under acidic conditions using a microfluidic method to obtain lipid nanoparticles, and then mixing the lipid nanoparticles with a pharmaceutically acceptable solvent to prepare the vaccine.

[0043] The preparation method of the present invention is simple, efficient, and has a high encapsulation rate.

[0044] Preferably, during the mixed encapsulation, the volumetric flow rate ratio of the aqueous phase to the lipid phase is 3:1, and the total flow rate is 12 mL / min.

[0045] The preparation method of the present invention comprises the following specific steps: (1) Ionizable lipids, helper phospholipids, cholesterol and PEG-modified lipids were dissolved in anhydrous ethanol at a molar ratio to prepare the lipid phase; the three S protein trimer mRNAs were mixed in an acidic buffer at a mass ratio of 1:1:1 to prepare the aqueous phase.

[0046] Preferably, the acidic buffer is a citrate buffer, wherein the concentration of citric acid is 50 mM; the pH value of the citrate buffer is 4.0.

[0047] (2) The initial mRNA-LNP product was rapidly formed by using a microfluidic mixing device with a flow rate ratio of 3:1 between the aqueous phase and the lipid phase and a total flow rate of 12 mL / min.

[0048] (3) The initial mRNA-LNP product was concentrated and buffer replaced using ultrafiltration centrifuge tubes, and finally prepared using final buffer to obtain a trivalent mRNA-LNP vaccine formulation that can be delivered in vivo.

[0049] Preferably, the final buffer solution is Tris-HCl with a concentration of 20 mM and a pH of 7.4.

[0050] The vaccine of this invention can induce cross-neutralizing antibodies against multiple β-coronaviruses and local IgA mucosal immunity in the respiratory tract.

[0051] Preferably, the vaccine of the present invention can employ a heterologous immunization strategy combining intramuscular injection and intranasal administration, and can achieve dual induction of systemic humoral immunity and respiratory mucosal immunity.

[0052] The effects of the present invention will be illustrated by specific embodiments below.

[0053] In each embodiment, the full-length amino acid sequence of the S protein is derived from: SARS-CoV-2 (original strain, GenBank:NC_045512.2), SARS-CoV-1 (GenBank:AAP51227.1) and MERS-CoV (GenBank: YP_009047204.1).

[0054] Example 1: Design and Construction of Trimeric / Monomer S Protein Antigen mRNA This embodiment uses the full-length S protein sequences of SARS-CoV-2, SARS-CoV-1, and MERS-CoV as templates to perform structural engineering modifications on the three sequences, as detailed below: (1) Frin protease recognition sequence mutation: For SARS-CoV-2 and MERS-CoV, the furin protease recognition sequence at the S1 / S2 junction was mutated respectively. Specifically, RRAR of SARS-CoV-2 was mutated to GSAS, and RSVR of MERS-CoV was mutated to ASVG. Since the SARS-CoV-1 S protein does not contain a typical furin protease cleavage site at the S1 / S2 junction, this mutation is not required.

[0055] (2) Proline stable mutations: Six proline stable mutations (6P: F817P, A892P, A899P, A942P, K986P and V987P) were introduced into the S2 subunit of SARS-CoV-2, six proline stable mutations (6P: F799P, A874P, A881P, A924P, K968P and V969P) were introduced into the S2 subunit of SARS-CoV, and two proline stable mutations (2P: V1060P and L1061P) were introduced into MERS-CoV to lock the pre-fusion conformation.

[0056] (3) Insertion of T4-Fd trimerization domain: A T4-Fd trimerization domain was inserted between the C-terminus and the transmembrane region of the extracellular domain of the three mutated S proteins, respectively, while retaining the transmembrane region and the cytoplasmic tail region. At the same time, monomeric mRNA constructs without T4-Fd were designed as controls for each mutated protein.

[0057] The specific structures of each protein trimer mRNA construct and the monomeric mRNA construct without T4-Fd are as follows: Figure 1 As shown.

[0058] The modified SARS-CoV-2 S protein in this embodiment is denoted as SARS-CoV-2-S. GSAS / 6P -T4-Fd, whose amino acid sequence is shown in SEQ ID NO: 1, and whose nucleotide sequence of the trimer mRNA is shown in SEQ ID NO: 2.

[0059] The modified SARS-CoV-1 S protein in this embodiment is denoted as SARS-CoV-1-S. 6P -T4-Fd, whose amino acid sequence is shown in SEQ ID NO: 3, and whose nucleotide sequence of the trimer mRNA is shown in SEQ ID NO: 4.

[0060] The modified MERS-CoV S protein in this embodiment is denoted as MERS-CoV-S. ASVG / 2P -T4-Fd, whose amino acid sequence is shown in SEQ ID NO: 5, and whose nucleotide sequence of the trimer mRNA is shown in SEQ ID NO: 6.

[0061] SARS-CoV-2 S protein monomer (SARS-CoV-2-S) GSAS / 6P The amino acid sequence of SARS-CoV-1 is shown in SEQ ID NO: 7, and the nucleotide sequence is shown in SEQ ID NO: 8; the SARS-CoV-1 S protein monomer (SARS-CoV-1-S 6P The amino acid sequence of the MERS-CoV S protein monomer is shown in SEQ ID NO: 9, and the nucleotide sequence is shown in SEQ ID NO: 10; ASVG / 2P The amino acid sequence of the compound is shown in SEQ ID NO: 11, and the nucleotide sequence is shown in SEQ ID NO: 12.

[0062] After optimizing all target sequences with human codons, they were cloned into the pGEM-3zf vector backbone containing the T7 promoter, 5'UTR, 3'UTR and Poly(A) tail sequences, respectively, to obtain three recombinant plasmids containing the corresponding modified protein genes.

[0063] The 5'UTR nucleotide sequence of Moderna mRNA-1273 in this embodiment is shown in SEQ ID NO: 13, and the 3'UTR nucleotide sequence of Moderna mRNA-1273 is shown in SEQ ID NO: 14.

[0064] For each recombinant plasmid, it was transformed into NEB®Stable E. coli competent cells, positive clones were selected and expanded, and plasmid DNA was extracted using an endotoxin-free plasmid extraction kit.

[0065] Recombinant plasmid enzyme digestion identification electrophoresis image as shown below Figure 2 As shown, Figure 2 Lanes 1-3 of the A-band contain pGEM-SARS2-Trimer, pGEM-SARS1-Trimer, and pGEM-MERS-Trimer plasmids, respectively. Figure 2 In the BD model, lane 1 of each electrophoresis pattern is the undigested plasmid, lane 2 is the plasmid digested with SpeI, and lane 3 is the plasmid digested with SpeI / EcoRI.

[0066] like Figure 2 As shown, after double digestion with SpeI / EcoRI, all three recombinant plasmids showed a target fragment of approximately 4000 bp and a vector backbone band of approximately 3000 bp, consistent with the expected target fragment size; the linearized product from single digestion with EcoRI was approximately 7000 bp. The band positions were consistent with expectations, confirming correct construction.

[0067] Example 2: mRNA in vitro transcription and quality assessment In this embodiment, linearized recombinant plasmids were used as templates to synthesize mRNAs containing the constructs described in Example 1 using an in vitro transcription system with T7 RNA polymerase. Simultaneously, a Cap1 co-transcriptional capping strategy was employed, with N1-mΨTP completely replacing the natural uridine substrate. The reaction was carried out at 37°C for 2 h to obtain the first, second, and third nucleic acids of this invention.

[0068] After transcription, the DNA template was digested with DNase I and purified using magnetic beads. The concentration of the purified product was determined by UV spectrophotometry, and the integrity was assessed by formaldehyde denaturing agarose gel electrophoresis.

[0069] like Figure 3 As shown, the positions of the SARS-CoV-2 in lane 3, the SARS-CoV-1 in lane 1, and the MERS-CoV mRNA in lane 2 are consistent with their theoretical lengths of approximately 3837 nt, 3783 nt, and 4368 nt, respectively. The bands are single and show no obvious degradation.

[0070] Example 3: In vitro expression verification The three mRNAs obtained in Example 2 were transfected into human embryonic kidney 293T cells (HEK293T) using Lipofectamine 3000. Cell lysates were collected at 24 h, 48 h and 72 h after transfection for Western blot analysis.

[0071] Incubation was performed with SARS-CoV-2, SARS-CoV-1, and MERS-CoV S protein-specific primary antibodies and horseradish peroxidase (HRP)-labeled secondary antibodies, respectively. Enhanced chemiluminescence (ECL) was used for imaging, with β-actin as an internal control.

[0072] The Western blot analysis of trimeric mRNA expression in vitro is shown in the figure below. Figure 4 As shown in the figure, M represents the protein molecular weight standard, NC represents the negative control (HEK293T cell lysate without mRNA transfection), and 1d, 2d, and 3d represent cell lysates collected on days 1, 2, and 3 after transfection, respectively; β-actin is the internal reference protein, and the arrow indicates the expected band position of the S protein (approximately 180 kDa).

[0073] like Figure 4 As shown, each construct exhibited a clear, specific band at approximately 180 kDa, consistent with the expected apparent molecular weight of the S protein after glycosylation.

[0074] Example 4 Preparation and Physicochemical Characterization of Trivalent mRNA-LNP Formulation This embodiment uses microfluidic mixing technology to prepare mRNA-LNP formulations.

[0075] The lipid phase of this embodiment was prepared by dissolving SM-102, DSPC, cholesterol, and PEG2000-DMG in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5. The aqueous phase contained the three S protein trimer mRNAs obtained in Example 2. These three S protein trimer mRNAs were mixed at a mass ratio of 1:1:1 and dissolved in a 50 mM citrate buffer with a pH of 4.0, controlling the N / P ratio to be 8. The N / P ratio (nitrogen-to-phosphate ratio) refers to the molar ratio of ionizable lipid nitrogen atoms to mRNA phosphate groups.

[0076] Rapidly mix the aqueous phase and lipid phase using a microfluidic mixing device at a flow rate ratio of 3:1 and a total flow rate of 12 mL / min. Then, use ultrafiltration centrifugation to replace the mixture with a 20 mM Tris-HCl (pH 7.4) buffer solution.

[0077] The morphological and physicochemical characterization diagrams of mRNA-LNP are shown below. Figure 5 As shown. Figure 5 The values ​​above the PDI bar chart for B are the particle size (nm) and PDI value, respectively, and the dashed line represents the upper limit of acceptable particle size (150 nm). Figure 5 C: Zeta potential of each component; Figure 5 In the encapsulation efficiency (EE%) of each component in D, the dashed line represents the minimum acceptable level (80%).

[0078] like Figure 5 The physicochemical characterization results show that transmission electron microscopy (TEM) reveals that the LNP particles are generally spherical and morphologically intact. Dynamic light scattering (DLS) analysis indicates that the particle size of each group is mainly distributed between 77 and 125 nm, with the trivalent trimer group having an average particle size of approximately 86 nm and a PDI of 0.10–0.19. The zeta potential of each group is close to neutral and slightly negative, and the encapsulation efficiency remains between 97% and 98%.

[0079] Example 5: Mouse Immunization Experiment and Evaluation of Immune Interference Six-week-old specific pathogen-free (SPF) female BALB / c mice were randomly divided into groups (n=6 per group). The groups included a trivalent trimer group (Mix Trimer, total dose 30 μg, each antigen mRNA 10 μg), a trivalent monomer group (Mix Monomer, total dose 30 μg), each monovalent trimer and monomer group (10 μg), and a phosphate-buffered saline (PBS) negative control group. All groups received a primary immunization via intramuscular injection at week 0, followed by a booster injection of the same dose intramuscularly at week 2 (IM+IM regimen). Blood was collected via the mandibular vein at week 2 post-primary immunization (before booster) and week 2 post-booster (week 4 post-primary immunization), and serum was separated. The mouse groups for evaluating the systemic immunogenicity of different mRNA vaccine conformations are shown in Table 1.

[0080] Table 1. Mouse grouping for systemic immunogenicity evaluation of mRNA vaccines with different conformations Note: In the table, SARS2 represents SARS-CoV-2, SARS1 represents SARS-CoV-1, and MERS represents MERS-CoV; the dosages in the table are single-dose doses, and the initial immunization (week 0) and booster immunization (week 2) doses are the same.

[0081] (1) Specific IgG antibody response: In this embodiment, serum-specific IgG was detected using an indirect enzyme-linked immunosorbent assay (ELISA). Two weeks after the booster immunization, the IgG titers against SARS-CoV-2, SARS-CoV-1, and MERS-CoV S proteins in the trivalent trimeric group were 1,840,000, 269,339, and 182,268, respectively, all of which were not lower than those in the corresponding monovalent trimeric group (419,740, 189,679, and 147,673), indicating that trivalent co-delivery did not inhibit the IgG response of any component.

[0082] Figure 6 In the graph showing the titer of specific IgG antibodies in the serum of immunized mice, the horizontal axis represents the groups in order: PBS control group, trivalent trimer group, trivalent monomer group, each monovalent trimer group, and each monovalent monomer group. All data comparisons between groups were assessed using two-way ANOVA. p >0.05, * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001.

[0083] like Figure 6 As shown, the IgG titers in the trivalent trimeric group were approximately 2.5 times higher than those in the trivalent monomer group (SARS-CoV-2: 1840000 vs 723253). p <0.01; SARS-CoV-1: 269339 vs 134276, approximately 2.0 times, p >0.05; MERS-CoV: 182268 vs 96667, approximately 1.9 times, p <0.0001), confirming that the trimerization design can enhance IgG levels, with consistent trends across groups in the second week after primiparity.

[0084] (2) Assessment of pseudovirus neutralizing antibody response and immune interference: This embodiment uses a sham virus neutralization test to detect functional neutralizing activity. Figure 7 This is a graph showing the neutralization inhibition curves of immunized mouse serum against SARS-CoV-2 (original strain, XBB.1.5), SARS-CoV-1, and MERS-CoV pseudoviruses. The X-axis represents the sample dilution factor (Log10), and the Y-axis represents the neutralization inhibition rate (%). The red curve represents the trivalent group, and the blue, orange, and green curves represent the monovalent groups, respectively (see legend). The horizontal dashed line represents the 50% neutralization inhibition rate. The data were fitted using a four-parameter nonlinear regression model.

[0085] Figure 8 Antibody titer (pVNT) 50 (Comparison chart, the number at the top of the bar represents pVNT) 50 The values ​​are shown in the figure, and the group names corresponding to each bar are labeled on the horizontal axis. All comparisons between groups were performed using two-way ANOVA, with the symbol **** indicating statistical differences between groups. p < 0.0001, *** p <0.001,** p <0.01, * p <0.05).

[0086] It can be seen that in the second week after booster immunization, the trivalent trimeric group showed reduced pVNT levels against the original SARS-CoV-2 strain, SARS-CoV-1, and MERS-CoV. 50 The values ​​were 4596, 4018, and 4875, respectively, with corresponding monovalent trimeric groups of 2218, 2667, and 3334. The trivalent group showed resistance to the SARS-CoV-2 pVNT... 50 Significantly higher than the unit price group ( p <0.001), pVNT for SARS-CoV-1 and MERS-CoV 50 It was also higher than the unit price group, but the difference was not statistically significant. p >0.05).

[0087] Based on the combined data of IgG and neutralizing antibodies, no immune interference was observed after the three heterologous S protein mRNAs were co-delivered in equal mass ratios within the same LNP, and each component induced an effective immune response.

[0088] (3) The enhancing effect of trimerization on neutralizing antibodies: like Figure 8 As shown, in all vaccine groups, the neutralizing activity of the trimer construct was higher than that of the corresponding monomer construct. In the monovalent group, the SARS-CoV-2 trimer pVNT... 50 (2218) is approximately 2.1 times that of monomer (1057). p <0.001), SARS-CoV-1 (2667 vs 1832) was approximately 1.5 times higher ( p >0.05), MERS-CoV (3334 vs 2009) is about 1.7 times higher ( p <0.05); In the trivalent group, the trivalent trimeric group showed resistance to pVNT of SARS-CoV-2, SARS-CoV-1, and MERS-CoV. 50 (4596, 4018, 4875) are approximately 1.6 times larger than the trivalent monomer groups (2891, 2259, 2958). p <0.05), 1.8 times ( p <0.01) and 1.6 times ( p <0.05). The T4-Fd trimerization design can enhance the neutralizing antibody response in both monovalent and trivalent systems.

[0089] (4) Cross-neutralizing activity against SARS-CoV-2 variants: like Figure 8 As shown, the trivalent trimer group antagonizes the pVNT of the non-target mutant strain Omicron XBB.1.5. 50 The value was 1224, approximately 7.4 times that of the monovalent SARS-CoV-2 trimeric group (166). p <0.0001), which is also about 2.3 times that of the trivalent monomer group (526). p <0.001), indicating that trivalent co-delivery can significantly enhance the cross-neutralization ability against mutants, and trimerization further amplifies this effect.

[0090] Example 6: Induction of respiratory mucosal IgA via heterologous immune pathway Based on the trivalent trimer group design in Example 5, this embodiment adjusts the booster dose to 3 μg / animal to match the dose of the intranasal booster group. The animals are divided into the IM+IN group (intramuscular injection primary immunization - intranasal booster group) and the IM+IM group (intramuscular injection primary immunization - intramuscular injection booster group) to verify the effects of different immunization routes.

[0091] As shown in Table 2, the initial immunization method was the same for all groups (intramuscular injection, 30 μg, week 0); the booster method varied depending on the group: the IM+IN group received an intranasal booster (3 μg / animal) in week 2, and the IM+IM group received an intramuscular booster of the same dose in week 2. At the experimental endpoint, endotracheal intubation was performed, and the lungs were lavaged with pre-cooled sterile 800 μL PBS to collect BALF. Specific IgA levels were detected by indirect ELISA.

[0092] Table 2. Grouping of mice in the mucosal immune pathway exploration experiment. Note: IM stands for Intramuscular injection, and IN stands for Intranasal injection.

[0093] Figure 9 This is a comparison of BALF-specific IgA levels induced by different immunization routes of the trivalent mRNA vaccine. In the figure, IM+IM represents the intramuscular injection primary immunization-intramuscular injection booster group; IM+IN represents the intramuscular injection primary immunization-intranasal booster group; and BALF represents bronchoalveolar lavage fluid. The bars, from left to right, represent BALF-specific IgA OD against SARS-CoV-2, SARS-CoV-1, and MERS-CoV S proteins. 450 Values. All inter-group data were compared using two-way ANOVA to assess statistical differences (****). p <0.0001, *** p <0.001,** p <0.01, * p <0.05).

[0094] like Figure 9 As shown, the IM+IN group exhibits BALF-specific IgA OD against the S proteins of SARS-CoV-2, SARS-CoV-1, and MERS-CoV. 450 The mean values ​​were approximately 2.5, 1.8, and 2.1, respectively, all significantly higher than the IM+IM group. p (All <0.0001). The traditional IM+IM approach failed to effectively induce local respiratory-specific IgA, while the IM+IN strategy significantly increased local IgA levels against the three coronaviruses, demonstrating the effectiveness of heterologous immunization strategies in the multivalent trimeric mRNA-LNP system.

[0095] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition, characterized in that, The composition comprises a first nucleic acid, a second nucleic acid, and a third nucleic acid, wherein the first nucleic acid comprises mRNA encoding the SARS-CoV-2 S protein, the second nucleic acid comprises mRNA encoding the SARS-CoV-1 S protein, and the third nucleic acid comprises mRNA encoding the MERS-CoV S protein; The SARS-CoV-2 S protein, the SARS-CoV-1 S protein, and the MERS-CoV S protein all contain stable proline mutations, and an exogenous trimerized domain is inserted between the C-terminus of the extracellular domain and the transmembrane region of each S protein. The SARS-CoV-2 S protein and the MERS-CoV S protein also include inactivation mutations at the furin cleavage site.

2. The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition according to claim 1, characterized in that, In the amino acid sequence of the SARS-CoV-2 S protein, the furin protease cleavage site inactivation mutation is changed from RRAR to GSAS, the proline stable mutation is changed to 6P proline stable mutation, and the mutation sites are F817P, A892P, A899P, A942P, K986P, and V987P; the exogenous trimerization domain is a T4 phage fibrin fold; the amino acid sequence of the SARS-CoV-2 S protein has a primitive transmembrane region and a cytoplasmic tail region.

3. The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition according to claim 1, characterized in that, In the amino acid sequence of the SARS-CoV-1 S protein, the proline is stably mutated to a 6P proline stable mutation, with mutation sites F799P, A874P, A881P, A924P, K968P, and V969P; the exogenous trimerization domain is a T4 phage fibrin fold; and the amino acid sequence of the SARS-CoV-1 S protein has a primitive transmembrane region and a cytoplasmic tail region.

4. The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition according to claim 1, characterized in that, In the amino acid sequence of the MERS-CoV S protein, the furin protease cleavage site inactivation mutation is RSVR to ASVG, the proline stable mutation is 2P proline stable mutation, and the mutation sites are V1060P and L1061P; the exogenous trimerization domain is a T4 phage fibrin fold; the amino acid sequence of the MERS-CoV S protein has a primitive transmembrane region and a cytoplasmic tail region.

5. A trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition according to any one of claims 1-4, characterized in that, The amino acid sequence of the SARS-CoV-2 S protein is shown in SEQ ID NO:1, the amino acid sequence of the SARS-CoV-1 S protein is shown in SEQ ID NO:3, and the amino acid sequence of the MERS-CoV S protein is shown in SEQ ID NO:

5.

6. The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition according to claim 5, characterized in that, The mRNA nucleotide sequence for encoding the SARS-CoV-2 S protein is shown in SEQ ID NO:2, the mRNA nucleotide sequence for encoding the SARS-CoV-1 S protein is shown in SEQ ID NO:4, and the mRNA nucleotide sequence for encoding the MERS-CoV S protein is shown in SEQ ID NO:

6.

7. The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition according to claim 6, characterized in that, The 5'UTR sequences of the first nucleic acid, the second nucleic acid, and the third nucleic acid are identical, as are the 3'UTR sequences of the first nucleic acid, the second nucleic acid, and the third nucleic acid.

8. The trivalent β-coronavirus S protein trimer mRNA-LNP vaccine composition according to claim 5, characterized in that, In the composition, the mass ratio of the first nucleic acid, the second nucleic acid, and the third nucleic acid is 1:1:

1.

9. A trivalent β-coronavirus S protein trimer mRNA-LNP vaccine, characterized in that, The invention includes lipid nanoparticles comprising the trivalent β-coronavirus S protein trimer mRNA composition as described in any one of claims 1-8 and liposomes for encapsulating the composition.

10. A method for preparing a trivalent β-coronavirus S protein trimer mRNA-LNP vaccine as described in claim 9, characterized in that, The aqueous phase containing the trivalent β-coronavirus S protein trimer mRNA composition and the lipid phase containing the liposomes are mixed and encapsulated under acidic conditions using a microfluidic method to obtain the lipid nanoparticles. The lipid nanoparticles are then mixed with a pharmaceutically acceptable solvent to prepare the vaccine.

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