MRNA OR MRNA COMPOSITION, AND PREPARATION METHOD THEREFOR AND APPLICATION THEREOF

Abstract

Provided are an mRNA or an mRNA composition, and an mRNA vaccine comprising the mRNA or the mRNA composition. The mRNA or the mRNA composition comprises an mRNA sequence encoding an S protein of a novel coronavirus SARS-CoV-2 or a variant thereof, and an mRNA sequence encoding an RBD in the S protein or a variant thereof. Further provided are the applications of the mRNA or the mRNA composition, and the mRNA vaccine comprising the mRNA or the mRNA composition in preparation of a medication for preventing and/or treating a disease caused by a novel coronavirus SARS-CoV-2 infection.

Claims

1-24. (canceled)

25. An mRNA or mRNA composition, comprising: an mRNA sequence encoding an S protein of SARS-CoV-2 or a variant thereof, and an mRNA sequence encoding an RBD in the S protein or a variant thereof.

26. The mRNA or mRNA composition according to claim 25, wherein the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof and the mRNA sequence encoding the RBD in the S protein or the variant thereof are derived from the same SARS-CoV-2 mutant or different SARS-CoV-2 mutants.

27. The mRNA or mRNA composition according to claim 25, wherein the S protein or the variant thereof comprises a wild-type full-length S protein or a full-length S protein fixed in a pre-fusion conformation.

28. The mRNA or mRNA composition according to claim 25, wherein the full-length S protein fixed in the pre-fusion conformation comprises a mutation at positions 682RRAR685 and/or a mutation at positions 986KV987.

29. The mRNA or mRNA composition according to claim 27, wherein the wild-type full-length S protein comprises an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to an amino acid sequence as set forth in SEQ ID NO: 1; the full-length S protein fixed in the pre-fusion conformation comprises an amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 15, or an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 2 or 15.

30. The mRNA or mRNA composition according to claim 25, wherein the S protein or the variant thereof does not comprise a signal peptide, comprises a signal peptide of the wild-type S protein or comprises a signal peptide of the wild-type S protein and a preceding strong signal peptide.

31. The mRNA or mRNA composition according to claim 25, wherein the RBD comprises an amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 13, or an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 3 or 13.

32. The mRNA or mRNA composition according to claim 25, wherein the RBD or the variant thereof does not comprise a signal peptide, comprises a signal peptide of the wild-type S protein or comprises a signal peptide of the wild-type S protein and a preceding strong signal peptide.

33. The mRNA or mRNA composition according to claim 25, wherein the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof and the mRNA sequence encoding the RBD in the S protein or the variant thereof are two separate mRNA sequences or are ligated in one mRNA sequence.

34. The mRNA or mRNA composition according to claim 33, wherein the ligation order for the one mRNA sequence from 5′ to 3′ is: the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof to the mRNA sequence encoding the RBD in the S protein or the variant thereof, or the mRNA sequence encoding the RBD in the S protein or the variant thereof to the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof.

35. The mRNA or mRNA composition according to claim 25, wherein the mRNA is a conventional mRNA, a self-amplifying mRNA, or a trans-amplifying mRNA.

36. The mRNA or mRNA composition according to claim 25, wherein the mRNA or mRNA composition comprises an mRNA sequence consisting of the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof and the mRNA sequence encoding the RBD in the S protein or the variant thereof selected from any one of the following: A) consisting of the 5′ cap, the 5′ non-coding region, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the 3′ non-coding region and the polyA tail; B) consisting of the 5′ cap, the 5′ non-coding region, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the 3′ non-coding region and the polyA tail; C) consisting of the 5′ cap, the 5′ non-coding region, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the internal ribosome entry site (IRES), the mRNA sequence encoding the RBD in the S protein or the variant thereof, the 3′ non-coding region and the polyA tail; D) consisting of the 5′ cap, the 5′ non-coding region, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the IRES, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the 3′ non-coding region and the polyA tail; E) consisting of the 5′ cap, the 5′ conserved sequence element, the RNA replicase coding region, the subgenomic promoter, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the 3′ conserved sequence element and the polyA tail; F) consisting of the 5′ cap, the 5′ conserved sequence element, the RNA replicase coding region, the subgenomic promoter, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the 3′ conserved sequence element and the polyA tail; G) consisting of the 5′ cap, the 5′ conserved sequence element, the RNA replicase coding region, the subgenomic promoter, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the IRES, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the 3′ conserved sequence element and the polyA tail; and H) consisting of the 5′ cap, the 5′ conserved sequence element, the RNA replicase coding region, the subgenomic promoter, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the IRES, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the 3′ conserved sequence element and the polyA tail.

37. The mRNA or mRNA composition according to claim 25, wherein the mRNA or mRNA composition comprises a combination of two mRNA sequences selected from any one of the following: a) an mRNA consisting of the 5′ cap, the 5′ non-coding region, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the 3′ non-coding region and the polyA tail, combined with an mRNA consisting of the 5′ cap, the 5′ non-coding region, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the 3′ non-coding region and the polyA tail; b) an mRNA consisting of the 5′ cap, the 5′ conserved sequence element, the RNA replicase coding region, the subgenomic promoter, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the 3′ conserved sequence element and the polyA tail, combined with an mRNA consisting of the 5′ cap, the 5′ conserved sequence element, the RNA replicase coding region, the subgenomic promoter, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the 3′ conserved sequence element and the polyA tail; c) an mRNA consisting of the 5′ cap, the 5′ conserved sequence element, the subgenomic promoter, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the IRES, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the 3′ conserved sequence element and the polyA tail, combined with an mRNA consisting of the 5′ cap, the 5′ non-coding region, the RNA replicase coding region, the 3′ non-coding region and the polyA tail; d) an mRNA consisting of the 5′ cap, the 5′ conserved sequence element, the subgenomic promoter, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the IRES, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the 3′ conserved sequence element and the polyA tail, combined with an mRNA consisting of the 5′ cap, the 5′ non-coding region, the RNA replicase coding region, the 3′ non-coding region and the polyA tail; e) an mRNA consisting of the 5′ cap, the 5′ conserved sequence element, the subgenomic promoter, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the 3′ conserved sequence element and the polyA tail, combined with an mRNA consisting of the 5′ cap, the 5′ non-coding region, the RNA replicase coding region, the 3′ non-coding region and the polyA tail; and f) an mRNA consisting of the 5′ cap, the 5′ conserved sequence element, the subgenomic promoter, the mRNA sequence encoding the RBD in the S protein or the variant thereof, the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof, the 3′ conserved sequence element and the polyA tail, combined with an mRNA consisting of the 5′ cap, the 5′ non-coding region, the RNA replicase coding region, the 3′ non-coding region and the polyA tail.

38. The mRNA or mRNA composition according to claim 37, wherein the RNA replicase coding region is selected from alphavirus, picornavirus, flavivirus, paramyxovirus and calicivirus.

39. An mRNA vaccine comprising the mRNA or mRNA composition according to claim 25.

40. The mRNA vaccine according to claim 39, wherein in the mRNA vaccine, the mass ratio of the mRNA sequence encoding the S protein of SARS-CoV-2 or the variant thereof to the mRNA sequence encoding the RBD in the S protein or the variant thereof is (1-5) : (1-5).

41. The mRNA vaccine according to claim 39, wherein the mRNA vaccine is a liposome, a lipid complex or a lipid nanoparticle.

42. A method for preventing or treating a disease caused by SARS-CoV-2 infection or resisting SARS-CoV-2 infection, comprising administering the mRNA or mRNA composition according to claim 25.

43. A method for screening an antibody, comprising administering to an individual the mRNA or mRNA composition according to claim 25.

44. A method for inducing a neutralizing antigen-specific immune response in an individual, comprising administering to the individual the mRNA or mRNA composition according to claim 25.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0109] FIG. 1: sequencing result of the sequence encoding the wild-type SARS-CoV-2 RBD with tPA signal peptide.

[0110] FIG. 2: sequencing result of the sequence encoding the wild-type SARS-CoV-2 S protein, combining FIG. 2A and FIG. 2B.

[0111] FIG. 3: sequence map of a basic plasmid template comprising T7 promoter, 5′ UTR, 3′ UTR and polyA tail.

[0112] FIG. 4: detection of capped mRNA in denaturing formaldehyde gel after purification, wherein M denotes marker, 1 denotes the mRNA encoding the wild-type SARS-CoV-2 RBD with tPA signal peptide, and 2 denotes the mRNA encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987.

[0113] FIG. 5: WB (western blot) results, wherein 1 denotes the mRNA encoding the wild-type SARS-CoV-2 RBD with tPA signal peptide, and 2 denotes the negative control.

[0114] FIG. 6: immunofluorescence result.

[0115] FIG. 7: particle size and the particle size distribution result of mRNA-LNP by DLS (dynamic light scattering), wherein A denotes RBD+S-1-LNP, B denotes RBD+S-2-LNP, C denotes RBD+S-3-LNP, D denotes RBD-LNP, and E denotes S-LNP.

[0116] FIG. 8: mRNA integrity result of the packaged sample by formaldehyde denatured gel, wherein 1 denotes the mRNA encoding the wild-type SARS-CoV-2 RBD with tPA signal peptide, 2 denotes RBD+S-1, 3 denotes RBD+S-2, 4 denotes RBD+S-3, and 5 denotes the mRNA encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987.

[0117] FIG. 9: S protein-specific antibody titer after primary and secondary immunizations, wherein Negative denotes the negative control.

[0118] FIG. 10: interferon gamma (IFN-y) detection by ELISpot (enzyme linked immunospot), wherein the ordinate denotes spot forming unit (SFU) per million splenocytes, and Negative denotes the negative control.

[0119] FIG. 11: IFN-y, interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) detections by CD4 CK intracellular staining (ICS).

[0120] FIG. 12: IFN-y, interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) detections by CD8 CK intracellular staining (ICS).

[0121] FIG. 13: detection of capped mRNA in denaturing formaldehyde gel after purification, wherein M denotes marker, 1 denotes the mRNA encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987 prepared in Example 1, 2 denotes the mRNA encoding the full-length S protein of 501Y.V2 strain fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987, and 3 denotes the mRNA encoding the wild-type SARS-CoV-2 RBD sequence of 501Y.V2 strain with IgE signal peptide. The results suggested that the mRNA was of the correct size and was essentially free of degradation.

[0122] FIG. 14: WB assay results, wherein 1 denotes the expression supernatant of the mRNA encoding the wild-type SARS-CoV-2 RBD of 501Y.V2 strain with IgE signal peptide, 2 denotes the expression supernatant of the mRNA encoding the full-length S protein of 501Y.V2 strain fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987, 3 denotes the cell supernatant of the negative control, 4 denotes the expression cell pellet of the mRNA encoding the wild-type SARS-CoV-2 RBD sequence of 501Y.V2 strain with IgE signal peptide, 5 denotes the expression cell pellet of the mRNA encoding the full-length S protein of 501Y.V2 strain fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987, and 6 denotes the cell pellet of the negative control.

[0123] FIG. 15: particle size and the particle size distribution result of mRNA-LNP by DLS (dynamic light scattering).

[0124] FIG. 16: titer of S protein-specific antibody for 501Y.V2 strain after primary and secondary immunizations.

[0125] FIG. 17: titer of S protein-specific antibody for Wuhan-Hu-1 isolate after primary and secondary immunizations.

[0126] FIG. 18: alternative neutralizing antibody titers, wherein the 4 groups on the left are the alternative neutralizing antibody titer against Wuhan-Hu-1 isolate and the 4 group on the right are the alternative neutralizing antibody titer against the 501Y.V2 strain.

[0127] FIG. 19: cellular immune response detection in CD4+ T cell Th1 subtype.

[0128] FIG. 20: cellular immune response detection in CD8+ T cell Th1 subtype.

DETAILED DESCRIPTION

[0129] Technical schemes in the examples of the present invention will be described clearly and completely in conjunction with the accompanying drawings. It is apparent that the examples described herein are only some examples of the present invention, but not all of them. Based on the examples of the present invention, all other examples obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0130] Sources of reagents used in the examples:

[0131] Neutralizing antibodies in the serum of immunized mice were detected by alternative neutralizing antibody assay through competitive binding of ACE2 protein and RBD protein. Specific antibodies in the serum of immunized mice were detected by specific antibody detection using RBD protein.

[0132] The RBD protein used for Wuhan-Hu-1 isolate was a wild-type RBD protein (manufacturer: Genscript, Cat. No.: Z03483-1).

[0133] The RBD protein used for 501Y.V2 strain was an RBD protein with 501Y.V2 strain mutations (manufacturer: Novoprotein, Cat. No.: DRA125).

Example 1: Preparation and Detection of mRNA

[0134] 1. Designed gene sequences of the antigens were artificially synthesized.

[0135] 2. Short nucleotide chains (primers) were synthesized through the solid phase phosphoramiditetriester method.

[0136] 3. The primers were mutually used as templates for PCR amplification.

[0137] 4. The amplification product in step 3 was ligated into pUC57 vector, transformed and sequenced.

[0138] 5. The sequence was confirmed consistent as expected by sequencing, and the results are shown in FIGS. 1-2. Specifically, FIG. 1 shows the sequencing result of the sequence encoding the wild-type SARS-CoV-2 RBD with tPA signal peptide, with the nucleotide sequence set forth in SEQ ID NO: 4 and the amino acid sequence set forth in SEQ ID NO: 3. FIG. 2 shows the sequencing results of the sequences encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987, with the nucleotide sequence set forth in SEQ ID NO: 5 and the amino acid sequence set forth in SEQ ID NO: 2.

[0139] 6. A base plasmid template comprising T7 promoter, 5′ UTR, 3′ UTR and polyA tail were prepared, with the sequence set forth in FIG. 3 (SEQ ID NO: 8).

[0140] 7. The template was subjected to PCR with homologous primers, and the result suggested a correct outcome.

[0141] The basic plasmid template was linearized with restriction endonuclease BsmBI. The PCR products were ligated to basic plasmid templates by homologous recombination, and were used for transforming an Xl1-Blue strain. The sequencing result confirmed that the sequence was correct and the transcription template was successfully constructed. The strain was cultured in shake flasks, and purified by a large-extraction kit free of endotoxin to obtain a transcription template.

[0142] The transcription template was linearized using restriction endonuclease BbsI. A T7 in-vitro transcription kit was used for transcription to obtain uncapped mRNA sequences set forth in SEQ ID NOs: 4-5 (the specific mRNA sequences are set forth in SEQ ID NOs: 16-17, respectively). The transcription templates were digested with DNaseI, and the mRNA was purified by precipitation. mRNA was capped with Cap1 capping kit and the capped mRNA was purified with mRNA purification kit. The purified mRNA was dissolved in an acidic sodium citrate buffer for later use.

[0143] The capped mRNA was detected in denaturing formaldehyde gel after purification, as shown in FIG. 4, wherein M denotes marker, 1 denotes the mRNA encoding the wild-type SARS-CoV-2 RBD with tPA signal peptide, and 2 denotes the mRNA encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987. The results suggested that the mRNA was of the correct size and was essentially free of degradation.

[0144] HEK293 cells were transferred into 3 wells on a 24-well plate. Cells in the wells 1 and 2 were transfected using lipofectamine 2000 transfection agent with 0.5 .Math.g of the capped and purified mRNA encoding the wild-type SARS-CoV-2 RBD with tPA signal peptide and the mRNA encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987, respectively, and lipofectamine 2000 transfection agent was added in well 3 as the negative control. After 24 h of transfection, cell supernatants of wells 1 and 3 were subjected to WB assay, and cells of wells 2 and 3 were fixed and subjected to immunofluorescent assay with anti-S protein polyclonal antibody. The WB assay results are shown in FIG. 5, wherein 1 denotes the mRNA encoding the wild-type SARS-CoV-2 RBD with tPA signal peptide, and 2 denotes the negative control. The results showed that the expressed protein was of correct size. The immunofluorescent assay results are shown in FIG. 6, demonstrating that the mRNA encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987 can be normally expressed.

Example 2: Preparation of mRNA Vaccines and Immunization

1. Material Preparation

[0145] 1) Cationic lipid D-Lin-MC3-DMA, distearoylphosphatidylcholine (DSPC), cholesterol, and PEGylated lipid PEG-DMG were dissolved and mixed in ethanol in a molar ratio of 50:10:38.5:1.5.

[0146] 2) The mRNA encoding the wild-type SARS-CoV-2 RBD with tPA signal peptide and the mRNA encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987 prepared in Example 1 were mixed in mass ratios of 1:1, 2:1, 1:2 to obtain mRNA mixtures, respectively marked as RBD+S-1, RBD+S-2 and RBD+S-3.

2. Procedures

[0147] RBD+S-1, RBD+S-2, RBD+S-3, the mRNA encoding the wild-type SARS-CoV-2 RBD with tPA signal peptide and the mRNA encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987 were packaged in Precision Nanosystems Ignite instrument in a flow ratio of lipid mixture:mRNA = 1:3. The packaged mRNA-LNP (LNP refers to lipid nanoparticle) was dialyzed into DPBS, ultrafiltered and concentrated, and a sample for subsequent animal studies was obtained after sterile filtration. The particle size and the particle size distribution of mRNA-LNP were detected by DLS, and the detection result is shown in FIG. 7. The particle sizes of the packaged samples were 70-100 nm, and the PDI was less than 0.2. Among these, RBD+S-1-LNP had an average particle size of 77.15 nm, a PDI of 0.038 and an intercept of 0.958, as shown in Table 1; RBD+S-2-LNP had an average particle size of 77.04 nm, a PDI of 0.055 and an intercept of 0.959, as shown in Table 2; RBD+S-3-LNP had an average particle size of 91.43 nm, a PDI of 0.049 and an intercept of 0.974, as shown in Table 3; RBD-LNP had an average particle size of 77.92 nm, a PDI of 0.036 and an intercept of 0.954, as shown in Table 4; S-LNP had an average particle size of 76.89 nm, a PDI of 0.031 and an intercept of 0.977, as shown in Table 5.

TABLE-US-00001 RBD+S-1-LNP Particle size (nm) Strength (%) Standard deviation Peak 1 81.06 100 18.64 Peak 2 0 0 0 Peak 3 0 0 0

TABLE-US-00002 RBD+S-2-LNP Particle size (nm) Strength (%) Standard deviation Peak 1 81.93 100 20.68 Peak 2 0 0 0 Peak 3 0 0 0

TABLE-US-00003 RBD+S-3-LNP Particle size (nm) Strength (%) Standard deviation Peak 1 97 100 24.54 Peak 2 0 0 0 Peak 3 0 0 0

TABLE-US-00004 RBD-LNP Particle size (nm) Strength (%) Standard deviation Peak 1 82.01 100 19.41 Peak 2 0 0 0 Peak 3 0 0 0

TABLE-US-00005 S-LNP Particle size (nm) Strength (%) Standard deviation Peak 1 80.72 100 18.76 Peak 2 0 0 0 Peak 3 0 0 0

mRNA integrity of the packaged sample was detected in formaldehyde denatured gel, as shown in FIG. 8, wherein 1 denotes the mRNA encoding the wild-type SARS-CoV-2 RBD with tPA signal peptide, 2 denotes RBD + S-1, 3 denotes RBD + S-2, 4 denotes RBD + S-3, and 5 denotes the mRNA encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987. The results suggested that the mRNA was essentially free of degradation.

[0148] BALB/c female mice aged about 6 weeks were randomized into 6 groups each containing 6 mice. The mice were intramuscularly administered at 10 .Math.g on days 0 and 28. S protein-specific antibody titer was measured on days 28 and 42. On day 42, the mice were sacrificed and the cytokine was measured.

3. Results

[0149] The S protein-specific antibody titers after the primary and secondary immunizations are shown in FIG. 9. It can be seen that after the secondary immunization, the specific antibody titer of full-length S protein immunization alone was significantly lower than that of RBD immunization alone; in the three groups receiving combined S+RBD immunization, two groups had no significant difference from RBD immunization alone in the specific antibody titer, and only the RBD+S-1 group showed a synergistic effect and a specific antibody titer significantly higher than that of RBD immunization alone. The results of interferon gamma (IFN-y) detection by ELISpot are shown in FIG. 10. It can be seen that the IFN-γ secretion of full-length S protein immunization alone was significantly higher than that of RBD immunization alone; in the three groups receiving combined S+RBD immunization, two groups had no significant difference from full-length S protein immunization alone in IFN-γ secretion, and only RBD+S-3 showed a synergistic effect and an IFN-γ secretion level significantly higher than that of full-length S protein immunization alone. IFN-y, interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) detection results by CK intracellular staining (ICS) are shown in FIGS. 11 and 12. CD4+ T cells showed low response and great individual difference, thus suggesting less significance; the result of CD8+ T cells was basically consistent with that of ELISpot. In the three groups receiving combined S+RBD immunization, two groups had no significant difference from full-length S protein immunization alone in secretions of IFN-y, IL-2 and TNF-α, and only RBD+S-3 showed significant synergistic effect. The results suggested that the combination of the full-length S protein and the RBD can integrate the cellular immune advantages of the full-length S protein and the humoral immune advantages of the RBD, achieving synergistic effect of gain and superior effect in the prevention of 2019-nCoV infection.

Example 3: Preparation and Detection of mRNA With Sequence Derived From Different SARS-CoV-2 Mutants

[0150] 1. The sequence of the wild-type SARS-CoV-2 RBD with IgE signal peptide was synthesized by amplification with primers used as templates of each other, wherein the RBD sequence contained K417N, E484K and N501Y mutations of 501Y.V2 strain, with the nucleotide sequence set forth in SEQ ID NO: 12 and the amino acid sequence set forth in SEQ ID NO: 13.

[0151] 2. The sequence encoding the full-length S protein of 501Y.V2 strain fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987 was synthesized by amplification with primers used as templates of each other, wherein the sequences contained L18F, D80A, D215G, L242-L244 deletions (L242-244del), R246I, K417N, E484K, N501Y and A701V of 501Y.V2 strain, with the nucleotide sequence set forth in SEQ ID NO: 14 and the amino acid sequence set forth in SEQ ID NO: 15.

[0152] 3. A base plasmid template comprising T7 promoter, 5′ UTR, 3′ UTR and polyA tail were prepared, with the sequence set forth in FIG. 3 (SEQ ID NO: 8).

[0153] 4. The template was subjected to PCR with homologous primers, and the result suggested a correct outcome.

[0154] The basic plasmid template was linearized with restriction endonuclease BsmBI. The PCR products were ligated to basic plasmid templates by homologous recombination, and were used for transforming an Xl1-Blue strain. The sequencing result confirmed that the sequence was correct and the transcription template was successfully constructed. The strain was cultured in shake flasks, and purified by a large-extraction kit free of endotoxin to obtain a transcription template.

[0155] The transcription template was linearized using restriction endonuclease BbsI. A T7 in-vitro transcription kit was used for transcription to obtain uncapped mRNA sequences set forth in SEQ ID NOs: 12 and 14 (the specific mRNA sequences are set forth in SEQ ID NOs: 18 and 19, respectively). The transcription templates were digested with DNaseI, and the mRNA was purified by precipitation. mRNA was capped with Cap 1 capping kit and the capped mRNA was purified with mRNA purification kit. The purified mRNA was dissolved in an acidic sodium citrate buffer for later use.

[0156] The capped mRNA was detected in denaturing formaldehyde gel after purification, as shown in FIG. 13, wherein M denotes marker, 1 denotes the mRNA encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987 prepared in Example 1, 2 denotes the mRNA encoding the full-length S protein of 501Y.V2 strain fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987, and 3 denotes the mRNA encoding the wild-type SARS-CoV-2 RBD sequence of 501Y.V2 strain with IgE signal peptide. The results suggested that the mRNA was of the correct size and was essentially free of degradation.

[0157] HEK293 cells were transferred into 3 wells on a 24-well plate. Cells in the wells 1 and 2 were transfected using lipofectamine 2000 transfection agent with 0.5 .Math.g of the capped and purified mRNA encoding the wild-type SARS-CoV-2 RBD of 501Y.V2 strain with IgE signal peptide and the mRNA encoding the full-length S protein of 501Y.V2 strain fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987, respectively, and cells in well 3 were the negative control. After 24 h of transfection, the cell supernatant and cell precipitate were separated by centrifugation and subjected to WB assay. The WB assay results are shown in FIG. 14, wherein 1 denotes the expression supernatant of the mRNA encoding the wild-type SARS-CoV-2 RBD of 501Y.V2 strain with IgE signal peptide, 2 denotes the expression supernatant of the mRNA encoding the full-length S protein of 501Y.V2 strain fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987, 3 denotes the cell supernatant of the negative control, 4 denotes the expression cell pellet of the mRNA encoding the wild-type SARS-CoV-2 RBD sequence of 501Y.V2 strain with IgE signal peptide, 5 denotes the expression cell pellet of the mRNA encoding the full-length S protein of 501Y.V2 strain fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987, and 6 denotes the cell pellet of the negative control. The results showed that the expressed protein was of correct size.

Example 4: Preparation of Combined mRNA Vaccines With Sequence Derived From Different SARS-CoV-2 Mutants and Immunization

1. Material Preparation

[0158] 1) Cationic lipid D-Lin-MC3-DMA, distearoylphosphatidylcholine (DSPC), cholesterol, and PEGylated lipid PEG-DMG were dissolved and mixed in ethanol in a molar ratio of 50:10:38.5:1.5.

[0159] 2) The mRNA encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987 prepared in Example 1, the mRNA encoding the full-length S protein of 501Y.V2 strain fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987 prepared in Example 3, and the mRNA encoding the wild-type SARS-CoV-2 RBD sequence of 501Y.V2 strain with IgE signal peptide prepared in Example 3 were prepared.

[0160] 3) The mRNA encoding the full-length S protein of 501Y.V2 strain fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987 prepared in Example 3 and the mRNA encoding the wild-type SARS-CoV-2 RBD sequence of 501Y.V2 strain with IgE signal peptide prepared in Example 3 were mixed in a mass ratio of 1:2 to obtain an mRNA mixture, abbreviated as combo A.

[0161] 4) The mRNA encoding the full-length S protein fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987 prepared in Example 1 and the mRNA encoding the wild-type SARS-CoV-2 RBD sequence of 501Y.V2 strain with IgE signal peptide prepared in Example 3 were mixed in a mass ratio of 1:2 to obtain an mRNA mixture, abbreviated as combo B.

2. Procedures

[0162] Combo A, combo B, the mRNA encoding the full-length S protein of 501Y.V2 strain fixed in the pre-fusion conformation with a mutation to GSAG at 682RRAR685 and a mutation to PP at 986KV987 prepared in Example 3 and the mRNA encoding the wild-type SARS-CoV-2 RBD sequence of 501Y.V2 strain with IgE signal peptide prepared in Example 3 were packaged in Precision Nanosystems Ignite instrument in a flow ratio of lipid mixture:mRNA = 1:3, abbreviated as combo A-LNP, combo B-LNP, S-SA-LNP and RBD-SA-LNP, respectively. The packaged mRNA-LNP was dialyzed into DPBS, ultrafiltered and concentrated, and a sample for subsequent animal studies was obtained after sterile filtration. The particle size and the particle size distribution of mRNA-LNP were detected by DLS, and the detection result is shown in FIG. 15. The particle sizes of the packaged samples were 70-100 nm, and the PDI was less than 0.2. Among these, combo A-LNP had an average particle size of 79.87 nm, a PDI of 0.132 and an intercept of 0.962, as shown in Table 6; combo B-LNP had an average particle size of 80.61 nm, a PDI of 0.123 and an intercept of 0.958, as shown in Table 7; S-SA-LNP had an average particle size of 81.13 nm, a PDI of 0.159 and an intercept of 0.939, as shown in Table 8; RBD-SA-LNP had an average particle size of 82.74 nm, a PDI of 0.112 and an intercept of 0.960, as shown in Table 9.

TABLE-US-00006 Combo A-LNP Particle size (nm) Strength (%) Standard deviation Peak 1 91.43 100 32.02 Peak 2 0 0 0 Peak 3 0 0 0

TABLE-US-00007 Combo B-LNP Particle size (nm) Strength (%) Standard deviation Peak 1 91.05 100 30.06 Peak 2 0 0 0 Peak 3 0 0 0

TABLE-US-00008 S-SA-LNP Particle size (nm) Strength (%) Standard deviation Peak 1 93.36 100 33.28 Peak 2 0 0 0 Peak 3 0 0 0

TABLE-US-00009 RBD-SA-LNP Particle size (nm) Strength (%) Standard deviation Peak 1 92.91 100 30.56 Peak 2 0 0 0 Peak 3 0 0 0

[0163] BALB/c female mice aged about 6 weeks were randomized into 5 groups each containing 6 mice. The mice were intramuscularly administered at 5 .Math.g on days 0 and 14. S protein-specific antibody titer was measured on days 14 and 28. On day 28, the mice were sacrificed and the cytokine was measured.

3. Results

[0164] The titers of S protein-specific antibody for 501Y.V2 strain after primary and secondary immunizations are shown in FIG. 16. The groups showed no significant difference. The S protein-specific antibody titers against Wuhan-Hu-1 isolate after the primary and secondary immunizations are shown in FIG. 17. The results showed that the two combined S+RBD immunizations had no significant difference in specific antibody titers, but the specific antibody titers after secondary immunization of the full-length S protein alone and RBD alone were significantly lower than those of the combination of mRNAs derived from different SARS-CoV-2 mutants (combo B). The alternative neutralizing antibody titer (as in FIG. 18, the 4 groups on the left are the alternative neutralizing antibody titer against Wuhan-Hu-1 isolate and the 4 group on the right are the alternative neutralizing antibody titer against the 501Y.V2 strain) also showed that the alternative neutralizing antibody titer against Wuhan-Hu-1 isolate of combo B was significantly higher than the mRNA combination derived from the same SARS-CoV-2 mutant (501Y.V2 strain) (combo A), full-length S protein alone and RBD alone; the alternative neutralizing antibody titer against the 501Y.V2 strain of combo B had no significant difference from combo A and RBD alone, and was significantly higher than full-length S protein alone. The results suggested that the combination of full-length S protein and RBD had humoral immunity advantages over full-length S protein alone, and that mRNA combination derived from different SARS-CoV-2 mutants can provide superior cross-protection in humoral immunity than mRNA combination derived from the same SARS-CoV-2 mutant.

[0165] The cellular immune response detection results in the CD4+ T cell Th1 subtype and the CD8+ T cell Th1 subtype are shown in FIGS. 19 and 20. The results suggested that the combination of full-length S protein and RBD has cellular immunity advantages over RBD alone. The superiority of the combined design of full-length S protein and RBD in the aspect of vaccine application is confirmed.

[0166] The preferred embodiments of the present invention are described in detail above, which, however, are not intended to limit the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, all of which will fall within the protection scope of the present invention.

[0167] In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, such combinations will not be illustrated separately.