CN114181962A

CN114181962A - Novel coronavirus mRNA vaccine and preparation method and application thereof

Info

Publication number: CN114181962A
Application number: CN202210046527.2A
Authority: CN
Inventors: 陈润生; 程铧; 张栋栋; 张欢; 于洋; 骆健俊
Original assignee: Beijing Yibo Puhui Biotechnology Development Co ltd; Institute of Biophysics of CAS
Current assignee: Beijing Yibo Puhui Biotechnology Development Co ltd; Institute of Biophysics of CAS
Priority date: 2022-01-17
Filing date: 2022-01-17
Publication date: 2022-03-15
Anticipated expiration: 2042-01-17
Also published as: CN114181962B

Abstract

The invention provides a novel coronavirus mRNA vaccine and a preparation method and application thereof. The invention firstly provides an mRNA molecule which encodes IL15 SA-antigen-Fc fusion protein, wherein the IL15 SA-antigen-Fc fusion protein sequentially comprises an amino acid sequence of IL15SA, an amino acid sequence of an antigen and an amino acid sequence of Fc from N end to C end, wherein the antigen is an antigen from a virus. The invention also provides a vaccine comprising said mRNA molecule, which induces high neutralizing antibodies and simultaneously induces high-strength specific T cell immunity, useful for preventing infection of novel coronavirus variants and original strains.

Description

Novel coronavirus mRNA vaccine and preparation method and application thereof

Technical Field

The invention relates to a virus mRNA vaccine and a preparation method and application thereof, in particular to a mRNA vaccine aiming at a novel coronavirus (SARS-CoV-2) and a preparation method and application thereof.

Background

The mRNA vaccine is one of novel coronavirus vaccines which are most concerned in recent two years, and the mRNA vaccine rapidly gets the first opportunity by the advantages of short preparation period, controllable cost, high protection efficiency and the like. The FDA of the United states approved two mRNA vaccines of Pfizer-BioNTech and Moderna successively in 12 months of 2020. However, cross-protection, long-lasting protection of mRNA vaccines is still a problem.

Disclosure of Invention

An object of the present invention is to provide an mRNA molecule, which can be used for further preparation of a viral vaccine.

Another object of the present invention is to provide a method for producing the mRNA molecule.

It is another object of the invention to provide related uses of the mRNA molecules, for example in the preparation of vaccines.

The invention provides that when designing mRNA virus vaccine, the combination of humoral immunity and cellular immunity is emphasized, and high-titer neutralizing antibody and specific T cell immunity are induced to enhance the protection rate; meanwhile, the induction of memory B cells and T cells is emphasized, thereby being beneficial to the long-term protective effect of the vaccine.

In particular, in one aspect, the invention provides an mRNA molecule encoding an IL15 SA-antigen-Fc fusion protein, said IL15 SA-antigen-Fc fusion protein comprising, in order from N-terminus to C-terminus, the amino acid sequence of IL15SA, the amino acid sequence of an antigen, the amino acid sequence of Fc, wherein said antigen is an antigen from a virus.

According to a particular embodiment of the invention, the mRNA molecule provided by the invention may comprise an antigen selected from the group consisting of: pres or X or C antigen of hepatitis b virus HBV, RBD segment or nucleocapsid protein NP on spike protein S or S protein of coronavirus, E6 or E7 antigen of HPV virus, EBNA antigen of EBV virus, or pp65 antigen of CMV virus. The coronavirus may be various coronavirus, including novel coronavirus (SARS-CoV-2, original strain or variant). The HPV virus may be HPV16 or HPV18 type.

According to a particular embodiment of the invention, the mRNA molecule is provided which encodes the amino acid sequence shown in SEQ ID number 2.

According to a particular embodiment of the invention, the mRNA molecule provided by the invention comprises, in order from the 5 'end to the 3' end: a 5 'UTR sequence, a sequence encoding the IL15 SA-antigen-Fc fusion protein, a 3' UTR sequence, and a poly A sequence.

According to a particular embodiment of the invention, the mRNA molecule provided by the invention is unmodified or modified, said modification comprising: one or more of a 5' end capping modification and a pseudouridine triphosphate modification.

On the other hand, the invention also provides a fusion protein, and the amino acid sequence of the fusion protein is the amino acid sequence obtained by encoding the mRNA molecule.

According to a specific embodiment of the present invention, the fusion protein provided by the present invention, that is, IL15 SA-antigen-Fc fusion protein, comprises the amino acid sequence of IL15SA, the amino acid sequence of an antigen, and the amino acid sequence of Fc in order from N-terminus to C-terminus, wherein the antigen is an antigen IL15 SA-antigen-Fc fusion protein derived from a virus.

IL15SA is also called IL15 superagonist, is a fusion protein containing IL-15 gene N72D mutation and IL-15R alpha Sushi region, and the safety of the application thereof has been verified clinically. In the present invention, IL15SA is linked to viral antigens, allowing it to target endogenous DC cells, which in turn induces better B-cell and T-cell immunity. The Fc part can increase the half-life and stability of the fusion protein, and simultaneously has high affinity with FcR or FcRn, can target antigen presenting cells such as DC cells and macrophages, and increases the antigen presenting efficiency.

According to a particular embodiment of the invention, the fusion protein provided by the invention is an isolated protein.

According to some embodiments of the present invention, in the fusion protein of the present invention, the amino acid sequence of IL15SA has an amino acid sequence consisting of amino acids 1 to 229 as shown in SEQ ID number 2.

According to some embodiments of the invention, in the fusion protein of the invention, the Fc may be an Fc fragment of IgG1, IgG2, IgG3 or IgG 4. In some more specific embodiments of the present invention, the amino acid sequence of the Fc has an amino acid sequence consisting of amino acids 559-775 as shown in SEQ ID number 2.

According to some embodiments of the present invention, the IL15SA amino acid sequence and the antigen amino acid sequence, and/or the antigen amino acid sequence and the Fc amino acid sequence of the fusion protein of the present invention may be independently linked by a linker or a hinge as required. There are various options for the linker or hinge suitable for use in the invention, and besides those used in the embodiments of the invention, there may be selected IgG2 hinge (erkccvecppcp), IgG3 hinge (elktpldtthtcprcp) or IgG4 hinge (eskygpcppscp).

According to some embodiments of the invention, the antigen is an RBD fragment of the S protein of the spike protein of a coronavirus. In some more specific embodiments, the amino acid sequence of the RBD segment has an amino acid sequence consisting of amino acids 242-541 as shown in SEQ ID number 2.

According to some embodiments of the invention, there is provided a fusion protein having an amino acid sequence as shown in SEQ ID number 2.

According to some embodiments of the invention, there is provided an mRNA molecule encoding the amino acid sequence shown as SEQ ID number 2.

In another aspect, the invention also provides a DNA molecule encoding the mRNA molecule of the invention.

According to some embodiments of the invention, the DNA molecule is provided having the nucleotide sequence shown as SEQ ID number 1.

In another aspect, the invention also provides a recombinant plasmid comprising the DNA molecule of the invention. The recombinant plasmid can be prepared by loading the DNA of the present invention onto an empty vector according to the prior art in the art.

In another aspect, the invention also provides lipid nanoparticles loaded with the mRNA molecules of the invention. The lipid nanoparticles can be prepared by mixing the mRNA molecules of the present invention with LNPs according to the prior art in the field. In general, LNP is used in which the proportion of cationic lipids in the total lipids is not less than 50 mol%. The loading of the mRNA molecules in the lipid nanoparticles is about 50% to 80%.

In another aspect, the present invention also provides a viral vaccine comprising: the mRNA molecule, the fusion protein, the DNA molecule, the recombinant plasmid or the lipid nanoparticle of the present invention.

In another aspect, the present invention also provides a neutralizing antibody induced and isolated from the viral vaccine of the present invention.

According to some embodiments of the invention, the mRNA molecule provided by the invention has a structural gene IL15SA-RBD δ -FC1 comprising three parts (see fig. 1), IL15SA, the RBD region of the δ mutant spike protein, and a FC1 fragment, respectively. Wherein: IL15SA and RBD gene are connected by flexible linker or IgG hinge to ensure that two proteins can be folded into natural spatial conformation to normally play respective functions, namely IL15SA activates cell responses such as DC, T, B, NK and the like and induces the generation of memory T, B cells, and RBD is correctly recognized by antigen presenting cells. The hinge structure of an IgG antibody is reserved between the RBD and the IgG1FC segment, the IgG1 hinge region ensures the flexibility of the spatial conformation of an antigen part, and the disulfide bond enables the protein to form a dimer so as to ensure the more complex conformation of the antigen protein and more effectively present, simultaneously, the stability of the protein is increased, and the purification is facilitated.

The invention is proved by experiments that: the IL15SA-RBD delta-FC 1 can induce high neutralizing antibody which is 20-80 times higher than that reported in the literature, and can synchronously induce high-strength specific T cell immunity which is 20-40 times higher than that reported in the literature. The IL15SA-RBD delta-FC 1 of the present invention is useful for preventing infection of novel coronavirus variants and original strains.

According to some embodiments of the present invention, the RBD δ region of IL15SA-RBD δ -FC1 of the present invention can be replaced with an antigen gene of other virus which can induce neutralizing antibody, such as the oricron mutant RBD region or Pres antigen of hepatitis b virus, etc., for the prevention of other virus.

According to some embodiments of the invention, the RBD δ region of IL15SA-RBD δ -FC1 of the present invention can be replaced with structural proteins of various viruses, such as X or C antigen of hepatitis b virus HBV, nucleocapsid protein NP of various coronaviruses (including novel coronaviruses), E6 or E7 antigen of HPV viruses such as HPV16 or HPV18 type, EBNA antigen of EBV virus, or pp65 antigen of CMV virus, etc., for the treatment and long-term protection of viral infections.

Drawings

FIG. 1 is a schematic structural diagram of IL15SAA-RBD delta-FC 1 according to the present invention.

FIG. 2 shows the results of detection of mRNA products of IL15 SA-RBD. delta. -FC1 transcribed in vitro.

FIG. 3 shows the results of particle size, homogeneity and integrity tests of LNP-encapsulated mRNA.

FIG. 4 shows the results of detection of IL15SA-RBD delta-FC 1 mRNA vaccine induced binding to antibodies.

FIG. 5 shows the results of detection of IL15SA-RBD delta-FC 1 mRNA vaccine-induced neutralizing antibodies.

FIG. 6 shows the results of detection of cytokine secretion by T cells stimulated by the polypeptide.

FIG. 7 shows the results of the detection of specific CD8+ T cell ratios following polypeptide stimulation.

FIG. 8 shows the results of the mRNA vaccine tested for cardiotoxicity, nephrotoxicity and hepatotoxicity.

Detailed Description

In order to clearly understand the technical features, objects and advantages of the present invention, the following detailed description of the technical solutions of the present invention is made with reference to the specific embodiments and the accompanying drawings, which are understood to be merely illustrative of the present invention and not limiting the scope of the present invention. Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. The method operations not specifically mentioned in the examples were carried out according to the conventional operations of the prior art or the operations suggested by the manufacturer's specifications.

Example 1

This example provides a novel coronavirus mRNA vaccine whose structural genes consist essentially of three parts (see FIG. 1), IL15SA, the RBD region of the delta mutant spike protein, and a fragment of FC 1. Wherein:

IL15SA and RBD gene are connected by flexible linker or IgG hinge to ensure that two proteins can be folded into natural spatial conformation, and then the respective functions are normally exerted, IL15SA activates cell reactions such as DC, T, B, NK and the like, and induces the generation of memory T, B cells, and RBD is correctly recognized by antigen presenting cells;

the hinge structure of an IgG antibody is reserved between the RBD and the IgG1FC segment, the IgG1 hinge region ensures the flexibility of the space conformation of an antigen part, and the disulfide bond ensures that the protein can form a dimer to ensure the more complex conformation of the antigen protein and be presented more effectively;

the IgG1FC part increases the half-life and stability of the fusion protein, has high affinity with FcR or FcRn, can target antigen presenting cells such as DC cells and macrophages, and increases the antigen presenting efficiency.

1. Construction of IL15SA-RBD delta-FC 1 vector

Both IL15SA and RBD delta gene sequences were synthesized by codon optimization by Kingchi corporation. The IgG1FC gene was derived from the invitogen pFase-IgG 1FC plasmid, and in this example, the IgG1FC gene was cloned into a lentiviral vector by PCR. It is to be noted that any sequence in which the amino acid is not changed but only the nucleotide is changed is within the scope of the present invention. Meanwhile, the vector carrying the target gene is a lentiviral vector pCDH series, and the target gene can also be constructed on other expression vectors.

Synthetic gene IL15SA (SEQ ID number 3):

GAATTCGCCACCATGGCACCTAGAAGAGCAAGAGGATGCAGAACACTTGGACTTCCTGCACTTCTTCTTCTTCTTCTTCTTAGACCTCCTGCAACAAGAGGAATCACATGCCCTCCTCCTATGTCAGTGGAACACGCAGATATCTGGGTGAAATCATACTCACTTTACTCAAGAGAAAGATACATCTGCAACTCAGGATTTAAAAGAAAAGCAGGAACATCATCACTTACAGAATGCGTGCTTAACAAAGCAACAAACGTGGCACACTGGACAACACCTTCACTTAAATGCATCAGAGATGGTGGCGGTGGCTCCGGCGGTGGCGGGTCAGGCGGTGGAGGCTCTAATTGGGTGAACGTGATCTCAGATCTTAAGAAGATTGAGGATCTTATCCAATCAATGCACATCGATGCAACACTTTACACAGAATCAGATGTGCACCCTTCATGCAAAGTGACAGCAATGAAATGCTTTCTTCTTGAACTTCAAGTGATCTCACTTGAATCAGGAGATGCATCAATCCACGATACAGTGGAGAATCTGATCATCCTTGCAAACGATTCACTTTCATCAAACGGAAACGTGACAGAATCAGGATGCAAAGAATGCGAAGAACTTGAAGAGAAGAATATAAAGGAGTTCCTTCAATCATTTGTGCACATCGTGCAAATGTTTATCAACACATCAGGATCC

synthetic gene RBD δ (SEQ ID number 4) (linker structure in underlined part):

GGATCCGGCGGGGGCGGGAGCGGCGGGGGCGGCAGCTGCACCCTGAAGAGCTTCACCGTGGAGAAGGGCATCTATCAGACAAGCAACTTCAGAGTGCAGCCCACCGAGAGCATCGTGAGATTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACAAGATTCGCTAGCGTGTACGCCTGGAACAGAAAGAGAATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCTAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAGGTGAGACAGATCGCCCCCGGGCAGACCGGCAAGATCGCCGATTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAATAGCAACAACCTGGACAGCAAGGTGGGCGGCAACTACAACTACAGATACAGACTGTTCAGAAAGAGCAACCTGAAGCCCTTCGAGAGAGACATCAGCACCGAGATCTACCAAGCCGGCAGCAAGCCCTGCAACGGCGTGGAGGGCTTCAACTGCTACTTCCCCCTGCAGAGCTACGGCTTTCAGCCCACCAACGGCGTGGGCTATCAGCCCTACAGAGTGGTCGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACCGTGTGCGGCCCCAAGAAGAGCACCAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACCGAGAGCAACAAGAAGTTCCTGCCCTTTCAGCAGTTCGGCAGAGACATCGCCGACACCACCGACGCCGTGAGAGACCCTCAGACCCTGGAGATCCTGGACATCACCCCCTGCAGCTTCGGCGGCGTGAGCGTGATCACCCCCCTCGAG

FC1 (SEQ ID number 5) (underlined structure):

ctcgaggagccaaagtcatgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcacgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgatctaga

the specific construction method of the IL15SA-RBD delta-FC 1 comprises the following steps:

(1) the slow virus skeleton vector is pCDH series, and the polyclonal enzyme cutting sites of the slow virus skeleton vector sequentially comprise EcoRI, BamHI, XhoI and XbaI.

(2) An IgG1FC primer segment was designed, and the primer sequences were 5 'GCCtcgaggagccaaagtcatgtgac (SEQ ID number 6) and 3' Gctctagatcatttacccggagacag (SEQ ID number 7), respectively. The FC gene was cloned into pCDH vectors XhoI and XbaI using pFase-IgG 1FC as a template to obtain pCDH-FC 1.

(3) The RBD delta synthetic gene is cut by BamHI and XhoI and cloned to a pCDH-FC1 vector to obtain pCDH-RBD delta-FC 1.

(4) The synthetic gene of IL15SA was digested with EcoRI and BamHI and cloned into pCDH-RBD delta-FC 1 to obtain pCDH-IL15SA-RBD delta-FC 1.

2. In vitro transcription preparation of mRNA for IL15SA-RBD delta-FC 1

(1) The Shanghai chemical company was entrusted with the synthesis of UTR sequence cloned into the commercial vector pCDNA3.1, the synthesized vector sequence comprising the following elements:

t7 promoter sequence: TAATACGACTCACTATAGG (SEQ ID number 8);

5' -UTR sequence:

GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCGCCACC（SEQ ID No. 9）；

3' -UTR sequence:

GCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA（SEQ ID No. 10）；

the sequence after UTR is 30nt polA + GCATATGACT +70nt polA.

Meanwhile, the 5 '-UTR sequence contains Nhe1 restriction site (GCTAGC) in front of the sequence, and the 3' -terminal polA sequence contains Apal restriction site (GGGCCC) behind the sequence.

(2) The IL15SA-RBD delta-FC 1 fragment on the pCDH-IL15SA-RBD delta-FC 1 plasmid is cut by enzyme and then is connected with pCDNA3.1 to obtain the vector for in vitro transcription.

(3) The vector was digested for 2h with Apa1 to obtain a linear vector. The linear vector was recovered after agarose gel electrophoresis.

(4) A commercial in vitro reverse Transcription Kit T7 High Yield RNA Transcription Kit (N-Me-Pseudo UTP) was purchased from Novonoprazan, 1. mu.g of linear plasmid was used for Transcription of mRNA according to the instruction, and all uridine triphosphates in the sequence were modified by replacing them with pseudouridine triphosphates.

(5) Commercial Capping kit, Vaccinium Capping System and mRNA Cap 2 '-O-Methyransferase, purchased from Novozam, accomplished Cap1 modification at the 5' end of mRNA by two catalytic reactions according to the instructions.

(6) The size and integrity of the transcribed sequence were checked by formaldehyde denaturing agarose gel electrophoresis, and the results are shown in FIG. 2, with a single transcribed mRNA band and no significant degradation.

3. LNP Loading modified mRNA expressing IL15SA-RBD delta-FC 1 protein

(1) LNP and fusion protein mRNA were mixed at a ratio of 1: 3. Wherein the LNP is prepared according to the prior art, and the proportion of the cationic lipid in the total lipid is about 50 mol%.

(2) Dialysis against PBS solution and concentration gave LNP particles loaded with mRNA.

(3) The particle size, uniformity and integrity of the particles were analyzed by a dynamic light scattering instrument, and as a result, as shown in fig. 3, the particle diameter was around 100nm, and the uniformity was excellent.

4. Immunization of mice

(1) 5-week-old female Balb/c mice were randomly grouped into 3 dose groups of 0.01. mu.g, 1. mu.g and 10. mu.g, respectively.

(2) The mRNA LNP particles prepared as described above were subjected to concentration measurement, and groups of mice were immunized at the 0 th and 14 th days with doses of 0.01. mu.g (in terms of mRNA, the same shall apply hereinafter), 1. mu.g and 10. mu.g, respectively, and were inoculated with vaccines by left and right hind limb muscle injections, respectively.

(3) Two weeks after the first immunization (day 14) and two weeks after the booster immunization (day 28), serum of each group was collected and frozen for use; meanwhile, the spleen of the mouse is taken two weeks after the boosting immunization, and the lymphocyte is separated to carry out the cell immunization experiment.

5. Bound antibody and neutralizing antibody assays

(1) The ELISA method was used to detect the level of bound antibody in mice by coating ELISA plates with Byddelta protein (cat # P2341) purified from Bycnanthus.

The results are shown in figure 4, where mice immunized in both the 1ug dose group and the 10ug dose group produced higher titers of bound antibody, the 10ug group induced higher titers of bound antibody. The dilution ratio of the binding antibody in the 1. mu.g dose group was 1:52000 at two weeks after the first immunization and 1:200000 at two weeks after the booster immunization, which is substantially identical to that of the Moderna mRNA vaccine (mRNA-1273) under the same conditions. More importantly, at week 9, the amount of bound antibody was not significantly different from week 4 (two weeks after booster immunization), and it was concluded from the week 4 data that the amount of neutralizing antibody at week 9 should also be comparable to the week 4 level, suggesting that long-lasting protection may occur.

(2) Commercial detection kit for neutralizing antibodies against novel coronavirus RBD, DD3101, purchased from Novopopam (detection kit for original strain since antibody kit for delta mutant strain was not available in the market at the time of research), was used for detecting the titer of neutralizing antibodies in serum. The main principle of the kit is that purified ACE2 protein is coated on a 96-well plate, and the influence of an antibody generated after mRNA immunization on the binding capacity of ACE2 protein is detected. The level of neutralizing antibodies produced in the mice was measured by ELISA according to the instructions.

The results are shown in FIG. 5, where both low and high dose immunized mice produced neutralizing antibodies, the 10 μ g dose produced higher titers of neutralizing antibodies. The dilution ratio of neutralizing antibody in two weeks after boosting immunization at a dose of 1 μ g is 1:6400, which is much higher than the geometric mean titer of 1:819 at the same dose of mRNA-1273 vaccine. In addition, in a neutralizing antibody test experiment, the original strain RBD antigen is adopted by the competitive ELISA kit, so that on one hand, the mRNA vaccine can generate cross protection reaction on the original strain, and on the other hand, the mRNA vaccine can reasonably speculate that stronger reaction can be generated if the antigen is RBD delta. In summary, the mRNA vaccines used in the present application can produce higher titers of neutralizing antibodies and thus higher protection rates.

6. T cell immune response detection

(1) And (4) recovering the separated mouse spleen lymphocytes.

(2) According to 1 × 10⁶Cells/well were plated in 48-well plates, 2 wells per sample, and lymphocyte stimulation was performed for 16 hours by adding polypeptide fragments of RBD δ protein (purchased from kasey).

(3) One of the wells was used to collect 200 μ l of supernatant for detecting the levels of IFN- γ, TNF- α and IL2 secreted into the culture medium, the results are shown in fig. 6, the polypeptide stimulation could significantly induce the immune response of T cells, secrete the corresponding stimulating factors, and consistent with the antibody results, could significantly induce cytokines such as IFN γ, TNF α, IL2 at 1 μ g dose.

(4) The other well was incubated for 5 hours with the addition of transport inhibitor.

(5) Cells were collected, stained intracellularly and extracellularly with various flow antibodies, and the proportion of CD8+ T cells secreting IFN γ was determined. The results are shown in fig. 7, and the polypeptide stimulation can obviously cause the immune response of T cells, and corresponding stimulating factors are secreted, which is consistent with the antibody results.

The Th1/Th2 cell subsets and their mutual balance play a key role in the regulation of immune responses. Previous studies on SARS-CoV have shown that Th1 biased immune responses enhance protection against viral infection, whereas Th2 type immune responses are associated with enhanced lung pathology after challenge. The data show that mRNA vaccines tend to induce Th1 type T cell responses, which are beneficial against viral infections. Meanwhile, the number of CD8+ T cells producing IFN γ after polypeptide stimulation was as high as 40% at 10 μ g dose, showing a very strong T cell immune response.

The data show that the mRNA vaccine has excellent effect in humoral and cellular immunity and has great clinical application value.

7. Safety evaluation of mRNA vaccines

Serum was collected at day 14 after the booster immunization and the mice were tested for cardiac, hepatic, and renal toxicity using a commercial kit. All manipulations were performed according to the instructions, 3 replicates per sample.

Before and after the mRNA vaccine immunization, the mice were observed to have no obvious abnormalities, including stress response, tremors, hair changes, body surface damage, nail loss, skin inflammation, etc. Mouse sera were collected on the 14 th day after the booster immunization and used for toxicity studies of organs such as heart, liver, and kidney of mice.

The clinical data of the mRNA vaccine indicate that the mRNA vaccine has certain cardiotoxicity. Here, creatinine creatine isoenzyme (CK-MB) assay was used to determine whether the mRNA vaccines used in the present application would cause heart damage in mice. The results are shown in FIG. 8. The results show that the creatinine creatine isozymes were slightly elevated in the 1. mu.g and 10. mu.g dose groups, but there was no significant difference, indicating that this vaccine did not cause severe cardiotoxicity. The liver/kidney is an important metabolic tissue in the body, and is often used as an important test object for clinical drug administration test. The metabolic damage of the liver tissue of the mouse can be detected by analyzing the activity of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT). As can be seen from FIG. 8, the dose group of 10. mu.g had the lowest glutamic-oxaloacetic transaminase, and there was no significant difference between the control and the 1. mu.g group; the glutamic-pyruvic transaminase of the 1ug and 10ug dose groups was slightly increased compared with the control group, but the difference was not obvious, which suggests that the mRNA vaccine had no obvious hepatotoxicity. Metabolic damage to mouse kidney tissue was measured by Creatinine (CR) secretion levels, and as can be seen in figure 8, the creatinine levels were slightly elevated for the 1 μ g and 10 μ g dose groups. Clinically, a small increase in CR levels may be due to a variety of factors, such as obesity, diet, and a 2-fold increase in CR remains a safe margin, thus suggesting that this mRNA vaccine does not potentially cause systemic toxicity to the kidney.

SEQUENCE LISTING

<110> Beijing assist Bopu Biotechnology development Limited

Institute of biophysics of Chinese academy of sciences

<120> novel coronavirus mRNA vaccine, preparation method and application thereof

<130> GAI21CN8637

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 2328

<212> DNA

<213> Artificial Sequence

<220>

<223> IL15SA-RBD delta-FC 1 coding sequence

<220>

<221> CDS

<222> (1)..(2328)

<400> 1

gaa ttc gcc acc atg gca cct aga aga gca aga gga tgc aga aca ctt 48

Glu Phe Ala Thr Met Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu

1 5 10 15

gga ctt cct gca ctt ctt ctt ctt ctt ctt ctt aga cct cct gca aca 96

Gly Leu Pro Ala Leu Leu Leu Leu Leu Leu Leu Arg Pro Pro Ala Thr

20 25 30

aga gga atc aca tgc cct cct cct atg tca gtg gaa cac gca gat atc 144

Arg Gly Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile

35 40 45

tgg gtg aaa tca tac tca ctt tac tca aga gaa aga tac atc tgc aac 192

Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn

50 55 60

tca gga ttt aaa aga aaa gca gga aca tca tca ctt aca gaa tgc gtg 240

Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val

65 70 75 80

ctt aac aaa gca aca aac gtg gca cac tgg aca aca cct tca ctt aaa 288

Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys

85 90 95

tgc atc aga gat ggt ggc ggt ggc tcc ggc ggt ggc ggg tca ggc ggt 336

Cys Ile Arg Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

100 105 110

gga ggc tct aat tgg gtg aac gtg atc tca gat ctt aag aag att gag 384

Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu

115 120 125

gat ctt atc caa tca atg cac atc gat gca aca ctt tac aca gaa tca 432

Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser

130 135 140

gat gtg cac cct tca tgc aaa gtg aca gca atg aaa tgc ttt ctt ctt 480

Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu

145 150 155 160

gaa ctt caa gtg atc tca ctt gaa tca gga gat gca tca atc cac gat 528

Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp

165 170 175

aca gtg gag aat ctg atc atc ctt gca aac gat tca ctt tca tca aac 576

Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asp Ser Leu Ser Ser Asn

180 185 190

gga aac gtg aca gaa tca gga tgc aaa gaa tgc gaa gaa ctt gaa gag 624

Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu

195 200 205

aag aat ata aag gag ttc ctt caa tca ttt gtg cac atc gtg caa atg 672

Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met

210 215 220

ttt atc aac aca tca gga tcc ggc ggg ggc ggg agc ggc ggg ggc ggc 720

Phe Ile Asn Thr Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

agc tgc acc ctg aag agc ttc acc gtg gag aag ggc atc tat cag aca 768

Ser Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr

245 250 255

agc aac ttc aga gtg cag ccc acc gag agc atc gtg aga ttc ccc aac 816

Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn

260 265 270

atc acc aac ctg tgc ccc ttc ggc gag gtg ttc aac gcc aca aga ttc 864

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

275 280 285

gct agc gtg tac gcc tgg aac aga aag aga atc agc aac tgc gtg gcc 912

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

290 295 300

gac tac agc gtg ctg tac aac agc gct agc ttc agc acc ttc aag tgc 960

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

305 310 315 320

tac ggc gtg agc ccc acc aag ctg aac gac ctg tgc ttc acc aac gtg 1008

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

325 330 335

tac gcc gac agc ttc gtg atc aga ggc gac gag gtg aga cag atc gcc 1056

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

340 345 350

ccc ggg cag acc ggc aag atc gcc gat tac aac tac aag ctg ccc gac 1104

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

355 360 365

gac ttc acc ggc tgc gtg atc gcc tgg aat agc aac aac ctg gac agc 1152

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

370 375 380

aag gtg ggc ggc aac tac aac tac aga tac aga ctg ttc aga aag agc 1200

Lys Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser

385 390 395 400

aac ctg aag ccc ttc gag aga gac atc agc acc gag atc tac caa gcc 1248

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

405 410 415

ggc agc aag ccc tgc aac ggc gtg gag ggc ttc aac tgc tac ttc ccc 1296

Gly Ser Lys Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

420 425 430

ctg cag agc tac ggc ttt cag ccc acc aac ggc gtg ggc tat cag ccc 1344

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro

435 440 445

tac aga gtg gtc gtg ctg agc ttc gag ctg ctg cac gcc ccc gcc acc 1392

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

450 455 460

gtg tgc ggc ccc aag aag agc acc aac ctg gtg aag aac aag tgc gtg 1440

Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val

465 470 475 480

aac ttc aac ttc aac ggc ctg acc ggc acc ggc gtg ctg acc gag agc 1488

Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser

485 490 495

aac aag aag ttc ctg ccc ttt cag cag ttc ggc aga gac atc gcc gac 1536

Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp

500 505 510

acc acc gac gcc gtg aga gac cct cag acc ctg gag atc ctg gac atc 1584

Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile

515 520 525

acc ccc tgc agc ttc ggc ggc gtg agc gtg atc acc ccc ctc gag gag 1632

Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Leu Glu Glu

530 535 540

cca aag tca tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct 1680

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

545 550 555 560

gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag 1728

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

565 570 575

gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg 1776

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

580 585 590

gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac 1824

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

595 600 605

ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac 1872

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

610 615 620

aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac 1920

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

625 630 635 640

tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc 1968

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

645 650 655

cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 2016

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

660 665 670

gaa cca cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag 2064

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

675 680 685

aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac 2112

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

690 695 700

atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 2160

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

705 710 715 720

acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 2208

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

725 730 735

aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca 2256

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

740 745 750

tgc tcc gtg atg cac gag gct ctg cac aac cac tac acg cag aag agc 2304

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

755 760 765

ctc tcc ctg tct ccg ggt aaa tga 2328

Leu Ser Leu Ser Pro Gly Lys

770 775

<210> 2

<211> 775

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 2

Glu Phe Ala Thr Met Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu

1 5 10 15

Gly Leu Pro Ala Leu Leu Leu Leu Leu Leu Leu Arg Pro Pro Ala Thr

20 25 30

Arg Gly Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile

35 40 45

Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn

50 55 60

Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val

65 70 75 80

Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys

85 90 95

Cys Ile Arg Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

100 105 110

Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu

115 120 125

Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser

130 135 140

Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu

145 150 155 160

Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp

165 170 175

Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asp Ser Leu Ser Ser Asn

180 185 190

Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu

195 200 205

Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met

210 215 220

Phe Ile Asn Thr Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr

245 250 255

Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn

260 265 270

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

275 280 285

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

290 295 300

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

305 310 315 320

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

325 330 335

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

340 345 350

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

355 360 365

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

370 375 380

Lys Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser

385 390 395 400

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

405 410 415

Gly Ser Lys Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

420 425 430

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro

435 440 445

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

450 455 460

Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val

465 470 475 480

Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser

485 490 495

Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp

500 505 510

Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile

515 520 525

Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Leu Glu Glu

530 535 540

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

545 550 555 560

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

565 570 575

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

580 585 590

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

595 600 605

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

610 615 620

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

625 630 635 640

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

645 650 655

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

660 665 670

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

675 680 685

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

690 695 700

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

705 710 715 720

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

725 730 735

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

740 745 750

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

755 760 765

Leu Ser Leu Ser Pro Gly Lys

770 775

<210> 3

<211> 693

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesis of Gene IL15SA

<400> 3

gaattcgcca ccatggcacc tagaagagca agaggatgca gaacacttgg acttcctgca 60

cttcttcttc ttcttcttct tagacctcct gcaacaagag gaatcacatg ccctcctcct 120

atgtcagtgg aacacgcaga tatctgggtg aaatcatact cactttactc aagagaaaga 180

tacatctgca actcaggatt taaaagaaaa gcaggaacat catcacttac agaatgcgtg 240

cttaacaaag caacaaacgt ggcacactgg acaacacctt cacttaaatg catcagagat 300

ggtggcggtg gctccggcgg tggcgggtca ggcggtggag gctctaattg ggtgaacgtg 360

atctcagatc ttaagaagat tgaggatctt atccaatcaa tgcacatcga tgcaacactt 420

tacacagaat cagatgtgca cccttcatgc aaagtgacag caatgaaatg ctttcttctt 480

gaacttcaag tgatctcact tgaatcagga gatgcatcaa tccacgatac agtggagaat 540

ctgatcatcc ttgcaaacga ttcactttca tcaaacggaa acgtgacaga atcaggatgc 600

aaagaatgcg aagaacttga agagaagaat ataaaggagt tccttcaatc atttgtgcac 660

atcgtgcaaa tgtttatcaa cacatcagga tcc 693

<210> 4

<211> 942

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic Gene RBD. delta

<400> 4

ggatccggcg ggggcgggag cggcgggggc ggcagctgca ccctgaagag cttcaccgtg 60

gagaagggca tctatcagac aagcaacttc agagtgcagc ccaccgagag catcgtgaga 120

ttccccaaca tcaccaacct gtgccccttc ggcgaggtgt tcaacgccac aagattcgct 180

agcgtgtacg cctggaacag aaagagaatc agcaactgcg tggccgacta cagcgtgctg 240

tacaacagcg ctagcttcag caccttcaag tgctacggcg tgagccccac caagctgaac 300

gacctgtgct tcaccaacgt gtacgccgac agcttcgtga tcagaggcga cgaggtgaga 360

cagatcgccc ccgggcagac cggcaagatc gccgattaca actacaagct gcccgacgac 420

ttcaccggct gcgtgatcgc ctggaatagc aacaacctgg acagcaaggt gggcggcaac 480

tacaactaca gatacagact gttcagaaag agcaacctga agcccttcga gagagacatc 540

agcaccgaga tctaccaagc cggcagcaag ccctgcaacg gcgtggaggg cttcaactgc 600

tacttccccc tgcagagcta cggctttcag cccaccaacg gcgtgggcta tcagccctac 660

agagtggtcg tgctgagctt cgagctgctg cacgcccccg ccaccgtgtg cggccccaag 720

aagagcacca acctggtgaa gaacaagtgc gtgaacttca acttcaacgg cctgaccggc 780

accggcgtgc tgaccgagag caacaagaag ttcctgccct ttcagcagtt cggcagagac 840

atcgccgaca ccaccgacgc cgtgagagac cctcagaccc tggagatcct ggacatcacc 900

ccctgcagct tcggcggcgt gagcgtgatc acccccctcg ag 942

<210> 5

<211> 711

<212> DNA

<213> Artificial Sequence

<220>

<223> FC1

<400> 5

ctcgaggagc caaagtcatg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 60

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 240

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420

tcccgggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 600

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcacga ggctctgcac 660

aaccactaca cgcagaagag cctctccctg tctccgggta aatgatctag a 711

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 6

gcctcgagga gccaaagtca tgtga 25

<210> 7

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 7

gctctagatc atttacccgg agacag 26

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> T7 promoter

<400> 8

taatacgact cactataggg 20

<210> 9

<211> 57

<212> DNA

<213> Artificial Sequence

<220>

<223> 5'-UTR

<400> 9

gggaaataag agagaaaaga agagtaagaa gaaatataag accccggcgc cgccacc 57

<210> 10

<211> 111

<212> DNA

<213> Artificial Sequence

<220>

<223> 3'-UTR

<400> 10

gctggagcct cggtggccta gcttcttgcc ccttgggcct ccccccagcc cctcctcccc 60

ttcctgcacc cgtacccccg tggtctttga ataaagtctg agtgggcggc a 111

Claims

1. An mRNA molecule encoding IL15 SA-antigen-Fc fusion protein, wherein the IL15 SA-antigen-Fc fusion protein comprises an amino acid sequence of IL15SA, an amino acid sequence of an antigen and an amino acid sequence of Fc from N end to C end, wherein the antigen is an antigen from a virus.

2. The mRNA molecule according to claim 1, wherein the antigen is selected from the group consisting of: pres or X or C antigen of hepatitis b virus HBV, RBD segment or nucleocapsid protein NP on spike protein S or S protein of coronavirus, E6 or E7 antigen of HPV virus, EBNA antigen of EBV virus, or pp65 antigen of CMV virus.

3. The mRNA molecule according to claim 1, wherein the mRNA molecule encodes the amino acid sequence shown in SEQ ID number 2.

4. The mRNA molecule according to claim 1, wherein the mRNA molecule comprises, in order from the 5 'end to the 3' end: a 5 'UTR sequence, a sequence encoding the IL15 SA-antigen-Fc fusion protein, a 3' UTR sequence, and a poly a sequence;

the mRNA molecule is unmodified or modified, the modifications comprising: one or more of a 5' end capping modification and a pseudouridine triphosphate modification.

5. A fusion protein having an amino acid sequence encoded by the mRNA molecule of any one of claims 1 to 4.

6. A DNA molecule encoding the mRNA molecule of any one of claims 1 to 4.

7. A recombinant plasmid comprising the DNA molecule of claim 6.

8. A lipid nanoparticle loaded with the mRNA molecule of any one of claims 1-4.

9. A viral vaccine, comprising: the mRNA molecule of any one of claims 1 to 4, the fusion protein of claim 5, the DNA molecule of claim 6, the recombinant plasmid of claim 7, or the lipid nanoparticle of claim 8.

10. A neutralizing antibody induced by and isolated from the viral vaccine of claim 9.