CN113151312B

CN113151312B - Novel coronavirus SARS-CoV-2mRNA vaccine and its preparation method and application

Info

Publication number: CN113151312B
Application number: CN202110224383.0A
Authority: CN
Inventors: 严景华; 黄庆瑞; 马素芳; 高福
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-03-02
Filing date: 2021-03-01
Publication date: 2022-12-09
Anticipated expiration: 2041-03-01
Also published as: CN113151312A

Abstract

The invention provides a novel coronavirus SARS-CoV-2mRNA vaccine and a preparation method and application thereof. The present invention provides three mRNA vaccines, namely, RBD, S1 and S vaccines. The RBD vaccine can induce high-titer antigen-specific IgG antibodies and virus neutralizing antibodies after one-needle immunization, the high-titer neutralizing antibodies can be maintained for at least 26 weeks, and remarkable immune protection can be provided for human ACE2 transgenic mice in a serum adoptive transfer protection experiment. The RBD and S vaccine of the invention can induce immune protection which can completely resist SARS-CoV-2 virus infection in a human ACE2 transgenic mouse after two-needle immunization. A large number of experimental results show that the mRNA vaccine has good immunogenicity, forms strong immune protection after immunizing organisms, and has great development potential.

Description

Novel coronavirus SARS-CoV-2mRNA vaccine and its preparation method and application

Technical Field

The invention relates to the fields of biological medicine and virology, in particular to a novel coronavirus SARS-CoV-2mRNA vaccine and a preparation method and application thereof.

Background

At present, SARS-CoV-2 is a patient infected by new corona virus, but asymptomatic infected persons can also become the source of infection, the virus is mainly spread by respiratory droplets and contact, the transmission routes such as aerosol and digestive tract are still clear, and the virus can be vertically spread by mother and infant with the latent period of 1-14 days, mostly 3-7 days. Most patients are mainly febrile, hypodynamia and dry cough, few patients are accompanied with symptoms such as nasal obstruction, watery nasal discharge, angina and diarrhea, severe patients mostly have dyspnea or hypoxemia in 1 week or 2 weeks of attack, and severe patients can rapidly progress into acute respiratory distress syndrome, septic shock, and even metabolic acidosis and blood coagulation dysfunction which are difficult to correct. At present, the clinical treatment mainly aims at the symptomatic support treatment, and no specific medicine or vaccine exists. The new type of epidemic situation of coronary pneumonia causes great loss to our country and the global economy, society and people's life health. The rapid development of effective vaccines against SARS-CoV-2 has become an important task for the majority of medical and scientific researchers.

Disclosure of Invention

The invention provides three kinds of mRNA vaccines, namely RBD, S1 and S vaccines, in order to solve the problem that a special drug and a vaccine aiming at the infection of a novel coronavirus SARS-CoV-2 are not developed at present. The RBD vaccine can induce high-titer antigen-specific IgG antibodies and virus neutralizing antibodies after one-needle immunization, the high-titer neutralizing antibodies can be maintained for at least 26 weeks, and remarkable immune protection can be provided for human ACE2 transgenic mice in a serum adoptive transfer protection experiment. The RBD and S vaccine of the invention can induce immune protection which can completely resist SARS-CoV-2 virus infection in a human ACE2 transgenic mouse after two-needle immunization. A large number of experimental results show that the three mRNA vaccines have good immunogenicity, form strong immune protection after immunizing organisms, and have great development potential.

To this end, the present invention provides in a first aspect an mRNA comprising a coding region comprising an antigenic polypeptide encoding SARS-CoV-2 or an antigenic fragment, variant or derivative thereof, wherein said antigenic polypeptide is selected from the group consisting of the receptor binding domain RBD of SARS-CoV-2, the spike protein S1 subunit of SARS-CoV-2 or the spike protein S full-length sequence of SARS-CoV-2,

preferably, the antigenic polypeptide has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to the receptor binding domain RBD of SARS-CoV-2, the spike protein S1 subunit of SARS-CoV-2, or the spike protein S full-length sequence of SARS-CoV-2,

wherein some or all of the uracil and/or cytosine in the mRNA is chemically modified to increase the stability of the mRNA in vivo.

In some embodiments of the invention, the chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the uracils in the mRNA,

wherein the substance for replacing uracil is selected from at least one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, or 5-methoxy-pseudouridine, and 2' -O-methyluridine, preferably pseudouridine or N1-methylpseudidine or N1-ethylpseudidine, further preferably N1-methylpseudidine;

and/or, the chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the cytosines in the mRNA with 5-methylcytosine.

In other embodiments of the invention, the antigenic polypeptide encoded by the mRNA is the receptor binding domain RBD protein of SARS-CoV-2 as represented by the amino acid sequence shown in SEQ ID NO. 1, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to the amino acid sequence shown in SEQ ID NO. 1.

In some embodiments of the invention, the nucleotide sequence encoding the receptor binding domain RBD of SARS-CoV-2 is set forth in SEQ ID NO. 2.

The receptor binding domain RBD of SARS-CoV-2 is the Receptor Binding Domain (RBD) in the S1 subunit of the spike protein (S protein) of SARS-CoV-2 virus, SARS-CoV-2 can infect human body by the binding of RBD and ACE2 receptor on human cell surface, therefore, RBD, S1 subunit and S can be used as immunogen in vaccine preparation.

In other embodiments of the invention, the antigenic polypeptide encoded by the mRNA is the spike protein S1 subunit of SARS-CoV-2 as set forth in the amino acid sequence set forth in SEQ ID NO. 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to the amino acid sequence set forth in SEQ ID NO. 5.

In some embodiments of the invention, the nucleotide sequence encoding the S1 subunit of the spike protein is set forth in SEQ ID NO 6.

In other embodiments of the invention, the antigenic polypeptide encoded by the mRNA is the spike protein S full length of SARS-CoV-2 having the amino acid sequence set forth in SEQ ID NO. 7, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to the amino acid sequence set forth in SEQ ID NO. 7.

In some embodiments of the invention, the full-length nucleotide sequence encoding the spike protein S is set forth in SEQ ID NO 8.

In other embodiments of the invention, the mRNA further comprises:

1) A 5' cap structure;

2) 5' UTR sequence;

3) 3' UTR sequence; and

4) (ii) a sequence of a poly (A) A,

wherein the mRNA comprises the following elements in the 5'→ 3' direction in that order: 5' cap structure, 5' UTR sequence, antigenic polypeptide of SARS-CoV-2 or an antigenic fragment, variant or derivative thereof, 3' UTR sequence and polyadenylation sequence.

In some embodiments of the invention, the 5' cap structure is selected from m ⁷ GpppG、 m ₂ ⁷ , ^3′-O GpppG、m ⁷ Gppp (5') N1 or m ⁷ Gppp(m ^2′-O ) At least one of N1.

In some preferred embodiments of the invention, the 5' cap structure is m ⁷ Gppp (5') N1 or m ⁷ Gppp(m ² ^′-O )N1。

According to the requirements of different mRNAs, different 5 'cap structures can be flexibly added at the 5' end of the mRNA.

“m ⁷ G "represents a 7-methylguanosine cap nucleoside," ppp "represents the triphosphate linkage between the 5 'carbon of the cap nucleoside and the first nucleotide of the primary RNA transcript, N1 is the most 5' nucleotide," G "represents a guanosine, and" m7 "represents a methyl group at the 7-position of guanine," m ^2′-O "represents a methyl group at the 2' -O position of the nucleotide.

In some embodiments of the invention the 5' UTR sequence is selected from an RNA sequence corresponding to the nucleic acid sequence set forth in any one of SEQ ID NOS 9-11 or homologues, fragments or variants thereof.

In other embodiments of the invention the 3' UTR sequence is selected from an RNA sequence corresponding to the nucleic acid sequence of any one of SEQ ID Nos 12 to 14 or homologues, fragments or variants thereof.

In some embodiments of the invention, the polyadenylation sequence comprises a sequence of 25 to 400 adenylates.

In some preferred embodiments of the invention, the polyadenylation sequence comprises a sequence of 50 to 400 adenylates.

In some further preferred embodiments of the invention, the poly A sequence comprises a sequence of 50-300 adenylates.

In yet still further preferred embodiments of the invention, the poly A sequence comprises a sequence of 50-250A.

In yet still further preferred embodiments of the present invention, the poly A sequence comprises a sequence of 60-200A.

In some embodiments of the invention, the mRNA has an mRNA selected from the group consisting of the mRNAs shown in SEQ ID Nos. 15-50, or an mRNA having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to the mRNA of SEQ ID Nos. 15-50.

In a second aspect, the invention provides a pharmaceutical composition comprising at least one of the mrnas of the first aspect, and optionally a delivery vehicle.

In some embodiments of the invention, the delivery vehicle is a nanoparticle.

In some preferred embodiments of the invention, the delivery vehicle is a lipid nanoparticle.

In other embodiments of the invention, the lipid nanoparticle has a net neutral charge at neutral pH.

In some embodiments of the invention, the lipid nanoparticle has an average diameter of 50-200nm and has a polydispersity index value of less than 0.4.

The nanoparticle provided by the invention can efficiently deliver mRNA, and has the following characteristics and advantages: for example, in the case of encapsulating mRNA, acidic pH conditions make ionizable cationic lipids carry positive charges, and compress negatively charged mRNA molecules, thereby achieving higher encapsulation efficiency; under the physiological pH condition, the ionizable lipid nanoparticle has neutral electricity, does not act with a negatively charged cell membrane, and has high biocompatibility; after the ionizable lipid nanoparticle forms an endosome through endocytosis and enters a cell, the acidic condition in the endosome enables the nanoparticle to be charged with positive charges again and to generate electrostatic interaction with an endosome membrane with negative charges, so that mRNA can be released.

In some embodiments of the invention, the mass ratio of mRNA to delivery vehicle is a: b, wherein A is selected from 0.05 to 2, B is selected from 1 to 100, preferably A is 0.05, B is 1; or A is 1, B is 100; or A is 2, B is 1; or A is 1 and B is 50; or A is 1 and B is 5.

In other embodiments of the present invention, the lipid nanoparticle comprises a cationic lipid and at least one selected from the group consisting of a non-cationic lipid, a sterol, a PEG-modified lipid.

In some embodiments of the invention, the lipid nanoparticle is a cationic lipid, a non-cationic lipid, a sterol, and a PEG-modified lipid in a molar ratio of 20-60.

In other embodiments of the present invention, the cationic lipid is an ionizable cationic lipid selected from one or more of the following: 2, 2-dioleylene-4-dimethylaminoethyl- [1,3] -dioxolane, dioleylene-methyl-4-dimethylaminobutyrate and di ((Z) -non-2-en-1-yl) 9- ((4- (dimethylamino) butanoyl) oxy) heptadecaheptanedionate, preferably dioleylene-methyl-4-dimethylaminobutyrate. In some embodiments of the invention, the non-cationic lipid is a neutral lipid selected from at least one of Distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC) and Dioleoylphosphatidylserine (DOPS), preferably DSPC.

In other embodiments of the invention, the sterol is cholesterol.

In some embodiments of the invention, the PEG-modified lipid is selected from at least one of PEG-DMG, PEG-DSG, and PEG-DMPE, preferably PEG-DMPE.

In other embodiments of the invention, the PEG of the PEG-modified lipid is 0.5 to 200kDa, preferably 1 to 50kDa, more preferably 1 to 5kDa, and even more preferably 2kDa in length.

In some embodiments of the invention, the pharmaceutical composition optionally contains an adjuvant.

In a third aspect, the invention provides a kit comprising an mRNA according to the first aspect of the invention and/or a pharmaceutical composition according to the second aspect of the invention.

In a fourth aspect, the invention provides mRNA of the first aspect, a pharmaceutical composition of the second aspect and a kit of the third aspect, and application of the kit in preparing a medicament for preventing and/or treating SARS-CoV-2 virus infection is provided.

A fifth aspect of the present invention provides the mRNA according to the first aspect of the present invention, the pharmaceutical composition according to the second aspect of the present invention, the method for preparing the kit according to the third aspect of the present invention, comprising the step of chemically modifying uracil and/or cytosine in the mRNA contained therein, which comprises part or all of the coding region comprising an antigenic polypeptide encoding SARS-CoV-2, wherein said antigenic polypeptide is selected from the group consisting of the receptor binding domain RBD of SARS-CoV-2, the spike protein S1 subunit of SARS-CoV-2 and the spike protein S full-length sequence of SARS-CoV-2, or an antigenic fragment, variant or derivative thereof.

In some embodiments of the invention, the chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the uracil in the coding region of said mRNA with a substance selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio T-methyl-pseudouridine, 2-thio-5-azauridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydrouridine or 5-methoxy-pseudouridine and 2' -O-methylpseudouridine, preferably with at least one of N1-methyl-pseudouridine or N-pseudoethyl-1, N-methyl-pseudouridine;

The key technology of mRNA vaccines has advanced in the last few years. mRNA vaccines have subversive advantages in terms of safety, rapid preparation and immunogenicity. The mRNA vaccine has high safety, does not have the risk of potential back mutation of attenuated vaccines, does not have the risk of incomplete inactivation of inactivated vaccines, and does not have the possibility that the DNA vaccine is integrated into a host genome to cause gene mutation. Generally, at least 5-6 months is needed for traditional vaccine development, and mRNA vaccine can realize standardized production, can produce the required vaccine within 10 days, and has incomparable advantages in dealing with new outbreak infectious diseases including SARS-CoV-2. Meanwhile, the mRNA vaccine can induce T cell and B cell immune response, has good immunogenicity, and can induce high-level neutralizing antibody generation through one-needle immunization.

The invention constructs and prepares SARS-CoV-2mRNA vaccine based on Receptor Binding Domain (RBD), S1 subunit and S full length of SARS-CoV-2 spike protein (S protein), provides mRNA which can code antigenic polypeptide or immunogenic fragment of new type coronavirus SARS-CoV-2 and pharmaceutically acceptable carrier, and selects nano particles to package and deliver, thus obtaining messenger ribonucleic acid (mRNA) vaccine for preventing or treating new type coronavirus SARS-CoV-2 infection.

The present invention synthesizes the plasmid template required for encoding SARS-CoV-2 antigenic polypeptide or mRNA of immunogenic fragment. In some embodiments of the invention, the mRNA plasmid template pHRT key elements comprise, in order 5'→ 3': t7 promoter, 5'UTR sequence, coding region of SARS-CoV-2 antigenic polypeptide or immunogenic fragment, 3' UTR sequence, polyadenylation sequence and linearized enzyme cleavage site.

The characteristic and the advantage of the preparation method of the mRNA which can code the SARS-CoV-2 antigenic polypeptide or the immunogenic fragment: (1) The linearized plasmid is selected as an mRNA transcription template, compared with a PCR product as a template, the error by-product generated in the transcription process is less, and the plasmid is easy to obtain in large quantity and has stable quality; (2) The introduction of chemically modified ribonucleotides in mRNA transcription synthesis can reduce the immunogenicity of mRNA in organisms, prolong the half-life of mRNA, further improve the expression level of target antigens and finally improve the immune protection effect of mRNA vaccines; (3) Because the plasmid template contains a polyadenylic acid sequence, the mRNA tailing step can be completed in the transcription at the same time, and the vaccine production process is simpler; (4) The yield is high, and 100-150 mug of mRNA can be finally obtained by 1 mug of template.

The present invention provides a preparation method for encapsulating mRNA using the above-described ionizable nanoparticle. The method is developed based on the microfluidic technology and has the following characteristics and advantages: rapidly, 24L nanoparticles can be prepared within 4 hours; the prepared nano particles have uniform particle size, and the polydispersity coefficient value is less than 0.4; easy scale-up, laboratory scale preparation parameters and conditions are essentially straightforward for both pilot and large scale production.

The invention has the beneficial effects that: the SARS-CoV-2mRNA vaccine of the invention has short development cycle, and is particularly suitable for the development of new sudden infectious disease vaccines including SARS-CoV-2; the safety is high, mRNA only needs to synthesize antigen in cytoplasm and then is degraded, and the mRNA can not enter cell nucleus and has no risk of inducing gene mutation; the immunogenicity is high, and high-level neutralizing antibodies can be induced by one-needle immunization and can be maintained for at least 26 weeks; the protective effect is good, and the vaccine can induce immune protection which can completely resist SARS-CoV-2 infection after being immunized by two needles.

Drawings

The invention will be explained below with reference to the drawings.

FIG. 1 is a diagram of an mRNA transcription template empty vector pHRNT plasmid;

FIG. 2 is a diagram of the mRNA transcription template pHRNT-COVID-19 plasmid;

FIG. 3 shows the linearized cleavage of the mRNA transcription template, lane 1, pHRNT-RBD; lane 2, linearized pHRNT-RBD; lane 3, pHRNT-S1; lane 4, linearized pHRNT-S1; lane 5, pHRNT-S; lane 6, linearized pHRNT-S;

figure 4 mrna vaccine dynamic light scattering size analysis;

FIG. 5 is a cryoelectron micrograph of mRNA vaccine;

FIG. 6 IgG antibody titers induced by different constructed vaccines;

FIG. 7 shows the neutralizing antibody titer of pseudovirus induced by different vaccines;

FIG. 8, SARS-CoV-2 euvirus neutralizing antibody titers induced by different constructed vaccines;

FIG. 9 enzyme-linked immunospot assay;

FIG. 10 intracellular cytokine staining of CD4 positive T cells;

FIG. 11 intracellular cytokine staining of CD8 positive T cells;

FIG. 12 IgG antibody titers induced by different needles of the vaccine;

FIG. 13 SARS-CoV-2 euvirus neutralizing antibody titers induced by different needle times of vaccine;

FIG. 14. Lung tissue viral load;

FIG. 15H & E staining pathology analysis of lung tissue;

FIG. 16. Lung tissue viral load;

FIG. 17 Long term monitoring of vaccine induced IgG antibody levels;

FIG. 18 Long term monitoring of vaccine induced pseudovirus neutralizing antibody levels;

figure 19 lung tissue viral load.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for the purpose of illustration only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions such as the molecular cloning protocol (third edition published by scientific publishers) compiled by J. SammBruker et al or according to conditions recommended by the manufacturer. Reagents used in the experiment can be purchased by reagent companies unless otherwise specified.

Example 1: mRNA vaccine preparation

(1) Obtaining a target gene: the full-length amino acid sequences of the SARS-CoV-2 receptor binding domain RBD, the spike protein S1 subunit and the spike protein S are derived from Genebank MN908947, after codon optimization, the nucleic acid sequences are respectively shown as

SEQ ID NO

2, 6 and 8, after being synthesized by Beijing Okagaku New Biotechnology Co., ltd, the nucleic acid sequences are respectively cloned to mRNA transcription template pHRNT vectors (for example, purchased from Beijing Okagaku New Biotechnology Co., ltd) (FIG. 1), mRNA transcription template plasmids pHRNT-RBD, pHRNT-S1 and pHRNT-S (FIG. 2) are obtained.

(2) Transcription of mRNA

The mRNA transcription templates pHRNT-RBD, pHRNT-S1 and pHRNT-S were subjected to enzyme digestion linearization using the restriction enzyme BamHI, respectively, run through agarose gel electrophoresis (FIG. 3) to confirm whether linearization was complete, and then the linearized transcription templates were recovered using a gel recovery kit.

The following table was used to formulate an mRNA in vitro transcription reaction system (enzymes and reagents used were purchased from NEB, USA):

after 4 hours reaction at 37 ℃, 1. Mu.l RNase-free DNase I was added and the reaction was carried out at 37 ℃ for 15 minutes.

Then, the RNA is isolated and purified. There are various methods for separating and purifying RNA, such as ammonium acetate precipitation, liCl precipitation, organic solvent extraction-ammonium acetate precipitation, and RNA binding column purification. The LiCl precipitation method is taken as an example to illustrate that:

a) Adding 7.5M LiCl into the RNA solution to ensure that the final concentration of LiCl is 2.5M;

b) Overnight at-20 ℃;

c) Centrifuging at 12000rpm/min for 15 min, and discarding the solution;

d) Adding pre-cooled 75% ethanol at-20 ℃ into the precipitate, cleaning the precipitate, centrifuging at 12000rpm/min for 1 minute, removing the ethanol solution, and repeatedly cleaning for three times;

e) The RNA pellet was air-dried at room temperature and then the RNA was dissolved with RNase-free water. After determination of RNA concentration using Nanodrop, the cells were stored at-80 ℃.

(2) mRNA capping

mRNA can be capped by the following method ⁷ Gppp(m ^2′-O ) N1, the specific method is as follows:

a) In vitro transcribed mRNA,50-60 μ g, diluted to 67 μ l with RNase-free water;

b) Incubating at 65 ℃ for 5-10 minutes, and then cooling on ice;

c) The following table was used to prepare the reaction mixture (enzymes and reagents used were purchased from NEB, USA);

d) The cooled mRNA in b) was added to the mixture of c) before the reaction started, and 4. Mu.l of capping enzyme was added thereto and reacted at 37 ℃ for half an hour.

The capped mRNA is then isolated and purified. The specific method is as described above, and finally mRNA-RBD, mRNA-S1 and mRNA-S are obtained respectively.

(3) mRNA nanoparticle packaging

And packaging the mRNA by a nano-particle through a microfluidic technology. The aqueous phase was mRNA solution (50 mM sodium acetate buffer, pH 4.0), the ethanol phase was lipid mixture, formulated from dioleyl-methyl-4-dimethylaminobutyrate, distearoylphosphatidylcholine, cholesterol and PEG2K-DMPE in a molar ratio of 50. The total flow rate of the water phase and the ethanol phase is 12ml/min during packaging, and the volume ratio of the water phase to the ethanol phase is 3:1. after replacing the buffer solution with PBS using a dialysis bag, mRNA vaccines RBD, S1, and S were obtained, respectively. The encapsulated mRNA concentration was then determined using RiboGreen reagent and stored at 4 ℃ until use.

(4) mRNA vaccine nanoparticle morphology characterization

The vaccine solution, lipid nanoparticle size determination using a dynamic light scattering particle size analyzer, showed that the average diameter of the lipid nanoparticles in the vaccine was 78.4 nm and the uniformity was good (fig. 4). Cryo-electron microscopy results also demonstrated that the vaccine particles were relatively uniform in size (figure 5).

Example 2: evaluation of vaccine-induced humoral immune response

36 female 6-8 week old BALB/c mice were divided into 6 groups of 6 mice each, and were each intramuscularly injected with placebo (packaged in the same manner as the vaccine groups but encapsulated with polycytidylic acid from Sigma), RBD (0.3. Mu.g, 2. Mu.g and 15. Mu.g), S1 (15. Mu.g) or S (15. Mu.g) mRNA vaccines, and bled at 4 weeks after immunization, serum was separated at 4 ℃ and inactivated at 56 ℃ for 30 minutes and stored at-80 ℃ until use.

(1) Antigen specific antibody titer determination

The SARS-CoV-2S extracellular domain protein was diluted to 1. Mu.g/ml with an ELISA coating solution, and 100. Mu.l was added to each well of a 96-well plate, which was then left at 4 ℃ overnight. The next day the ELISA plate was blocked, mouse serum was diluted according to a 2-fold gradient, added to the ELISA plate and incubated for 1 hour at 37 ℃, followed by three washes with PBS (PBST) containing 0.05% tween 20, followed by addition of goat anti-mouse HRP secondary antibody (purchased from sequoia kuchenensis biotechnology, beijing), after 1 hour incubation at 37 ℃, PBST washed three times, followed by addition of TMB developing solution for visualization, and stopped with 2M hydrochloric acid and read on an ELISA plate with OD 450.

The results show that the RBD mRNA vaccine induced a dose-dependent effect of the new coronavirus specific antibodies, with higher doses giving higher antibody titers (fig. 6). At 15 μ g dose, a needle immunization of the RBD and S mRNA vaccines induced titers of up to 10 ⁵ Antigen-specific antibody reaction, which indicates that the two vaccines have good immunogenicity; the S1 vaccine group antibody titer is less than 10 ⁴ It was shown that the S1mRNA vaccine was slightly less immunogenic than the other two mRNA vaccines (FIG. 6).

(2) Pseudovirus neutralizing antibody titer assay

Pseudovirus packaging: plasmid pCAGGS-SARS-CoV-2S (for example, SARS-CoV-2 spike protein S full-length gene synthesized by Beijing Optimus Engine New technology Co., ltd., and cloned into pCAGGS vector) was co-transfected with pseudovirus backbone plasmid HIV Pnl4-3.Luc. RE (Invitrogen) using Lipofectamine2000 into 293T cells. 6 hours after transfection, cells were washed 2 times with PBS and replaced with serum-free DMEM. After 48 hours, the cell supernatant was collected, centrifuged to remove cell debris, and the cells were stored in a freezer at-80 ℃.

TCID50 assay: huh7 cells (commercially available,for example, from shanghai enzyme linked organisms) were plated in 96-well plates one day in advance. After 24 hours of growth, the harvested virus fluid was diluted 10-fold the next day, in order of 10 ^-1 、10 ^-2 ……10 ^-10 And added to a 96-well plate, at which time the confluency of Huh7 cells is 80% -100%. After 4 hours of infection, the virus solution was discarded, and the cells were washed 2 times with PBS and replaced with 10% serum in complete medium DMEM. After 48 hours, the culture medium was discarded, the cells were washed 2 times with PBS, cell lysate was added, and luciferase activity values were measured using a GloMax 96Microplate Luminometer (Promega). TCID50 was calculated by the Reed-Muench method.

Determination of pseudovirus neutralizing antibody titer: huh7 cells were plated on a 96-well plate one day in advance, and the mouse serum was diluted in a 2-fold gradient the next day, mixed with 1000TCID50 pseudovirus, incubated at 37 ℃ for 30 minutes, and then the mixture was added to the Huh7 cells. After incubation for 4 hours at 37 ℃, the virus solution was discarded, and the cells were washed 2 times with PBS and replaced with complete medium DMEM containing 10% serum. After 48 hours, the culture solution is discarded, the cells are washed for 2 times by PBS, cell lysate is added, and the luciferase activity value is detected. Calculating the serum dilution factor of the cells which are neutralized by 90 percent when the cells are infected by a Reed-Muench method, namely obtaining the NT of the neutralizing antibody titer of the pseudovirus ₉₀ The value is obtained.

The result of the neutralization of the pseudovirus shows that similar to the result of the SARS-CoV-2 specific antibody, when the RBD vaccine is immunized by 2 mug, the titer of the neutralizing antibody NT of the pseudovirus ₉₀ 190, NT when immunized at 15. Mu.g ₉₀ Significantly increased to 700 (fig. 7). At the same time, at 15 mug dose, the S vaccine induces the pseudovirus neutralizing antibody NT ₉₀ 653, which is comparable to the RBD group, whereas the S1 vaccine group was only 57, indicating that both the RBD and S vaccines are well immunogenic, whereas the S1 vaccine is weaker (fig. 7).

(3) Euvirus neutralizing antibody titer assay

Vero E6 cells were plated on a 96-well plate one day in advance, and the mouse serum or the serum of a Xinguan convalescent patient was subjected to gradient dilution at a 2-fold ratio the next day, mixed with 100TCID50 wild-type SARS-CoV-2 true virus (strain HB01, purchased from, for example, the institute for microbiology P3 laboratory of China academy of sciences), incubated at 37 ℃ for 30 minutes, and then the mixture was added to Vero E6 cells. After incubation for 48 hours at 37 ℃, 100 μ l of culture supernatant is taken to be extracted by virus RNA in a full-automatic nucleic acid extractor, the operation method is completely carried out according to the instructions of the instrument and the kit, and finally 80 μ l of eluent is used for elution. Then, a one-step fluorescence quantitative kit is used for carrying out Real-Time fluorescence RT-PCR reaction on the sample on a Light Cycler 480Real-Time PCR system instrument, the CT value of the measured sample is substituted into a standard curve, and then the virus TCID in the sample is calculated ₅₀ The value is obtained. Viral replication inhibition (%) = (viral control-sample control)/viral control 100%. Finally, calculating the serum dilution times of 50% neutralized cells when infected by a Reed-Muench method, namely obtaining the NT titer of the neutralizing antibody of the euvirus ₅₀ The value is obtained. The primers and probes used in the RT-PCR reaction were as follows:

a forward primer: the sequence is shown as SEQ ID NO. 3

Reaction primers: the sequence is shown in SEQ ID NO. 4

And (3) probe: 5'-FAM-TCCTCACTGCCGTCTTGTTGACCA-BHQ1-3'.

The results show that 2 μ g and 15 μ g induced titer of euvirus neutralizing antibodies after RBD one-needle immunization was 108 and 920, respectively, with 15 μ g dose group antibody titer levels comparable to serum neutralizing titer values (429) in convalescent patients (figure 8).

Example 3: evaluation of vaccine-induced cellular immune response

12 female 6-8 week-old C57BL/6 mice were divided into 2 groups of 6 mice each, and each was intramuscularly injected with placebo and RBD (15. Mu.g), and the mice were euthanized at 4 weeks after immunization, and spleens were taken and placed in pre-cooled 1640 medium. A40 μm mesh was placed on a 50ml centrifuge tube, the spleen was ground using a 5ml syringe plunger, cells were dropped into the 50ml tube by adding 1640 medium, and impurities and large cell clumps were filtered off. All cells were transferred to a 15ml centrifuge tube, centrifuged at 2000rpm at room temperature to collect cells, washed once with 12ml of 1640 medium, and centrifuged again to collect cells. Adding 4ml of erythrocyte lysate and suspension cells, standing at room temperature for 5-10 minutes, adding 8ml of 1640 culture medium, centrifuging to collect cells, adding 12ml of 1640 culture medium, washing for one time, and centrifuging again to collect cells. 10ml of 1640 medium was added, the cells were suspended,total number of cells was calculated using a cell counting plate. Centrifuging again to collect cells, adding 10% FSB 1640 medium to give a cell concentration of 1 × 10 ⁷ One per ml.

Enzyme-linked immunospot assay: flat-bottomed 96-well plates were pre-coated with 10. Mu.g/ml anti-mouse IFN-. Gamma.antibody (BD Biosciences) and incubated overnight at 4 ℃. The next day the liquid was discarded and 10% FBS in 1640 medium was added and blocked for 2 hours at room temperature. 2 x 10 per well ⁵ Mouse spleen cells, supplemented with medium to make a total volume of 100. Mu.l per well, were added to the RBD polypeptide pool (concentration of each polypeptide was 2. Mu.g/ml). Phytohemagglutinin (PHA) was used as a positive control. Cells without any stimulus served as negative controls. The cells were cultured in a cell incubator for 18 hours. The detection procedure was performed exactly as described in the ELISA spot-read kit (ELISA) provided by BD Inc. The number of spots formed was read using an enzyme-linked immunospot reader.

Intracellular cytokine staining experiment: add 1 × 10 per well in round bottom 96-well plates ⁶ And (3) adding RBD polypeptide library into spleen cells of each mouse, stimulating the cells (the concentration of each polypeptide is 2 mug/ml), and setting a group added with PHA as a positive control and a group not added with any stimulant as a negative control respectively. After 4 hours of stimulation, golgiStop was added and then cultured in a cell incubator for 14 hours. The cells were then harvested by centrifugation and stained with antibodies, the procedure being exactly as for Cytofix/Cytoperm from BD ^TM Instructions for the Fixation/Permeabilization kit. The cellular fluorescence was then detected on a FACSCanton flow cytometer.

The results of the ELISA spots show that, compared with the control group, the RBD vaccine can significantly stimulate the T cells to secrete gamma interferon after one-needle immunization (figure 9). The results of intracellular cytokine staining experiments show that the proportion of CD4 and CD8 positive T cells positive for interferon gamma in the RBD vaccine group is significantly higher than that of the control (fig. 10 and 11). Therefore, the results show that the RBD vaccine can induce a significant T cell immune response after one-needle immunization.

Example 4: evaluation of immune Effect of vaccines at different needle counts

15 female 6-8 week old BALB/c mice were divided into 3 groups of 5 mice each, and at week 0 of priming, placebo, RBD (15. Mu.g) and S (15. Mu.g) were intramuscular injected, serum was isolated by orbital bleeding at week 4 after immunization, followed by boosting once, and at week 8 after priming, blood was taken and serum was isolated, and RBD-specific antibody and S-specific antibody titers were determined in the prime and boost sera using ELISA method. The SARS-CoV-2 true virus neutralizing antibody titer in the primary immune and the enhanced serum is determined by using a micro-neutralization method based on cytopathic effect (CPE), and the specific method is as follows:

vero E6 cells were plated on a 96-well plate one day in advance, and the mouse serum was diluted stepwise at a ratio of 2 times the next day, mixed with 100TCID50 wild-type SARS-CoV-2 Euvirus (strain HB 01) at 4 duplicate wells per well, incubated at 37 ℃ for 30 minutes, and then the mixture was added to Vero E6 cells. Virus wells containing no serum and only virus and negative wells containing no virus and serum were set. CPE was observed and recorded for each well after three days in 37 ℃ incubator. The serum dilution inhibiting 50% of virus infection is the NT50 value of the titer of the neutralizing antibody of the true virus.

ELISA results showed that both RBD and S vaccines induced high levels of SARS-CoV-2 virus specific antibodies after one-needle immunization, antibody levels were further increased by 170 and 50-fold, respectively, after booster immunization, and binding of approximately 20% of RBD monomer in S-induced S specific antibodies, indicating the presence of a large number of antibodies targeting non-RBD regions or antibodies targeting specific steric conformational epitopes in S trimer protein (FIG. 12). The results of the experiment on neutralizing antibodies of the true viruses show that compared with the primary vaccine, the neutralizing antibody levels of the RBD and S vaccines after boosting are remarkably improved and respectively reach 58,000 and 159,000, and the vaccines are proved to have good immunogenicity (figure 13).

Example 5: evaluation of vaccine immunity attack protection effect

(1) Evaluation of virus attack protection effect of RBD vaccine

18 female 6-8 week old human ACE2 transgenic mice were divided into 3 groups (placebo, prime and boost) of 6 mice each. Placebo and prime mice were immunized with one placebo and one RBD vaccine (15 μ g), respectively, at week 0, and boost mice were immunized with one RBD vaccine (15 μ g), respectively, at

weeks

0 and 2. All mice were challenged with wild-type SARS-CoV-2 by nasal drops at 4 weeks after primary immunization at a dose of 10^5FFU/50 μ l/mouse. On day 5 after challenge, mice were euthanized and the lungs were harvested. After the lungs of four mice in each group were weighed and recorded, DMEM medium was added and homogenized using a tissue homogenizer, the supernatant was centrifuged to determine the amount of SARS-CoV-2 virus in the sample by the RT-PCR method in example 2, and finally the number of copies of virus per gram of lung tissue was calculated. After the lungs of the remaining 2 mice in each group were fixed with 4% paraformaldehyde solution for 72 hours, paraffin sections (3-4 μm) were prepared, and then H & E staining was performed on the paraffin sections using a hematoxylin eosin (H & E) staining kit to analyze pathological changes in the lungs.

The lung virus load result shows that the virus load of mice in the placebo group is as high as nearly 10^8.5 copies/g lung tissue, one mouse in one needle of RBD immune group can detect 10^7 copies/g lung tissue, the virus load is reduced by 97 percent compared with the placebo group, and the rest mice can not detect the virus; after two-needle RBD vaccine immunizations, no virus was detected in all mice (fig. 14). Pathological analysis of lung tissues H & E shows that the mice in the placebo group have severe bronchopneumonia and interstitial pneumonia and a large amount of immune cell infiltration; one-needle immunization group of mice only has little infiltration of immune cells and no other pathological changes, and two-needle immunization group of mice have normal lungs. The results indicated that one needle of the RBD vaccine induced nearly complete immune protection, and two needles completely protected mouse lung tissue against infection by the new coronavirus (fig. 15).

(2) Evaluation of protective effect of S vaccine

12 female 6-8 week old human ACE2 transgenic mice were divided into 2 groups of 6 mice each. Placebo and S vaccines (15 μ g) were immunized separately at week 0, and a boost was performed at week 2. All mice were challenged with wild-type SARS-CoV-2 by nasal drip at week 4 after primary immunization at a dose of 10^5FFU/50 μ l/mouse. On day 5 after challenge, mice were euthanized and the lungs were harvested. The SARS-CoV-2 viral load in lung tissue was determined according to the method in (1).

The results show that the virus load of the placebo group mice is about 10^8 copies/gram of lung tissue, and no virus can be detected in the S vaccine group mice, which shows that the S vaccine can provide complete immune protection for human ACE2 transgenic mice after two-needle immunization (figure 16).

Example 6: evaluation of vaccine immunity long-term protection effect

15 female 6-8 week old BALB/c mice were divided into 3 groups (placebo, short term and long term) of 5 mice each. Placebo and long-term mice were immunized with placebo and RBD (15 μ g) at week 0, and their sera were isolated by orbital bleeding at

weeks

2, 4, 6, 8, 10, 12, 16, and 20, and their mice were euthanized at week 26 and their sera isolated by copious bleeding for subsequent serum adoptive transfer challenge-protection experiments. Short-term group mice were immunized with RBD vaccine (15 μ g) at week 0, euthanized at week 8, and serum was isolated in large amounts for subsequent serum adoptive transfer challenge protection experiments. All sera were assayed for RBD-specific antibody titer and pseudovirus neutralizing antibody titer using the method in example 2. After mixing the sera of all mice collected in large amounts in each group, the sera were adoptively transferred to human ACE2 transgenic mice by intraperitoneal injection in an amount of 350 μ l/mouse. All human ACE2 transgenic mice were challenged with wild type SARS-CoV-2 by nasal drip at a dose of 10^5FFU/50 μ l/mouse. On day 5 after challenge, mice were euthanized and the lungs were harvested. The SARS-CoV-2 viral load in lung tissue was determined as in example 5.

The results of antibody long-term monitoring showed that after RBD immunization, both RBD-specific and pseudovirus-neutralizing antibodies peaked at week 8 with titers of 10^5 and 10^3, respectively, and then remained essentially stable for the next 8-26 weeks, and at week 26, the level of pseudovirus-neutralizing antibodies NT90 remained around 600 (FIGS. 17 and 18).

The results of the serum adoptive transfer challenge protection experiment show that the virus load of mice in the two vaccine groups is significantly lower than that of the placebo group, and the RBD (basic fibroblast antigen) immunized by one injection can provide significant immune protection for the mice for up to 26 weeks (figure 19).

Claims

Mrna comprising the following elements in order in the 5'→ 3' direction: a 5' cap structure, a 5' UTR sequence, a coding region for an antigenic polypeptide of SARS-CoV-2 or an antigenic fragment thereof, a 3' UTR sequence and a polyadenylation sequence, wherein the antigenic polypeptide is selected from the receptor binding domain RBD of SARS-CoV-2, or the full-length sequence of the spike protein S of SARS-CoV-2,

wherein, the coding region of the antigenic polypeptide of SARS-CoV-2 or the antigenic fragment thereof is the RNA sequence coded by SEQ ID NO. 2 or 8; the 5' UTR sequence is selected from the RNA sequence of any one of SEQ ID NO 9-11; the 3' UTR sequence is selected from the RNA sequence of any one of SEQ ID NO 12-14,

wherein some or all of the uracil and/or cytosine in the mRNA is chemically modified to increase the stability of the mRNA in vivo.
2. The mRNA of claim 1, wherein the chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of uracil in the mRNA,

wherein the substance which replaces uracil is at least one selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxy-uridine and 2' -O-methyluridine;

and/or, the chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the cytosines in the mRNA with 5-methylcytosine.
3. The mRNA of claim 2, wherein the chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the uracils in the mRNA with pseudouridine or N1-methylpseuduridine or N1-ethylpseuduridine.
4. The mRNA of claim 1 or 2, wherein the antigenic polypeptide encoded by the mRNA is the receptor binding domain RBD protein of SARS-CoV-2 as shown in the amino acid sequence of SEQ ID NO. 1.
5. The mRNA of claim 1 or 2, wherein the antigenic polypeptide encoded by the mRNA is the spike protein S full length of SARS-CoV-2 having the amino acid sequence of SEQ ID NO. 7.
6. The mRNA of claim 1, wherein the 5' cap structure is selected from the group consisting of m ⁷ GpppG、m ₂ ^7,3′-O GpppG、m ⁷ Gppp (5') N1 or m ⁷ Gppp(m ^2′-O ) At least one of N1.
7. The mRNA of claim 1, wherein the 5' cap structure is selected from the group consisting of m ⁷ Gppp (5') N1 or m ⁷ Gppp(m ^2′-O )N1。
8. The mRNA of claim 1, wherein the polyadenylation sequence comprises a sequence of 25 to 400 adenylates.
9. The mRNA of claim 1, wherein the polyadenylation sequence comprises a sequence of 50 to 400 adenylates.
10. The mRNA of claim 1, wherein the polyadenylation sequence comprises a sequence of 50 to 300 adenylates.
11. The mRNA of claim 1 wherein the polyadenylation sequence comprises a sequence of 50 to 250 adenylates.
12. The mRNA of claim 1, wherein the poly A sequence comprises a sequence of 60-200 adenylates.
13. The mRNA of claim 1, wherein the mRNA has an mRNA selected from the group consisting of SEQ ID NOS 15-50.
14. A pharmaceutical composition comprising at least one of the mrnas of any one of claims 1-13, and a delivery vehicle.
15. The pharmaceutical composition of claim 14, wherein the delivery vehicle is a nanoparticle.
16. The pharmaceutical composition of claim 14, wherein the delivery vehicle is a lipid nanoparticle.
17. The pharmaceutical composition of claim 15, wherein the lipid nanoparticle has a net neutral charge at neutral pH.
18. The pharmaceutical composition of claim 15, wherein the lipid nanoparticles have an average diameter of 50-200nm and a polydispersity index value of less than 0.4.
19. The pharmaceutical composition of any one of claims 14-17, wherein the mRNA to delivery vehicle mass ratio is a: b, wherein A is selected from 0.05 to 2, and B is selected from 1 to 100.
20. The pharmaceutical composition of any one of claims 14-17, wherein the mRNA to delivery vehicle mass ratio is a: b, wherein A is 0.05 and B is 1; or A is 1, B is 100; or A is 2, B is 1; or A is 1, B is 50; or A is 1 and B is 5.
21. The pharmaceutical composition of any one of claims 15-17, wherein the lipid nanoparticle comprises a cationic lipid and at least one selected from the group consisting of a non-cationic lipid, a sterol, and a PEG-modified lipid.
22. The pharmaceutical composition according to any one of claims 15 to 17, wherein the lipid nanoparticle is a cationic lipid, a non-cationic lipid, a sterol and a PEG-modified lipid in a molar ratio of 20-60.
23. The pharmaceutical composition according to any one of claims 15 to 17, wherein the lipid nanoparticle is a cationic lipid, a non-cationic lipid, a sterol, and a PEG-modified lipid in a molar ratio of 30-60.
24. The pharmaceutical composition of claim 21 or 22, wherein the cationic lipid is an ionizable cationic lipid selected from one or more of the following: 2, 2-dioleylene-4-dimethylaminoethyl- [1,3] -dioxolane, dioleylene-methyl-4-dimethylaminobutyrate and di ((Z) -non-2-en-1-yl) 9- ((4- (dimethylamino) butanoyl) oxy) heptadecanedioate.
25. The pharmaceutical composition of claim 21 or 22, wherein the cationic lipid is dioleyl-methyl-4-dimethylaminobutyrate.
26. The pharmaceutical composition of claim 21, wherein the non-cationic lipid is a neutral lipid selected from at least one of distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, and dioleoylphosphatidylserine.
27. The pharmaceutical composition of claim 21, wherein the non-cationic lipid is distearoylphosphatidylcholine.
28. The pharmaceutical composition of claim 21, wherein the sterol is cholesterol.
29. The pharmaceutical composition of claim 21, wherein the PEG-modified lipid is selected from at least one of PEG-DMG, PEG-DSG, and PEG-DMPE.
30. The pharmaceutical composition of claim 21, wherein the PEG-modified lipid is PEG-DMPE.
31. The pharmaceutical composition of claim 21, wherein the PEG of the PEG-modified lipid is 0.5-200KDa in length, alternatively 1-50KDa in length, alternatively 1-5KDa in length.
32. The pharmaceutical composition of any one of claims 14-17, wherein the pharmaceutical composition comprises an adjuvant.
33. A kit comprising the mRNA of any one of claims 1 to 13 and/or the pharmaceutical composition of any one of claims 14 to 32.
34. Use of the mRNA of any one of claims 1 to 13, the pharmaceutical composition of any one of claims 14 to 32, the kit of claim 33 for the preparation of a medicament for the prevention and/or treatment of a SARS-CoV-2 viral infection.
35. The mRNA of any one of claims 1 to 13, the pharmaceutical composition of any one of claims 14 to 32, the method of making the kit of claim 33, comprising the step of chemically modifying uracil and/or cytosine in the mRNA contained therein, including in part or all of the coding region comprising an antigenic polypeptide encoding SARS-CoV-2, or an antigenic fragment, variant or derivative thereof, wherein the antigenic polypeptide is selected from the receptor binding domain RBD of SARS-CoV-2, or the full-length sequence of spike protein S of SARS-CoV-2.
36. The method of claim 35, wherein said chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the uracil in the coding region of said mRNA with at least one member selected from the group consisting of pseudouridine, N1-methylpseuduridine, N1-ethylpseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine or 5-methoxy-and 2' -O-methyluridine;

and/or, the chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the cytosines in the mRNA with 5-methylcytosine.
37. The method of claim 35, wherein the chemical modification comprises replacing at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of uracil in a coding region of the mRNA with pseudouridine or N1-methylpseuduridine or N1-ethylpseuduridine.