CN116200403A

CN116200403A - Novel coronavirus mRNA vaccine for preventing mutant strain

Info

Publication number: CN116200403A
Application number: CN202211516868.8A
Authority: CN
Inventors: 魏立帆; 杨思聪; 吴磊彬; 范超; 胡学文; 王凌宇; 淡墨; 路丽艳; 李永丰; 王雅鹃; 李彦辉; 孙召朋; 唐昭娜; 胡喜新
Original assignee: Jushi Biopharmaceutical Co ltd
Current assignee: Jushi Biopharmaceutical Co ltd
Priority date: 2021-11-30
Filing date: 2022-11-29
Publication date: 2023-06-02
Also published as: WO2023098679A1

Abstract

The invention relates to a novel coronavirus mRNA vaccine for preventing mutant strains, which mainly comprises mRNA with mutation sites and lipid nano particles; the mRNA vaccine obtained by the invention has good immune effect against a plurality of novel coronavirus mutant strains.

Description

Novel coronavirus mRNA vaccine for preventing mutant strain

The present application claims the priority of the prior application entitled "novel coronavirus mRNA vaccine for preventing mutant" filed by the national intellectual property agency of china on the date of 2021, 11 and 30, with patent application No. 202111445859. X. The entirety of the prior application is incorporated by reference into this application.

Technical Field

The application belongs to the technical field of biological medicine, and particularly relates to a novel coronavirus mRNA vaccine and a preparation method thereof.

Background

The novel coronavirus (SARS-CoV-2, hereinafter referred to as novel coronavirus) is a single-stranded RNA virus, has a genome size of 29.9Kb, and consists of 11 genomes, and encodes 12 proteins including spike protein (S protein), envelope protein, capsid protein, and the like. Among them, the spinous process protein (S protein) is considered as the most important viral surface membrane protein for use by coronaviruses in invading cells, and contains two subunits (subnit), S1 and S2. Wherein the S1 subunit is responsible for binding to the host cell receptor ACE2 and the S2 subunit is responsible for anchoring the S protein to the host cell membrane and mediating fusion of the viral envelope with the host cell membrane. The S1 subunits include N-Terminal Domain (NTD) and Receptor Binding Domain (RBD), wherein RBD is involved in binding to host cell receptor ACE2; the S2 subunit includes Heptad Repeat (HR), central Helix (CH) and Connector Domain (CD), and in addition, there is a Furin cleavage site at the interface of the S1 subunit and the S2 subunit; studies have shown that: the S protein is considered the most likely vaccine antigen choice; depending on the interaction of the S protein with human angiotensin converting enzyme 2 (ACE 2), the new coronavirus invades human airway epithelial cells, inducing lung disease, which is designated as covd-19 by the world health organization.

The new coronavirus belongs to one of Coronaviruses (CoVs), belongs to the family Coronaviridae (coroneviridae) of the order nidoviridae (Nidovirales), is widely available in nature, and has spherical or elliptic virus particles with polymorphism and a diameter of about 60-220 nm. The virus has an envelope (envelope) on which there is a spike, the internal genome being single-stranded positive strand RNA (+ssRNA), and the whole virus particle being under electron microscopy like a crown or coronal crown and hence the name.

The coronaviridae subfamilies are divided into four genera, α, β, γ and δ, based on serotype and genomic characteristics. To date, a total of 7 coronaviruses can infect humans: including genus alpha 229E and NL63, genus beta OC43 and HKU1, middle east respiratory syndrome associated coronavirus (MERSr-CoV), severe acute respiratory syndrome associated coronavirus (SARSr-CoV) and novel coronavirus (SARS-CoV-2).

The new coronavirus has extremely strong infectivity, no specific medicine exists at present, and vaccination is still the most effective method for resisting the new coronavirus diseases. Candidate new crown vaccines in the clinical study stage are classified according to the WHO classification into the following categories: attenuated vaccines, inactivated vaccines, protein subunit vaccines, viral vector vaccines, nucleic acid vaccines (including mRNA, DNA) and virus-like particle vaccines.

At present, 25 new coronavirus vaccines enter a clinical test stage in China, wherein 4 vaccines are approved by the drug administration of China to be marketed under the condition, 3 vaccines are recently used in emergency in China, 14 vaccines are obtained outside the country to develop a III-phase clinical test, and the progress is totally smooth.

The FDA currently approved or emergency authorised use 3 new crown vaccines (Moderna mRNA vaccine, BIOTECH/PFIZER mRNA vaccine and Janssen's non-replicating viral vector vaccine), with the vaccine of pyroxene formally fully approved in 2021, 8 months for use in people 16 years and older.

The European Union current requirements approve 4 new crown vaccines, two of which are mRNA vaccines, and two of which are non-replicating viral vector vaccines by AstraZeneca and Janssen.

Based on the obtained vaccine, the mRNA vaccine has unique advantages compared with the traditional vaccine as the third-generation vaccine, firstly, the mRNA vaccine is obtained by production through the process of enzymatic in-vitro transcription, and the amplification of cells is not relied on, so that the monitoring and quality control of all production processes can be easily realized, the processes of cell culture, antigen extraction, purification and the like are saved, the production time is greatly shortened, the mass production can be easily realized, the productivity of the vaccine is improved, the annual productivity of hundreds of millions and even billions of doses is easily realized, and the method is very important for rapidly coping with new infectious diseases worldwide. Secondly, mRNA vaccine can induce organism to produce humoral immunity and cellular immunity at the same time, protect organism through multiple mechanisms, the protection efficiency is higher, the existing data show that the effective rate of mRNA vaccine on the market at present is above 90%.

However, although mRNA vaccines have the advantages of rapid development, high safety, easy industrialization, etc., 2 versions of mRNA vaccines have been obtained worldwide: research results in multiple countries such as the united states, the cartol, the israel and the like show that the protection effect of the existing vaccine against the infection of the new crown variant strain is reduced; beginning at 7 in 2021, there is a rapid increase in the number of newly increased covd-19 cases in countries such as israel, uk and united states where the vaccination rate is very high; mRNA vaccination rates in the population 12 years old and older by israel have reached nearly 80%. In addition, the international journal of top-level medicine, journal of the american society of medicine (JAMA), published data from over 364 ten thousand mRNA vaccinators reported autonomously (Bardales et al 2021), and the results indicated that: adverse reactions after vaccination with mRNA new coronaries were more common, the first 5 most common (after completion of the second dose) were injection site pain (72.3%), sensory fatigue (53.9%), headache (46.7%), myalgia (44.0%) and chills (31.3%), respectively; in addition to the above adverse reactions, there are some rare adverse reactions such as: the FDA contemplates deferring approval of the mRNA vaccine of Moderna for adolescent new crown vaccine booster needle vaccination due to the increased risk that may lead to rare inflammatory heart disease. It follows that although a number of mRNA vaccines have emerged, there is an unmet need for an efficient, low side effect vaccine in the marketplace.

In addition, since 2020, there have been many SARS-CoV-2 mutant strains that have begun to be prevalent worldwide, including B.1.1.7 (British variant, alpha strain, key mutation: D614G, N501Y, P681H), B.1.351 (south Africa variant, beta strain, key mutation: E484K, N501Y, K417N), B.1.617.1 (first generation Indian variant, kappa strain, key mutation: L452R, E484Q, D614G) and B.1.617.2 (second generation Indian variant, delta strain, key mutation: L452R, E484Q, D G), P.1 (Brazil variant, key mutation: K417T, E484K, N Y), and B.1.1.529 strain (south Africa variant Omicron, involving 40 more point mutations) that occur at the end of month 2021. Except that the newly appeared B.1.1.529 strain is not deeply studied, whether the strain enhances the immunity escape capability of the virus or not can not be determined, and other variant strains enhance the immunity escape capability of the new coronavirus and simultaneously enhance the transmission capability of the virus. The current reported data shows that the new coronavirus Delta strain has become the most prevalent strain worldwide, and simultaneously increases the risk of hospitalization of infected patients. At present, no mRNA vaccine aiming at a plurality of mutants of the novel coronavirus exists, most of traditional mRNA vaccines only aim at wild type SARS-CoV-2, and the protection efficiency of the plurality of mutants is limited.

Based on the consensus of the scientific community, an ideal mRNA vaccine should have the following characteristics:

1. translating the nucleic acid at the injection/inoculation site (e.g., muscle);

2. very effective at inducing antigen-specific immune responses against SARS-CoV-2 protein encoded at very low doses and dosing regimens;

3. suitable for vaccinating infants and/or newborns or the elderly, in particular the elderly;

4. the composition/vaccine is suitable for intramuscular administration;

5. simultaneously inducing specific and functional humoral and T-cell immune responses against coronaviruses, such as SARS-COV-2;

6. induce specific B cell memory against coronaviruses, such as SARS-COV-2;

7. inducing a functional antibody effective to neutralize the virus;

8. inducing functional antibodies that effectively neutralize the presence of SARS-CoV-2 variants;

9. induce protective immunity against coronavirus infection, e.g., against SARS-CoV-2 or emerging variants thereof;

10. can provide immune protection of SARS-CoV-2 rapidly and relatively long-term without enhancing SARS-CoV-2 infection due to vaccination or immunopathology;

11. there was no Antibody Dependence Enhancement (ADE) caused by the nucleic acid-based SARS-CoV-2 vaccine; does not excessively induce systemic cytokine or chemokine responses after administration of the vaccine;

12. The vaccine has good tolerance and no toxic or side effect for all people;

13. an advantageous vaccination regimen, which requires only one or two low doses of vaccination to obtain sufficient protection.

Aiming at the defects in the prior art, the patent aims at designing an mRNA vaccine by aiming at an S protein coding region of SARS-CoV-2 virus and uses in vitro transcribed synthesized mRNA. The inventor fully examines the current epidemic strains, analyzes mutation sites of the strains, considers factors such as broad spectrum of the vaccine, stability of three-dimensional structure of antigen protein, effectiveness of expression in human cells and the like, designs and optimizes the nucleic acid sequence of the mRNA vaccine, and develops the mRNA vaccine for efficiently and stably translating and expressing antigen S protein. The mRNA of the present application is delivered into the body and translated into SARS-CoV-2S protein (antigen protein), which causes the body to produce humoral and cellular immunity and memory B cells and T cells, and when contacting SARS-CoV-2 virus, can effectively prevent the infection of various mutants of SARS-CoV-2. The animal can be induced to produce protective effect on various SARS-CoV-2 mutants by using smaller dose. The safety and effectiveness are obviously superior to the existing vaccine technology, and unexpected technical effects are obtained.

Disclosure of Invention

The present application provides an immune composition (e.g., an mRNA vaccine) comprising RNA encoding a highly immunogenic antigen capable of eliciting an effective neutralizing antibody response against a coronavirus antigen such as SARS-CoV-2;

in a first aspect, the present application provides a polynucleotide (e.g., mRNA) encoding at least one antigenic peptide or protein derived from SARS-CoV-2 coronavirus or an immunogenic fragment or immunogenic variant thereof, said nucleotide comprising at least one heterologous untranslated region (UTR) having a nucleotide sequence of at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or at least 99% or 100% identity to the nucleotide sequence of any one of SEQ ID NOs 1-6;

in some embodiments, the polynucleotide is suitable for use in the production of a vaccine;

in some embodiments, the at least one antigenic peptide or protein comprises or consists of at least one fragment or immunogenic variant derived from a structural, accessory or replicative protein or any of the above proteins.

In some embodiments, the structural protein is or is derived from spike protein (S), envelope protein (E), membrane protein (M) or nucleocapsid protein (nucleocapsid protein, N) or an immune fragment or immune variant thereof.

In some embodiments, the spike protein (S) is selected from spike protein S1 protein or an immune fragment thereof and variants thereof, S2 protein or an immune fragment thereof and variants thereof.

In some embodiments, a polynucleotide of the present application may comprise or consist of a sequence that hybridizes to SEQ ID NO: 1. 2, 3, 4, 5, 6, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity.

In some embodiments, at least one coding region of a polynucleotide of the present application encodes a mutation comprising K986P and V987P proteins, said coding region comprising a nucleotide sequence optimized for G/C content.

In some embodiments, the heterologous untranslated region (UTR) comprises at least one heterologous 3'UTR and/or 5' UTR.

In some embodiments, the polynucleotide comprises at least the following structure:

(a) A 5' -cap structure;

(b) The 3' polyadenylation sequence is preferably 50-200A, more preferably 80-200A;

(c) 5'UTR, 10-200 nucleotides in length, preferably 15-150 nucleotides in length, preferably alpha-1-globin5' UTR, KOZAK sequences;

(d) 3'UTR, preferably comprising human gp130, DH143, hHBB and hHBA 1' UTR sequences;

In some embodiments, the 3'UTR may also be selected from PSMB3, ALB7, α -globin, CASP1, COX6B1, GNAS, NDUFA1, DH143, gp130, hHBB, hHBA1, CYBA (cytochrome B-245alpha chain), rabbit β -globin, hepatitis B Virus (HBV), VEEV (Venezuelan equine encephalitis virus) virus, rps9 (Ribosomal Protein S9), FIG4 (FIG 4 Phosphoinositide 5-phosphophase), human albumin hHBB (human hemoglobin subunit beta), 3' UTR of HBA1 (human Hemoglobin Subunit Alpha 1) or homologues, fragments or variants from any of these genes.

In some embodiments, the 5' UTR is selected from Xenopus or a humanized alpha-or beta-globin, a human cytochrome B-245a polypeptide, a hydroxysteroid 17B dehydrogenase (17B) and a tobacco etch virus (Tobacco etch virus), alpha-1-globin (alpha-1-globin), HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, or a homologue, fragment or variant from any of these genes.

In some embodiments, the nucleotide of any one of the preceding claims, wherein the nucleotide comprises at least one 3 '-polyadenylation sequence, preferably comprises 30 to 200 adenosine nucleotides and/or at least one 3' -polyadenylation sequence.

In some embodiments, the present application relates to an mRNA encoding a coronavirus antigen comprising amino acids having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or at least 99% or 100% identity to the amino acid sequence of SEQ ID NO. 15.

In some embodiments, the mRNA comprises a 5' untranslated region (UTR), a 3' UTR, and a 3' -polyadenylation.

In some embodiments, the mRNA further comprises a 5' guanosine cap selected from the group consisting of: m7Gppp _(2'OMeA) pG、m7GpppApA、m7GpppApC、m7GpppApG、m7GpppApU、m7GpppCpA、m7GpppCpC、m7GpppCpG、m7GpppCpU、m7GpppGpA、m7GpppGpC、m7GpppGpG、m7GpppGpU、m7GpppUpA、m7GpppUpC、m7GpppUpG、m7GpppUpU、m7Gpppm6ApG、m7G _3’Ome pppApA、m7G _3’Ome pppApC、m7G _3’Ome pppApU、m7G _3’Ome pppApG、m7G _3’Ome pppCpA、m7G _3’Ome pppCpC、m7G _3’Ome pppCpG、m7G _3’Ome pppCpU、m7G _3’ _Ome pppUpA、m7G _3’Ome pppUpC、m7G _3’Ome pppUpG、m7G _3’Ome pppUpU、m7G _3’Ome pppA _2’Ome pG、m7G _3’ _Ome pppA _2’Ome pC、m7G _3’Ome pppA _2’Ome pU、m7G _3’Ome pppA _2’Ome pA、m7G _3’Ome pppC _2’Ome pA、m7G _3’Ome pppC _2’ _Ome pU、m7G _3’Ome pppC _2’Ome pG、m7G _3’Ome pppC _2’Ome pC、m7G _3’Ome pppG _2’Ome pA、m7G _3’Ome pppG _2’Ome pU、m7G _3’Ome pppG _2’Ome pG、m7G _3’Ome pppG _2’Ome pC、m7G _3’Ome pppU _2’Ome pA、m7G _3’Ome pppU _2’Ome pU、m7G _3’ _Ome pppU _2’Ome pG、m7G _3’Ome pppU _2’Ome pC。

In some embodiments, the mRNA encoding a coronavirus antigen comprises at least one mutation relative to the S protein of the wild-type strain: L452R, E484Q, D614G, K986P, V987P.

In some embodiments, the mRNA comprises chemically modified bases or analogs including 5-methoxymethyl uridine (5-methoxymethyl uridine), 5-methylthiouridine (5-methylthio uridine), 1-methoxymethyl pseudouridine (1-methoxymethyl pseudouridine), 5-methylcytidine (5-methylcytodine), 5-methoxycytidine (5-methoxy cytodine), 1-methylpseudouridine (N1-Methyl-Pseudo-UTP), pseudouridine; preferably 1-methyl pseudouridine partially replaces uridine; most preferably, uracil is completely replaced with 1-methyl pseudouridine, such that each uracil in the sequence is replaced with 1-methyl pseudouridine.

In some embodiments, the 3' -polyadenylation comprises the nucleotide sequence of SEQ ID No. 10.

In some embodiments, the 5' UTR comprises the nucleotide sequence of SEQ ID NO. 9;

in some embodiments, the 3' UTR comprises the nucleotide sequence of SEQ ID NOS: 11-14.

In some embodiments, the foregoing spike protein (S) may comprise or consist of an amino acid sequence having at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID No. 15.

In some embodiments, the foregoing polynucleotides are DNA or RNA.

In some embodiments, the foregoing polynucleotide is a coding RNA.

In some embodiments, the coding RNA is mRNA, self-replicating RNA, circular RNA, or replicon RNA, preferably mRNA.

In some embodiments, the mRNA comprises at least one 3' -polyadenylation sequence, the 3' -polyadenylation sequence comprising 30 to 200 adenosine nucleotides and the 3' terminal nucleotide is adenosine.

In some embodiments, wherein the mRNA preferably comprises a 5' -cap structure, the 5' -cap structure is preferably an m7G, cap0, cap1, cap2, modified cap0, or modified cap1 structure, preferably a cap1 structure, most preferably an m7G (5 ') ppp (5 ') (2 ' ome) pG.

In a second aspect, the present application provides a composition, preferably an immunogenic composition, comprising at least one nucleotide of the first aspect. Suitably, the composition may comprise at least one nucleotide, for example at least one coding RNA, complexed with, encapsulated in or associated with one or more lipids, thereby forming a lipid nanoparticle.

In some embodiments, the present application relates to a novel coronavirus nucleic acid vaccine characterized in that the vaccine carrier is a Lipid Nanoparticle (LNP) comprising an ionizable cationic lipid, a structural lipid, a helper lipid, and a surfactant, in some embodiments, the molar content of the ionizable cationic lipid, structural lipid, helper lipid, and surfactant add up to 100% in terms of mole percent (mol%);

in some embodiments, the lipid nanoparticle comprises 20-60mol% ionizable cationic lipid, 25-55mol% structural lipid, 5-25mol% helper lipid, and 0.5-15mol% surfactant.

In some embodiments, the cationic lipid is selected from the group consisting of SM-102, ALC-0315, ALC-0519, dlin-MC3-DMA, DODMA, C-200, dlin DMA, preferably SM-102; the SM-102 structure is as follows:

In some embodiments, the structural lipid is selected from cholesterol, and cholesterol derivatives, preferably cholesterol;

in some embodiments, the helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DSPC;

in some embodiments, the surfactant is selected from the group consisting of PEG2000-DMG, PEG-DSPE, DTDA-PEG2000, TPGS, preferably PEG2000-DMG.

In some embodiments, the lipid nanoparticle comprises 20-50mol% ionizable cationic lipid. For example, the lipid nanoparticle may comprise 41, 42, 43, 44, 45, 46, 47, 48, 49, 50mol% ionizable cationic lipid.

In other embodiments, the lipid nanoparticle comprises 50-60mol% ionizable cationic lipid. For example, the lipid nanoparticle may comprise 51, 52, 53, 54, 55, 56, 57, 58, 59, 60mol% ionizable cationic lipid.

In some embodiments, the lipid nanoparticle comprises 5-25mol% dspc, preferably 2-15mol% dspc; for example, the lipid nanoparticle may comprise 3, 4, 5,6,7,8,9, 10, 11, 12, 13, 14 or 15mol% dspc.

In some embodiments, the lipid nanoparticle comprises 25-55 mole% cholesterol, preferably 30-40 mole% cholesterol. For example, the lipid nanoparticle may comprise 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40mol% cholesterol.

In some embodiments, the lipid nanoparticle comprises 0.5 to 15 mole% DMG-PEG, preferably 1 to 2 mole% DMG-PEG. For example, the lipid nanoparticle may comprise 1,1.5 or 2mol% dmg-PEG.

In some embodiments, the lipid nanoparticle comprises 50mol% ionizable cationic lipid, 10mol% dspc,38.5mol% cholesterol, and 1.5mol% dmg-PEG.

In some embodiments, the lipid nanoparticle comprises 50mol% SM-102, 10mol% DSPC,38.5mol% cholesterol, and 1.5mol% DMG-PEG.

In some embodiments, the lipid nanoparticle of the present application comprises about 2:1 to about 30: n of 1: p ratio.

In some embodiments, the lipid nanoparticle of the present application comprises about 6: n of 1: p ratio.

In some embodiments, the lipid nanoparticle of the present application comprises about 3: n of 1: p ratio.

In some embodiments, the lipid nanoparticle of the present application comprises about 10:1 to about 100:1 to RNA.

In some embodiments, the lipid nanoparticle of the present application comprises about 20:1 to RNA.

In some embodiments, the lipid nanoparticle of the present application comprises about 10:1 to RNA. In some embodiments, the lipid nanoparticle of the present application has an average diameter from about 50nm to about 150 nm.

In some embodiments, the lipid nanoparticle of the present application has an average diameter of about 70nm to about 120 nm. Preferably 100-120nm, most preferably 100nm.

In some embodiments, the novel coronavirus nucleic acid vaccine of the present application further comprises: buffer components and cryoprotectants;

in some embodiments, the buffer may be selected from: examples of buffers include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydrogen phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium phosphate, dibasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, magnesium hydroxide, aluminum hydroxide, alginic acid, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, month Gui Jimei sulfate, sodium lauryl sulfate, and combinations thereof.

In some embodiments, the cryoprotectant may be selected from the group consisting of sugars/polyols, polymers, surfactants, azoacids, and salts, wherein the sugar may be selected from the group consisting of: lactose, sucrose, trehalose, galactose, etc.

In some embodiments, the amount of cryoprotectant is from 1 to 50% w/w, such as from 2 to 50% w/w, or from 4 to 45% w/w, or from 6 to 12% w/w, or preferably from 6 to 10% w/w, or most preferably from 7 to 9% w/w.

In some embodiments, the pharmaceutical compositions of the present application comprise the aforementioned lipid nanoparticle composition and an external phase buffer.

In some embodiments, the aqueous buffer comprises: tromethamine, sodium acetate, sucrose, ph=7-8.

In some embodiments, the tromethamine is present in an amount selected from the group consisting of 10-30mmol/L, preferably 15-25mmol/L, preferably 15mmol/L, 15.5mmol/L, 16mmol/L, 16.5mmol/L, 17mmol/L, 17.5mmol/L, 18mmol/L, 18.5mmol/L, 19mmol/L, 19.5mmol/L, 20mmol/L, 20.5mmol/L, 21mmol/L, 21.5mmol/L, 22mmol/L, 22.5mmol/L, 23mmol/L, 23.5mmol/L, 24mmol/L, 24.5mmol/L, 25mmol/L, most preferably 20mmol/L.

In some embodiments, the sodium acetate is present in an amount selected from 0-20mmol/L, preferably 5-11mmol/L, preferably 5mmol/L, 5.5mmol/L, 6mmol/L, 6.5mmol/L, 7mmol/L, 7.5mmol/L, 8mmol/L, 8.5mmol/L, 9mmol/L, 9.5mmol/L, 10mmol/L, 10.5mmol/L, 10.6mmol/L, 10.7mmol/L, 10.8mmol/L, 10.9mmol/L, 11mmol/L, 11.5mmol/L, 12mmol/L, 12.5mmol/L, 13mmol/L, most preferably 10.7mmol/L.

In some embodiments, the sucrose is present in an amount selected from the group consisting of: 5-15%, preferably 7.5-10%, more preferably 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 9%, 9.5%, 10%, most preferably 8.7%.

In some embodiments, the pharmaceutical composition of the present application comprises mRNA having the sequence shown in SEQ ID NO:1, a lipid nanoparticle composition comprising 50mol% SM-102, 10mol% DSPC,38.5mol% cholesterol and 1.5mol% PEG2000-DMG, N: P=6, 20mmol/L tromethamine, 10.7mmol/L sodium acetate, 8.7% sucrose, and pH 7.0-8.0.

In a third aspect, the present application provides novel coronavirus vaccines, preferably antigenic polypeptides comprising a SARS-CoV-2 composition or vaccine.

In some embodiments, comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID No. 14.

In some embodiments, the antigenic proteins of the present application include one or more of the following mutations relative to the amino acid sequence of the spike protein of the novel coronavirus strain (NCBI SEQ ID NO: nc_ 045512.2): L452R, E484Q, D614G, K986P, V987P, A1020W, P1069F, H1048Q, T887W, P1069F;

In a fourth aspect, the present application provides a novel coronavirus vaccine, preferably a SARS-CoV-2 vaccine, wherein the vaccine comprises at least one nucleotide of the first aspect, or a composition of the second aspect, or at least one polypeptide of the third aspect.

In some embodiments, the vaccine is administered by intravenous injection, intramuscular injection, or subcutaneous injection, preferably intramuscular injection;

in some embodiments, the vaccine may be in a dosage form selected from the group consisting of a lyophilized powder for injection, a liquid injection, an inhalation formulation;

in a fifth aspect, the present application provides a kit of parts or kit of parts comprising at least one nucleotide of the first aspect, and/or at least one composition of the second aspect, and/or at least one polypeptide of the third aspect, and/or at least one vaccine of the fourth aspect.

In a sixth aspect, the present application provides a composition comprising at least two isolated components, wherein the at least two isolated components are selected from the two nucleotides of the first aspect, and/or the at least two compositions of the second aspect, and/or the at least two polypeptides of the third aspect, and/or the at least two vaccines of the fourth aspect. Other aspects of the present application relate to methods of treating or preventing coronavirus infection, preferably SARS-CoV-2 infection, in a subject, and to the first and second medical uses of the nucleotides, compositions and vaccines. Methods of making the nucleic acids, compositions or vaccines are also provided.

In some embodiments, the present application relates to a method comprising administering to a subject a composition of claims 7-16 effective to induce a neutralizing antibody response against SARS-CoV-2 in the subject.

In a seventh aspect, the present application relates to a method for preparing a novel coronavirus vaccine, characterized in that a vaccine vector and mRNA are mixed to obtain the novel coronavirus vaccine.

In some embodiments, the vaccine carrier is a cationic lipid nanoparticle, and the preparation method specifically comprises the steps of:

(1) The protonatable cationic lipid, the structural lipid, the auxiliary lipid and the surfactant are dissolved in an organic solution according to the formula proportion to obtain an organic phase;

(2) Dissolving optimized mRNA in citrate buffer solution or sodium acetate solution to obtain water phase;

(3) Uniformly mixing the organic phase in the step (1) and the water phase in the step (2) to generate a mixed solution, thereby obtaining a novel coronavirus vaccine;

in some embodiments, the organic solution comprises absolute ethanol;

in some embodiments, the total concentration of protonatable cationic lipids, structural lipids, co-lipids, and surfactant in the organic phase is from 10 to 15mg/ml;

in some embodiments, the mRNA concentration is 0.01-1mg/ml, preferably 0.1-0.2mg/ml;

In some embodiments, the volume ratio of the organic phase to the aqueous phase is 1:2-4;

in some embodiments, the mixing is performed by a microfluidic device, and the flow rate is controlled to be more than or equal to 12ml/min;

in some embodiments, the present application also relates to the use of said mRNA or said composition in the preparation of a vaccine.

In some embodiments, the vaccine comprises a multiple vaccine and a multivalent vaccine.

Preferably, the vaccine is a SARS-CoV-2 virus mRNA vaccine, preferably the vaccine is a SARS-CoV-2 virus mutant vaccine, preferably the mutant is B.1.1.7 (British variant, alpha strain, critical mutation: D614G, N501Y, P681H), B.1.351 (south African variant, beta strain, critical mutation: E484K, N501Y, K417N), B.1.617.1 (first generation Indian mutant, kappa strain, critical mutation: L452R, E484Q, D614G), and B.1.617.2 (second generation Indian mutant, delta strain, critical mutation: L452R, E484Q, D G), P.1 (Brazil mutant, critical mutation: K417T, E484K, N Y), and B.1.1.529 strain (south African mutant Omicron, involving 40 more point mutations).

In an eighth aspect, the present application also contemplates a combination vaccine comprising a first vaccine selected from the mRNA, composition or pharmaceutical composition described previously and a second vaccine for sequential use.

In some embodiments, the combination vaccine, the second vaccine is selected from the group consisting of: attenuated or inactivated vaccines, adenovirus vaccines, mRNA vaccines, DNA vaccines, recombinant protein vaccines.

In some embodiments, the second vaccine is selected from the group consisting of: moderna (mRNA-1273), cureVac (CVnCoV), johnson & Johnson (COVID-19 Vaccine Janssen), astraZeneca (Vaxzevria), pfizer/BioNTech (Comirnaty), sputnik (Gam-COVID-Vac), sinovac (COVID-19 Vaccine (Vero Cell), novax (NVX-CoV 2373), kang Xinuo New crown Vaccine, beijing national drug New crown Vaccine, proc.

In some embodiments, the vaccines described herein are suitable for sequential vaccination with one or more vaccines selected from the group consisting of. The vaccine may be based on any technical route of vaccine including, but not limited to, attenuated or inactivated vaccines, adenovirus vaccines, mRNA vaccines, DNA vaccines, recombinant protein vaccines, and the like.

In some embodiments, the one or more vaccines are selected from the group consisting of: moderna (mRNA-1273), cureVac (CVnCoV), johnson & Johnson (COVID-19 Vaccine Janssen), astraZeneca (Vaxzevria), pfizer/BioNTech (Comirnaty), sputnik (Gam-COVID-Vac), sinovac (COVID-19 Vaccine (Vero Cell), novavax (NVX-CoV 2373), kang Xinuo New coronal Vaccine, beijing national drug New coronal Vaccine, proc.Navigator-Zhi recombinant New coronavirus Vaccine;

In some embodiments, one relatively complete vaccination, the number of vaccinations required to complete an immunization may be 1, 2, 3, or 4, each vaccination may be selected at 21, 28, 35, 2, 3, 4, 5, 6 months intervals;

in some embodiments, the vaccines of the present application may be used as booster needles for subjects who have been injected with two doses of inactivated vaccine;

the novel coronavirus mRNA vaccine obtained by the method can effectively inhibit the infection of a plurality of current epidemic strains, and greatly reduce the infection rate (the infection rate of partial strains is lower than 10 percent); and under the 21-day immunization strategy, the bound antibody titer of the serum 6 months after the first immunization is still 10 ⁶ About, the long-term immune protection effect is very good. In addition, the vaccine has moderate storage conditions, can be stably stored for a long time at the temperature of minus 20 ℃, is relatively convenient to transport, has low requirements on equipment, and is more suitable for areas needing most urgent vaccination such as underdeveloped economy or underdeveloped.

Drawings

Fig. 1: bioanalyzer analyzed the purity and size of S protein mRNA.

Fig. 2: transmission electron microscopy results for mRNA and Lipid Nanoparticle (LNP) complexes.

Fig. 3: results of immunoblotting experiments on the expression of S protein in host cells.

Fig. 4: mRNA transfection HEK293T cell flow cytometry detection results.

Fig. 5: antibody neutralization pseudovirus experiments.

Fig. 6: pseudovirus infection rate experiments.

Fig. 7: antibody binding affinity experiments.

Note that: strain b.1.1.7 (uk variant); b.1.351 (south Africa variant); p.1 (Brazil variant); b.1.617.1 (first generation indian mutant strain);

fig. 8: toxicity test after muscle immunization of mice.

Note that: mice transfected with hACE2 protein were immunized twice on days 0 and 21 at doses of 5 and 20 μg mRNA LNP lipid nanoparticles, respectively, buffer group as negative control, model group as non-immunized group.

Fig. 9: SARS-Cov2 specific T cell activity results.

Note that: medium belongs to negative control, and no immunostimulation is performed; canavalia gladiata protein belongs to a positive control and can activate all T cells in a nonspecific manner

Fig. 10: ELISPot evaluates the results of SARS-Cov-2 specific memory cell activity.

Note that: PBS was the uncoated antigen group, the negative control group; RBD and S proteins are specific antibody detection groups; igG is the total antibody detection group.

Fig. 11: and (3) carrying out an experiment on neutralizing antibody effect of the Omikovia strain.

Fig. 12: effect experiments on the protection rate of mice.

Fig. 13: safety efficacy experiments against mouse immunity.

Examples:

definition:

unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For definitions and terms in the art, the expert may refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

Notwithstanding that the numerical ranges and approximations of the parameters set forth in the broad scope of the present application, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range recited as "1 to 10" should be considered to include any and all subranges between (inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood as being incorporated in its entirety.

It will be appreciated by those skilled in the art that many different polynucleotides may encode the same polypeptide due to the degeneracy of the genetic code. It will also be appreciated that the skilled artisan can make nucleotide substitutions using conventional techniques that do not affect the polypeptide sequence encoded by the nucleic acid molecule to reflect codon usage of any particular host organism in which the polypeptide is expressed. Thus, unless otherwise indicated, "a polynucleotide encoding a protein or immunogenic fragment of the present application" includes: all polynucleotide sequences which are degenerate to each other and which encode identical amino acid sequences.

Antigens

An antigen as used herein is a protein capable of inducing an immune response (e.g., eliciting an immune system to produce antibodies to the antigen). In this context, unless otherwise indicated, the use of the term "antigen" includes immunogenic proteins and immunogenic fragments (immunogenic fragments that induce or are capable of inducing an immune response to at least one coronavirus). It is understood that the term "protein" includes peptides and the term "antigen" includes antigenic fragments. Other molecules may also be antigenic, such as bacterial polysaccharides or combinations of proteins and polysaccharide structures, and viral vaccine antigens described herein include viral proteins, viral protein fragments, and proteins derived from the design and/or mutation of the coronavirus SARS-CoV-2.

Furthermore, the term "antigen" as used herein will be recognized and understood by one of ordinary skill in the art to mean a substance that can be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, for example. By forming antibodies and/or antigen-specific T cells as part of an adaptive immune response. The antigen may be or may comprise a peptide or protein, which may be presented to T cells by MHC. Fragments, variants and derivatives derived from, for example, peptides or proteins are also included. The spike protein (S) of a coronavirus comprising at least one epitope, preferably the spike protein from SARS-CoV-2 (nCoV-2019) or a variant thereof, is designed as an antigen.

Antigenic peptides or proteins: the term "antigenic peptide or protein" or "immunogenic peptide or protein" will be recognized and understood by one of ordinary skill in the art; by a peptide, a protein is meant derived from a (antigenic or immunogenic) protein which stimulates the adaptive immune system of the body to provide an adaptive immune response. Thus, the antigenic/immunogenic peptide or protein comprises at least one epitope (as defined herein) or antigen (as defined herein) from a protein from which it is derived (e.g., the spike protein (S) of a coronavirus), preferably from SARS-CoV-2 (2019 neocoronavirus).

As recognized and well known to those skilled in the art: protein fragments, functional protein domains and homologous proteins are also considered to be within the scope of the coronavirus antigen of interest. For example: any protein fragment of a novel coronavirus or mutant thereof provided that the fragment is immunogenic and confers a protective immune response to the coronavirus; in addition to variants that are identical to the reference protein but truncated, in some embodiments, the antigen includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, and the antigen/antigen polypeptide may range in length from about 4, 6 or 8 amino acids to the full-length protein.

Epitope(s)

Epitope: the term "epitope" (also referred to in the art as an "antigenic determinant") as used herein will be recognized and understood by one of ordinary skill in the art to mean a T cell epitope and a B cell epitope. T cell epitopes or portions of antigenic peptides or proteins and may comprise fragments, e.g., preferably having a length of about 6 to about 20 or more amino acids. Fragments processed and presented by MHC class I molecules preferably have a length of about 8 to about 10 amino acids, for example: 8. fragments of 9 or 10 (or 11 or 12 amino acids) or processed and presented by MHC class II molecules preferably have a length of about 13 to about 20 or more amino acids. These fragments are usually recognized by T cells in the form of complexes consisting of peptide fragments and MHC molecules, i.e. these fragments are not usually recognized in their natural form. B cell epitopes are typically fragments located on the outer surface of a (native) protein or peptide antigen, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their native form. Such epitopes of the proteins or peptides may also be selected from any variants of such proteins or peptides mentioned herein. In this context, an epitope may be a conformational or discontinuous epitope consisting of fragments of a protein or peptide as defined herein, which fragments are discontinuous in the amino acid sequence of the protein or peptide as defined herein, but which are aggregated together in a three-dimensional structure or a continuous or linear epitope consisting of a single polypeptide chain.

Nucleic acid

The term "nucleic acid" or "nucleic acid molecule" will be recognized and understood by one of ordinary skill in the art. As used herein, the term "nucleic acid" or "nucleic acid molecule" preferably refers to DNA (molecule) or RNA (molecule). It is preferably used synonymously with the term polynucleotide. Preferably, the nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers covalently linked to each other by phosphodiester linkages of a sugar/phosphate backbone. The term "nucleic acid molecule" also includes modified nucleic acid molecules, such as base-, sugar-, or backbone-modified DNA or RNA molecules as defined herein.

In particular, the compositions of the present application comprise (at least one) RNA having an Open Reading Frame (ORF) encoding a coronavirus antigen (e.g., a variant trimeric spike protein, e.g., a stable pre-fusion spike protein). In some embodiments, the RNA is messenger RNA (mRNA).

In some embodiments, the nucleic acid comprises at least one heterologous untranslated region (UTR). The term "untranslated region" or "UTR element" will be recognized and understood by one of ordinary skill in the art to mean a portion of a nucleic acid molecule, typically located 5 'or 3' of a coding sequence. Is referred to as the 5'UTR at the 5' end and as the 3'UTR at the 3' end. In general, UTRs do not translate into proteins; UTRs may be part of nucleic acids, such as DNA or RNA. UTRs may contain elements for controlling gene expression, also known as regulatory elements. Such regulatory elements may be ribosome binding sites, miRNA binding sites and the like; the RNA (e.g., mRNA) can further comprise a 5' utr, a 3' -poly a, and/or a 5' cap analog.

In some embodiments, the 5' UTR is a heterologous UTR, i.e., a UTR associated with a different ORF found in nature; in another embodiment, the 5' UTR is a synthetic UTR; the 5'UTR is the region of mRNA upstream (5') of the start codon (the first codon of the mRNA transcript translated by the ribosome). The 5' UTR does not encode proteins. The natural 5' UTR has features that play a role in translation initiation, such as a Kozak sequence with a consensus CCR (A/G) CCAUGG; exemplary 5 'UTRs also include Xenopus or human-derived alpha-or beta-globin, human cytochrome b-245a polypeptides, hydroxysteroid 17b dehydrogenases (17 b) dehydrogenises, and tobacco etch virus (Tobacco etch virus), alpha-1-globin (alpha-1-globin) 5' UTRs, and the like.

In some embodiments, the 3' utr may be heterologous or synthetic; for example: globin UTRs, including Xenopus laevis beta-globin UTRs and human beta-globin UTRs; other 3' UTRs may also be CYBA (cytochrome b-245alpha chain), rabbit beta-globin, hepatitis B Virus (HBV), alpha-globin 3' UTR and VEEV (Venezuelan equine encephalitis virus) virus 3' UTR sequences. In some embodiments, rps9 (Ribosomal Protein S9) 3'UTR, FIG4 (FIG 4 Phosphoinositide 5-phosphopatase), gp130, DH143 and human albumin hHBB (human hemoglobin subunit beta), 3' UTR of HBA1 (human Hemoglobin Subunit Alpha 1) may also be used.

In some embodiments, 3' -poly a, also known as poly a tail; the poly (A) tail is the region of mRNA downstream, e.g., immediately downstream (i.e., 3 '), of the 3' UTR that contains multiple consecutive adenosine monophosphates. The poly (a) tail may comprise 10 to 300 adenosine monophosphates, may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some preferred embodiments, the poly (A) tail contains 50 to 250 adenosine monophosphates, more preferably 50-100 adenosine monophosphates; most preferably 100 adenosine monophosphates; in a related biological environment (e.g., in a cell, in vivo), the function of the 3' -polyadenylation tail is to protect the mRNA from enzymatic degradation, e.g., in the cytoplasm, and to aid in transcription termination and/or export of mRNA and translation from the nucleus.

In some embodiments, the RNA (e.g., mRNA) further comprises a 5' guanosine cap; the 5 'guanosine cap is transcribed from eukaryotic mRNA, the 5' cap consists of inverted 7-methylguanosine, connected to the rest of the eukaryotic mRNA through a 5'-5' triphosphate bridge, so-called cap0 (cap 0), mainly acting as a quality control for correct mRNA processing and helping to stabilize eukaryotic mRNA; 2' -OH methylation was performed at the first nucleotide on the basis of cap0, called cap1 (cap 1); in addition to cap0 and cap1, further methylation modifications can be made to the second nucleotide, referred to as cap 2; in general, the 5' -cap may be synthesized by: different synthetic routes for 5' capped mRNA based on enzymatic, chemical or chemoenzymatic methods;

In some embodiments, in vitro transcription, a cap analog (5' cap analog) is added directly to an In Vitro Transcription (IVT) system, including but not limited to: m is m ⁷ Gppp(2'OMeA)pG、m ⁷ GpppApA、m ⁷ GpppApC、m ⁷ GpppApG、m ⁷ GpppApU、m ⁷ GpppCpA、m ⁷ GpppCpC、m ⁷ GpppCpG、m ⁷ GpppCpU、m ⁷ GpppGpA、m ⁷ GpppGpC、m ⁷ GpppGpG、m ⁷ GpppGpU、m ⁷ GpppUpA、m ⁷ GpppUpC、m ⁷ GpppUpG、m ⁷ GpppUpU、m ⁷ Gpppm ⁶ ApG、m ⁷ G _3’Ome pppApA、m ⁷ G _3’Ome pppApC、m ⁷ G _3’Ome pppApU、m ⁷ G _3’Ome pppApG、m ⁷ G _3’Ome pppCpA、m ⁷ G _3’Ome pppCpC、m ⁷ G _3’Ome pppCpG、m ⁷ G _3’Ome pppCpU、m ⁷ G _3’Ome pppUpA、m ⁷ G _3’Ome pppUpC、m ⁷ G _3’ _Ome pppUpG、m ⁷ G _3’Ome pppUpU、m ⁷ G _3’Ome pppA _2’Ome pG、m ⁷ G _3’Ome pppA _2’Ome pC、m ⁷ G _3’Ome pppA _2’Ome pU、m ⁷ G _3’ _Ome pppA _2’Ome pA、m ⁷ G _3’Ome pppC _2’Ome pA、m ⁷ G _3’Ome pppC _2’Ome pU、m ⁷ G _3’Ome pppC _2’Ome pG、m ⁷ G _3’Ome pppC _2’ _Ome pC、m ⁷ G _3’Ome pppG _2’Ome pA、m ⁷ G _3’Ome pppG _2’Ome pU、m ⁷ G _3’Ome pppG _2’Ome pG、m ⁷ G _3’Ome pppG _2’Ome pC、m ⁷ G _3’ _Ome pppU _2’Ome pA、m ⁷ G _3’Ome pppU _2’Ome pU、m ⁷ G _3’Ome pppU _2’Ome pG、m ⁷ G _3’Ome pppU _2’Ome pC, etc.

In some embodiments, the capping analog may also be other structures, such as: tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, etc. The specific sequence of which can be determined according to the condition of the template.

It should also be appreciated that the novel coronavirus vaccines of the present application may include any 5 'untranslated region (UTR) and/or any 3' untranslated region (UTR). Nucleic acids comprise polymers of nucleotides (nucleotide monomers). Thus, a nucleic acid is also referred to as a polynucleotide. The nucleic acid may be or include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose Nucleic Acid (TNA), ethylene Glycol Nucleic Acid (GNA), peptide Nucleic Acid (PNA), locked Nucleic Acid (LNAs), ethylene Nucleic Acid (ENA), cyclohexenyl nucleic acid (CeNA), and/or chimeras and/or combinations thereof.

Messenger RNA (mRNA) is any RNA, in situ or ex vivo that encodes a (at least one) protein (a naturally occurring, non-naturally occurring or modified amino acid polymer) and can be translated in vitro, in vivo to produce the encoded protein. The skilled artisan will appreciate that unless otherwise indicated, the nucleic acid sequences listed in this application may refer to "T" in a representative DNA sequence, but when the sequence represents RNA (e.g., mRNA), the "T" will be replaced with "U". Thus, any DNA disclosed and identified herein by a particular sequence identifier also discloses a corresponding RNA (e.g., mRNA) sequence complementary to the DNA, wherein each "T" of the DNA sequence is substituted with a "U".

Open reading frame

An Open Reading Frame (ORF) is a continuous stretch of DNA or RNA that begins with an initiation codon (e.g., methionine (ATG or AUG)) and with a termination codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA), typically an ORF encodes a protein. It is to be understood that the sequences disclosed herein may also comprise additional elements, such as 5 'and 3' utrs, but unlike ORFs, these elements are not necessarily present in the RNA polynucleotides of the present application.

In some embodiments, the composition comprises an RNA (e.g., mRNA) comprising a nucleotide sequence of any of SEQ ID NOs 1-6 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity.

In some embodiments, the open reading frame is preferably at least partially codon optimized. Codon optimisation is based on the finding that: translation efficiency can be determined by the different frequencies at which transferred RNA (tRNA) occurs in the cell. Thus, if an increased degree of so-called "rare codons" are present in the coding region of a nucleic acid of the present application as defined herein, translation of the correspondingly modified nucleic acid sequence is less efficient than if codons were present encoding a relatively "common" tRNA. One skilled in the art may be able to make codon optimisation for the sequence to be translated based on the characteristics of its in vitro expression system.

Chemically modified or unmodified nucleotides

In some embodiments, the RNA (e.g., mRNA) is not chemically modified, but rather comprises standard ribonucleotides consisting of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides disclosed herein comprise standard nucleoside residues, such as those found in transcribed RNAs (e.g., a, G, C or U). In some embodiments, the nucleotides and nucleosides disclosed herein include standard deoxyribonucleosides, such as deoxyribonucleosides present in DNA (e.g., dA, dG, dC, or dT);

in some embodiments, the compositions of the present application comprise RNA having an open reading frame encoding a coronavirus antigen, wherein the nucleic acid comprises nucleotides and/or nucleosides, which may be standard (unmodified) or modified as known in the art. In some embodiments, the nucleotides and nucleosides of the present application include modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications may include modifications of the nucleotides and/or sugar, backbone or nucleobase portions of the nucleosides known in the art.

In some embodiments, modified nucleobases in a nucleic acid (e.g., RNA nucleic acid, e.g., mRNA nucleic acid) include 1-methyl-pseudouridine (1-methyl-pseudouridine), 1-ethyl-pseudouracil (1-methyl-pseudouridine), 5-methoxy-uridine (5-methoxy-uridine), 5-methyl-cytidine (5-methyl-cytodine), and/or pseudouracil (pseudouridine), pseudouridine.

In vitro transcription system (IVT)

In vitro transcription is to use DNA as a template to simulate in vivo transcription processes to generate mRNA in an in vitro cell-free system with components such as RNA polymerase and NTP. In general, the capped RNA synthesized in the in vitro transcription reaction can be used for subsequent experiments such as microinjection, in vitro translation, transfection, etc. In vitro transcription systems typically include a transcription buffer, nucleotide Triphosphates (NTPs), an rnase inhibitor, and a polymerase. NTP may be chosen at self-synthesis or from a vendor. The NTP may be a natural or unnatural NTP. Alternative polymerases include, but are not limited to, phage RNA polymerases, such as T7RNA polymerase, T3RNA polymerase, SP6 RNA polymerase and/or polymerase mutants thereof, such as: but are not limited to, polymerases capable of incorporating modified nucleic acids and/or modified nucleotides, including chemically modified nucleic acids and/or nucleotides. Some embodiments exclude the use of dnase. In some embodiments, the RNA comprises a 5' guanosine cap.

In addition to in vitro transcription system synthesis, chemical synthesis methods may be employed, including solid phase chemical synthesis and liquid phase chemical synthesis; with respect to solid phase chemical synthesis, the nucleic acids disclosed herein may be prepared in whole or in part using solid phase techniques; solid-phase chemical synthesis of nucleic acids is an automated process in which molecules are immobilized on a solid support and synthesized stepwise in a reactant solution. Solid phase synthesis can be used for site-specific introduction of chemical modifications in nucleic acid sequences; with respect to liquid phase chemical synthesis, the synthesis of the nucleic acids of the present application by sequential addition of monomer constructs may be performed in the liquid phase. Furthermore, the above synthetic methods may also be used in combination, since each of the synthetic methods discussed above has its own advantages and limitations, and attempts may be made to combine these methods together, overcoming the limitations described above. Combinations of these methods are within the scope of the present application.

Antigen variants

In some embodiments, the compositions of the present application comprise RNA encoding a variant coronavirus antigen (e.g., a variant trimeric spike protein, e.g., a stable pre-fusion spike protein). An antigenic variant or other polypeptide variant refers to a molecule whose amino acid sequence differs from the wild-type, natural or reference sequence. An antigen/polypeptide variant may have substitutions, deletions and/or insertions at certain positions within the amino acid sequence compared to the native or reference sequence. Typically, the variant has at least 50% identity to a wild-type, natural or reference sequence. In some embodiments, the variant has at least 80% or at least 90% identity to a wild-type, native or reference sequence.

Variant antigens/polypeptides encoded by the nucleic acids of the present disclosure may comprise amino acid changes that confer any of a variety of desirable properties, e.g., enhancing their immunogenicity, enhancing their expression, and/or improving their stability or PK/PD properties. Variant antigens/polypeptides may generally be prepared using conventional mutagenesis techniques and analyzed as appropriate to determine whether they have the desired properties. Assays for determining expression levels and immunogenicity are well known in the art, and exemplary such assays are set forth in the examples section. Similarly, PK/PD characteristics of protein variants may be measured using art-recognized techniques, for example, by determining expression of antigen over time in an vaccinated subject and/or by observing the persistence of an induced immune response. The stability of the protein encoded by the variant nucleic acid may be measured by determining the thermal stability or stability at the time of urea denaturation, or may be measured using computer prediction. Methods for such experiments and computer determinations are known in the art.

The term "identity" refers to the relationship between the sequences of two or more polypeptides (e.g., antigens) or polynucleotides (nucleic acids) as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or among sequences determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identical matches between smaller sequences in two or more sequences, with gap alignments (if any) being solved by a specific mathematical model or computer program (e.g., an "algorithm"). The identity of the relevant antigen or nucleic acid can be readily calculated by known methods. "percent (%) identity" for a polypeptide or polynucleotide sequence is defined as the percent sequence of the candidate amino acid or nucleic acid sequence that is the same residue as the residue in the amino acid (amino acid residue or nucleic acid residue) or the nucleic acid sequence of the second sequence after aligning the sequences and introducing gaps, if desired, to achieve the greatest percent identity. Methods and computer programs for alignment are well known in the art. It will be appreciated that identity depends on the calculation of percent identity, but its value may be different due to differences and penalties introduced in the calculation. Typically, variants of a particular polynucleotide or polypeptide (e.g., antigen) have 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity as determined by the sequence alignment procedures and parameters described herein and are known to those of skill in the art.

Lipid Nanoparticle (LNP)

The RNAs (e.g., mrnas) of the present application are formulated in Lipid Nanoparticles (LNPs). Lipid nanoparticles typically include ionizable cationic lipids, helper lipids, cholesterol, and PEG lipid components, and nucleic acids of interest. Lipid nanoparticles of the present application may be produced using components, compositions, and methods generally known in the art.

Multivalent vaccine

The compositions provided herein may include RNA or RNAs encoding two or more antigens of the same or different species. In some embodiments, the composition comprises an RNA or RNAs encoding two or more coronavirus antigens. In some embodiments, the RNA can encode 1,2,3,4,5,6,7,8,9, 10, 11, 12 or more coronavirus antigens.

Two or more different RNAs (e.g., mrnas) encoding antigens may be formulated in the same lipid nanoparticle. In other embodiments, two or more different RNAs encoding antigens may be formulated in separate lipid nanoparticles (each RNA formulated in a single lipid nanoparticle). The lipid nanoparticles can then be combined and administered as a single vaccine composition (e.g., comprising multiple RNAs encoding multiple antigens), or can be administered separately.

Multigang vaccine

The compositions provided herein may include RNA or RNAs encoding two or more antigens of the same or different viral strains. Also provided herein are combination vaccines comprising RNA encoding one or more coronaviruses and antigens of one or more different organisms. Thus, the vaccine of the present application may be a combination vaccine targeting one or more antigens of the same strain/species, or one or more antigens of different strains/species, e.g. antigens inducing immunity to organisms found in the same geographical area where the risk of coronavirus infection is high or organisms to which an individual may be exposed when exposed to coronavirus.

Sequential inoculation

Sequential vaccination refers to the spaced vaccination of vaccines of different technical routes, including basic immune sequencing and booster immune sequencing; if the first needle is an inactivated vaccine, the second needle is an adenovirus vaccine or an mRNA vaccine or any other vaccine that is not inactivated, this mode of vaccination is called basic immune sequence; if two doses of inactivated vaccination have been previously completed, and a follow-up need for booster injection, any other non-inactivated route of vaccination is used, this mode of vaccination is referred to as booster sequential.

Pharmaceutical preparation

Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for preventing or treating, for example, human and other mammalian coronaviruses. The compositions provided herein are useful as therapeutic or prophylactic agents. They are useful in medicaments for the prophylaxis and/or treatment of coronavirus infections.

The term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use in vivo or in vitro. The "pharmaceutically acceptable carrier" does not cause an undesirable physiological effect after administration to a subject or after administration to a subject. The carrier in the pharmaceutical composition must be "acceptable" in the sense that it is also compatible with the active ingredient and capable of stabilizing it. One or more solubilizing agents can be used as a drug carrier for delivery of the active agent. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible carriers, adjuvants, additives and diluents to obtain compositions useful as dosage forms. Examples of other carriers include colloidal silica, magnesium stearate, cellulose and sodium lauryl sulfate. Other suitable pharmaceutical carriers and diluents, and pharmaceutical necessities therefor, are described in Remington's Pharmaceutical Sciences.

The related sequences referred to in this application are as follows:

SF-1 SEQ ID NO:1

gggagacccaagcuggcuagcguuuaaacuuaagcuugguaccgagcucggauccacuaguccagugugguggaauucgaauaaacuaguauucuu

cugguccccacagacucagagagaacccgccaccauguucguguuccuggugcugcugcccuuagugagcagccagugcgucaaccuaacuacuaga

acacagcugccuccugccuacacaaacagcuucaccagaggcguguauuaccccgauaagguguuccggucuucugugcugcacagcacccaggaccu

guuucugccuuucuucagcaaugugaccugguuccacgccauccacgugucuggcacaaacggcacaaaaagguucgacaaccccguucugccuuuc

aaugacggcguguacuucgcuagcacagaaaagagcaacaucaucagaggauggaucuucggaaccacccuggauuccaagacacagucccuguuga

ucguuaauaaugccaccaauguggugaucaaggugugugaguuccaguuuugcaacgauccuuuucugggcguguauuaccacaagaauaacaaaa

gcuggauggaaagcgaguuuagaguauacucuagcgccaacaacugcaccuucgaguacgucagccagccuuuucugauggaccuggaaggcaagca

gggcaacuucaagaaucugagagaguucguguucaaaaacauugacggauauuucaaaaucuacagcaagcacacaccuaucaaucuggugcgggac

cugccacagggguuuagcgcauuggagccucuggucgaucugccuaucggcaucaacaucacccgguuccagacccugcuagcccugcauagaagcu

aucugaccccuggcgacagcucaagcggcuggaccgccggcgccgccgccuacuacgugggcuaccugcagccuagaaccuuucugcugaaguacaa

cgagaacggcaccauaaccgacgcuguggacugcgcccuggacccccugucugaaaccaagugcacccugaagagcuuuaccguggaaaagggcaucu

accaaacaagcaauuuccgggugcagccuaccgagucuaucgugcgguuucccaacaucaccaaccugugcccauucggagaaguguucaacgccacc

agauucgccagcguguacgccuggaaccggaagagaaucucuaacugcguggccgauuacagcgugcucuauaacucggcuagcuucagcacauuca

agugcuacggcgugagccccacaaagcugaacgaccugugcuucaccaacgucuacgccgacagcuucgugauuagaggcgaugagguccgacagau

cgccccuggccaaaccggcaagauugcugacuacaacuacaagcugccugaugacuucacuggaugugugaucgcuuggaacuccaacaaccuggac

ucuaaagugggcggaaacuacaacuaccgguaccggcuguuuagaaagaguaaccuuaaaccuuucgagagagauaucagcacagaaaucuaccaggc

uggcagcacaccuuguaacggcgugcaaggcuucaacugcuauuucccucuccagucuuauggcuuccagccuaccaacggcgugggcuaucagccc

uaccgagugguggugcuguccuucgagcugcugcaugccccugcuaccgugugcggcccuaagaaaucgaccaaccuggugaagaacaagugcguga

auuuuaauuucaacggccucaccggcaccggagugcugaccgagagcaacaagaaguuccugcccuuccagcaguucggaagagacaucgccgauacc

acagacgccgugcgggacccccaaacacuggaaauccuggacaucacaccuugcagcuucggcggcgugagcgugaucaccccuggcaccaacacaag

caaccagguggccgugcucuaccagggcgugaacugcaccgaagugccaguggccauccacgccgaucagcugaccccuaccuggagaguguacagc

accggcagcaacguguuccagacaagggcugguugucugaucggcgccgagcacgugaauaauuccuacgagugcgacaucccuauuggcgccggaa

ucugugccagcuaccagacacagacaaacuccccacguagagccagauccgucgccagccagagcaucaucgcuuacaccaugagccugggcgcugaa

aacagcguggcuuacagcaacaauucuaucgccauaccuaccaauuuuacaaucucggugacaaccgagauccugccuguuagcaugaccaagaccag

cguggacugcacaauguacaucugcggagacagcaccgagugcuccaaccugcugcuucaauacggguccuucuguacacagcugaauagagcccug

acaggcaucgcuguugagcaggacaagaacacccaggagguguuugcccaagugaagcagaucuacaagacuccgccuaucaaggacuucggcggcu

ucaacuucagccagauccugccagauccuucuaagccuagcaagcgcaguuucaucgaggaccuguuguucaacaaggugacccuggccgacgccgg

cuuuaucaagcaguacggcgauugccugggcgacaucgccgcuagagaucugaucugcgcccagaaguucaauggacugacagugcugccgccccug

cugaccgaugagaugaucgcucaguacaccucugcccugcuggccgggacaaucaccagcggauggacauucggggccggcgccgcccugcagaucc

ccuuugccaugcagauggccuacagauucaacgguauuggcgugacccaaaacgugcuguacgagaaucagaaguuaaucgcaaaccaguucaacag

cgccaucggcaagauccaggauucccugaguuccacggccagcgcucuggguaagcugcaagacguggugaaccagaacgcacaggcccugaacaccc

uggucaaacagcugagcuccaacuucggagcgaucagcagcgugcuuaaugacauccugagcagacuggauccccccgaggccgaaguccaaaucgac

cggcugaucacaggcagguugcagagccugcagaccuacgugacccaacagcugaucagagcagccgagaucagagccucagccaaucuggcugcuac

gaagaugagcgagugugugcugggacagagcaagcggguggauuuuugcggcaaaggguaccaccugaugagcuucccucagagcgccccucacgg

cguuguguuccugcacgugacguacgugcccgcccaagagaagaacuucaccacagccccugccaucugccacgacggaaaagcccacuucccucgug

agggcgucuucgugagcaacggcacacacugguucgugacucagagaaacuucuacgagccucagaucaucaccaccgacaacaccuuugugagcgg

caacugcgacguggugaucgggaucgugaacaacaccguguacgacccccugcagccugagcuggauagcuucaaagaggaacuggacaaguauuuc

aagaaucacaccucuccagacguggaccugggcgacaucagcggcauuaacgccucuguggugaacauccagaaggaaaucgaucggcugaacgagg

uggccaagaacuugaacgagagccugaucgaccugcaggagcugggcaaguacgagcaguacaucaaguggccuugguacaucuggcugggauucau

cgccggccugauugccaucgucauggugaccaucaugcuguguugcaugacaagcugcugcagcugucugaagggauguugcucuuguggcucuug

cuguaaauucgacgaagacgacucggaacccgugcugaagggcguuaagcuccacuacaccugaugacucgagcugguacugcaugcacgcaaugcu

agcugccccuuucccguccuggguaccccgagucucccccgaccucgggucccagguaugcucccaccuccaccugccccacucaccaccucugcuag

uuccagacaccucccaagcacgcagcaaugcagcucaaaacgcuuagccuagccacacccccacgggaaacagcagugauuaaccuuuagcaauaaacg

aaaguuuaacuaagcuauacuaaccccaggguuggucaauuucgugccagccacacccuggagcuagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaag

cauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

SF-2 SEQ ID NO:2

cugguccccacagacucagagagaacccgccaccauguucguguuccugguccugcugccccuggugucuagucagugugugaaccugacaacccgc

acccagcugcccccugccuacacuaacucuuuuaccaggggaguguacuacccagacaaaguguuuagaucuucugugcugcacucuacacaggauc

uguuucugccauucuucucuaacgugacaugguuccacgccauucacgucaguggaacuaacggcacuaagagauucgacaaccccgugcugccuuu

caacgacggaguguacuucgcgagcacugagaaguccaacaucaucagaggcuggaucuuuggcaccacacuggauagcaagacacagagucugcug

auugugaacaacgccacuaauguugugaucaaggugugugaguuccaguucugcaacgauccuuuucuggggguguauuaccacaaaaacaacaaga

guuggauggagucagaguuuaggguguauucuuccgcaaacaacuguacauucgaauacgucucccagccauuucugauggaccuggaggggaagc

agggcaacuuuaaaaaucugcgcgaguuuguguucaagaacaucgauggguacuuuaagauuuacuccaaacacacaccuaucaaccuggugagaga

ucugccacagggauucagcgcccuggagccacugguggaucugccaaucggaaucaauaucacaagauuccagacauuacucgcucugcacaggucc

uaucugacuccaggugauagcagcagcgguuggacugcuggagcagcugccuacuaugugggauaccugcagccaagaacauuccuguugaaauaca

acgagaauggaacaaucacagaugcuguggacugugcacuggauccacugucugagaccaagugcacacugaagagcuucacugucgagaagggcau

cuaucagacaucuaacuuuagagugcagcccacagagagcaucgugcgauuccccaacaucacaaaccuguguccuuuuggcgagguguucaacgcc

acaagauucgcuucuguguacgcauggaacaggaagcgaaucucuaacuguguggccgauuacagcgugcuguacaacucugcuucuuucuccacau

ucaaguguuacggagugucaccuacuaaacugaacgaccugugcuucacuaacguguaugccgauaguuucgugauccggggagaugaggugaggc

agaucgccccaggacagaccgggaaaaucgccgauuacaacuauaagcugccagacgauuucaccgguugugugaucgccuggaacucaaacaaccug

gauagcaaaguggggggcaacuauaacuacagauaccgacuguucaggaaaagcaaccugaaaccuuuugagcgcgauaucuccacugagaucuacca

ggcaggaucuacaccauguaauggagugcagggauucaacuguuacuucccccuccagucuuauggcuuucagccuacuaacggagugggauaucag

cccuacagaguggugguccuguccuucgagcugcugcacgcacccgcuaccgugugcggaccuaagaagucaaccaaccuggugaaaaacaagugug

uaaacuuuaacuucaacggacucaccggcaccggaguucugacagagucuaacaaaaaguuccugcccuuccagcaguucggcagggauaucgcuga

caccacugacgcugugagagacccacagacccuggagauccuggauaucaccccuuguucuuucggcggugucagugugaucacaccaggaacuaac

accaguaaccagguggccgugcucuaccagggagugaacuguaccgaggugccuguggcuauccacgccgaccagcugaccccuaccuggagggugu

auagcaccggaucuaacguguuccagacacgagccggcugccucaucggcgccgagcacgugaacaacucuuacgagugugacaucccuaucggagc

uggaaucugugcuuccuaccagacacagaccaauucuccuagaagagccagaucugucgcaagccagaguaucaucgcuuacaccaugagccuggga

gcugagaacagcguggcuuacagcaacaacagcaucgcaaucccuaccaauuucacaaucucagugacuaccgagauccugcccgugagcaugacuaa

aacaucaguggacuguacaauguauaucugcggagacuccacagagugcucaaaccugcugcugcaguauggaucuuucuguacucagcugaacaga

gcucugacaggaaucgcaguggagcaggacaagaacacucaggagguguuugcccaggugaagcagaucuacaaaaccccuccuaucaaagauuuug

ggggauucaacuucagccagauccugccugauccuuccaaaccuuccaaaagaucauuuaucgaggaccuucuguucaacaaggucacccuggcuga

cgcuggcuuuaucaagcaguauggcgauugucugggugauaucgcagcaagagaucugauuugugcucagaaguuuaacggacugacaguucugcc

uccgcugcugaccgaugagaugaucgcccaguacacaagugcacugcuggcuggcaccaucacauccggcuggaccuucggagccggcgccgcccug

cagauuccuuucgcuaugcagauggcauaucgcuuuaacggaauuggcgugacucagaacguccuguacgagaaccagaaacugaucgcaaaccagu

ucaacagcgcuaucggaaaaauccaggauagccugucaucuacagccagugcccugggaaagcugcaggacguggugaaucagaacgcacaggcccu

gaacacacuggugaaacagcugagcucaaacuuuggagcuaucuccucgguccugaacgacauccuguccagacuggauccgccugaggcugaggug

cagaucgacagacugaucacaggaagacugcagagucugcaaaccuacgugacccagcagcucauccgcgcugcugagaucagagcguccgcaaaccu

ggcagccaccaaaaugagugagugugugcucggacagagcaaacgcguggauuucuguggcaagggauaccaucugaugagcuucccacagagcgcc

ccacacggagugguguuccugcaugugacuuacguccccgcucaggagaaaaauuucacaacugcgccugcuaucugccacgaugguaaggcucacu

uuccaagagagggaguguuugugagcaaugggacacauugguucgucacccagagaaacuucuacgagccucagaucaucacuaccgacaauacuuu

cgugucaggaaacugcgacguggugaucggcaucgucaacaauaccguuuacgacccucugcagccugagcuggacaguuucaaagaggagcuggau

aaauacuucaaaaaccacacuagcccagacguggaccugggagauaucagcggcaucaaugcaucuguggugaacauccagaaagagauugacagacu

gaacgagguggccaagaaccugaacgaaagucugaucgaucugcaagagcugggaaaauaugagcaguacaucaaauggccuugguacauuuggcug

ggauucaucgcuggacugaucgcaauugugauggugacaauuaugcugugcugcaugacaagcugcuguucuugucucaaaggaugcugcucuugc

gguagcugcuguaaauuugaugaggacgauagugagccugugcucaagggagucaaacugcacuauaccuaaugacucgagcugguacugcaugca

cgcaaugcuagcugccccuuucccguccuggguaccccgagucucccccgaccucgggucccagguaugcucccaccuccaccugccccacucaccac

cucugcuaguuccagacaccucccaagcacgcagcaaugcagcucaaaacgcuuagccuagccacacccccacgggaaacagcagugauuaaccuuua

gcaauaaacgaaaguuuaacuaagcuauacuaaccccaggguuggucaauuucgugccagccacacccuggagcuagcaaaaaaaaaaaaaaaaaaaaa

aaaaaaaaagcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

SF-3 SEQ ID NO:3

cuguaaauucgacgaagacgacucggaacccgugcugaagggcguuaagcuccacuacaccugaugacaagcacgcagcaaugcagcucaaaacgcuu

agccuagccacacccccacgggaaacagcagugauuaaccuuuagcaauaaacgaaaguuuaacuaagcuauacuaaccccaggguuggucaauuucg

ugccagccaccucgagcugguacugcaugcacgcaaugcuagcugccccuuucccguccuggguaccccgagucucccccgaccucgggucccaggu

augcucccaccuccaccugccccacucaccaccucugcuaguuccagacaccuccgcucgcuuucuugcuguccaauuucuauuaaagguuccuuug

uucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuucauugcaauugcc

auguguauguggguucgcccacauacucugaugauccccaaucguggcgugucggccugcuucggcaggcacuggcgccgggaucauucauggcaa

gcuggagccucgguggccaugcuucuugccccuugggccuccccccagccccuccuccccuuccugcacccguacccccguggucuuugaauaaagu

cugagugggcggcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

aaaaaaaaaaaaaaaaaaaaaaa

SF-4 SEQ ID NO:4

cuguaaauucgacgaagacgacucggaacccgugcugaagggcguuaagcuccacuacaccugaugagcucgcuuucuugcuguccaauuucuauua

aagguuccuuuguucccuaaguccaacuacuaaacugggggauauuaugaagggccuugagcaucuggauucugccuaauaaaaaacauuuauuuuc

auugcaauugccauguguauguggguucgcccacauacucugaugauccccaaucguggcgugucggccugcuucggcaggcacuggcgccgggau

cauucauggcaagcuggagccucgguggccaugcuucuugccccuugggccuccccccagccccuccuccccuuccugcacccguacccccgugguc

uuugaauaaagucugagugggcggcacucgagcugguacugcaugcacgcaaugcuagcugccccuuucccguccuggguaccccgagucucccccg

accucgggucccagguaugcucccaccuccaccugccccacucaccaccucugcuaguuccagacaccuccaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

agcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

SF-5 SEQ ID NO:5

cauucauggcaacucgagcugguacugcaugcacgcaaugcuagcugccccuuucccguccuggguaccccgagucucccccgaccucgggucccag

guaugcucccaccuccaccugccccacucaccaccucugcuaguuccagacaccucc

SF-6 SEQ ID NO:6

uuugaauaaagucugagugggcggcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

SF-0 SEQ ID NO:7

cugguccccacagacucagagagaacccgccaccauguuuguuuuucuuguuuuauugccacuagucucuagucaguguguuaaucuuacaaccaga

acucaauuacccccugcauacacuaauucuuucacacgugguguuuauuacccugacaaaguuuucagauccucaguuuuacauucaacucaggacu

uguucuuaccuuucuuuuccaauguuacuugguuccaugcuauacaugucucugggaccaaugguacuaagagguuugauaacccuguccuaccau

uuaaugaugguguuuauuuugcuuccacugagaagucuaacauaauaagaggcuggauuuuugguacuacuuuagauucgaagacccagucccuac

uuauuguuaauaacgcuacuaauguuguuauuaaagucugugaauuucaauuuuguaaugauccauuuuuggguguuuauuaccacaaaaacaaca

aaaguuggauggaaagugaguucagaguuuauucuagugcgaauaauugcacuuuugaauaugucucucagccuuuucuuauggaccuugaaggaa

aacaggguaauuucaaaaaucuuagggaauuuguguuuaagaauauugaugguuauuuuaaaauauauucuaagcacacgccuauuaauuuagugc

gugaucucccucaggguuuuucggcuuuagaaccauugguagauuugccaauagguauuaacaucacuagguuucaaacuuuacuugcuuuacaua

gaaguuauuugacuccuggugauucuucuucagguuggacagcuggugcugcagcuuauuauguggguuaucuucaaccuaggacuuuucuauua

aaauauaaugaaaauggaaccauuacagaugcuguagacugugcacuugacccucucucagaaacaaaguguacguugaaauccuucacuguagaaaa

aggaaucuaucaaacuucuaacuuuagaguccaaccaacagaaucuauuguuagauuuccuaauauuacaaacuugugcccuuuuggugaaguuuuu

aacgccaccagauuugcaucuguuuaugcuuggaacaggaagagaaucagcaacuguguugcugauuauucuguccuauauaauuccgcaucauuu

uccacuuuuaaguguuauggagugucuccuacuaaauuaaaugaucucugcuuuacuaaugucuaugcagauucauuuguaauuagaggugaugaa

gucagacaaaucgcuccagggcaaacuggaaagauugcugauuauaauuauaaauuaccagaugauuuuacaggcugcguuauagcuuggaauucua

acaaucuugauucuaagguuggugguaauuauaauuaccuguauagauuguuuaggaagucuaaucucaaaccuuuugagagagauauuucaacug

aaaucuaucaggccgguagcacaccuuguaaugguguugaagguuuuaauuguuacuuuccuuuacaaucauaugguuuccaacccacuaauggug

uugguuaccaaccauacagaguaguaguacuuucuuuugaacuucuacaugcaccagcaacuguuuguggaccuaaaaagucuacuaauuugguuaa

aaacaaaugugucaauuucaacuucaaugguuuaacaggcacagguguucuuacugagucuaacaaaaaguuucugccuuuccaacaauuuggcaga

gacauugcugacacuacugaugcuguccgugauccacagacacuugagauucuugacauuacaccauguucuuuugguggugucaguguuauaaca

ccaggaacaaauacuucuaaccagguugcuguucuuuaucaggauguuaacugcacagaagucccuguugcuauucaugcagaucaacuuacuccua

cuuggcguguuuauucuacagguucuaauguuuuucaaacacgugcaggcuguuuaauaggggcugaacaugucaacaacucauaugagugugaca

uacccauuggugcagguauaugcgcuaguuaucagacucagacuaauucuccucggcgggcacguaguguagcuagucaauccaucauugccuacac

uaugucacuuggugcagaaaauucaguugcuuacucuaauaacucuauugccauacccacaaauuuuacuauuaguguuaccacagaaauucuacca

gugucuaugaccaagacaucaguagauuguacaauguacauuuguggugauucaacugaaugcagcaaucuuuuguugcaauauggcaguuuuugu

acacaauuaaaccgugcuuuaacuggaauagcuguugaacaagacaaaaacacccaagaaguuuuugcacaagucaaacaaauuuacaaaacaccacca

auuaaagauuuuggugguuuuaauuuuucacaaauauuaccagauccaucaaaaccaagcaagaggucauuuauugaagaucuacuuuucaacaaag

ugacacuugcagaugcuggcuucaucaaacaauauggugauugccuuggugauauugcugcuagagaccucauuugugcacaaaaguuuaacggcc

uuacuguuuugccaccuuugcucacagaugaaaugauugcucaauacacuucugcacuguuagcggguacaaucacuucugguuggaccuuuggug

caggugcugcauuacaaauaccauuugcuaugcaaauggcuuauagguuuaaugguauuggaguuacacagaauguucucuaugagaaccaaaaauu

gauugccaaccaauuuaauagugcuauuggcaaaauucaagacucacuuucuuccacagcaagugcacuuggaaaacuucaagauguggucaaccaaa

augcacaagcuuuaaacacgcuuguuaaacaacuuagcuccaauuuuggugcaauuucaaguguuuuaaaugauauccuuucacgucuugacaaagu

ugaggcugaagugcaaauugauagguugaucacaggcagacuucaaaguuugcagacauaugugacucaacaauuaauuagagcugcagaaaucaga

gcuucugcuaaucuugcugcuacuaaaaugucagaguguguacuuggacaaucaaaaagaguugauuuuuguggaaagggcuaucaucuuaugucc

uucccucagucagcaccucaugguguagucuucuugcaugugacuuaugucccugcacaagaaaagaacuucacaacugcuccugccauuugucaug

auggaaaagcacacuuuccucgugaaggugucuuuguuucaaauggcacacacugguuuguaacacaaaggaauuuuuaugaaccacaaaucauuac

uacagacaacacauuugugucugguaacugugauguuguaauaggaauugucaacaacacaguuuaugauccuuugcaaccugaauuagacucauuc

aaggaggaguuagauaaauauuuuaagaaucauacaucaccagauguugauuuaggugacaucucuggcauuaaugcuucaguuguaaacauucaaa

aagaaauugaccgccucaaugagguugccaagaauuuaaaugaaucucucaucgaucuccaagaacuuggaaaguaugagcaguauauaaaauggcc

augguacauuuggcuagguuuuauagcuggcuugauugccauaguaauggugacaauuaugcuuugcuguaugaccaguugcuguaguugucuca

agggcuguuguucuuguggauccugcugcaaauuugaugaagacgacucugagccagugcucaaaggagucaaauuacauuacacauaaacccugga

gcuagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

aaaaaaaaaaaaaaucuaga

note that: u in the above sequence may be replaced in whole or in part by a modified base, for example: 1-methyl pseudouridine or pseudouridine

KOZAK sequence SEQ ID NO. 8

gccaccatg

alpha-1-globin5’UTR SEQ ID NO:9

gggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggatccactagtccagtgtggtggaattcgaataaactagtattcttctggtccccacaga

ctcagagagaacccgccacc

3' -Poly A sequence SEQ ID NO 10

aaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcatatgactaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

aaaaaa

gp130 3’UTR SEQ ID NO:11

ctcgagctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccccgagtctcccccgacctcgggtcccaggtatgctcccacctccacctgcc

ccactcaccacctctgctagttccagacacctcc

DH143 3’UTR SEQ ID NO:12

caagcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaactaagctatactaa

ccccagggttggtcaatttcgtgccagccac

hHBA1 3’UTR SEQ ID NO:13

gctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcgg

ca

hHBB 3’UTR SEQ ID NO:14

gctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaa

acatttattttcattgcaattgccatgtgtatgtgggttcgcccacatactctgatgatccccaatcgtggcgtgtcggcctgcttcggcaggcactggcgccgggatcatt

catggcaa

Antigen S protein amino acid sequence: SEQ ID NO. 15

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVS

GTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG

VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIN

LVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNEN

GTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAW

NRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNY

KLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVQGFNCYFPLQ

SYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPF

QQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTW

RVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENS

VAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDK

NTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARD

LICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYE

NQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEA

EVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHG

VVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVV

IGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL

QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKL

HYT

(underlined are relative to WT strain-related mutation sites)

The specific embodiment is as follows:

embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustration of the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: codon optimization and In Vitro Transcription (IVT) of S protein antigens

1.1 codon optimization experiments

The SF mRNA sequence is designed according to the natural coding region sequence of the novel coronavirus S protein, and mutation and optimization are carried out on the basis of the natural coding region sequence. In addition to the coding region (ORF), SF mRNA sequence features include alpha-1-globin 5'UTR, gp130 and DH143 3' UTR and polyA. For example: SF-1 and SF-3mRNA sequences were optimized in the S protein coding region, and the GC content was increased compared to SF-0 mM, while remaining consistent in the UTR region.

The following is a brief experimental procedure:

based on literature reports, the present study designed a variety of humanized UTR sequences, ligated to both ends of a luciferase reporter gene (Nluc), and cloned into vector pvax.1 (gold biotechnology ltd). mRNA was synthesized in vitro using linearization vector (custom made by Sony Biotechnology Co., ltd., structure see FIG. 1) as a template, and HEK293T cells were transfected. At the set time, luciferase expression levels in the cells were observed and the effect of UTR and codon optimisation on mRNA expression levels and stability was assessed. The expression level results were as follows:

table 1: optimization of mRNA relative expression amount data by different codons

From the above experimental results, it can be seen that: codon optimization has a relatively large influence on the relative expression amount of mRNA, taking SF-0, SF-1 and SF-2 as examples, and under the condition that other conditions are unchanged (5 'UTR and 3' UTR), the expression amount of antigen protein can be greatly provided through the codon optimization; we selected SF-1 for subsequent experiments.

1.2 in vitro transcription (IVT)

1. According to the instructions of IVT kit (E131, novoprotein), an IVT reaction system WAs configured, namely, 10X Transcription Buffer, ATP, GTP, CTP, 1-N-Me-pseudoUTP (cat# WA0992, megadimension technology), 5 'cap analogue m7G (5') ppp (5 ') (2' OMeA) pG (cat# GAGNH23C2L1B, megadimension technology), water for injection, linearized plasmid templates for T7 promoters (custom made by Kirsrui Biotechnology Co., ltd., structure see FIG. 1) and Enzyme Mix, respectively;

2. The mixed reaction system is placed at 37 ℃ for reaction for 40min;

3. the reaction was terminated by adding the corresponding proportion of DNase I.

S protein mRNA is synthesized in vitro, and then is subjected to hydrophobic chromatography and ultrafiltration concentration, separation and purification to obtain high-purity mRNA (> 95%), and the specific result is shown in FIG. 2. The experimental results show that: high purity mRNA was obtained by the above procedure, which can be used in subsequent experiments. And (3) injection: internal standard (25 nt Agilent, RNA 6000Nano Kit (reorder-no 5067-1511)

Example 2: preparation and confirmation of protein antigen mRNA-LNP preparation

1. Precisely weighing a certain amount of lipid with molar mass ratio of 50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG2000, and dissolving with appropriate amount of absolute ethanol to obtain lipid working solution (final concentration of lipid working solution 20 mg/mL);

2. separately, a citrate buffer solution (10 mM, pH 4.0) containing 130mM sodium chloride, a Tris-NaOAc buffer solution (20mM,10.7mM,pH 7.5), a Tris-NaOAc buffer solution (20mM,10.7mM,pH 7.5) containing 60% sucrose was prepared;

3. an appropriate amount of the mRNA stock solution synthesized and purified in example 1 was diluted with the sodium chloride-citric acid buffer solution prepared as described above, and the final concentration of the mRNA working solution was adjusted to 0.18mg/mL.

4. The lipid working solution and the mRNA working solution are mixed according to the volume ratio of 1:3 by using a microfluidic instrument and a matched chip (shm, michaer's instrument technology Co., ltd.) to prepare the LNP solution carrying mRNA.

5. The prepared LNP solution was diluted by adding 9 volumes of Tris-NaOAc buffer solution, and concentrated and purified by tangential flow ultrafiltration (TFF), and the ethanol solution was removed from the system.

6. And detecting the mRNA content in the LNP solution by using an ultraviolet spectrophotometry, adding a proper amount of Tris-NaOAc buffer solution (20mM,10.7mM,pH 7.5) containing 60% of sucrose, and adjusting the final concentration of the mRNA in the LNP solution to 100 mug/mL, wherein the sucrose content in an external water phase system is 8.7%.

7.S protein mRNA and lipid working fluid are mixed by micro-flow control to form LNP complex.

The transmission electron microscope results showed (fig. 3): the S protein mRNA and Lipid Nanoparticle (LNP) complex form is shown, the particle size is about 100nm, and the uniformity is good.

Example 3: immunoblotting experiments of S protein expression in host cells

S protein mRNA was transfected into COS-7 cells (African green monkey kidney fibroblasts, available from ATCC) and after 24h of transfection, the cells were collected and subjected to immunoblotting. Cells not transfected with S protein (enzyme hydrolysis of the forskolin site) mRNA served as negative controls (B). Beta-action is used as an internal reference. Results show (see fig. 4): both the full length of the S protein and the S1 subunit protein can be detected, and the purity meets the requirement of further experiments.

Example 4: FACS method for detecting expression of antigen protein on cell surface

1. To examine the expression of novel coronavirus S protein mRNA in human cells and localization on cell membranes, we digested HEK293T (purchased from ATCC) cells cultured for more than 24 hours and transferred to 6 well plates to control cell density at 400000 cells per well.

2. After incubation of the six well plates at 37 degrees for 24 hours, the cell status was observed with a microscope. mRNA transfection can be performed when the cell confluency reaches more than 80 percent.

3. The corresponding mRNA was transfected into HEK293T cells (2. Mu.g of mRNA per well) using Lipofectamine 2000 kit (11668027, invitrogen) and specific procedures were described with reference to the kit. And the culture was continued at 37℃for 24 hours.

4. After the mRNA was expressed for 24 hours, the expression amount was close to the peak, at which time the cell supernatant was removed, washed once with PBS, 200. Mu.L of trypsin was added to digest the cells, and 3mL of complete medium was used to neutralize the pancreatin. The cell suspension was transferred into a 15mL centrifuge tube, centrifuged at 1400rpm for 5 minutes, the supernatant was discarded, the cells were resuspended with 3mL of 1% BSA solution (BSA dissolved in PBS), and then centrifugation at 1400rpm was continued for 5 minutes, and the supernatant was discarded.

5. Cells were resuspended with 0.5mL of 1% BSA solution, centrifuged at 1400rpm for 5 minutes, and the supernatant was discarded. At this point, cells were resuspended with 150. Mu.L of a solution of the antibodies to the novel coronavirus S protein (1:250 dilution of the antibodies with 1% BSA solution, genScript, cat# CI 1534) and transferred to a 1.5ml centrifuge tube. Incubate at room temperature for 2 hours.

6. 1mL of PBS solution was added and centrifuged at 1400rpm for 5 minutes, the supernatant was discarded, 0.5mL of PBS solution was added to resuspend the cells, and the supernatant was discarded after centrifugation at 1400rpm for 5 minutes. 0.5mL of PE fluorescent-labeled goat anti-rabbit secondary antibody diluted with PBS (1:250 dilution) was added and incubated at room temperature for 1 hour in the dark.

7. 1mL of PBS solution was added and centrifuged at 1400rpm for 5 minutes, the supernatant was discarded, 0.5mL of PBS solution was added to resuspend the cells, and the supernatant was discarded after centrifugation at 1400rpm for 5 minutes. Cells were resuspended in 0.5mL PBS solution for examination.

8. Flow cytometry detection: the cells not transfected with mRNA are set as a negative control group, the negative control cells are detected by a flow cytometer, target cell signals are circled on a dot pattern, FITC fluorescent signals are read by using a histogram, and the sensitivity and voltage of a detector are adjusted to determine the range of positive cell fluorescent signals on the histogram, so that the cell is circled.

9. The percentage of positive cells was recorded for each sample in turn, and 10000 signals were read for each sample.

After transfection of S protein mRNA into HEK293T cells, expression of the antigen protein of interest on the cell surface was detected using FACS flow cytometry, and cells not transfected with S protein mRNA served as negative controls (B). Experimental results show (see fig. 5): the positive rate of S protein mRNA transfection and expression was 98.11% relative to control B. It can be seen that the antigen protein encoded by the mRNA prepared and transfected in the present application can be effectively expressed on the cell surface and effectively combined with the antibody.

Example 5: antibody neutralization pseudovirus assay

5.1 pseudovirus preparation of novel coronaviruses the following steps are followed:

1. HEK293T cells were simultaneously transfected with expression plasmids of the novel coronavirus S protein and mutants thereof, and with HIV-1 backbone plasmid (Kirschner Biotechnology Co.) using Polyethylenimine (PEI). The HIV-1 backbone plasmid simultaneously expresses viral packaging proteins and luciferase reporter genes.

2. After 48 or 72h of transfection, the supernatant containing pseudoviruses was collected and filtered through a 0.45 μm filter to remove cell debris.

3. The pseudovirus supernatant obtained by filtration was split-packed and stored at-196 ℃.

The p24 ELISA kit (632200, takara) detected pseudovirus titers and was confirmed to meet the requirements of further experiments.

5.2 in vitro pseudovirus neutralization experiments

Luciferase-labeled SASR-CoV-2 pseudovirus (see section 5.1 for preparation) was co-incubated with serum from S protein mRNA immunized mice. The incubated culture broth was then infected with HEK293T cells (XCC 14, novoprotein) constitutively expressing ACE2 protein, and the fluorescent value of intracellular pseudovirus was detected after 48h of transfection. The results of fig. 6 show that: post-immunization mouse serum versus pseudovirus IC ₅₀ A value of 203.4 is effective in generating an immune response.

5.3 experiments for neutralizing various SARS-CoV-2 Strain antibodies

S protein mRNA-LNP preparation serum from immunized and non-immunized mice was incubated with pseudoviruses of SARS-CoV-2 strains B.1617.1, P.1, B.1.1.7, WT (NCBI SEQ ID NO: NC_ 045512.2) and B.1.351, respectively. And then, infecting HEK293T cells which are constitutively used for expressing ACE2 protein by the incubated culture solution, and detecting the fluorescence value of the intracellular pseudovirus after 48 hours of transfection to calculate the infection rate of the pseudovirus. The results (see fig. 7) show: compared with the control group, the immune group can greatly reduce the infection rate, the average infection rate of the immune groups WT, B.1.617.1, P.1 and B.1.1.7 except the B.1.351 strain is lower than 10 percent, and the effect on the B.1.351 strain is not inferior to that of the current similar products on the market, such as: the protection of the strain by the coronavirus of Asp Li Kangxin was not effective (Shabir A Madhi et al, efficacy of the ChAdOx, 1 nCoV-19 Covid-19 Vaccine against the B.1.351 Variant,NEJM,2021.5.20), and the protection of the strain by the novel coronavirus vaccine of Novavax was only 49% (Vivek Shinde et al, efficiency of NVX-CoV2373 Covid-19 Vaccine against the B.1.351 Variant,NEJM,2021.5.20). The vaccine obtained by the application has broad-spectrum obvious immunity effect of reducing the infection of a plurality of novel coronavirus mutant strains.

Example 6: antibody binding experiments with S protein

Serum from mice immunized with S protein mRNA-LNP preparation was co-incubated with S proteins (gold Style custom) of SARS-CoV-2 strains B.1.617.1, P.1, B.1.1.7, WT and B.1.351, respectively, and ELISA was used to examine the mouse serum neutralizing antibody titer and neutralizing ability against S proteins of the different strains. Experimental results showed (see fig. 8): antibodies produced by immunized mice have extremely strong binding capacity with the current common SARS-CoV-2 strain. In contrast, binding was stronger with indian mutant b.1.617.1.

Example 7 toxicity test results

In order to further verify the immune effect of the mRNA vaccine prepared in the present application, an immune challenge experiment was performed against mice transfected with hACE2 protein, specifically as follows:

the test uses 46 female hACE2 transgenic mice divided into 6 groups by body weight. 1 to 4 groups are main test groups (including low dose group, high dose group, negative control group and model control group), and 5 to 6 groups are satellite groups (without any treatment, only for eliminating individual differences of experimental animals). The negative control group and the model control group were given vehicle control, and the low and high dose groups and the corresponding satellite groups were given 5 and 20 μg/dose of vaccine, respectively. Animals were immunized 1 time for 21 days and 2 times in total. Animals of groups 1-4 were transferred to biosafety tertiary laboratory (BSL-3) for treatment after immunization 2. The animals of the model control group, 5 and 20 mug/vaccine group were challenged by nasal drip at 13 days post 2 immunization, and the challenge dose of Delta strain (from the national center for disease control) was 10≡5PFU/animal. Animal body weight was measured daily before and after challenge. All animals were dissected 7 days after challenge, lung tissue viral loads (N gene and ORF1ab gene) were determined by fluorescent quantitative PCR, and lung histopathological examination was performed. The serum was periodically monitored for Delta S1 protein-specific antibodies during the assay.

The experimental results show that the titers of specific antibodies to Delta S1 protein in the sera of animals of the vaccine group of 5 and 20. Mu.g/animal prior to challenge (day 29 of first immunization, day 8 of second immunization) were approximately 1:10 ⁶ . Antibody titer was stable from the period of challenge to the end of the test, and remained at 1:10 ⁶ Left and right;

the results of the mouse lung tissue virus titer show that the ORF1ab genes of the animals in the model group are 6log10 copies/mL on average and the N genes are 6.38log10 copies/mL on average, which indicates that the Delta virus replicates well in the mice after the mice are infected. Only lower copies were detected for

vaccine groups

5 and 20. Mu.g/ORF 1ab gene was on average 2.81log10 copies/mL, no N gene was detected. Compared with the model animals, the viral load of the vaccine animals is reduced by >2log10, which suggests that the administration of 5 and 20 mug/vaccine can effectively reduce the viral load of Delta variant infected hACE2 transgenic mouse model.

7 days after challenge, lung histopathological results showed that lung tissue of model group animals mice was seen with alveolar space, alveolar septum, peribronchiolar and/or slight to moderate macrophage aggregation, characterized by increased macrophage numbers, increased volume and/or increased eosinophilia. Indicating that the Delta contamination model meets the requirements. Only 1 animal lung bronchiole surrounding slight macrophage aggregation was seen in the 5 μg/dose group, and the lung was unchanged in the 20 μg/dose group. Therefore, the vaccine obtained by the application can effectively prevent the lung pathological damage of mice caused by Delta virus infection.

In a comprehensive view, compared with an unimmunized group, the immunity group of the mRNA vaccine can greatly enhance the resistance of mice to the novel coronavirus Delta, and a remarkable technical effect of preventing the novel coronavirus infection is achieved. Comparing the current two marketed mRNA Vaccines, the results of a document (Mark G et al, prevention and Attenuation of Covid-19with the BNT162b2 and mRNA-1273 Vaccines) reported in the New England journal show that: the average viral load of the non-vaccinated infected person is 3.8log 10 copies per ml, the average viral load of the partially or fully vaccinated infected person is 2.3log10 copies per ml; (40% lower viral RNA load in vivo compared to the non-vaccinated infected subjects after partial or complete mRNA vaccination); the vaccine obtained by the method can reduce the viral load by more than 53%, and is significantly superior to BNT162b2 and mRNA-1273.

Example 8: cell immunity effect experiment

8.1T cell immunization

BALB/c mice were given intramuscular injections at doses of 5, 10 and 20 μg/min mRNA-LNP formulation, 1 immunization per 21 days, 2 immunization. Spleens were collected 14 days after the 2 nd immunization. Spleen cells of mice were isolated, assayed using IFN-. Gamma.ELISPot.kit, and the coated plates were added to SARS-CoV-2 polypeptide libraries 1 and 2 (customized by Nanjing gold St. Biotechnology Co., ltd.) for immunostimulation to detect SARS-CoV-2 specific T cells. Results show (see fig. 9): significant SARS-CoV-2S protein specific T cell responses were detected in spleen cells of all three groups, which indicated that the vaccines obtained in this application were successful in eliciting cellular immunity in the organism.

8.2B cell immunization

BALB/c mice were given intramuscular doses (vaccine groups) of 5, 10 and 20 μg/LNP preparation, 1 immunization per 21 days, 2 immunization. Spleens were collected 7 days after the 2 nd immunization and the situation of memory B cells production at different doses under the optimal immunization strategy was studied. After separating spleen immune cells of mice, adding R848 and IL-2, performing in vitro co-stimulation amplification culture for 4 days, paving into a pre-coated SARS-CoV-2RBD or S protein plate, and detecting SARS-CoV-2 specific B cells by using Mouse IgG ELISpot kit. The results show (see figure 10) that the vaccine of the present application was able to significantly induce the production of SARS-CoV-2RBD and S protein specific memory B cells in the spleen of mice in each of the three dose groups.

Example 9: protection effect experiment for Omikovia strain

9.1 neutralizing antibody experiments

After the mRNA-LNP preparation prepared by the application is immunized on a mouse for 21 days, serum of the mouse is extracted, neutralization effects of the serum on pseudoviruses of Wild Type (WT) and amikacin (B.1.1.529) strains of the novel coronavirus are observed, and a half-binding rate (IC is calculated ₅₀ ) Specific results indicate (see fig. 11): the neutralization effect is better for the strain of the amikacin.

9.2 Effect experiments on the protection Rate of mice

The test uses 45 male hACE2 transgenic mice, which are divided into 4 groups according to body weight, 9 negative control groups, and 12 groups of the other 3 groups (9 main test animals for toxicity test, and 3 satellite animals for antibody detection, the satellite animals are not treated at all, and only used for eliminating individual differences of experimental animals). The negative control and model (challenge) control were given vehicle, and the low and high doses were given 5 and 20 μg/min respectively of the mRNA-LNP formulation prepared herein. Animals were immunized 3 times total, i.e., iD0 (first immunization was denoted as iD 0), iD21, and iD109, respectively. 8 days after 3 rd immunization (iD 117), model control groups and 5 and 20. Mu.g/vaccine group test animals of the present application were transferred to BSL-3 laboratory and challenged by nasal drip, with an Omicron strain dose of 10000 PFU/animal. Recording death condition and weight change of animals after toxin attack, and calculating average survival days and death rate; dead animals and euthanized animals (all surviving animals were euthanized at day 8 post-infection) tissues such as lung were dissected and examined for virus titers and lung histopathology.

The challenge results showed that only 1 animal survived the model group 8 days after challenge, and that the survival rates of both 5 and 20 μg/vaccine group of the present application were 100% (see fig. 12), indicating that: the vaccine group has a protective effect on mice infected by Omicron, and can obviously reduce the death rate of animals; and the weight of the animals in the vaccine group has an increasing trend at the end of the challenge period (see figure 13). The results show that the vaccine group has better preventive effect on Omicron virus infection and illness and death.

In addition, a long-term immunity effect verification experiment is also carried out, and the specific steps are as follows:

BALB/c mice, 10 in each group, male and female halves, were respectively intramuscular injected with PBS (blank), vaccine mRNA-LNP formulations of the present application (three

dose groups

5, 10 and 20. Mu.g), immunized 2 times at 21-day intervals, and the concentration of neutralizing antibodies was measured multiple times during 7 to 175 days after 2 immunizations. The experimental results show that: under the 21-day immunization strategy, the bound antibody titer of serum after the first 6 months of immunization of the three dose groups was still 10 ⁶ About, the long-term immune protection effect is proved to be very good.

Example 10: stability of mRNA vaccines for prolonged storage at different temperatures

The purpose of the experiment is as follows: the law of the change of the mRNA vaccine finished product along with time under long-term stability and acceleration stability and the law of the change along with the freeze thawing times are inspected, scientific basis is provided for the production, storage, packaging and transportation conditions of the sample, and meanwhile, basis is provided for the subsequent quality standard formulation of the related finished product.

Product information: the product is prepared by combining mRNA molecular bulk drug with liposome auxiliary materials to form a Tris-NaOAc aqueous solution final product of liposome nano-particles.

Table 2 mainly examines the index and quality criteria

Detecting items	Quality standard
		Appearance of	Should be a slightly milky clear liquid
pH value of	Should be 6.0 to 8.0
		Osmotic pressureMolar concentration	Should be 240-420mOsmol/kg
Particle size	＜200nm
		Zeta potential	-10～+10mV
Nanoparticle dispersion coefficient (PDI)	<0.3
		Encapsulation efficiency	≥80％
RNA concentration
		80～130μg/mL

The storage stability of the mRNA vaccine finished product at different temperatures was investigated according to the above criteria. First, the standard is tested for the first time on day 0, and then stored for 12 weeks under three temperature conditions (5+ -3 ℃, -20 ℃, -80 ℃), and then tested for the second time; the specific results are shown in Table 3.

TABLE 3 stability data for mRNA vaccines stored at different temperatures over time

From the above results, it can be seen that: after the vaccine is stored for 12 weeks at the temperature of 5+/-3 ℃ which is relatively high, each detection item is maintained at a relatively high level, and similarly, each detection index is very good at the temperature of-20 ℃, so that the storage requirement of the mRNA vaccine can be met.

Claims

1. An mRNA comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or at least 99% or 100% identity to the nucleotide sequence of any one of SEQ ID NOs 1-6.

2. An mRNA encoding a coronavirus antigen comprising amino acids having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or at least 99% or 100% identity to the amino acid sequence of SEQ ID No. 15.

3. The mRNA of any one of claims 1-2, wherein the mRNA comprises a 5' untranslated region (UTR), a 3' UTR, and a 3' -poly a.

4. The mRNA of any one of claims 1-3, wherein the 3' -poly a comprises the nucleotide sequence of SEQ ID No. 10.

5. An mRNA according to any of claims 1 to 3, wherein the 5'UTR comprises the nucleotide sequence of SEQ ID NO. 9 and/or the 3' UTR is selected from the nucleotide sequences of SEQ ID NO. 11 to 14.

6. The mRNA of any one of claims 1-5, further comprising a 5' guanosine cap selected from the group consisting of: m7Gppp (2' OMeA) pG, m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppC, m7GpppCpG, m7GpppCpU, m7GpppGpA, m7GpppGpC, m7GpppG, m7GpppU, m7GpppUpA, m7GpppUpC, m7GpppUpG, m7GpppUpU, m7Gpppm6ApG, m7G _3’Ome pppApA、m7G _3’Ome pppApC、m7G _3’ _Ome pppApU、m7G _3’Ome pppApG、m7G _3’Ome pppCpA、m7G ₃ ^’ _Ome pppCpC、m7G ₃ ^’ _Ome pppCpG、m7G ₃ ^’ _Ome pppCpU、m7G ₃ ^’ _Ome pppUpA、m7G ₃ ^’ _Ome pppUpC、m7G ₃ ^’ _Ome pppUpG、m7G ₃ ^’ _Ome pppUpU、m7G ₃ ^’ _Ome PppA ₂ ^’ _Ome pG、m7G ₃ ^’ _Ome pppA ₂ ^’ _Ome pC、m7G _3’Ome pppA _2’Ome pU、m7G _3’Ome pppA _2’Ome pA、m7G _3’Ome pppC _2’Ome pA、m7G _3’Ome pppC _2’ _Ome pU、m7G ₃ ^’ _Ome pppC ₂ ^’ _Ome pG、m7G ₃ ^’ _Ome pppC ₂ ^’ _Ome pC、m7G ₃ ^’ _Ome pppG ₂ ^’ _Ome pA、m7G ₃ ^’ _Ome pppG ₂ ^’ _Ome pU、m7G ₃ ^’ _Ome pppG ₂ ^’ _Ome pG、m7G ₃ ^’ _Ome pppG ₂ ^’ _Ome pC、m7G ₃ ^’ _Ome pppU ₂ ^’ _Ome pA、m7G ₃ ^’ _Ome pppU ₂ ^’ _Ome pU、m7G _3’ _Ome pppU _2’Ome pG、m7G _3’Ome pppU _2’Ome pC。

7. The mRNA of any one of claims 1-6, which encodes a coronavirus antigen comprising at least one mutation relative to the S protein of a wild-type strain: L452R, E484Q, D614G, K986P, V987P.

8. A composition comprising a lipid nanoparticle and messenger RNA (mRNA) of any one of claims 1-7.

9. The composition of claim 8, comprising an ionizable cationic lipid, a structural lipid, a helper lipid, and a surfactant.

10. The composition of claim 8 or 9, wherein the lipid nanoparticle comprises 20-60mol% ionizable cationic lipid, 25-55mol% structural lipid, 5-25mol% helper lipid, and 0.5-15mol% surfactant, in mole percent (mol%).

11. The composition of any one of claims 8-10, said structural lipid being selected from cholesterol, and cholesterol derivatives, preferably cholesterol.

12. The composition of any one of claims 8-11, wherein the cationic lipid is selected from the group consisting of: SM-102, ALC-0315, ALC-0519, dlin-MC3-DMA, DODMA, C12-200, dlin DMA, preferably SM-102.

13. A composition according to any one of claims 8 to 12, wherein the helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DSPC.

14. The composition according to any one of claims 8-13, wherein the surfactant is selected from PEG2000-DMG, PEG-DSPE, DTDA-PEG2000, TPGS, preferably PEG2000-DMG.

15. The composition of any one of claims 8-14, wherein the lipid nanoparticle comprises 50mol% sm-102, 10mol% dspc,38.5mol% cholesterol, and 1.5mol% dmg-PEG.

16. The mRNA of claims 1-7 or the composition of claims 8-15, wherein the mRNA comprises a chemically modified base or analog selected from the group consisting of 5-methoxymethyl uridine (5-methoxymethyl uridine), 5-methylthiouridine (5-methylthio uridine), 1-methoxymethyl pseudouridine (1-methoxymethyl pseudouridine), 5-methylcytidine (5-methylcytidine), 5-methoxycytidine (5-methoxy-cytodine), 1-Methyl pseudouridine (N1-Methyl-pseudouridine-UTP), pseudouridine; preferably 1-methyl pseudouridine; most preferably, uracil is completely replaced with 1-methyl pseudouridine, such that each uracil in the sequence is replaced with 1-methyl pseudouridine.

17. Use of an mRNA according to any one of claims 1 to 7 or a composition according to any one of claims 8 to 16 for the preparation of a vaccine.

18. The use of claim 17, wherein the vaccine is selected from the group consisting of a multiple vaccine and a multivalent vaccine.

19. The use according to claim 17, said vaccine being a SARS-CoV-2 virus mRNA vaccine, preferably said vaccine being a SARS-CoV-2 virus mutant vaccine, said mutant preferably being b.1.1.7 (british variant, alpha strain, key mutation: D614G, N Y, P681H), b.1.351 (south african variant, beta strain, key mutation: E484K, N501Y, K417N), b.1.617.1 (first generation indian mutant, kappa strain, key mutation: L452R, E484Q, D G) and b.1.617.2 (second generation indian mutant, delta strain, key mutation: L452R, E484Q, D G), p.1 (brazil mutant, key mutation: K417T, E K, N Y), and b.1.529 strain (south african mutant Omicron, involving 40 point mutations).

20. A method comprising administering to a subject a composition of claims 8-16 effective to induce a neutralizing antibody response against SARS-CoV-2 in the subject.

21. A pharmaceutical composition comprising the composition of claims 8-16, tris buffer, sodium acetate and 8.7% sucrose, pH 7.4.

22. A combination vaccine comprising a first vaccine selected from the group consisting of the mRNA of claims 1-7, the composition of claims 8-16, the pharmaceutical composition of claim 21 and a second vaccine for sequential use.

23. The combination vaccine of claim 22, wherein the second vaccine is selected from the group consisting of: attenuated or inactivated vaccines, adenovirus vaccines, mRNA vaccines, DNA vaccines, recombinant protein vaccines.

24. The combination vaccine of claim 22 or 23, wherein the second vaccine is selected from the group consisting of: moderna (mRNA-1273), cureVac (CVnCoV), johnson & Johnson (COVID-19 Vaccine Janssen), astraZeneca (Vaxzevria), pfizer/BioNTech (Comirnaty), sputnik (Gam-COVID-Vac), sinovac (COVID-19 Vaccine (Vero Cell), novax (NVX-CoV 2373), kang Xinuo New crown Vaccine, beijing national drug New crown Vaccine, proc.