Combination vaccine against coronavirus and influenza virus

Abstract

The invention provides an mRNA (messenger Ribonucleic Acid) molecule aiming at a coronavirus SARS-CoV-2 and / or an influenza virus, an mRNA vaccine containing the molecule and application of the mRNA vaccine. According to the combined vaccine, mRNA molecules respectively aim at Spike protein of an SARS-CoV-2 strain subjected to stable mutation or hemagglutinin protein of an influenza strain, optimized mRNA sequences can play an effective immune role, and the combined vaccine provided by the invention can activate specific immune response of an organism immune system aiming at various virus antigens; the immunoreaction can be synergistically enhanced through combined stimulation of various virus antigens.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, the present invention relates to a combined vaccine against coronavirus and influenza virus, especially an mRNA vaccine. Background Art

[0002] The novel coronavirus infection is an acute respiratory infectious disease caused by the novel coronavirus (SARS-CoV-2). After the patient recovers, he or she will basically not be infected again in the short term. However, as the virus continues to mutate and the antibodies in the bodies of recovered patients or vaccinated people gradually decrease or even disappear, there is still a possibility of secondary infection. Therefore, people may need to continuously inject the new coronavirus vaccine or booster shot against a specific strain every year to achieve the purpose of continuous immunity.

[0003] Influenza, also known as flu, is an acute respiratory infection caused by influenza viruses. Annual flu vaccination is the most effective way to prevent influenza, which can significantly reduce the risk of influenza and serious complications.

[0004] The "Technical Guidelines for Influenza Vaccination in China (2022-2023)" points out that, based on the existing research and recommendations, after careful consideration, it is considered acceptable for people aged 18 and above to receive both inactivated influenza vaccines and new crown vaccines at the same time. When vaccinated at the same time, the vaccination sites on both sides of the limbs should be performed separately. The research results of Sinopharm Group China Biologic show that simultaneous vaccination with the new crown inactivated vaccine and the quadrivalent influenza vaccine can increase the chance of immunity in susceptible people, reduce the risk of co-infection and the risk of cross-infection in fever clinics, and improve the vaccination efficiency of vaccination clinics. However, if a new crown influenza combined vaccine can be developed to prevent infection with both viruses at the same time with one vaccination, a lot of manpower, material and financial resources can be saved.

[0005] Therefore, developing a new coronavirus influenza combination vaccine with good immunogenicity, high safety, and no immune interference between immunogens is an urgent problem to be solved by technical personnel in this field. Summary of the invention

[0006] To solve the above problems, in a first aspect, the present invention provides an mRNA molecule comprising a coding region, wherein the coding region encodes a Spike protein of a first SARS-CoV-2 strain and / or a second SARS-CoV-2 strain that has undergone a stabilizing mutation, or a variant thereof having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto, wherein the stabilizing mutation is selected from a double proline stabilizing mutation or a six proline stabilizing mutation, and wherein the first SARS-CoV-2 strain is SARS-CoV-2BA5.2 and the second SARS-CoV-2 strain is SARS-CoV-2B.1.617.2.

[0007] In some embodiments, the double proline stabilizing mutation is at positions corresponding to K986 and V987 of the Spike protein of wild-type SARS-CoV-2; and / or the six proline stabilizing mutation is at positions corresponding to F817, A892, A899, A942, K986, V987 of the Spike protein of wild-type SARS-CoV-2.

[0008] In some embodiments, the Spike protein of the first SARS-CoV-2 strain that has undergone a stabilizing mutation comprises the amino acid sequence shown in SEQ ID NO.1, and / or the Spike protein of the second SARS-CoV-2 strain that has undergone a stabilizing mutation comprises the amino acid sequence shown in SEQ ID NO.4.

[0009] In a second aspect, the present invention provides an mRNA molecule comprising a coding region encoding a hemagglutinin protein of a first influenza strain and / or a hemagglutinin protein of a second influenza strain, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95% sequence identity thereto, wherein the first influenza strain is H1N1 and the second influenza strain is H3N2.

[0010] In some embodiments, the hemagglutinin protein of the first influenza strain comprises the amino acid sequence shown in SEQ ID NO.2, and / or the hemagglutinin protein of the second influenza strain comprises the amino acid sequence shown in SEQ ID NO.3.

[0011] In a third aspect, the present invention provides an mRNA molecule comprising a coding region, wherein:

[0012] (1) comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs. 25-29, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs. 25-29 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. 1; and / or

[0013] (2) comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.39-40, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.39-40 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. 4. In a fourth aspect, the present invention provides an mRNA molecule comprising a coding region, wherein:

[0014] (1) comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.30-34, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.30-34 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.2; and / or

[0015] (2) comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.35-38, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.35-38 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.3.

[0016] In some embodiments, the mRNA molecule further comprises a 5' untranslated region, a 3' untranslated region, and / or a polyA tail. Preferably, the polyA tail has 20 to 200, preferably 50 to 150 adenylate nucleotides.

[0017] In some embodiments, the mRNA molecule comprises a chemical modification, preferably a 1-methyl-pseudouridine modification, and also preferably, each U in the mRNA molecule is a 1-methyl-pseudouridine.

[0018] In some embodiments, the mRNA molecule comprises or is composed of a nucleotide sequence as shown in any one of SEQ ID NOs.9-13, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.9-13 and encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1.

[0019] In some embodiments, the mRNA molecule comprises or is composed of a nucleotide sequence as shown in any one of SEQ ID NOs.23-24, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.23-24 and encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.4.

[0020] In some embodiments, the mRNA molecule comprises or is composed of a nucleotide sequence as shown in any one of SEQ ID NOs.14-18, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.14-18 and encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.2.

[0021] In some embodiments, the mRNA molecule comprises or is composed of a nucleotide sequence as shown in any one of SEQ ID NOs.19-22, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.19-22 and encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.3.

[0022] In a fifth aspect, the present invention provides an mRNA composition comprising at least one mRNA molecule as described in the present invention.

[0023] In some embodiments, the composition comprises at least one, at least two, at least three, or at least four mRNA molecules as described in the third aspect, and at least one, at least two, at least three, or at least four mRNA molecules as described in the fourth aspect.

[0024] In some embodiments, the composition comprises: a first mRNA molecule comprising a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.25-29, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.25-29 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1, preferably, the first mRNA molecule comprises a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.29, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.29 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1; and / or a second mRNA molecule comprising a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.39-40, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO. NOs.39-40 has a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.4, preferably, the second mRNA molecule comprises a coding region, the coding region comprises the nucleotide sequence as shown in SEQ ID NO.39 or consists of it, or comprises a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO.39 or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.4; and / or a third mRNA molecule, which comprises a coding region, the coding region comprises the nucleotide sequence as shown in any one of SEQ ID NOs.30-34 or consists of it, or comprises a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity to any one of SEQ ID NOs.30-34 or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.2, preferably, the third mRNA comprises a coding region, the coding region molecule comprises or consists of the nucleotide sequence as shown in SEQ ID NO.34, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.34 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.2; and / or a fourth mRNA molecule, which comprises a coding region, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs.35-38, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NOs.35-38 has a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3. Preferably, the fourth mRNA molecule comprises a coding region, the coding region comprises or consists of the nucleotide sequence as shown in SEQ ID NO.38, or comprises or consists of a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.38 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3.

[0025] In some embodiments, the composition comprises: a first mRNA molecule comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.9-13, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.9-13 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1, preferably, the first mRNA molecule comprises or consists of a nucleotide sequence as shown in SEQ ID NO.13, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.13 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1; and / or a second mRNA molecule comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.23-24, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO. NOs.23-24 has a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.4, preferably, the second mRNA molecule comprises the nucleotide sequence as shown in SEQ ID NO.23 or consists of it, or comprises a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO.23 or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.4; and / or a third mRNA molecule comprises the nucleotide sequence as shown in any one of SEQ ID NOs.14-18 or consists of it, or comprises a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity to any one of SEQ ID NOs.14-18 or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.2, preferably, the third mRNA molecule comprises the nucleotide sequence as shown in SEQ ID NO. NO.18 or consists of the nucleotide sequence thereof, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.18 and encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.2; and / or a fourth mRNA molecule, which comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs.19-22, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.19-22 and encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3, preferably, the fourth mRNA molecule comprises or is composed of the nucleotide sequence as shown in SEQ ID NO.22, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.22 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3.

[0026] In some embodiments, the composition comprises: a first mRNA molecule comprising a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.29, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.29 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1; a second mRNA molecule comprising a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.39, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.39 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.4; a third mRNA molecule comprising a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.34, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.34 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.2; and a fourth mRNA molecule, which comprises a coding region, wherein the coding region comprises or is composed of the nucleotide sequence as shown in SEQ ID NO.38, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO.38 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3.

[0027] In some embodiments, the composition comprises: a first mRNA molecule comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.13, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.13 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1; a second mRNA molecule comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.23, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.23 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.4; a third mRNA molecule comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.18, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.18 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.2; and a fourth mRNA molecule comprising or consisting of a nucleotide sequence as shown in SEQ ID NO. The nucleotide sequence shown in NO.22 or is composed thereof, or contains or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO.22 and encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO.3.

[0028] In some embodiments, the composition comprises: a first mRNA molecule comprising a coding region, wherein the coding region comprises a nucleotide sequence as shown in any one of SEQ ID NOs.25-29 and a nucleotide sequence as shown in any one of SEQ ID NOs.39-40, or is composed of the same; and / or a second mRNA molecule comprising a coding region, wherein the coding region comprises a nucleotide sequence as shown in any one of SEQ ID NOs.30-34 and a nucleotide sequence as shown in any one of SEQ ID NOs.35-38, or is composed of the same.

[0029] In some embodiments, the composition is a vaccine.

[0030] In some embodiments, at least one of the mRNAs is complexed with lipids to form one or more liposomes, lipid complexes, or lipid nanoparticles.

[0031] In some embodiments, the composition further comprises an adjuvant.

[0032] In some embodiments, the composition is formulated for oral administration, transdermal administration, or parenteral administration; preferably, the composition is formulated as a solution, emulsion, suspension, tablet, capsule, powder, pill or aerosol.

[0033] In a sixth aspect, the present invention provides a kit comprising at least one mRNA molecule as described in the present invention.

[0034] In some embodiments, the kit comprises at least one, at least two, at least three, or at least four mRNA molecules as described in the third aspect, and at least one, at least two, at least three, or at least four mRNA molecules as described in the fourth aspect.

[0035] In some embodiments, the kit comprises: a first mRNA molecule comprising a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.25-29, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.25-29 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1, preferably, the first mRNA molecule comprises a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.29, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.29 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1; and / or a second mRNA molecule comprising a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.39-40, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO. NOs.39-40 has a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.4, preferably, the second mRNA molecule comprises a coding region, the coding region comprises the nucleotide sequence as shown in SEQ ID NO.39 or consists of it, or comprises a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO.39 or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.4; and / or a third mRNA molecule, which comprises a coding region, the coding region comprises the nucleotide sequence as shown in any one of SEQ ID NOs.30-34 or consists of it, or comprises a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity to any one of SEQ ID NOs.30-34 or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.2, preferably, the third mRNA comprises a coding region, the coding region molecule comprises or consists of the nucleotide sequence as shown in SEQ ID NO.34, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.34 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.2; and / or a fourth mRNA molecule, which comprises a coding region, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs.35-38, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NOs.35-38 has a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3. Preferably, the fourth mRNA molecule comprises a coding region, the coding region comprises or consists of the nucleotide sequence as shown in SEQ ID NO.38, or comprises or consists of a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.38 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3.

[0036] In some embodiments, the kit comprises: a first mRNA molecule comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.9-13, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.9-13 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1, preferably, the first mRNA molecule comprises or consists of a nucleotide sequence as shown in SEQ ID NO.13, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.13 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1; and / or a second mRNA molecule comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.23-24, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO. NOs.23-24 has a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.4, preferably, the second mRNA molecule comprises the nucleotide sequence as shown in SEQ ID NO.23 or consists of it, or comprises a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO.23 or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.4; and / or a third mRNA molecule comprises the nucleotide sequence as shown in any one of SEQ ID NOs.14-18 or consists of it, or comprises a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity to any one of SEQ ID NOs.14-18 or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.2, preferably, the third mRNA molecule comprises the nucleotide sequence as shown in SEQ ID NO. NO.18 or consists of the nucleotide sequence thereof, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.18 and encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.2; and / or a fourth mRNA molecule, which comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs.19-22, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.19-22 and encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3, preferably, the fourth mRNA molecule comprises or is composed of the nucleotide sequence as shown in SEQ ID NO.22, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.22 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3.

[0037] In some embodiments, the kit comprises: a first mRNA molecule comprising a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.29, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.29 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1; a second mRNA molecule comprising a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.39, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.39 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.4; a third mRNA molecule comprising a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.34, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.34 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. NO.2; and a fourth mRNA molecule, which comprises a coding region, wherein the coding region comprises or is composed of the nucleotide sequence as shown in SEQ ID NO.38, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO.38 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3.

[0038] In some embodiments, the kit comprises: a first mRNA molecule comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.13, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.13 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1; a second mRNA molecule comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.23, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.23 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.4; a third mRNA molecule comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.18, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.18 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.2; and a fourth mRNA molecule comprising or consisting of a nucleotide sequence as shown in SEQ ID NO. The nucleotide sequence shown in NO.22 or is composed thereof, or contains or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO.22 and encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO.3.

[0039] In some embodiments, the kit comprises: a first mRNA molecule comprising a coding region, wherein the coding region comprises a nucleotide sequence as shown in any one of SEQ ID NOs.25-29 and a nucleotide sequence as shown in any one of SEQ ID NOs.39-40, or is composed of the same; and / or a second mRNA molecule comprising a coding region, wherein the coding region comprises a nucleotide sequence as shown in any one of SEQ ID NOs.30-34 and a nucleotide sequence as shown in any one of SEQ ID NOs.35-38, or is composed of the same.

[0040] In a seventh aspect, a nucleic acid molecule is provided, which encodes an mRNA molecule according to the first aspect, the second aspect, the third aspect and the fourth aspect.

[0041] In an eighth aspect, a vector is provided, which comprises a nucleic acid molecule according to the seventh aspect of the invention.

[0042] In a ninth aspect, a cell is provided, comprising the nucleic acid molecule according to the seventh aspect of the invention or the vector according to the eighth aspect of the invention.

[0043] In a tenth aspect, the present invention provides a method for inducing an immune response against SARS-CoV-2 and / or influenza virus in a subject, the method comprising administering to the subject the mRNA, composition, nucleic acid, vector or cell of the present invention. The subject may be a human or an animal, including but not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, etc.

[0044] Beneficial Effects

[0045] The experimental results of this application prove that the Spike antigen proteins of two different novel coronavirus strains selected by the present invention can achieve a broad-spectrum anti-novel coronavirus effect. The optimized mRNA sequence disclosed in the present invention can produce higher expression in the body of the immunized person and produce a more effective and lasting immune response. The mRNA combination vaccine disclosed in the present invention can be suitable for a variety of administration methods, and can activate specific humoral immunity and cellular immune responses against a variety of viral antigens, while producing cross-immune protection against a variety of novel coronavirus variants. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figures 1A to 1D The protein expression levels of different sequence mRNA immunizations at 24h and 48h are shown, among which, Figure 1A The electrophoresis diagrams of protein expression of mRNA with different sequences at 24h and 48h are shown. Figures 1B to 1D Schematic diagram of the 24h and 48h protein expression levels of SARS-CoV-2BA5.2 Spike, H1N1 HA and H3N2 HA mRNA after grayscale measurement.

[0047] Figure 2A The humoral immunity levels of animals induced by SEQ ID NO: 23 and SEQ ID NO: 24 on days 20 and 35 of animal immunization are shown.

[0048] Figure 2B The figure shows the cellular immunity level of animals induced by SEQ ID NO: 23 and SEQ ID NO: 24 on day 35 of animal immunization.

[0049] Figure 3A The data showed that 20 days after the initial immunization, the experimental and control groups had specific IgG antibody titers against the S protein of four new coronaviruses (SARS-CoV-2BA5.2, SARS-CoV-2B.1.617.2, SARS-CoV-2 (2019-nCoV) and SARS-CoV-2 (XBB1.16)) and the HA protein of two influenza A viruses (H1N1 and H3N2).

[0050] Figure 3BThe results showed that 14 days after the booster immunization, the experimental and control groups had specific IgG antibody titers against the S proteins of four new coronaviruses (SARS-CoV-2BA5.2, SARS-CoV-2B.1.617.2, SARS-CoV-2 (2019-nCoV) and SARS-CoV-2 (XBB1.16)) and the HA proteins of two influenza A viruses (H1N1 and H3N2).

[0051] Figure 4A and Figure 4B The hemagglutination inhibition test is shown to detect the hemagglutination antibody titer, where Figure 4A Hemagglutination inhibition antibody titers of influenza virus A / Guangdong-Maonan / SWL1536 / 2019 (CNIC-1909) (H1N1) antigen are shown; Figure 4B Hemagglutination inhibition antibody titers for influenza virus A / Darwin / 9 / 2021 (IVR-228) (H3N2) antigen are shown.

[0052] Figure 5 The data show the neutralizing antibody titers against the new coronaviruses SARS-CoV-2B.1.617.2, SARS-CoV-2BA5.2, SARS-CoV-2 (XBB 1.16) and SARS-CoV-2 (2019-nCoV) in the serum of experimental mice on the 35th day of animal immunization.

[0053] Fig. 6A The spot number levels of spleen cells after stimulation with peptide pools are shown.

[0054] Figure 6B Shows the activity levels of spleen cells after stimulation with peptide pools.

[0055] Figure 7 It shows the proportion of TNFα+CD4+ T cells, IFNγ+CD4+ T cells, IL-2+CD4+ T cells, IL-4+CD4+ T cells, and IL-5+CD4+ T cells in the total CD4+ T cells after mouse spleen cells were stimulated with specific peptide library.

[0056] Figure 8 It shows the proportion of TNFα+CD8+ T cells, IFNγ+CD8+ T cells, IL-2+CD8+ T cells, IL-4+CD8+ T cells, and IL-5+CD8+ T cells in the total CD8+ T cells after mouse spleen cells were stimulated with specific peptide library. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0058] definition

[0059] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0060] As used herein, the terms "includes," "comprising," "having," "may," "containing," and variations thereof are generally intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The term "consisting of" generally indicates that no other components (or, similarly, features, integers, steps, etc.) can be present.

[0061] As used herein, the term "about" may be used to encompass variations of ±10% or less, ±5% or less, ±1% or less, ±0.5% or less, or ±0.1% or less from a particular value.

[0062] The term "nucleic acid" refers to any DNA or RNA molecule.

[0063] When used in the present application, the terms "mRNA" and "messenger RNA" can be used interchangeably. Artificial mRNA can generally be understood as mRNA molecules that are not naturally occurring, i.e., non-natural mRNA molecules. Such mRNA molecules may be considered non-natural due to their own sequence (non-naturally occurring) and / or due to other modifications (e.g., structural modifications of non-naturally occurring nucleotides). Artificial mRNA molecules can generally be designed and / or generated by genetic engineering methods to correspond to desired artificial nucleotide sequences. In this article, the artificial sequence is generally different from the wild-type sequence by at least one nucleotide. The term "wild type" can be understood as a sequence that occurs in nature. The mRNA molecule according to the present application is an artificial mRNA molecule.

[0064] mRNA is usually composed of the following structural elements: 5'-cap (5'-CAP) structure, 5'UTR (untranslated region), start codon, coding region, stop codon, 3'UTR (untranslated region) and poly A tail.

[0065] The 5'-cap structure is typically a modified nucleotide (cap analog), particularly a guanine nucleotide, which is located at the 5'-end of the mRNA molecule. Preferably the 5'-cap structure is a 5',5'-triphosphate linkage (also known as m7GPPPN). Other examples of 5'-cap structures include glyceryl, reverse deoxy abasic residues (parts), 1-(β-D-furanosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3', 4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5 dihydroxypentyl nucleotides, 3',3'-reverse nucleotide parts, 3',3'-reverse abasic parts, 3',2'-reverse nucleotide parts, 3',2'-reverse abasic parts, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3-phosphate, 3-phosphorothioate, dithiophosphate or bridged or non-bridged methylphosphonate parts. These modified 5'-cap structures can be used in the present invention to modify the mRNA sequence of the present invention. Other modified 5′-cap structures that can be used include CAP1 (additional methylation of the ribose of the adjacent nucleotide of m7GPPPN), CAP2 (additional methylation of the ribose of the second nucleotide downstream of m7GPPPN), CAP3 (additional methylation of the ribose of the third nucleotide downstream of m7GPPPN), CAP4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GPPPN), etc.

[0066] A cap analog refers to a non-polymerizable dinucleotide with a cap function that, when incorporated into the 5' end of an RNA molecule, promotes translation or localization, and / or protects the RNA molecule from degradation. Non-polymerizable means that the cap analog will only be incorporated at the 5' end, as it has no 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent RNA polymerase.

[0067] Cap analogs that can be used include, but are not limited to: m7GPPPG, m7GPPPA, m7GPPPC, unmethylated cap analogs (e.g., GPPPG), dimethylated cap analogs (e.g., m2,7GPPPG), trimethylated cap analogs (e.g., m2,2,7GPPPG), and the like.

[0068] The polyA tail or poly(A) sequence is typically a long sequence of about 400 adenosine nucleotides added to the 3' end of the RNA, for example, having about 25 to about 400, about 50 to about 400, about 50 to about 300, about 50 to about 250, or about 60 to about 250 adenosine nucleotides. In addition, the poly(A) sequence or poly(A) tail can be generated in vitro by enzymatic polyadenylation of RNA.

[0069] In a first aspect of the present invention, an mRNA molecule is provided, comprising a coding region, wherein the coding region encodes the full-length spike protein (Spike protein) S of SARS-CoV-2, the receptor binding motif of the S1 subunit of the spike protein, or the nucleocapsid protein N. More specifically, the coding region encodes the Spike protein of the first SARS-CoV-2 strain and / or the second SARS-CoV-2 strain that has undergone a stabilizing mutation, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95% sequence identity, wherein the stabilizing mutation is selected from a diproline stabilizing mutation or a hexaproline stabilizing mutation.

[0070] The double proline stabilizing mutations are usually located at positions corresponding to K986 and V987 of the Spike protein of wild-type SARS-CoV-2, while the hexaproline stabilizing mutations are usually located at positions corresponding to F817, A892, A899, A942, K986, and V987 of the Spike protein of wild-type SARS-CoV-2.

[0071] Advantageously, the first SARS-CoV-2 strain may be SARS-CoV-2BA5.2, and the second SARS-CoV-2 strain may be SARS-CoV-2B.1.617.2. In some embodiments, the coding region of the mRNA molecule according to the first aspect of the present invention may also be directed to other SARS-CoV-2 strains of the latest mutation.

[0072] When used in the present application, a fragment or variant of a polypeptide has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to the polypeptide itself, preferably at least 80%, more preferably at least 90%, most preferably at least 95% or even at least 98% sequence identity.

[0073] In a preferred embodiment, the Spike protein of the first SARS-CoV-2 strain that has undergone a stabilizing mutation comprises or consists of the amino acid sequence shown in SEQ ID NO.1, and / or the Spike protein of the second SARS-CoV-2 strain that has undergone a stabilizing mutation comprises or consists of the amino acid sequence shown in SEQ ID NO.4.

[0074] In a second aspect, the present invention provides an mRNA molecule comprising a coding region encoding a hemagglutinin protein of a first influenza strain and / or a hemagglutinin protein of a second influenza strain, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95% sequence identity thereto, wherein the first influenza strain is H1N1 and the second influenza strain is H3N2.

[0075] In some embodiments, the hemagglutinin protein of the first influenza strain comprises the amino acid sequence shown in SEQ ID NO.2, and / or the hemagglutinin protein of the second influenza strain comprises the amino acid sequence shown in SEQ ID NO.3.

[0076] In another aspect, the present invention provides an mRNA molecule comprising a coding region, wherein the coding region: (1) comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs.25-29, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.25-29 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.1; and / or (2) comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs.39-40, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.39-40 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.4.

[0077] In some embodiments, the mRNA molecule comprises or is composed of a nucleotide sequence as shown in any one of SEQ ID NOs.9-13, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.9-13 and encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1.

[0078] In some embodiments, the mRNA molecule comprises or is composed of a nucleotide sequence as shown in any one of SEQ ID NOs.23-24, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.23-24 and encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.4.

[0079] In another aspect, the present invention provides an mRNA molecule comprising a coding region, wherein the coding region: (1) comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs.30-34, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.30-34 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.2; and / or (2) comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs.35-38, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.35-38 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3.

[0080] In some embodiments, the mRNA molecule comprises or is composed of a nucleotide sequence as shown in any one of SEQ ID NOs.14-18, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.14-18 and encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.2.

[0081] In some embodiments, the mRNA molecule comprises or is composed of a nucleotide sequence as shown in any one of SEQ ID NOs.19-22, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.19-22 and encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.3.

[0082] The mRNA of the present invention can be a monocistronic mRNA, which can generally only include an open reading frame (coding sequence or coding region). An open reading frame is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein. The mRNA of the present invention can also be a bi / polycistronic mRNA, that is, it may have two (bicistronic) or more (polycistronic) open reading frames. The translation of this mRNA produces two (bicistronic) or more (polycistronic) different translation products (prerequisite is that ORF is not the same). In order to express in eukaryotes, such mRNA can include, for example, an internal ribosome entry site (IRES) sequence.

[0083] The mRNA of the present invention is preferably a stable mRNA, that is, it is modified to show increased resistance to degradation in vivo (e.g., by exonucleases or endonucleases) and / or in vitro (e.g., manufacturing processes prior to vaccine administration). Stabilization of the RNA can be achieved by providing a 5'-CAP structure, a polyA tail or other UTR modifications. Alternatively, stabilization of the RNA can be achieved by chemical modification or adjusting the G / C content of the nucleic acid.

[0084] The mRNA used in the present invention is preferably purified mRNA. That is, it has a higher purity than the starting material (e.g., in vitro transcribed RNA) after certain purification steps (e.g., HPLC, TFF, precipitation, etc.). Typical impurities that are substantially absent from the purified mRNA include peptides or proteins (e.g., enzymes derived from DNA-dependent RNA in vitro transcription, such as RNA polymerase, RNase, BSA, pyrophosphatase, restriction endonuclease, DNA enzyme), spermidine, RNA fragments, free nucleotides (modified nucleotides, conventional NTP, cap analogs), plasmid DNA fragments, buffer components (HEPES, TRIS, MgCl2), etc., and other impurities that may be derived from, for example, fermentation procedures include bacterial impurities (bioburden, bacterial DNA) or impurities derived from purification procedures (organic solvents, etc.). It would be ideal for "RNA purity" to be as close to 100% as possible. For RNA purity, it is also desirable for the amount of full-length RNA transcripts to be as close to 100% as possible. Thus, as used herein, "purified mRNA" has a purity greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Purity can be determined, for example, by analytical HPLC, the percentage corresponding to the ratio between the peak area of ​​the target RNA and the total area of ​​all peaks representing the various products; alternatively, the purity can be determined, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.

[0085] In other aspects of the present invention, a mRNA composition is provided. The composition according to the present invention is preferably provided as a pharmaceutical composition or as a vaccine. "Vaccine" is generally understood to be a preventive or therapeutic material that provides at least one epitope of an antigen, preferably an immunogen. "Providing at least one epitope" means, for example, that the vaccine comprises the epitope (or an antigen comprising the epitope) or the vaccine comprises, for example, a molecule encoding the epitope or an antigen comprising the epitope. The antigen preferably stimulates the adaptive immune system to provide an adaptive immune response. The composition or vaccine provided herein may further comprise at least one pharmaceutically acceptable excipient, adjuvant or other component (e.g., additive, auxiliary substance, etc.).

[0086] As used herein, the term "antigen" generally refers to a substance that can be recognized by the immune system, preferably recognized by the adaptive immune system, and can trigger an antigen-specific immune response, such as by forming antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen can be or can include a peptide or protein that can be presented to a T cell by MHC. In the sense of the present invention, an antigen can be a provided nucleic acid molecule, such as a translation product of an mRNA defined in the present application. In this article, fragments, variants and derivatives of peptides and proteins comprising at least one epitope are also understood to be antigens.

[0087] The term "vaccine" is generally understood to be a preventive or therapeutic material that provides at least one antigen or antigenic function. Antigens or antigenic functions can stimulate the body's adaptive immune system to provide an adaptive immune response. The mRNA that provides an antigen in the context of the present invention can generally be an mRNA having at least one open reading frame, which can be translated by a cell or organism provided with the mRNA. The product of this translation is a peptide or protein that can serve as an antigen, preferably an immunogen. The translation product can also be a fusion protein composed of more than one immunogen, such as a fusion protein composed of two or more epitopes, peptides or proteins derived from the same or different viral proteins, wherein the epitopes, peptides or proteins can be connected by a connecting sequence.

[0088] In some embodiments, the composition according to the invention comprises at least one mRNA molecule of the invention as described above.

[0089] In some embodiments, a composition according to the present invention comprises at least one mRNA molecule encoding an antigen derived from SARS-CoV-2 (e.g., a Spike protein or a fragment or variant thereof).

[0090] In some embodiments, a composition according to the invention comprises at least one mRNA molecule encoding an antigen derived from an influenza virus (eg, a hemagglutinin protein or a fragment or variant thereof).

[0091] In a preferred embodiment, the composition according to the invention comprises at least one mRNA molecule encoding an antigen derived from SARS-CoV-2 and at least one mRNA molecule encoding an antigen derived from influenza virus.

[0092] In some embodiments, the composition according to the present invention may also contain mRNA molecules encoding other antigens, such as other antigens derived from the SARS-CoV-2 virus or fragments or variants thereof, such as the receptor binding motif of the S1 subunit of the spike protein or the nucleocapsid protein N.

[0093] In some embodiments, the composition according to the present invention comprises at least 2 mRNA molecules, for example 3, 4 or more mRNA molecules. In a preferred embodiment, the composition according to the invention comprises at least one mRNA molecule encoding an antigen derived from SARS-CoV-2 and at least one mRNA molecule encoding an antigen derived from influenza virus. Therefore, in some embodiments, the composition according to the present invention can be a combined vaccine, especially a combined vaccine for at least one SARS-CoV-2 and at least one influenza virus. The combined vaccine according to the present invention can simultaneously activate specific immune responses to multiple viral antigens, providing a combined protective effect for subjects in need. In a preferred embodiment, the composition comprises at least 4 mRNA molecules.

[0094] In some embodiments, the composition comprises: a first mRNA molecule comprising a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.25-29, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.25-29 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1, preferably, the first mRNA molecule comprises a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in SEQ ID NO.29, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.29 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1; and / or a second mRNA molecule comprising a coding region, the coding region comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.39-40, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO. NOs.39-40 has a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.4, preferably, the second mRNA molecule comprises a coding region, the coding region comprises the nucleotide sequence as shown in SEQ ID NO.39 or consists of it, or comprises a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO.39 or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.4; and / or a third mRNA molecule, which comprises a coding region, the coding region comprises the nucleotide sequence as shown in any one of SEQ ID NOs.30-34 or consists of it, or comprises a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity to any one of SEQ ID NOs.30-34 or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.2, preferably, the third mRNA comprises a coding region, the coding region molecule comprises or consists of the nucleotide sequence as shown in SEQ ID NO.34, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.34 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.2; and / or a fourth mRNA molecule, which comprises a coding region, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs.35-38, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NOs.35-38 has a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity or consists of it and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3. Preferably, the fourth mRNA molecule comprises a coding region, the coding region comprises or consists of the nucleotide sequence as shown in SEQ ID NO.38, or comprises or consists of a nucleotide sequence with at least 80%, at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO.38 and encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO.3.

[0095] In a preferred embodiment, the composition of the present invention is a vaccine. At least one mRNA according to the present invention is complexed with one or more cationic or polycationic compounds, preferably with cationic or polycationic polymers, cationic or polycationic peptides or proteins, such as protamine, cationic or polycationic polysaccharides and / or cationic or polycationic lipids. In some embodiments, at least one mRNA according to the present invention can be complexed with lipids to form one or more liposomes, lipid complexes or lipid nanoparticles. Therefore, in one embodiment, the composition of the present invention comprises liposomes, lipid complexes and / or lipid nanoparticles containing at least one mRNA. In this context, the term "complexed" refers to that the mRNA is substantially stably combined with one or more of the above-mentioned compounds into a larger complex or assembly without covalent bonding. According to some preferred embodiments, the mRNA optionally contained in the composition or vaccine is complexed or associated with lipids (particularly cationic and / or neutral lipids) to form one or more liposomes, lipid complexes, or lipid nanoparticles (LNPs).

[0096] The lipid nanoparticle (LNP) comprises: (a) at least one mRNA according to the present invention, which is optionally contained in a composition or vaccine as defined herein, (b) a cationic lipid, (c) an aggregation reducing agent (e.g., a polyethylene glycol (PEG) lipid or a PEG-modified lipid), (d) optionally a non-cationic lipid (e.g., a neutral lipid), and (e) optionally a sterol. In the context of the present invention, the term "lipid nanoparticle" is not limited to any particular morphology, and includes any morphology produced when a cationic lipid and optionally one or more other lipids are combined, e.g., in an aqueous environment and / or in the presence of RNA. For example, liposomes, lipid complexes, emulsions, micelles, lipid nanocapsules, nanosuspensions, etc. are all within the scope of lipid nanoparticles. In some embodiments, the LNP comprises, in addition to at least one mRNA of the invention, (i) at least one cationic lipid; (ii) a neutral lipid; (iii) a sterol, such as cholesterol; and (iv) a PEG-lipid in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid; 25-55% sterol; 0.5-15% PEG-lipid.

[0097] LNP can comprise any cationic lipid that is suitable for forming lipid nanoparticles.Preferably, the cationic lipid carries a net positive charge at about physiological pH.The cationic lipid can be an amino lipid.As used herein, the term "amino lipid" means comprising those lipids having one or two fatty acids or fatty alkyl chains and an amino head group (comprising alkylamino or dialkylamino), which can be protonated at physiological pH to form a cationic lipid.

[0098] The cationic lipid can be, for example, N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearoyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium chloride propane (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleoyloxy-3-trimethylaminopropane chloride salt), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DOTAP). ,2-Dilinoleyl-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyl-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyl-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyl-3-trimethylaminopropane chloride (DLin-TMA.Ci), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Ci).CI), 1,2-dilinoleyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dilinoleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DM A), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadecane-9,12 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyldimethylaminomethyl-[1,3]-dioxolane (DLin-DMA), (6Z,9Z,28Z,31Z)-heptatrioxane- Other cationic lipids include, but are not limited to, N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(1-(2,3-dioleyl)propyl)-N(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamido glycylcarboxyspermine ( In addition, commercial preparations of cationic lipids such as LIPOFECTIN (including DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECTAMINE (including DOSPA and DOPE, available from GIBCO / BRL) can be used. Other synthetic lipids include 1,2-dioleoyl-sn-3-phosphoethanolamine (DOGS), 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-. In addition, commercial preparations of cationic lipids such as LIPOFECTIN (including DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECTAMINE (including DOSPA and DOPE, available from GIBCO / BRL) can be used. Suitable (cationic) lipids are disclosed in WO2009 / 086558, WO2009 / 127060, WO2010 / 048536, WO2010 / 054406, WO2010 / 088537, WO2010 / 129709, WO2011 / 153493, US2011 / 0256175, US2012 / 0128760, US2012 / 0027803 and US Pat. No. 8,158,601 (the disclosures of which are incorporated herein by reference).

[0099] Cationic lipids can also be amino lipids. Suitable amino lipids include those with alternative fatty acid groups and other dialkylamino groups, including those where the alkyl substituents are different (e.g., N-ethyl-N-methylamino- and N-propyl-N-ethylamino-). Typically, amino lipids with less saturated acyl chains are more easily sized, especially when used for the purpose of filtration sterilization, when the size of the complex must be less than about 0.3 microns, amino lipids containing unsaturated fatty acids with carbon chain lengths in the range of C14 to C22 can be used. Other scaffolds can also be used to separate the amino and fatty acid or fatty alkyl parts of amino lipids. Representative amino lipids include but are not limited to 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3 morpholinyl propane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-SD A), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.CI), 1,2-dilinoleyl-3-trimethylaminopropane chloride (DLin-TAP.CI), 1,2 and 2,2-dilinoleyldimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA).

[0100] In some embodiments, amino or cationic lipids have at least one protonable or deprotonable group, so that lipid is positively charged at or below the pH of physiological pH (for example, pH 7.4), and at a second pH, preferably neutral at or above the physiological pH. The lipid with more than one protonable or deprotonable group, or the lipid of zwitterion, can also be used for the present invention. In some embodiments, the pKa of the protonable group of protonable lipid is in the range of about 4 to about 11, for example, about 5 to about 7 pKa. LNP can include two or more cationic lipids. Different cationic lipids can be selected to obtain different favorable properties.

[0101] In some embodiments, the cationic lipid is present in a ratio of about 20 mol% to 75 mol% or about 45 to about 65 mol% of the total lipid present in the LNP, for example, about 20, 25, 30, 35, 40, 45, 50, 52.5, 55, 57.5, 60, 65, 70 or 75 mol% (based on 100% total moles of lipid in the lipid nanoparticle). In some embodiments, the ratio of cationic lipid to nucleic acid is about 3 to about 15, for example, about 5 to about 13 or about 7 to about 11. In one embodiment, these amounts are selected so that the N / P ratio in the nanoparticle or composition is in the range of about 0.1 to about 20. In this article, the N / P ratio is defined as the molar ratio of the nitrogen atom ("N") of the basic nitrogen-containing group of the lipid or lipidoid to the phosphate group ("P") of the RNA being carried.

[0102] In certain embodiments, the LNPs comprise one or more additional lipids that stabilize the formation of the particle during particle formation.

[0103] In some embodiments, non-cationic lipids, such as neutral lipids, anionic lipids or amphipathic lipids can be used. Neutral lipids can be any of many lipid species existing in the form of uncharged or neutral zwitterions at physiological pH. Such lipids include but are not limited to diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside. The selection of neutral lipids for particles described herein is usually guided by considering, for example, LNP size and the stability of LNP in blood flow. Preferably, neutral lipids are lipids (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine) with two acyl groups. In some embodiments, neutral lipids contain saturated fatty acids with carbon chain lengths in the range of C10 to C20. In other embodiments, neutral lipids with monounsaturated fatty acids or diunsaturated fatty acids with carbon chain lengths in the range of C10 to C20 are used. In addition, neutral lipids with a mixture of saturated and unsaturated fatty acid chains can be used. Suitable neutral lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), and dimyristoylphosphatidylethanolamine (DMPE). Anionic lipids suitable for use in LNPs include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups attached to neutral lipids.

[0104] In some embodiments, LNP comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of cationic lipid to neutral lipid is in the range of about 2: 1 to about 8: 1. Amphiphilic lipid refers to any suitable substance, wherein the hydrophobic part of the lipid substance is toward the hydrophobic phase, and the hydrophilic part is toward the aqueous phase. Such compounds include but are not limited to phospholipids, amino lipids and sphingolipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Other phosphorus-free compounds such as sphingolipids, glycosphingolipid families, diacylglycerols and β-acyloxy acids can also be used.

[0105] In some embodiments, non-cationic lipid is present in the ratio of about 5mol% to about 90mol%, about 5mol% to about 10mol%, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or about 90mol% of the total lipid present in LNP. In some embodiments, LNP comprises about 0% to about 25% by mole, for example, about 3% to about 15% or about 5% to about 12% neutral lipid. In some embodiments, LNP may include about 25%, 20%, 15%, about 10%, about 7.5% or about 7% neutral lipid by mole.

[0106] In some embodiments, sterol can be used, preferably cholesterol. Sterol can exist in a ratio of about 10mol% to about 60mol% or about 25mol% to about 40mol% of LNP. In some embodiments, sterol exists in a ratio of about 10, 15, 20, 25, 30, 32.5, 35, 37.5, 40, 45, 50, 55 or about 60mol% of the total lipid present in LNP (based on 100% total mole of lipid in LNP).

[0107] In some embodiments, aggregation reducing agents can be used, which can reduce lipid aggregation. The aggregation reducing agents that can be used can be selected from polyethylene glycol (PEG) lipids, including but not limited to PEG-diacylglycerol (DAG), PEG-dialkylglycerol, PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer) or its mixture (such as PEG-Cer14 or PEG-Cer20). PEG-DAA conjugates can be, for example, PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmitoxypropyl (C16) or PEG-distearyloxypropyl (C18). Other PEGylated lipids include, but are not limited to, polyethylene glycol-dimyristoylglycerol (C14-PEG or PEG-C14, wherein PEG has an average molecular weight of 2000 Da) (PEG-DMG), (R)-2,3-bis(octadecyloxy)propyl-1-(methoxypolyethylene glycol) 2000) propylcarbamate) (PEG-DSG), PEG-carbamoyl-1,2-dimyristyloxypropylamine, wherein PEG has an average molecular weight of 2000 Da (PEG-cDMA), N-acetylgalactosamine-(R)-2,3-bis(octadecyloxy)propyl-1-(methoxypolyethylene glycol) 2000) propylcarbamate) (GalNAc-PEG-DSG), mPEG (Mw 2000)-distearylphosphatidyl-ethanolamine (PEG-DSPE), polyethylene glycol-dipalmitoylglycerol (PEG-DPG). The concentration of the aggregation reducing agent may range from about 0.1 to about 15 mol % per 100% total moles of lipid in the LNP.

[0108] In some embodiments, the molar ratio of cationic lipid to PEGylated lipid is about 100:1 to about 25:1. In some embodiments, the composition of LNP is affected by the selection of cationic lipid component, cationic lipid saturation, PEGylation type, ratio of all components and biophysical parameters such as its size. In some embodiments, the LNP composition consists of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol and 1.4% PEG-c-DMA.

[0109] In some embodiments, LNP may comprise about 35% to about 45% cationic lipid, about 40% to about 50% cationic lipid, about 50% to about 60% cationic lipid and / or about 55% to about 65% cationic lipid. In some embodiments, the ratio of lipid to mRNA may be in the range of about 5:1 to about 20:1, about 10:1 to about 25:1, or about 15:1 to about 30:1. The average molecular weight of the PEG portion in the PEG-modified lipid may be in the range of about 500 to about 8000 Daltons, for example, 1000, 2000, 3000, 4000, 5000, 6000, 7000 or 8000 Daltons.

[0110] The total amount of nucleic acids in the lipid nanoparticles, in particular the total amount of one or more RNAs, varies and can be defined, for example, based on the w / w ratio of RNA to total lipids. In some embodiments of the invention, the RNA to total lipid ratio is less than 0.06 w / w, preferably 0.03 w / w to 0.04 w / w.

[0111] In some embodiments, LNP has about 50nm to about 300nm, such as about 50nm to about 250nm, for example, about 50nm to about 200nm median diameter size. In some embodiments, smaller LNP can be used. Such particle can include a diameter from 0.1pm to 100nm. In other embodiments, less LNP can be used to deliver nucleic acid, for example, diameter about 1nm to about 100nm, about 1nm to about 20nm, about 1nm to about 40nm, about 1nm to about 60nm, about 1nm to about 80nm, about 5nm to about 100nm, about 10 to about 50nm, about 20 to about 50nm, about 30 to about 50nm, about 30 to about 60nm, about 40 to about 60nm, about 20 to about 70nm, about 50 to about 70nm, about 60 to about 70nm, about 30 to about 80nm or about 20 to about 90nm. In some embodiments, the LNPs may have a diameter greater than 100 nm, greater than 200 nm, greater than 300 nm, greater than 400 nm, greater than 500 nm, greater than 600 nm, greater than 700 nm, greater than 800 nm, greater than 900 nm, or greater than 1000 nm.

[0112] Liposomes are usually composed of a lipid bilayer, which can be composed of cationic, anionic or neutral (phosphoric) lipids and cholesterol, which encapsulate an aqueous core. Both the lipid bilayer and the aqueous space can be incorporated with hydrophobic or hydrophilic compounds, respectively. Liposomes may have one or more lipid membranes. Liposomes may be unilamellar, referred to as unilamellar, or multilamellar, referred to as multilamellar. The characteristics and behavior of liposomes in vivo can be modified by adding a hydrophilic polymer coating to the liposome surface, such as polyethylene glycol (PEG), to impart steric stability. In addition, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surfaces or the ends of attached PEG chains (Front Pharmacol. 2015 Dec 1; 6: 286).

[0113] In some embodiments, the composition of the present invention comprises an adjuvant. The term "adjuvant" is generally understood to exclude agents that can confer immunity by themselves. Adjuvants non-specifically help the immune system to enhance antigen-specific immune responses by, for example, promoting the presentation of antigens to the immune system or the induction of non-specific innate immune responses. In addition, adjuvants can, for example, regulate antigen-specific immune responses by converting dominant Th2-based antigen-specific responses to more Th1-based antigen-specific responses, and vice versa. Therefore, adjuvants can advantageously regulate cytokine expression / secretion, antigen presentation, immune response types, etc.

[0114] In some embodiments, the composition of the present invention comprises at least one pharmaceutically acceptable carrier, excipient, or transfection agent (for example, for improving the transfection efficiency of the mRNA of the present invention). The selection of pharmaceutically acceptable carriers depends in principle on the mode of administration of the composition or vaccine according to the present invention. The composition or vaccine of the present invention can be administered systemically or topically, such as orally, percutaneously, or parenterally (including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injection and / or intranasal administration routes). Preferably, the composition or vaccine according to the present invention can be administered by intradermal, subcutaneous or intramuscular routes, preferably by injection. Therefore, the composition or vaccine of the present invention can be formulated into liquid or solid forms, such as solutions, emulsions, suspensions, tablets, capsules, powders, pills, etc. In some embodiments, the composition or vaccine of the present invention can also be formulated as an aerosol. In a preferred embodiment, the mRNA in the composition, vaccine or kit of the present invention is provided in a lyophilized form. Preferably, the lyophilized mRNA is reconstructed in a suitable buffer before administration, such as using an aqueous carrier, such as Ringer's lactate solution, Ringer's solution, phosphate buffer solution, etc. The vaccine or (pharmaceutical) composition according to the invention may generally comprise a pharmaceutically acceptable carrier. The expression "pharmaceutically acceptable carrier" as used herein preferably includes a liquid or non-liquid basis for the vaccine of the invention. If the vaccine of the invention is provided in liquid form, the carrier will be water, generally pyrogen-free water; isotonic saline or a buffered (aqueous) solution, such as a buffered solution such as phosphate, citrate, etc.

[0115] A therapeutically effective amount in the context of the present invention is generally understood to be an amount sufficient to induce an immune response.

[0116] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. Throughout the specification, reference to "one embodiment", "embodiment", "one example" or "example" means that the specific features, structures or characteristics described in conjunction with the embodiment or example are included in at least one embodiment of the present invention. Therefore, the phrases "one embodiment", "embodiment", "one example" or "example" appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. In addition, specific features, structures or characteristics can be combined in one or more embodiments or examples in any appropriate combination and / or sub-combination. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0117] Example

[0118] Example 1 Optimization of mRNA sequence of multi-combination vaccine

[0119] The mRNA multi-combination vaccine described in this application contains mRNA molecules that can express SARS-CoV-2BA5.2 Spike protein, H1N1 (A / Guangdong-Maonan / SWL1536 / 2019) HA protein, H3N2 (A / Darwin / 9 / 2021) HA protein, and SARS-CoV-2B.1.617.2 Spike protein. In order to obtain mRNA molecules that are more compatible with human codons, easier to translate, and have higher protein expression levels, the mRNA molecules expressing the above four proteins are optimized by considering codon preference (CAI), mRNA structure (including GC content, free energy, etc.), immunogenicity (CpG dinucleic acid content) and specific sequences (restriction sites, SD sequences, TATA boxes, etc.). The optimized sequences are as follows:

[0120] The SARS-CoV-2BA5.2 Spike sequence was subjected to double proline mutation (SEQ ID NO: 1), and five optimized SARS-CoV-2BA5.2 Spike mRNA sequences (SEQ ID NO: 9-13) were obtained from its coding sequence (SEQ ID NO: 5).

[0121] Five optimized H1N1 HA mRNAs (SEQ ID NOs: 14-18) and four optimized H3N2 HA mRNAs (SEQ ID NOs: 19-22) were obtained from the H1N1 (A / Guangdong-Maonan / SWL1536 / 2019) HA (SEQ ID NO: 2) coding sequence (SEQ ID NO: 6) and the H3N2 (A / Darwin / 9 / 2021) HA (SEQ ID NO: 3) coding sequence (SEQ ID NO: 7).

[0122] The SARS-CoV-2B.1.617.2Spike sequence (SEQ ID NO:4) was subjected to double proline mutation, and two optimized SARS-CoV-2B.1.617.2Spike mRNA sequences (SEQ ID NO:23-24) were obtained from its coding sequence (SEQ ID NO:8).

[0123] Table 1. Correspondence between sequences

[0124]

[0125] 1 μg of SARS-CoV-2BA5.2 Spike mRNA (SEQ ID NO: 9-13), H1N1 HA mRNA (SEQ ID NO: 14-18) and H3N2 HA mRNA (SEQ ID NO: 19-22) were transfected into Hek293 cells (Nanjing Kebai) using Messenger MAX (Thermo). Cell lysis solutions were prepared after 24 and 48 hours, and the expression levels of SARS-CoV-2BA5.2 Spike, H1N1 HA and H3N2 HA proteins in the cell lysis solutions were detected by Western blot (WB). Figure 1A ). The WB results were subjected to band grayscale analysis and background correction. The grayscale value of the target protein was divided by the grayscale value of the internal reference to correct the error. The obtained ratio represented the relative expression level of the target protein. The above ratio was subjected to data normalization and the expression trend of the target protein in each group of proteins within 48 hours was analyzed ( Figures 1B to 1D ). By comparing the protein expression trends from 24h to 48h of mRNA transfection, it can be seen from the results that the obtained optimized sequences (mRNA SEQ ID NO:9-13 expressing SARS-CoV-2BA5.2 Spike protein, mRNA SEQ ID NO:14-18 expressing H1N1 HA protein, and mRNA SEQ ID NO:19-22 expressing H3N2 HA protein) can all achieve the expression of the target protein. Among them, the mRNA sequence SEQID NO:13 expressing SARS-CoV-2BA.5.2Spike protein, the mRNA sequence SEQ ID NO:18 expressing H1N1 HA protein, and the mRNA sequence SEQID NO:22 expressing H3N2 HA protein have the highest and most stable expression levels.

[0126] SARS-CoV-2B.1.617.2Spike mRNA (SEQ ID NO: 23-24) was encapsulated in LNP and mice were immunized once on day 0 and day 21, with 5 μg mRNA injected each time. Blood was collected on day 20 and day 35 to detect humoral and cellular immunity levels. On day 20 and day 35 of immunization, the humoral immunity levels induced by SEQ ID NO: 23 and SEQ ID NO: 24 were comparable ( Figure 2A On day 35 of immunization, the cellular immunity induced by SEQ ID NO: 23 was stronger than that induced by SEQ ID NO: 24 ( Figure 2B ).

[0127] Based on the above results, the mRNA sequences with the best expression effect were selected from the optimized mRNA sequences: mRNA sequence encoding SARS-CoV-2BA5.2 spike SEQ ID NO: 13, mRNA sequence encoding H1N1 HA protein SEQ ID NO: 18, mRNA sequence encoding H3N2 HA protein SEQ ID NO: 22 and mRNA sequence encoding SARS-CoV-2

[0128] The mRNA sequence of B.1.617.2Spike protein SEQ ID NO:23 was used as a candidate representative sequence for in vivo efficacy evaluation.

[0129] Example 2 Animal Experiment Materials and Methods

[0130] 1. Sample Preparation

[0131] The modified mRNA was incorporated into lipid nanoparticles (LNPs) by PNI NanoAssemblr Ignite to prepare ARP01 injection, wherein the LNPs were prepared using conventional techniques in the art, such as those described in WO2021250263A1 and WO2021148511A1, and the prepared LNPs contained 4 different mRNAs, which were capable of expressing and producing 4 antigens. The 4 mRNAs were influenza virus Guangdong-Maonan / SWL1536 / 2019 (H1N1) HA protein mRNA (SEQ ID NO: 18), A / Darwin / 9 / 2021 (H3N2) HA protein mRNA (SEQ ID NO: 22), new coronavirus SARS-CoV-2BA5.2Spike protein mRNA (SEQ ID NO: 13), and SARS-CoV-2B.1.617.2Spike protein mRNA (SEQ ID NO: 23).

[0132] 2. Preparation of test substances

[0133] Prepare the test substances according to the scheme shown in Table 2.

[0134] Table 2. Preparation method of test substances

[0135]

[0136] 3. Immunization

[0137] (1) Vehicle control group (10% sucrose-Tris buffer): 200 μl of vehicle was injected intramuscularly.

[0138] (2) ARP01 injection for intramuscular injection of low, medium and high dose groups: 10 μg, 20 μg and 40 μg ARP01 were injected intramuscularly (injection volumes were 50, 100 and 200 μL, respectively).

[0139] (3) ARP01 intradermal injection group: 20 μg of ARP01 was intradermally injected (injection volume: 100 μl).

[0140] (4) Mice were vaccinated by intramuscular / intradermal injection on day 0 (primary immunization) and day 21 (boost immunization). The animals were weighed and observed 24 and 48 hours before and after administration, and once a week at other times, and body weight, health and survival status were recorded. Serum samples were collected on days 20 and 35. All animals were euthanized on day 35, and spleen tissues were collected.

[0141] The detailed protocol design is shown in Table 3 , and health monitoring is performed according to the indicators listed in Table 4 and humane endpoints are determined.

[0142] Table 3. Immunization schedule

[0143]

[0144] * Group G6 is mice that did not receive any medication

[0145] Table 4. Health monitoring and humanized endpoints

[0146]

[0147]

[0148] Example 3 Binding Antibody Titers after mRNA Immunization

[0149] On the 20th and 35th days after animal immunization using the method of Example 2, blood from the immunized animals in Example 2 was collected to prepare serum, and the specific IgG antibody titer in the serum samples was detected by ELISA, and four new coronavirus S proteins were detected respectively: SARS-CoV-2 (BA.4 / BA.5) Spike S1+S2 trimer Protein (Sino Biological-40589-V08H32), SARS-CoV-2 B.1.617.2 Spike S1+S2 trimer Protein (Sino Biological-40589-V08H10), SARS-CoV-2 (2019-nCoV) Spike S1+S2 ECD-His Recombinant Protein (Sino Biological-40589-V08H4) and SARS-CoV-2 XBB.1.16 (Omicron) Spike S1+S2 trimer Protein (Sino Biological-40589-V08H48), as well as two influenza A viruses Influenza A H1N1 (A / Guangdong-Maonan / SWL1536 / 2019) / (A / Hawaii / 70 / 2019) HA protein (Sino Biological-40717-V08H) and Influenza A H3N2 (A / Darwin / 9 / 2021) HA protein (Sino Biological-40859-V08H) IgG antibody levels.

[0150] Detection method:

[0151] 1) Coat 96-well plates with SARS-CoV-2 S protein and influenza A virus HA protein, respectively, at 4°C overnight;

[0152] 2) On the next day, take out the coated plate from 4°C, wash once with washing solution, and block with 3% BSA for 1 hour;

[0153] 3) Dilute the mouse serum sample to be tested, and test at 8 concentration points in duplicate wells;

[0154] 4) Pipette 50 μL of diluted serum sample into a 96-well plate and incubate at room temperature for 1-2 hours;

[0155] 5) Shake off the liquid in the hole and add washing liquid for washing;

[0156] 6) Add 50 μL of diluted HRP antibody (Abcam-ab97023) to each well and incubate at room temperature for 45-60 minutes;

[0157] 7) Shake off the liquid in the hole and wash with washing liquid;

[0158] 8) Add 50 μL of substrate solution to each well and incubate in the dark for 10-20 minutes, then add 50 μL of stop solution and mix gently;

[0159] 9) Measure the absorbance (OD) at 450 nm using a multi-function microplate reader;

[0160] 10) The highest dilution with an OD value 3 times higher than the background was determined as the endpoint antibody titer (EPT) of specific IgG in the mouse serum sample.

[0161] 1) Results:

[0162] 20 days after the first immunization, the titers of Spike S and HA-specific IgG antibodies in the experimental group were higher than those in the solvent control group ( Figure 3A ); 14 days after booster immunization, the titers of Spike S and HA-specific IgG antibodies in the test group were higher than those in the solvent control group, and higher than those in the primary immunization 20 days later ( Figure 3B ). The IgG level in each dose group showed dose correlation. And advantageously, in this experiment, four different S proteins of the new coronavirus were used to test the antibody titer, and for the above four different new coronavirus strains, the optimized mRNA sequences in this application can play an effective immune role.

[0163] Example 4: Hemagglutination inhibition test to detect hemagglutination antibody titer

[0164] On the 35th day after animal immunization using the method of Example 2, blood was collected from the immunized animals in Example 2 to prepare serum, and the titer of hemagglutination antibodies was detected by hemagglutination inhibition experiment, and the titer of hemagglutination antibodies against two influenza viruses was detected. The corresponding influenza virus HA protein was used for the experiment.

[0165] Experimental procedures

[0166] 1) Take fresh mouse serum, add receptor-destroying enzyme (Japan Seiken-340016) in a ratio of 1:4, mix, incubate at 37℃ for 17 hours, then incubate at 56℃ for 50 minutes, and then store at 4℃. Add concentrated red blood cell solution, neutralize overnight at 4℃, then centrifuge at 1500rpm for 10 minutes, and take the supernatant.

[0167] 2) Use fresh mouse blood to prepare mouse red blood cell solution, mix well and place on wet ice for later use;

[0168] 3) Dilute A / Guangdong-Maonan / SWL1536 / 2019(CNIC-1909)(H1N1)(NIBSC-19 / 312) and A / Darwin / 9 / 2021(IVR-228)(H3N2)(NIBSC-21 / 318) antigens at different dilution factors to determine the dilution factor of four hemagglutination units / 25 μL;

[0169] 4) Take a 96-well V-shaped plate, dilute the treated sample serum and standard antibody serum with physiological saline in a 2-fold ratio, add 4 hemagglutination units / 25μL of A / Guangdong-Maonan / SWL1536 / 2019(CNIC-1909)(H1N1)(NIBSC-19 / 312), A / Darwin / 9 / 2021(IVR-228)(H3N2)(NIBSC-21 / 318) antigens, mix well and incubate at room temperature for 1h, add 25μL of 1% red blood cell suspension to each well, gently shake and mix well, and incubate at room temperature for 40min. Observe the red blood cell agglutination phenomenon and record the results. The highest dilution at which complete hemagglutination inhibition occurs is the hemagglutination inhibition antibody titer, and the geometric average titer (GMT) is calculated.

[0170] Experimental Results

[0171] The results of the hemagglutination inhibition test showed that the serum of mice in the experimental groups 2-4 intramuscularly vaccinated and 5 intradermally injected with ARP01 could produce hemagglutination inhibition antibodies against two influenza A viruses, indicating that the ARP01 injection has good immunogenicity and can induce mice to produce high levels of neutralizing antibodies. According to the results, both intramuscular injection and intradermal injection can induce mice to produce neutralizing antibodies, indicating that the combined vaccine can be used in a variety of injection methods ( Figure 4A and Figure 4B ).

[0172] Example 5: Pseudovirus Neutralization Experiment

[0173] On the 20th and 35th days of animal immunization using the method of Example 2, the level of neutralizing antibodies against the new coronavirus in the serum of immunized mice was detected by pseudovirus neutralization experiment. The four pseudoviruses used in the experiment were SARS-CoV-2 (2019-nCoV), SARS-CoV-2BA5.2, SARS-CoV-2B.1.617.2, and SARS-CoV-2 (XBB 1.16) (the four new coronavirus pseudoviruses used in this experiment were provided by WuXi AppTec), and 8 concentration points were used for detection, and double-well detection was performed.

[0174] Specific experimental steps:

[0175] 1) Sample preparation: serum was inactivated in a 56°C water bath for 0.5-1 hour, and the initial antibody concentration was adjusted to 300 μg / mL;

[0176] 2) Add 260ul of autoclaved water to the 36 wells around the 96-well plate to seal the edges to reduce the error caused by evaporation of culture medium in the edge wells

[0177] 3) Add 150 μl / well of DMEM complete medium to column 2 (cell control CC), 100 μl / well of DMEM complete medium to column 3 (virus control VC), 142.5 μl / well of medium to wells B4-B11, and 100 μl / well of medium to the remaining wells

[0178] 4) Add 7.5 μl of the sample to be tested to wells B4-B11

[0179] 5) Gently pipette and aspirate the liquid in wells B4-B11 6-8 times, then transfer 50 μl of the liquid to the corresponding wells C4-C11. Then, dilute all wells 3-fold.

[0180] 6) Dilute the SARS-CoV-2 pseudovirus to 1.3×10 4 TCID50 / mL, add 50 μl to each well in columns 3 to 11

[0181] 7) Place the above 96-well plate in a cell culture incubator (37°C, 5% CO2) and incubate for 1 hour

[0182] 8) After incubation for 30 min, start digesting Huh7 cells and dilute the cell concentration to 2×10 5 cells / mL

[0183] 9) After the incubation, add 100 μl of cells to each well to make each well contain 2×10 4 Cells

[0184] 10) Place in a 37°C, 5% CO2 cell culture incubator for 24 hours

[0185] 11) After the culture is completed, discard 150 μl of supernatant and add 100 μl Bright-Glo TM Luciferase detection reagent, react at room temperature in the dark for 2 minutes, repeatedly pipette and transfer 150 μl of liquid to the white plate

[0186] 12) Read the luminescence value (RLU) using PerkinElmer EnSight multi-function imaging microplate reader

[0187] 13) Calculate the neutralization inhibition rate:

[0188] Inhibition rate = 1-(RLU mean of sample group-CC mean of blank control) / (VC mean-CC mean), and the neutralizing antibody titer (NAb titer) is expressed as the reciprocal of the serum dilution corresponding to the inhibition rate of 50%.

[0189] [Positive judgment value]

[0190] During the neutralization experiment, negative controls and positive controls need to be set up as references to determine whether the experiment is successful. The negative control ID50 is less than 30, and the positive ID50 is greater than 30 as the judgment value.

[0191] Experimental Results

[0192] The results of neutralizing antibody detection showed that compared with the solvent control group, the neutralizing antibody titers in the sera produced by mice in experimental groups 2-4 that were intramuscularly inoculated and group 5 that were intradermally injected with ARP01 were higher than those in the control group, and showed a certain dose correlation. Figure 5 The neutralizing antibody titers against SARS-CoV-2 (2019-nCoV), the neutralizing antibody titers against SARS-CoV-2B.1.617.2, the neutralizing antibody titers against SARS-CoV-2BA5.2 and the neutralizing antibody titers against SARS-CoV-2 (XBB 1.16) are disclosed.

[0193] Example 6: Elispot detection of spleen cells

[0194] ELISPOT analysis of spleen tissue T cell immunity (IFN-gamma). Splenic lymphocytes were stimulated by 7 stimulation conditions: 1. culture medium (no stimulation control) (Gibco-22400-089), 2. ConA (positive control) (Invitrogen-00-4978-93), 3. peptide library 1: H1N1-HA-CNIC1909 (Hefei Kesheng Jingpeptide Biotechnology Co., Ltd.), 4. peptide library 2: H3N2-HA-SAN010 (Hefei Kesheng Jingpeptide Biotechnology Co., Ltd.), 5. peptide library 3: SARA-Cov-S2P-BA.45 (Hefei Kesheng Jingpeptide Biotechnology Co., Ltd.), 6. peptide library 4: S-DeltaPep mixed peptide library (GenScript Biotech Co., Ltd.), 7. mixed peptide library (peptide library 1-4).

[0195] Experimental procedures

[0196] Mouse spleen was ground and filtered through a 70 μm cell strainer to obtain a single cell suspension. Red blood cells were lysed with 1×RBC and spleen cells were obtained after washing. Cells were counted using a cell counter. Based on the counting results, cells were diluted and counted at 1×10 6 100 μL / well was inoculated into Elispot plates.

[0197] Add 100 μL of the prepared 2× stimulant to the corresponding wells of the Elispot cell culture plate where cells have been seeded (7 stimulation conditions: culture medium (no stimulation control), ConA (positive control), peptide library 1: H1N1-HA-CNIC1909, peptide library 2: H3N2-HA-SAN010, peptide library 3: SARA-Cov-S2P-BA.45, peptide library 4: S-DeltaPep mixed peptide library, mixed peptide library (peptide library 1-4), triplicate wells), and incubate the cell plate in a 37°C, 5% CO2 incubator for 16 hours. Remove the Elispot plate from the incubator, wash 5 times with 200 μL / well PBS, add 100 μL R4-6A2-biotin antibody to each well, and incubate at RT for 2 hours. Discard the antibody and add 200 μL PBS to each well and wash 5 times. Add 100 μL / well streptavidin-ALP antibody and incubate at RT for 1 hour; 5) Discard the antibody and wash 5 times with 200 μL PBS per well. Add BCIP / NBT-plus substrate solution and incubate at RT for 9 minutes. Discard the substrate to terminate the reaction and wash the Elispot plate and membrane with running deionized water. Dry the Elispot plate and membrane at room temperature in the dark and read with the AID ELISPOT READER instrument.

[0198] Experimental Results

[0199] Compared with the solvent control group, the mean SFU values ​​of spleen cells of mice in experimental groups 2-4 that were intramuscularly inoculated and group 5 that were intradermally injected with ARP01 were significantly increased after stimulation with single viral antigen peptide library and mixed peptide library in a dose-dependent manner ( Fig. 6A and 6B ). This indicates that the combined vaccine can activate the body's immune system's specific immune response to multiple viral antigens, and has a synergistic immune response to the combined stimulation of multiple viral antigens. At the same dose (20ug / mouse), the subcutaneous administration method activated the same immune response as the intramuscular administration method, indicating that the combined vaccine can be applied to a variety of administration methods and provide more effective immune protection.

[0200] Example 7: Splenocyte ICS detection

[0201] Single splenocytes were isolated and stimulated by seven stimulation conditions during the experiment: 1. Culture medium (no stimulation control), 2. PMA + ionomycin (positive control), 3. Peptide library 1: H1N1-HA-CNIC1909 (Hefei Kesheng Jingpeptide Biotechnology Co., Ltd.), 4. Peptide library 2: H3N2-HA-SAN010 (Hefei Kesheng Jingpeptide Biotechnology Co., Ltd.), 5. Peptide library 3: SARA-Cov-S2P-BA.45 (Hefei Kesheng Jingpeptide Biotechnology Co., Ltd.), 6. Peptide library 4: S-DeltaPep mixed peptide library (GenScript Biotech Co., Ltd.), 7. Mixed peptide library (peptide library 1-4). Detection indicators: CD4+INFγ, CD4+TNFα, CD4+IL-2; CD4+IL-4, CD4+IL-5 and CD8+INFγ, CD8+TNFα, CD8+IL-2.

[0202] Experimental procedures

[0203] Freshly isolated spleen cells: Splenocytes were obtained by grinding mouse spleen tissue. The spleen cells of each group of mice were stimulated according to the above 7 conditions for 14 hours, and then blocked for 4 hours by adding a protein transport inhibitor (Invitrogen-00-4980-93). Finally, intracellular cytokine staining (ICS) was used to detect the expression of IFN-γ, TNF-α, IL-2, IL-4 and IL-5 in CD4+ T cells and IFN-γ, TNF-α, IL-2 in CD8+ T cells in mouse spleen samples.

[0204] GraphPad Prism was used to analyze the data, and One-way ANOVA was used to analyze the statistical differences in mouse serum neutralizing antibody titers.

[0205] ICS data were processed by FlowJo, and the final data were presented as mean±SD values ​​to present the expression of each group of samples.

[0206] Experimental Results

[0207] Splenocytes were stimulated with culture medium (negative control) and PMA + ionomycin (positive control). Compared with the culture medium control, the spleen cells of the experimental mice could significantly induce cytokines under the positive stimulation of PMA + ionomycin, and the results were in line with expectations. In order to evaluate the induction of cytokines by the peptide library, the cytokine level of each spleen sample under peptide library stimulation was subtracted from the cytokine level under the corresponding culture medium treatment condition to deduct the background expression of cytokines. The mean value of cytokines after background deduction is shown in the figure. Figure 7 , Figure 8The results showed that 14 days after booster immunization, the mouse spleen cells stimulated by the specific peptide library expressed IFNγ in each experimental group compared with the solvent control group. + CD4 + T cells, TNFα + CD4 + T, IL-2 + CD4 + T cells account for the total CD4 + The proportion of T cells increased; IFNγ in each experimental group + CD8 + T cells, TNFα + CD8 + T, IL-2 + CD8 + T cells account for the total CD8 + The proportion of T cells increased significantly.

[0208] Conclusion: The combined vaccine strain can produce high-titer binding antibodies and neutralizing antibodies against SARS-CoV-2B.1.617.2, SARS-CoV-2BA5.2, SARS-CoV-2 (XBB 1.16), SARS-CoV-2 (2019-nCoV), and two influenza A viruses Influenza A H1N1 and Influenza A H3N2 HA, and can stimulate Th1-biased T cell immunity and induce mice to produce antigen-specific CD4 + T, CD8 + T cells ( Figure 7 and 8 ). This indicates that the combined vaccine can activate specific humoral and cellular immune responses against multiple viral antigens, while producing cross-immune protection against multiple novel coronavirus variants.

[0209] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0210] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0211] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. An mRNA molecule comprising a coding region encoding a Spike protein of a first SARS-CoV-2 strain and / or a second SARS-CoV-2 strain that has undergone a stabilizing mutation, or a variant thereof having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity, wherein the stabilizing mutation is selected from a diproline stabilizing mutation or a hexaproline stabilizing mutation, and wherein the first SARS-CoV-2 strain is SARS-CoV-2BA5.2 and the second SARS-CoV-2 strain is SARS-CoV-2B.1.617.

2.

2. The mRNA molecule of claim 1, wherein the double proline stabilizing mutations are at positions corresponding to K986 and V987 of the Spike protein of wild-type SARS-CoV-2; and / or the six proline stabilizing mutations are at positions corresponding to F817, A892, A899, A942, K986, V987 of the Spike protein of wild-type SARS-CoV-2.

3. The mRNA molecule according to claim 1 or 2, wherein the Spike protein of the first SARS-CoV-2 strain that has undergone a stabilizing mutation comprises the amino acid sequence shown in SEQ ID NO.1, and / or the Spike protein of the second SARS-CoV-2 strain that has undergone a stabilizing mutation comprises the amino acid sequence shown in SEQ ID NO.

4.

4. The mRNA molecule according to claim 1 or 2, wherein the coding region: (1) comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs. 25-29, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs. 25-29 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. 1; and / or (2) comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.39-40, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.39-40 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.4; Preferably, the mRNA molecule comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs.9-13, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.9-13 and encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1; and / or The mRNA molecule comprises or is composed of a nucleotide sequence as shown in any one of SEQ ID NOs.23-24, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.23-24 and encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.

4.

5. An mRNA molecule comprising a coding region encoding a hemagglutinin protein of a first influenza strain and / or a hemagglutinin protein of a second influenza strain, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95% sequence identity thereto, wherein the first influenza strain is H1N1 and the second influenza strain is H3N2.

6. The mRNA molecule according to claim 5, wherein the hemagglutinin protein of the first influenza strain comprises the amino acid sequence shown in SEQ ID NO.2, and / or the hemagglutinin protein of the second influenza strain comprises the amino acid sequence shown in SEQ ID NO.

3.

7. The mRNA molecule according to claim 5 or 6, wherein the coding region: (1) comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.30-34, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.30-34 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.2; and / or (2) comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs.35-38, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.35-38 and encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.3; Preferably, the mRNA molecule comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs.14-18, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity with any one of SEQ ID NOs.14-18 and encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.2; and / or The mRNA molecule comprises or is composed of a nucleotide sequence as shown in any one of SEQ ID NOs.19-22, or comprises or is composed of a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NOs.19-22 and encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.

3.

8. The mRNA molecule according to any one of claims 1 to 7, wherein the mRNA molecule comprises a chemical modification, preferably a 1-methyl-pseudouridine modification, and further preferably, each U in the mRNA molecule is a 1-methyl-pseudouridine.

9. An mRNA composition, comprising: (1) at least one mRNA molecule targeting a SARS-CoV-2 strain, selected from the mRNA molecule according to any one of claims 1 to 4 and claim 8, and / or (2) at least one mRNA molecule against an influenza strain, selected from the mRNA molecule according to any one of claims 5 to 8.

10. The composition of claim 9, wherein the composition is a vaccine.

11. A nucleic acid molecule encoding the mRNA molecule according to any one of claims 1 to 8. A vector comprising the nucleic acid molecule according to claim 11 .

13. A cell comprising the nucleic acid molecule according to claim 11 or the vector according to claim 12.

14. A method for inducing an immune response against SARS-CoV-2 and / or influenza virus in a subject, the method comprising administering to the subject an mRNA molecule according to any one of claims 1 to 8, or a nucleic acid molecule according to claim 11, or a vector according to claim 12, or a cell according to claim 13.

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