MRNA vaccine against varicella-zoster virus

Abstract

The invention provides an mRNA (messenger Ribonucleic Acid) molecule aiming at varicella-zoster virus, an mRNA vaccine containing the molecule and application of the mRNA vaccine. The mRNA molecule disclosed by the invention is optimized aiming at glycoprotein E (VZV gE protein) of varicella-zoster virus, and the optimized mRNA molecule can play a role in effective immunization and is high in safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, the present invention relates to an mRNA vaccine against varicella-zoster virus. Background Art

[0002] Varicella-zoster virus (VZV) infection causes chickenpox and shingles. Chickenpox is one of the most common diseases caused by this virus and is characterized by pus-filled blisters on the skin, accompanied by symptoms such as itching and fever. After chickenpox infection, the virus lies dormant in the body and lurks in ganglion cells. When the body's immune system declines, especially when the body ages or has a compromised immune system, VZV may become active again. Nearly one-third of VZV-infected people will develop shingles in old age due to reactivation of VZV lurking in the nerves. Shingles usually presents as pain, tingling or burning sensation in the waist or one side of the chest, accompanied by the formation of blisters. These symptoms last for several weeks and may lead to complications, including encephalitis, pneumonia (direct viral pneumonia or secondary bacterial pneumonia), or bronchitis (viral bronchitis or secondary bacterial bronchitis).

[0003] Vaccines against varicella and herpes zoster have been widely used to prevent the occurrence of these diseases. Researchers are also working to improve the efficacy and safety of vaccines to provide better protection. Glycoprotein E (gE) of varicella-zoster virus is the main candidate protein for VZV subunit vaccine. Therefore, the development of a vaccine against varicella-zoster virus with good immunogenicity and high safety is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0004] To solve the above problems, the present invention provides a new mRNA vaccine against varicella-zoster virus with good immunogenicity and high safety.

[0005] In a first aspect, the present invention provides an mRNA molecule comprising a coding region, which encodes a fusion protein comprising a first domain and a second domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), and the second domain comprises a truncated varicella-zoster virus glycoprotein E (VZV gE protein), wherein the truncated varicella-zoster virus glycoprotein E is a protein lacking the intramembrane region of the VZV gE protein.

[0006] In some embodiments, the first domain comprises an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having 1, 2 or 3 amino acids inserted, substituted or deleted compared to the amino acid sequence shown in SEQ ID NO:8.

[0007] In some embodiments, the second domain does not contain the signal peptide of the VZV gE protein itself.

[0008] In some embodiments, the second domain comprises an amino acid sequence as shown in SEQ ID NO:119, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0009] In some embodiments, the first domain and the second domain are directly connected or connected by a connector. In some embodiments, the first domain is connected to the N-terminus of the second domain. In some other embodiments, the first domain is connected to the C-terminus of the second domain.

[0010] In some embodiments, the fusion protein comprises the amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

[0011] In some embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs:64-95, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:64-95. In some preferred embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 64, 65, 75, 77, 84, 86, 87, 89, 90, 92, 93 and 95, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of them. Preferably, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 65, 75, 92 and 95, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of them. Further preferably, the coding region comprises or consists of the nucleotide sequence as shown in SEQ ID NO:92, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.

[0012] 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.

[0013] In some embodiments, the mRNA molecule comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 9-40, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9-40. In some embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 9, 10, 20, 22, 29, 31, 32, 34, 35, 37, 38, 40, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of them. Preferably, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 10, 20, 37, 40, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of them. Further preferably, the coding region comprises or consists of the nucleotide sequence shown in SEQ ID NO:37, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.

[0014] In some embodiments, the mRNA molecule comprises a chemical modification, preferably a pseudouridine or 1-methyl-pseudouridine modification, and preferably, each U in the mRNA molecule is a pseudouridine or 1-methyl-pseudouridine.

[0015] In some embodiments, the coding region comprises or consists of a chemically modified nucleotide sequence as shown in any one of SEQ ID NOs: 64-95, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of SEQ ID NOs: 64-95. In some embodiments, the mRNA molecule comprises or consists of a chemically modified nucleotide sequence as shown in any one of SEQ ID NOs: 9-40, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of SEQ ID NOs: 9-40. Preferably, each U in the coding region is 1-methyl-pseudouridine.

[0016] In other embodiments, the coding region and mRNA molecules do not comprise chemical modifications. In some embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 64-95, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs: 64-95, wherein each U is uracil. In some embodiments, the mRNA molecule comprises or consists of a chemically modified nucleotide sequence as shown in any one of SEQ ID NOs: 9-40, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs: 9-40, wherein each U is uracil.

[0017] In a second aspect, the present invention provides an mRNA molecule comprising a coding region, which encodes a fusion protein comprising a first domain, a second domain and a third domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), the second domain comprises a truncated varicella-zoster virus glycoprotein E (VZV gE protein), the truncated varicella-zoster virus glycoprotein E is a protein lacking the intramembrane region of the VZV gE protein, and the third domain comprises a sequence derived from lysosomal associated membrane protein 3 (LAMP3, also known as DC-LAMP).

[0018] In some embodiments, the first domain comprises an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having 1, 2 or 3 amino acids inserted, substituted or deleted compared to the amino acid sequence shown in SEQ ID NO:8.

[0019] In some embodiments, the second domain does not contain the signal peptide of the VZV gE protein itself.

[0020] In some embodiments, the second domain comprises an amino acid sequence as shown in SEQ ID NO:119, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0021] In some embodiments, the third domain comprises an amino acid sequence as shown in SEQ ID NO:120, or an amino acid sequence having 1, 2 or 3 amino acids inserted, substituted or deleted compared to the amino acid sequence shown in SEQ ID NO:120.

[0022] In some embodiments, the first domain, the second domain and / or the third domain are directly connected or connected through a linker.

[0023] In some embodiments, the fusion protein includes a first domain, a second domain, and a third domain connected in any manner. In some embodiments, the fusion protein includes the first domain-second domain-third domain from N-terminus to C-terminus. In some embodiments, the fusion protein includes the first domain-third domain-second domain from N-terminus to C-terminus. In some embodiments, the fusion protein includes the second domain-first domain-third domain from N-terminus to C-terminus. In some embodiments, the fusion protein includes the second domain-third domain-first domain from N-terminus to C-terminus. In some embodiments, the fusion protein includes the third domain-first domain-second domain from N-terminus to C-terminus. In some embodiments, the fusion protein includes the third domain-second domain-first domain from N-terminus to C-terminus. In some embodiments, the fusion protein includes the third domain-second domain-first domain from N-terminus to C-terminus.

[0024] In some embodiments, the fusion protein comprises an amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO:3.

[0025] In some embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 96-107, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of them. In some preferred embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 96 and 98, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of them. Preferably, the coding region comprises or consists of the nucleotide sequence shown in SEQ ID NO: 98, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.

[0026] 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.

[0027] In some embodiments, the mRNA molecule comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 41-52, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of SEQ ID NOs: 41-52. In some embodiments, the mRNA molecule comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 41, 43, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of them. Preferably, the coding region comprises or consists of the nucleotide sequence as shown in SEQ ID NO:43, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.

[0028] 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.

[0029] In some embodiments, the coding region comprises or consists of a chemically modified nucleotide sequence as shown in any one of SEQ ID NOs: 96-107, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs: 96-107. In some embodiments, the mRNA molecule comprises or consists of a chemically modified nucleotide sequence as shown in any one of SEQ ID NOs: 41-52, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs: 41-52. Preferably, each U in the coding region is 1-methyl-pseudouridine.

[0030] In other embodiments, the coding region and mRNA molecules do not contain chemical modifications. In some embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 96-107, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of SEQ ID NOs: 96-107, wherein each U is uracil. In some embodiments, the mRNA molecule comprises or consists of a chemically modified nucleotide sequence as shown in any one of SEQ ID NOs:41-52, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs:41-52, wherein each U is uracil.

[0031] In a third aspect, the present invention provides an mRNA molecule comprising a coding region, which encodes a fusion protein comprising a first domain and a second domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), and the second domain comprises the extramembrane region of varicella-zoster virus glycoprotein E (VZV gE protein).

[0032] In some embodiments, the first domain comprises an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having 1, 2 or 3 amino acids inserted, substituted or deleted compared to the amino acid sequence shown in SEQ ID NO:8.

[0033] In some embodiments, the second domain does not contain the signal peptide of the VZV gE protein itself.

[0034] In some embodiments, the second domain comprises an amino acid sequence as shown in SEQ ID NO:121, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0035] In some embodiments, the first domain and the second domain are directly connected or connected by a connector. In some embodiments, the first domain is connected to the N-terminus of the second domain. In some other embodiments, the first domain is connected to the C-terminus of the second domain.

[0036] In some embodiments, the fusion protein comprises the amino acid sequence as shown in SEQ ID NO:4, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0037] In some embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 108-118, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of them. In some preferred embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 108, 109, 113, 117 and 118, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of them. Preferably, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 113 and 118, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of them. Further preferably, the coding region comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 113, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of them.

[0038] 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.

[0039] In some embodiments, the mRNA molecule comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs:53-63, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:53-63. In some embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 53, 54, 58, 62, and 63, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 53, 54, 58, 62, and 63. Preferably, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 54, 58, 63, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of SEQ ID NOs: 54, 58, 63. Further preferably, the coding region comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 58, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO: 58.

[0040] 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.

[0041] In some embodiments, the coding region comprises or consists of a chemically modified nucleotide sequence as shown in any one of SEQ ID NOs: 108-118, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of SEQ ID NOs: 108-118. In some embodiments, the mRNA molecule comprises or consists of a chemically modified nucleotide sequence as shown in any one of SEQ ID NOs: 53-63, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of SEQ ID NOs: 53-63. Preferably, each U in the coding region is 1-methyl-pseudouridine.

[0042] In other embodiments, the coding region and mRNA molecules do not comprise chemical modifications. In some embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 108-118, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs: 108-118, wherein each U is uracil. In some embodiments, the mRNA molecule comprises or consists of a chemically modified nucleotide sequence as shown in any one of SEQ ID NOs:53-63, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs:53-63, wherein each U is uracil.

[0043] In a fourth aspect, the present invention provides an mRNA composition comprising at least one mRNA molecule as described in the present invention.

[0044] In some embodiments, the composition comprises at least one, at least two, or at least three mRNA molecules as described above.

[0045] In some embodiments, the composition comprises: a first mRNA molecule comprising a coding region, the coding region encoding a fusion protein comprising a first domain and a second domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), and the second domain comprises a truncated varicella-zoster virus glycoprotein E (VZV gE protein), wherein the truncated varicella-zoster virus glycoprotein E is a protein lacking the intramembrane region of the VZV gE protein; and / or a second mRNA molecule comprising a coding region, the coding region encoding a fusion protein comprising a first domain, a second domain, and a third domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), and the second domain comprises a truncated varicella-zoster virus glycoprotein E (VZV gE protein), wherein the truncated varicella-zoster virus glycoprotein E is a protein lacking the intramembrane region of the VZV gE protein. gE protein, and the third domain comprises a sequence derived from lysosomal associated membrane protein 3 (LAMP3, also known as DC-LAMP); and / or a third mRNA molecule, which comprises a coding region, the coding region encoding a fusion protein comprising a first domain and a second domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), and the second domain comprises the extramembrane region of varicella-zoster virus glycoprotein E (VZV gE protein).

[0046] In some embodiments, the composition comprises: a first mRNA molecule, the coding region of which comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 64-95, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs: 64-95; and / or a second mRNA molecule, the coding region of which comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 96-107, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of them and encodes a nucleotide sequence as shown in SEQ ID NOs: NO:3; and / or a third mRNA molecule, whose coding region comprises or consists of the nucleotide sequence as shown in any one of SEQ ID NOs:108-118, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of them and encodes an amino acid sequence as shown in SEQ ID NO:4.

[0047] 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: 9-40, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of them and encoding an amino acid sequence as shown in SEQ ID NO: 2; and / or a second mRNA molecule comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs: 41-52, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of them and encoding an amino acid sequence as shown in SEQ ID NO: NO:3; and / or a third mRNA molecule comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs:53-63, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of them and encoding an amino acid sequence as shown in SEQ ID NO:4.

[0048] In some embodiments, the first mRNA molecule, the second mRNA molecule and / or the third mRNA molecule comprises a chemical modification, preferably a pseudouridine or 1-methyl-pseudouridine modification, and preferably, each U in the mRNA molecule comprising the chemical modification is 1-methyl-pseudouridine.

[0049] In some embodiments, the first mRNA molecule, the second mRNA molecule and / or the third mRNA molecule comprises a chemical modification, preferably a pseudouridine or 1-methyl-pseudouridine modification, and preferably, each U in the mRNA molecule comprising the chemical modification is 1-methyl-pseudouridine.

[0050] In some embodiments, the first mRNA molecule contains or does not contain a chemical modification; and / or the second mRNA molecule contains a chemical modification; and / or the third mRNA molecule contains a chemical modification, preferably, the chemical modification is a 1-methyl-pseudouridine modification, and also preferably, each U in the modified mRNA molecule is 1-methyl-pseudouridine.

[0051] In some embodiments, the composition is a vaccine.

[0052] In some embodiments, at least one of the mRNAs is complexed with lipids to form one or more liposomes, lipoplexes, or lipid nanoparticles. In some embodiments, the composition comprises liposomes, lipoplexes, and / or lipid nanoparticles containing at least one mRNA.

[0053] In some embodiments, the lipid nanoparticles comprise ionizable lipids, neutral lipids, cholesterol and PEG lipids. In some embodiments, the lipid nanoparticles comprise 40-60wt% ionizable lipids, 5-15wt% neutral lipids, 25-50wt% cholesterol and 0.5-3wt% PEG lipids.

[0054] In some embodiments, the composition further comprises an adjuvant.

[0055] 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.

[0056] In a fifth aspect, the present invention provides a kit comprising at least one mRNA molecule as described in the present invention or the composition as described in the present invention.

[0057] In some embodiments, the kit comprises at least one, at least two, or at least three mRNA molecules as described above.

[0058] In some embodiments, the composition comprises: a first mRNA molecule comprising a coding region, the coding region encoding a fusion protein comprising a first domain and a second domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), and the second domain comprises a truncated varicella-zoster virus glycoprotein E (VZV gE protein), wherein the truncated varicella-zoster virus glycoprotein E is a protein lacking the intramembrane region of the VZV gE protein; and / or a second mRNA molecule comprising a coding region, the coding region encoding a fusion protein comprising a first domain, a second domain, and a third domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), and the second domain comprises a truncated varicella-zoster virus glycoprotein E (VZV gE protein), wherein the truncated varicella-zoster virus glycoprotein E is a protein lacking the intramembrane region of the VZV gE protein. gE protein, and the third domain comprises a sequence derived from lysosomal associated membrane protein 3 (LAMP3, also known as DC-LAMP); and / or a third mRNA molecule, which comprises a coding region, the coding region encoding a fusion protein comprising a first domain and a second domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), and the second domain comprises the extramembrane region of varicella-zoster virus glycoprotein E (VZV gE protein).

[0059] In some embodiments, the composition comprises: a first mRNA molecule, the coding region of which comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 64-95, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs: 64-95; and / or a second mRNA molecule, the coding region of which comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 96-107, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of them and encodes a nucleotide sequence as shown in SEQ ID NOs: NO:3; and / or a third mRNA molecule, whose coding region comprises or consists of the nucleotide sequence as shown in any one of SEQ ID NOs:108-118, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of them and encodes an amino acid sequence as shown in SEQ ID NO:4.

[0060] 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: 9-40, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of them and encoding an amino acid sequence as shown in SEQ ID NO: 2; and / or a second mRNA molecule comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs: 41-52, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of them and encoding an amino acid sequence as shown in SEQ ID NO: NO:3; and / or a third mRNA molecule comprising or consisting of a nucleotide sequence as shown in any one of SEQ ID NOs:53-63, or comprising or consisting of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of them and encoding an amino acid sequence as shown in SEQ ID NO:4.

[0061] In some embodiments, the first mRNA molecule, the second mRNA molecule and / or the third mRNA molecule comprises a chemical modification, preferably a pseudouridine or 1-methyl-pseudouridine modification, and preferably, each U in the mRNA molecule comprising the chemical modification is 1-methyl-pseudouridine.

[0062] In some embodiments, the first mRNA molecule contains or does not contain a chemical modification; and / or the second mRNA molecule contains a chemical modification; and / or the third mRNA molecule contains a chemical modification, preferably, the chemical modification is a 1-methyl-pseudouridine modification, and also preferably, each U in the modified mRNA molecule is 1-methyl-pseudouridine.

[0063] In a sixth aspect, a nucleic acid molecule is provided, which encodes an mRNA molecule according to the first aspect, the second aspect and / or the third aspect.

[0064] In a seventh aspect, a vector is provided, which comprises the nucleic acid molecule according to the sixth aspect of the present invention.

[0065] In an eighth aspect, a cell is provided, comprising a nucleic acid molecule according to the sixth aspect of the invention or a vector according to the seventh aspect of the invention.

[0066] In a ninth aspect, the present invention provides a method for inducing an immune response against varicella-zoster 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The gE protein expression was detected by WB 24 hours after transfection with modified and unmodified VZV gE-truncated1 mRNA.

[0068] Figure 2 The gE protein expression was detected by WB 24, 48, and 72 hours after transfection with modified and unmodified VZV gE-truncated1 mRNA.

[0069] Figure 3 The gE protein expression was detected by WB 24 hours after transfection with modified and unmodified VZV gE-truncated1-DC mRNA.

[0070] Figure 4 The gE protein expression was detected by WB 24, 48, and 72 hours after VZV gE-truncated2 mRNA transfection.

[0071] Figure 5 The geometric mean titers (GMTs) of VZV gE-specific IgG antibodies in serum of different groups of mice are shown.

[0072] Figure 6 Shown are serum VZV gE-specific IgG1 (a), IgG2c (b) and IgG2c / IgG1 ratio (c) of mice in different groups.

[0073] Figure 7 Neutralizing antibody titers in sera of mice in different groups are shown.

[0074] Figure 8 The results of ELISPOT assay for IFN-γ (a) and IL-2 (b) secreting cells in spleen lymphocytes 14 days after secondary immunization are shown.

[0075] Fig. 9 Antigen-specific CD4 in spleen lymphocytes 14 days after secondary immunization + and CD8 +The results of secretory cytokine detection of T cells, where ac are TNF-α secreting, IFN-γ secreting and IL-2 secreting antigen-specific CD4 + T cell detection results, df, respectively, TNF-α secreting, IFN-γ secreting and IL-2 secreting antigen-specific CD8 + T cell test results.

[0076] Fig.10 Shown are the body weight changes of mice evaluated for immunogenicity. DETAILED DESCRIPTION

[0077] 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.

[0078] definition

[0079] 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.

[0080] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0081] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0082] The term "nucleic acid" refers to any DNA or RNA molecule.

[0083] 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.

[0084] 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.

[0085] The 5'-cap structure is typically a modified nucleotide (cap analog), in particular 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 moieties, 3', 3'-reverse abasic moieties, 3', 2'-reverse nucleotide moieties, 3', 2'-reverse abasic moieties, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3-phosphate, 3-phosphorothioate, phosphorodithioate or bridged or non-bridged methylphosphonate moieties. 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.

[0086] Cap analogs refer to non-polymerizable dinucleotides with cap functions that promote translation or localization when incorporated into the 5' end of an RNA molecule, and / or prevent RNA molecule degradation. Non-polymerizable means that the cap analog will only be incorporated at the 5' end because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent RNA polymerase. 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.

[0087] 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.

[0088] The term "fusion protein" refers to the use of genetic expression of polynucleotides encoding proteins or protein synthesis methods to connect two or more proteins or fragments thereof in a colinear manner through their respective peptide backbones. Preferably, the polypeptides or fragments thereof are from different sources.

[0089] The term "connector" as used herein refers to a (peptide) linker of natural and / or synthetic origin, consisting of linear amino acids. Each domain in the fusion protein of the present invention can be connected by a connection, wherein each connection is fused to at least two polypeptides or domains and / or connected in other ways (e.g., via a peptide bond). The connection should have a length suitable for connecting two or more monomer domains in this way, and the connection can ensure that the different domains connected to it are correctly folded and appropriately presented, thereby exerting the function of its biological activity. In different embodiments, the connection has a flexible conformation. Suitable flexible connection includes, for example, glycine, glutamine and / or serine residues. In some embodiments, the amino acid residues in the connection can be arranged in small repeating units of up to 5 amino acids, for example, with the amino acid sequence shown in GGGSGG or GGGSGGG.

[0090] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide or amino acid, gaps present in the target sequence are not counted. Likewise, since target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted, gaps present in the reference sequence are not counted.

[0091] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The determination of the sequence identity percentage between the comparison of the sequence and the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite that can be obtained from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. Government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0092] The present invention also relates to amino acid sequence variants, which can be prepared by introducing appropriate modifications into the nucleotide sequence of the encoding molecule or by peptide synthesis. Such modifications include, for example, deletions, insertions and / or substitutions of residues in the amino acid sequence. Any combination of deletions, insertions and substitutions can be performed to obtain a final construct having the desired properties, such as antigen binding activity. Sites for substitution generally include HVR and framework (FR). See the possible substitutions of amino acids in the table below.

[0093] Conservative amino acid substitution

[0094]

[0095]

[0096] Varicella zoster virus (VZV) particles are spherical with a diameter of 180-200 nanometers. These particles are mainly composed of a lipid envelope, a nucleocapsid, and a layer of loosely associated proteins (called the encapsid) between the nucleocapsid and the envelope. The proteins in these encapsids are involved in viral replication, assembly, and interaction with host cells. The encapsid is covered by a lipid envelope containing glycoproteins, which are displayed on the outside of the virus particles and are each about 8 nanometers long. The glycoproteins on the surface of VZV virus particles contain many immunogenic epitopes, the most important of which are glycoprotein E (gE) and glycoprotein I (gI). These glycoproteins are the main targets for recognition by the immune system and the production of antibodies.

[0097] The genome of VZV is a linear double-stranded DNA molecule with a length of about 125,000 nucleotides and a relative molecular mass of about 80 MDa. It has two main isomers: S (prototype) and IS (inverted S), which appear at equal frequencies with a total frequency of 90-95%. The VZV genome consists of two covalently linked long (L) and short (S) DNA fragments. In addition, the L (long) fragment can also be inverted, forming four linear isomers (IL and ILS). There are at least 71 open reading frames (ORFs) in the genome of VZV. These open reading frames are a series of continuous DNA sequences that can be read as protein coding sequences by the cell's transcription and translation machinery. These proteins play a key role in the life cycle of the virus, participating in processes including virus entry into host cells, replication, and assembly.

[0098] In one aspect of the present invention, an mRNA molecule is provided, which comprises a coding region, wherein the coding region encodes a fusion protein comprising a first domain and a second domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), and the second domain comprises a truncated varicella-zoster virus glycoprotein E (VZV gE protein), wherein the truncated varicella-zoster virus glycoprotein E is a protein lacking the intramembrane region of the VZV gE protein.

[0099] In some embodiments, the fusion protein comprises the amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

[0100] In some embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 64-95, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of them.

[0101] In another aspect of the present invention, an mRNA molecule is provided, which comprises a coding region, wherein the coding region encodes a fusion protein comprising a first domain, a second domain and a third domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), the second domain comprises a truncated varicella-zoster virus glycoprotein E (VZV gE protein), the truncated varicella-zoster virus glycoprotein E is a protein lacking the intramembrane region of the VZV gE protein, and the third domain comprises a sequence derived from lysosomal associated membrane protein 3 (LAMP3, also known as DC-LAMP).

[0102] In some embodiments, the fusion protein comprises an amino acid sequence as shown in SEQ ID NO:3, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO:3.

[0103] In some embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs:96-107, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of them.

[0104] In yet another aspect of the present invention, an mRNA molecule is provided, comprising a coding region, which encodes a fusion protein comprising a first domain and a second domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), and the second domain comprises the extramembrane region of varicella-zoster virus glycoprotein E (VZV gE protein).

[0105] In some embodiments, the fusion protein comprises the amino acid sequence as shown in SEQ ID NO:4, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0106] In some embodiments, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 108-118, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of them.

[0107] In some embodiments, the mRNA molecule of the present invention comprises a chemical modification, preferably a 1-methyl-pseudouridine modification, and preferably, each U in the mRNA molecule is 1-methyl-pseudouridine.

[0108] In some embodiments, the mRNA molecule of the present invention 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 residues.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.).

[0113] 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.

[0114] 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.

[0115] In some embodiments, the composition according to the invention comprises at least one mRNA molecule of the invention as described above.

[0116] In some embodiments, the composition according to the present invention comprises at least one mRNA molecule encoding a protein derived from varicella-zoster virus glycoprotein E (VZV gE protein).

[0117] 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).

[0118] 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, for example, 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 present 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. In some other embodiments, the lipid nanoparticles comprise ionizable lipids, neutral lipids, cholesterol and PEG lipids. Preferably, the lipid nanoparticles comprise 40-60wt% ionizable lipids, 5-15wt% neutral lipids, 25-50wt% cholesterol and 0.5-3wt% PEG lipids.

[0119] 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.

[0120] 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).

[0121] 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).

[0122] 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.

[0123] 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.

[0124] In certain embodiments, the LNPs comprise one or more additional lipids that stabilize the formation of the particle during particle formation.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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).

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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).

[0135] 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.

[0136] 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.

[0137] A therapeutically effective amount in the context of the present invention is generally understood to be an amount sufficient to induce an immune response.

[0138] 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.

[0139] Example

[0140] Example 1 Verification of VZV gE mRNA translation products

[0141] The intramembrane region of the VZV gE protein (SEQ ID NO: 1) was deleted, and the signal peptide of the VZV gE protein was replaced with LAMP-1 and Y598A mutation occurred to obtain VZV gE-truncated1 (SEQ ID NO: 2), that is, VZV gE-truncated1 comprises the amino acid sequence of LAMP1 (SEQ ID NO: 8) and the amino acid sequence of the intramembrane region of the VZV gE protein is deleted (SEQ ID NO: 119). Based on VZV gE-truncated1, the DC-LAMP functional sequence was added to the C-terminus to obtain VZV gE-truncated1-DC (SEQ ID NO: 3), that is, VZV gE-truncated1-DC comprises the amino acid sequence of LAMP1 (SEQ ID NO: 8), the amino acid sequence of the intramembrane region of the VZV gE protein is deleted (SEQ ID NO: 119) and the DC-LAMP functional sequence (SEQ ID NO: 120). Based on VZV gE-truncated1, the transmembrane region was further deleted to obtain VZV gE-truncated2 (SEQ ID NO: 4). VZV gE-truncated1 contained the amino acid sequence of LAMP1 (SEQ ID NO: 8) and the amino acid sequence of the extramembrane region of the VZV gE protein (SEQ ID NO: 121).

[0142] The VZV gE-truncated1 coding sequence (SEQ ID NO:5) was codon optimized to obtain mRNA sequences that met the human codon preference (SEQ ID NOs:9-18) and the mRNA sequences that met the human / mouse dual-species codon preference (SEQ ID NOs:19-40). Taking into account the minimum free energy and codon preference index (CAI), 12 VZV gE-truncated1 mRNAs (SEQ ID NOs:9, 10, 20, 22, 29, 31, 32, 34, 35, 37, 38, 40) were preliminarily screened.

[0143] The VZV gE-truncated1-DC coding sequence (SEQ ID NO: 6) was optimized by LinearDesign to obtain mRNA sequences (SEQ ID NOs: 41-52), and two VZVgE-truncated1-DC mRNAs (SEQ ID NOs: 41 and 43) were preliminarily screened out by comprehensively considering the minimum free energy and codon preference index (CAI).

[0144] The VZV gE-truncated2 coding sequence (SEQ ID NO:7) was optimized by LinearDesign to obtain mRNA sequences (SEQ ID NOs:53-63), and five VZVgE-truncated2 mRNAs (SEQ ID NOs:53, 54, 58, 62, 63) were preliminarily screened out by comprehensively considering the minimum free energy and codon preference index (CAI).

[0145] In the transcription process, uridine, one of the raw materials of mRNA, is replaced with N1-methyl pseudouridine in the reaction system, and the synthesized mRNA is a modified mRNA, which improves the stability of the transcription product mRNA and reduces the immunogenicity. The corresponding relationship of each sequence involved is shown in Table 1 below.

[0146] Table 1. Correspondence between sequences

[0147] serial number Amino acid sequence Coding sequence mRNA sequences mRNA coding region sequence 1 VZV gE-truncated1 SEQ ID NO:2 SEQ ID NO:5 SEQ ID NO:9-40 SEQ ID NO:64-95 2 VZVgE-truncated1-DC SEQ ID NO:3 SEQ ID NO:6 SEQ ID NO:41-52 SEQ ID NO:96-107 3 VZV gE-truncated2 SEQ ID NO:4 SEQ ID NO:7 SEQ ID NO:53-63 SEQ ID NO:108-118

[0148] 1 μg of VZV gE-truncated1 mRNA (SEQ ID NO: 9, 10, 20, 22, 29, 31, 32, 34, 35, 37, 38, 40), VZV gE-truncated1-DC mRNA (SEQ ID NO: 41, 43), and VZVgE-truncated2 mRNA (SEQ ID NO: 53, 54, 58, 62, 63) were transfected into Hek293 cells using MessengerMAX (Thermo). After 24, 48, and 72 hours of transfection, the culture medium was removed and washed with PBS, and then cell lysis buffer (Western and IP cell lysis buffer, Biyuntian P0013) was added to prepare cell lysates. The VZV gE protein content in the cell lysate was detected by Western blot (WB), and the in vitro expression efficiency of mRNA was compared, and mRNA with higher expression efficiency and more stable expression was selected as the candidate sequence.

[0149] After 24 hours of VZV gE-truncated1 mRNA transfection, four mRNA sequences (SEQ ID NO: 10, 20, 37, 40) with better expression efficiency in both modified and unmodified states were screened out from the above 12 optimized mRNA sequences for further screening ( Figure 1 ).

[0150] The expression stability of the selected mRNA sequences was further screened. After transfection of the modified and unmodified four mRNA sequences (SEQ ID NO: 10, 20, 37, 40) for 24, 48, and 72 hours, the cells were lysed and the expression level of VZV gE protein in the cell lysate was detected by WB ( Figure 2 ). By comparison, it was found that three mRNA sequences (SEQ ID NO: 37, 40, 20) could still stably and efficiently express gE protein after 72 hours in both modified and unmodified states, and the gE protein levels were higher than those of other mRNA sequences 72 hours after transfection. Therefore, the above three mRNA sequences (SEQ ID NO: 37, 40, 20) were determined as candidate sequences. Sequence 37 was specifically selected as an example for in vivo verification.

[0151] After 24 hours of VZV gE-truncated1-DC mRNA transfection, both modified mRNA sequences could express gE protein ( Figure 3 ). After grayscale analysis, one mRNA sequence (SEQ ID NO: 43) had a higher gE protein expression efficiency than another mRNA sequence (SEQ ID NO: 41), and the mRNA sequence (SEQ ID NO: 43) was used as a candidate sequence.

[0152] After 24 hours of transfection with VZV gE-truncated2 mRNA, the five modified mRNA sequences (SEQ ID NO: 53, 54, 58, 62, 63) all expressed a certain amount of protein. Among them, SEQ ID NO: 53, 54, 58, 63 had higher expression levels. After 48 hours, the cells were lysed and the expression level of VZV gE protein in the cell lysate was detected by WB. It was found that SEQ ID NO: 54, 58, 63 could still maintain a high expression level of protein. Among them, SEQ ID NO: 58, 63 could still maintain stable and effective expression after 72 hours ( Figure 4 ). SEQ ID NO: 58 was specifically selected as an example for in vivo verification. Example 2: Animal Experiment Materials and Methods

[0153] Sample preparation:

[0154] By PNI Ignite TMDifferent mRNA sequences are respectively incorporated into a system containing four lipid buffers mixed in proportion to assemble into different lipid nanoparticles (LNPs). Among them, LNPs are prepared using existing methods in the prior art, such as those described in WO2021250263A1 and WO2021148511A1, and the mRNA sequences are firefly luciferase-labeled mRNA-LNP, unmodified gE-truncated1 mRNA-LNP (SEQ ID NO: 37), modified gE-truncated1 mRNA-LNP (SEQ ID NO: 37), modified gE-truncated1-DC mRNA-LNP (SEQ ID NO: 43), and modified gE-truncated2 mRNA-LNP (SEQ ID NO: 58).

[0155] Immunizations:

[0156] Blank control (Group G1: saline group): An equal volume of saline was injected into bilateral intramuscular injections, 25 μL on each side.

[0157] Negative control (G2 group: Fluc group): 10 μg of mRNA-LNP labeled with firefly luciferase was injected into bilateral muscles, 25 μL on each side.

[0158] Positive control (Group G3: GSK Shingrix subunit vaccine): 5 μg of Shingrix subunit vaccine was injected intramuscularly on both sides, 25 μL on each side.

[0159] Four test groups (Group G4: unmodified gE-truncated 1 mRNA-LNP (SEQ ID NO: 37), Group G5: modified gE-truncated1 mRNA-LNP (SEQ ID NO: 37), Group G6: modified gE-truncated1-DC mRNA-LNP (SEQ ID NO: 43), Group G7: modified gE-truncated2 mRNA-LNP (SEQ ID NO: 58)): 10 μg of different test LNPs were injected bilaterally intramuscularly, 25 μL on each side.

[0160] Experimental steps:

[0161] 1) Two intramuscular injections are given 28 days apart, with the first vaccination being Day 0.

[0162] 2) 14 days after the second immunization, blood was collected from 6 mice in each group (C57BL / 6J mice, purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) to separate serum. a: Detection of VZV gE antigen-specific IgG, IgG1 and IgG2c antibody titers and analysis of IgG1 / IgG2c ratio. b: Detection of VZV virus neutralizing antibodies.

[0163] 3) 14 days after the second immunization, spleen lymphocytes were isolated from 6 mice in each group. a: The cells were stimulated with a gE protein peptide library (VZV (gE) Peptide Pool, purchased from Peptides / Elephants GmbH, LB01818) (10 μg / ml) for 24 hours, and the frequency of IFN-γ and IL-2 secreting cells was detected by ELISPOT method. b: The cells were stimulated with a gE protein peptide library (10 μg / ml) for 12 hours, and the level of IFN-γ, IL-2, and TNF secreting antigen-specific T cells was detected by FACS method.

[0164] Example 3: Neutralizing antibody titers after mRNA immunization

[0165] 14 days after the second immunization, blood was collected from 6 mice in each group to separate serum, and the VZV gE antigen-specific IgG, IgG1, and IgG2c antibody titers were detected (Endpoint titration assay), and the IgG1 / IgG2c ratio was analyzed.

[0166] The specific experimental method is as follows: use ELISA coating buffer to dilute gE protein antigen (purchased from ACROBiosystems), coat the 96-well plate with the antigen, and incubate overnight at 4°C; use a spray gun to add 200μL PBST washing solution to the 96-well plate and wash 3 times; add 100μL blocking solution to each well and place it in an air bath (25°C constant temperature box) for 2 hours; discard the blocking solution, and during the blocking process, prepare mouse serum samples, and use sample diluent to dilute the samples according to a certain starting dilution multiple, 8 gradients, and 2-fold ratio; add diluted mouse serum samples to the 96-well plate coated with antigen, 100μL per well, and place it in an air bath (30°C constant temperature box) for 2 hours; discard the serum samples, add 200μL to each well PBST washing solution, washed 3 times; using sample diluent, dilute the secondary antibody (goat anti-mouse IgG-HRP, goat anti-mouse IgG1-HRP, goat anti-mouse IgG2c-HRP (purchased from GenScript) according to the proportion, add 100 μL of secondary antibody diluent to each well after dilution, incubate in an air bath (25°C constant temperature box) for 1 hour; discard the secondary antibody, add 200 μL of PBST washing solution to each well, wash 5 times; after washing, add 100 μL of TMB color development solution to each well of the plate, protect from light, and observe the color development state in real time; add 100 μL of stop solution to each well for termination, and detect on the machine; read the OD value using a single wavelength detection (450nm wavelength) of an ELISA reader; 4.1 times the OD value of the blank well is determined as the threshold, and the dilution multiple corresponding to the OD value greater than or equal to the threshold is determined as the antibody titer.

[0167] The serum binding antibody titers of G1 and G2 groups were below the detection limit. In the secondary immunization 14 days later, the experimental groups G4-G7 were able to induce higher VZV gE-specific IgG antibodies ( Figure 5 ). Analysis of the IgG2c / IgG1 ratio 14 days after the second immunization showed that the G4-G7 groups were able to induce a more significant Th1 immune response compared to G3 ( Figure 6 ac).

[0168] Example 4: Neutralizing antibody titers after mRNA immunization

[0169] 14 days after the second immunization, blood was collected from 6 mice in each group to obtain serum, and the neutralizing antibody titer produced by each group of mice was determined using the real virus neutralization test method.

[0170] Experimental principle: Neutralizing antibodies make VZV lose its ability to infect cells through neutralization. VZV will express virus-specific proteins when it enters cells. Virus with infection and replication ability is displayed using virus protein-specific green fluorescent antibodies. By comparing the amount of infectious virus with that of the VZV control group, its ID50 (the dilution multiple of serum when VZV virus is 50% inhibited) is calculated to indicate the neutralizing activity of serum against VZV virus.

[0171] Experimental method: The density of MRC-5 cells (purchased from ATCC) was adjusted, inoculated into 96-well plates, and placed in a cell culture incubator (3°C, 5% CO2) for overnight culture. The experiment could be started when the cell confluence was about 90% on the next day. After the serum sample was inactivated in a water bath at 56°C for 30 minutes, it was diluted 20 times and diluted 3 times in a gradient, with a total of 8 dilutions (including the first well) and 2 replicates. After the serum sample was added to the diluted VZV virus (VZV-Oka strain purchased from ATCC) and neutralized in a 37°C, 5% CO2 incubator for about 1 hour, the virus and serum neutralization product, 50 μL per well, was added to the 96-well plate containing cells in 2 replicates, and the medium was changed after being cultured in a 37°C, 5% CO2 incubator for about 2 hours, and 100 μL of culture medium was added to each well to continue culturing for 48 hours. After that, the supernatant was discarded, the cells were fixed, and the fluorescently labeled detection antibody was added, and the plate was read using a CTL instrument.

[0172] The neutralizing antibody titers in the serum samples of G1 and G2 were all below the detection limit, while the neutralizing antibody titers in the serum samples of G4, G5, G6, and G7 were all high. Among them, the neutralizing antibody titer in the serum samples of G5 group was more obvious than that of other groups ( Figure 7 ).

[0173] Example 5: T cell responses after mRNA immunization

[0174] 1.ELISPOT method to detect the frequency of IFN-γ and IL-2 secreting cells in spleen lymphocytes 14 days after secondary immunization

[0175] 14 days after the secondary immunization, spleen immune cells of 6 mice in each group were isolated, culture medium was used as negative control, Staphylococcal enterotoxin B (SEB) was used as positive control, and gE peptide pool (VZV (gE) Peptide Pool, purchased from Peptides / Elephants GmbH, LB01818) was used for stimulation for 24 hours. Then, detection antibody was added and incubated at room temperature for 2 hours, and spots were counted after color development.

[0176] The specific experimental method is as follows: grind the mouse spleen on a clean bench to prepare a single cell suspension. Wash the cells once with 1 x PBS, centrifuge, and discard the supernatant; add red blood cell lysis buffer (purchased from Aibixin), mix by pipetting, lyse the red blood cells at room temperature, then add an appropriate amount of 1 x PBS to stop, filter the cells with a sieve, centrifuge, discard the supernatant to collect the cells; resuspend the cells in RPMI 1640 medium, count the cells, and adjust the concentration to 2x10 6cells / mL; rinse the ELISpot plate with sterile PBS, then block it with 1640 medium containing 10% FBS, 200 μL / well, place it in a cell culture incubator, and let it stand for more than 30 minutes; take out the ELISpot plate, discard the medium, add gE peptide library stimulation, 100 μL / well gE peptide library (final concentration 10 μg / mL), add 100 μL / well of cell suspension, and 2x10 5 cells, placed in a cell culture incubator for 24 hours; each sample was set up with a non-stimulation group and a stimulation group; after the stimulation, the ELISpot plate was taken out, the liquid was discarded, and it was washed 5 times with PBS, 200 μL / well; the primary antibody of the target factor was diluted with antibody diluent, 100 μL was added to each well, and incubated at room temperature for 2 hours; the primary antibody was discarded, and it was washed 5 times with PBS, 200 μL / well; the secondary antibody (streptavidin-ALP) was diluted with antibody diluent, 100 μL was added to each well, and incubated at room temperature for 1 hour; the secondary antibody was discarded, and it was washed 5 times with PBS, 200 μL / well; 100 μL of filtered color development solution BCIP / NBT-plus was added to each well, protected from light, and the color development state was observed in real time; the plate was rinsed under running water, dried, stored at room temperature and protected from light, and the plate was read using an ELISpot reader; the number of spots in the stimulated well of each sample was subtracted from the number of spots in the unstimulated well (background value), converted according to the number of cells, and the number of spots per million cells was used as the value for plotting.

[0177] The number of cells induced to secrete IFN-γ in G4-G7 groups under the stimulation of gE peptide library was higher than that in G3 group, and the number of cells that could induce higher levels of IL-2 ( Figure 8 ab).

[0178] 2. Flow cytometry was used to detect the levels of IFN-γ, IL-2, and TNF-α secreting antigen-specific T cells in spleen lymphocytes 14 days after the second immunization

[0179] After isolating the spleen immune cells of mice, the culture medium was used as a negative control and SEB as a positive control. After stimulating with the gE peptide library for 12 hours, a protein blocker (purchased from Aibixin) was added to block for 4 hours. Finally, the cells were collected and stained to detect antigen-specific CD4 + T and CD8 + Expression of IFN-γ, IL-2, and TNF-α in T cells.

[0180] The specific experimental method is as follows: grind the mouse spleen on a clean bench to prepare a single cell suspension. Wash the cells once with 1x PBS, centrifuge, and discard the supernatant; add red blood cell lysis buffer, pipette and mix, lyse the red blood cells at room temperature, then add an appropriate amount of 1x PBS to stop, filter the cells with a sieve, centrifuge, discard the supernatant to collect the cells; resuspend the cells in RPMI 1640 medium, count the cells, adjust the concentration, and then dispense into a 96-well plate, 3x10 per well. 6 cells, added gE protein peptide library (VZV (gE) Peptide Pool, purchased from Peptides / Elephants GmbH, LB01818) for stimulation, and added 1x Brefeldin A (BFA) to block cytokine secretion; each sample was designed with a stimulated group and an unstimulated group; after the stimulation, the cells were centrifuged, the supernatant was discarded to collect the cells, and they were washed once with 1xPBS, centrifuged again, and the supernatant was discarded; first, the live and dead dye antibody (purchased from Thermo Fisher) was stained for 5 minutes, and then the extracellular staining antibody mixture was added and stained for 20 minutes; after the cells were washed with 1xPBS, 200 μL / well of the fixation buffer was added, and the cells were fixed at room temperature in the dark for 20 minutes; after the cells were washed with 1xPBS, the cells were washed once with 1x permeabilization buffer prepared with triple distilled water; the intracellular staining antibody mixture was added and stained for 20 minutes; after the staining, the cells were washed once with 1xPBS, centrifuged, and the supernatant was discarded. Resuspend with an appropriate amount of 1xPBS and test on the machine; use Flowjo software for data analysis, subtract the value of each sample stimulation well from the value of the unstimulated well, and draw a graph.

[0181] FACS results showed that the IFN-γ-secreting antigen-specific CD4 + and CD8 + The proportion of T cells in the G5 group was higher than that in the G3 group. + T cells and G7 group induced IL-2 secreting antigen-specific CD8 + The proportion of T cells in the G3 group was higher than that in the G3 group, and the other groups were able to induce a considerable number of IL-2 and TNF-α secreting antigen-specific CD4 + and CD8 + T cells ( Fig. 9 af).

[0182] Example 6: Weight Change

[0183] The mice were observed once 24 hours before administration, 24 hours after administration, and 48 hours after administration, and once a week for the rest of the time. The weight of mice on day 0 was used as the initial weight to analyze the weight change trend of mice. The weight changes of mice in each group during the experiment are summarized in Fig.10After immunization on Day 0 and Day 28, the body weight of mice in each group decreased slightly and returned to normal weight within 7 days.

[0184] 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.

[0185] 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.

[0186] 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, wherein the coding region encodes a fusion protein comprising a first domain, a second domain and a third domain, wherein the first domain comprises at least a portion of the transmembrane domain of lysosomal associated membrane protein 1 (LAMP1), the second domain comprises a truncated varicella-zoster virus glycoprotein E (VZV gE protein), the truncated varicella-zoster virus glycoprotein E is a protein lacking the intramembrane region of the VZV gE protein, and the third domain comprises a sequence derived from lysosomal associated membrane protein 3 (LAMP3, also known as DC-LAMP).

2. The mRNA molecule according to claim 1, wherein the first domain comprises the amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having 1, 2 or 3 amino acids inserted, substituted or deleted compared to the amino acid sequence shown in SEQ ID NO:

8.

3. The mRNA molecule according to claim 1, wherein the second domain does not contain the signal peptide of the VZV gE protein itself, Preferably, the second domain comprises the amino acid sequence as shown in SEQ ID NO: 119, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity thereto.

4. The mRNA molecule according to claim 1, wherein the third domain comprises the amino acid sequence as shown in SEQ ID NO: 120, or an amino acid sequence having 1, 2 or 3 amino acids inserted, substituted or deleted compared to the amino acid sequence shown in SEQ ID NO:

120.

5. The mRNA molecule according to any one of claims 1 to 4, wherein the first domain, the second domain and / or the third domain are directly connected or connected through a linker.

6. The mRNA molecule according to any one of claims 1 to 4, wherein the fusion protein comprises the amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acid sequence as shown in SEQ ID NO:

3.

7. The mRNA molecule according to any one of claims 1 to 4, wherein the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 96 to 107, 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: 96 to 107, Preferably, the coding region comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 96 and 98, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any one of SEQ ID NOs: 96 and 98.

8. The mRNA molecule according to any one of claims 1 to 4, wherein the mRNA molecule comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 41 to 52, or comprises or consists of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs: 41 to 52, Preferably, the mRNA molecule comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs:41 and 43, 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:41 and 43.

9. The mRNA molecule according to any one of claims 1 to 4, wherein the mRNA molecule comprises a chemical modification, preferably a pseudouridine or 1-methyl-pseudouridine modification, and further preferably, each U in the mRNA molecule is a pseudouridine or 1-methyl-pseudouridine.

10. An mRNA composition comprising the mRNA molecule according to any one of claims 1-9.

11. The mRNA composition according to claim 10, wherein the composition is a vaccine, Preferably, the composition comprises liposomes, lipoplexes and / or lipid nanoparticles containing at least one mRNA, More preferably, the lipid nanoparticles comprise ionizable lipids, neutral lipids, cholesterol and PEG lipids. Further preferably, the lipid nanoparticles comprise 40-60 wt% ionizable lipids, 5-15 wt% neutral lipids, 25-50 wt% cholesterol and 0.5-3 wt% PEG lipids.

12. A nucleic acid molecule encoding the mRNA molecule according to any one of claims 1 to 9. A vector comprising the nucleic acid molecule according to claim 12 .

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

15. A method for inducing an immune response against varicella-zoster virus in a subject, the method comprising the step of administering to the subject an mRNA molecule according to any one of claims 1 to 9, or a nucleic acid molecule according to claim 12, or a vector according to claim 13, or a cell according to claim 14.

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