mRNA vaccine against herpes zoster and preparation method therefor

Abstract

The present invention relates to the field of nucleic acid vaccines, and in particular to an mRNA vaccine against the herpes zoster virus and a preparation method therefor. The main components of the vaccine of the present invention comprise mRNA encoding the varicella zoster virus glycoprotein gE or a variant thereof and a lipid nanoparticle. The glycoprotein gE variant mRNA comprises a coding region encoding a VZV glycoprotein E (gE protein) extracellular domain and transmembrane domain, or a coding region composed of the extracellular domain and the transmembrane domain, and does not encode an intracellular domain of the gE protein; or comprises mRNA encoding VZV gE protein having a mutation site.

Description

An mRNA vaccine against herpes zoster and its preparation method

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to a prior application, patent application number 202311503470.5, filed with the State Intellectual Property Office of China on November 13, 2023, entitled “An mRNA vaccine for resisting herpes zoster and method for its preparation.” The entire text of that prior application is incorporated herein by reference. Technical Field

[0003] The present application belongs to the field of biomedicine technology, and specifically relates to an mRNA vaccine for resisting herpes zoster and a preparation method thereof. Background Art

[0004] Herpes zoster (HZ) is an acute infectious skin disease caused by the reactivation of the latent varicella zoster virus (VZV). Initial VZV infection in childhood causes chickenpox. Following infection, the virus enters the sensory nerve endings of the skin and migrates centrally along the dorsal spinal roots or trigeminal ganglion nerve fibers, remaining in a persistent latent form within the sensory ganglia of the spinal or cranial nerves. When the body's immune system is weakened or exposed to nonspecific stimuli, the virus reactivates, multiplies, and causes inflammation or necrosis of the affected ganglia, resulting in neuralgia. Simultaneously, the reactivated virus spreads from one or several adjacent ganglia along the corresponding sensory nerve fibers to the skin, causing clusters of blisters on a unilaterally distributed erythematous base. The incidence of herpes zoster in the Asia-Pacific region is approximately 1%, and the incidence increases with age, reaching over 5% after the age of 40-50. Approximately 9% to 34% of patients with shingles experience postherpetic neuralgia, which can reach a pain rating of 7 or higher, considered severe and severely impacting patients' quality of life. There are no specific treatments for shingles and postherpetic neuralgia, so vaccination against shingles is an effective means of preventing shingles.

[0005] Globally available shingles vaccines include Zostavax (Merck), Shingrix (GSK), SkyZoster (SK), and Ganwei (Bai Ke Bio). Zostavax and SkyZoster are live attenuated vaccines, engineered to achieve structural changes and reduced toxicity while retaining immunogenicity, thereby establishing an immune response. Shingrix is ​​a recombinant subunit vaccine that uses the varicella-zoster glycoprotein E antigen and the AS01B adjuvant to generate and enhance the immune response in vivo. Clinical data show that Shingrix is ​​97% effective in individuals aged 50 to 69 years and remains as high as 91% in individuals aged 70 and above. Zostavax is 70% effective in individuals aged 50 to 69 years and 51% effective overall, making Shingrix more effective than Zostavax. A safer and more effective VZV vaccine remains a pressing need in the market.

[0006] Summary of the Invention

[0007] In order to meet the market's diverse needs for vaccines for varicella-zoster virus with multiple technical routes, the present application provides an immune composition (e.g., an mRNA vaccine) comprising RNA encoding a highly immunogenic antigen capable of inducing an effective neutralizing antibody response and cellular immunity against varicella-zoster virus. The present invention prepares a VZV mRNA vaccine based on the gE glycoprotein on the surface of varicella-zoster virus (VZV): mRNA encoding antigenic polypeptides or immunogenic fragments or variants thereof of the herpes zoster virus VZV is utilized, and lipid nanoparticles (LNP) technology is selected to encapsulate the mRNA and deliver it to immune cells in the body. LNP releases mRNA in the cell and translates it into immunogenic fragments or variants thereof of varicella-zoster virus (VZV), referred to as "VZV antigen peptides" or "antigenic peptides" in this application. The VZV antigen peptides expressed by immune cells further stimulate the body's humoral immunity, produce neutralizing antibodies against the VZV virus, and thus achieve a protective effect. mRNA vaccines can stimulate cellular immunity against viruses while stimulating humoral immunity. The activated T cells can kill virus-infected cells and produce memory cells to maintain continuous protective efficacy.

[0008] Specifically, the present application provides the following optional implementation scheme:

[0009] In a first aspect, the present application provides a nucleic acid molecule encoding a varicella-zoster virus (VZV) glycoprotein or a variant thereof, wherein the VZV glycoprotein is selected from the group consisting of: VZV gE, gI, gB, gH, gK, gL, gC, gN and gM.

[0010] In some embodiments, the nucleic acid molecule comprises a VZV RNA polynucleotide having an open reading frame encoding a VZV gE protein or a variant thereof that is a VZV antigenic peptide.

[0011] In some embodiments, the amino acid sequence of the VZV gE protein variant comprises one or more mutations selected from the following relative to SEQ ID NO.8, or the mutations relative to SEQ ID NO.8 are one or more mutations selected from the following: S593L, S595L, T596L, H362E, T598L, S593I, S595I, T596I, T598I, Del 560-623, wherein the positions of the amino acids are numbered with the amino acid sequence of SEQ ID NO.8 as the reference sequence, for example, S593L indicates that position 593 relative to the reference sequence SEQ ID NO: 8 is mutated from S to L.

[0012] In some embodiments, the amino acid sequence of the VZV gE protein variant comprises or is any one of the following mutations relative to SEQ ID NO. 8:

[0013] 1) S593L, S595L, T596L, and T598L;

[0014] 2) H362E, S593L, S595L, T596L, and T598L;

[0015] 3) H362E, S593I, S595I, T596I, and T598I;

[0016] 4) S593I, S595I, T596I, and T598I; and

[0017] 5) Del 560-623;

[0018] In addition, the positions of the amino acids are numbered with reference to the amino acid sequence of SEQ ID NO. 8.

[0019] In some embodiments, the VZV gE protein variant does not contain the signal peptide of the VZV gE protein, and the amino acid sequence of the portion thereof other than the signal peptide comprises or is any one of the following mutations relative to SEQ ID NO. 8:

[0020] 1) S593L, S595L, T596L, and T598L;

[0021] 2) H362E, S593L, S595L, T596L, and T598L;

[0022] 3) H362E, S593I, S595I, T596I, and T598I;

[0023] 4) S593I, S595I, T596I, and T598I; and

[0024] 5) Del 560-623;

[0025] In addition, the positions of the amino acids are numbered with reference to the amino acid sequence of SEQ ID NO. 8.

[0026] In some embodiments, the VZV gE protein variant comprises a signal peptide of the VZV gE protein, and the amino acid sequence of the portion thereof other than the signal peptide comprises or is any one of the following mutations relative to SEQ ID NO. 8:

[0027] 1) S593L, S595L, T596L, and T598L;

[0028] 2) H362E, S593L, S595L, T596L, and T598L;

[0029] 3) H362E, S593I, S595I, T596I, and T598I;

[0030] 4) S593I, S595I, T596I, and T598I; and

[0031] 5) Del 560-623;

[0032] Furthermore, the positions of the amino acids are numbered with reference to the amino acid sequence of SEQ ID NO. 8.

[0033] In some embodiments, the VZV gE protein variant comprises or consists of the extracellular and transmembrane regions of VZV glycoprotein E (may be referred to as gE protein or gE for short), and does not encode the intracellular region of the gE protein.

[0034] In some embodiments, the extracellular region comprises amino acids 31-538 of the varicella-zoster virus gE protein, and the transmembrane region comprises amino acids 539-559 of the varicella-zoster virus gE protein, and the positions of the amino acids are numbered with reference to the amino acid sequence of SEQ ID NO.8.

[0035] In some embodiments, the VZV gE protein variant comprises the extracellular region and transmembrane region of the VZV gE protein, but does not comprise the intracellular region, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, and the transmembrane region comprises amino acids 539-559 of the VZV gE protein, and the amino acid sequence of the gE protein is shown in SEQ ID NO.8.

[0036] In some specific embodiments, the antigenic peptide further comprises a signal peptide sequence, and the signal peptide sequence is located at the N-terminus of the extracellular region amino acid sequence.

[0037] In some embodiments, the signal peptide is the signal peptide of the VZV gE protein.

[0038] In some embodiments, the signal peptide comprises amino acids 1-30 or 2-30 of SEQ ID NO.8.

[0039] In some embodiments, the signal peptide is not the native signal peptide of the VZV gE protein.

[0040] In some embodiments, the signal peptide is a signal peptide derived from other viruses that can infect mammalian cells or a signal peptide derived from other mammalian proteins.

[0041] In some embodiments, the signal peptide is a signal peptide derived from an envelope protein of another enveloped virus.

[0042] In some embodiments, the VZV gE protein variant comprises the extracellular region and the transmembrane region of the VZV gE protein, but does not comprise the intracellular region, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, the transmembrane region comprises amino acids 539-559 of the VZV gE protein, and the intracellular region comprises amino acids 560-623 of the VZV gE protein, the amino acid sequence of the gE protein is shown in SEQ ID NO.8, and the VZV gE protein variant further comprises a signal peptide, which is not the natural signal peptide of the VZV gE protein.

[0043] In some embodiments, the VZV gE protein variant comprises the extracellular region and the transmembrane region of the VZV gE protein, but does not comprise the intracellular region, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, the transmembrane region comprises amino acids 539-559 of the VZV gE protein, and the intracellular region comprises amino acids 560-623 of the VZV gE protein. The amino acid sequence of the gE protein is shown in SEQ ID NO.8, and the VZV gE protein variant further comprises a signal peptide, which is not the natural signal peptide of the VZV gE protein, but is a signal peptide derived from other viruses that can infect mammalian cells or a signal peptide derived from other mammalian proteins.

[0044] In some embodiments, the VZV gE protein variant comprises the extracellular region of the VZV gE protein, the transmembrane region of the VZV gE protein, and the signal peptide of the VZV gE protein, but does not comprise the intracellular region of the VZV gE protein, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, the transmembrane region comprises amino acids 539-559 of the VZV gE protein, the signal peptide comprises amino acids 1-30 or 2-30 of the VZV gE protein, and the intracellular region comprises amino acids 560-623 of the VZV gE protein, and the extracellular region, the transmembrane region, and the signal peptide are each independently connected by 0, 1 or more amino acid residues, and the positions of the amino acids are numbered using the amino acid sequence of SEQ ID NO.8 as a reference sequence.

[0045] In some embodiments, the VZV gE protein variant comprises the extracellular region of the VZV gE protein, the transmembrane region of the VZV gE protein, and the signal peptide of the VZV gE protein, but does not comprise the intracellular region of the VZV gE protein, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, the transmembrane region comprises amino acids 539-559 of the VZV gE protein, the signal peptide comprises amino acids 1-30 or 2-30 of the VZV gE protein, and the intracellular region comprises amino acids 560-623 of the VZV gE protein. From N-terminus to C-terminus, the extracellular region, the transmembrane region, and the signal peptide are each independently connected by 0, 1 or more amino acid residues, and the positions of the amino acids are numbered using the amino acid sequence of SEQ ID NO.8 as a reference sequence.

[0046] In some embodiments, the VZV gE protein variant comprises the extracellular region of the VZV gE protein, the transmembrane region of the VZV gE protein, and the signal peptide of the VZV gE protein, but does not comprise the intracellular region of the VZV gE protein, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, the transmembrane region comprises amino acids 539-559 of the VZV gE protein, the signal peptide comprises amino acids 1-30 or 2-30 of the VZV gE protein, and the intracellular region comprises amino acids 560-623 of the VZV gE protein, the extracellular region, the transmembrane region, and the signal peptide are each independently connected by 0, 1 or more amino acid residues, and the amino acid sequence of the VZV gE protein is shown in SEQ ID NO.8.

[0047] In some embodiments, the VZV gE protein variant comprises the extracellular region of the VZV gE protein, the transmembrane region of the VZV gE protein, and the signal peptide of the VZV gE protein, but does not comprise the intracellular region of the VZV gE protein, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, the transmembrane region comprises amino acids 539-559 of the VZV gE protein, the signal peptide comprises amino acids 1-30 or 2-30 of the VZV gE protein, and the intracellular region comprises amino acids 560-623 of the VZV gE protein, from N-terminus to C-terminus, the extracellular region, the transmembrane region, and the signal peptide are each independently connected by 0, 1 or more amino acid residues, and the amino acid sequence of the VZV gE protein is shown in SEQ ID NO.8.

[0048] In some embodiments, the VZV gE protein variant comprises the extracellular region, transmembrane region, and intracellular region of the VZV gE protein, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, the transmembrane region comprises amino acids 539-559 of the VZV gE protein, the intracellular region comprises amino acids 560-623 of the VZV gE protein, the amino acid sequence of the gE protein is shown in SEQ ID NO.8, and the VZV gE protein variant further comprises a signal peptide, which is not the native signal peptide of the VZV gE protein.

[0049] In some embodiments, the VZV gE protein variant comprises the extracellular region, transmembrane region, and intracellular region of the VZV gE protein, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, the transmembrane region comprises amino acids 539-559 of the VZV gE protein, and the intracellular region comprises amino acids 560-623 of the VZV gE protein, and the amino acid sequence of the gE protein is shown in any one of SEQ ID NOs. 14-17, and the VZV gE protein variant further comprises a signal peptide, which is not the natural signal peptide of the VZV gE protein, but is a signal peptide derived from other viruses that can infect mammalian cells or a signal peptide derived from other mammalian proteins.

[0050] In some embodiments, the VZV gE protein variant comprises the extracellular region of the VZV gE protein, the transmembrane region of the VZV gE protein, the intracellular region of the VZV gE protein, and the signal peptide of the VZV gE protein, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, the transmembrane region comprises amino acids 539-559 of the VZV gE protein, the signal peptide comprises amino acids 1-30 or 2-30 of the VZV gE protein, and the intracellular region comprises amino acids 560-623 of the VZV gE protein, and the extracellular region, transmembrane region, intracellular region and signal peptide are each independently connected by 0, 1 or more amino acid residues, and the positions of the amino acids are numbered using the amino acid sequence of SEQ ID NO.8 as a reference sequence.

[0051] In some embodiments, the VZV gE protein variant comprises the extracellular region of the VZV gE protein, the transmembrane region of the VZV gE protein, the intracellular region of the VZV gE protein, and the signal peptide of the VZV gE protein, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, the transmembrane region comprises amino acids 539-559 of the VZV gE protein, the signal peptide comprises amino acids 1-30 or 2-30 of the VZV gE protein, and the intracellular region comprises amino acids 560-623 of the VZV gE protein, from N-terminus to C-terminus, and the extracellular region, transmembrane region, intracellular region and signal peptide are each independently connected by 0, 1 or more amino acid residues, and the positions of the amino acids are numbered using the amino acid sequence of SEQ ID NO.8 as a reference sequence.

[0052] In some embodiments, the VZV gE protein variant comprises the extracellular region of the VZV gE protein, the transmembrane region of the VZV gE protein, the intracellular region of the VZV gE protein, and the signal peptide of the VZV gE protein, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, the transmembrane region comprises amino acids 539-559 of the VZV gE protein, the signal peptide comprises amino acids 1-30 or 2-30 of the VZV gE protein, and the intracellular region comprises amino acids 560-623 of the VZV gE protein, the extracellular region, the transmembrane region, the intracellular region, and the signal peptide are each independently connected by 0, 1 or more amino acid residues, and the amino acid sequence of the VZV gE protein is shown in any one of SEQ ID NOs. 14-17.

[0053] In some embodiments, the VZV gE protein variant comprises an extracellular region of a VZV gE protein, a transmembrane region of a VZV gE protein, an intracellular region of a VZV gE protein, and a signal peptide of a VZV gE protein, wherein the extracellular region comprises amino acids 31-538 of the VZV gE protein, the transmembrane region comprises amino acids 539-559 of the VZV gE protein, the signal peptide comprises amino acids 1-30 or 2-30 of the VZV gE protein, and the intracellular region comprises amino acids 560-623 of the VZV gE protein, from N-terminus to C-terminus, the extracellular region, the transmembrane region, the intracellular region, and the signal peptide are each independently connected by 0, 1 or more amino acid residues, and the amino acid sequence of the VZV gE protein is shown in any one of SEQ ID NOs. 14-17.

[0054] In some embodiments, the gE protein or its variant comprises amino acids that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs. 13-17, or the amino acid sequence of the antigen is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 13-17.

[0055] In some embodiments, the amino acid sequence of the gE protein or its variant is shown in any one of SEQ ID NO. 13-17.

[0056] In some embodiments, the amino acid sequence of the gE protein or its variant is shown in SEQ ID NO.13.

[0057] In some embodiments, the sequence encoding the aforementioned antigenic peptide in the nucleic acid molecule (coding sequence) is codon-optimized to increase the GC content.

[0058] In some embodiments, the sequence encoding the aforementioned antigenic peptide in the nucleic acid molecule (coding sequence) is codon-optimized so that the codons therein are high-frequency codons in mammalian cells.

[0059] In some embodiments, the sequence encoding the aforementioned antigenic peptide in the nucleic acid molecule (coding sequence) is codon-optimized so that the codons therein are high-frequency codons in human cells.

[0060] In some embodiments, the sequence encoding the aforementioned antigenic peptide in the nucleic acid molecule (coding sequence) is codon-optimized so that the codons therein are high-frequency codons in human neural cells.

[0061] In some embodiments, the sequence encoding the aforementioned antigenic peptide in the nucleic acid molecule (coding sequence) is codon-optimized so that the codons therein are high-frequency codons in human immune cells.

[0062] In some embodiments, the aforementioned nucleic acid molecule is DNA.

[0063] In some embodiments, the aforementioned nucleic acid molecule is RNA.

[0064] In some embodiments, the nucleic acid molecule is mRNA, self-replicating RNA, circular RNA, or replicon RNA, preferably mRNA.

[0065] In some embodiments, the mRNA molecule comprises at least the following structure:

[0066] (a) 5'-cap structure;

[0067] (b) a 5'UTR of 10-200 nucleotides, preferably 15-150 nucleotides in length;

[0068] (c) 3' polyadenylation sequence;

[0069] (d) 3'UTR.

[0070] In some embodiments, the aforementioned mRNA molecule comprises a 5'-cap structure, wherein the 5'-cap structure is m7G, cap0, cap1, cap2, modified cap0 or modified cap1 structure, and the 5'-cap structure is preferably m7G, cap0, cap1, cap2, modified cap0 or modified cap1 structure, which is selected from any one of the following: m7Gppp(2'OMeA)pG, m7GpppApA, m7Gp ppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GpppCpG, m7GpppCpU, m7GpppGpA, m7Gp ppGpC, m7GpppGpG, m7GpppGpU, m7GpppUpA, m7GpppUpC, m7GpppUpG, m7GpppUpU, m7Gpppm6ApG, m7G 3’Ome pppApA、m7G 3’Ome pppApC、m7G 3’Ome pppApU、m7G 3’Ome pppApG、m7G 3’Ome pppCpA、m7G 3’Ome pppCpC、m7G 3’Ome pppCpG、m7G 3’Ome pppCpU、m7G 3’Ome pppUpA、m7G 3’Ome pppUpC、m7G 3’Ome pppUpG、m7G 3’Ome pppUpU、 m7G 3’Ome PppA 2’Ome pG、m7G 3’Ome pppA 2’Ome pC、m7G 3’Ome pppA 2’Ome pU、m7G 3’Ome pppA 2’Ome pA、m7G 3’Ome ppC 2’Ome pA、m7G 3’Ome ppC 2’Ome pU、m7G 3’Ome ppC 2’Ome pG、m7G 3’Ome ppC 2’Ome pC、m7G 3’Ome pppG 2’Ome pA、m7G 3’Ome pppG 2’Ome pU、m7G 3’Ome pppG 2’Ome pG、m7G3’Ome pppG 2’Ome pC、m7G 3’Ome pppU 2’Ome pA、m7G 3’Ome pppU 2’Ome pU、m7G 3’Ome pppU 2’Ome pG、m7G 3’Ome pppU 2’Ome pC; most preferably m7G(5')ppp(5')(2'OMeA)pG.

[0071] In some embodiments, the mRNA molecule comprises a 5'UTR or a coding sequence for a 5'UTR selected from Xenopus or human α-globin or β-globin, human cytochrome b-245a polypeptide, hydroxysteroid (17b) dehydrogenase, and tobacco etch virus, alpha-1-globin, Kozak sequence, HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or a homolog, fragment, or variant from any of these genes;

[0072] Preferably, the 5'UTR comprises or is a tobacco etch virus (TEV) 5'UTR; further preferably, the nucleotide sequence of the TEV 5'UTR comprises SEQ ID NO. 18 or has at least 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with the nucleotide sequence as shown in SEQ ID NO: 18;

[0073] or,

[0074] wherein the nucleotide sequence of the 5'UTR comprises a Kozak sequence as shown in SEQ ID NO: 9 or has at least 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity to the nucleotide sequence as shown in SEQ ID NO: 9;

[0075] or,

[0076] The nucleotide sequence of the 5'UTR comprises the nucleotide sequence of pVAX.1+TEV as shown in SEQ ID NO.10 or has at least 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with the nucleotide sequence as shown in SEQ ID NO:10.

[0077] In some embodiments, the mRNA molecule comprises a 3'UTR or a coding sequence for a 3'UTR, and the 3'UTR can also be selected from the group consisting of PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS, NDUFA1, DH143, gp130, hHBB, hHBA1, CYBA (cytochrome b-245alpha chain), rabbit beta-globin, hepatitis B virus (HBV), VEEV (Venezuelan equine encephalitis virus) virus, rps9 (Ribosomal Protein S9), FIG4 (FIG4 Phosphoinositide 5-Phosphatase), human albumin hHBB (human hemoglobin subunit beta), HBA1 (human Hemoglobin Subunit Alpha 1) 3'UTR, or a homolog, fragment, or variant from any of these genes;

[0078] Preferably, the 3'UTR comprises or is hemoglobin-1 (hHBA1) 3'UTR; further preferably, the nucleotide sequence of the hHBA1 3'UTR comprises the nucleotide sequence shown in SEQ ID NO.12 or has at least 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with the nucleotide sequence shown in SEQ ID NO:12.

[0079] In some embodiments, the mRNA molecule comprises a 3'-poly(A) or a coding sequence for a 3'-poly(A), wherein the 3'-poly(A) is 90-120 nt in length and contains non-A bases; preferably, the nucleotide sequence of the 3'-poly(A) comprises the nucleotide sequence shown in SEQ ID NO.11 or has at least 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with the nucleotide sequence shown in SEQ ID NO:11.

[0080] In some embodiments, some or all of the uridine (U) in the mRNA molecule are base-modified uridine or uridine analogs; preferably, the chemically modified uridine is selected from any one or more of 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, 1-methyl pseudouridine (N1-Methyl-Pseudo-UTP) and pseudouridine; more preferably, each U in the sequence is 1-methyl pseudouridine.

[0081] In some embodiments, the mRNA molecule comprises or consists of a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs. 1-7.

[0082] In some embodiments, the mRNA molecule comprises or consists of a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs. 2-7.

[0083] In some embodiments, the mRNA molecule comprises or consists of a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO.3.

[0084] In some embodiments, the mRNA molecule is formed by capping a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs. 1-7.

[0085] In some embodiments, the mRNA molecule is formed by capping a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs. 2-7.

[0086] In some embodiments, the mRNA molecule is formed by capping a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleotide sequence of SEQ ID NO. 3.

[0087] In a second aspect, the present application provides a composition, preferably an immunogenic composition comprising at least one nucleic acid molecule of the first aspect. Suitably, the composition may comprise at least one nucleic acid molecule, such as at least one encoding RNA, complexed with one or more lipids, encapsulated in one or more lipids, or associated with one or more lipids to form lipid nanoparticles.

[0088] In some embodiments, the composition relates to a nucleic acid vaccine against varicella-zoster virus VZV, characterized in that the vaccine carrier is a lipid nanoparticle (LNP) comprising ionizable cationic lipids, structural lipids, auxiliary lipids and surfactants.

[0089] In some embodiments, the molar content of the ionizable cationic lipid, structural lipid, helper lipid, and surfactant totals 100% on a molar percentage (mol %) basis.

[0090] In some embodiments, the lipid nanoparticles comprise 20-60 mol% ionizable cationic lipid, 25-55 mol% structural lipid, 5-25 mol% helper lipid, and 0.5-15 mol% surfactant.

[0091] In some embodiments, the cationic lipid is selected from SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200, DlinDMA, preferably SM-102; the structure of SM-102 is as follows:

[0092] In some embodiments, the structured lipid is selected from cholesterol, and cholesterol derivatives, preferably cholesterol.

[0093] In some embodiments, the helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DSPC.

[0094] In some embodiments, the surfactant is selected from PEG2000-DMG, PEG-DSPE, DTDA-PEG2000, TPGS, preferably PEG2000-DMG.

[0095] In some embodiments, the lipid nanoparticles comprise 20-50 mol% ionizable cationic lipids. For example, the lipid nanoparticles may comprise 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mol% ionizable cationic lipids.

[0096] In other embodiments, the lipid nanoparticles comprise 50-60 mol% ionizable cationic lipids. For example, the lipid nanoparticles may comprise 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 mol% ionizable cationic lipids.

[0097] In some embodiments, the lipid nanoparticle comprises 5-25 mol% DSPC, preferably 2-15 mol% DSPC; for example, the lipid nanoparticle may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mol% DSPC.

[0098] In some embodiments, the lipid nanoparticles comprise 25-55 mol% cholesterol, preferably 30-40 mol% cholesterol. For example, the lipid nanoparticles may comprise 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 mol% cholesterol.

[0099] In some embodiments, the lipid nanoparticles comprise 0.5-15 mol% DMG-PEG, preferably 1-2 mol% DMG-PEG. For example, the lipid nanoparticles may comprise 1, 1.5 or 2 mol% DMG-PEG.

[0100] In some embodiments, the lipid nanoparticles comprise 50 mol% ionizable cationic lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.

[0101] In some embodiments, the lipid nanoparticles comprise 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.

[0102] In some embodiments, the lipid nanoparticles of the present application comprise an N:P ratio of about 2:1 to about 30:1.

[0103] In some embodiments, the lipid nanoparticles of the present application comprise an N:P ratio of about 6:1.

[0104] In some embodiments, the lipid nanoparticles of the present application comprise an N:P ratio of about 3:1.

[0105] In some embodiments, the lipid nanoparticles of the present application comprise a wt / wt ratio of the ionizable cationic lipid component to the RNA of about 10:1 to about 100:1.

[0106] In some embodiments, the lipid nanoparticles of the present application comprise a wt / wt ratio of the ionizable cationic lipid component to the RNA of about 20:1.

[0107] In some embodiments, the lipid nanoparticles of the present application comprise a wt / wt ratio of an ionizable cationic lipid component to RNA of about 10: 1. In some embodiments, the lipid nanoparticle compositions of the present application have an average diameter of from about 50 nm to about 150 nm.

[0108] In some embodiments, the lipid nanoparticles of the present application have an average diameter of about 70 nm to about 120 nm, preferably 100-120 nm, and most preferably 100 nm.

[0109] In some embodiments, the mRNA solution is diluted in water for injection.

[0110] In some embodiments, the mass ratio of the lipid nanoparticles to the mRNA is between 1: 1 and 30: 1. The lipid carrier is preferably an LNP composition, and the mass ratio is preferably 20:1.

[0111] In some embodiments, the varicella-zoster virus nucleic acid vaccine of the present application further comprises: a buffer component and a cryoprotectant.

[0112] In some embodiments, the buffer can be selected from: Examples of buffering agents include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium gluconate, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, dibasic calcium phosphate, phosphoric acid, tricalcium phosphate, dibasic calcium phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, dibasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, magnesium hydroxide, aluminum hydroxide, alginic acid, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, lauryl magnesium sulfate, sodium lauryl sulfate, and combinations thereof.

[0113] In some embodiments, the cryoprotectant can be selected from substances such as sugars / polyols, polymers, surfactants, amino acids and salts, wherein the sugar can be selected from lactose, sucrose, trehalose, galactose and the like.

[0114] In some embodiments, the amount of the cryoprotectant is 1 to 50% w / w, such as from 2 to 50% w / w, or from 4 to 45% w / w, or from 6 to 12% w / w, or preferably from 6 to 10% w / w, or most preferably from 7 to 9% w / w.

[0115] In some embodiments, the pharmaceutical composition of the present application includes the aforementioned lipid nanoparticles and an external phase buffer.

[0116] In some embodiments, the aqueous buffer comprises: tromethamine, sodium acetate, sucrose, pH=7-8.

[0117] In some embodiments, the content of tromethamine is selected from 10-30mmol / L, preferably 15-25mmol / L, preferably 15mmol / L, 15.5mmol / L, 16mmol / L, 16.5mmol / L, 17mmol / L, 17.5mmol / L, 18mmol / L, 18.5mmol / L, 19mmol / L, 19.5mmol / L, 20mmol / L, 20.5mmol / L, 21mmol / L, 21.5mmol / L, 22mmol / L, 22.5mmol / L, 23mmol / L, 23.5mmol / L, 24mmol / L, 24.5mmol / L, 25mmol / L, and most preferably 20mmol / L.

[0118] In some embodiments, the content of sodium acetate is selected from 0-20mmol / L, preferably 5-11mmol / L, preferably 5mmol / L, 5.5mmol / L, 6mmol / L, 6.5mmol / L, 7mmol / L, 7.5mmol / L, 8mmol / L, 8.5mmol / L, 9mmol / L, 9.5mmol / L, 10mmol / L, 10.5mmol / L, 10.6mmol / L, 10.7mmol / L, 10.8mmol / L, 10.9mmol / L, 11mmol / L, 11.5mmol / L, 12mmol / L, 12.5mmol / L, 13mmol / L, and most preferably 10.7mmol / L.

[0119] In some embodiments, the sucrose content is selected from: 5-15%, preferably 7.5-10%, more preferably 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 9%, 9.5%, 10%, and most preferably 8.7%.

[0120] In some embodiments, the pharmaceutical composition of the present application includes mRNA having a sequence as shown in SEQ ID NO.1-7, lipid nanoparticles, 20 mmol / L tromethamine, 10.7 mmol / L sodium acetate, 8.7% sucrose, pH 7.0-8.0, and the lipid nanoparticles contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% PEG2000-DMG, N:P=6.

[0121] In a third aspect, the present application provides varicella-zoster virus antigen peptides.

[0122] In some embodiments, the antigenic peptide is encoded by the nucleic acid molecule of the first aspect.

[0123] In some embodiments, the amino acid sequence of the antigenic peptide is identical to the amino acid sequence encoded by the nucleic acid molecule of the first aspect.

[0124] In some embodiments, the antigenic peptide comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs. 13-17.

[0125] In a fourth aspect, the present application provides a varicella-zoster virus vaccine, wherein the vaccine comprises at least one nucleic acid molecule of the first aspect, or at least one composition of the second aspect, or at least one antigenic peptide of the third aspect.

[0126] In some embodiments, the vaccine is a varicella-zoster virus VZV RNA (e.g., mRNA) vaccine, which contains a VZV gE antigenic peptide coding sequence, wherein the antigenic peptide comprises, from N-terminus to C-terminus, a VZV gE protein signal peptide sequence, a VZV gE protein extracellular region sequence, and a VZV gE protein transmembrane region sequence.

[0127] In some embodiments, the VZV RNA (eg, mRNA) vaccine contains at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a VZV gE antigenic peptide.

[0128] In some embodiments, the vaccine is administered by intravenous injection, intramuscular injection or subcutaneous injection, preferably intramuscular injection.

[0129] In some embodiments, the dosage form of the vaccine can be selected from lyophilized powder injection, liquid injection, and inhalation preparation.

[0130] In a fifth aspect, the present application provides a kit or a kit of parts comprising at least one nucleic acid molecule described in the first aspect, and / or at least one composition described in the second aspect, and / or at least one antigenic peptide described in the third aspect, and / or at least one vaccine described in the fourth aspect.

[0131] In the sixth aspect, the present application provides a composition comprising at least two separated components, wherein the at least two separated components are selected from the two nucleic acid molecules described in the first aspect, and / or the two compositions described in the second aspect, and / or the two antigenic peptides described in the third aspect, and / or the at least two vaccines described in the fourth aspect.

[0132] In the seventh aspect, the present application provides a method for treating or preventing varicella-zoster virus infection in a subject, comprising administering to the subject at least one nucleic acid molecule described in the first aspect, and / or at least one composition described in the second aspect, and / or at least one antigenic peptide described in the third aspect, and / or at least one vaccine described in the fourth aspect, which can effectively induce a neutralizing antibody response against varicella-zoster virus (VZV) in the subject.

[0133] In the eighth aspect, the present application provides the use of the nucleic acid molecule of the first aspect, or the composition of the second aspect, or the antigenic peptide or protein of the third aspect, or the vaccine of the fourth aspect in the preparation of a medicament for treating or preventing varicella-zoster virus infection.

[0134] In a ninth aspect, the present application relates to a method for preparing a varicella-zoster virus vaccine, characterized in that a vaccine vector and the mRNA described in the first aspect are mixed to obtain a varicella-zoster virus vaccine.

[0135] In some embodiments, the vaccine carrier is a cationic lipid nanoparticle, and the preparation method specifically comprises the following steps:

[0136] (1) dissolving an ionizable cationic lipid, a structural lipid, an auxiliary lipid, and a surfactant in an organic solution according to a formula ratio to obtain an organic phase;

[0137] (2) dissolving the mRNA in citrate buffer or sodium acetate solution to obtain an aqueous phase;

[0138] (3) The organic phase of step (1) and the aqueous phase of step (2) are mixed to generate a mixed solution to obtain a varicella-zoster virus vaccine.

[0139] In some embodiments, the organic solution comprises anhydrous ethanol.

[0140] In some embodiments, the total concentration of ionizable cationic lipids, structural lipids, helper lipids, and surfactants in the organic phase is 10-15 mg / ml.

[0141] In some embodiments, the concentration of the mRNA is 0.01-1 mg / ml, preferably 0.1-0.2 mg / ml.

[0142] In some embodiments, the volume ratio of the organic phase to the aqueous phase is 1:2-4.

[0143] In some embodiments, the mixing is performed using a microfluidic device with a flow rate controlled to be ≥12 ml / min.

[0144] In the tenth aspect, the present application also relates to the use of the nucleic acid molecule described in the first aspect, the composition described in the second aspect, or the antigenic peptide described in the third aspect in the preparation of a vaccine.

[0145] In some embodiments, the vaccines include combination vaccines and multivalent vaccines.

[0146] In the eleventh aspect, the present application also designs a combination vaccine, comprising a first vaccine and a second vaccine used sequentially, wherein the first vaccine is selected from the nucleic acid molecule described in the first aspect or the composition described in the second aspect, or the antigen peptide described in the third aspect, or the vaccine described in the fourth aspect.

[0147] In some embodiments, in the combination vaccine, the second vaccine is selected from: attenuated or inactivated vaccines, adenovirus vaccines, mRNA vaccines, DNA vaccines, and recombinant protein vaccines.

[0148] In some embodiments, the second vaccine is selected from the group consisting of: Zostavax, Shingrix, and NBP608.

[0149] In some embodiments, the mRNA of the first vaccine and the second vaccine are selected from the group consisting of the nucleotide sequences shown in any two of SEQ ID NOs: 1-7.

[0150] In some embodiments, the vaccine described in the present application is suitable for sequential vaccination with one or more vaccines selected from the following groups, and the vaccine can be based on vaccines of any technical route, including but not limited to attenuated or inactivated vaccines, adenovirus vaccines, mRNA vaccines, DNA vaccines, recombinant protein vaccines, etc.

[0151] In some embodiments, in one relatively complete vaccination, the number of vaccinations of the one or more vaccines required to complete immunization can be 1 time, 2 times, 3 times or 4 times, and the interval between each vaccination can be 0 days, 7 days, 21 days, 28 days, 35 days, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0152] In a twelfth aspect, the present application also relates to a method for inducing an antigen-specific immune response in a subject, comprising administering to the subject the VZV vaccine according to the fourth aspect in an amount effective to produce an antigen-specific immune response.

[0153] In some embodiments, the antigen-specific immune response comprises a T cell response.

[0154] In some embodiments, the antigen-specific immune response comprises a B cell response.

[0155] In some embodiments, wherein the subject is about 5 years of age or younger, wherein the subject is between about 1 year of age and about 5 years of age, wherein the subject is between about 6 months of age and about 1 year of age, wherein the subject is about 6 months of age or younger, or wherein the subject is about 12 months of age or younger.

[0156] In some embodiments, the subject is an elderly subject who is about 60 years old, about 70 years old, or older (e.g., about 60, 65, 70, 75, 80, 85, or 90 years old).

[0157] In some embodiments, the subject is a young adult between the ages of about 20 and about 50 years old (e.g., about 20, 25, 30, 35, 40, 45, or 50 years old).

[0158] In some embodiments, the subject is a full-term or premature infant.

[0159] In some embodiments, the subject is a pregnant woman.

[0160] In some embodiments, the subject is exposed to, infected with, or at risk of being infected with VZV.

[0161] In some embodiments, the subject is immunocompromised (has a compromised immune system, eg, suffers from an immune disorder or an autoimmune disorder).

[0162] Beneficial effects of the present invention:

[0163] The present invention is based on an antigenic peptide derived from VZV glycoprotein E, and an mRNA sequence is designed and optimized. Compared with the original coding sequence of the VZV gE protein, the GC content of the optimized sequence is improved. It is cloned into the vector pVAX.1, mRNA is synthesized in vitro, and COS7 cells are transfected. The relative expression level of the antigenic peptide is significantly increased relative to VZV-WT. For example, the protein expression level of the mRNA encoding VZV-trunc (SEQ ID NO.3) is 6.5 times the protein expression level of VZV-WT mRNA (i.e., the original mRNA of the VZV gE protein). Mice immunized with the VZV mRNA vaccine of the present application can produce high-titer binding antibodies, which is higher than GSK's Shingrix. Mice immunized with the VZV mRNA vaccine of the present application can produce high-titer specific protective antibodies, which is higher than Shingrix. Mice immunized with the VZV mRNA vaccine of the present application can produce high-titer neutralizing antibodies, which is higher than Shingrix. Mice immunized with the VZV mRNA vaccine of the present application produce strong specific T cell immunity in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0164] Figure 1: Bioanalyzer analysis of the purity and size of VZV gE protein mRNA.

[0165] Figure 2: Results of LNP formulation optimization experiment.

[0166] Figure 3: Transmission electron microscopy shows the morphology of the VZV gE protein mRNA and lipid nanoparticle (LNP) complex.

[0167] Figure 4: Immunoblotting results of the expression of VZV gE protein in host cells.

[0168] Figure 5: Experimental results of the expression of target antigen protein in cells detected by FACS flow cytometry.

[0169] Figure 6: Results of the serum binding antibody detection experiment in mice after secondary immunization with different VZV mRNA vaccines (3 μg) and different doses of Shingrix vaccine.

[0170] Figure 7: Results of the specific protective antibody detection experiment (FAMA method) in mouse serum after secondary immunization with different VZV mRNA vaccines (3 μg) and different doses of Shingrix vaccine.

[0171] Figure 8: Experimental results of neutralizing antibody detection in mouse serum after secondary immunization with different VZV mRNA vaccines (3 μg) and Shingrix vaccine.

[0172] Figure 9: Experimental results of specific T cell detection in mice after secondary immunization with different VZV mRNA vaccines (3 μg) and different doses of Shingrix vaccine. DETAILED DESCRIPTION

[0173] definition:

[0174] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0175] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" should be considered to include any and all sub-ranges between a minimum of 1 and a maximum of 10 (including endpoints); that is, all sub-ranges starting with a minimum of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0176] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. It will also be understood that the skilled artisan can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleic acid molecule to reflect the codon usage of any particular host organism in which the polypeptide is expressed. Therefore, unless otherwise indicated, "polynucleotides encoding the protein or immunogenic fragment of the present application" include all polynucleotide sequences that are degenerate to each other and encode the same amino acid sequence.

[0177] As used herein, the term "reference sequence" is a standard sequence used for homologous sequence alignment, which can be used to define the sequence of amino acid positions in homologous protein or polypeptide sequences. For example, the amino acids in the target sequence are "numbered as the reference sequence" using the amino acid sequence of the reference sequence. This means that after the reference sequence and the target sequence have the same amino acid residues at as many positions as possible by introducing gaps or deleting amino acids into the reference sequence, the reference sequence is numbered consecutively in sequence order starting from the first nucleotide at the 5' end, and the amino acid positions in the target sequence that correspond to the reference sequence by comparison are defined with the same number. In this application, unless otherwise specified, the "reference sequence" is SEQ ID NO.8. It should be understood that "the extracellular region comprises amino acids 31-538 of the gE protein" means that the extracellular region comprises the amino acid sequence consisting of all amino acids between positions 31 and 538 of the gE protein numbered according to the reference sequence. The length of the sequence may be longer or shorter than positions 31-538 (a total of 508mers) of the reference sequence due to the addition or deletion of amino acids. "Amino acids 539-559 of the gE protein" should also be interpreted in the same manner.

[0178] The term "signal peptide" refers to a short peptide chain that directs the localization or translocation of newly synthesized proteins. Under natural conditions, it is typically located at the N-terminus of the protein precursor, guiding the ribosome to the endoplasmic reticulum, thereby allowing the continuously synthesized polypeptide to pass through the endoplasmic reticulum. Signal peptides typically consist of a positively charged N-region, a hydrophobic H-region, and a neutral polar C-region. The C-region contains a slightly conserved enzyme cleavage site. After the signal peptide completes protein localization, it is typically recognized and removed by a signal peptidase on the membrane. In mRNA, the coding sequence for the signal peptide is typically located after the start codon of the protein it encodes.

[0179] In the present application, "N-terminal side" is used to describe the relative position relationship between two sequences, an amino acid and a sequence, or two amino acids in the same amino acid sequence. Among them, "N-terminal" refers to the end of the amino acid sequence that contains a free amino group. For example, "the signal peptide sequence is located on the N-terminal side of the extracellular region" means that the "signal peptide sequence" is closer to the N-terminus of the amino acid sequence in which it is located relative to the "extracellular region". Similarly, "C-terminal side" is also used to describe the relative position relationship between two sequences, an amino acid and a sequence, or two amino acids in the same amino acid sequence. Among them, "C-terminal" refers to the end of the amino acid sequence that contains a free carboxyl group. The sequence or amino acid located on the N-terminal side or C-terminal side of a sequence or an amino acid can be directly connected to the sequence or amino acid, or separated by one or more amino acid residues.

[0180] As used herein, "coding sequence" may refer to a ribonucleotide sequence in a mature mRNA that can be translated into a protein, or may refer to the complementary sequence of a deoxyribonucleotide (DNA) sequence that serves as a template for transcribing the ribonucleotide (RNA) sequence. Furthermore, the "coding sequence" of the present application may further include sequences encoding functional nucleic acids or elements, such as polynucleotide sequences in DNA that can be transcribed to form a 5'UTR, a 3'UTR, or a polyadenosine tail.

[0181] The term "5' cap" is located at the 5' end of the mRNA and contains methylated guanylate, which is linked to the 5' end of the mRNA via pyrophosphate to form a 5',5'-triphosphate linkage with its adjacent nucleotide. There are usually three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), which are called type O, type I and type II, respectively. Type O refers to the unmethylated ribose of the terminal nucleotide, type I refers to the methylation of the ribose of one terminal nucleotide, and type II refers to the methylation of the ribose of both terminal nucleotides. In this article, "CleanCap AG" is used to refer to the m7G(5')ppp(5')(2'-OMeA)pG cap.

[0182] As used herein, the term "Poly (A) tail" or "Poly A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues typically located at the 3'-end of an RNA molecule. Poly-A tails or Poly-A sequences are known to those skilled in the art and can be selected according to actual needs. In mRNA, when a 3'-UTR is present, the Poly-A sequence is attached to the 3' end of the 3'-UTR. An uninterrupted poly-A tail is characterized by continuous adenylate residues. The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, or consists of, at least 20, at least 30, at least 40, at least 80, or at least 100 and at most 500, at most 400, at most 300, at most 200, or at most 150 adenylate residues (A), in particular about 120 A's. Typically, the vast majority of nucleotides in the poly (A) tail are adenosine, and the vast majority refers to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%. Or at least 99% of the nucleotides, but the remaining nucleotides are allowed to be nucleotides other than A, such as U (uridylic acid), G (guanylic acid) or C (cytidylic acid). Unless otherwise specified, the "nucleotide" of this application, in addition to referring to naturally occurring ribonucleotides or deoxyribonucleotide monomers, should also be understood herein to refer to related structural variants, including derivatives and analogs, which are functionally equivalent with respect to the specific context in which the nucleotide is used, unless the context clearly indicates otherwise. For example, "nucleotide" refers to a deoxyribonucleotide or a ribonucleotide. The nucleotide can be a standard nucleotide (i.e., adenosine, guanosine, cytidine, thymidine and uridine), a nucleotide isomer or a nucleotide analog. Nucleotide analogs refer to nucleotides with modified purine or pyrimidine bases or modified ribose moieties. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine, pseudouridine, etc.) or non-naturally occurring nucleotides. Non-limiting examples of modifications on the sugar or base moiety of the nucleotide include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphoryl, and thiol groups, and the substitution of the carbon and nitrogen atoms of the bases with other atoms (e.g., 7-deazapurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholino oligonucleotides.

[0183] In this application, "peptide" has its broadest definition. Since proteins themselves contain polypeptides, "peptide" covers the meaning of polypeptides with only primary structure, as well as proteins with secondary or tertiary structure. Therefore, in this application, for example, the term "antigenic peptide" covers peptide chains with only primary structure that can induce an immune response in the body, as well as proteins with secondary or tertiary structure formed by shearing, splicing, folding and / or stacking of the peptide chain. It can represent a fragment of a protein (such as a truncation) or a complete protein, and since proteins can contain components such as sugars and lipids, the antigenic peptides of this application can also cover glycoproteins, lipoproteins or fragments thereof; similarly, since proteins themselves can have chemical modifications, the antigenic peptides of this application can also have modifications, such as phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, etc. The term "VZV antigenic peptide" may refer to any VZV-derived protein (e.g., gE protein) that is immunogenic and can induce an immune response against VZV in the body, a peptide fragment of a VZV-derived protein, a partial or complete protein of a VZV-derived protein, a partial or complete protein of a mutant of a VZV-derived protein, a peptide fragment of the mutant, and a truncation. It should be understood that the "peptide," "antigenic peptide," and "VZV antigenic peptide" of the present application may include amino acids, non-natural amino acids, modified amino acids (e.g., with modified side chains and / or backbones), and amino acid analogs. To further illustrate, amino acids are generally organic acids comprising a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, for example, thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halogen, hydrazide, alkenyl, alkynyl, ether, borate, boronate, phospho, phosphono, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising photosensitive crosslinkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, amino acids comprising metals, amino acids containing novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photolabile (photocaged) and / or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids comprising carbon-linked sugars, redox-active amino acids, amino thioacid-containing amino acids, and amino acids comprising one or more toxic moieties.The amino acids described in the present application include, but are not limited to, 20 natural amino acids and 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), beta-alanine or beta-aminoalanine (bAla), 2-aminobutyric acid (Abu), 4-aminobutyric acid or pipecolic acid (4Abu), 6-aminohexanoic acid (Acp), 2-aminoheptaneic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisomethacrylic acid (bAib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), methamphetamine (Des), 2,2'-diaminopimelic acid (Dp m), 2,3-diaminopropanesulfonic acid (Dpr), ethylglycine (EtGly), N-ethylaspartic acid (EtAsn), hydroxylysine (Hyl), isohydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), isoleucine (aIle), N-methylglycine or sarcosine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle), and ornithine (Orm).

[0184] As used in this application, the terms "gE protein extracellular region", "gE protein transmembrane region", "gE protein intracellular region", and "gE protein signal peptide" refer to the extracellular region, transmembrane region, intracellular region, and signal peptide of the VZV gE protein or a variant thereof that retains its immunogenicity, respectively, unless otherwise specified. Those skilled in the art will appreciate that, since the VZV gE protein is an envelope protein, it will enter the secretory pathway and localize to the cell membrane of the host cell after expression from nucleic acid, or ultimately form a viral envelope together with the cell membrane. Based on its position relative to the cell membrane or viral envelope at this time, it can be divided into an extracellular region (extracellular portion), a transmembrane region (transmembrane portion), and an intracellular region (intracellular portion); wherein the extracellular region is a hydrophilic segment (excluding the signal peptide portion, unless otherwise specified), the intracellular region is a hydrophobic segment, and the transmembrane region is located between the extracellular and intracellular regions and is a helical segment. In some embodiments, the extracellular region comprises or is a partial segment or full length of positions 31-538 relative to the reference sequence SEQ ID NO. 8. In some embodiments, the intracellular region is a hydrophobic segment comprising or is a partial segment or full length of positions 560-623 relative to the reference sequence SEQ ID NO. 8. In some embodiments, the transmembrane region comprises or is a partial segment or full length of positions 539-559 relative to the reference sequence SEQ ID NO. 8.

[0185] As used herein, it should be understood that, unless otherwise specified, the term "uridine" herein encompasses natural uridine and its derivatives, including but not limited to: 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethyl pseudouridine, 5-methylcytidine, 5-methoxycytidine, 1-methylpseudo-UTP, pseudouridine, 1-ethyl-pseudouridine, and 5-methoxy-uridine. In other embodiments, all or part of the nucleic acids of the present invention may be replaced with modified bases, such as 1-methylpseudouridine or pseudouridine.

[0186] The term "N:P ratio," also referred to herein as "N / P" or "N:P," represents the molar ratio of the protonated nitrogen of the ionizable cationic lipid to the phosphate groups of the mRNA. The N:P ratio describes the ratio between the cationic charge of the amino groups (N+) in the ionizable cationic lipid and the anionic charge of the phosphate groups (PO4-) in the nucleic acid backbone and is the basis for the electrostatic complexation of the ionizable cationic lipid and the nucleic acid. The N:P ratio is a key formulation factor for LNPs, influencing their physicochemical properties and in vivo drug release.

[0187] As used herein, the term "about" refers to the usual error range of each value that is readily known to those skilled in the art. Reference to an "about" value or parameter herein includes embodiments for the value or parameter itself. As used herein, when the term "about" precedes a numerical value, it generally means within a range of 10% above or below that numerical value. For example, "about 100" encompasses 90 and 110. In some embodiments, when the number "about" is followed by an integer not greater than 10, the number "about" includes decimals, which are obtained by rounding off the decimal points of these decimals. For example, about 9 includes the endpoint values ​​of the range 8.5 to 9.5 and all values ​​therebetween. When the number after "about" is a ratio, such as 3:1, it can refer to a ratio between 2.5:1 and 3.5:1, or, for example, 7:1, it can refer to a ratio between 6.5:1 and 7.5:1. In this application, unless otherwise specified, a value is an integer or an exact decimal value, the value includes any value that can be obtained by rounding off, and the normal error range allowed for obtaining or using the value in the art.

[0188] antigen

[0189] Antigens used herein are proteins that can induce an immune response (e.g., cause the immune system to produce antibodies against the antigen). In this article, unless otherwise indicated, the use of the term "antigen" includes immunogenic proteins and immunogenic fragments (immunogenic fragments that induce or can induce an immune response to at least one varicella-zoster virus). It should be understood that the term "protein" includes peptides and the term "antigen" includes antigenic fragments. Other molecules may also be antigenic, such as bacterial polysaccharides or a combination of protein and polysaccharide structures, and the viral vaccine antigens described herein include viral proteins, viral protein fragments, and proteins designed and / or mutated from varicella-zoster virus.

[0190] Furthermore, the term "antigen," as used herein, will be recognized and understood by those of ordinary skill in the art to mean a substance that can be recognized by the immune system, preferably by the adaptive immune system, and that is capable of triggering an antigen-specific immune response, for example, by forming antibodies and / or antigen-specific T cells as part of an adaptive immune response. An antigen can be or include a peptide or protein that can be presented to T cells by MHC. Also included are fragments, variants, and derivatives derived from, for example, a peptide or protein, such as a varicella-zoster fusion protein containing at least one epitope.

[0191] As will be appreciated and understood by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of varicella-zoster virus antigens of interest. For example, any protein fragment of varicella-zoster virus or a mutant thereof, provided that the fragment is immunogenic and confers a protective immune response against varicella-zoster virus; in addition to variants that are identical to a reference protein but truncated, in some embodiments, the antigen comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, and the length of the antigen / antigenic polypeptide may range from about 4, 6, or 8 amino acids to the full-length protein.

[0192] Epitope

[0193] Epitope: As used herein, the term "epitope" (also referred to in the art as "antigenic determinant") will be recognized and understood by those of ordinary skill in the art to refer to both T cell and B cell epitopes. A T cell epitope refers to an antigenic epitope recognized by the T cell receptor (TCR). The epitope component is a peptide that is degraded from a protein and is often found within the antigen molecule. It must be processed by antigen presenting cells (APCs) and then bound to an MHC molecule to form a complex for TCR recognition. It can generally include fragments preferably having a length of about 6 to about 20 or more amino acids, for example. Fragments processed and presented by MHC class I molecules are preferably about 8 to about 10 amino acids in length, such as 8, 9, or 10 (or 11 or 12 amino acids), or fragments processed and presented by MHC class II molecules are preferably about 13 to about 20 or more amino acids in length. These fragments are typically recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule; i.e., these fragments are not typically recognized in their native form. B cell epitopes are generally fragments located on the outer surface of a (native) protein or peptide antigen, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which can be recognized by antibodies, i.e. recognized in their native form. Such epitopes of proteins or peptides can also be selected from any variants of such proteins or peptides mentioned herein. In this context, epitopes can be conformational or discontinuous epitopes, consisting of fragments of proteins or peptides as defined herein, which are discontinuous in the amino acid sequence of the proteins or peptides as defined herein, but are aggregated together in a three-dimensional structure or are continuous or linear epitopes consisting of a single polypeptide chain.

[0194] Nucleic Acids

[0195] The term "nucleic acid" or "nucleic acid molecule" will be recognized and understood by those of ordinary skill in the art. As used herein, the term "nucleic acid" or "nucleic acid molecule" preferably refers to a DNA (molecule) or RNA (molecule). It is preferably used synonymously with the term polynucleotide. Preferably, a nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other via phosphodiester bonds of a sugar / phosphate backbone. The term "nucleic acid molecule" also includes modified nucleic acid molecules, such as base-modified, sugar-modified, or backbone-modified DNA or RNA molecules as defined herein.

[0196] Specifically, the composition of the present application comprises (at least one) RNA having an open reading frame (ORF) encoding a varicella-zoster virus antigen (e.g., F protein). In some embodiments, the RNA is a messenger RNA (mRNA).

[0197] In some embodiments, the nucleic acid comprises at least one heterologous untranslated region (UTR). The term "untranslated region" or "UTR" or "UTR element" will be recognized and understood by those of ordinary skill in the art to mean a portion of a nucleic acid molecule, typically located 5' or 3' to a coding sequence. The 5' end is referred to as a 5'UTR, and the 3' end is referred to as a 3'UTR. Generally speaking, UTRs are not translated into proteins; UTRs can be part of a nucleic acid, such as DNA or RNA. UTRs can comprise elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements can be ribosome binding sites, miRNA binding sites, etc.; RNA (e.g., mRNA) can further comprise a 5'UTR, a 3'UTR, a 3'-poly(A) and / or a 5' cap analog.

[0198] In some embodiments, the 5'UTR is a heterologous UTR, i.e., a UTR found in nature that is associated with a different ORF; in another embodiment, the 5'UTR is a synthetic UTR; the 5'UTR is a region of the mRNA that is located upstream (5') of the start codon (the first codon of the mRNA transcript translated by the ribosome). The 5'UTR does not encode a protein. The natural 5'UTR has characteristics that play a role in translation initiation, such as the Kozak sequence, which has a consensus of CCR(A / G)CCAUGG; exemplary 5'UTRs also include African clawed frog or human α-globin or β-globin, human cytochrome b-245a polypeptide, hydroxysteroid (17b) dehydrogenase, tobacco etch virus, alpha-1-globin 5'UTR, and the like.

[0199] In some embodiments, the 3'UTR can be heterologous or synthetic; for example, globin UTRs, including African clawed frog β-globin UTRs and human β-globin UTRs; other 3'UTRs can also be CYBA (cytochrome b-245alpha chain), rabbit β-globin, hepatitis B virus (HBV), α-globin 3'UTR, and VEEV (Venezuelan equine encephalitis virus) virus 3'UTR sequences. In some embodiments, rps9 (Ribosomal Protein S9) 3'UTR, FIG4 (FIG4 Phosphoinositide 5-Phosphatase), gp130, DH143, and human albumin hHBB (human hemoglobin subunit beta), HBA1 (human Hemoglobin Subunit Alpha 1) 3'UTRs can also be used.

[0200] In some embodiments, the 3'-poly (A) tail is also called a poly (A) tail; the poly (A) tail is located downstream of the 3' UTR, for example, the mRNA region immediately downstream (i.e., 3'), which contains multiple consecutive adenosine monophosphates. The poly (A) tail may contain 10 to 300 adenosine monophosphates, and may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some preferred embodiments, the poly (A) tail contains 50 to 250 adenosine monophosphates, more preferably 50-100 adenosine monophosphates; most preferably 100 adenosine monophosphates; in relevant biological environments (e.g., in cells, in vivo), the function of the 3'-poly (A) tail is to protect the mRNA from enzymatic degradation, for example in the cytoplasm, and to facilitate transcription termination and / or export of the mRNA from the nucleus and translation.

[0201] In some embodiments, the RNA (e.g., mRNA) further comprises a 5' guanosine cap; the 5' guanosine cap is a eukaryotic mRNA transcript, and the 5' cap is composed of an inverted 7-methylguanosine, connected to the rest of the eukaryotic mRNA via a 5'-5' triphosphate bridge, namely cap 0 (cap 0), which primarily serves as a quality control for correct mRNA processing and helps stabilize the eukaryotic mRNA; based on cap 0, the first nucleotide is methylated with 2'-OH, referred to as cap 1 (cap 1); in addition to cap 0 and cap 1, the second nucleotide can also be further methylated, referred to as cap 2; generally speaking, the 5'-cap can be synthesized by different synthetic routes of 5'-capped mRNA based on enzymatic, chemical, or chemoenzymatic methods;

[0202] In some embodiments, during in vitro transcription, a cap analog is directly added to the in vitro transcription (IVT) system, and the 5' cap analog includes but is not limited to: m 7 Gppp(2'OMeA)pG、m 7 GpppApA、m 7 GpppApC、m 7 GpppApG、m 7 GpppApU、m 7 GpppCpA、m 7 GpppCpC、m 7 GpppCpG、m 7 GpppCpU、m 7 GpppGpA、m 7 GpppGpC、m 7 GpppGpG、m 7 GpppGpU、m 7 GpppUpA、m 7 GpppUpC、m 7 GpppUpG、m 7 GpppUpU、m 7 Gpppm 6 ApG、m 7 G 3’Ome pppApA、m 7 G 3’Ome pppApC、m 7 G 3’Ome pppApU、m 7 G 3’Ome pppApG、m 7 G 3’Ome pppCpA、m 7 G 3’Ome pppCpC、m7 G 3’Ome pppCpG、m 7 G 3’Ome pppCpU、m 7 G 3’Ome pppUpA、m 7 G 3’Ome pppUpC、m 7 G 3’Ome pppUpG、m 7 G 3’Ome pppUpU、m 7 G 3’Ome pppA 2’Ome pG、m 7 G 3’Ome pppA 2’Ome pC、m 7 G 3’Ome pppA 2’Ome pU、m 7 G 3’Ome pppA 2’Ome pA、m 7 G 3’Ome pppC 2’Ome pA、m 7 G 3’Ome pppC 2’Ome pU、m 7 G 3’Ome pppC 2’Ome pG、m 7 G 3’Ome pppC 2’Ome pC、m 7 G 3’Ome pppG 2’Ome pA、m 7 G 3’Ome pppG 2’Ome pU、m 7 G 3’Ome pppG 2’Ome pG、m 7 G 3’Ome pppG 2’Ome pC、m 7 G 3’Ome pppU 2’Ome pA、m 7 G 3’Ome pppU 2’Ome pU、m 7 G 3’Ome pppU 2’Ome pG、m 7 G 3’Ome pppU 2’Ome etc.

[0203] In some embodiments, the capped analogs may also be other structures, such as tetramers, pentamers, hexamers, heptamers, octamers, nonamers, or decamers, etc. The specific sequence thereof may be determined according to the conditions of the template.

[0204] It should also be understood that the respiratory syncytial virus mRNA vaccine of the present application may include any 5' untranslated region (UTR) and / or any 3' untranslated region (UTR). Nucleic acid comprises a polymer (nucleotide monomer) of nucleotides. Therefore, nucleic acid is also referred to as a polynucleotide. Nucleic acid can be or can include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNAs), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimera and / or a combination thereof.

[0205] Messenger RNA (mRNA) is any RNA that encodes (at least one) protein (a naturally occurring, non-naturally occurring, or modified amino acid polymer) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein. The skilled person will understand that, unless otherwise indicated, the nucleic acid sequences listed in this application may reference "T" in the representative DNA sequence, but when the sequence represents RNA (e.g., mRNA), the "T" will be replaced with "U". Therefore, any DNA disclosed and identified herein by a specific sequence identification number also discloses an RNA (e.g., mRNA) sequence that is complementary to the DNA or has the same base sequence, in which case each "T" of the nucleotide sequence represents a "U".

[0206] open reading frame

[0207] An open reading frame (ORF) is a continuous stretch of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). Generally speaking, an ORF typically encodes a protein. It will be understood that the sequences disclosed herein may also contain additional elements, such as 5' and 3' UTRs, but unlike ORFs, these elements are not necessarily present in the RNA polynucleotides of the present application.

[0208] In some embodiments, the composition comprises RNA (e.g., mRNA) comprising the nucleotide sequence of any one of SEQ ID NOs. 1-3 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity.

[0209] In some embodiments, the open reading frame is preferably at least partially codon optimized. Codon optimization is based on such discovery: translation efficiency can be determined by the different frequencies of transfer RNA (tRNA) occurring in the cell. Therefore, if there is so-called "rare codon" of increasing degree in the coding region of the nucleic acid of the application defined herein, the translation of the corresponding modified nucleotide sequence is less efficient than when there is a codon for encoding relatively "common" tRNA. Those skilled in the art can carry out codon optimization for sequence to be translated with the characteristics of its in vitro expression system.

[0210] Chemically modified or unmodified nucleotides

[0211] In some embodiments, RNA (e.g., mRNA) is not chemically modified, but rather comprises standard ribonucleotides consisting of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides disclosed herein comprise standard nucleoside residues, such as those present in transcribed RNA (e.g., A, G, C, or U). In some embodiments, the nucleotides and nucleosides disclosed herein include standard deoxyribonucleosides, such as those present in DNA (e.g., dA, dG, dC, or dT);

[0212] In some embodiments, the compositions of the present application include RNA with an open reading frame encoding a respiratory syncytial virus antigen, wherein the nucleic acid includes standard (unmodified) or modified nucleotides and / or nucleosides known in the art. In some embodiments, the nucleotides and nucleosides of the present application include modified nucleotides or nucleosides. The nucleotides and nucleosides of this modification can be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include modifications of the sugar, backbone or core base moieties of nucleotides and / or nucleosides well known in the art.

[0213] In some embodiments, the modified nucleic acid base in the nucleic acid (e.g., RNA nucleic acid, e.g., mRNA nucleic acid) includes 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methoxy-uridine, 5-methyl-cytidine and / or pseudouridine, pseudouridine.

[0214] In vitro transcription system (IVT)

[0215] In vitro transcription is the process of using DNA as a template in an in vitro cell-free system containing components such as RNA polymerase and NTP to mimic the in vivo transcription process to generate mRNA. Generally speaking, the capped RNA synthesized in the in vitro transcription reaction can be used for subsequent experiments such as microinjection, in vitro translation, and transfection. The in vitro transcription system usually includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase. NTPs can be synthesized by oneself or selected from a supplier. NTPs can be natural or non-natural NTPs. Optional polymerases include, but are not limited to, phage RNA polymerases, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or polymerase mutants thereof, such as, but not limited to, polymerases capable of incorporating modified nucleic acids and / or modified nucleotides, including chemically modified nucleic acids and / or nucleotides. Some embodiments exclude the use of DNA enzymes. In some embodiments, the RNA contains a 5' guanosine cap.

[0216] In addition to synthesis by in vitro transcription systems, chemical synthesis methods can also be used, including solid-phase chemical synthesis and liquid-phase chemical synthesis; with respect to solid-phase chemical synthesis, the nucleic acids disclosed in this application can be prepared in whole or in part using solid-phase technology; solid-phase chemical synthesis of nucleic acids is an automated method in which molecules are fixed on a solid support and synthesized stepwise in a reactant solution. Solid-phase synthesis can be used for site-specific introduction of chemical modifications in nucleotide sequences; with respect to liquid-phase chemical synthesis, the nucleic acids of this application can be synthesized in liquid phase by sequentially adding monomer constructs. In addition, the above-mentioned synthesis methods can also be used in combination, because the synthesis methods discussed above each have their own advantages and limitations, and attempts can be made to combine these methods to overcome the above-mentioned limitations. Combinations of these methods are within the scope of this application.

[0217] Antigenic variants

[0218] In some embodiments, the compositions of the present application comprise RNA encoding varicella-zoster virus antigen variants (e.g., variant trimeric F proteins, e.g., stable pre-fusion F proteins). Antigen variants or other polypeptide variants refer to molecules whose amino acid sequences are different from wild-type, natural or reference sequences. Compared to natural or reference sequences, antigen / polypeptide variants may have substitutions, deletions and / or insertions at certain positions within the amino acid sequence. Typically, variants have at least 50% identity with wild-type, natural or reference sequences. In some embodiments, variants have at least 80% or at least 90% identity with wild-type, natural or reference sequences.

[0219] Variant antigens / polypeptides encoded by the nucleic acid of the present application can include amino acid changes that confer any of a variety of desired properties, for example, enhancing their immunogenicity, enhancing their expression and / or improving their stability or PK / PD properties. Conventional mutagenesis techniques can generally be used to prepare variant antigens / polypeptides, and analysis is performed as appropriate to determine whether they have the desired properties. Determining the expression level and immunogenicity assay is well known in the art, and exemplary such assays are described in the Examples section. Similarly, the PK / PD properties of protein variants can be measured using techniques generally recognized in the art, for example, by determining the expression of the antigen in the inoculated subject over time and / or by observing the persistence of the induced immune response. The stability of the protein encoded by the variant nucleic acid can be measured by measuring the thermal stability or stability during urea denaturation, or can be measured using computer prediction. Methods for such tests and computer determinations are known in the art.

[0220] The term "identity" refers to the relationship between the sequences of two or more polypeptides (e.g., antigens) or polynucleotides (nucleic acids) determined by comparing sequences. Identity also refers to the degree of sequence relatedness between or among sequences determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences, where gap alignments (if any) are solved by a specific mathematical model or computer program (e.g., an "algorithm"). The identities of the related antigens or nucleic acids can be easily calculated by known methods. "Percent (%) identity" for polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleotide sequence that are identical to the residues in the amino acid sequence or the nucleotide sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to obtain maximum percent identity. Methods and computer programs for comparison are well known in the art. It is understood that identity is determined by the calculation of percent identity, but its value may vary due to the gaps and penalties introduced in the calculation. Typically, variants of a particular polynucleotide or polypeptide (e.g., an antigen) have 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a particular reference polynucleotide or polypeptide as determined by the sequence alignment programs and parameters described herein and known to those of skill in the art.

[0221] Lipid nanoparticles (LNPs)

[0222] The RNA of the present application (for example, mRNA) is formulated in lipid nanoparticles (LNPs).Lipid nanoparticles generally include ionizable cationic lipids, helper lipids, cholesterol and PEG lipid components and nucleic acid of interest. The lipid nanoparticles of the present application can use components generally known in the art, and compositions and methods are generated.

[0223] Multivalent vaccines

[0224] The compositions provided herein may include RNA or multiple RNAs encoding two or more antigens of the same or different types. In some embodiments, the compositions include RNA or multiple RNAs encoding two or more respiratory syncytial virus antigens. In some embodiments, the RNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more respiratory syncytial virus antigens.

[0225] Two or more different RNAs (e.g., mRNA) encoding antigens can be formulated in the same lipid nanoparticle. In other embodiments, two or more different RNAs encoding antigens can be formulated in separate lipid nanoparticles (each RNA is formulated in a single lipid nanoparticle). The lipid nanoparticles can then be combined and administered as a single vaccine composition (e.g., comprising a variety of RNAs encoding multiple antigens), or can be administered separately.

[0226] Combined vaccines

[0227] The compositions provided herein may include RNA or multiple RNAs encoding two or more antigens of the same or different virus strains. Combination vaccines are also provided herein, which include RNA encoding one or more varicella-zoster viruses and one or more antigens of different organisms. Therefore, the vaccine of the present application can be a combined vaccine targeting one or more antigens of the same strain / species, or one or more antigens of different strains / species, such as other microorganisms found in geographical areas where the risk of varicella-zoster virus infection is high or other antigens that may be contacted at the same time when an individual is exposed to varicella-zoster virus.

[0228] Sequential inoculation

[0229] Sequential vaccination refers to the interval vaccination of vaccines with different technical routes, including basic immunization sequence and booster immunization sequence; if the first shot is an inactivated vaccine and the second shot is an adenovirus vaccine or mRNA vaccine or any other non-inactivated vaccine, this vaccination method is called basic immunization sequence; if two doses of inactivated vaccine have been completed and a booster shot is needed subsequently, any other non-inactivated vaccine is used instead, this vaccination method is called booster immunization sequence.

[0230] pharmaceutical preparations

[0231] Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for preventing or treating, for example, varicella-zoster virus in humans and other mammals. The compositions provided herein can be used as therapeutic or prophylactic agents. They can be used in medicaments for preventing and / or treating varicella-zoster virus infection.

[0232] The term "pharmaceutical composition" refers to the combination of an active agent and an inert or active carrier such that the composition is particularly suitable for in vivo or in vitro diagnostic or therapeutic use. A "pharmaceutically acceptable carrier" does not cause undesirable physiological effects upon administration to or after administration to a subject. The carrier in a pharmaceutical composition must be "acceptable" in the sense that it is compatible with the active ingredient and capable of stabilizing it. One or more solubilizing agents may be used as pharmaceutical carriers for delivering the active agent. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible carriers, adjuvants, additives, and diluents to obtain a composition that can be used as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Other suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for them, are described in Remington's Pharmaceutical Sciences.

[0233] Methods for preparing mRNA vaccines are known in the art. In particular, the mRNA in the mRNA vaccine, while containing the antigen peptide coding sequence, further contains the coding sequences of multiple necessary functional components to express, regulate, or enhance the expression level of the above-mentioned HPV antigen polypeptide. The functional components include, but are not limited to, a 5' cap, a 5' UTR, a 3' UTR, a poly tail, etc. The functional components are known in the art, and those skilled in the art can select and combine them according to actual needs. Both the 5' UTR and the 3' UTR are usually transcribed from genomic DNA and are elements possessed by pre-mature mRNA (or mRNA precursor or pre-mRNA). The characteristic structural features of mature mRNA (such as the 5'-cap and the 3'-poly (A) tail) are usually added to the transcribed (pre-mature) mRNA during mRNA processing. Therefore, in some embodiments, the mRNA is an mRNA precursor. In some embodiments, the mRNA is a mature mRNA.

[0234] The relevant sequences involved in this application are as follows.

[0235] It should be understood that the following sequences are merely exemplary sequences of embodiments of the present application and are not intended to limit the present application. The nucleic acid sequences in the following sequences may represent DNA sequences or RNA sequences. When representing RNA sequences, "T" and "U" are used interchangeably to represent uridine unless otherwise specified. The sequences used in the specific examples later in this application correspond to the following sequences by sequence number or name, and when representing mRNA sequences in the specific examples, the uridines therein are all 1-methylpseudouridine.

[0236] SEQ ID NO.1

[0237] VZV gE protein nucleotide original sequence VZV-WT

[0238] SEQ ID NO.2

[0239] VZV gE protein nucleotide sequence VZV-2

[0240] SEQ ID NO.3

[0241] VZV gE protein nucleotide sequence VZV-trunc

[0242] SEQ ID NO.4

[0243] VZV gE protein nucleotide sequence VZV-mut-Leu

[0244] SEQ ID NO.5

[0245] VZV gE protein nucleotide sequence VZV-mut-Ile

[0246] SEQ ID NO.6

[0247] VZV gE protein nucleotide sequence VZV-mut-H362E-Leu

[0248] SEQ ID NO.7

[0249] VZV gE protein nucleotide sequence VZV-mut-H362E-Ile

[0250] Note: U in the above sequence can be completely (100%) or partially replaced with modified bases, such as 1-methylpseudouridine or pseudouridine.

[0251] SEQ ID NO.8

[0252] VZV gE protein antigen original amino acid sequence

[0253] SEQ ID NO. 9KOZAK sequence

[0254] SEQ ID NO.10

[0255] pVAX.1+TEV 5'UTR sequence

[0256] SEQ ID NO.11

[0257] 3'-polyadenylation sequence

[0258] SEQ ID NO.12

[0259] 3'-UTR hemoglobin-1 (hHBA1) sequence

[0260] SEQ ID NO.13

[0261] VZV-trunc

[0262] SEQ ID NO.14

[0263] VZV-mut-Ile

[0264] SEQ ID NO.15

[0265] VZV-mut-Ile-H362E

[0266] SEQ ID NO.16

[0267] VZV-mut-Leu

[0268] SEQ ID NO.17

[0269] VZV-mut-Leu-H362E

[0270] SEQ ID NO.18

[0271] TEV 5'UTR

[0272] The embodiments of the present application will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present application and should not be considered as limiting the scope of the present application. In the examples, if no specific conditions are specified, the conditions according to conventional conditions or manufacturer recommendations are used. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0273] Example 1: Codon Optimization of VZV gE Protein or Its Antigenic Fragments or Immunogenic Variant Protein Antigens

[0274] mRNA sequences were designed based on the native coding region sequence of the varicella-zoster virus gE protein, its antigenic fragments, or immunogenic variant protein antigens. In addition to the coding region, mRNA sequence features also included the pVAX.1+TEV 5'UTR, hHBA1 3'UTR, and 100 polyA residues. The VZV-2, VZV-trunc, VZV-mut-Leu, VZV-mut-Ile, VZV-mut-H362E-Leu, and VZV-mut-H362E-Ile mRNA sequences were optimized within the VZV gE protein coding region. Compared to the native VZV gE protein coding sequence, the GC content of these optimized sequences increased while maintaining consistency within the UTR region. The sequence design optimization strategy and relative expression results are shown in Table 1.

[0275] Table 1: Relative expression data of different codon-optimized mRNAs

[0276] In this study, multiple optimized sequences were designed and cloned into the vector pVAX.1 (GenScript Biotech Co., Ltd.). Using the linearized vector (customized by GenScript Biotech Co., Ltd.) as a template, mRNA was synthesized in vitro and transfected into COS7 cells. At a set time, the expression level of VZV gE protein in the cells was detected by ELISA to evaluate the effect of codon optimization on mRNA expression level and stability. The expression level results are shown in Table 1. The results show that the expression level of mRNA can be significantly improved by codon optimization. According to the above experimental results, the relative expression level of VZV-trunc antigen peptide mRNA (SEQ ID NO.3) is high, which is the preferred mRNA sequence for subsequent research and development of vaccines.

[0277] The in vitro transcription (IVT) steps for VZV gE protein antigen are as follows:

[0278] 1) Prepare the IVT reaction system according to the instructions of the IVT kit (Novozymes kit catalog number: DD4201-P-01), namely, mix 10x Transcription Buffer, ATP, GTP, CTP, PseudoUTP (1-methylpseudouracil, Zhaowei Technology, catalog number R5-064), 5' cap analog (m7G(5')ppp(5')(2'OMeA)pG, Zhaowei Technology, catalog number ON-134), water for injection, plasmid template (linearized plasmid with T7 promoter, the template is the DNA sequence corresponding to SEQ ID NO. 3), and Enzyme Mix.

[0279] 2) The mixed reaction system was placed at 37°C for 40 minutes.

[0280] 3) Add the corresponding proportion of DNase I to terminate the reaction.

[0281] 4) Add an equal volume of a certain concentration (7.5 M) LiCl solution and allow to settle twice. Then, wash twice with 75% ethanol solution, air-dry at room temperature in a laminar flow hood, and dissolve with an appropriate amount of RNase-free H2O.

[0282] The purity of the obtained mRNA was analyzed using a Bioanalyzer. The experimental results showed (see Figure 1) that the VZV-trunc antigen mRNA was synthesized in vitro and then separated and purified to obtain high-purity mRNA.

[0283] Example 2: Formulation screening experiment

[0284] The lipid ratio of the formulation was optimized based on comprehensive considerations, with the specific formulations selected as shown in Table 2. Normal mice were immunized once with 3 μg of mRNA (encapsulated using LNPs of different formulations 1-11), and the binding antibodies in mouse serum were measured 14 days later. Based on considerations such as binding antibody levels and formulation stability, Formulation 9 was ultimately determined to be the preferred formulation. Figure 2 shows the results of LNPs encapsulating the VZV-trunc mRNA shown in Table 1.

[0285] Table 2. LNP formulation ingredients list

[0286] Note: The chemical name of HUO is: Heptadec-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate), also known as SM-102.

[0287] Example 3: Preparation and confirmation of antigen peptide mRNA-LNP formulation

[0288] According to the formulation selected in Example 2 (i.e., Formulation 9), a VZV gE protein or an antigenic fragment or immunogenic variant protein antigen mRNA-LNP formulation was prepared. The specific preparation steps are as follows:

[0289] 1. Accurately weigh a certain amount of SM-102, 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG2000 lipids with a molar mass ratio of 50% and add appropriate amount of anhydrous ethanol to dissolve them to prepare a lipid working solution (final concentration of lipid working solution 12 mg / mL).

[0290] 2. Prepare citric acid buffer solution (10 mM, pH 4.0) containing 130 mM sodium chloride, Tris-NaOAc buffer solution (20 mM, 10.7 mM, pH 7.4), and Tris-NaOAc buffer solution (20 mM, 10.7 mM, pH 7.4) containing 60% sucrose.

[0291] 3. Take an appropriate amount of mRNA stock solution and dilute it with the sodium chloride-citrate buffer solution prepared above to adjust the final concentration of the mRNA working solution to 0.18 mg / mL.

[0292] 4. Use a microfluidic instrument and supporting chip to mix the lipid working solution and mRNA working solution in a volume ratio of 1:3 to prepare an mRNA-loaded LNP solution.

[0293] 5. Dilute the prepared LNP solution by adding 9 times the volume of Tris-NaOAc buffer solution, and concentrate and purify it by TFF to remove the ethanol solution in the system.

[0294] 6. Use UV spectrometry to detect the mRNA content in the LNP solution and add an appropriate amount of Tris-NaOAc buffer solution (20 mM, 10.7 mM, pH 7.5) containing 60% sucrose to adjust the final mRNA concentration in the finished LNP solution to 100 g / mL and the sucrose content (w / v, i.e., concentration) in the external aqueous phase system to 8.7%.

[0295] The results showed that microfluidic mixing of VZV gE protein, its antigenic fragment, or immunogenic variant protein antigen mRNA and lipid working solution formed LNP complexes with uniform particle size and morphology, mostly around 100 nm, meeting the basic requirements for further experiments. For example, the structure of VZV-trunc mRNA-LNP is shown in Figure 3.

[0296] Example 4: Immunoblotting of VZV gE protein antigen peptides expressed in host cells

[0297] COS-7 cells (Note: African green monkey kidney fibroblasts, purchased from Nanjing Kebai Biotechnology Co., Ltd.) were transfected with mRNA expressing the VZV gE protein antigen. 24 hours after transfection, the cells were harvested and analyzed by immunoblotting. Cells not transfected with VZV gE antigen mRNA served as a negative control (Blank), and GAPDH was used as an internal control. Figure 4 shows representative results, showing the protein expression levels of VZV-trunc. This demonstrates that the mRNA encoding the VZV gE antigen peptide obtained in this application is capable of effectively and stably expressing the target antigen in COS-7 cells.

[0298] The specific steps of immunoblotting are as follows:

[0299] 1. Remove the transfected cells and discard the supernatant. Wash once with PBS. Add 100 μL 1x SDS-PAGE protein loading buffer to each well. After shaking and lysing for 3 minutes, collect the sample for later use.

[0300] 2. Heat the collected samples at 95°C for 7 minutes and then load them onto the plate. Perform electrophoresis at 80 V. The loading volume for each sample is 10 μL.

[0301] 3. After electrophoresis, assemble the methanol-activated PVDF membrane into a transfer device according to the "sandwich method", and then transfer the membrane at 100V in an ice bath for 1.5 hours.

[0302] 4. Block the PVDF membrane in PBST buffer containing 5% skim milk powder at room temperature for 1 hour, wash three times with PBST, and then incubate the PVDF membrane with VZV gE Antibody at 4°C overnight.

[0303] 5. On the next day, the PVDF membrane was washed three times with PBST and incubated with GAPDH antibody (mouse monoclonal antibody) at room temperature for 1 hour.

[0304] 6. After washing away unbound secondary antibody with PBST, add ECL developer and use Proteinsimple / FluorChem E to collect chemiluminescence images.

[0305] Example 5: Flow cytometry detection of antigen protein expression in cells

[0306] After transfecting COS-7 cells with VZV gE antigen peptide mRNA, flow cytometry was used to detect the expression of the target antigen protein in the cells. Cells not transfected with VZV gE antigen peptide mRNA served as a negative control (Blank). The specific method is as follows:

[0307] 1. To detect the expression of VZV gE antigen peptide mRNA in cells, COS-7 cells cultured for more than 24 hours were digested and transferred into 6-well plates. The cell density was controlled at 300,000 cells per well.

[0308] 2. Incubate the six-well plate at 37°C for 24 hours and observe the cells under a microscope. mRNA transfection can be performed when the cell confluence reaches 80% or above.

[0309] 3. Transfect the corresponding mRNA into COS-7 cells using the Lipofectamine 2000 kit (4 μg of mRNA per well). Refer to the kit instructions for specific procedures. Continue to culture at 37°C for 24 hours.

[0310] 4. After 24 hours of sustained mRNA expression, when expression levels approach peak, remove the cell supernatant, wash once with PBS, digest with 0.05% trypsin for 1 minute, neutralize with complete culture medium, and harvest the cells. Centrifuge the harvested cells at 350g for 5 minutes, and discard the supernatant. Resuspend the cells in 2 ml of PBS, harvest the cells, centrifuge at 350g for 5 minutes, and discard the supernatant. Finally, resuspend the cells in 100 μl of PBS, and control the cell count to between 200,000 and 1,000,000.

[0311] 5. Use 150μl Cyto-Fast TM Fix the collected cells with Fix / Perm Buffer and incubate on ice for 20 minutes.

[0312] 6. Add 1 ml of 1×Cyto-Fast TM Perm Wash solution, 350g, centrifuge for 5 minutes. Discard the supernatant and use 1ml 1×Cyto-Fast TM Resuspend in Perm Wash solution, rinse once more, and centrifuge to discard the supernatant.

[0313] 7. Use 100 μl 1× Cyto-Fast TM Dilute the primary antibody to 1:100 in Perm Wash solution, add it to the cells, shake to mix, and incubate on ice for 30 minutes.

[0314] 8. Add 1 ml of 1× Cyto-Fast to the cells after incubation with the primary antibody. TM Perm Wash solution, 350g, centrifuge for 5 min, discard the supernatant and use 1 ml 1× Cyto-Fast TM Resuspend in Perm Wash solution, rinse once more, and centrifuge to discard the supernatant.

[0315] 9. Use 250 μl 1× Cyto-Fast TM Dilute the secondary antibody to 1:1000 in Perm Wash solution, add it to the cells, shake to mix, and incubate on ice for 30 minutes.

[0316] 10. Add 1 ml of 1× Cyto-Fast to the cells after incubation with the secondary antibody. TM Perm Wash solution, 350g, centrifuge for 5 min, discard the supernatant and use 1 ml 1× Cyto-Fast TM Resuspend in Perm Wash solution, rinse once more, and centrifuge to discard the supernatant.

[0317] 11. Dilute the 4% paraformaldehyde fixative to 1% paraformaldehyde using PBS. Resuspend the cells in 200 μl of 1% paraformaldehyde fixative and analyze the expression of VZV gE protein in the cells using flow cytometry.

[0318] 12. Flow cytometry: Set cells without mRNA transfection as the negative control group. Analyze each sample sequentially, and read the FITC fluorescence intensity signal using a histogram. Read 10,000 signals for each sample.

[0319] The experimental results show that the mRNA encoding the VZV gE antigen obtained in this application can effectively and stably express the target protein in COS-7 cells. For example, Figure 5 shows the protein expression level of the antigen peptide of the VZV-trunc mRNA.

[0320] Example 6: Mouse serum binding antibody and detection experiment

[0321] 6-8 week old BALB / c mice were randomly divided into 10 groups, half male and half female, and immunized by intramuscular injection of vehicle, test substance VZV mRNA-LNP preparation (3 μg / mouse), and marketed vaccine Shingrix (3 μg / mouse or 5 μg / mouse) on day 0 and day 28, respectively. Serum samples were collected on days 14 and 35 after immunization, and the serum binding antibody titer on day 14 was tested. The results are shown in Figure 6: Mice immunized with the VZV mRNA vaccine of the present application were able to produce high titers of binding antibodies. The geometric mean titer (GMT) of binding antibodies in the serum of mice immunized with VZV mRNA vaccines (VZV-2, VZV-trunc, VZV-mut-Leu, VZV-mut-H362E-Leu, VZV-mut-Ile, and VZV-mut-H362E-Ile) was higher than that of the marketed vaccine Shingrix. Among them, the serum binding antibody GMTs of VZV-2, VZV-trunc, VZV-mut-Ile and VZV-mut-H362E-Ile were significantly higher than those of VZV-mut-Leu and VZV-mut-H362E-Leu, and the Bar values ​​were smaller, with smaller differences among different individuals and more stable immune effects.

[0322] The method for detecting mouse serum binding antibodies is as follows:

[0323] A high-binding capacity 96-well plate coated with VZV gE protein was prepared one day in advance. The next day, serum diluted into different gradients was added to the coated 96-well plate and incubated for 2 hours. The binding antibodies in the serum were then detected using a universal ELISA method.

[0324] Example 7: Mouse serum specific protective antibody detection experiment (FAMA method)

[0325] BALB / c mice aged 6-8 weeks were randomly divided into 10 groups, half male and half female, and immunized on days 0 and 28 by intramuscular injection with vehicle, the test VZV gE mRNA formulation (3 μg / mouse), and the marketed vaccine Shingrix (3 μg / mouse or 5 μg / mouse). Serum samples were collected on days 14, 35, and 63 after immunization, and the specific protective antibody titer in the serum on day 63 was measured. The results are shown in Figure 7: Mice immunized with the VZV vaccine of the present application were able to produce high titers of specific protective antibodies. The geometric mean titer (GMT) of specific protective antibodies in the serum of mice immunized with the VZV mRNA vaccines (VZV-2, VZV-trunc, VZV-mut-Leu, VZV-mut-H362E-Leu, VZV-mut-Ile, and VZV-mut-H362E-Ile) was higher than that of the marketed vaccine Shingrix at the same dose (3 μg / mouse). Furthermore, VZV-trunc and VZV-mut-Leu demonstrated significantly higher specific protective antibody GMTs compared to VZV-2, VZV-mut-H362E-Leu, VZV-mut-Ile, and VZV-mut-H362E-Ile. Furthermore, the Bar value for the VZV-trunc group was also lower, indicating less variability among individuals and a more stable immune response.

[0326] The method for detecting specific protective antibodies in mouse serum is as follows:

[0327] The FAMA assay uses varicella-zoster virus (VZV)-infected cells as antigens to prepare fixed antigen slides. Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (H+L) is used as a secondary antibody to detect VZV-specific IgG antibodies in mouse serum. The resulting antigen-antibody complex is primarily localized on the surface of infected cells, where a distinctive membranous ring of fluorescence is observed under a fluorescence microscope. Infected cells are classified as positive if a yellow-green fluorescent ring appears on the surface; those with no or no complete fluorescent ring are classified as negative. The sample is serially diluted, and the highest dilution at which the fluorescent ring appears in the test group is recorded. The FAMA antibody titer is the highest dilution at which the fluorescent ring appears in the test sample.

[0328] Example 8: Mouse serum true virus neutralizing antibody detection experiment

[0329] BALB / c mice aged 6-8 weeks were randomly divided into 10 groups, half male and half female, and were immunized by intramuscular injection of vehicle, test VZV mRNA-LNP preparation (3 μg / mouse), and marketed vaccine Shingrix (3 μg / mouse or 5 μg / mouse) on days 0 and 28, respectively. Serum samples were collected on days 14, 35, and 63 after immunization, and the serum neutralizing antibody titer on day 63 was tested. The results are shown in Figure 8: Mice immunized with the VZV vaccine (VZV-trunc and VZV-2) of this application were able to produce high-titer neutralizing antibodies, and the geometric mean titer (GMT) of neutralizing antibodies in the serum of mice immunized with the VZV mRNA vaccine was higher than that of the marketed vaccine Shingrix. Among them, the neutralizing antibody GMT of VZV-trunc was significantly higher than that of VZV-2.

[0330] The method for detecting neutralizing antibodies in mouse serum is as follows:

[0331] 1. Cell preparation: Adjust the density of MRC-5 cells, seed them into 96-well plates, and culture them in a cell culture incubator (37°C, 5% CO2) overnight. Ensure that the cell confluence is about 90% on the next day before starting the experiment.

[0332] 2. Serum inactivation: Inactivate in a water bath at 56°C for 30 minutes.

[0333] 3. Gradient dilution: The sample is diluted 30 times initially, and a 3-fold gradient dilution is performed, with a total of 8 dilutions (including the first well) and 2 replicates.

[0334] 4. Virus dilution: Input the appropriate amount of virus according to the PFU value of the virus.

[0335] 5. Neutralization reaction: Add diluted virus (VR-1832 TM The cells were diluted downwards in a 2-fold gradient in the back-drip wells for a total of 3 dilutions and then neutralized in a 37°C, 5% CO2 incubator for approximately 1 hour.

[0336] 6. Virus adsorption: Add the above viruses, serum neutralization products, and positive and back-tick well viruses to the prepared cells, 50ul per well, 2 replicates, and culture in a 37°C, 5% CO2 incubator for about 2 hours. After changing the medium, add 100ul of culture medium to each well and continue to culture for about 48 hours.

[0337] 7. Plate assay: Discard the supernatant, fix the cells, add fluorescently labeled detection antibodies, and read the plate using a CTL instrument.

[0338] Example 9: Specific T cell response detection experiment in mice

[0339] Female BALB / c mice aged 6-8 weeks were randomly divided into groups, and 3 mice in each group were immunized by intramuscular injection of vehicle, test substance VZV mRNA-LNP preparation (3 μg / mouse) and marketed vaccine Shingrix (3 μg / mouse or 5 μg / mouse) on day 0 and day 28, respectively. Spleen samples were collected on day 42 after immunization to detect the response of specific T cells in the mice. The results are shown in Figure 9: Strong specific T cell immunity was produced in the mice immunized with the VZV vaccine of the present application. The proportion of specific T cells in the mice in the VZV mRNA vaccine (VZV-2, VZV-trunc, VZV-mut-Leu, VZV-mut-H362E-Leu, VZV-mut-Ile and VZV-mut-H362E-Ile) group was significantly higher than that in the Shingrix group with the same dose (3ug) or even higher dose (5μg) (P<0.05). Among them, the specific T cell ratios of VZV-trunc, VZV-mut-Leu, VZV-mut-H362E-Leu, VZV-mut-Ile and VZV-mut-H362E-Ile were higher than those of VZV-2; and further, at the same dose, VZV-trunc was also higher than VZV-mut-Leu, VZV-mut-H362E-Leu, VZV-mut-Ile and VZV-mut-H362E-Ile.

[0340] The method for detecting specific T cell responses in mice is as follows:

[0341] 1. Isolation of Mouse Splenocytes

[0342] Mouse spleens were placed on a 70 μm cell sieve and ground. The cells on the sieve were rinsed with Dulbecco's phosphate-buffered saline (DPBS) containing 5% fetal bovine serum (FBS). After centrifugation, erythrocyte lysis buffer was added to lyse the erythrocytes. After complete lysis, lysis was terminated by adding DPBS containing 5% FBS. After centrifugation, the cells were resuspended in RPMI-1640 medium containing 10% FBS, 1% GlutaMax™, and 1% penicillin-streptomycin (PS). Cells were counted using a cell counter for subsequent ICS analysis.

[0343] 2. ICS (Intracellular Factor Staining Based on Flow Cytometry) Detection of Mouse Spleen Cells

[0344] Mouse spleen cells were seeded into 96-well cell culture plates at 1,000,000 cells / well, and stimulators (control group: medium; test group: VZV peptide library (PepMix TM VZV (gE), JPT, a short peptide library covering the full length of the gE amino acid sequence), incubated at 37°C, 5% CO2 for 16 hours. Protein transport inhibitors were then added and incubated for another 4 hours. After incubation, the cells were stained in a 96-well V-well plate. Fixable Viability Stain 700 and purified rat anti-mouse CD16 / CD32 mouse Fc blocker were incubated at room temperature for 10 minutes, and then CD3 and CD8 antibodies were added and incubated at 4°C for 30 minutes. The cells were washed twice with Staining buffer and fixed and permeabilized at 4°C for 30 minutes. After washing the cells twice with Wash buffer, intracellular cytokine staining was performed. Intracellular antibodies CD4, IFN-γ, TNF-α, and IL-2 were mixed and incubated at 4°C for 30 minutes. After washing the cells with Wash buffer, the cells were resuspended and transferred to a flow tube for analysis by flow cytometry.

[0345] In summary, the mRNA vaccine obtained in this application can fully activate the immune system, induce high titers of neutralizing antibodies and cellular immunity, and achieve unexpected technical effects. In addition, the various immune responses caused by VZV-trunc, VZV-mut-Leu, VZV-mut-H362E-Leu, VZV-mut-Ile and VZV-mut-H362E-Ile are significantly stronger than those caused by VZV-2. The most outstanding VZV-trunc has smaller individual differences than VZV-mut-Leu, VZV-mut-H362E-Leu, VZV-mut-Ile and VZV-mut-H362E-Ile, and the cellular immunity caused is stronger, and it can always cause a relatively stronger humoral immune response.

Claims

1. A nucleic acid molecule comprising a varicella-zoster virus (VZV) antigenic peptide coding sequence, wherein the antigenic peptide comprises an extracellular region and a transmembrane region of VZV glycoprotein E (gE protein); the extracellular region comprises amino acids 31-538 of the gE protein, and the transmembrane region comprises amino acids 539-559 of the gE protein, and the positions of the amino acids are numbered with reference to the amino acid sequence of SEQ ID NO.8; and the antigenic peptide is mutated to or comprises one or more selected from the following relative to the amino acid of SEQ ID NO.8: S593L, S595L, T596L, H362E, T598L, S593I, S595I, T596I, T598I and Del 560-623.

2. The nucleic acid molecule according to claim 1, wherein the amino acid sequence of the VZV gE protein variant comprises or is any one of the following mutations relative to SEQ ID NO.8: 1) S593L, S595L, T596L and T598L; 2) H362E, S593L, S595L, T596L and T598L; 3) H362E, S593I, S595I, T596I and T598I; 4) S593I, S595I, T596I and T598I; and 5) Del 560-623; Furthermore, the positions of the amino acids are numbered using the amino acid sequence of SEQ ID NO.8 as a reference sequence.

3. The nucleic acid molecule according to claim 1 or 2, wherein the antigenic peptide further comprises a signal peptide sequence, and the signal peptide sequence is located at the N-terminal side of the extracellular region. 4 . The nucleic acid molecule according to claim 3 , wherein the signal peptide is the signal peptide of VZV gE protein, preferably, the signal peptide sequence comprises or is the amino acid sequence at positions 1-30 or 2-30 of SEQ ID NO.

8.

5. The nucleic acid molecule according to any one of claims 1 to 4, wherein the antigenic peptide sequence is as shown in any one of SEQ ID NOs. 13 to 17, or comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs. 13 to 17. The nucleic acid molecule according to any one of claims 1 to 5, comprising a 3'-poly(A) sequence or a tailing signal sequence.

7. The nucleic acid molecule according to claim 6, wherein the 3'-poly(A) nucleotide sequence is as shown in SEQ ID NO. 11 or comprises a nucleotide sequence having at least 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with the nucleotide sequence as shown in SEQ ID NO:

11.

8. The nucleic acid molecule according to any one of claims 1 to 7, comprising a 5'UTR or a coding sequence for a 5'UTR, wherein the 5'UTR comprises or is a Tobacco Etch Virus (TEV) 5'UTR.

9. The nucleic acid molecule according to claim 8, wherein the nucleotide sequence of the 5'UTR is as shown in SEQ ID NO.18 or SEQ ID NO.10 or comprises a nucleotide sequence having at least 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with the nucleotide sequence as shown in SEQ ID NO:18 or SEQ ID NO.

10. 10 . The nucleic acid molecule according to claim 1 , comprising a 3′UTR or a coding sequence for a 3′UTR, wherein the 3′UTR comprises or is the 3′UTR of hemoglobin-1 (hHBA1).

11. The nucleic acid molecule according to claim 10, wherein the nucleotide sequence of the hHBA1 3'UTR is as shown in SEQ ID NO. 12 or comprises a nucleotide sequence having at least 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with the nucleotide sequence as shown in SEQ ID NO:

12.

12. A nucleic acid molecule according to any one of claims 1 to 11, wherein the nucleotide sequence is as shown in any one of SEQ ID NOs. 2 to 7, or comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence as shown in any one of SEQ ID NOs. 2 to 7.

13. The nucleic acid molecule according to any one of claims 1 to 12, which is an mRNA molecule.

14. The nucleic acid molecule according to any one of claims 1 to 13, wherein the mRNA further comprises a 5' guanosine cap selected from any one of the following: m7Gppp(2'OMeA)pG, m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GpppCpG, m7GpppCpU, m7GpppGpA, m7GpppGpC, m7GpppGpG, m7GpppGpU, m7GpppUpA, m7GpppUpC, m7GpppUpG, m7GpppUpU, m7G 3’Ome pppApA, m7G 3’Ome pppApC, m7G 3’Ome pppApU、m7G 3’Ome pppApG, m7G 3’Ome pppCpA、m7G 3’Ome pppCpC, m7G 3’Ome pppCpG、m7G 3’Ome pppCpU、m7G 3’Ome pppUpA、m7G 3’Ome pppUpC、m7G 3’Ome pppUpG、m7G 3’Ome pppUpU、m7G 3’Ome PpA 2’Ome pG、m7G 3’Ome pppA 2’Ome pC、m7G 3’Ome pppA 2’Ome pU、m7G 3’Ome pppA 2’Ome pA, m7G 3’Ome pppC 2’Ome pA, m7G 3’Ome pppC 2’Ome pU、m7G 3’Ome pppC 2’Ome pG、m7G 3’Ome pppC 2’Ome pC、m7G 3’Ome pppG 2’Ome pA, m7G 3’Ome pppG 2’Ome pU、m7G 3’Ome pppG 2’Ome pG、m7G 3’Ome pppG 2’Ome pC、m7G 3’Ome U 2’Ome pA, m7G 3’Ome pppU 2’Ome pU, m7G 3’Ome pppU 2’Ome pG, m7G 3’Ome pppU 2’Ome pC, preference m7G(5')ppp(5')(2'OMeA)pG.

15. The nucleic acid molecule according to claim 13 or 14, wherein one or more of the uridines (U) in the nucleic acid molecule are base-modified uridines; preferably, the base-modified uridines are selected from any one or more of 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, 1-methyl pseudouridine (N1-Methyl-Pseudo-UTP) and pseudouridine; more preferably, each U in the nucleic acid molecule is 1-methyl pseudouridine.

16. A composition comprising a liposome and the nucleic acid molecule according to any one of claims 1 to 15.

17. The composition of claim 16, wherein the liposome is a lipid nanoparticle (LNP), and in terms of molar percentage (mol%), the LNP consists of 20-60 mol% ionizable cationic lipids, 25-55 mol% structural lipids, 5-25 mol% auxiliary lipids and 0.5-15 mol% surfactant.

18. The composition according to claim 16 or 17, wherein the lipid nanoparticles consist of 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG.

19. The composition according to any one of claims 16-18, wherein the molar ratio of the cationic tertiary amine in the lipid nanoparticle to the anionic phosphate group from the nucleic acid molecule according to any one of claims 1-15 is about 3:1 to 7:1, for example 6:

1.

20. The composition according to any one of claims 16 to 19, wherein the pH value thereof is equivalent to the normal physiological pH value of the human body, preferably about 7.3 to 7.5, for example 7.

4.

21. The composition according to any one of claims 16 to 20, further comprising Tris-NaOAc buffer and 8.7% sucrose.

22. A composition according to any one of claims 16-21, wherein the nucleic acid molecule is an mRNA having a sequence as shown in any one of SEQ ID NO.1-7, the lipid nanoparticles comprise 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% PEG2000-DMG, the molar ratio of the cationic tertiary amine in the LNP to the anionic phosphate group from the nucleic acid molecule is 6:1, and the pH value of the composition is 7.

4.

23. The antigenic peptide according to any one of claims 1-15.

24. An engineered cell comprising the nucleic acid molecule of any one of claims 1 to 15, or the antigenic peptide of claim 23.

25. Use of the nucleic acid molecule of any one of claims 1 to 15, the composition of any one of claims 16 to 22, the antigenic peptide of claim 23, or the engineered cell of claim 24 in the preparation of a vaccine.

26. A method for preventing VZV infection or symptoms caused by VZV, comprising administering to a subject an effective amount of the nucleic acid molecule of any one of claims 1 to 15, the composition of any one of claims 16 to 22, the antigenic peptide of claim 23, or the engineered cell of claim 24 to induce an immune response against varicella-zoster virus in the subject.

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