Varicella-zoster virus gE and gI fusion protein and application thereof

Abstract

The present invention provides fusion proteins (or variants thereof) of gE and gI useful for the prevention or treatment of varicella-zoster virus infection, and uses of such fusion proteins (or variants thereof).

Description

A fusion protein of varicella-zoster virus gE and gI and its application

[0001] This application is based on and claims priority to an application with CN application number 2023100792531 and application date January 19, 2023. The contents of the CN application are hereby incorporated into this application as a whole. Technical Field

[0002] The present invention relates to the fields of immunology and molecular virology, in particular to the prevention and treatment of varicella-zoster virus. Specifically, the present invention relates to a fusion protein of gE and gI (or a variant thereof) that can be used to prevent or treat varicella-zoster virus infection, and the use of such a fusion protein (or a variant thereof). Background Art

[0003] Varicella-zoster virus (VZV) is a DNA virus with an envelope. VZV is a spherical virus particle, composed of 150 hexamers and 12 pentamers assembled into a regular icosahedral structure with T=16. VZV virus particles can be roughly divided into four parts radiating outward from the center of the sphere, namely, viral nucleic acid (virus DNA), nucleocapsid (capsid), tegument protein (tegument), and membrane protein (envelop protein). The core of the virus is DNA, which controls the metabolic activity of the virus. The icosahedral nucleocapsid of VZV is very stable and plays an important role in protecting the genetic material inside. The outer layer of the viral capsid is tegument protein, which is composed of multiple proteins encoded by viral DNA and plays a role in viral infection, expression in host cells, immune response, assembly and exocytosis. The outer surface of VZV particles is a capsule formed by a variety of glycoproteins and lipids. The main glycoproteins include gB, gC, gE, gH, gL, gI, etc. They play an important role in identifying host cells, immune response, and enhancing the stability of viral particles.

[0004] When people are infected with VZV, they are prone to developing papules and chickenpox, often complicated by pneumonia, and have a high mortality rate (more common in children). After the host recovers, a small amount of VZV will still remain in its neuronal cells. When activated again, it will induce symptoms such as herpes zoster and post-synuclein pain. In severe cases, it will cause malignant complications such as encephalomyelitis and conjunctivitis (more common in immunodeficient or immunocompromised people aged ≥50 years). Vaccination is the best solution to prevent diseases such as chickenpox and herpes zoster, but there are currently few vaccines available. Shingrix, the world's first recombinant herpes zoster vaccine TMThe main protein component of VZV is the envelope glycoprotein gE of VZV, indicating that gE is an important target for the study of herpes zoster vaccines. As the glycoprotein with the highest content on the VZV envelope, gE contains a total of 623 amino acids, and some of its amino acid sequences play an important role in the propagation of VZV. Among them, the 24-71 and 163-208 amino acid sequences of gE are the key regions for its recognition of the IDE receptor and formation of heterodimers with gI, respectively. Whether gE can form the correct secondary structure is also the key to its interaction with IDE. Currently, some subunit or preclinical vaccines used to prevent VZV infection are designed around the gE protein. The content of the envelope glycoprotein gI on the VZV virus particle is second only to gE. gI contains a total of 354 amino acids, and its amino acid sequence can be roughly divided into three parts: the extracellular segment 1-271 amino acid sequence (the 1-20 amino acid sequence is a signal peptide), the hydrophobic transmembrane region, and the intracellular segment (including the C-terminus). When gI is missing, the replication rate of VZV is greatly slowed, and the synthesis of viral syncytia is severely affected. It plays a crucial role in the replication and amplification of VZV in culture models such as Vero cells, T cells, and human skin tissue. Furthermore, gI, as a molecular chaperone protein, promotes the maturation of gE protein. When the gI gene is knocked out, gE protein colocalizes with Golgi marker molecules in the perinuclear region and is unable to properly transport to the cytoplasmic membrane and participate in the assembly of VZV viral particles.

[0005] Current research on VZV infection and immune mechanisms has shown that a potent VZV-specific CD4+ T cell immune response is closely related to protective immunity against herpes zoster. TM Clinical studies of the vaccine have shown that it can induce gE-specific CD4+ T cell responses that are 10 times or more higher than those of the ZOSTAVAX vaccine, and the protection rate in people aged 60-60 years old reached 97.4%, higher than the 64% of the ZOSTAVAX vaccine.

[0006] However, there is still a need in the art for VZV vaccines with higher protective efficacy.

[0007] Summary of the Invention

[0008] The present invention designs a gE-gI fusion protein, demonstrates the protein preparation method and property identification results, and confirms that the gE-gI fusion protein can induce the body to produce high levels of neutralizing antibodies and cytokines, which is superior to the single gE protein, the combination of gE protein and gI protein, the gE-gI heterodimer protein and the marketed Shingrix TM The vaccine has a good immune stimulation effect. The fusion protein of the present invention has the advantage of being a diagnostic antigen or a vaccine candidate molecule in preventing and / or treating varicella-zoster virus infection.

[0009] Fusion protein

[0010] Therefore, in one aspect, the present application provides a fusion protein comprising a first peptide segment and a second peptide segment, wherein the first peptide segment comprises the extracellular region of varicella-zoster virus (VZV) gE protein or a fragment thereof, or is composed of the same; and the second peptide segment comprises the extracellular domain of VZV gI protein or a fragment thereof, or is composed of the same.

[0011] In certain embodiments, the first peptide segment comprises one or more of amino acids 24-71 and 163-208 of the VZV gE protein; and / or the second peptide segment comprises one or more of amino acids 95 and 105-125 of the VZV gI protein.

[0012] In certain embodiments, the first peptide segment comprises at least 100, at least 150, at least 200 or at least 250 consecutive amino acid residues of the amino acid residues corresponding to positions 150-410 of SEQ ID NO: 11 in the gE protein; and / or, the second peptide segment comprises at least 50, at least 100, at least 150 or at least 180 consecutive amino acid residues of the amino acid residues corresponding to positions 45-240 of SEQ ID NO: 12 in the gI protein.

[0013] In certain embodiments, the first peptide segment comprises a peptide segment corresponding to positions 150-410 (or 150-417, or 150-425, or 150-437, or 150-445, or 150-457, or 150-465, or 150-477, or 150-485, or 150-497, or 150-507) of the gE protein in SEQ ID NO: 11. , or 150-517, or 150-525, or 150-537, or 143-410, or 143-417, or 143-425, or 143-437, or 143-445, or 143-457, or 143-465, or 143-477, or 143-485, or 143-497, or 143-507, or 143-517, or 143-525, or 143-537, or 120-410, or 120-417, or 120-425, or 120-437, or 120-445, or 120-457, or 120-465, or 120-477, or 120-485, or 120-497, or 120-507, or 120-517, or 120-525, or 120-537, or 103-410, or 103-417, or 103-425, or 103-437, or 103-445, or 103-457, or 103-465, or 1 03-477, or 103-485, or 103-497, or 103-507, or 103-517, or 103-525, or 103-537, or 90-410, or 90-417, or 90-425, or 90-437, or 90-445, or 90-457, or 90-465, or 90-477, or 90-485, or 90-497, or 90-507, or 90-517, or 90-525, or 90-53 7, or 83-410, or 83-417, or 83-425, or 83-437, or 83-445, or 83-457, or 83-465, or 83-477, or 83-485, or 83-497, or 83-507, or 83-517, or 83-525, or 83-537, or 70-410, or 70-417, or 70-425, or 70-437, or 70-445, or 70-457, or 70-465,or 70-477, or 70-485, or 70-497, or 70-507, or 70-517, or 70-525, or 70-537, or 63-410, or 63-417, or 63-425, or 63-437, or 63-445, or 63-457, or 63-465, or 63-477, or 63-485, or 63-497, or 63-507, or 63-517, or 63- 525, or 63-537, or 50-410, or 50-417, or 50-425, or 50-437, or 50-445, or 50-457, or 50-465, or 50-477, or 50-485, or 50-497, or 50-507, or 50-517, or 50-525, or 50-537, or 43-410, or 43-417, or 43-425, or 43-437, or 43-445, or 43-457, or 43-465, or 43-477, or 43-485, or 43-497, or 43-507, or 43-517, or 43-525, or 43-537, or 31-410, or 31-417, or 31-425, or 31-437, or 31-445, or 31-457, or 31-465, or 31-477, or 31-485, or 31-4 97, or 31-507, or 31-517, or 31-525, or 31-537, or 23-410, or 23-417, or 23-425, or 23-437, or 23-445, or 23-457, or 23-465, or 23-477, or 23-485, or 23-497, or 23-507, or 23-517, or 23-525, or 23-537).

[0014] In certain embodiments, the first peptide segment comprises the 150th to 410th peptide segments of the gE protein corresponding to the peptide segments of SEQ ID NO: 11. 150-417, or 150-425, or 150-437, or 150-445, or 150-457, or 150-465, or 150-477, or 150-485, or 150-497, or 150-507, or 150-517, or 150-525, or 150-537, or 143-410, or 143-417, or 143-425, or 143-437, or 143-445, or 143-457, or 143-465, or 143-477, or 143 -485, or 143-497, or 143-507, or 143-517, or 143-525, or 143-537, or 120-410, or 120-417, or 120-425, or 120-437, or 120-445, or 120-457, or 120-465, or 120-477, or 120-485, or 120-497, or 120-507, or 120-517, or 120-525, or 120-537, or 103-410, or 103-417, or 1 03-425, or 103-437, or 103-445, or 103-457, or 103-465, or 103-477, or 103-485, or 103-497, or 103-507, or 103-517, or 103-525, or 103-537, or 90-410, or 90-417, or 90-425, or 90-437, or 90-445, or 90-457, or 90-465, or 90-477, or 90-485, or 90-497, or 90-507, or 90-517, or 90-525, or 90-537, or 83-410, or 83-417, or 83-425, or 83-437, or 83-445, or 83-457, or 83-465, or 83-477, or 83-485, or 83-497, or 83-507, or 83-517, or 83-525, or 83-537, or 70-410, or 70-417, or 70-425, or 70-437, or 70-445, or 70-457, or 70-465,or 70-477, or 70-485, or 70-497, or 70-507, or 70-517, or 70-525, or 70-537, or 63-410, or 63-417, or 63-425, or 63-437, or 63-445, or 63-457, or 63-465, or 63-477, or 63-485, or 63-497, or 63-507, or 63-517, or 63-5 25, or 63-537, or 50-410, or 50-417, or 50-425, or 50-437, or 50-445, or 50-457, or 50-465, or 50-477, or 50-485, or 50-497, or 50-507, or 50-517, or 50-525, or 50-537, or 43-410, or 43-417, or 43-425, or 43-437, or 43 -445, or 43-457, or 43-465, or 43-477, or 43-485, or 43-497, or 43-507, or 43-517, or 43-525, or 43-537, or 31-410, or 31-417, or 31-425, or 31-437, or 31-445, or 31-457, or 31-465, or 31-477, or 31-485, or 31-497, or 31-507, or 31-517, or 31-525, or 31-537, or 23-410, or 23-417, or 23-425, or 23-437, or 23-445, or 23-457, or 23-465, or 23-477, or 23-485, or 23-497, or 23-507, or 23-517, or 23-525, or 23-537) or consisting of the amino acid residues at the corresponding positions.

[0015] In certain embodiments, the first peptide segment comprises the same peptide segment as that of positions 70-485 (or 70-497, or 70-507, or 70-517, or 70-525, or 70-537, or 63-485, or 63-497, or 63-507, or 63-517, or 63-525, or 63-537, or 50-485, or 50-497, or 50-507, or 50-517, or 50-525, or 50-537, or 4 or 23-485, or 23-497, or 23-507, or 23-517, or 23-525, or 23-537).

[0016] In certain embodiments, the first peptide segment comprises the same peptide segment as positions 70-485 (or 70-497, or 70-507, or 70-517, or 70-525, or 70-537, or 63-485, or 63-497, or 63-507, or 63-517, or 63-525, or 63-537, or 50-485, or 50-497, or 50-507, or 50-517, or 50-525, or 50-537, or 43-4 85, or 43-497, or 43-507, or 43-517, or 43-525, or 43-537, or 31-485, or 31-497, or 31-507, or 31-517, or 31-525, or 31-537, or 23-485, or 23-497, or 23-507, or 23-517, or 23-525, or 23-537).

[0017] In certain embodiments, the first peptide segment comprises the same peptide segment as that of positions 63-497 (or 63-507, or 63-517, or 63-525, or 63-537, or 50-497, or 50-507, or 50-517, or 50-525, or 50-537, or 43-497, or 43-507, or 43-517) of the gE protein as that of positions 63-497 (or 63-507, or 63-517, or 63-525, or 63-537, or 50-497, or 50-507, or 50-517, or 50-525, or 50-537, or 43-497, or 43-507, or 43-517 or 43-525, or 43-537, or 31-497, or 31-507, or 31-517, or 31-525, or 31-537, or 23-497, or 23-507, or 23-517, or 23-525, or 23-537).

[0018] In certain embodiments, the first peptide segment comprises the same peptide segment as that of positions 63-497 (or 63-507, or 63-517, or 63-525, or 63-537, or 50-497, or 50-507, or 50-517, or 50-525, or 50-537, or 43-497, or 43-507, or 43-517, or or consisting of amino acid residues at positions 43-525, or 43-537, or 31-497, or 31-507, or 31-517, or 31-525, or 31-537, or 23-497, or 23-507, or 23-517, or 23-525, or 23-537).

[0019] In certain embodiments, the first peptide segment comprises amino acid residues at positions corresponding to positions 31-537 of SEQ ID NO: 11 in the gE protein.

[0020] In certain embodiments, the first peptide segment comprises or consists of amino acid residues at positions corresponding to positions 31-537 or 23-537 of SEQ ID NO: 11 in the gE protein.

[0021] In certain embodiments, the first peptide segment consists of the extracellular region of the gE protein. In certain embodiments, the second peptide segment is optionally connected to the C-terminus of the first peptide segment via a linker (e.g., a peptide linker, e.g., a peptide linker set forth in SEQ ID NO: 15), and the first peptide segment consists of the extracellular region of the gE protein.

[0022] In certain embodiments, the first peptide segment consists of the extracellular region of the gE protein without a signal peptide or containing a partial signal peptide sequence.

[0023] In certain embodiments, the first peptide segment comprises VZV or consisting of amino acids 23-350 (or 23-358, or 23-365, or 23-375, or 23-385, or 23-401, or 23-410, or 23-420, or 23-430, or 23-440, or 23-500, or 23-510, or 23-520, or 23-537, or 23-538, or 23-539, or 23-540, or 23-541, or 23-542, or 23-543, or 23-544, or 23-545, or 23-546) of the gE protein.

[0024] In certain embodiments, the first peptide segment comprises VZV or consisting of amino acids 27-350 (or 27-358, or 27-365, or 27-375, or 27-385, or 27-401, or 27-410, or 27-420, or 27-430, or 27-440, or 27-500, or 27-510, or 27-520, or 27-537, or 27-538, or 27-539, or 27-540, or 27-541, or 27-542, or 27-543, or 27-544, or 27-545, or 27-546) of the gE protein.

[0025] In certain embodiments, the first peptide segment comprises VZV or consisting of amino acids 31-350 (or 31-358, or 31-365, or 31-375, or 31-385, or 31-401, or 31-410, or 31-420, or 31-430, or 31-440, or 31-500, or 31-510, or 31-520, or 31-537, or 31-538, or 31-539, or 31-540, or 31-541, or 31-542, or 31-543, or 31-544, or 31-545, or 31-546) of the gE protein.

[0026] In certain embodiments, the first peptide segment comprises or consists of amino acid residues 31-510 of the VZV gE protein.

[0027] In certain embodiments, the first peptide segment comprises or consists of amino acids 23-537 (or 23-538, or 23-539, or 23-540, or 23-541, or 23-542, or 23-543, or 23-544, or 23-545, or 23-546) of the VZV gE protein.

[0028] In certain embodiments, the first peptide segment comprises or consists of amino acids 23-537 of the VZV gE protein.

[0029] In certain embodiments, the gE protein has: (a) an amino acid sequence as shown in any one of SEQ ID NOs: 11, 46-55; (b) an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to the amino acid sequence shown in any one of SEQ ID NOs: 11, 46-55; or, (c) a sequence having one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence shown in any one of SEQ ID NOs: 11, 46-55.

[0030] In certain embodiments, the amino acid sequence of the gE protein is shown in SEQ ID NO:11.

[0031] In certain embodiments, the first peptide segment comprises an amino acid sequence as shown in any one of SEQ ID NOs: 13, 33-42, 64-69, or has at least 90%, for example, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in any one of SEQ ID NOs: 13, 33-42, 64-69, or has one or several (for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions, or consists of a sequence thereof.

[0032] In certain embodiments, the second peptide segment comprises amino acid residues at positions corresponding to positions 45-240 (or 45-251, or 45-260, or 45-271, or 41-240, or 41-251, or 41-260, or 41-271, or 30-240, or 30-251, or 30-260, or 30-271, or 21-240, or 21-251, or 21-260, or 21-271) of the gI protein.

[0033] In certain embodiments, the second peptide segment comprises or consists of amino acid residues at positions corresponding to positions 45-240 (or 45-251, or 45-260, or 45-271, or 41-240, or 41-251, or 41-260, or 41-271, or 30-240, or 30-251, or 30-260, or 30-271, or 21-240, or 21-251, or 21-260, or 21-271) of the gI protein.

[0034] In certain embodiments, the second peptide segment comprises amino acid residues at positions corresponding to positions 30-260 (or positions 30-271, or positions 21-260, or positions 21-271) of SEQ ID NO:12 in the gI protein.

[0035] In certain embodiments, the second peptide segment comprises or consists of amino acid residues at positions corresponding to positions 30-260 (or positions 30-271, or positions 21-260, or positions 21-271) of SEQ ID NO: 12 in the gI protein.

[0036] In certain embodiments, the second peptide segment comprises amino acid residues at positions 21-271 of the gI protein corresponding to positions 21-271 of SEQ ID NO:12.

[0037] In certain embodiments, the second peptide segment consists of the extracellular region of the gI protein. In certain embodiments, the second peptide segment is optionally linked to the N-terminus of the first peptide segment via a linker (e.g., a peptide linker, e.g., a peptide linker set forth in SEQ ID NO: 15), and the second peptide segment consists of the extracellular region of the gI protein.

[0038] In certain embodiments, the second peptide segment consists of the extracellular region of the gI protein without a signal peptide or containing a partial signal peptide sequence.

[0039] In certain embodiments, the second peptide segment comprises or consists of amino acid residues at positions corresponding to positions 21-271 of SEQ ID NO: 12 in the gI protein.

[0040] In certain embodiments, the second peptide segment comprises or consists of amino acids 21-220 (or 21-230, or 21-235, or 21-250, or 21-260, or 21-271) of the VZV gI protein.

[0041] In certain embodiments, the second peptide segment comprises or consists of amino acids 30-220 (or 30-230, or 30-235, or 30-250, or 30-260, or 30-271) of the VZV gI protein.

[0042] In certain embodiments, the second peptide segment comprises or consists of amino acids 36-220 (or 36-230, or 36-235, or 36-250, or 36-260, or 36-271) of the VZV gI protein.

[0043] In certain embodiments, the first peptide segment comprises the same peptide segment as that at positions 63-497 (or 63-507, or 63-517, or 63-525, or 63-537, or 50-497, or 50-507, or 50-517, or 50-525, or 50-537, or 43-497, or 43-507, or 43-517, or 43-525) of the gE protein as that at positions 63-497 (or 63-507, or 63-517, or 63-525, or 63-537, or 50-497, or 50-507, or 50-517, or 50-525, or 50-537, or 43-497, or 43-507, or 43-517, or 43-525) of SEQ ID NO: 11. , or 43-537, or 31-497, or 31-507, or 31-517, or 31-525, or 31-537, or 23-497, or 23-507, or 23-517, or 23-525, or 23-537) of the gI protein; and the second peptide segment comprises the amino acid residues at positions corresponding to positions 30-260 (or 30-271, or 21-260, or 21-271) of SEQ ID NO: 12 in the gI protein.

[0044] In certain embodiments, the first peptide segment comprises the same peptide segment as that at positions 63-497 (or 63-507, or 63-517, or 63-525, or 63-537, or 50-497, or 50-507, or 50-517, or 50-525, or 50-537, or 43-497, or 43-507, or 43-517, or 43-525, or 43-537) of the gE protein as that at positions 63-497 (or 63-507, or 63-517, or 63-525, or 63-537, or 50-497, or 50-507, or 50-517, or 50-525, or 50-537, or 43-497, or 43-507, or 43-517, or 43-525, or 43-537) of SEQ ID NO: 11. : The second peptide segment comprises or consists of the amino acid residues at the positions corresponding to positions 30-260 (or positions 30-271, or positions 21-260, or positions 21-271) of SEQ ID NO: 12 in the gI protein.

[0045] In certain embodiments, the gI protein has: (a) an amino acid sequence as shown in any one of SEQ ID NOs: 12, 56-58; (b) an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to the amino acid sequence shown in any one of SEQ ID NOs: 12, 56-58; or, (c) one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence shown in any one of SEQ ID NOs: 12, 56-58.

[0046] In certain embodiments, the amino acid sequence of the gI protein is shown in SEQ ID NO:12.

[0047] In certain embodiments, the second peptide segment comprises or consists of amino acids 36-235 of the VZV gI protein.

[0048] In certain embodiments, the second peptide segment comprises or consists of amino acids 21-271 of the VZV gI protein.

[0049] In certain embodiments, the second peptide segment comprises an amino acid sequence as shown in any one of SEQ ID NOs: 14, 43-45, or has at least 90%, for example, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any one of SEQ ID NOs: 14, 43-45, or has a sequence having one or several (for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions, or consists of the same.

[0050] In certain embodiments, the second peptide segment is optionally linked to the N-terminus or C-terminus of the first peptide segment via a linker (eg, a peptide linker, eg, a peptide linker shown in SEQ ID NO: 15).

[0051] In certain embodiments, the linker is a peptide linker comprising one or more (eg, 5-20) flexible amino acids.

[0052] In certain embodiments, the linker is a peptide linker comprising one or more glycines and / or one or more serines.

[0053] In certain embodiments, the fusion protein comprises or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 6-7, 20-32, 59-63, and 70.

[0054] It is readily understood by those skilled in the art that, to facilitate protein expression, the fusion protein optionally includes a signal peptide sequence. The signal peptide sequence may be present within the first peptide segment or the second peptide segment, or may be present outside the first and second peptide segments (e.g., the first and second peptide segments do not contain a signal peptide sequence, and the fusion protein additionally includes a signal peptide sequence outside the first and second peptide segments).

[0055] In certain embodiments, the second peptide segment is optionally linked to the N-terminus of the first peptide segment via a linker (e.g., a peptide linker, e.g., a peptide linker set forth in SEQ ID NO: 15), and the second peptide segment comprises a signal peptide sequence at its N-terminus (e.g., a signal peptide sequence derived from the VZV gI protein). In certain embodiments, the second peptide segment is optionally linked to the C-terminus of the first peptide segment via a linker (e.g., a peptide linker, e.g., a peptide linker set forth in SEQ ID NO: 15), and the first peptide segment comprises a signal peptide sequence at its N-terminus (e.g., a signal peptide sequence derived from the VZV gE protein). In certain embodiments, neither the first peptide segment nor the second peptide segment comprises a signal peptide sequence, and the fusion protein further comprises a signal peptide sequence at its N-terminus.

[0056] In certain embodiments, the fusion protein comprises a signal peptide and / or a detectable label (eg, a tag protein).

[0057] In certain embodiments, the fusion protein comprises a signal peptide at its N-terminus (e.g., the native signal peptide of VZV gE protein, the native signal peptide of VZV gI protein, gp67 signal peptide, melittin signal peptide (MSP), or tissue plasminogen activator signal peptide (tPA signal peptide)).

[0058] In certain embodiments, the fusion protein comprises a detectable label at its C-terminus.

[0059] On the other hand, the present application also provides an RNA molecule comprising a nucleotide sequence encoding the fusion protein as described above.

[0060] In certain embodiments, the nucleotide sequence encoding the fusion protein is codon-optimized or non-optimized according to the codon preference of the host cell (eg, insect cell or mammalian cell).

[0061] In certain embodiments, the RNA molecule further comprises one or more selected from the group consisting of a 5'UTR, a Kozak sequence, a start codon, a stop codon, a 3'UTR, and a poly-A tail.

[0062] In certain embodiments, the RNA molecule comprises, from the 5' end to the 3' end, a 5'UTR, a Kozak sequence, a start codon, a nucleotide sequence encoding the fusion protein, a stop codon, a 3'UTR, and a poly-A tail.

[0063] In certain embodiments, the RNA molecule has one or more characteristics selected from the group consisting of:

[0064] (1) the nucleotide sequence encoding the fusion protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO:71; in certain embodiments, the nucleotide sequence encoding the fusion protein is shown in SEQ ID NO:71;

[0065] (2) There are one or more stop codons at the 3' end of the nucleotide sequence encoding the fusion protein;

[0066] (3) the 5'UTR has a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 17; in certain embodiments, the 5'UTR has the nucleotide sequence of SEQ ID NO: 17;

[0067] (4) the 3'UTR has a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 18; in certain embodiments, the 3'UTR has the nucleotide sequence of SEQ ID NO: 18;

[0068] (5) the Kozak sequence is shown in SEQ ID NO: 72;

[0069] (6) The poly-A tail comprises one or more poly(A) sequences, each of which independently consists of 20-120 consecutive adenine nucleotides; preferably, the poly-A tail comprises multiple poly(A) sequences, and adjacent poly(A) sequences are connected by a non-A spacer sequence; preferably, the poly-A tail has a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19; in certain embodiments, the poly-A tail has the nucleotide sequence shown in SEQ ID NO: 19;

[0070] (7) The 5' end of the RNA molecule is modified with a 5' cap (e.g., CAP type 0, CAP type 1, CAP type 2).

[0071] On the other hand, the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein or the RNA molecule as described above.

[0072] In certain embodiments, the nucleotide sequence encoding the fusion protein is codon-optimized or non-optimized according to the codon preference of the host cell (eg, insect cell or mammalian cell).

[0073] On the other hand, the present application also provides a vector comprising the isolated nucleic acid molecule as described above.

[0074] In another aspect, the present application provides a delivery composition comprising a delivery vehicle and an RNA molecule, an isolated nucleic acid molecule or a vector as described above.

[0075] In certain embodiments, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, and viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses).

[0076] In certain embodiments, the delivery composition comprises an RNA molecule as described above.

[0077] In certain embodiments, the delivery vehicle is a lipid nanoparticle (LNP).

[0078] In certain embodiments, the lipid nanoparticles comprise lipids ALC-0315, ALC-0519, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), and cholesterol.

[0079] In certain embodiments, the molar percentage of ALC-0315 in the lipid nanoparticles is in the range of 40%-55% (e.g., 40%-46.3%, 40%-48%, 40%-50%, 40%-52%, 43%-46.3%, 43%-58%, 43%-50%, 43%-52%, 43%-55%). In certain embodiments, the molar percentage of ALC-0315 in the lipid nanoparticles is 46.3%.

[0080] In certain embodiments, the lipid nanoparticles have a molar percentage of cholesterol in the range of 35%-50% (e.g., 35%-42.7%, 35%-45%, 40%-42.7%, 40%-45%, 40%-50%). In certain embodiments, the lipid nanoparticles have a molar percentage of cholesterol of 42.7%.

[0081] In certain embodiments, the molar percentage of DSPC in the lipid nanoparticles is in the range of 5%-12% (e.g., 5%-9.4%, 5%-10%, 5%-11%, 8%-9.4%, 8%-10%, 8%-11%, 8%-12%, 9%-9.4%, 9%-10%, 9%-11%, 9%-12%). In certain embodiments, the molar percentage of DSPC in the lipid nanoparticles is 46.3%.

[0082] In certain embodiments, the molar percentage of ALC-0519 in the lipid nanoparticles is in the range of 1.0%-2.0% (e.g., 1.0%-1.6%, 1.0%-1.8%, 1.3%-1.6%, 1.3%-1.8%, 1.3%-2.0%). In certain embodiments, the molar percentage of ALC-0519 in the lipid nanoparticles is 1.6%.

[0083] In certain embodiments, the delivery composition is a vaccine (eg, a nucleic acid vaccine).

[0084] On the other hand, the present application also provides a host cell comprising the RNA molecule, isolated nucleic acid molecule or vector as described above.

[0085] In certain embodiments, the host cell is selected from prokaryotic cells (eg, E. coli cells), and eukaryotic cells (eg, yeast cells, insect cells, plant cells, mammalian cells).

[0086] In certain embodiments, the host cell is an insect cell.

[0087] In certain embodiments, the host cell is a mammalian cell.

[0088] On the other hand, the present application also provides a method for preparing the fusion protein as described above, which comprises culturing the host cell as described above under conditions allowing protein expression, and recovering the fusion protein from the cell culture.

[0089] Therapeutic uses

[0090] On the other hand, the present application also provides an immunogenic composition comprising the fusion protein, RNA molecule, isolated nucleic acid molecule, vector or delivery composition as described above, and optionally a pharmaceutically acceptable carrier and / or excipient (eg, adjuvant).

[0091] In certain embodiments, the immunogenic composition comprises a fusion protein, RNA molecule, isolated nucleic acid molecule, carrier or delivery composition and adjuvant as described above, wherein the adjuvant is selected from: aluminum salt adjuvants, zinc-aluminum mixed adjuvants (e.g., FH002C), Freund's adjuvant, oil emulsion adjuvants (e.g., MF59 adjuvant), cytokines, TLR agonists, CpG adjuvants, nucleic acid adjuvants, liposomes, saponin adjuvants, AS01B adjuvants and any combination thereof.

[0092] In certain embodiments, the immunogenic composition comprises a fusion protein or delivery composition as described above, and the adjuvant.

[0093] In certain embodiments, the immunogenic composition comprises a fusion protein as described above, and an AS01B adjuvant.

[0094] In certain embodiments, the immunogenic composition comprises a delivery composition as described above, and a CpG adjuvant.

[0095] In certain embodiments, the immunogenic composition is a vaccine.

[0096] The immunogenic compositions described above can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The immunogenic compositions of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0097] Immunogenic compositions as above can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, inguinal, intravesical, local (such as, powder, ointment or drops), or nasal route.But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection).Technician will appreciate that route of administration and / or mode will change according to intended purpose.

[0098] The immunogenic composition described above should be administered in an amount sufficient to induce an immune response against VZV. The appropriate amount of immunogen can be determined based on the specific disease or condition to be treated or prevented, its severity, the age of the subject, and other personal attributes of the specific subject (e.g., the subject's general state of health and the robustness of the subject's immune system). The determination of an effective dose is also guided by animal model studies, followed by human clinical trials, and by an administration regimen that significantly reduces the occurrence or severity of the target disease symptoms or conditions in the subject.

[0099] On the other hand, the present application also provides the use of the fusion protein, or RNA molecule, or isolated nucleic acid molecule, or vector, or delivery composition, or host cell, or immunogenic composition as described above in the preparation of an immunogenic composition, which is used to induce an immune response against VZV in a subject and / or to prevent and / or treat VZV infection or a disease associated with VZV infection in a subject.

[0100] In certain embodiments, the immunogenic composition is a vaccine.

[0101] In certain embodiments, the VZV infection is a primary VZV infection or a recurrent infection.

[0102] In certain embodiments, the disease associated with VZV infection is selected from the group consisting of: herpes zoster, varicella, and complications thereof (eg, neuropathic pain, pneumonia, encephalomyelitis, conjunctivitis).

[0103] In certain embodiments, the subject is a mammal, such as a human.

[0104] On the other hand, the present application also provides a method for inducing an immune response against VZV in a subject and / or for preventing and / or treating VZV infection or a disease associated with VZV infection in a subject, comprising: administering to a subject in need thereof an effective amount of the fusion protein, RNA molecule, isolated nucleic acid molecule, vector, delivery composition, host cell or immunogenic composition as described above.

[0105] In certain embodiments, the VZV infection is a primary VZV infection or a recurrent infection.

[0106] In certain embodiments, the disease associated with VZV infection is selected from the group consisting of: herpes zoster, varicella, and complications thereof (eg, neuropathic pain, pneumonia, encephalomyelitis, conjunctivitis).

[0107] In certain embodiments, the subject is a mammal, such as a human.

[0108] Detection purpose

[0109] On the other hand, the present application also provides a method for detecting the presence of antibodies specific to VZV gI protein and / or VZV gE protein in a sample, which comprises using the fusion protein as described above.

[0110] In certain embodiments, the method is an immunological assay, such as immunoblotting, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0111] In certain embodiments, the method comprises: (1) contacting the sample with the fusion protein as described above; (2) detecting the formation of a fusion protein-antibody immune complex or detecting the amount of the immune complex; the formation of the immune complex indicates the presence of VZV gI protein and / or VZV gE protein-specific antibodies in the sample.

[0112] In certain embodiments, the method further comprises using a secondary antibody with a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin) to detect the presence of antibodies specific for the VZV gI protein and / or VZV gE protein in the sample.

[0113] In certain embodiments, the second antibody is specific for a constant region contained in an antibody of the species (eg, human) from which the test sample originates.

[0114] In certain embodiments, the second antibody is an anti-immunoglobulin (eg, human immunoglobulin) antibody, such as an anti-IgG antibody.

[0115] In certain embodiments, the sample is a bodily fluid sample (eg, whole blood, plasma, serum, salivary fluid, or urine) from a subject (eg, a mammal, preferably a human).

[0116] In certain embodiments, the methods are used for diagnostic purposes or non-diagnostic purposes. In certain embodiments, the methods are used for non-diagnostic purposes.

[0117] On the other hand, the present application also provides the use of the fusion protein, or RNA molecule, or isolated nucleic acid molecule, or vector, or delivery composition or host cell as described above in the preparation of a detection reagent for detecting the presence of VZV gI protein and / or VZV gE protein-specific antibodies in a sample.

[0118] In certain embodiments, the detection reagent detects the presence of VZV gI protein and / or VZV gE protein-specific antibodies in a sample by the method for detecting the presence of VZV gI protein and / or VZV gE protein-specific antibodies in a sample as described above.

[0119] In certain embodiments, the sample is a bodily fluid sample (eg, whole blood, plasma, serum, salivary fluid, or urine) from a subject (eg, a mammal, preferably a human).

[0120] Reagent test kit

[0121] On the other hand, the present application also provides a kit comprising the fusion protein, or RNA molecule, or isolated nucleic acid molecule, or vector, or delivery composition, or host cell as described above.

[0122] In certain embodiments, the kit comprises a fusion protein as described above, and a second antibody, wherein the second antibody is as defined above.

[0123] Definition of terms

[0124] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0125] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0126] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0127] As used herein, the term "VZV" is short for "Varicella-zoster virus," which is a DNA virus.

[0128] As used herein, the terms "gE protein," "gE," and "glycoprotein gE" refer to a VZV envelope glycoprotein and have the same meaning and are used interchangeably. The specific amino acid sequence of the wild-type gE protein can be obtained from public databases (e.g., the GenBank database), for example, the amino acid sequence shown in GenBank Accession Nos. AB097933 (pOka strain), AB097932 (vOka strain), NC001348 (Dumas strain), AY548170 (MSP strain), QCA45176.1, AEW89412.1, ANS12941.1, QCA43570.1, QWE79571.1, AEW89124.1, AQT34120.1, or AEW88980.1.

[0129] In the present invention, when referring to the amino acid sequence of the gE protein, the description is made with reference to the sequence set forth in SEQ ID NO: 11. For example, the expression "amino acid residues at positions 150-410 of the gE protein corresponding to positions 150-410 of SEQ ID NO: 11" refers to the amino acid sites / residues in the compared sequence that are at equivalent positions to amino acid residues 150-410 of SEQ ID NO: 11 when the sequence is optimally aligned with SEQ ID NO: 11, i.e., when the sequence is aligned with SEQ ID NO: 11 to achieve the highest percentage identity.

[0130] Those skilled in the art will appreciate that VZV may include multiple isolates, and that differences may exist between the amino acid sequences of the gE proteins of various isolates. Furthermore, those skilled in the art will appreciate that, despite possible sequence differences, the gE proteins of different VZV isolates share a very high degree of identity in amino acid sequence (typically greater than 95%, such as greater than 96%, greater than 97%, greater than 98%, or greater than 99%) and possess substantially the same biological function. Therefore, in the present invention, the term "gE protein" includes not only the protein set forth in any one of SEQ ID NOs: 11, 46-55, but also the gE proteins of various VZV isolates. Furthermore, when describing a sequence fragment of a gE protein, it includes not only a sequence fragment of any one of SEQ ID NOs: 11, 46-55, but also the corresponding sequence fragments in the gE proteins of various VZV isolates. For example, the expression "amino acid residues 23-537 of the gE protein" includes amino acid residues 23-537 of any one of SEQ ID NOs: 11, 46-55, and corresponding fragments in the gE proteins of various VZV isolates.

[0131] As used herein, "gE protein extracellular region" refers to the part of the gE protein that does not include the intracellular region and the transmembrane region. For example, from the analysis of the specific sequence, in certain embodiments, the gE protein extracellular region refers to the gE protein at least comprising the amino acid residues at the corresponding amino acid positions with the 1-537 amino acids of SEQ ID NO: 11. Currently, studies have also shown that the amino acid residues at the corresponding amino acid positions with the 1-546 amino acids of SEQ ID NO: 11 in the gE protein can also serve as the corresponding functions of the gE protein extracellular region. Therefore, in certain embodiments, the gE protein extracellular region refers to the amino acid residues at the corresponding amino acid positions with the 1-546 (or 1-538, or 1-539, or 1-540, or 1-541, or 1-542, or 1-543, or 1-544, or 1-545) amino acids of SEQ ID NO: 11 in the gE protein. Those skilled in the art will appreciate that mutations or variations (including but not limited to substitutions, deletions and / or additions) may be naturally generated or artificially introduced into SEQ ID NO: 11 without affecting the biological properties of the gE protein. For example, the gE proteins of different strains of VZV may differ naturally in amino acid sequence but have substantially the same biological properties. Therefore, when describing the extracellular region of the gE protein, it includes not only amino acid residues 1-537 (or 1-538, or 1-539, or 1-540, or 1-541, or 1-542, or 1-543, or 1-544, or 1-545, or 1-546) of SEQ ID NO: 11, but also amino acid residues 1-537 (or 1-538, or 1-539, or 1-540, or 1-541, or 1-542, or 1-543, or 1-544, or 1-545, or 1-546) of SEQ ID NO: 11, and ... The amino acids 1-537 (or 1-538, or 1-539, or 1-540, or 1-541, or 1-542, or 1-543, or 1-544, or 1-545, or 1-546) of NO:11 are at the amino acid residues at the corresponding amino acid positions.

[0132] As used herein, the terms "gI protein," "gI," and "glycoprotein gI" refer to a VZV envelope glycoprotein and have the same meaning and are used interchangeably. The specific amino acid sequence of the wild-type gI protein can be obtained from public databases (e.g., the GenBank database), for example, the amino acid sequences set forth in GenBank Accession Nos. AB097933 (pOka strain), AB097932 (vOka strain), NC001348 (Dumas strain), AY548170 (MSP strain), AEW89483.1, AGY34059.1, or AEW89051.1.

[0133] In the present invention, when referring to the amino acid sequence of the gI protein, the description is made with reference to the sequence set forth in SEQ ID NO: 12. For example, the expression "amino acid residues at positions 45-240 of the gI protein corresponding to positions 45-240 of SEQ ID NO: 12" refers to the amino acid sites / residues in the compared sequence that are at equivalent positions to amino acid residues 45-240 of SEQ ID NO: 12 when the sequence is optimally aligned with SEQ ID NO: 12, i.e., when the sequence is aligned with SEQ ID NO: 12 to achieve the highest percentage identity.

[0134] Those skilled in the art will appreciate that VZV may include multiple isolates, and that there may be differences in the amino acid sequences of the gI proteins of various isolates. Furthermore, those skilled in the art will appreciate that, despite possible sequence differences, the gI proteins of different VZV isolates have extremely high amino acid sequence identity (typically greater than 95%, such as greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or greater than 99.8%) and have substantially the same biological function. Therefore, in the present invention, the term "gI protein" includes not only the protein shown in any one of SEQ ID NOs: 12 and 56-58, but also the gI proteins of various VZV isolates. Furthermore, when describing a sequence fragment of the gI protein, it includes not only the sequence fragment of any one of SEQ ID NOs: 12 and 56-58, but also the corresponding sequence fragments in the gI proteins of various VZV isolates. For example, the expression "amino acid residues 21-271 of gI protein" includes amino acid residues 21-271 of any one of SEQ ID NOs: 12, 56-58, and corresponding fragments in gI proteins of various VZV isolates.

[0135] As used herein, the "gI protein extracellular region" refers to the part that does not include the intracellular region and the transmembrane region in the gI protein. For example, from the analysis of the specific sequence, in certain embodiments, the gI protein extracellular region refers to the amino acid residues in the gI protein that are in the corresponding amino acid positions with the 1st-271st amino acid of SEQ ID NO:12. It will be understood by those skilled in the art that mutations or variations (including but not limited to, replacement, deletion and / or addition) can be naturally produced or artificially introduced in SEQ ID NO:12 without affecting the biological properties of the gI protein. For example, the gI protein of the different strains of VZV can naturally have differences in amino acid sequence, but has substantially the same biological properties. Therefore, when describing the extracellular region of the gI protein, it not only includes the 1st-271st amino acid residues of SEQ ID NO:12, but also includes the amino acid residues in the corresponding amino acid positions with the 1st-271st amino acid of SEQ ID NO:12 in the natural or artificial variants (for example, the gI protein as shown in any one of SEQ ID NO:56-58).

[0136] According to the present invention, the expressions "corresponding sequence fragments" or "corresponding amino acid positions" refer to fragments or amino acid sites / residues that are located at equivalent positions in the compared sequences when the sequences are optimally aligned, i.e. when the sequences are aligned to obtain the highest percentage identity.

[0137] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0138] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0139] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material elements it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain an initiation of replication site.

[0140] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0141] Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the desired expression level, etc. A vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including proteins, isolated nucleic acid molecules, etc., as described herein.

[0142] According to the present invention, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; adjuvants include, but are not limited to, aluminum salt adjuvants, zinc-aluminum mixed adjuvants (e.g., FH002C), Freund's adjuvant, oil emulsion adjuvant, cytokines, TLR agonists, CpG adjuvants, liposomes, AS01B adjuvants, or combinations thereof; ionic strength enhancers include, but are not limited to, sodium chloride.

[0143] According to the present invention, the term "adjuvant" refers to a nonspecific immunopotentiator that, when delivered to the body together with an antigen or in advance, can enhance the body's immune response to the antigen or change the type of immune response. There are many types of adjuvants, including but not limited to aluminum salt adjuvants, zinc-aluminum mixed adjuvants (e.g., FH002C), Freund's adjuvant, oil emulsion adjuvants (e.g., MF59 adjuvant), cytokines, TLR agonists, CpG adjuvants, liposomes, AS01B adjuvants, or any combination thereof. In the present invention, it is particularly preferred that the adjuvant is AS01B adjuvant, CpG adjuvant, or any combination thereof.

[0144] According to the present invention, the term "effective amount" refers to an amount that is effective for achieving the intended purpose. For example, an effective amount for preventing or treating a disease (e.g., VZV infection) refers to an amount that is effective for preventing, arresting, or delaying the onset of a disease (e.g., VZV infection), or alleviating, reducing, or treating the severity of an existing disease (e.g., a disease caused by VZV infection). Determining such an effective amount is within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered treatments.

[0145] In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0146] As used herein, "subject" refers to an animal, such as a vertebrate. Preferably, the subject is a mammal, such as a human, bovine, equine, feline, canine, rodent, or primate. Particularly preferably, the subject is a human. This term is used interchangeably with "patient" herein.

[0147] Advantageous Effects of the Invention

[0148] The VZV gE-gI fusion protein of the present invention exhibits excellent reactivity with gE and / or gI-specific murine monoclonal antibodies. This design, unlike previously reported gE-gI dimer designs, achieves fusion expression of gE and gI through a single plasmid and single clone, while retaining the reactivity of both gE and gI proteins. It can be used as a detection antigen in diagnostic reagents and an immunogen in vaccines, demonstrating its potential and value in varicella-zoster virus diagnosis.

[0149] In particular, compared to gE protein alone, a combination of gE protein and gI protein, gE-gI heterodimer protein, the marketed Shingrix TM The vaccine, the gE-gI fusion protein of the present invention has significantly excellent inducing neutralizing antibody and T cell reactivity, showing its application prospect and value in the prevention and / or treatment of herpes zoster.

[0150] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0151] Figure 1 shows the SDS-polyacrylamide gel electrophoresis (left) and Western Blot identification results (right) of the insect cell-derived gE-gI fusion protein samples at each purification stage (after purification using a Q-FF column (Figure 1A), a DEAE-FF column (Figure 1B), and a Butyl column (Figure 1C)) in Example 1, wherein lane M: molecular weight marker; lane 1: gE-gI fusion protein.

[0152] Figure 2 shows the SDS-polyacrylamide gel electrophoresis (left) and Western Blot identification results (right) of the gE-gI fusion protein samples from the CHO source at each purification stage (Ni-6FF column, Figure 2A) and the molecular sieve Superdex200 Increase (Figure 2B) in Example 1, wherein lane M: molecular weight marker; lane 1: gE-gI fusion protein.

[0153] FIG3 shows the results of high performance size exclusion chromatography (HPSEC) of the insect cell-derived gE-gI fusion protein in Example 2.

[0154] FIG4 shows the results of high performance size exclusion chromatography (HPSEC) of the CHO-derived gE-gI fusion protein in Example 2.

[0155] FIG5 shows the analytical ultracentrifugation results of the insect cell-derived gE-gI fusion protein in Example 3.

[0156] FIG6 shows the analytical ultracentrifugation results of the CHO-derived gE-gI fusion protein in Example 3.

[0157] Figure 7 shows the results of enzyme-linked immunosorbent assay (ELISA) of the insect cell-derived gE-gI fusion protein in Example 4 with gE protein-specific antibodies (6G7, 6H6, 9H9, 10H6, 11B11, 11B12, 11E3, 12E12, 13B6, 14G1, 17B7) and gI protein-specific antibodies (12D2, 13B2, 14B11, 18B2, 19F2).

[0158] Figure 8 shows the results of enzyme-linked immunosorbent assay (ELISA) of the CHO-derived gE-gI fusion protein in Example 4 with gE protein-specific antibodies (1B11, 4G4, 6G7, 6H6, 11B11, 11B12, 11E3, 14G1) and gI protein-specific antibodies (7E7, 18B2, 9D11, 16F1, 13B2).

[0159] FIG9 shows the test results of antigen-specific IgG titers induced in mouse serum by the gE-gI fusion protein in Example 5 with FH002C adjuvant, Freund's complete adjuvant (Freund's) and aluminum adjuvant (Al).

[0160] FIG10 shows the results of detecting the neutralizing antibody titers in mouse sera induced by gE-gI fusion protein with FH002C adjuvant, Freund's complete adjuvant (Freund's) and aluminum adjuvant (Al) using the Elispot method and the plaque method in Example 5, respectively.

[0161] Figure 11 shows the results of T cell immune response induction in Example 6 in a saline blank group (Saline) without gE-gI fusion protein and gE-gI fusion protein combined with FH002C adjuvant, Freund's complete adjuvant (Freund's) and aluminum adjuvant (Al), respectively. The left figure shows the proportion of induced IL2+ cytokine-positive CD4+ T cells, and the right figure shows the proportion of induced IFNγ+ cytokine-positive CD4+ T cells.

[0162] FIG12 shows the binding antibody and neutralizing antibody titers of two different vaccines (the vaccine containing insect cell-derived gE-gI fusion protein and AS01B adjuvant, and Shingrix vaccine) after two injections in Example 7.

[0163] Figure 13 shows the results of CD4+ T cell responses stimulated by the saline blank group (Saline) without gE-gI fusion protein and two different vaccines (a vaccine containing insect cell-derived gE-gI fusion protein and AS01B adjuvant, and Shingrix vaccine) in Example 7, wherein the left figure shows the proportion of induced IFNγ+ cytokine-positive CD4+ T cells, and the right figure shows the proportion of induced IL2+ cytokine-positive CD4+ T cells.

[0164] Figure 14 shows the results of CD8+ T cell responses stimulated by the saline blank group (Saline) without gE-gI fusion protein and two different vaccines (a vaccine containing insect cell-derived gE-gI fusion protein and AS01B adjuvant, and Shingrix vaccine) in Example 7, wherein the left figure shows the proportion of induced IFNγ+ cytokine-positive CD8+ T cells, and the right figure shows the proportion of induced IL2+ cytokine-positive CD8+ T cells.

[0165] Figure 15 shows the results of Elispot-IFN-γ (left) and IL-2 (right) cytokine level detection produced by the saline blank group (Saline) without gE-gI fusion protein and two different vaccines (a vaccine containing insect cell-derived gE-gI fusion protein with AS01B adjuvant, and Shingrix vaccine) in Example 7.

[0166] Figure 16 shows the binding antibodies (left panel), binding antibody curves (middle panel) and neutralizing antibody titers (right panel) of two different vaccines (CHO-derived gE-gI fusion protein vaccine with AS01B adjuvant and Shingrix vaccine) after two injections in Example 8.

[0167] Figure 17 shows the results of CD4+ T cell responses stimulated by a saline blank group (Saline) containing no gE-gI fusion protein and two different vaccines (CHO-derived gE-gI fusion protein vaccine with AS01B adjuvant, and Shingrix vaccine) in Example 8, wherein the left figure shows the proportion of induced IFNγ+ cytokine-positive CD4+ T cells, and the right figure shows the proportion of induced IL2+ cytokine-positive CD4+ T cells.

[0168] Figure 18 shows the results of CD8+ T cell responses stimulated by the saline blank group (Saline) without gE-gI fusion protein and two different vaccines (CHO-derived gE-gI fusion protein vaccine with AS01B adjuvant, and Shingrix vaccine) in Example 8, wherein the left figure shows the proportion of induced IFNγ+ cytokine-positive CD8+ T cells, and the right figure shows the proportion of induced IL2+ cytokine-positive CD8+ T cells.

[0169] Figure 19 shows the results of Elispot-IFN-γ (left) and IL-2 (right) cytokine level detection produced by the saline blank group (Saline) without gE-gI fusion protein and two different vaccines (CHO-derived gE-gI fusion protein vaccine with AS01B adjuvant, and Shingrix vaccine) in Example 8.

[0170] Figure 20 shows the IgG binding antibody titers generated by different forms of gE-gI protein (gE protein alone, gE protein + gI protein, gE-gI fusion protein, and co-expressed gE-gI heterodimer protein) in combination with an AS01b-like adjuvant in Example 9. The left graph shows the antibody titers at week 3, and the right graph shows the antibody titers at week 5.

[0171] Figure 21 shows the flow cytometry-detected T cell responses generated by different forms of gE-gI protein (gE protein alone, gE protein + gI protein, gE-gI fusion protein, and co-expressed gE-gI heterodimer protein) with AS01b-like adjuvant in Example 9. The upper left graph shows the proportion of induced IFNγ+ cytokine-positive CD4+ T cells, the upper right graph shows the proportion of induced IL2+ cytokine-positive CD4+ T cells, the lower left graph shows the proportion of induced IFNγ+ cytokine-positive CD8+ T cells, and the lower right graph shows the proportion of induced IL2+ cytokine-positive CD8+ T cells.

[0172] Figure 22 shows the results of Elispot-IFN-γ (left panel) and IL-2 (right panel) cytokine level detection produced by different forms of gE-gI protein (gE protein alone, gE protein + gI protein, gE-gI fusion protein, and co-expressed gE-gI heterodimer protein) in Example 9 combined with AS01b-like adjuvant.

[0173] FIG23 shows the gEgI-specific IgG antibody titers produced after two injections of fusion proteins constructed based on different mutant strains in Example 10.

[0174] FIG24 shows the gEgI-specific CD4+ and CD8+ cellular immunity levels generated after two injections of fusion proteins constructed based on different mutant strains in Example 10.

[0175] FIG25 shows the Western Blot detection results of the fusion proteins constructed based on different gE truncations in Example 11.

[0176] FIG26 shows the capillary electrophoresis results of the mRNA encoding the gEgI fusion protein in Example 12.

[0177] FIG. 27 shows the particle size measurement results of the mRNA encoding the gEgI fusion protein and LNP encapsulation in Example 12.

[0178] FIG. 28 shows the IgG binding antibody titers generated by immunization with gEgI mRNA / LNP and gEgI mRNA / LNP combined with CpG adjuvant in Example 12.

[0179] FIG29 shows the results of CD4+ T and CD8+ T cell responses generated by immunostimulation with gEgI mRNA / LNP and gEgI mRNA / LNP combined with CpG adjuvant in Example 12.

[0180] FIG30 shows the monoclonality of the Top1 clone (CB4019-clone-16) in Example 13.

[0181] FIG31 shows an SDS-PAGE image of the gEgI protein obtained in Example 13.

[0182] FIG32 shows the HPLC identification chart of the gE-gI fusion protein derived from the CHO stable cell line in Example 13.

[0183] FIG33 shows the IgG binding antibody titer produced by the gE-gI fusion protein derived from the CHO stable cell line in Example 13, combined with AS01B adjuvant and Shingrix vaccine.

[0184] FIG34 shows the neutralizing antibody titer produced by the gE-gI fusion protein derived from the CHO stable cell line in Example 13, combined with the AS01B adjuvant and Shingrix vaccine.

[0185] FIG35 shows the CD4+ T cell and CD8+ T cell response results generated by the gE-gI fusion protein derived from the CHO stable cell line in Example 13, combined with AS01B adjuvant and Shingrix vaccine.

[0186] FIG36 shows the levels of Elispot-IFN-γ and IL-2 cytokines produced by the gE-gI fusion protein derived from the CHO stable cell line in Example 13, combined with AS01B adjuvant and Shingrix vaccine.

[0187] Sequence information

[0188] A description of the sequences involved in this application is provided in the table below.

[0189] Table 1: Sequence information DETAILED DESCRIPTION

[0190] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0191] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples illustrate the present invention by way of example and are not intended to limit the scope of the present invention.

[0192] Example 1: Large-scale expression of fusion protein

[0193] The designed fusion protein structure is shown in Table 2:

[0194] Table 2: Exemplary structures of fusion proteins

[0195] Gene synthesis: The gene sequences shown in SEQ ID NOs: 1, 2 and 3 were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0196] The following experiments were performed using gE-gI fusion protein as an example.

[0197] Transfection of insect cells

[0198] (1) Determine whether sf9 cells (purchased from Invitrogen, catalog number: 11496-015) or sf21 cells (purchased from Invitrogen, catalog number: 11497-013) are in the logarithmic growth phase (1.5-2.5×10 6 / mL), the survival rate was maintained above 90%. 200 μL of culture medium ESF 921 containing 2% FBS (purchased from Expression systems, catalog number: 96-001-01), 0.1 μg of Baculovirus DNA (purchased from Expression systems, catalog number: 91-002) and 1 ug of pAc-gEgI plasmid (the plasmid is constructed with a gE-gI encoding nucleotide sequence, SEQ ID NO: 4) were added to a 24-well plate and mixed evenly. 1 μL of transfection reagent (purchased from Expression systems, catalog number: 95-055-075) was added to 50 μL of ESF921 culture medium (purchased from Expression systems, catalog number: 96-001-01) and mixed evenly. Combine the above two into one tube, mix evenly, and let stand at room temperature for 30 minutes. Wash the cells during the resting period (do this just before the timer expires): After the cells have fully adhered, remove the culture medium with a pipette and add 300 μL of ESF921 culture medium. Move quickly to avoid dehydrating the cells. After gently rocking, remove the culture medium and add another 300 μL of ESF921 culture medium. When the timer expires, add approximately 100 μL of this mixture dropwise to each well of cells, dispensing evenly. Incubate at 27°C for 6 hours, discard the supernatant, and add 500 μL of complete culture medium (50% CCM3 + 50% TNM-FH (SIGMA-ALDRICH, T1032) + 10% FBS).

[0199] (2) Collect the cell supernatant obtained in step (1), centrifuge at 500g for 5 minutes, remove cell debris and debris, and store the supernatant at 4°C in the dark. This is the P1 virus seed solution.

[0200] (3) Amplification of baculovirus

[0201] Determine whether sf9 cells or sf21 cells are in the logarithmic growth phase (1.5-2.5×10 6 / mL), the survival rate was maintained above 90%. 8-10mL of the solution was spread on a 10cm plate at a density of 6×10 5 / mL cells. Let it stand for 15 minutes to allow the cells to adhere. Add about 600μL of P1 virus solution and drip evenly. Incubate at 27℃ for 3-4 days. Observe the cytopathic effect. Collect the cell supernatant, centrifuge at 1000rpm for 5 minutes, remove cell debris and debris, and filter with a 0.22um filter membrane. Store the supernatant at 4℃ in the dark. This is the P2 virus seed solution. The P2 virus titer is about 10 6 -10 7 P3 can be amplified by volume in a 250 mL shake flask according to this method.

[0202] Insect cell protein expression

[0203] 250 mL of ESF921 medium was added to a 1 L shake flask and the culture density was 2 × 10 6 / mL, H5 cells with a survival rate of more than 90% (purchased from Invitrogen, catalog number: B855-02). Add the virus according to the corresponding MOI, seal the bottle mouth with sealing film, and culture it in a 27°C shaker at 120rpm. Take out the cells in the shake flask every day, observe and count them, and record the relevant data. The appropriate MOI can ensure that more than 70% of the cells are diseased on the first day. On the second day, all the cells are diseased, and the survival rate is about 80%. On the third day, the cells ruptured and the survival rate dropped to 30-50%. At this time, you can consider collecting the cells. Use 10000rpm and centrifuge for 10min to collect the cells, then separate the supernatant and purify it to obtain a gE-gI fusion protein with a monomer sequence of SEQ ID NO: 10, and the C-terminus of the fusion protein contains a 10×HIS amino acid sequence.

[0204] Mammalian cell protein expression operations

[0205] Preparation: ExpiCHO TM Expression Medium (purchased from Thermo Scientific, product number A2910002) was preheated at 37°C and 10 ml of ExpiCHO was taken in a sterile tube. TM Expression Medium refrigerator pre-cooled at 4℃, plasmid 13.3×10 3 Centrifuge at g for 10 min; cells with a viability of more than 95% were diluted to 6 × 10 in a 250 ml shake flask with preheated ExpiCHO™ Expression Medium. 6 / ml in a 6-well plate, first dilute 50ug plasmid with 2ml of 4℃ pre-cooled culture medium, mix well, and then dilute 160ul ExpiFectamine with 1.84ml of pre-cooled culture medium TMCHO Reagent (purchased from Thermo Scientific, product number A29133), mix well, then add the diluted transfection reagent to the diluted plasmid and mix well, let it stand at room temperature for 1-5 minutes, add the plasmid and transfection reagent mixture to the diluted cells, and incubate at 37°C, 5% CO2; after 24 hours, add 300ul ExpiCHO TM Enhancer, 12ml ExpiCHO TM Feed(ExpiFectamine TM CHO Reagent transfection kit), cultured at 37°C, 5% CO2, for 7 days.

[0206] Tag-free purification of gE-gI fusion protein

[0207] Chromatographic purification was performed using an AKTA system;

[0208] Instrument system: AKTA Pure preparative liquid chromatograph;

[0209] Purification using a Q-FF column (Cytiva)

[0210] (1) Collect the expression sample, centrifuge at 7000 RPM for 10 min, dialyze the supernatant into 50 mM TB8.0, and incubate at 4°C overnight;

[0211] (2) The protein sample was passed through the column at 8 mL / min and then equilibrated with 50 mM TB8.0;

[0212] (3) Use 50mM TB8.0 and 1M NaCl to perform gradient elution of the target protein, and collect the 0.3M NaCl elution fraction.

[0213] DEAE-FF column (Cytiva) purification

[0214] (1) The protein sample was passed through the chromatography column at 5 mL / min and then equilibrated with 50 mM TB8.0;

[0215] (2) Use 50mM TB8.0 and 1M NaCl to perform gradient elution of the target protein, and collect the 0.3M NaCl elution fraction.

[0216] Butyl column (Cytiva) purification

[0217] (1) The eluted sample was dialyzed to 50 mM TB8.0 / 2 M NaCl;

[0218] (2) The protein sample was passed through the chromatography column at 5 mL / min, and then the column was equilibrated with 50 mM TB8.0 / 2 M NaCl;

[0219] (3) Use 50 mM TB8.0 to perform gradient elution of the target protein.

[0220] Purification of gE-gI fusion protein by affinity chromatography

[0221] Ni affinity chromatography purification was performed using the AKTA system;

[0222] Instrument system: AKTA Pure preparative liquid chromatograph;

[0223] Purification medium: Ni Sepharose 6 Fast Flow affinity medium; Buffer: divided into A pump and B pump buffer, generally A pump is 1× PBS buffer (160g / L NaCl, 8.1mmol / LNa2HPO4, 1.5mmol / L KH2PO4, 2.7mmol / L KCl, pH 7.4), B pump is 1× PBS + 250mmol / L imidazole buffer;

[0224] System flow rate: 5mL / min; detection wavelength: UV@280nm

[0225] Elution conditions: elution of contaminants with 50 mM imidazole buffer (250 mmol / L imidazole buffer diluted with 1× PBS buffer), followed by washing with 1× PBS, and then elution of the target protein (gE-gI fusion protein) with 250 mM imidazole buffer;

[0226] The product eluted with 250 mM imidazole was collected to obtain 40 mL of a purified sample, and the supernatant was purified by centrifugation.

[0227] Purification of gE-gI fusion protein by molecular sieve

[0228] Instrument system: AKTA explorer 100 preparative liquid chromatography system produced by GE Healthcare (formerly Amershan Pharmacia).

[0229] Chromatographic medium: Superdex 200 increase (cytiva).

[0230] Column volume: 20 cm × 20 mm.

[0231] Buffer: 20 mM phosphate buffer pH 7.4.

[0232] Flow rate: 0.7 mL / min.

[0233] Detector wavelength: 280nm.

[0234] The samples were purified by Ni affinity chromatography.

[0235] The elution procedure is: collect the penetration peak in segments.

[0236] The product that penetrated through Supedex 200 increase was collected to obtain 5 mL of purified sample.

[0237] SDS-PAGE

[0238] Take 50 μL of each elution product, add 30 μL of 6× Loading Buffer and mix well. After incubating in an 80°C water bath for 10 min, take 10 μL and run electrophoresis on a 10% SDS-polyacrylamide gel at 120 V for 120 min. Then stain with Coomassie Brilliant Blue to visualize the electrophoresis bands.

[0239] Western Blot Identification

[0240] (1) Transfer: After electrophoresis, use a vertical protein transfer apparatus to transfer the protein to the nitrocellulose membrane.

[0241] (2) Blocking: Soak the nitrocellulose membrane in 50 mL of 1× blocking solution and incubate at 37°C for 1 h.

[0242] (3) Incubation with primary antibody: Recover the blocking solution, rinse with deionized water three times, soak the nitrocellulose membrane with 50 mL of primary antibody (VZV gE protein-specific monoclonal antibody 1B11, stored in this laboratory), and incubate at 37°C for 1 h.

[0243] (4) Incubation with secondary antibody: Recover the primary antibody, rinse the nitrocellulose membrane 4 times with 1×PBST, 5 min each time, soak the nitrocellulose membrane with 50 mL of secondary antibody (GAH-HRP), and incubate at 37°C for 1 h.

[0244] (5) Color development: Recover the secondary antibody and rinse the nitrocellulose membrane four times with 1× PBST for 5 min each time. Mix equal volumes of color development solution A and B and soak the membrane surface for color development and imaging.

[0245] Figure 1 shows SDS-polyacrylamide gel electrophoresis (left) and Western blot analysis (right) of insect cell-derived gE-gI fusion protein samples at each purification stage (after purification using a Q-FF column (Figure 1A), a DEAE-FF column (Figure 1B), and a Butyl column (Figure 1C). Lane M: Molecular weight marker; Lane 1: gE-gI fusion protein. The results indicate that the gE-gI fusion protein obtained after three-column purification is approximately 90% pure. Western blot analysis using a VZV gE-specific monoclonal antibody was positive.

[0246] Figure 2 shows SDS-polyacrylamide gel electrophoresis (left) and Western blot analysis (right) of CHO-derived gE-gI fusion protein samples from various purification stages (Ni-6FF column, Figure 2A) and Superdex200 Increase (Figure 2B). Lane M: Molecular weight marker; Lane 1: gE-gI fusion protein. The results demonstrate that the gE-gI fusion protein obtained after two-column purification is approximately 90% pure. Western blot analysis using a VZV gE-specific monoclonal antibody demonstrates positive results.

[0247] Example 2: High Performance Size Exclusion Chromatography (HPSEC) Analysis of gE-gI Fusion Protein

[0248] Instrument: Waters. System flow rate: G3000PW XL The flow rate is 0.5 mL / min. The wavelength is 190-600 nm, and the column wavelengths are 280 nm and 254 nm.

[0249] Buffer: PBS

[0250] Procedure: Pre-equilibrate the column for 30-60 minutes until there is no significant change in the absorbance at 280 nm. Return the detector absorbance to zero. Edit the chromatography run method. Centrifuge the sample first. Inject the sample to be analyzed (gE-gI fusion protein obtained in Example 1) into a 100 μL sample loop. Set the automatic sample loading function and run for 30 minutes.

[0251] The results showed that the retention volume of the gE-gI fusion protein expressed by insect cells was approximately 6.2 mL, the sample showed a single peak, and the homogeneity and purity were greater than 90% ( Figure 3 ); the retention volume of the gE-gI fusion protein expressed by CHO cells was approximately 6.3 mL, the sample showed a single peak, and the homogeneity and purity were greater than 90% ( Figure 4 ).

[0252] Example 3: Calculation of sample sedimentation coefficient by analytical ultracentrifugation method

[0253] The instrument used was a Beckman XL-A analytical ultracentrifuge equipped with an optical detection system and an An-60Ti rotor.

[0254] The sample pool was installed according to the operating instructions, 400 μL of sample buffer (the same buffer as the sample) was added to the control pool, and 380 μL of sample (gE-gI fusion protein prepared in Example 1, OD280 of about 0.8) was added to the sample pool. The sample pool was balanced so that the weight difference was within 0.1 g.

[0255] The sample pool was placed in the An-60Ti rotor, and the rotor was placed in the cavity of the Beckman XL-A analytical ultracentrifuge, and an optical path detector was installed.

[0256] Parameter settings: temperature (20°C), Rmin (6.0 cm), Rmax (7.2 cm), wavelength (280 nm), step speed (0.003 cm), scan mode (continuous), data interval (30 sec), and number of data (150 scans). Set the centrifugal speed to 30,000 rpm.

[0257] After the experiment, SENDTERP software was used to calculate the density and viscosity of the buffer, as well as the partial microvolume of the known proteins. Sedimentation coefficients were analyzed using the Origin versions of Nonlin and SEDFIT. The friction ratio f / f0 was set to 1.2 for globular proteins. The analysis range was set based on the molecular weight and basic properties of the sample protein, and the calculation resolution was set to 100. The RMSD value was generally required to be no greater than 0.01, and the residue map fluctuation was within 0.05.

[0258] The results of ultracentrifugation analysis of the insect cell-derived gE-gI fusion protein are shown in FIG5 . The results show that the sedimentation coefficient of the gE-gI fusion protein is approximately 4.5S and the molecular weight is approximately 84.1 kDa, which is consistent with expectations and exists in a monomeric form.

[0259] The ultracentrifugation results of the CHO-derived gE-gI fusion protein are shown in FIG6 . The results show that the sedimentation coefficient of the gE-gI fusion protein is approximately 5.3S and the molecular weight is approximately 107 kDa, which is consistent with expectations and exists in a monomeric form.

[0260] Example 4: gE-gI fusion protein activity evaluation experiment

[0261] ELISA operation process:

[0262] (1) The gE-gI fusion protein prepared in Example 1 was coated on the plate (200 ng / well), incubated at 37°C for 2 h, washed once, and blocked with bovine serum albumin diluent (ED, 200 μL / well) at room temperature for 2 h;

[0263] (2) Wash the plate once, dilute gE protein-specific monoclonal antibodies (1B11, 4G4, 6H6, 6G7, 11B11, 11B12, 11E3, 14G1, 12E12, 13B6, 10H6, 17B7, 9H9; preserved in this laboratory) and gI protein-specific monoclonal antibodies (18B2, 7E7, 9D11, 16F1, 13B2, 19F2, 14B11, 12D2; preserved in this laboratory) with SD-1 diluent, add 5 μg / mL to the first well, and then serially dilute by 2 times. Incubate at 37°C for 1 h.

[0264] (3) Wash the plate five times, add the secondary antibody GAH-HRP (1:5000) to a 96-well plate at 100 μL / well, and incubate at room temperature for 1 h.

[0265] (4) The plate was washed five times, developed at 37°C for 10 min, and then detected at a wavelength of 450 nm using a microplate reader. Data were analyzed using GraphPad Prism 5 (GraphPad, USA) software.

[0266] The ELISA results are shown in FIG7 , which shows that the insect cell-derived gE-gI fusion protein prepared in Example 1 has good reactivity with both the gE protein-specific monoclonal antibody and the gI protein-specific monoclonal antibody.

[0267] The ELISA results are shown in FIG8 , which indicates that the CHO-derived gE-gI fusion protein prepared in Example 1 has good reactivity with both the gE protein-specific monoclonal antibody and the gI protein-specific monoclonal antibody.

[0268] Example 5: gE-gI fusion protein immunogenicity evaluation experiment

[0269] This experimental protocol was approved by the Experimental Animal Care Ethics Committee of Xiamen University. All operations were performed in strict accordance with animal ethics guidelines and approved protocols.

[0270] Six-week-old Balb / C mice were selected and divided into 4 groups of 5 mice each. Each group used the gE-gI fusion protein prepared in Example 1 (immune dose 1 μg) and was combined with FH002C adjuvant (FH002C adjuvant, see PCT application No. PCT / CN2021 / 130588 (the full text of which is incorporated herein by reference) or journal literature Wu Y, et al. A recombinant spike protein subunit vaccine confers protective immunity against SARS-CoV-2 infection and transmission in hamsters. Sci Transl Med. 2021 Aug 11; 13(606): eabg1143. DOI: 10.1126 / scitranslmed.abg1143 (the full text of which is incorporated herein by reference)) or aluminum adjuvant (prepared in this laboratory) or 50% Freund's complete adjuvant (purchased from Sigma) Mice were immunized with 150 μL of IgG (Aldrich, Cat. No. F5506) intramuscularly into the left or right hind limb at 0, 1, and 4 weeks. Ocular venous blood was collected at 0, 1, 2, 3, 4, and 5 weeks, with blood drawn before injection at 0, 1, and 4 weeks. Blood samples were centrifuged at 13,000 g for 10 minutes, and serum samples were stored at -20°C. Antigen-specific IgG and neutralizing antibody titers were measured using an end-point enzyme-linked immunosorbent assay and a vOka virus neutralization assay, respectively.

[0271] Neutralization operation process (ELISPOT method):

[0272] (1) Guinea pig serum (purchased from Beijing Borsi Technology Co., Ltd., catalog number: BM361Y) and vOka virus (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) dry powders were reconstituted with virus protection solution, and the complement was filtered through a 0.22 μm filter for later use;

[0273] (2) Serum was diluted 50-fold with virus protection solution and added to the first well of a 24-well plate. Four 2-fold serial dilutions were performed and incubated with vOka virus at 37°C for 1 h.

[0274] (3) The serum-virus mixture was transferred to a 24-well plate pre-plated with ARPE-19 cells and incubated at 37°C for 1 h. After 1 h, the liquid was discarded and supplemented with F12 medium, and cultured at 37°C for 3 days.

[0275] (4) After 3 days, the culture medium was discarded and the cells were washed once with PBS. The cells were fixed with the fixative solution at room temperature for 5 min, the fixative solution was discarded, and the cells were permeabilized with the permeabilization solution at room temperature for 10 min.

[0276] (5) Add the primary antibody 1B11-HRP (1:2000) to a 24-well plate and incubate at 37°C for 1 h;

[0277] (6) The plate was washed five times, developed at room temperature for 5 min, and the spots were read and counted using an enzyme-linked dot-spot image analysis system. Data were analyzed using GraphPad Prism 5 (GraphPad, USA) software.

[0278] Neutralization operation process (plaque method):

[0279] (1) Guinea pig serum and vOka virus powder were reconstituted with virus protection solution, and complement was filtered through a 0.22 μm filter for later use;

[0280] (2) Serum was diluted 50-fold with virus protection solution and added to the first well of a 24-well plate. Four 2-fold serial dilutions were performed and incubated with vOka virus at 37°C for 1 h.

[0281] (3) Transfer the serum-virus mixture to a 24-well plate pre-plated with ARPE-19 cells and incubate at 37°C for 1 h. After 1 h, discard the liquid and add F12 medium. Incubate at 37°C for 7-10 days until plaques form.

[0282] (4) Discard the culture medium, wash once with PBS, and fix with fixative at room temperature for 5 min;

[0283] (5) The samples were stained with crystal violet dye for 10 min, washed with deionized water, and counted manually. Data were analyzed using GraphPad Prism 5 (GraphPad, USA) software.

[0284] The results of the antigen-specific IgG and neutralizing antibody titers in the serum induced by the insect cell-derived fusion protein with different adjuvants are shown in Figures 9 and 10, respectively. The results in Figure 9 show that after three injections of immunization, all mice showed IgG serum conversion in the second week, and the IgG titer increased over time. At week 5, all mice showed high IgG antibody titers. Among them, the IgG antibody titer in the aluminum adjuvant group was the lowest, and the IgG antibody titer in the FH002C adjuvant group was relatively high. Figure 10 shows that all mice in each adjuvant group showed neutralizing antibody titers, among which the neutralization titer in the FH002C adjuvant group was higher than that in the aluminum adjuvant group and the Freund's adjuvant group. Figures 9 and 10 indicate that the gE-gI fusion protein can provide higher immunogenicity at a lower immunization dose.

[0285] Example 6: Detection of cytokines by flow cytometry

[0286] Mice were immunized according to the method described in Example 6, and the subsequent experimental procedures were as follows:

[0287] a) Spleen removal: Mice were sacrificed by cervical dislocation and soaked in 75% ethanol for 3-5 minutes. The mice were then placed in right lateral recumbency, and the spleen was removed aseptically (fat removed as much as possible).

[0288] b) Grinding: Place a 6-well plate on a mesh and add 1640 medium containing 10% FBS. Place the spleen on the mesh (completely immersed in the medium). Grind with a 2 mL syringe until no red tissue is visible. Transfer the ground cells to a 50 mL tube and place on ice.

[0289] c) Centrifuge at 400g for 5 min at 4°C, discard the supernatant, and tap the bottom of the tube with hemostats to loosen the cell pellet.

[0290] d) Add 10 mL of pre-chilled RBC solution to the cell pellet, resuspend, and place on ice for 5 minutes (invert and mix thoroughly).

[0291] e) Centrifuge at 400 g for 5 min at 4°C, discard the supernatant, and tap the bottom of the tube with hemostats to loosen the cell pellet.

[0292] f) Resuspend the cell pellet in 10 mL of pre-chilled culture medium, wash away any red blood cell pellet or fat, and aspirate to homogenize the cell pellet. Take 50 μL of the aliquot for counting.

[0293] g) Centrifuge at 400g for 5 min at 4°C, discard the supernatant, tap the bottom of the tube with hemostats to loosen the cell pellet, add a certain amount of culture medium, and dilute the cell count to 2 × 10 7 / mL;

[0294] h) Plating: 96-well U-bottom plate, 200 μL (2-4×10 6 cells), centrifuged at 400 g for 5 min at 4 °C, and the supernatant was discarded;

[0295] i) Resuspend the cells in 100 μL of culture medium containing peptides ((gE / gI) overlapping peptides, diluted with FACS solution, with a final peptide concentration of 2 μg / mL) and stimulate for 18 h;

[0296] j) Add 20 μL of Golgi inhibitor (1:1000, diluted in culture medium) and incubate for 6 h;

[0297] k) Centrifuge at 400g for 2 min at 4°C, remove the supernatant, add 200 μL FACS solution (1× PBS + 10% FBS) to resuspend the cells, centrifuge again, and remove the supernatant;

[0298] (All the following operations were carried out in the dark and kept at 4°C)

[0299] l) Cell surface staining: FITC-conjugated anti-mouse CD4 antibody (purchased from Biolegend, catalog number: 100406), PE-Cy7-conjugated anti-mouse CD8α antibody (purchased from Biolegend, catalog number: 100722), and LIVE / DEAD TM Fixable Aqua Dead Cell Stain Reagent (Biolegend, Cat. No. 103248) was used for staining. 40 μL of surface (AQUA / CD4 / CD8 antibody, diluted in FACS solution) was added to each well. The cells were resuspended and incubated at 4°C in the dark for 30–60 min. 200 μL of FACS solution was added and pipetted 7–8 times.

[0300] m) Fixation / Permeabilization: Centrifuge at 2000 rpm for 2 min at 4°C, remove the supernatant, add 75 μL of Fixation / Permeabilization solution to each well to resuspend the cells, and incubate at 4°C in the dark for 60 min.

[0301] n) Centrifuge at 2000 rpm, 4°C for 2 min, and discard the supernatant;

[0302] o) Add 200 μL of 1× BD Perm / Wash solution to each well to resuspend the cells. Centrifuge at 2000 rpm at 4°C for 2 min and discard the supernatant.

[0303] p) Intracellular staining: For staining, PE-conjugated anti-mouse IL-2 antibody (BD, Catalog No. 554428) and APC-conjugated anti-mouse IFN-γ antibody (BD, Catalog No. 554413) were used. 50 μL of cytokine antibody (IL-2 / IFN-γ antibody, diluted in 1× BD Perm / Wash solution) was added to each well and the cells were resuspended. The cells were incubated at 4°C in the dark for 60 min. 200 μL of Perm Buffer was then added and the cells were resuspended.

[0304] q) Centrifuge at 2000 rpm, 4°C for 2 min, remove the supernatant, and add 200 μL of 1× BD Perm / Wash solution to each well to resuspend the cells;

[0305] r) Filter the treated cells through a 200-mesh sieve (place the sieve strips on the surface of the well and pipette the cell suspension vertically and slowly into the well), and transfer them to a flow cytometer tube;

[0306] s) Samples were measured using a BD LSRFortessa X-20 flow cytometer, and the data were analyzed by FlowJo V10.

[0307] The experimental results are shown in FIG11 . The results in FIG11 indicate that the gE-gI fusion protein combined with different adjuvants can induce T cell immune responses.

[0308] Example 7: Parallel comparison of insect cell-derived gE-gI fusion protein with AS01B adjuvant and Shingrix vaccine

[0309] This experimental protocol was approved by the Experimental Animal Care Ethics Committee of Xiamen University. All operations were performed in strict accordance with animal ethics guidelines and approved protocols.

[0310] Six-week-old Balb / C mice were selected and divided into three groups of 8 mice each. The experimental group was immunized with the insect cell-derived gE-gI fusion protein (immunization dose 5 μg) prepared in Example 1 and the purchased AS01B adjuvant (purchased from GSK, batch number: 4N2AB). The control group was immunized with Shingrix vaccine (purchased from GSK, batch number: 4N2AB). The blank group was immunized with normal saline Saline. The tibial muscle of the mice was injected intramuscularly (50 μL) at 0 and 4 weeks, respectively. Ocular venous blood was collected at 0 weeks, 2 weeks, 4 weeks, 6 weeks and 8 weeks, respectively. Blood was collected before injection at 0 and 4 weeks. The blood samples were centrifuged at 13000g for 10 minutes, and the serum samples were stored at -20°C. The antigen-specific IgG and neutralizing antibody titers were determined by end-point enzyme-linked immunosorbent assay and vOka virus neutralization method, respectively.

[0311] The results are shown in Figure 12. The results in Figure 12 indicate that the IgG binding antibodies and neutralizing antibodies produced by the gE-gI fusion protein combined with the AS01B adjuvant are higher than those of the Shingrix vaccine; among them, the neutralizing antibody titer produced by the gE-gI fusion protein combined with the AS01B adjuvant is about 1.8 times that of the Shingrix vaccine.

[0312] At week 8, spleens were harvested for flow cytometry and Elispot assays of T cell responses. The flow cytometry assay was as described in Example 6, and the ELISPOT assay protocol for cytokines (using kits purchased from MABTECH, catalog numbers 3441-4HPW-10 and 3321-4HPW-10) was as follows:

[0313] (1) Splenocytes were isolated and 500,000 cells were plated per well. The supernatant was removed by centrifugation and 100 μL of culture medium containing peptides (gE / gI overlapping peptides) was added to resuspend the cells and stimulated at 37°C for 20 h.

[0314] (2) Discard the culture medium, wash with sterile PBS five times, dilute the detection antibodies R4-6A2-biotin (IFN-γ) and 5H4-biotin (IL-2) to 1 μg / mL, add 100 μL to each well, and incubate at room temperature for 2 h;

[0315] (3) Wash with sterile PBS five times, add 100 μL of Streptavidin-ALP (1:1000) to each well, and incubate at room temperature for 1 h;

[0316] (4) Wash with sterile PBS five times, add 100 μL of substrate solution (BCIP / NBT-plus) to each well for color development, and continue washing until spots appear;

[0317] (5) The plate was dried and the spots were read and counted using an enzyme-linked dot-spot image analysis system; data were analyzed using GraphPad Prism 5 (GraphPad, USA) software.

[0318] The experimental results are shown in Figures 13-15, where Figure 13 shows the CD4+T cell response results stimulated by two different vaccines in this example. The results show that the levels of gE-gI-specific CD4+IFN-γT cells and CD4+IL-2T cells produced by the insect cell-derived gE-gI fusion protein combined with AS01B adjuvant are higher than those of the Shingrix vaccine, and the proportion of CD4+IFN-γT cells is twice that of the Shingrix vaccine, which is a significant difference.

[0319] FIG14 shows the results of CD8+ T cell responses stimulated by two different vaccines in this example. The results show that the gE-gI fusion protein combined with AS01B adjuvant produces more gE-gI-specific CD8+ IFN-γ T cells than the Shingrix vaccine.

[0320] Figure 15 shows the Elispot-IFN-γ and IL-2 cytokine level detection results produced by the two different vaccines in this example. The results show that the IFN-γ and IL-2 levels produced by the gE-gI fusion protein combined with the AS01B adjuvant are higher than those of the Shingrix vaccine, and the differences are significant.

[0321] Example 8: Parallel comparison experiment of CHO-derived gE-gI fusion protein with AS01B adjuvant and Shingrix vaccine

[0322] This experimental protocol was approved by the Experimental Animal Care Ethics Committee of Xiamen University. All operations were performed in strict accordance with animal ethics guidelines and approved protocols.

[0323] Six-week-old Balb / C mice were selected and divided into three groups, with 8 mice in each group. The experimental group was immunized with the CHO-derived gE-gI fusion protein (immunization dose 5 μg) prepared in Example 1 and the purchased AS01B adjuvant (purchased from GSK, batch number: 4N2AB). The control group was immunized with Shingrix vaccine (purchased from GSK, batch number: 4N2AB), and the blank group was immunized with normal saline Saline. The tibial muscle of the mice was injected intramuscularly (50 μL) at 0 and 4 weeks, respectively. Ocular venous blood was collected at 0 weeks, 2 weeks, 4 weeks, 6 weeks and 8 weeks, respectively. Blood was collected before injection at 0 and 4 weeks. The blood samples were centrifuged at 13000g for 10 minutes, and the serum samples were stored at -20°C. The antigen-specific IgG and neutralizing antibody titers were determined by end-point enzyme-linked immunosorbent assay and vOka virus neutralization method, respectively.

[0324] The results are shown in Figure 16. The results in Figure 16 indicate that the IgG binding antibodies produced by the CHO-derived gE-gI fusion protein combined with AS01B adjuvant are comparable to those of the Shingrix vaccine; the neutralizing antibody titer produced by the gE-gI fusion protein combined with AS01B adjuvant is approximately twice that of the Shingrix vaccine.

[0325] Flow cytometry was performed as described in Examples 6 and 7.

[0326] The experimental results are shown in Figure 17, which shows the results of the CD4+T cell response stimulated by the two different vaccines in this example. The results show that the levels of gE-gI-specific CD4+IFN-γT cells and CD4+IL-2T cells produced by the CHO-derived gE-gI fusion protein combined with the AS01B adjuvant are higher than those of the Shingrix vaccine, with significant differences. The proportion of CD4+IFN-γT cells is three times that of the Shingrix vaccine.

[0327] Figure 18 shows the results of CD8+T cell responses stimulated by two different vaccines in this example. The results show that the gE-gI fusion protein combined with AS01B adjuvant produced more gE-gI-specific CD8+IFN-γT cells than the Shingrix vaccine, with a significant difference, which is about seven times that of the Shingrix vaccine.

[0328] Figure 19 shows the Elispot-IFN-γ and IL-2 cytokine level detection results produced by two different vaccines in this example. The results show that the gE-gI fusion protein combined with AS01B adjuvant produces higher gE-gI-specific IFN-γ T cells and specific IL-2 T cells than the Shingrix vaccine, with significant differences.

[0329] Example 9: Parallel comparison experiment of different forms of gE-gI protein with AS01b-like adjuvant

[0330] This experimental protocol was approved by the Experimental Animal Care Ethics Committee of Xiamen University. All operations were performed in strict accordance with animal ethics guidelines and approved protocols.

[0331] Six-week-old C57BL / 6 mice were selected and divided into five groups, with five mice in each group. The experimental group was administered with the gE-gI fusion protein prepared in Example 1 (immune dose 8.3 μg) and an adjuvant similar to AS01b (prepared in our laboratory, for preparation methods, see Didierlaurent AM, et al. Enhancement of adaptive immunity by the human vaccine adjuvant AS01 depends on activated dendritic cells. J Immunol. 2014 Aug 15; 193(4): 1920-30. doi: 10.4049 / jimmunol.1400948. Epub 2014 Jul 14.; Dendouga N, et al. Cell-mediated immune responses to a varicella-zoster virus glycoprotein E vaccine using both a TLR agonist and QS21 in mice. Vaccine. 2012 Apr 26;30(20):3126-35.doi:10.1016 / j.vaccine.2012.01.088.Epub 2012 Feb 10., the entire text of which is incorporated herein by reference). Control groups were immunized with gE protein alone (immunization dose 5 μg), gE protein + gI protein (immunization dose 5 μg + 3.3 μg), and co-expressed gE-gI heterodimeric protein (preparation method refers to the literature pmid:9697709, immunization dose 8.3 μg) with AS01b-like adjuvant (prepared in our laboratory). The blank group was immunized with normal saline. The tibialis muscle of the mice was injected intramuscularly (50 μL) at 0 and 3 weeks, respectively. Ocular venous blood was collected at 0, 3, and 5 weeks, respectively. Blood was collected before injection at 0 and 3 weeks. The blood samples were centrifuged at 13,000 g for 10 minutes, and the serum samples were stored at -20°C. The antigen-specific IgG and neutralizing antibody titers were measured by end-point enzyme-linked immunosorbent assay and vOka virus neutralization assay, respectively. The results are shown in FIG20 . The results in FIG20 indicate that the IgG binding antibodies produced by the gE-gI fusion protein combined with an AS01b-like adjuvant were significantly higher than those of the gE protein alone, gE protein + gI protein, and co-expressed gE-gI heterodimer protein.

[0332] At week 8, spleens were harvested for flow cytometry and Elispot detection of T cell responses, wherein the flow cytometry method was as described in Example 6, and the ELISPOT cytokine detection process was as described in Example 7.

[0333] The experimental results are shown in Figures 21-22, where Figure 21 shows the results of CD4+T and CD8+T cell responses stimulated by the four different vaccines in this example. The results show that the level of gE-gI-specific CD4+IFN-γT cells produced by the gE-gI fusion protein combined with an AS01b-like adjuvant was significantly higher than that of gE protein alone, gE protein + gI protein, and co-expressed gE-gI protein. The levels of CD4+IL-2T and CD8+IFN-γT cells were significantly higher than those of gE protein alone and gE protein + gI protein, and slightly higher than those of co-expressed gE-gI protein.

[0334] Figure 22 shows the Elispot-IFN-γ and IL-2 cytokine level detection results produced by the four different vaccines in this example. The results show that the IFN-γ and IL-2 levels produced by the gE-gI fusion protein combined with the AS01b-like adjuvant are higher than those of the single gE protein, gE protein + gI protein and co-expressed gE-gI protein, and the differences are significant.

[0335] Example 10: Parallel comparison experiment of gE-gI proteins of different strains with AS01b-like adjuvant

[0336] The gE full-length amino acid sequence (SEQ ID NO. 11) involved in Examples 1-9 of the present application is completely consistent with the gE protein sequence of the pOka strain (GenBank: AB097933) and the vOka strain (GenBank: AB097932). The gI sequence involved in Examples 1-9 of the present application is completely consistent with the gI protein sequence of the pOka strain (GenBank: AB097933), the vOka strain (GenBank: AB097932), the Dumas strain (GenBank: NC001348), and the MSP strain (GenBank: AY548170).

[0337] This example further evaluated the immunogenicity of gE-gI fusion proteins constructed based on gE and gI from different strains. The information of the fusion protein constructs involved is shown in the following table:

[0338] Table 3 Fusion protein structure information

[0339] Among them, gE (aa.23-537, I40T) is derived from the Dumas strain (GenBank: NC001348), which contains the I40T mutation compared to SEQ ID NO: 13; gE (aa.23-537, I40T&D150N) is derived from the MSP strain (GenBank: AY548170), which contains the I40T&D150N mutation compared to SEQ ID NO: 13; gE (aa.23-537, T512M&L536I) is derived from the gE protein shown in GenBank: AEW88980, which contains the T512M&L536I mutation compared to SEQ ID NO: 13; gE (aa.23-537, Q404K) is derived from the gE protein shown in GenBank: AEW89124, which contains the T512M&L536I mutation compared to SEQ ID NO: 13. NO: 13 contains the Q404K mutation; gE (aa.23-537, W319R) is derived from the gE protein shown in GenBank: QCA43570, which contains the W319R mutation compared to SEQ ID NO: 13; gE (aa.23-537, E124D) is derived from the gE protein shown in GenBank: QCA45176, which contains the E124D mutation compared to SEQ ID NO: 13; gE (aa.23-537, L359S) is derived from the gE protein shown in GenBank: QWE79571, which contains the L359S mutation compared to SEQ ID NO: 13; gE (aa.23-537, L162R) is derived from the gE protein shown in GenBank: AEW89412, which contains the L359S mutation compared to SEQ ID NO: 13. NO:13 contains the L162R mutation; gE (aa.23-537, A186V&L536I) is derived from the gE protein shown in GenBank: ANS12941, which contains the A186V&L536I mutation compared to SEQ ID NO:13; gE (aa.23-537, L536I) is derived from the gE protein shown in GenBank: AQT34120, which contains the L536I mutation compared to SEQ ID NO:13. gI (aa.21-271, K246Q) is derived from the gI protein shown in GenBank: AEW89051, which contains a K246Q mutation compared to SEQ ID NO: 14; gI (aa.21-271, T211P) is derived from the gI protein shown in GenBank: AEW89483, which contains a T211P mutation compared to SEQ ID NO: 14; gI (aa.21-271, N267S) is derived from the gI protein shown in GenBank: AGY34059, which contains an N267S mutation compared to SEQ ID NO: 14.

[0340] In this example, the fusion proteins shown in Table 3 were purified and immunoassayed using the methods described in Example 1, 5, and 6.

[0341] Figure 23 shows the gEgI-specific IgG antibody titers produced after two injections of fusion proteins constructed based on different mutant strains in this example. The results show that there is no significant difference in the IgG antibody levels produced by fusion proteins constructed based on different mutant strains combined with AS01b-like adjuvants, which is equivalent to the effect of the gEgI fusion protein shown in SEQ ID NO: 6.

[0342] Figure 24 shows the gEgI-specific T cell response generated after two injections of fusion proteins constructed based on different mutants in this embodiment. The results show that the gEgI fusion proteins constructed based on different mutants can produce CD4+T cell and CD8+T cell responses higher than Shingrix, among which some strains can also produce CD4+IFN-γ (such as gEgI (gE-Q404K) and gEgI (gE-I40T, D150N)) and CD4+IL2+ (such as gEgI (gE-L359S)), CD8+IFN-γ+T (such as gEgI (gE-Q404K)) cellular immune responses higher than the gEgI fusion proteins shown in SEQ ID NO: 6. This shows that the gEgI fusion proteins constructed based on gE and gI of different strains have significantly excellent immunogenicity and can produce immune responses higher than Shingrix.

[0343] Example 11: Design and preparation of truncated gEgI fusion protein

[0344] In this experiment, the length of gE in the gEgI fusion protein was truncated, while the length of gI was kept unchanged. Both the N-terminus and C-terminus of gE were truncated to construct the gEgI fusion protein, and the immunological activity of the gEgI fusion protein was evaluated. The following five clones were designed: gE(43-517)gI, gE(63-497)gI, gE(83-477)gI, gE(103-457)gI, and gE(143-417)gI. The information of the above five fusion proteins is as follows:

[0345] Table 4 Fusion protein molecular information

[0346] The fusion protein was prepared as described in Example 1. The fusion protein constructed from the truncated gE protein was detected by Western Blot.

[0347] FIG25 shows the results of this example, which show that although the five fusion protein constructs have different expression levels, they all have good immune activity.

[0348] Example 12: Parallel comparison experiment of gEgI mRNA vaccine combined with CpG adjuvant

[0349] Linearization to obtain mRNA transcription template

[0350] 25 μg of pUC-gEgI plasmid (the plasmid pUC57 was purchased from Sangon Biotechnology, catalog number: B522201, on which the mRNA expression framework and gE-gI encoding nucleotide sequence were constructed, the framework being the T7 promoter (SEQ ID NO: 16) + the DNA sequence corresponding to the 5'UTR (SEQ ID NO: 17) + the coding sequence of the kozak sequence (SEQ ID NO: 72) + the start codon + the gEgI fusion protein encoding nucleotide sequence (DNA sequence corresponding to SEQ ID NO: 71) + the stop codon + the DNA sequence corresponding to the 3'UTR (SEQ ID NO: 18) + the DNA sequence corresponding to PolyA (SEQ ID NO: 19)) was added to a 1.5 mL Eppendorf tube, 5 μL of BspQⅠ restriction endonuclease (purchased from New England Biolabs, catalog number: R0712S), 15 μL of NEBuffer r3.1 (purchased from New England Biolabs, catalog number: Biolabs, Catalog No.: #B6003S), supplemented with ddH2O to a total volume of 150 μL, and mixed thoroughly. Enzyme digestion reaction was carried out in a 50°C water bath for 30 min. The digestion system was recovered in a clean bench between cells according to the instructions of the gel recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., Catalog No.: DP209-03). The recovered product was identified by agarose gel electrophoresis to obtain the linearized plasmid, which was used as a transcription template.

[0351] in vitro transcription of mRNA

[0352] (1) Prepare mRNA using a T7 in vitro transcription kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd., catalog number: DD4202). Add 2 μL of ATP, 2 μL of CTP, 2 μL of GTP, 2 μL of LTP, 2 μL of Cap, 2 μL of RNase inhibitor, and 2 μg of linearized gEgI plasmid template to a 1.5 mL RNase-free Eppendorf tube, supplemented with DEPC water (purchased from Shanghai Biotech, catalog number: B501005-0500) to a total volume of 20 μL, and incubate in a 37°C metal water bath for 4 h.

[0353] (2) Add 2 μL of DNase I (purchased from New England Biolabs, catalog number: M0303L) and incubate at 37°C for 20 min to digest the residual DNA template in the system;

[0354] (3) Add 30 μL of DEPC water to terminate the digestion reaction;

[0355] (4) Add 30 μL of LiCl (purchased from Invitrogen, catalog number: AM9480) and incubate at -20°C for 30 min to precipitate mRNA;

[0356] (5) Centrifuge at 13,000 rpm for 10 min at 4°C and carefully remove the supernatant with a pipette.

[0357] (6) Add 1 mL of pre-cooled 75% ethanol, centrifuge at 13,000 rpm for 5 min at 4°C, and carefully remove the supernatant with a pipette to remove residual protein in the system;

[0358] (7) Open the centrifuge tube and air-dry for 2 min. Add 30 μL of DEPC water to resuspend the precipitate to obtain gEgI mRNA.

[0359] Identification of mRNA molecules by capillary electrophoresis

[0360] (1) Take 90 μL of DEPC water and dilute 10 μL of Dilution Buffer 10× Dilution Buffer (Bioptic Inc., C104408-10X) to make 1× Dilution Buffer;

[0361] (2) Mix 1 μL of mRNA sample with 19 μL of 1× Dilution Buffer;

[0362] (3) Capillary electrophoresis was performed using Qsep100 to analyze the size of mRNA.

[0363] The results of the capillary electrophoresis experiment are shown in Figure 26. The gEgI mRNA size determined by capillary electrophoresis is 2403 nt, which is slightly smaller than the theoretical value of 2676 nt, but within the error range. The capillary electrophoresis results show a single peak for gEgI mRNA, indicating good mRNA homogeneity.

[0364] mRNA mixed with LNP

[0365] (1) Prepare the mRNA aqueous phase. Dilute the mRNA to 80 μg / mL using 50 mM citrate buffer, pH 3.0.

[0366] (2) Prepare a stock solution of a lipid mixture with a total lipid concentration of 100 mM according to the proportions in the table below and store at room temperature. Incubate at 37°C until no precipitate is visible before use. Dilute with anhydrous ethanol to a final working concentration of 5 mM or 10 mM.

[0367] Table 5 Lipid formulation

[0368] (3) Mixing mRNA and lipid phase. Using a microfluidic instrument, clean the channels with ethanol, and pre-rinse both channels with ethanol and citric acid buffer, respectively. After sufficient rinsing, adjust the parameters and prepare for sample loading. The lipid mixed phase: nucleic acid phase = 1:3 (v / v), the flow rate ratio is 1:3, and the total flow rate is 12 mL / min.

[0369] (4) The mixed mRNA / LNP was immediately diluted with 5 times the volume of 1×PBS and concentrated by ultrafiltration twice using a 10KD ultrafiltration tube. The replacement buffer was 1×PBS and concentrated to the initial mRNA / LNP mixing volume.

[0370] Determination of mRNA / LNP concentration and encapsulation efficiency

[0371] (1) Using Quant-iT TM RiboGreen TM mRNA concentration was determined using the RNA Reagent and Kit (Thermo Fisher Scientific, Cat. No. R11490). Dilute 250 μL of 20× TE (4.75 mL DEPC water) to 5 mL of 1× TE. Dilute 4 μL of RNA standard with 196 μL of 1× TE to create Solution A. Dilute 7.5 μL of Ribogreen with 1492.5 μL of 1× TE to create Solution B.

[0372] (2) In a 96-well U-bottom plate, add 100 μL, 98 μL, 90 μL, 50 μL, and 0 μL of 1×TE and 0 μL, 2 μL, 10 μL, 50 μL, and 100 μL of solution A, and add 100 μL of solution B to each well and mix well.

[0373] (3) Demulsification: Use 1×TE to prepare 2% Triton buffer, dilute mRNA / LNP 100-fold, mix well, and let it stand at room temperature for 5 minutes; for the other well, use 1×TE to dilute mRNA / LNP 100-fold; add 100 μL of solution B to each well, mix well, and let it stand at room temperature for 5 minutes;

[0374] (4) Read the fluorescence using a multi-label detection imaging analysis system instrument, draw a standard curve according to step (2), and calculate the mRNA sample concentration.

[0375] mRNA / LNP particle size determination (DLS)

[0376] (1) Prepare a sample cup (UVette cuvette), add 45 μL PBS and 5 μL mRNA / LNP, mix well, and add to the cuvette;

[0377] (2) Determine the particle size according to the instrument operating instructions;

[0378] (3) Record the measurement results, select the appropriate distribution method based on the actual results, and process the measurement data.

[0379] The DLS measurement results are shown in Figure 27. The results show that the particle size of mRNA / LNP is about 74 nm, which is consistent with the theoretical size, and the sample has a single sharp peak, indicating that the mRNA / LNP has good uniformity.

[0380] Immunological evaluation experiment of gEgI mRNA / LNP combined with CpG adjuvant

[0381] This experimental protocol was approved by the Experimental Animal Care Ethics Committee of Xiamen University. All operations were performed in strict accordance with animal ethics guidelines and approved protocols.

[0382] Six-week-old C57 mice were divided into four groups of five. The experimental groups received either gEgI mRNA / LNP or gEgI mRNA / LNP plus 10 μg CpG (10 μg / 100 μL per mouse). The control group received Shingrix vaccine (purchased from GSK, lot number 4N2AB), and the blank group received saline. The mice were injected intramuscularly into the tibialis muscle at 0 and 3 weeks. Ocular venous blood was collected before injection at 0, 2, and 5 weeks. Blood samples were centrifuged at 13,000 g for 10 minutes, and serum samples were stored at -20°C. Antigen-specific IgG binding antibody titers were determined by ELISA, and cytokine levels were analyzed by flow cytometry.

[0383] The ELISA results are shown in FIG28 . The results in FIG28 indicate that the IgG binding antibody titers generated by gEgI mRNA / LNP and gEgI mRNA / LNP combined with CpG immunization were comparable to those of Shingrix vaccine, with no significant difference.

[0384] Figure 29 shows the results of T cell responses stimulated by two different vaccines in this example. The results show that gEgI mRNA / LNP and gEgI mRNA / LNP combined with CpG immunization produce gEgI-specific CD8+IFN-γT cells that are higher than Shingrix vaccine, with significant differences. This shows that the mRNA vaccine corresponding to the gEgI fusion protein of the present application has the ability to induce high levels of cellular immunity, is superior to commercial vaccines, and has good application prospects.

[0385] Example 13 Preparation and evaluation of gEgI fusion protein derived from a stable cell line

[0386] In order to facilitate subsequent production and transformation applications, the present application prepared a gEgI fusion protein derived from a stably transfected CHO cell line and performed an immune assessment.

[0387] Preparation of gEgI fusion protein from stable CHO cell lines:

[0388] To facilitate subsequent generation and transformation, the stable cell line uses the original signal peptide of gE. Therefore, the sequence of gE is 1-537aa of the full-length gE protein (SEQ ID NO: 11), and the original signal peptide is approximately 1-30aa. During the expression of the fusion protein, the signal peptide 1-30aa will be removed. Therefore, after enzyme cleavage, the fusion protein contains the gE extracellular region of 31-537aa (SEQ ID NO: 69) and the gI extracellular region of 21-271 (SEQ ID NO: 14). The amino acid sequence of the fusion protein after enzyme cleavage and removal of the signal peptide is shown in SEQ ID NO: 70.

[0389] The specific preparation method is as follows:

[0390] gE (1-537) and gI (21-271) were also connected using a linker (SEQ ID NO: 15). Using a tag-free design, the gEgI sequence was constructed into a stable cell line plasmid, Han Teng PMGT vector (Guangzhou Han Teng Biotechnology Co., Ltd.), and the constructed recombinant plasmid was transfected into CHOZN CHOK1 GSKO (purchased from Merck, USA, Cat# CHOGS) host cells. Cell pools were obtained by MSX (Sigma, Cat# GSS-1015-F) pressure screening. The expression levels of the target protein in different cell pools were compared by Fed-batch, and the cell pool with high expression was finally selected for monoclonal plating. 238 well-growing single clones were obtained by SINGLE CELL PRINTER (Cytena, model F.sight) plating combined with cell imaging, and the culture was expanded step by step. The top 20 clones were screened based on the expression levels of the clones in 24-well plates and 6-well plates. The expression levels of the target protein of the top 20 clones were compared by Fed-batch. The top 5 clones were screened based on cell growth data, ELISA, SDS-page, and Western blot results. A research cell bank (RCB) was established, and 35 cells were frozen for each clone. The top 1 clone was selected for expression culture using EX-CELL CD CHO Fusion Medium under the following conditions: 37°C, 140rpm, 5% CO2, 85% humidity, and a 25mm rotation radius (Kuhner, model ISF1-XC). After 6 days of culture, the cell supernatant was harvested by centrifugation at 200g for 8 minutes and purified using the tag-free purification method for the gEgI fusion protein described in Example 1.

[0391] Table 6 shows the comprehensive data of cell passage stability, yield changes, etc. in the stability study of the top 5 clones. The best top 1 clone is CB4019-clone-16.

[0392] Table 6 Summary of Top5 cell line clones

[0393] FIG30 shows the results of the monoclonality confirmation of the Top1 clone (CB4019-clone-16), which showed that CB4019-clone-16 was derived from a single cell division.

[0394] FIG31 shows the gEgI protein obtained by the tag-free purification method of Example 1, with a purity greater than 95%.

[0395] The results in Figure 32 show that the retention volume of the gE-gI fusion protein derived from the CHO stable cell line was approximately 6.9 mL, the sample presented a single peak, and the homogeneity and purity were higher than 90%.

[0396] Immunological evaluation of gEgI fusion protein derived from stable CHO cell line:

[0397] The obtained gEgI fusion protein was immunologically evaluated according to the method of Example 8. The results of the immunological evaluation are shown in Figures 33-36.

[0398] As shown in Figure 33, the IgG binding antibodies produced by the gE-gI fusion protein derived from the CHO stable cell line and the AS01B adjuvant were 5 times that of the Shingrix vaccine.

[0399] Figure 34 shows that the neutralizing antibody titer generated by gE-gI fusion protein combined with AS01B adjuvant is about twice that of Shingrix vaccine.

[0400] Figure 35 shows the results of CD4+T and CD8+T cell responses stimulated by two different vaccines in this example. The results show that the level of gE-gI-specific CD4+IFN-γT cells produced by gE-gI fusion protein combined with AS01b-like adjuvant is significantly higher than Shingrix, about 1.4 times that of Shingrix.

[0401] Figure 36 shows the Elispot-IFN-γ and IL-2 cytokine level detection results produced by two different vaccines in this example. The results show that the IFN-γ and IL-2 levels produced by the gE-gI fusion protein combined with an AS01b-like adjuvant are higher than those of Shingrix, among which IFN-γ is 2.4 times that of Shingrix.

[0402] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A fusion protein comprising a first peptide segment and a second peptide segment, wherein: The first peptide segment comprises the extracellular region of varicella-zoster virus (VZV) gE protein or a fragment thereof, or consists of the same; the second peptide segment comprises the extracellular domain of VZV gI protein or a fragment thereof, or consists of the same.

2. The fusion protein of claim 1, wherein The first peptide segment comprises at least 100, at least 150, at least 200 or at least 250 consecutive amino acid residues of the amino acid residues at positions corresponding to positions 150-410 of SEQ ID NO: 11 in the gE protein; and / or the second peptide segment comprises at least 50, at least 100, at least 150 or at least 180 consecutive amino acid residues of the amino acid residues at positions corresponding to positions 45-240 of SEQ ID NO: 12 in the gI protein.

3. The fusion protein of claim 1 or 2, wherein The first peptide segment comprises amino acid residues at positions corresponding to positions 150-410 of SEQ ID NO: 11 in the gE protein; Preferably, the first peptide segment comprises amino acid residues at positions corresponding to positions 70-485 of SEQ ID NO: 11 in the gE protein; Preferably, the first peptide segment comprises amino acid residues at positions corresponding to positions 63-497 of SEQ ID NO: 11 in the gE protein; Preferably, the first peptide segment comprises the amino acid residues at positions corresponding to positions 31-537 of SEQ ID NO: 11 in the gE protein; Preferably, the gE protein has: (a) an amino acid sequence as shown in any one of SEQ ID NOs: 11, 46-55; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in any one of SEQ ID NOs: 11, 46-55; or, (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence shown in any one of SEQ ID NOs: 11, 46-55; Preferably, the first peptide segment comprises an amino acid sequence as shown in any one of SEQ ID NOs: 13, 33-42, 64-69, or has at least 90%, for example at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the amino acid sequence shown in any one of SEQ ID NOs: 13, 33-42, 64-69, or has a sequence with one or several (for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions, or consists of the same.

4. The fusion protein according to any one of claims 1 to 3, wherein The second peptide segment comprises amino acid residues at positions corresponding to positions 45-240 of SEQ ID NO: 12 in the gI protein; Preferably, the second peptide segment comprises the amino acid residues at positions corresponding to positions 30-260 of SEQ ID NO: 12 in the gI protein; Preferably, the second peptide segment comprises the amino acid residues at positions corresponding to positions 21-271 of SEQ ID NO: 12 in the gI protein; Preferably, the gI protein has: (a) an amino acid sequence as shown in any one of SEQ ID NOs: 12, 56-58; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in any one of SEQ ID NOs: 12, 56-58; or, (c) one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions compared to the amino acid sequence shown in any one of SEQ ID NOs: 12, 56-58; Preferably, the second peptide segment comprises an amino acid sequence as shown in any one of SEQ ID NOs: 14, 43-45, or has at least 90%, for example at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the amino acid sequence shown in any one of SEQ ID NOs: 14, 43-45, or has a sequence with one or several (for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions, or consists of the same.

5. The fusion protein according to any one of claims 1 to 4, wherein The second peptide segment is optionally connected to the N-terminus or C-terminus of the first peptide segment via a linker (e.g., a peptide linker, for example, a peptide linker shown in SEQ ID NO: 15); Preferably, the linker is a peptide linker comprising one or more (e.g., 5-20) flexible amino acids; Preferably, the linker is a peptide linker comprising one or more glycines and / or one or more serines; Preferably, the fusion protein comprises or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 6-7, 20-32, 59-63, 70.

6. The fusion protein according to any one of claims 1 to 5, wherein The fusion protein comprises a signal peptide and / or a detectable label (e.g., a tag protein); Preferably, the fusion protein comprises a signal peptide at its N-terminus (e.g., the natural signal peptide of VZV gE protein, the natural signal peptide of VZV gI protein, gp67 signal peptide, melittin signal peptide (MSP) or tissue-type plasminogen activator signal peptide (tPA signal peptide)); Preferably, the fusion protein comprises a detectable label at its C-terminus.

7. An RNA molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1 to 6.

8. The RNA molecule of claim 7, further comprising one or more selected from the group consisting of a 5'UTR, a Kozak sequence, a start codon, a stop codon, a 3'UTR, and a poly-A tail; Preferably, the RNA molecule comprises, from the 5' end to the 3' end, 5'UTR, Kozak sequence, start codon, nucleotide sequence encoding the fusion protein, stop codon, 3'UTR, and poly-A tail.

9. The RNA molecule of claim 7 or 8, having one or more features selected from the group consisting of: (1) the nucleotide sequence encoding the fusion protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:71; preferably, the nucleotide sequence encoding the fusion protein is shown in SEQ ID NO:71; (2) There are one or more stop codons at the 3' end of the nucleotide sequence encoding the fusion protein; (3) the 5'UTR has a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 17; preferably, the 5'UTR has the nucleotide sequence shown in SEQ ID NO: 17; (4) the 3'UTR has a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 18; preferably, the 3'UTR has the nucleotide sequence shown in SEQ ID NO: 18; (5) the Kozak sequence is shown in SEQ ID NO:72; (6) The poly-A tail comprises one or more poly(A) sequences, each of which independently consists of 20-120 consecutive adenylic acids; preferably, the poly-A tail comprises multiple poly(A) sequences, and adjacent poly(A) sequences are connected by a non-A spacer sequence; preferably, the poly-A tail has a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 19; preferably, the poly-A tail has the nucleotide sequence shown in SEQ ID NO: 19; (7) The 5' end of the RNA molecule is modified with a 5' cap (eg, CAP type 0, CAP type 1, CAP type 2).

10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1-6 or the RNA molecule of any one of claims 7-9.

11. A vector comprising the isolated nucleic acid molecule of claim 10.

12. A delivery composition comprising a delivery vector and the RNA molecule of any one of claims 7 to 9, or the isolated nucleic acid molecule of claim 10, or the vector of claim 11; Preferably, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles and viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses or adeno-associated viruses); Preferably, the delivery composition comprises the RNA molecule of any one of claims 7-9.

13. The delivery composition of claim 12, wherein The delivery vehicle is a lipid nanoparticle (LNP).

14. A host cell comprising the RNNA molecule of any one of claims 7 to 9, or the isolated nucleic acid molecule of claim 10, or the vector of claim 11; Preferably, the host cell is selected from prokaryotic cells (such as E. coli cells), and eukaryotic cells (such as yeast cells, insect cells, plant cells, mammalian cells); Preferably, the host cell is an insect cell; Preferably, the host cell is a mammalian cell.

15. A method for preparing the fusion protein of any one of claims 1 to 6, comprising culturing the host cell of claim 14 under conditions allowing protein expression, and recovering the fusion protein from the cell culture.

16. An immunogenic composition comprising the fusion protein of any one of claims 1 to 6, or the RNA molecule of any one of claims 7 to 9, or the isolated nucleic acid molecule of claim 10, or the vector of claim 11, or the delivery composition of claim 12 or 13, and optionally a pharmaceutically acceptable carrier and / or excipient (e.g., adjuvant); Preferably, the immunogenic composition comprises the fusion protein of any one of claims 1 to 6, or the RNA molecule of any one of claims 7 to 9, or the isolated nucleic acid molecule of claim 10, or the vector of claim 11, or the delivery composition of claim 12 or 13 and an adjuvant, wherein, The adjuvant is selected from the group consisting of aluminum salt adjuvants, zinc-aluminum mixed adjuvants (e.g., FH002C), Freund's adjuvant, oil emulsion adjuvants (e.g., MF59 adjuvant), cytokines, TLR agonists, CpG adjuvants, nucleic acid adjuvants, liposomes, saponin adjuvants, AS01B adjuvants, and any combination thereof; Preferably, the immunogenic composition comprises the fusion protein of any one of claims 1 to 6 or the delivery composition of claim 12 or 13, and the adjuvant; Preferably, the immunogenic composition is a vaccine.

17. Use of the fusion protein of any one of claims 1 to 6, or the RNA molecule of any one of claims 7 to 9, or the isolated nucleic acid molecule of claim 10, or the vector of claim 11, or the delivery composition of claim 12 or 13, or the host cell of claim 14, or the immunogenic composition of claim 16 in the preparation of an immunogenic composition for inducing an immune response against VZV in a subject and / or for preventing and / or treating VZV infection or a disease associated with VZV infection in a subject; Preferably, the immunogenic composition is a vaccine; Preferably, the VZV infection is a primary VZV infection or a recurrent infection; Preferably, the disease associated with VZV infection is selected from: herpes zoster, chickenpox, and complications thereof (e.g., neuropathic pain, pneumonia, encephalomyelitis, conjunctivitis); Preferably, the subject is a mammal, such as a human.

18. A method for inducing an immune response against VZV in a subject and / or for preventing and / or treating VZV infection or a disease associated with VZV infection in a subject, comprising: Administering an effective amount of the fusion protein of any one of claims 1 to 6, or the RNA molecule of any one of claims 7 to 9, or the isolated nucleic acid molecule of claim 10, or the vector of claim 11, or the delivery composition of claim 12 or 13, or the host cell of claim 14, or the immunogenic composition of claim 16 to a subject in need thereof; Preferably, the VZV infection is a primary VZV infection or a recurrent infection; Preferably, the disease associated with VZV infection is selected from: herpes zoster, chickenpox, and complications thereof (e.g., neuropathic pain, pneumonia, encephalomyelitis, conjunctivitis); Preferably, the subject is a mammal, such as a human.

19. A method for detecting the presence of antibodies specific for VZV gI protein and / or VZV gE protein in a sample, comprising using the fusion protein of any one of claims 1 to 6; Preferably, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (eg ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay; Preferably, the method comprises: (1) contacting the sample with the fusion protein according to any one of claims 1 to 6; (2) detecting the formation of a fusion protein-antibody immune complex or detecting the amount of the immune complex; the formation of the immune complex indicates the presence of VZV gI protein and / or VZV gE protein-specific antibodies in the sample; Preferably, the method further comprises using a detectable marker (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent to detect the presence of antibodies specific for VZV gI protein and / or VZV gE protein in the sample using a secondary antibody using a light dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin; Preferably, the second antibody is specific to the constant region contained in the antibody of the species (e.g., human) from which the test sample originates; Preferably, the second antibody is an anti-immunoglobulin (eg, human immunoglobulin) antibody, such as an anti-IgG antibody; Preferably, the sample is a body fluid sample (eg, whole blood, plasma, serum, salivary secretions or urine) from a subject (eg, a mammal, preferably a human).

20. Use of the fusion protein of any one of claims 1 to 6, or the RNA molecule of any one of claims 7 to 9, or the isolated nucleic acid molecule of claim 10, or the vector of claim 11, or the delivery composition of claim 12 or 13, or the host cell of claim 14 in the preparation of a detection reagent for detecting the presence of antibodies specific for VZV gI protein and / or VZV gE protein in a sample; Preferably, the detection reagent detects the presence of antibodies specific for VZV gI protein and / or VZV gE protein in a sample by the method of claim 19; Preferably, the sample is a body fluid sample (eg, whole blood, plasma, serum, salivary secretions or urine) from a subject (eg, a mammal, preferably a human).

21. A kit comprising the fusion protein of any one of claims 1-6, or the RNA molecule of any one of claims 7-9, or the isolated nucleic acid molecule of claim 10, or the vector of claim 11, or the delivery composition of claim 12 or 13, or the host cell of claim 14; Preferably, the kit comprises the fusion protein of any one of claims 1 to 6, and a second antibody, wherein: The second antibody is as defined in claim 19.

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