Varicella-zoster virus gE and gI fusion proteins and uses

Abstract

The present invention provides fusion proteins of gE and gI (or variants thereof) that can be used to prevent or treat varicella-zoster virus infection, and uses of such fusion proteins (or variants thereof).

Description

[Technical Field]

[0001] This application claims the benefit of Chinese Application No. 2023100792531, filed on January 19, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to the fields of immunology and molecular virology, particularly the prevention and treatment of varicella-zoster virus. Specifically, the present disclosure 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 uses of the fusion protein or variant. [Background technology]

[0003] Varicella-zoster virus (VZV) is a DNA virus with an envelope. VZV particles are spherical and composed of 150 hexamers and 12 pentamers, which assemble into a T=16 icosahedral structure. VZV particles can be broadly divided into four regions, radially outward from the spherical center, containing the viral nucleic acid (viral DNA), capsid, tegument proteins, and envelope proteins, respectively. The viral core is DNA, which controls the viral life cycle. The icosahedral nucleocapsid of VZV is highly stable and plays an important role in protecting the internal genetic material. The outer portion of the viral capsid is the tegument protein, which is composed of various proteins encoded by the viral DNA and plays a role in viral infection, host cell expression, immune response, assembly, and exocytosis. The outer surface of VZV particles is an envelope formed by various glycoproteins and lipids, the main glycoproteins of which are gB, gC, gE, gH, gL, gI, etc., which play important roles in host cell identification, immune response, and enhancing the stability of virus particles.

[0004] VZV infection is associated with a high incidence of papular rash and chickenpox, often complicated by pneumonia, and a high mortality rate (mainly in children). Even after recovery, small amounts of VZV remain in neuronal cells, and reactivation of VZV can cause symptoms such as varicella zoster and postherpetic neuralgia. In severe cases, VZV can cause malignant complications such as encephalomyelitis and conjunctivitis (primarily in immunocompromised or immunosuppressed patients aged 50 years or older). Vaccination is the best way to prevent diseases such as chickenpox and varicella zoster, but few vaccines are currently available. The main protein component of Shingrix™, the world's first recombinant varicella zoster vaccine, is the VZV envelope glycoprotein E (gE), indicating that gE is an important target for varicella zoster vaccine research. As the most abundant glycoprotein on the VZV envelope, gE contains a total of 623 amino acid residues, some of which play important roles in VZV transit. Among these, amino acids 24-71 and 163-208 of gE are key regions for recognizing the IDE (insulin-degrading enzyme) receptor and forming heterodimers with gI, respectively. Therefore, the ability of gE to form the correct secondary structure is key to its interaction with IDE. Currently, several subunit vaccines or preclinical vaccines used to prevent VZV infection are designed around the gE protein. The content of envelope glycoprotein I (gI) on VZV particles is second only to gE. gI consists of a total of 354 amino acid residues, which can be broadly divided into three parts: the extracellular segment (amino acids 1-271, including the signal peptide from amino acids 1-20), the hydrophobic transmembrane region, and the intracellular segment including the C-terminus. Deficiency of gI significantly reduces the replication rate of VZV and severely affects the viral syncytium synthesis process, suggesting that gI plays an important role in VZV replication and amplification in culture models such as Vero cells, T cells, and human skin tissue. Furthermore, gI acts as a molecular chaperone protein and promotes the maturation of gE. When the gI gene is knocked out, gE colocalizes with Golgi marker molecules in the perinuclear region and is unable to transport to the cytoplasmic membrane and participate in VZV particle assembly.

[0005] Current research into VZV infection and immune mechanisms has demonstrated that strong VZV-specific CD4 + This indicates that T cell immune responses are closely associated with protective immunity against varicella zoster. In current clinical studies of the Shingrix™ vaccine, which uses gE as the immunogen, the vaccine stimulates gE-specific CD4 + It can generate a T cell response that is more than 10 times stronger than that of the ZOSTAVAX vaccine, and the protection rate in people aged 60 to 69 is 97.4%, which is higher than the 64% of the ZOSTAVAX vaccine.

[0006] However, there remains a need in the art for more protective VZV vaccines. Summary of the Invention

[0007] The present disclosure provides a gE-gI fusion protein, and through its preparation and characterization, demonstrates that the gE-gI fusion protein can induce high levels of neutralizing antibodies and cytokines in organisms, which is superior to a single gE protein, a blend of gE and gI proteins, a gE-gI heterodimer, and the commercially available Shingrix™ vaccine, and has a strong immunostimulatory effect. The fusion protein of the present disclosure has the advantage of being a diagnostic antigen or vaccine candidate for the prevention and / or treatment of varicella-zoster virus infection.

[0008] fusion proteins Thus, in one aspect, the present disclosure provides a fusion protein comprising a first peptide segment and a second peptide segment, wherein the first peptide segment comprises or consists of the extracellular domain of a varicella-zoster virus (VZV) gE protein or a fragment thereof, and the second peptide segment comprises or consists of the extracellular domain of a VZV gI protein or a fragment thereof.

[0009] In certain embodiments, the first peptide segment comprises one or more amino acids at positions 24-71, 163-208 of the VZV gE protein, and / or the second peptide segment comprises one or more amino acids at positions 95, 105-125 of the VZV gI protein.

[0010] In some embodiments, the first peptide segment comprises at least 100, at least 150, at least 200, or at least 250 consecutive amino acid residues within the amino acid residues of the gE protein at positions corresponding to positions 150 to 410 of SEQ ID NO: 11, and / or the second peptide segment comprises at least 50, at least 100, at least 150, or at least 180 consecutive amino acid residues within the amino acid residues of the gI protein at positions corresponding to positions 45 to 240 of SEQ ID NO: 12.

[0011] In some embodiments, the first peptide segment is located at positions 150-410 (or positions 150-417, or positions 150-425, or positions 150-437, or positions 150-445, or positions 150-457, or positions 150-465, or positions 150-477, or positions 150-485, or positions 150-497, or positions 150-507, or positions 150-517, or positions 150-525, or positions 150-537, or positions 143-410, or positions 143-417, or positions 143-425, or positions 143-437) of SEQ ID NO: 11. 7, or positions 143 to 445, or positions 143 to 457, or positions 143 to 465, or positions 143 to 477, or positions 143 to 485, or positions 143 to 497, or positions 143 to 507, or positions 143 to 517, or positions 143 to 525, or positions 143 to 537, or positions 120 to 410, or positions 120 to 417, or positions 120 to 425, or positions 120 to 437, or positions 120 to 445, or positions 120 to 457, or positions 120 to 465, or positions 120 to 477, or positions 120 to 485, or positions 120 to 497, or positions 12 0 to 507, or positions 120 to 517, or positions 120 to 525, or positions 120 to 537, or positions 103 to 410, or positions 103 to 417, or positions 103 to 425, or positions 103 to 437, or positions 103 to 445, or positions 103 to 457, or positions 103 to 465, or positions 103 to 477, or positions 103 to 485, or positions 103 to 497, or positions 103 to 507, or positions 103 to 517, or positions 103 to 525, or positions 103 to 537, or positions 90 to 410, or positions 90 to 417, or positions 90 to 425, or position 9 0 to 437, or positions 90 to 445, or positions 90 to 457, or positions 90 to 465, or positions 90 to 477, or positions 90 to 485, or positions 90 to 497, or positions 90 to 507, or positions 90 to 517, or positions 90 to 525, or positions 90 to 537, or positions 83 to 410, or positions 83 to 417, or positions 83 to 425, or positions 83 to 437, or positions 83 to 445, or positions 83 to 457, or positions 83 to 465, or positions 83 to 477, or positions 83 to 485, or positions 83 to 497, or positions 83 to 507, or positions 83 to 517,or positions 83 to 525, or positions 83 to 537, or positions 70 to 410, or positions 70 to 417, or positions 70 to 425, or positions 70 to 437, or positions 70 to 445, or positions 70 to 457, or positions 70 to 465, or positions 70 to 477, or positions 70 to 485, or positions 70 to 497, or positions 70 to 507, or positions 70 to 517, or positions 70 to 525, or positions 70 to 537, or positions 63 to 410, or positions 63 to 417, or positions 63 to 425, or positions 63 to 437, or positions 63 to 445, or positions 63 to 457, or is positions 63 to 465, or positions 63 to 477, or positions 63 to 485, or positions 63 to 497, or positions 63 to 507, or positions 63 to 517, or positions 63 to 525, or positions 63 to 537, or positions 50 to 410, or positions 50 to 417, or positions 50 to 425, or positions 50 to 437, or positions 50 to 445, or positions 50 to 457, or positions 50 to 465, or positions 50 to 477, or positions 50 to 485, or positions 50 to 497, or positions 50 to 507, or positions 50 to 517, or positions 50 to 525, or positions 50 to 537, or positions Positions 43 to 410, or positions 43 to 417, or positions 43 to 425, or positions 43 to 437, or positions 43 to 445, or positions 43 to 457, or positions 43 to 465, or positions 43 to 477, or positions 43 to 485, or positions 43 to 497, or positions 43 to 507, or positions 43 to 517, or positions 43 to 525, or positions 43 to 537, or positions 31 to 410, or positions 31 to 417, or positions 31 to 425, or positions 31 to 437, or positions 31 to 445, or positions 31 to 457, or positions 31 to 465, or positions 31 to 477, or positions and the amino acid residues of the gE protein are located at positions corresponding to positions 31 to 485, or 31 to 497, or 31 to 507, or 31 to 517, or 31 to 525, or 31 to 537, or positions 23 to 410, or positions 23 to 417, or positions 23 to 425, or positions 23 to 437, or positions 23 to 445, or positions 23 to 457, or positions 23 to 465, or positions 23 to 477, or positions 23 to 485, or positions 23 to 497, or positions 23 to 507, or positions 23 to 517, or positions 23 to 525, or positions 23 to 537.

[0012] In certain embodiments, the first peptide segment is located at positions 150-410 (or positions 150-417, or positions 150-425, or positions 150-437, or positions 150-445, or positions 150-457, or positions 150-465, or positions 150-477, or positions 150-485, or positions 150-497, or positions 150-507, or positions 150-517, or positions 150-525, or positions 150-537, or positions 143-410, or positions 143-417, or positions 143-425, or positions 143-437) of SEQ ID NO: 11. 7, or positions 143 to 445, or positions 143 to 457, or positions 143 to 465, or positions 143 to 477, or positions 143 to 485, or positions 143 to 497, or positions 143 to 507, or positions 143 to 517, or positions 143 to 525, or positions 143 to 537, or positions 120 to 410, or positions 120 to 417, or positions 120 to 425, or positions 120 to 437, or positions 120 to 445, or positions 120 to 457, or positions 120 to 465, or positions 120 to 477, or positions 120 to 485, or positions 120 to 497, or positions 12 0 to 507, or positions 120 to 517, or positions 120 to 525, or positions 120 to 537, or positions 103 to 410, or positions 103 to 417, or positions 103 to 425, or positions 103 to 437, or positions 103 to 445, or positions 103 to 457, or positions 103 to 465, or positions 103 to 477, or positions 103 to 485, or positions 103 to 497, or positions 103 to 507, or positions 103 to 517, or positions 103 to 525, or positions 103 to 537, or positions 90 to 410, or positions 90 to 417, or positions 90 to 425, or position 9 0 to 437, or positions 90 to 445, or positions 90 to 457, or positions 90 to 465, or positions 90 to 477, or positions 90 to 485, or positions 90 to 497, or positions 90 to 507, or positions 90 to 517, or positions 90 to 525, or positions 90 to 537, or positions 83 to 410, or positions 83 to 417, or positions 83 to 425, or positions 83 to 437, or positions 83 to 445, or positions 83 to 457, or positions 83 to 465, or positions 83 to 477, or positions 83 to 485, or positions 83 to 497, or positions 83 to 507, or positions 83 to 517,or positions 83 to 525, or positions 83 to 537, or positions 70 to 410, or positions 70 to 417, or positions 70 to 425, or positions 70 to 437, or positions 70 to 445, or positions 70 to 457, or positions 70 to 465, or positions 70 to 477, or positions 70 to 485, or positions 70 to 497, or positions 70 to 507, or positions 70 to 517, or positions 70 to 525, or positions 70 to 537, or positions 63 to 410, or positions 63 to 417, or positions 63 to 425, or positions 63 to 437, or positions 63 to 445, or positions 63 to 457, or positions 6 positions 3 to 465, or positions 63 to 477, or positions 63 to 485, or positions 63 to 497, or positions 63 to 507, or positions 63 to 517, or positions 63 to 525, or positions 63 to 537, or positions 50 to 410, or positions 50 to 417, or positions 50 to 425, or positions 50 to 437, or positions 50 to 445, or positions 50 to 457, or positions 50 to 465, or positions 50 to 477, or positions 50 to 485, or positions 50 to 497, or positions 50 to 507, or positions 50 to 517, or positions 50 to 525, or positions 50 to 537, or positions 43 to 410 , or positions 43 to 417, or positions 43 to 425, or positions 43 to 437, or positions 43 to 445, or positions 43 to 457, or positions 43 to 465, or positions 43 to 477, or positions 43 to 485, or positions 43 to 497, or positions 43 to 507, or positions 43 to 517, or positions 43 to 525, or positions 43 to 537, or positions 31 to 410, or positions 31 to 417, or positions 31 to 425, or positions 31 to 437, or positions 31 to 445, or positions 31 to 457, or positions 31 to 465, or positions 31 to 477, or positions 31 to 485, or positions or positions 31 to 497, or positions 31 to 507, or positions 31 to 517, or positions 31 to 525, or positions 31 to 537, or positions 23 to 410, or positions 23 to 417, or positions 23 to 425, or positions 23 to 437, or positions 23 to 445, or positions 23 to 457, or positions 23 to 465, or positions 23 to 477, or positions 23 to 485, or positions 23 to 497, or positions 23 to 507, or positions 23 to 517, or positions 23 to 525, or positions 23 to 537).

[0013] In certain embodiments, the first peptide segment is located at positions 70-485 (or positions 70-497, or positions 70-507, or positions 70-517, or positions 70-525, or positions 70-537, or positions 63-485, or positions 63-497, or positions 63-507, or positions 63-517, or positions 63-525, or positions 63-537, or positions 50-485, or positions 50-497, or positions 50-507, or positions 50-517, or positions 50-525, or positions 50-537) of SEQ ID NO: 11. 37, or positions 43 to 485, or positions 43 to 497, or positions 43 to 507, or positions 43 to 517, or positions 43 to 525, or positions 43 to 537, or positions 31 to 485, or positions 31 to 497, or positions 31 to 507, or positions 31 to 517, or positions 31 to 525, or positions 31 to 537, or positions 23 to 485, or positions 23 to 497, or positions 23 to 507, or positions 23 to 517, or positions 23 to 525, or positions 23 to 537).

[0014] In certain embodiments, the first peptide segment is located at position 70-485 (or position 70-497, or position 70-507, or position 70-517, or position 70-525, or position 70-537, or position 63-485, or position 63-497, or position 63-507, or position 63-517, or position 63-525, or position 63-537, or position 50-485, or position 50-497, or position 50-507, or position 50-517, or position 50-525, or position 50 ...537, of SEQ ID NO: 11. or positions 43-485, or 43-497, or 43-507, or 43-517, or 43-525, or 43-537, or positions 31-485, or positions 31-497, or positions 31-507, or positions 31-517, or positions 31-525, or positions 31-537, or positions 23-485, or positions 23-497, or positions 23-507, or positions 23-517, or positions 23-525, or positions 23-537.

[0015] In certain embodiments, the first peptide segment is located at positions 63-497 (or positions 63-507, or positions 63-517, or positions 63-525, or positions 63-537, or positions 50-497, or positions 50-507, or positions 50-517, or positions 50-525, or positions 50-537, or positions 43-497, or positions 43-507, or positions 43-507, or positions 50-517, or positions 50-525, or positions 50-537, of SEQ ID NO: 11. The amino acid sequence of the gE protein includes amino acid residues of the gE protein at positions corresponding to positions 43 to 517, or positions 43 to 525, or positions 43 to 537, or positions 31 to 497, or positions 31 to 507, or positions 31 to 517, or positions 31 to 525, or positions 31 to 537, or positions 23 to 497, or positions 23 to 507, or positions 23 to 517, or positions 23 to 525, or positions 23 to 537.

[0016] In certain embodiments, the first peptide segment is located at positions 63-497 (or positions 63-507, or positions 63-517, or positions 63-525, or positions 63-537, or positions 50-497, or positions 50-507, or positions 50-517, or positions 50-525, or positions 50-537, or positions 43-497, or positions 43-507, or positions 43-517) of SEQ ID NO: 11. 7, or positions 43 to 525, or positions 43 to 537, or positions 31 to 497, or positions 31 to 507, or positions 31 to 517, or positions 31 to 525, or positions 31 to 537, or positions 23 to 497, or positions 23 to 507, or positions 23 to 517, or positions 23 to 525, or positions 23 to 537).

[0017] In certain embodiments, the first peptide segment comprises amino acid residues of the gE protein at positions corresponding to positions 31-537 of SEQ ID NO:11.

[0018] In certain embodiments, the first peptide segment comprises or consists of amino acid residues of the gE protein located at positions corresponding to positions 31-537 or positions 23-537 of SEQ ID NO:11.

[0019] In certain embodiments, the first peptide segment comprises the extracellular region of a gE protein. In some 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., the peptide linker set forth in SEQ ID NO: 15), and the first peptide segment comprises the extracellular region of a gE protein.

[0020] In some embodiments, the first peptide segment consists of the extracellular region of the gE protein that does not contain a signal peptide or that contains a partial signal peptide sequence.

[0021] In some embodiments, the first peptide segment comprises or consists of amino acid residues at positions 23-350 (or positions 23-358, or positions 23-365, or positions 23-375, or positions 23-385, or positions 23-401, or positions 23-410, or positions 23-420, or positions 23-430, or positions 23-440, or positions 23-500, or positions 23-510, or positions 23-520, or positions 23-537, or positions 23-538, or positions 23-539, or positions 23-540, or positions 23-541, or positions 23-542, or positions 23-543, or positions 23-544, or positions 23-545, or positions 23-546) of a VZV gE protein.

[0022] In certain embodiments, the first peptide segment comprises or consists of amino acid residues at positions 27-350 (or positions 27-358, or positions 27-365, or positions 27-375, or positions 27-385, or positions 27-401, or positions 27-410, or positions 27-420, or positions 27-430, or positions 27-440, or positions 27-500, or positions 27-510, or positions 27-520, or positions 27-537, or positions 27-538, or positions 27-539, or positions 27-540, or positions 27-541, or positions 27-542, or positions 27-543, or positions 27-544, or positions 27-545, or positions 27-546) of a VZV gE protein.

[0023] In certain embodiments, the first peptide segment comprises or consists of amino acid residues at positions 31-350 (or positions 31-358, or positions 31-365, or positions 31-375, or positions 31-385, or positions 31-401, or positions 31-410, or positions 31-420, or positions 31-430, or positions 31-440, or positions 31-500, or positions 31-510, or positions 31-520, or positions 31-537, or positions 31-538, or positions 31-539, or positions 31-540, or positions 31-541, or positions 31-542, or positions 31-543, or positions 31-544, or positions 31-545, or positions 31-546) of a VZV gE protein.

[0024] In some embodiments, the first peptide segment comprises or consists of amino acid residues at positions 31 to 510 of the VZV gE protein.

[0025] In some embodiments, the first peptide segment comprises or consists of amino acid residues at positions 23-537 (or positions 23-538, or positions 23-539, or positions 23-540, or positions 23-541, or positions 23-542, or positions 23-543, or positions 23-544, or positions 23-545, or positions 23-546) of the VZV gE protein.

[0026] In some embodiments, the first peptide segment comprises or consists of amino acid residues from positions 23 to 537 of the VZV gE protein.

[0027] In certain embodiments, the gE protein has (a) an amino acid sequence set forth in any one of SEQ ID NOs: 11, 46 to 55; (b) an amino acid sequence having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to the amino acid sequence set forth in any one of SEQ ID NOs: 11, 46 to 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 to the amino acid sequence set forth in any one of SEQ ID NOs: 11, 46 to 55.

[0028] In certain embodiments, the amino acid sequence of the gE protein is set forth in SEQ ID NO:11.

[0029] In certain embodiments, the first peptide segment comprises or consists of the amino acid sequence set forth in any one of SEQ ID NO: 13, SEQ ID NO: 33 to SEQ ID NO: 42, SEQ ID NO: 64 to SEQ ID NO: 69, or a sequence having at least 90%, for example at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or 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 set forth in any one of SEQ ID NO: 13, SEQ ID NO: 33 to SEQ ID NO: 42, SEQ ID NO: 64 to SEQ ID NO: 69.

[0030] In certain embodiments, the second peptide segment comprises amino acid residues of the gI protein at positions corresponding to positions 45-240 (or positions 45-251, or positions 45-260, or positions 45-271, or positions 41-240, or positions 41-251, or positions 41-260, or positions 41-271, or positions 30-240, or positions 30-251, or positions 30-260, or positions 30-271, or positions 21-240, or positions 21-251, or positions 21-260, or positions 21-271) of SEQ ID NO: 12.

[0031] In certain embodiments, the second peptide segment comprises or consists of amino acid residues of the gI protein located at positions corresponding to positions 45-240 (or positions 45-251, or positions 45-260, or positions 45-271, or positions 41-240, or positions 41-251, or positions 41-260, or positions 41-271, or positions 30-240, or positions 30-251, or positions 30-260, or positions 30-271, or positions 21-240, or positions 21-251, or positions 21-260, or positions 21-271) of SEQ ID NO: 12.

[0032] In certain embodiments, the second peptide segment comprises amino acid residues of the gI protein at positions corresponding to positions 30-260 (or positions 30-271, or positions 21-260, or positions 21-271) of SEQ ID NO:12.

[0033] In certain embodiments, the second peptide segment comprises or consists of amino acid residues of the gI protein located at positions corresponding to positions 30-260 (or positions 30-271, or positions 21-260, or positions 21-271) of SEQ ID NO: 12.

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

[0035] In certain embodiments, the second peptide segment comprises the extracellular domain of a gI protein. In some embodiments, the second peptide segment is optionally connected to the N-terminus of the first peptide segment via a linker (e.g., a peptide linker, e.g., the peptide linker set forth in SEQ ID NO: 15), and the second peptide segment comprises the extracellular domain of a gI protein.

[0036] In some embodiments, the second peptide segment consists of the extracellular region of the gI protein that does not contain a signal peptide or that contains a partial signal peptide sequence.

[0037] In certain embodiments, the second peptide segment comprises or consists of amino acid residues of the gI protein located at positions corresponding to positions 21-271 of SEQ ID NO:12.

[0038] In some embodiments, the second peptide segment comprises or consists of amino acid residues at positions 21-220 (or positions 21-230, or positions 21-235, or positions 21-250, or positions 21-260, or positions 21-271) of the VZV gI protein.

[0039] In some embodiments, the second peptide segment comprises or consists of amino acid residues at positions 30-220 (or positions 30-230, or positions 30-235, or positions 30-250, or positions 30-260, or positions 30-271) of the VZV gI protein.

[0040] In some embodiments, the second peptide segment comprises or consists of amino acid residues at positions 36-220 (or positions 36-230, or positions 36-235, or positions 36-250, or positions 36-260, or positions 36-271) of the VZV gI protein.

[0041] In certain embodiments, the first peptide segment is located at positions 63-497 (or positions 63-507, or positions 63-517, or positions 63-525, or positions 63-537, or positions 50-497, or positions 50-507, or positions 50-517, or positions 50-525, or positions 50-537, or positions 43-497, or positions 43-507, or positions 43-517, or positions 43-525, or positions 43-537, or positions 31-497, or positions 31-497, or positions 31-497, of SEQ ID NO: 11. the first peptide segment comprises amino acid residues of gE protein at positions corresponding to positions 30 to 260 (or positions 30 to 271, or positions 21 to 260, or positions 21 to 271) of SEQ ID NO: 12; and the second peptide segment comprises amino acid residues of gI protein at positions corresponding to positions 30 to 260 (or positions 30 to 271, or positions 21 to 260, or positions 21 to 271) of SEQ ID NO: 12.

[0042] In certain embodiments, the first peptide segment is located at positions 63-497 (or positions 63-507, or positions 63-517, or positions 63-525, or positions 63-537, or positions 50-497, or positions 50-507, or positions 50-517, or positions 50-525, or positions 50-537, or positions 43-497, or positions 43-507, or positions 43-517, or positions 43-525, or positions 43-537, or positions 31-497, or positions 31-507, or positions 31-507, or positions 31-517, or positions 43-525, or positions 43-537, or positions 31-497, or positions 31-507, or positions 31-507, or positions 31-517, or positions 31-525, or positions 31-53 ... the first peptide segment comprises or consists of amino acid residues of gE protein located at positions corresponding to positions 30 to 260 (or positions 30 to 271, or positions 21 to 260, or positions 21 to 271) of SEQ ID NO: 12; and the second peptide segment comprises or consists of amino acid residues of gI protein located at positions corresponding to positions 30 to 260 (or positions 30 to 271, or positions 21 to 260, or positions 21 to 271) of SEQ ID NO: 12.

[0043] In certain embodiments, the gI protein has (a) the amino acid sequence set forth in any one of SEQ ID NOs: 12, 56 to 58, (b) an amino acid sequence having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) to the amino acid sequence set forth in any one of SEQ ID NOs: 12, 56 to 58, 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 to the amino acid sequence set forth in any one of SEQ ID NOs: 12, 56 to 58.

[0044] In certain embodiments, the amino acid sequence of the gI protein is set forth in SEQ ID NO:12.

[0045] In some embodiments, the second peptide segment comprises or consists of amino acid residues from positions 36 to 235 of the VZV gI protein.

[0046] In some embodiments, the second peptide segment comprises or consists of amino acid residues from positions 21 to 271 of the VZV gI protein.

[0047] In certain embodiments, the second peptide segment comprises or consists of an amino acid sequence set forth in any one of SEQ ID NO: 14, SEQ ID NO: 43 to SEQ ID NO: 45, or a sequence having at least 90%, e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to, or 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 set forth in any one of SEQ ID NO: 14, SEQ ID NO: 43 to SEQ ID NO: 45.

[0048] In certain embodiments, 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, e.g., the peptide linker shown in SEQ ID NO: 15).

[0049] In certain embodiments, the linker is a peptide linker that includes one or more (eg, 5-20) flexible amino acids.

[0050] In certain embodiments, the linker is a peptide linker comprising one or more glycines and / or one or more serines.

[0051] In certain embodiments, the fusion protein comprises or consists of an amino acid sequence set forth in any one of SEQ ID NO: 6 to SEQ ID NO: 7, SEQ ID NO: 20 to SEQ ID NO: 32, SEQ ID NO: 59 to SEQ ID NO: 63, and SEQ ID NO: 70.

[0052] It is readily apparent to those skilled in the art that the fusion protein optionally contains a signal peptide sequence to facilitate protein expression. The signal peptide sequence may be present in the first peptide segment or the second peptide segment, or the signal peptide sequence may be present outside the first and second peptide segments (e.g., neither the first peptide segment nor the second peptide segment contains a signal peptide sequence, and the fusion protein further contains a signal peptide sequence outside the first and second peptide segments).

[0053] In certain embodiments, the second peptide segment is connected to the N-terminus of the first peptide segment, optionally via a linker (e.g., a peptide linker, e.g., the 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 a VZV gI protein). In certain embodiments, the second peptide segment is connected to the C-terminus of the first peptide segment, optionally via a linker (e.g., a peptide linker, e.g., the 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 a 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.

[0054] In certain embodiments, the fusion protein comprises a signal peptide and / or a detectable label (eg, a tag protein).

[0055] In certain embodiments, the fusion protein comprises a signal peptide at its N-terminus (e.g., the native signal peptide of the VZV gE protein, the native signal peptide of the VZV gI protein, the gp67 signal peptide, the melittin signal peptide (MSP), or the tissue-type plasminogen activator signal peptide (tPA signal peptide)).

[0056] In certain embodiments, the fusion protein comprises a detectable label at its C-terminus.

[0057] In another aspect, the present disclosure also provides an RNA molecule comprising a nucleotide sequence encoding a fusion protein as described above.

[0058] In some embodiments, the nucleotide sequence encoding the fusion protein is codon-optimized or non-optimized according to the codon preferences of the host cell (e.g., insect cells or mammalian cells).

[0059] In some 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 polyA tail.

[0060] In some embodiments, the RNA molecule comprises, from 5' to 3': a 5' UTR, a Kozak sequence, a start codon, a nucleotide sequence encoding the fusion protein, a stop codon, a 3' UTR, and a polyA tail.

[0061] In certain embodiments, the RNA molecule has one or more of the following characteristics: (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 compared to the nucleotide sequence set forth in SEQ ID NO:71, and in certain embodiments, the nucleotide sequence encoding the fusion protein is set forth in SEQ ID NO:71; (2) the presence of 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 having 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 compared to the nucleotide sequence set forth in SEQ ID NO: 17, and in certain embodiments, the 5' UTR has the nucleotide sequence set forth in SEQ ID NO: 17; (4) the 3' UTR has a nucleotide sequence having 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 compared to the nucleotide sequence set forth in SEQ ID NO: 18, and in certain embodiments, the 3' UTR has the nucleotide sequence set forth 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 polyadenylate sequences, each of which is independently composed of 20 to 120 consecutive adenylates, preferably the poly-A tail comprises multiple polyadenylate sequences, adjacent polyadenylate sequences being 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 compared to the nucleotide sequence set forth in SEQ ID NO: 19, and in some embodiments the poly-A tail has the nucleotide sequence set forth in SEQ ID NO: 19; (7) The 5' end of the RNA molecule is modified with a 5' cap (e.g., CAP-0, CAP-1, CAP-2).

[0062] In another aspect, the present disclosure also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein described above, or an RNA molecule described above.

[0063] In some embodiments, the nucleotide sequence encoding the fusion protein is codon-optimized or non-optimized according to the codon preferences of the host cell (e.g., insect cells or mammalian cells).

[0064] In another aspect, the present disclosure also provides a vector comprising the above-described isolated nucleic acid molecule.

[0065] In another aspect, the present disclosure provides a delivery composition comprising a delivery vehicle and an RNA molecule, isolated nucleic acid molecule, or vector described above.

[0066] In certain embodiments, the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microvesicle, and a viral vector (e.g., a replication-defective retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus).

[0067] In certain embodiments, the delivery composition comprises an RNA molecule as described above.

[0068] In certain embodiments, the delivery vehicle is a lipid nanoparticle (LNP).

[0069] In certain embodiments, the lipid nanoparticles comprise the lipids ALC-0315, ALC-0519, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol.

[0070] In certain embodiments, the molar percentage of ALC-0315 in the lipid nanoparticles ranges from 40% to 55% (e.g., 40% to 46.3%, 40% to 48%, 40% to 50%, 40% to 52%, 43% to 46.3%, 43% to 58%, 43% to 50%, 43% to 52%, 43% to 55%). In certain embodiments, the molar percentage of ALC-0315 in the lipid nanoparticles is 46.3%.

[0071] In certain embodiments, the molar percentage of cholesterol in the lipid nanoparticles ranges from 35% to 50% (e.g., 35% to 42.7%, 35% to 45%, 40% to 42.7%, 40% to 45%, 40% to 50%). In certain embodiments, the molar percentage of cholesterol in the lipid nanoparticles is 42.7%.

[0072] In some embodiments, the molar percentage of DSPC in the lipid nanoparticles is in the range of 5% to 12% (e.g., 5% to 9.4%, 5% to 10%, 5% to 11%, 8% to 9.4%, 8% to 10%, 8% to 11%, 8% to 12%, 9% to 9.4%, 9% to 10%, 9% to 11%, 9% to 12%). In some embodiments, the molar percentage of DSPC in the lipid nanoparticles is 46.3%.

[0073] In some embodiments, the molar percentage of ALC-0519 in the lipid nanoparticles ranges from 1.0% to 2.0% (e.g., 1.0% to 1.6%, 1.0% to 1.8%, 1.3% to 1.6%, 1.3% to 1.8%, 1.3% to 2.0%). In some embodiments, the molar percentage of ALC-0519 in the lipid nanoparticles is 1.6%.

[0074] In some embodiments, the delivery composition is a vaccine (eg, a nucleic acid vaccine).

[0075] In another aspect, the present disclosure also provides a host cell comprising the above-described RNA molecule, isolated nucleic acid molecule, or vector.

[0076] In certain embodiments, the host cell is selected from the group consisting of a prokaryotic cell (e.g., an E. coli cell) and a eukaryotic cell (e.g., a yeast cell, an insect cell, a plant cell, a mammalian cell).

[0077] In certain embodiments, the host cell is an insect cell.

[0078] In certain embodiments, the host cell is a mammalian cell.

[0079] In another aspect, the present disclosure also provides a method of preparing the above-described fusion protein, comprising culturing a host cell as described above under conditions that allow for protein expression, and recovering the fusion protein from the cell culture.

[0080] therapeutic use In another aspect, the present disclosure also provides an immunogenic composition comprising the above-described fusion protein, RNA molecule, isolated nucleic acid molecule, vector, or delivery composition, and optionally a pharmaceutically acceptable carrier and / or excipient (e.g., an adjuvant).

[0081] In certain embodiments, the immunogenic composition comprises the fusion protein, RNA molecule, isolated nucleic acid molecule, vector, or delivery composition described above, and an adjuvant, wherein the adjuvant is selected from the group consisting of an aluminum salt adjuvant, a zinc-aluminum hybrid adjuvant (e.g., FH002C), Freund's adjuvant, an oil emulsion adjuvant (e.g., MF59 adjuvant), a cytokine, a TLR agonist, a CpG adjuvant, a nucleic acid adjuvant, a liposome, a saponin adjuvant, an AS01B adjuvant, and any combination thereof.

[0082] In certain embodiments, the immunogenic composition comprises the fusion protein or delivery composition described above and an adjuvant.

[0083] In certain embodiments, the immunogenic composition comprises the fusion protein described above and an AS01B adjuvant.

[0084] In certain embodiments, the immunogenic composition comprises the delivery composition described above and a CpG adjuvant.

[0085] In certain embodiments, the immunogenic composition is a vaccine.

[0086] The immunogenic compositions described above can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, 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 disclosure should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such an injection can be a sterile solution for injection. Furthermore, the sterile solution for injection can be prepared for storage and use as a sterile lyophilized powder (e.g., by vacuum drying or lyophilization). This sterile, lyophilized powder 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% (weight / volume) NaCl), glucose solution (e.g., 5% glucose), a surfactant-containing solution (e.g., a solution containing 0.01% polysorbate 20), a pH buffer (e.g., phosphate buffer), Ringer's solution, and any combination thereof.

[0087] The immunogenic compositions described above can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or intranasal routes. However, for many therapeutic applications, the preferred route / mode of administration is parenteral (e.g., intravenous or bolus, subcutaneous, intraperitoneal, intramuscular). One skilled in the art will appreciate that the route and / or mode of administration will vary depending on the intended purpose.

[0088] The immunogenic compositions 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 particular disease or condition being treated or prevented, its severity, the subject's age, and other personal attributes of the particular subject (e.g., the subject's general health and the robustness of the subject's immune system). Determination of an effective dose is also guided by animal model studies, subsequent human clinical trials, and administration regimens that significantly reduce the occurrence or severity of the target disease symptoms or condition in the subject.

[0089] In another aspect, the present disclosure also provides the use of the above-described fusion protein, or RNA molecule, or isolated nucleic acid molecule, or vector, or delivery composition, or host cell, or immunogenic composition in the manufacture 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.

[0090] In certain embodiments, the immunogenic composition is a vaccine.

[0091] In certain embodiments, the VZV infection is a primary or recurrent VZV infection.

[0092] In certain embodiments, the disease associated with VZV infection is selected from the group consisting of shingles, chickenpox, and their complications (e.g., postherpetic neuralgia, pneumonia, encephalomyelitis, conjunctivitis).

[0093] In certain embodiments, the subject is a mammal, such as a human.

[0094] In another aspect, the present disclosure also provides a method for inducing an immune response against VZV in a subject and / or preventing and / or treating VZV infection or a disease associated with VZV infection in a subject, the method 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 described above.

[0095] In certain embodiments, the VZV infection is a primary or recurrent VZV infection.

[0096] In certain embodiments, the disease associated with VZV infection is selected from the group consisting of shingles, chickenpox, and their complications (e.g., postherpetic neuralgia, pneumonia, encephalomyelitis, conjunctivitis).

[0097] In certain embodiments, the subject is a mammal, such as a human.

[0098] Detection Applications In another aspect, the present disclosure also provides a method for detecting the presence of VZV gI protein-specific antibodies and / or VZV gE protein-specific antibodies in a sample, which comprises using the fusion protein described above.

[0099] In certain embodiments, the method is an immunological detection method, such as an immunoblot, an enzyme-linked immunosorbent assay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0100] In certain embodiments, the method comprises: (1) contacting a sample with the fusion protein; and (2) detecting the formation of a fusion protein-antibody immune complex or detecting the amount of the immune complex, wherein the formation of the immune complex indicates the presence of VZV gI protein-specific antibodies and / or VZV gE protein-specific antibodies in the sample.

[0101] In certain embodiments, the method further comprises detecting the presence of VZV gI protein-specific antibodies and / or VZV gE protein-specific antibodies in the sample using a second antibody having a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin).

[0102] In certain embodiments, the second antibody is specific for a constant region contained in an antibody of the species (eg, human) from which the sample being tested originates.

[0103] In certain embodiments, the second antibody is an anti-immunoglobulin (eg, human immunoglobulin) antibody, such as an anti-IgG antibody.

[0104] In certain embodiments, the sample is a bodily fluid sample (eg, whole blood, plasma, serum, saliva, or urine) from a subject (eg, a mammal, preferably a human).

[0105] In certain embodiments, the method is used for diagnostic or non-diagnostic purposes. In certain embodiments, the method is used for non-diagnostic purposes.

[0106] In another aspect, the present disclosure also provides use of the above-described fusion protein, or RNA molecule, or isolated nucleic acid molecule, or vector, or delivery composition, or host cell in the manufacture of a detection reagent, wherein the detection reagent is used to detect the presence of VZV gI protein-specific antibodies and / or VZV gE protein-specific antibodies in a sample.

[0107] In certain embodiments, the detection reagent detects the presence of VZV gI protein-specific antibodies and / or VZV gE protein-specific antibodies in a sample by the methods described above.

[0108] In certain embodiments, the sample is a bodily fluid sample (eg, whole blood, plasma, serum, saliva, or urine) from a subject (eg, a mammal, preferably a human).

[0109] kit In another aspect, the present disclosure also provides a kit comprising the fusion protein, or RNA molecule, or isolated nucleic acid molecule, or vector, or delivery composition, or host cell described above.

[0110] In certain embodiments, the kit comprises the fusion protein described above and a second antibody, wherein the second antibody is as defined above.

[0111] Definition of Terms In this disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures of virology, biochemistry, and immunology used herein are all routine procedures widely used in the corresponding fields. At the same time, in order to better understand this disclosure, the definitions and explanations of relevant terms are provided below.

[0112] When the terms "for example," "eg," "such as," "including," "comprising," or variations thereof are used herein, these terms are not to be considered limiting terms and should be interpreted as meaning "without being limited to" or "limited to."

[0113] Unless otherwise specified herein or clearly contradicted by context, the singular forms "a," "an," and "the" and similar referents shall be construed to include both the singular and the plural in the context of describing this disclosure, particularly in the context of the appended claims.

[0114] As used herein, the term "VZV" is an abbreviation for "Varicella-Zoster Virus," a DNA virus.

[0115] As used herein, the terms "gE protein," "gE," and "glycoprotein E" refer to a type of VZV envelope glycoprotein, have the same meaning, and can be used interchangeably. The specific amino acid sequence of the wild-type gE protein can be obtained from a public database (e.g., the GenBank database), for example, from the amino acid sequences set forth 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.

[0116] In the present disclosure, references to the amino acid sequence of gE protein are made with reference to the sequence set forth in SEQ ID NO: 11. For example, the phrase "amino acid residues in gE protein at positions corresponding to positions 150-410 of SEQ ID NO: 11" refers to the amino acid sites / residues in the sequence that are at positions corresponding to amino acid residues 150-410 of SEQ ID NO: 11 when 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.

[0117] Those skilled in the art will understand that VZV comprises multiple isolates, and that there may be differences between the amino acid sequences of the gE proteins of various isolates. Furthermore, those skilled in the art will understand that, despite possible sequence differences, the gE proteins of different VZV isolates share a high degree of amino acid sequence identity (usually greater than 95%, e.g., greater than 96%, 97%, 98%, or 99%) and have substantially the same biological function. Therefore, in the present disclosure, the term "gE protein" includes not only the protein set forth in SEQ ID NO: 11, SEQ ID NO: 46-55, but also the gE proteins of various VZV isolates. Furthermore, when a fragment of a gE protein is described, the fragment includes not only the fragment of SEQ ID NO: 11, SEQ ID NO: 46-55, but also the corresponding fragment in the gE proteins of various VZV isolates. For example, the phrase "amino acid residues at positions 23 to 537 of the gE protein" includes the amino acid residues at positions 23 to 537 of any one of SEQ ID NO: 11, SEQ ID NO: 46 to SEQ ID NO: 55, and corresponding fragments in the gE protein of various VZV isolates.

[0118] As used herein, the term "extracellular region of a gE protein" refers to a portion of a gE protein that does not include the intracellular region and the transmembrane region. For example, based on a specific sequence analysis, in some embodiments, the extracellular region of a gE protein refers to a region of a gE protein that includes at least the amino acid residues at positions corresponding to amino acid positions 1-537 of SEQ ID NO: 11. Current research has shown that the amino acid residues in the gE protein at positions corresponding to amino acid positions 1-546 of SEQ ID NO: 11 can also be used as the extracellular region of the gE protein to perform the corresponding functions of the extracellular region of the gE protein. Thus, in certain embodiments, the extracellular region of a gE protein refers to the amino acid residues in the gE protein at positions corresponding to amino acid positions 1-546 (or positions 1-538, or positions 1-539, or positions 1-540, or positions 1-541, or positions 1-542, or positions 1-543, or positions 1-544, or positions 1-545) of SEQ ID NO: 11. It will be understood by those skilled in the art that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can occur naturally or can be artificially introduced into SEQ ID NO: 11 without affecting the biological properties of the gE protein. For example, gE proteins of different VZV strains can naturally differ in amino acid sequence but have substantially the same biological properties. Therefore, when describing the extracellular region of a gE protein, the extracellular region not only includes the amino acid residues at positions 1 to 537 (or positions 1 to 538, or positions 1 to 539, or positions 1 to 540, or positions 1 to 541, or positions 1 to 542, or positions 1 to 543, or positions 1 to 544, or positions 1 to 545, or positions 1 to 546) of SEQ ID NO: 11, but also includes amino acid residues in natural or artificial variants of SEQ ID NO: 11 (e.g., gE proteins set forth in any one of SEQ ID NOs: 46 to 55) that are located at positions corresponding to amino acid positions 1 to 537 (or positions 1 to 538, or positions 1 to 539, or positions 1 to 540, or positions 1 to 541, or positions 1 to 542, or positions 1 to 543, or positions 1 to 544, or positions 1 to 545, or positions 1 to 546) of SEQ ID NO: 11.

[0119] As used herein, the terms "gI protein," "gI," and "glycoprotein I" refer to the envelope glycoprotein of VZV, and have the same meaning and can be used interchangeably. The specific amino acid sequence of the wild-type gI protein can be obtained from a public database (e.g., the GenBank database), and may be, for example, the amino acid sequence set forth in GenBank accession numbers AB097933 (pOka strain), AB097932 (vOka strain), NC001348 (Dumas strain), AY548170 (MSP strain), AEW89483.1, AGY34059.1, or AEW89051.1.

[0120] In this disclosure, references to the amino acid sequence of gI protein are made with reference to the sequence set forth in SEQ ID NO: 12. For example, the phrase "amino acid residues in gI protein at positions corresponding to positions 45-240 of SEQ ID NO: 12" refers to the amino acid positions / residues in the sequence that are at positions corresponding to amino acid residues 45-240 of SEQ ID NO: 12 when 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.

[0121] Those skilled in the art will understand that VZV comprises multiple isolates, and that there may be differences between the amino acid sequences of the gI proteins of various isolates. Furthermore, those skilled in the art will understand that, despite possible sequence differences, the gI proteins of different VZV isolates share a high degree of identity in amino acid sequence (typically greater than 95%, e.g., greater than 96%, 97%, 98%, 99%, 99.5%, or greater than 99.8%) and have substantially the same biological function. Thus, in the present disclosure, the term "gI protein" includes not only the protein set forth in SEQ ID NO: 12 and any one of SEQ ID NOs: 56-58, but also the gI proteins of various VZV isolates. Furthermore, when a fragment of a gI protein is described, the fragment includes not only the fragment of SEQ ID NO: 12 and any one of SEQ ID NOs: 56-58, but also the corresponding fragment in the gI protein of various VZV isolates. For example, the phrase "amino acid residues at positions 21 to 271 of the gI protein" includes the amino acid residues at positions 21 to 271 of SEQ ID NO: 12 and any one of SEQ ID NO: 56 to SEQ ID NO: 58, as well as corresponding fragments in the gI protein of various VZV isolates.

[0122] As used herein, the "extracellular region of a gI protein" refers to a portion of a gI protein that does not include the intracellular region and the transmembrane region. For example, based on a specific sequence analysis, in some embodiments, the extracellular region of a gI protein refers to the amino acid residues in the gI protein at positions corresponding to positions 1-271 of SEQ ID NO: 12. Those skilled in the art will appreciate that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally or artificially introduced into SEQ ID NO: 12 without affecting the biological properties of the gI protein. For example, gI proteins of different VZV strains may naturally differ in amino acid sequence but have substantially the same biological properties. Thus, when describing the extracellular region of a gI protein, the extracellular region includes not only the amino acid residues at positions 1-271 of SEQ ID NO: 12, but also the amino acid residues in natural or artificial variants of SEQ ID NO: 12 (e.g., gI proteins set forth in any one of SEQ ID NOs: 56-58) at positions corresponding to positions 1-271 of SEQ ID NO: 12.

[0123] According to the present disclosure, the expressions "corresponding fragment" or "corresponding amino acid position" refer to the fragment or amino acid site / residue that is at the corresponding position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity.

[0124] As used herein, the term "identity" refers to the sequence match between two polypeptides or two nucleic acids. If a position in both compared sequences is occupied by the same nucleotide or amino acid residue (e.g., if each position in two DNA molecules is occupied by an adenine nucleotide, or each position in two polypeptides is occupied by a lysine), 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, multiplied by 100. For example, if six out of ten positions in two sequences are identical, the two sequences are 60% identical. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six total positions are identical). Typically, comparisons are performed when the two sequences are aligned to maximize identity. Such alignments can be accomplished, for example, by using the method of Needleman et al. (1970), J. Mol. Biol. 48:443-453, which can be conveniently implemented by computer programs 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.Furthermore, the percent identity between two amino acid sequences can be determined using the Needleman-Wunsch algorithm (J Mol Biol. 48:444-453 (1970)), which is incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0125] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing 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 substitution of an amino acid residue with an amino acid residue having a similar side chain, e.g., substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., similar in size, shape, charge, chemical properties including the ability to form covalent 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace a 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).

[0126] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. When the vector is introduced into a host cell by transformation, transduction, or transfection, the genetic material elements carried by the vector can 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 λ 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, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors can contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication.

[0127] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as E. 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.

[0128] Those skilled in the art will understand that the design of the expression vector can depend on factors such as the choice of the host cell to be transformed, the level of expression desired, etc. Upon introduction into a host cell, the vector can produce a transcript, protein, or peptide, including the proteins, isolated nucleic acid molecules, etc. described herein.

[0129] According to the present disclosure, 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 are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are 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 hybrid adjuvants (e.g., FH002C), Freund's adjuvant, oil emulsion adjuvants, cytokines, TLR adjuvants, CpG adjuvants, liposomes, AS01B adjuvants, or combinations thereof; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0130] According to the present disclosure, the term "adjuvant" refers to a nonspecific immune-enhancing agent that can enhance or change the type of immune response of the body to an antigen when delivered to the body together with or beforehand to an antigen. There are many types of adjuvants, including, but not limited to, aluminum salt adjuvants, zinc-aluminum hybrid adjuvants (e.g., FH002C), Freund's adjuvant, oil emulsion adjuvants (e.g., MF59 adjuvant), cytokines, TLR agonists, CpG adjuvants, liposomes, AS01B adjuvant, or any combination thereof. In the present disclosure, it is particularly preferred that the adjuvant is AS01B adjuvant, CpG adjuvant, or any combination thereof.

[0131] According to the present disclosure, the term "effective amount" refers to an amount that can effectively achieve an intended purpose. For example, an effective amount for preventing or treating a disease (e.g., VZV infection) refers to an amount that can effectively prevent, stop, or delay the onset of the disease (e.g., VZV infection), or an amount that can alleviate, reduce, or treat the severity of an existing disease (e.g., a disease caused by VZV infection). Determining such an effective amount is within the ability of one skilled in the art. For example, the effective amount for therapeutic use depends 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 therapeutic agents administered simultaneously.

[0132] In this disclosure, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Also in this disclosure, amino acids are generally represented by their one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0133] As used herein, the term "subject" refers to an animal, such as a vertebrate. Preferably, the subject is a mammal, such as a human, cow, horse, cat, dog, rodent, or primate. Particularly preferably, the subject is a human. As used herein, this term can be used interchangeably with "patient." [Effects of the Invention]

[0134] As disclosed herein, the VZV gE-gI fusion protein exhibits good reactivity with both gE-specific mouse antibodies and gI-specific mouse monoclonal antibodies. Unlike previously reported gE-gI dimers, the gE-gI fusion protein can be expressed from a single plasmid and a single clone. It simultaneously retains the properties of both the gE and gI proteins and can be used as a detection antigen for diagnostic reagents and an immunogen for vaccines, demonstrating its potential application in the diagnosis of varicella-zoster virus.

[0135] In particular, compared to a single gE protein, a blend of gE and gI proteins, a gE-gI heterodimer, and the commercially available Shingrix™ vaccine, the gE-gI fusion protein of the present disclosure has significantly superior activity in inducing neutralizing antibody and T cell responses, indicating the promise and value of its promising application in the prevention and / or treatment of shingles.

[0136] Although embodiments of the present disclosure will be described in detail below in conjunction with figures and examples, those skilled in the art will understand that the following figures and examples are used only to illustrate the present disclosure and do not limit the scope of the present disclosure. Various objects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description of the figures and preferred embodiments. [Brief explanation of the drawings]

[0137] [Figure 1]FIG. 1 shows the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (left panel) and Western blot (right panel) of insect cell-derived gE-gI fusion protein samples at various purification stages in Example 1 (after sequential chromatographic purification on a Q-FF column (panel A), a DEAE-FF column (panel B), and a butyl column (panel C)). (In the figure, lane M: molecular weight marker; lane 1: gE-gI fusion protein). [Figure 2] FIG. 1 shows the results of SDS-polyacrylamide gel electrophoresis (left panel) and Western blot (right panel) of a CHO cell-derived gE-gI fusion protein sample at various purification stages in Example 1 (after sequential purification by chromatography on a Ni-6FF column (panel A) and size-exclusion chromatography with Superdex200 Increase (panel B)). (In the figure, lane M: molecular weight marker; lane 1: gE-gI fusion protein.) [Figure 3] FIG. 1 shows the results of high performance size exclusion chromatography (HPSEC) of insect cell-derived gE-gI fusion protein in Example 2. [Figure 4] FIG. 1 shows the results of high performance size exclusion chromatography (HPSEC) of the CHO cell-derived gE-gI fusion protein in Example 2. [Figure 5] FIG. 1 shows the results of analytical ultracentrifugation of insect cell-derived gE-gI fusion protein in Example 3. [Figure 6] FIG. 1 shows the results of analytical ultracentrifugation of a gE-gI fusion protein derived from CHO cells in Example 3. [Figure 7] This figure shows the results of enzyme immunoassay of insect cell-derived gE-gI fusion protein using 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) in Example 4. [Figure 8]This figure shows the results of enzyme immunoassay of CHO cell-derived gE-gI fusion protein using gE protein-specific antibodies (1B11, 4G4, 6G7, 6H6, 11B11, 11B12, 11E3, 14G1) and gI protein-specific antibodies (7E7, 18B2, 9D11, 16F1, 13B2) in Example 4. [Figure 9] FIG. 10 shows antigen-specific IgG titers in mouse serum induced by gE-gI fusion protein using FH002C adjuvant, Freund's complete adjuvant (Freund's), and aluminum adjuvant (Al) in Example 5. [Figure 10] FIG. 1 shows neutralizing antibody titers in mouse serum induced by gE-gI fusion protein using FH002C adjuvant, Freund's complete adjuvant (Freund's), and aluminum adjuvant (Al), as detected by Elispot assay and plaque assay, respectively, in Example 5. [Figure 11] FIG. 1 shows the T cell immune responses induced by a saline blank (saline) containing no gE-gI fusion protein, and gE-gI fusion protein combined with FH002C adjuvant, Freund's complete adjuvant (Freund's), and aluminum adjuvant (Al) in Example 6. The left panel shows the percentage of induced IL2+ CD4+ T cells, and the right panel shows the percentage of induced IFNγ+ CD4+ T cells. [Figure 12] FIG. 1 shows the binding and neutralizing antibody titers induced by two different vaccines (a vaccine containing insect cell-derived gE-gI fusion protein and AS01B adjuvant, and a Shingrix vaccine) after two injections in Example 7. [Figure 13]FIG. 1 shows CD4+ T cell responses stimulated by a saline blank (saline) containing no gE-gI fusion protein and two different vaccines (a vaccine containing insect cell-derived gE-gI fusion protein and AS01B adjuvant, and a Shingrix vaccine) in Example 7. The left panel shows the percentage of induced IFNγ+ CD4+ T cells, and the right panel shows the percentage of induced IL2+ CD4+ T cells. [Figure 14] FIG. 1 shows CD8+ T cell responses stimulated by a saline blank (saline) containing no gE-gI fusion protein and two different vaccines (a vaccine containing insect cell-derived gE-gI fusion protein and AS01B adjuvant, and a Shingrix vaccine) in Example 7. The left panel shows the percentage of induced IFNγ+ CD8+ T cells, and the right panel shows the percentage of induced IL2+ CD8+ T cells. [Figure 15] FIG. 1 shows the levels of IFN-γ (left panel) and IL-2 (right panel) detected by Elispot assay stimulated by a saline blank (saline) without gE-gI fusion protein and two different vaccines (a vaccine containing insect cell-derived gE-gI fusion protein and AS01B adjuvant, and a Shingrix vaccine) in Example 7. [Figure 16] Figure 1 shows the binding antibody titers (left panel), the curves of binding antibody titers (middle panel), and the neutralizing antibody titers (right panel) induced by two different vaccines (a vaccine containing CHO-derived gE-gI fusion protein and AS01B adjuvant, and the Shingrix vaccine) after two injections in Example 8. [Figure 17] FIG. 1 shows CD4+ T cell responses stimulated by a saline blank (saline) containing no gE-gI fusion protein and two different vaccines (a vaccine containing CHO-derived gE-gI fusion protein and AS01B adjuvant, and a Shingrix vaccine) in Example 8. The left panel shows the percentage of induced IFNγ+ CD4+ T cells, and the right panel shows the percentage of induced IL2+ CD4+ T cells. [Figure 18] FIG. 1 shows CD8+ T cell responses stimulated by a saline blank (saline) containing no gE-gI fusion protein and two different vaccines (a vaccine containing CHO-derived gE-gI fusion protein and AS01B adjuvant, and a Shingrix vaccine) in Example 8. The left panel shows the percentage of induced IFNγ+ CD8+ T cells, and the right panel shows the percentage of induced IL2+ CD8+ T cells. [Figure 19] FIG. 1 shows the levels of IFN-γ (left panel) and IL-2 (right panel) induced by a saline blank (saline) without gE-gI fusion protein and two different vaccines (a vaccine containing CHO-derived gE-gI fusion protein and AS01B adjuvant, and a Shingrix vaccine) as detected by Elispot assay in Example 8. [Figure 20] This figure shows the binding antibody IgG titers induced by different forms of gE-gI protein (gE protein alone, gE protein + gI protein, gE-gI fusion protein, and co-expressed gE-gI heterodimer) combined with an AS01B-like adjuvant in Example 9. The left panel shows the antibody titers at week 3, and the right panel shows the antibody titers at week 5. [Figure 21] This figure shows the T cell responses detected by flow cytometry induced by different forms of gE-gI protein (gE protein only, gE protein + gI protein, gE-gI fusion protein, and co-expressed gE-gI heterodimer) combined with an AS01B-like adjuvant in Example 9. The upper left panel shows the percentage of induced IFNγ+CD4+ T cells, the upper right panel shows the percentage of induced IL2+CD4+ T cells, the lower left panel shows the percentage of induced IFNγ+CD8+ T cells, and the lower right panel shows the percentage of induced IL2+CD8+ T cells. [Figure 22]In Example 9, this figure shows the levels of IFN-γ (left panel) and IL-2 (right panel) detected by Elispot induced by different forms of gE-gI protein (gE protein only, gE protein + gI protein, gE-gI fusion protein, and co-expressed gE-gI heterodimer) combined with AS01B-like adjuvant. [Figure 23] FIG. 1 shows the antigen-specific IgG titers induced after two injections of fusion proteins constructed based on the different mutants in Example 10. [Figure 24] FIG. 1 shows antigen-specific CD4+ and CD8+ cell responses induced after two injections of fusion proteins constructed based on the different mutants in Example 10. [Figure 25] FIG. 1 shows the results of Western blot of fusion proteins constructed based on different gE truncates in Example 11. [Figure 26] FIG. 1 shows the results of capillary electrophoresis of mRNA encoding the gE-gI fusion protein in Example 12. [Figure 27] FIG. 12 shows the particle size of LNP-encapsulated mRNA encoding a gE-gI fusion protein in Example 12. [Figure 28] FIG. 12 shows the titers of binding antibody IgG induced by immunization with gE-gI mRNA / LNP and gE-gI mRNA / LNP combined with CpG adjuvant in Example 12. [Figure 29] FIG. 12 shows the CD4+ T cell and CD8+ T cell responses induced by immunization with gE-gI mRNA / LNP and gE-gI mRNA / LNP combined with CpG adjuvant in Example 12. [Figure 30] FIG. 1 shows the monoclonality of the Top1 clone (CB4019-clone-16) in Example 13. [Figure 31] FIG. 1 shows an SDS-PAGE diagram of the gE-gI fusion protein obtained in Example 13. [Figure 32]FIG. 1 shows an HPLC diagram of the gE-gI fusion protein derived from the CHO stable cell line in Example 13. [Figure 33] FIG. 13 shows the titers of binding antibody IgG induced by gE-gI fusion protein derived from a CHO stable cell line in combination with AS01B adjuvant and Shingrix vaccine in Example 13. [Figure 34] FIG. 13 shows the titers of neutralizing antibodies induced by the gE-gI fusion protein derived from a CHO stable cell line combined with AS01B adjuvant and Shingrix vaccine in Example 13. [Figure 35] FIG. 12 shows the CD4+ T cell and CD8+ T cell responses induced by the gE-gI fusion protein derived from a CHO stable cell line in combination with AS01B adjuvant and Shingrix vaccine in Example 13. [Figure 36] FIG. 1 shows the levels of IFN-γ and IL-2 induced by the gE-gI fusion protein derived from a CHO stable cell line in combination with AS01B adjuvant and Shingrix vaccine in Example 13, as detected by Elispot assay. DETAILED DESCRIPTION OF THE INVENTION

[0138] Sequence information A description of the sequences relevant to this disclosure is provided in the table below.

[0139] [Table 1-1]

[0140] [Table 1-2]

[0141] [Table 1-3]

[0142] Table 1-4

[0143] Table 1-5

[0144] Table 1-6

[0145] Table 1-7

[0146] Table 1-8

[0147] Table 1-9

[0148] Table 1-10

[0149] Table 1-11

[0150] Table 1-12

[0151] Table 1-13

[0152] [Table 1-14]

[0153] [Table 1-15]

[0154] [Table 1-16]

[0155] [Table 1-17]

[0156] [Table 1-18]

[0157] [Table 1-19]

[0158] [Table 1-20]

[0159] [Table 1-21]

[0160] [Table 1-22]

[0161] Specific Modes for Carrying Out the Disclosure The present disclosure will now be described with reference to the following examples that are intended to illustrate, but not limit, the disclosure.

[0162] Unless otherwise specified, molecular biological experimental methods and immunoassays used in the present disclosure are essentially performed according to 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., Compiled Molecular Biology Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. The use of restriction endonucleases is in accordance with the conditions recommended by the product manufacturer. Those skilled in the art will understand that the examples serve to illustrate the present disclosure and do not limit the scope of protection claimed by the present disclosure. [Example]

[0163] Example 1: Overexpression of fusion proteins The structures of the designed fusion proteins are shown in Table 2.

[0164] [Table 2]

[0165] Gene synthesis: The genes shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0166] The following exemplary experiments were carried out using the gE-gI fusion protein as an example.

[0167] Insect cell transfection (1) sf9 cells (purchased from Invitrogen, catalog number 11496-015) or sf21 cells (purchased from Invitrogen, catalog number 11497-013) were grown in the logarithmic growth phase (1.5 × 10 6 cells / mL~2.5×10 6The cell population was confirmed to be >90% (cells / mL), and viability was maintained. 200 μL of ESF 921 culture medium (Expression systems, catalog number 96-001-01) containing 2% FBS, 0.1 μg of baculovirus DNA (Expression systems, catalog number 91-002), and 1 μg of pAc-gEgI plasmid (this plasmid was constructed to have the nucleotide sequence encoding a gE-gI fusion protein, SEQ ID NO: 4) was added to a 24-well plate and mixed well. 1 μL of transfection reagent (Expression systems, catalog number 95-055-075) was added to 50 μL of ESF921 medium (Expression systems, catalog number 96-001-01) and mixed well. The two were combined into one tube, mixed well, and allowed to stand at room temperature for 30 minutes. The cells were washed during the resting period (this was done before the resting process was completed): after the cells had completely attached to the wall, the medium was removed using a pipette, and then 300 μL of ESF921 medium was added. These movements were performed quickly to avoid cell dehydration. After gentle shaking, the medium was discarded and 300 μL of ESF921 medium was added. When the time was reached, approximately 100 μL of the above mixture was added dropwise evenly to the cells in each well. The cells were then incubated at 27 °C for 6 hours, the supernatant was discarded, and 500 μL of complete culture medium (50% CCM3 + 50% TNM-FH (SIGMA-ALDRICH, T1032) + 10% FBS) was added. (2) The cell supernatant obtained in step (1) was collected and centrifuged at 500 g for 5 minutes to remove cell debris and fragments, and the supernatant was stored in the dark at 4° C. This was used as the P1 virus seed solution. (3) Baculovirus amplification sf9 cells or sf21 cells were in the logarithmic growth phase (1.5 × 10 6 cells / mL~2.5×10 6 cells / mL), maintain viability above 90%, and maintain a concentration of 6 x 10 58-10 mL of cells at a density of 1000 cells / mL were coated onto a 10 cm plate. The cells were left to stand for 15 minutes to allow them to adhere to the wall. Approximately 600 μL of P1 virus solution was added dropwise evenly and incubated at 27°C for 3-4 days. A cytopathic effect was observed. The cell supernatant was collected, centrifuged at 1000 rpm for 5 minutes to remove cell debris and fragments, and filtered through a 0.22 μm filter membrane. The supernatant was stored in the dark at 4°C. This was used as the P2 virus seed solution. The P2 virus titer was approximately 10 6 pfu / mL ~10 7 pfu / mL. P3 virus could be obtained by volume culture in 250 mL shake flasks according to this method.

[0168] Protein expression in insect cells H5 cells (purchased from Invitrogen, catalog number B855-02) were cultured in 250 mL of ESF921 medium at a density of 2 × 10 6 A total of 100 cells / mL (with a viability of over 90%) were added to a 1 L shake flask. Virus was added in an amount according to the corresponding MOI, the flask neck was sealed with a sealing film, and the culture was performed in a shaker at 120 rpm at 27°C. The cells in the shake flask were sampled, observed, counted, and the relevant data were recorded daily. An appropriate MOI ensured that over 70% of the cells were infected on the first day, all cells were infected on the second day with a viability of approximately 80%, and on the third day, the cells burst and the viability dropped to 30%-50%. At this point, the cells could be harvested. The cells were collected by centrifugation at 10,000 rpm for 10 minutes, and the supernatant was then separated and purified to obtain a gE-gI fusion protein having the sequence shown in SEQ ID NO: 10. The C-terminus of the fusion protein contained 10xHIS.

[0169] Protein expression in mammalian cells Preparation: Preheat ExpiCHO™ Expression Medium (purchased from Thermo Scientific, Catalog No. A2910002) to 37°C, take 10 mL of ExpiCHO™ Expression Medium, place in a sterile tube, pre-cool in a 4°C refrigerator, and infuse 13.3 x 10 plasmids.3 The cells were centrifuged at 4°C for 10 min at 500 x g and cells with viability >95% were cultured at 6 x 10 s in a 250 ml shake flask using pre-warmed ExpiCHO™ Expression Medium. 6 The plasmid was diluted to 1000 cells / ml and then coated onto a 6-well plate. 50 μg of plasmid was diluted with 2 mL of pre-chilled culture medium at 4°C and mixed thoroughly. 160 μL of ExpiFectamine™ CHO Reagent (purchased from Thermo Scientific, catalog number A29133) was diluted with 1.84 mL of pre-chilled culture medium and mixed thoroughly. The diluted transfection reagent was then added to the diluted plasmid and mixed thoroughly. The mixture was then incubated at room temperature for 1 to 5 minutes. The mixture of plasmid and transfection reagent was then added to the diluted cells and cultured at 37°C and 5% CO2. After 24 hours, 300 μL of ExpiCHO™ Enhancer and 12 mL of ExpiCHO™ Feed (provided with the ExpiFectamine™ CHO Reagent Transfection Kit) were added, and the cells were cultured at 37°C and 5% CO2 for 7 days.

[0170] Tag-free purification of gE-gI fusion proteins Chromatographic purification was performed using the AKTA system; Instrument system: AKTA Pure preparative liquid chromatograph;

[0171] Purification by chromatography on a Q-FF column (Cytiva) (1) The expression samples were collected and centrifuged at 7000 rpm for 10 minutes, and the supernatant was dialyzed against 50 mM TB8.0 and left at 4°C overnight. (2) The protein sample was passed through the column at 8 mL / min, and the column was then equilibrated with 50 mM TB8.0. (3) The target protein was gradient eluted using 50 mM TB8.0 and 1 M NaCl, and the elution fractions at 0.3 M NaCl were collected.

[0172] Purification by chromatography on a DEAE-FF column (Cytiva) (1) The eluted sample was passed through the column at 5 mL / min, and then the column was equilibrated with 50 mM TB8.0. (2) The target protein was subjected to gradient elution using 50 mM TB8.0 and 1 M NaCl, and the elution fractions at 0.3 M NaCl were collected.

[0173] Purification by chromatography on a butyl column (Cytiva) (1) The eluted sample was dialyzed against 50 mM TB8.0 / 2M NaCl. (2) The protein sample was passed through the column at 5 mL / min, and the column was then equilibrated with 50 mM TB8.0 / 2 M NaCl. (3) The target protein was gradient eluted using 50 mM TB8.0.

[0174] Purification of gE-gI fusion protein by affinity chromatography The AKTA system was used for Ni-affinity chromatography purification. Instrument system: AKTA Pure preparative liquid chromatograph; Purification medium: Ni Sepharose 6 Fast Flow affinity medium; Buffer: Including A pump buffer and B pump buffer; generally, A pump buffer was 1x PBS buffer (160 g / L NaCl, 8.1 mmol / L Na2HPO4, 1.5 mmol / L KH2PO4, 2.7 mmol / L KCl, pH 7.4), and B pump buffer was 1x PBS + 250 mmol / L imidazole buffer; System flow rate: 5 mL / min, detection wavelength: UV at 280 nm Elution conditions: impurity proteins were eluted using 50 mM imidazole buffer (obtained by diluting 250 mmol / L imidazole buffer with 1× PBS buffer), followed by washing with 1× PBS, and then the target protein (gE-gI fusion protein) was eluted with 250 mM imidazole buffer.

[0175] The product eluted with 250 mM imidazole was collected to give a 40 mL purified sample, and the supernatant was centrifuged for purification.

[0176] Purification of gE-gI fusion protein by size exclusion chromatography Instrument system: AKTA explorer 100 preparative liquid chromatography system manufactured by GE Healthcare (formerly Amershan Pharmacia). Chromatography medium: Superdex 200 increase (cytiva) Column volume: 20cm x 20mm Buffer solution: 20 mM phosphate buffer, pH 7.4 Flow rate: 0.7mL / min Detector wavelength: 280nm The loading sample was purified by Ni affinity chromatography. The elution procedure was as follows: the permeate peak was fractionated and collected. The permeate of the chromatography on Supedex 200 increase was collected to give a 5 mL purified sample.

[0177] SDS-PAGE 50 μL of each eluate was added to 30 μL of 6x loading buffer and mixed thoroughly. After incubation in an 80°C water bath for 10 minutes, 10 μL of the mixture was electrophoresed on a 10% SDS-polyacrylamide gel at 120 V for 120 minutes. The electrophoretic bands were then stained with Coomassie Brilliant Blue to reveal the bands.

[0178] Western blot (1) Transfer to membrane: After electrophoresis, a vertical protein transfer instrument was used to transfer the proteins to a nitrocellulose membrane. (2) Blocking: The nitrocellulose membrane was immersed in 50 mL of 1× blocking solution and incubated at 37° C. for 1 hour. (3) Incubation with primary antibody: The blocking solution was removed, and the nitrocellulose membrane was rinsed three times with deionized water. It was then immersed in 50 mL of primary antibody (VZV gE protein-specific monoclonal antibody 1B11, stored in our laboratory) and incubated at 37°C for 1 hour. (4) Incubation with secondary antibody: The primary antibody was collected, and the nitrocellulose membrane was rinsed with 1x PBST four times for 5 minutes each time. The nitrocellulose membrane was then immersed in 50 mL of secondary antibody (GAH-HRP) and incubated at 37°C for 1 hour. (5) Color development: After the secondary antibody was removed, the nitrocellulose membrane was washed four times with 1x PBST for 5 minutes each time. Equal volumes of color development solution A and B were thoroughly mixed, and the membrane surface was immersed in the solution for color development and imaging.

[0179] Protein expressed in insect cells: SDS-polyacrylamide gel electrophoresis (left panel) and Western blot (right panel) results of gE-gI fusion protein samples at various purification stages (purification by sequential chromatography on a Q-FF column (Panel A in Figure 1), a DEAE-FF column (Panel B in Figure 1), and a butyl column (Panel C in Figure 1)) are shown in Figure 1 (in the figure, lane M: molecular weight marker; lane 1: gE-gI fusion protein). The results showed that the purity of the obtained gE-gI fusion protein after three-column chromatography purification was approximately 90%, and Western blot detection using a VZV gE-specific monoclonal antibody was positive.

[0180] Protein expressed in CHO cells: SDS-polyacrylamide gel electrophoresis (left panel) and Western blot (right panel) of the gE-gI fusion protein sample at various purification stages (sequential purification by chromatography on a Ni-6FF column (Panel A in Figure 2) and size-exclusion chromatography on a Superdex 200 Increase column (Panel B in Figure 2)) are shown in Figure 2 (in the figure, lane M: molecular weight marker; lane 1: gE-gI fusion protein). The results showed that after two-column chromatography purification, the purity of the obtained gE-gI fusion protein was approximately 90%, and Western blot detection using a VZV gE-specific monoclonal antibody was positive.

[0181] Example 2: High-Performance Size-Exclusion Chromatography (HPSEC) Analysis of gE-gI Fusion Proteins Equipment: Waters. System flow rate: G3000PW XL The flow rate was 0.5 mL / min. The wavelength was 190 nm to 600 nm, and the column wavelengths were 280 nm and 254 nm. Buffer: PBS Procedure: The chromatography column was pre-equilibrated for 30-60 minutes until there was no significant change in absorbance at 280 nm, and the detector absorbance was set to zero. The chromatography procedure was edited to first centrifuge the sample, then inject the sample to be analyzed (gE-gI fusion protein obtained in Example 1) into a 100 μL sample loop. The instrument was set for automatic loading and run for 30 minutes.

[0182] 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 both the homogeneity and purity were higher than 90% (Figure 3), and 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 both the homogeneity and purity were higher than 90% (Figure 4).

[0183] Example 3: Calculation of sample sedimentation coefficients by analytical ultracentrifugation The instrument used was a Beckman XL-A analytical ultracentrifuge equipped with an optical detection system and an An-60Ti rotor.

[0184] The sample pool was set up according to the operating instructions, 400 μL of sample buffer (the same buffer as used for the sample) was added to the control pool, and 380 μL of sample (gE-gI fusion protein prepared in Example 1, OD280 was approximately 0.8) was added to the sample pool, and the sample pools were adjusted to have a weight difference of within 0.1 g.

[0185] The sample pool was placed in an An-60Ti rotor, and the rotor was then placed in the cavity of a Beckman XL-A analytical ultracentrifuge and an optical path detector was installed. The parameters were set as follows: 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 s), and data number (150 scans). The centrifuge speed was set to 30,000 rpm.

[0186] After the experiment was completed, the density and viscosity of the buffer solution, as well as the partial specific volume of the known protein, were calculated using SENDTERP software. The sedimentation coefficients were analyzed using Nonlin and the Origin version of SEDFIT software. The globulin friction ratio f / f0 was preset to 1.2. The analysis range was set according to the molecular weight and basic properties of the sample protein. The calculation resolution was set to 100. Generally, RMSD values ​​were expected to be less than 0.01, and the variation of the residual graph was within 0.05.

[0187] The analytical ultracentrifugation results of the insect cell-derived gE-gI fusion protein are shown in Figure 5. The results showed that the gE-gI fusion protein had a sedimentation coefficient of approximately 4.5 S and a molecular weight of approximately 84.1 kDa, as expected, and existed in a monomeric form.

[0188] The analytical ultracentrifugation results of the CHO-derived gE-gI fusion protein are shown in Figure 6. The results showed that the gE-gI fusion protein had a sedimentation coefficient of approximately 5.3 S and a molecular weight of approximately 107 kDa, as expected, and existed in a monomeric form.

[0189] Example 4: Evaluation of gE-gI fusion protein activity ELISA operation process: (1) The gE-gI fusion protein prepared in Example 1 was coated onto a plate (200 ng / well) and left to stand at 37°C for 2 hours. The plate was washed once and blocked with a diluted bovine serum albumin solution (ED, 200 μL / well) at room temperature for 2 hours. (2) The plate was washed once, and gE protein-specific monoclonal antibodies (1B11, 4G4, 6H6, 6G7, 11B11, 11B12, 11E3, 14G1, 12E12, 13B6, 10H6, 17B7, 9H9; stored in this laboratory) and gI protein-specific monoclonal antibodies (18B2, 7E7, 9D11, 16F1, 13B2, 19F2, 14B11, 12D2; stored in this laboratory) were separately diluted with SD-1 diluent to 5 μg / mL in the first well, then diluted with a 2-fold gradient dilution method and incubated at 37°C for 1 hour. (3) The plate was washed five times, and the secondary antibody GAH-HRP (1:5000) was added to the 96-well plate (100 μL / well) and allowed to stand at room temperature for 1 hour. (4) The plate was washed five times, and the color was developed at 37°C for 10 minutes. After stopping, detection was performed at a wavelength of 450 nm using an ELISA reader, and data analysis was performed using GraphPad Prism 5 (GraphPad, USA) software.

[0190] The results of the ELISA are shown in Figure 7. Figure 7 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.

[0191] The results of the ELISA are shown in Figure 8. Figure 8 shows that the CHO-derived gE-gI fusion protein prepared in Example 1 has good reactivity with both gE protein-specific monoclonal antibodies and gI protein-specific monoclonal antibodies.

[0192] Example 5: Evaluation of gE-gI fusion protein immunogenicity This experimental scheme was approved by the Ethics Committee for Laboratory Animal Care of Xiamen University, and all procedures were performed in strict accordance with animal ethics guidelines and approved procedures.

[0193] Six-week-old Balb / C mice were selected and divided into four groups, with five mice in each group. In each group, the gE-gI fusion protein prepared in Example 1 (the immunization dose was 1 μg) and FH002C adjuvant (FH002C adjuvant, see PCT Application No. PCT / CN2021 / 130588 (the entire text of which is incorporated herein by reference) or the academic journal article 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 entire text of which is incorporated herein by reference)) or aluminum adjuvant (prepared in this laboratory) or 50% Freund's complete adjuvant (Sigma) were administered. The combination of 1000mg ribonucleotides (purchased from Aldrich, catalog number F5506) was used for immunization, and mice were intramuscularly injected (150 μL) into the left or right hind limb at weeks 0, 1, and 4. Ocular vein blood was collected at weeks 0, 1, 2, 3, 4, and 5, respectively, and blood was drawn before injection at weeks 0, 1, and 4. Blood samples were centrifuged at 13,000 g for 10 minutes, and serum samples were stored at -20°C. Antigen-specific IgG titers and neutralizing antibody titers were determined by endpoint enzyme-linked immunosorbent assay and vOka virus-based neutralization assay, respectively.

[0194] Neutralization procedure (ELISPOT assay): (1) Dried powders of guinea pig serum (purchased from Beijing Bersee Science and Technology Co., Ltd., catalog number BM361Y) and vOka virus (purchased from Beijing Wantai Biological Pharmacy Enterprise Co., Ltd.) were reconstituted with virus protection solution, and the complement was filtered through a 0.22 μm miniature filter for subsequent use. (2) The serum was diluted 50-fold with virus protection solution, added to the first well of a 24-well plate, subjected to two-fold serial dilutions in a 4-gradient, and incubated with vOka virus at 37°C for 1 hour. (3) The serum-virus mixture was transferred to a 24-well plate precoated with ARPE-19 cells and incubated at 37°C for 1 hour. The liquid was discarded after 1 hour, replenished with F12 culture medium, and cultured at 37°C for 3 days. (4) After 3 days, the culture medium was discarded, the plate was washed once with PBS, and the cells were fixed with fixative at room temperature for 5 minutes. The fixative was discarded, and the cells were permeabilized with permeabilization solution at room temperature for 10 minutes. (5) The primary antibody 1B11-HRP (1:2000) was added to a 24-well plate and allowed to stand at 37°C for 1 hour. (6) The plate was washed five times, allowed to develop at room temperature for 5 minutes, and the spots were read and counted using an enzyme-linked immunospot image analysis system. GraphPad Prism 5 (GraphPad, USA) software was used for data analysis.

[0195] Neutralization procedure (plaque assay): (1) The dried powders of guinea pig serum and vOka virus were reconstituted in a virus protection solution, and the complement was filtered through a 0.22 μm microfilter for subsequent use. (2) The serum was diluted 50-fold with virus protection solution, added to the first well of a 24-well plate, subjected to two-fold serial dilutions in a 4-gradient, and incubated with vOka virus at 37°C for 1 hour. (3) The serum-virus mixture was transferred to a 24-well plate pre-coated with ARPE-19 cells and incubated at 37°C for 1 hour. The liquid was discarded and replenished with F12 culture medium after 1 hour. The cells were then cultured at 37°C for 7 to 10 days until plaques formed. (4) The culture medium was discarded, the cells were washed once with PBS, and then fixed with a fixative solution at room temperature for 5 minutes. (5) Staining was performed with crystal violet dye for 10 minutes, washing was performed with deionized water, spots were counted manually, and data analysis was performed using GraphPad Prism 5 (GraphPad, USA) software.

[0196] The antigen-specific IgG titers and neutralizing antibody titers in serum induced by the insect cell-derived fusion protein in combination with different adjuvants are shown in Figures 9 and 10, respectively. The results in Figure 9 indicate that after three immunization injections, all mice showed IgG seroconversion at week 2, and the IgG titers increased over time. By week 5, all mice showed high IgG antibody titers, with the aluminum adjuvant group showing the lowest IgG titers and the FH002C adjuvant group showing relatively high IgG antibody titers. Figure 10 shows that all mice in each adjuvant group showed neutralizing antibody titers, with the FH002C adjuvant group showing higher neutralizing antibody titers than the aluminum adjuvant and Freund's adjuvant groups. Figures 9 and 10 demonstrate that the gE-gI fusion protein has high immunogenicity even at low immunization doses.

[0197] Example 6: Detection of cytokines by flow cytometry Mice were immunized according to the method described in Example 6, and the subsequent experimental procedures were as follows: a) Spleen collection: Mice were sacrificed by cervical dislocation and immersed in 75% ethanol for 3 to 5 minutes. Mice were placed in the right lateral position, and the spleen was aseptically removed (fat was removed as much as possible). b) Trituration: Place a 6-well cell plate into a mesh, add 1640 culture medium containing 10% FBS, place the spleen into the mesh (completely immersed in the culture medium), and triturate with a 2 mL syringe head until no red tissue is visible. Transfer the triturated cells into a 50 mL tube and place on ice. c) The cells were centrifuged at 400 g and 4° C. for 5 minutes, the supernatant discarded, and the cell pellet loosened by tapping the bottom of the tube with a hemostat. d) The cell pellet was resuspended in 10 mL of pre-chilled RBC solution and placed on ice for 5 minutes for lysis (mixing by inversion during this time). e) The cells were centrifuged at 400 g and 4° C. for 5 minutes, the supernatant discarded, and the cell pellet loosened by tapping the bottom of the tube with a hemostat. f) 10 mL of pre-chilled culture medium was added to the cell pellet, and the pellet was resuspended. Red blood cell pellet or fat was washed away, and after pipetting evenly, 50 μL of cells were taken and counted. g) Centrifuge the cells at 400 g and 4°C for 5 minutes, discard the supernatant, and loosen the cell pellet by tapping the bottom of the tube with a hemostat until the cell density is 2 x 10 7 A predetermined volume of culture medium was added to dilute the cells / mL. h) Plating: 96-well U-bottom plate with 200 μL (2 × 10) per well 6 cells ~4×10 6 cells), centrifuged at 400 g and 4° C. for 5 minutes, and the supernatant was discarded. i) 100 μL of culture medium containing polypeptides (gE / gI overlapping polypeptides (diluted in FACS solution, final polypeptide concentration was 2 μg / mL) was added, the cells were resuspended, and stimulation was carried out for 18 hours. j) 20 μL of Golgi inhibitor (1:1000, diluted in culture medium) was incubated for 6 hours. k) Centrifuge at 400g and 4°C for 2 minutes, discard the supernatant, add 200 μL of FACS solution (1x PBS + 10% FBS) to resuspend the cells, then centrifuge the cells and discard the supernatant. (All the following operations were carried out in the dark and kept at 4°C.) l) Cell surface staining: Staining was performed using 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™ fixed Aqua cell staining reagent (purchased from Biolegend, catalog number 103248). 40 μL of surface staining reagent (AQUA / CD4 / CD8 antibody, diluted with FACS solution) was added to each well to resuspend the cells, and the treatment was carried out for 30-60 minutes at 4°C in the dark, followed by adding 200 μL of FACS solution and pipetting 7-8 times. m) Fixation / Permeabilization: Centrifuge at 2000 rpm and 4°C for 2 minutes, discard the supernatant, add 75 μL of fixation / permeabilization solution to each well to resuspend the cells, and perform the treatment in the dark at 4°C for 60 minutes. n) Centrifuge at 2000 rpm and 4°C for 2 minutes and discard the supernatant. o) 200 μL of 1×BD Perm / Wash solution was added to each well to resuspend the cells, centrifuged at 2000 rpm and 4° C. for 2 minutes, and the supernatant was discarded. p) Intracellular staining: Staining was performed using PE-conjugated anti-mouse IL-2 antibody (purchased from BD, catalog number 554428) and APC-conjugated anti-mouse IFN-γ antibody (purchased from BD, catalog number 554413), 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 treatment was carried out in the dark at 4° C. for 60 minutes, and 200 μL of Perm Buffer was added and the cells were resuspended. q) Centrifuge at 2000 rpm and 4° C. for 2 minutes, discard the supernatant, and add 200 μL of 1×BD Perm / Wash solution to each well to resuspend the cells. r) The treated cells were subjected to filtration through a 200 mesh screen (the screen strip was placed on the surface of the well, and the cell suspension was added slowly by pipetting it vertically) and transferred to a flow cytometer tube. s) Samples were measured using a BD LSRFortessa X-20 flow cytometer and data were analyzed using FlowJo V10.

[0198] The experimental results are shown in Figure 11 and demonstrate that the gE-gI fusion proteins combined with different adjuvants are all capable of inducing T cell immune responses.

[0199] Example 7: Side-by-side comparison of insect cell-derived gE-gI fusion protein and Shingrix vaccine combined with AS01B adjuvant This experimental scheme was approved by the Ethics Committee for Laboratory Animal Care of Xiamen University, and all procedures were performed in strict accordance with animal ethics guidelines and approved procedures.

[0200] Six-week-old Balb / C mice were selected and divided into three groups, each with eight mice. The experimental group was immunized with the insect cell-derived gE-gI fusion protein prepared in Example 1 (immunization dose was 5 μg) and 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 saline. Mice were intramuscularly injected (50 μL) into the tibialis muscle at weeks 0 and 4, respectively. Ophthalmic vein blood was collected at weeks 0, 2, 4, 6, and 8, respectively, with blood samples taken at weeks 0 and 4 before injection. 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 determined by end-point enzyme-linked immunosorbent assay and vOka virus-based neutralization assay, respectively.

[0201] The results are shown in Figure 12. The results in Figure 12 show that both the binding antibody IgG titer and the neutralizing antibody titer induced by the gE-gI fusion protein combined with AS01B adjuvant were higher than those of the Shingrix vaccine, and the neutralizing antibody titer induced by the gE-gI fusion protein combined with AS01B adjuvant was approximately 1.8-fold higher than that of the Shingrix vaccine.

[0202] At week 8, spleens were collected for flow cytometry and Elispot assay of T cell response detection, and flow cytometry was performed in the same manner as in Example 6, and the ELISPOT cytokine detection process (the kits used were purchased from MABTECH, catalog numbers 3441-4HPW-10 and 3321-4HPW-10) was as follows: (1) Splenocytes were isolated and then plated at 500,000 cells per well. The supernatant was removed by centrifugation, and the cells were resuspended in 100 μL of culture medium containing the polypeptide (gE / gI overlapping polypeptide). Stimulation was carried out at 37°C for 20 hours. (2) The culture medium was discarded, and the cells were washed five times with sterile PBS. The detection antibodies R4-6A2-biotin (for IFN-γ detection) and 5H4-biotin (for IL-2 detection) were diluted to 1 μg / mL and added at 100 μL per well. The cells were incubated at room temperature for 2 hours. (3) The cells were washed five times with sterile PBS, and 100 μL of streptavidin-ALP (1:1000) was added to each well and incubated at room temperature for 1 hour. (4) The cells were washed five times with sterile PBS, and 100 μL of substrate solution (BCIP / NBT-plus) was added to each well for color development until spots appeared. The reaction was then stopped by washing the plate. (5) The plates were inverted and dried, and then the spots were read and counted using an enzyme-linked immunospot image analysis system, and GraphPad Prism 5 (GraphPad, USA) software was used for data analysis.

[0203] The experimental results are shown in Figures 13 to 15. Figure 13 shows the CD4 + The results of T cell responses were shown. The results showed that antigen-specific CD4 T cells were induced by insect cell-derived gE-gI fusion protein combined with AS01B adjuvant. + IFN-γ + T cells and CD4 + IL-2 + T cell levels were higher than those of the Shingrix vaccine, and CD4 + IFN-γ + The percentage of T cells was twice as high as that of the Shingrix vaccine, showing a significant difference.

[0204] Figure 14 shows the CD8 stimulated by two different vaccines in this example. + The results of T cell responses were shown. The results showed that gE-gI-specific CD8 T cells were induced by the gE-gI fusion protein in combination with the AS01B adjuvant. + IFN-γ + T cells were shown to be higher than with the Shingrix vaccine.

[0205] Figure 15 shows the levels of cytokines IFN-γ and IL-2 induced by the two different vaccines in this example, as detected by Elispot assay. The results showed that the levels of IFN-γ and IL-2 induced by the gE-gI fusion protein combined with AS01B adjuvant were significantly higher than those of the Shingrix vaccine.

[0206] Example 8: Side-by-side comparison of CHO-derived gE-gI fusion protein and Shingrix vaccine combined with AS01B adjuvant This experimental scheme was approved by the Ethics Committee for Laboratory Animal Care of Xiamen University, and all procedures were performed in strict accordance with animal ethics guidelines and approved procedures.

[0207] Six-week-old Balb / C mice were selected and divided into three groups, each with eight mice. The experimental group was immunized with the CHO-derived gE-gI fusion protein prepared in Example 1 (immunization dose was 5 μg) and 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 saline. The mice were intramuscularly injected (50 μL) into the tibialis muscle at weeks 0 and 4, respectively. Ophthalmic vein blood was collected at weeks 0, 2, 4, 6, and 8, respectively. Blood samples at weeks 0 and 4 were collected before injection. 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 determined by end-point enzyme-linked immunosorbent assay and vOka virus-based neutralization assay, respectively.

[0208] The results are shown in Figure 16. The results in Figure 16 show that the binding antibody IgG induced by the CHO-derived gE-gI fusion protein combined with AS01B adjuvant was equivalent to that of the Shingrix vaccine, and the titer of neutralizing antibodies induced by the gE-gI fusion protein combined with AS01B adjuvant was approximately twice that of the Shingrix vaccine.

[0209] For the flow cytometry method, see Examples 6 and 7.

[0210] The experimental results are shown in Figure 17, which shows the CD4 + The results of T cell responses were shown. The results showed that antigen-specific CD4 T cells were induced by CHO-derived gE-gI fusion protein combined with AS01B adjuvant. + IFN-γ + T cells and CD4 + IL-2 + T cells were significantly higher than Shingrix vaccine, and CD4 + IFN-γ + The percentage of T cells was three times higher than with the Shingrix vaccine.

[0211] Figure 18 shows the CD8 stimulated by two different vaccines in this example. + The results of T cell responses were shown. The results showed that gE-gI-specific CD8 T cells were induced by the gE-gI fusion protein in combination with the AS01B adjuvant. + IFN-γ + T cells were significantly higher than Shingrix vaccine, and CD8 + IFN-γ + The percentage of T cells was shown to be approximately seven times higher than with the Shingrix vaccine.

[0212] Figure 19 shows the levels of cytokines IFN-γ and IL-2 induced by the two different vaccines in this example. The results show that gE-gI-specific IFN-γ induced by the gE-gI fusion protein combined with AS01B adjuvant was significantly higher than that of the control group. + T cells and IL-2 + The percentage of T cells was significantly higher than that of the Shingrix vaccine.

[0213] Example 9: Side-by-side comparison of different forms of gE-gI protein in combination with AS01B-like adjuvants This experimental scheme was approved by the Ethics Committee for Laboratory Animal Care of Xiamen University, and all procedures were performed in strict accordance with animal ethics guidelines and approved procedures.

[0214] Six-week-old C57BL / 6 mice were selected and divided into five groups with five mice in each group. The experimental group received the gE-gI fusion protein prepared in Example 1 (immunization dose: 8.3 μg) and an AS01B-like adjuvant (prepared in this laboratory; for the preparation method, 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 contents of which are incorporated herein by reference.)) was used. The control group was 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 heterodimer (preparation method: see reference pmid:9697709 (immunization dose: 8.3 μg)) in combination with an AS01B-like adjuvant (prepared in our laboratory). The blank group was immunized with saline. Mice were intramuscularly injected (50 μL) into the tibialis muscle at weeks 0 and 3, respectively. Ophthalmic vein blood samples were collected at weeks 0, 3, and 5, respectively, with the blood samples collected at weeks 0 and 3 before injection. 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 determined by end-point enzyme-linked immunosorbent assay and vOka virus-based neutralization assay, respectively. The results are shown in Figure 20.The results in Figure 20 showed that the titers of binding antibody IgG induced by the gE-gI fusion protein combined with AS01B-like adjuvant were significantly higher than those of gE protein alone, gE protein + gI protein, and the co-expressed gE-gI heterodimer.

[0215] At 8 weeks, spleens were harvested for flow cytometry and Elispot assay of T cell responses, flow cytometry was performed as in Example 6, and the ELISPOT cytokine detection process was the same as in Example 7.

[0216] The experimental results are shown in Figures 21 and 22. Figure 21 shows the CD4 + T cells and CD8 + The results of T cell responses were shown. The results showed that gE-gI-specific CD4 T cells were induced by gE-gI fusion proteins combined with AS01B-like adjuvants. + IFN-γ + The levels of T cells were significantly higher than those of gE protein alone, gE protein + gI protein, and co-expressed gE-gI heterodimer, and CD4 + IL-2 + T cells and CD8 + IFN-γ + The levels of T cells were significantly higher than those of gE protein alone and gE protein plus gI protein, and slightly higher than those of the co-expressed gE-gI heterodimer.

[0217] Figure 22 shows the levels of cytokines IFN-γ and IL-2 induced by stimulation with the four different vaccines in this example, as detected by Elispot assay. The results showed that the levels of IFN-γ and IL-2 induced by the gE-gI fusion protein combined with the AS01B-like adjuvant were significantly higher than those induced by gE protein alone, gE protein + gI protein, and co-expressed gE-gI heterodimer.

[0218] Example 10: Side-by-side comparison of different strain-based gE-gI fusion proteins in combination with AS01B-like adjuvants The full-length gE amino acid sequence (SEQ ID NO: 11) involved in Examples 1 to 9 of the present application was completely identical to the gE protein sequences of the pOka strain (GenBank: AB097933) and the vOka strain (GenBank: AB097932). The gI sequences involved in Examples 1 to 9 of the present disclosure were completely identical to the gI protein sequences of the pOka strain (GenBank: AB097933), the vOka strain (GenBank: AB097932), the Dumas strain (GenBank: NC001348), and the MSP strain (GenBank: AY548170).

[0219] In this example, the immunogenicity of gE-gI fusion proteins constructed based on gE and gI from different strains was further evaluated, and the information of the relevant fusion protein constructs is shown in the table below:

[0220] [Table 3]

[0221] Among them, gE (aa. 23-537, I40T) is derived from the Dumas strain (GenBank: NC001348) and contains the I40T mutation compared to SEQ ID NO: 13; gE (aa. 23-537, I40T and D150N) is derived from the MSP strain (GenBank: AY548170) and contains the I40T and D150N mutations compared to SEQ ID NO: 13; gE (aa. 23-537, T512M and L536I) is derived from the gE protein shown in GenBank: AEW88980 and contains the T512M and L536I mutations compared to SEQ ID NO: 13. gE(aa.23-537, Q404K) is derived from the gE protein shown in GenBank: AEW89124 and contains a Q404K mutation compared to SEQ ID NO: 13; gE(aa.23-537, W319R) is derived from the gE protein shown in GenBank: QCA43570 and contains a W319R mutation compared to SEQ ID NO: 13; gE(aa.23-537, E124D) is derived from the gE protein shown in GenBank: QCA45176 and contains an E124D mutation compared to SEQ ID NO: 13; g gE(aa.23-537, L359S) is derived from the gE protein shown in GenBank: QWE79571 and 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 and contains the L162R mutation compared to SEQ ID NO: 13; gE(aa.23-537, A186V and L536I) is derived from the gE protein shown in GenBank: ANS12941 and contains the A186V and L536I mutations compared to SEQ ID NO: 13. gE (aa. 23-537, L536I) is derived from the gE protein shown in GenBank: AQT34120 and contains a L536I mutation compared to SEQ ID NO: 13; gI (aa. 21-271, K246Q) is derived from the gI protein shown in GenBank: AEW89051 and 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 and 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 and contains the N267S mutation compared to SEQ ID NO: 14.

[0222] In this example, the fusion proteins shown in Table 3 were purified and immunologically evaluated according to the methods described in Examples 1, 5, and 6.

[0223] Figure 23 shows the titers of antigen-specific IgG induced after two injections of the fusion proteins constructed based on the different mutant strains in this example. The results showed that there was no significant difference in the level of IgG induced by the fusion proteins constructed based on the different mutant strains combined with the AS01B-like adjuvant, which was comparable to the level of the gE-gI fusion protein set forth in SEQ ID NO: 6. .

[0224] Figure 24 shows the antigen-specific T cell responses induced after two injections of the fusion proteins constructed based on different mutant strains in this example. The results show that the gE-gI fusion proteins constructed based on different mutant strains induced higher CD4 T cell responses compared to Shingrix. + T cells and CD8 + Among these, certain strain-based fusion proteins were able to generate a higher percentage of CD4 T cell responses compared to the gE-gI fusion protein set forth in SEQ ID NO:6. + IFN-γ + T cells (e.g., gE-gI (gE-Q404K), gE-gI (gE-I40T, D150N)), CD4 + IL2 + T cells (e.g., gE-gI(L359S)), and CD8 + IFN-γ + The results showed that gE-gI fusion proteins constructed based on different strains of gE and gI had significantly superior immunogenicity and induced stronger immune responses than Shingrix.

[0225] Example 11: Design and preparation of truncated gE-gI fusion proteins In this experiment, gE-gI fusion proteins were expressed with shortened gE length, while the length of gI remained unchanged. The N- and C-termini of gE were simultaneously shortened to construct gE-gI fusion proteins, and the immunoreactivity of the gE-gI fusion proteins 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 for these five fusion proteins was as follows:

[0226] [Table 4]

[0227] The fusion proteins were prepared according to Example 1. The resulting fusion proteins constructed with the truncated gE protein were subjected to Western blotting.

[0228] FIG. 25 shows the results of this example, which demonstrate that although the above five fusion protein constructs have different expression levels, they all have good immunoreactivity.

[0229] Example 12: Side-by-side comparative study of gE-gI mRNA vaccine combined with CpG adjuvant Linearization to obtain mRNA transcription templates In a 1.5 mL Eppendorf tube, 25 μg of pUC-gEgI plasmid (plasmid pUC57 was purchased from Sangon Biotechnology, catalog number B522201, on which an mRNA expression framework and a nucleotide sequence encoding gE-gI were constructed, the framework was as follows: T7 promoter (SEQ ID NO: 16) + DNA sequence corresponding to 5' UTR (SEQ ID NO: 17) + coding sequence for Kozak sequence (SEQ ID NO: 72) + start codon + nucleotide sequence encoding gE-gI fusion protein (DNA sequence corresponding to SEQ ID NO: 71) + stop codon + DNA sequence corresponding to 3' UTR (SEQ ID NO: 18) + DNA sequence corresponding to polyA (SEQ ID NO: 19)), 5 μL of BspQI restriction endonuclease (purchased from New England Biolabs, catalog number R0712S), and 15 μL of NEBuffer r3.1 (New England Biolabs, catalog number R0712S). The enzyme digestion reaction was carried out in a water bath at 50°C for 30 minutes. The resulting enzyme digestion product was collected in a cell-free clean bench using a gel collection kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number DP209-03) according to the manufacturer's instructions. The collected product was identified by agarose gel electrophoresis, and the linearized plasmid was used as a transcription template.

[0230] In vitro transcription of mRNA (1) mRNA was prepared using a T7 in vitro transcription kit (purchased from Nanjing Vazyme Biotech Co., Ltd., catalog number DD4202). 2 μL of ATP, 2 μL of CTP, 2 μL of fo GTP, 2 μL of UTP, 2 μL of Cap, 2 μL of RNase inhibitor, and 2 μg of linearized gE-gI plasmid template were added separately to a 1.5 mL RNase-free Eppendorf tube. The mixture was supplemented with DEPC water (purchased from Shanghai Sangon, catalog number B501005-0500) to a total volume of 20 μL, and incubated in a metal water bath at 37°C for 4 hours. (2) 2 μL of DNase I (purchased from New England Biolabs, catalog number M0303L) was added and incubated at 37° C. for 20 minutes to digest the remaining DNA template in situ. (3) 30 μL of DEPC water was added to terminate the digestion reaction. (4) 30 μL of LiCl (purchased from Invitrogen, catalog number AM9480) was added, and the mixture was left to stand at −20° C. for 30 minutes to precipitate mRNA. (5) Centrifugation was carried out at 4°C and 13,000 rpm for 10 minutes, and the supernatant was carefully removed with the tip of a pipette. (6) 1 mL of pre-cooled 75% ethanol was added, centrifuged at 4°C and 13,000 rpm for 5 minutes, and the supernatant was carefully removed with the tip of a pipette to remove residual proteins in the system. (7) The centrifuge tube was opened and air-dried for 2 minutes, and 30 μL of DEPC water was added to resuspend the precipitate, thereby obtaining gE-gI mRNA.

[0231] Identification of mRNA by capillary electrophoresis (1) 90 μL of DEPC water was taken and used to dilute 10 μL of 10× dilution buffer (Bioptic Inc., C104408-10X), thereby preparing 1× dilution buffer. (2) 1 μL of mRNA sample was taken and mixed with 19 μL of 1× dilution buffer. (3) Capillary electrophoresis was performed using Qsep100 to analyze the size of mRNA.

[0232] The results of the capillary electrophoresis experiment are shown in Figure 26. The size of the gE-gI mRNA measured by capillary electrophoresis was 2403 nt, and the theoretical size of the designed molecule was 2676 nt. The mRNA size was slightly smaller than the theoretical value, but within the error range. The results of capillary electrophoresis showed that the gE-gI mRNA showed a single peak, indicating good mRNA homogeneity.

[0233] Mixture of mRNA and LNP (1) To dilute mRNA to 80 μg / mL, an aqueous mRNA phase was prepared using 50 mM citrate buffer at pH 3.0. (2) A lipid mixture stock solution with a total lipid concentration of 100 mM was prepared according to the ratios in the table below and stored at room temperature. Before use, it was placed in a 37°C water bath until no precipitate was visible. Dilution was performed with absolute ethanol to reach a final working concentration of 5 mM or 10 mM.

[0234] [Table 5]

[0235] (3) The mRNA and lipid phase were mixed using a microfluidic device. The pipeline was washed with ethanol, and the dual channel was pre-rinsed with ethanol and citrate buffer, respectively. After thorough rinsing, the sample loading parameters were set as follows: mixed lipid phase:nucleic acid phase = 1:3 (volume / volume), flow rate ratio 1:3, and total flow rate 12 mL / min. (4) The mixed mRNA / LNP was immediately diluted with 5 times the volume of 1x PBS and ultrafiltered twice using a 10 kDa ultrafiltration tube. The replacement buffer was 1x PBS, and the mixture was concentrated to reach the initial volume of the mixed mRNA / LNP.

[0236] Determination of mRNA / LNP concentration and encapsulation efficiency (1) mRNA concentration was determined using Quant-iT™ RiboGreen™ RNA Reagent and Kit (purchased from Thermo Fisher, catalog number R11490). 250 μL of 20×TE was diluted with 4.75 mL of DEPC water to obtain 5 mL of 1×TE. 196 μL of 1×TE was diluted with 4 μL of RNA standard solution to obtain solution A. 7.5 μL of Ribogreen was diluted with 1492.5 μL of 1×TE to obtain solution B. (2) In a 96-well U-bottom plate, 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 were added to the corresponding wells, and 100 μL of Solution B was added to each well and mixed well. (3) Demulsification: A 2% Triton buffer solution was prepared in 1x TE, and the mRNA / LNP was diluted 100-fold with this solution. After thorough mixing, the mixture was allowed to stand at room temperature for 5 minutes. The mRNA / LNP was diluted 100-fold with 1x TE, and 100 μL of solution B was added to each well. The mixture was then thoroughly mixed and allowed to stand at room temperature for 5 minutes. (4) A multi-label detection / imaging analysis system instrument was used to read the fluorescence, and a standard curve was drawn according to step (2) to calculate the mRNA sample concentration.

[0237] Determination of mRNA / LNP particle size (DLS) (1) A sample cup (UVette) was prepared, and 45 μL of PBS was added together with 5 μL of mRNA / LNP, mixed well, and placed in the cuvette. (2) Particle size was measured according to the instrument's instruction manual. (3) To record the measurement results and process the measurement data, appropriate distribution methods were selected according to the actual results.

[0238] The DLS measurement results are shown in Figure 27. The results showed that the mRNA / LNP particle diameter was approximately 74 nm, which was consistent with the theoretical size, and the sample had a single peak, indicating that the mRNA / LNP had good uniformity.

[0239] Immunological evaluation experiment of gE-gI mRNA / LNP combined with CpG adjuvant This experimental scheme was approved by the Ethics Committee for Laboratory Animal Care of Xiamen University, and all procedures were performed in strict accordance with animal ethics guidelines and approved procedures.

[0240] Six-week-old C57BL / 6 mice were selected and divided into four groups, each with five mice. The experimental group was immunized with gE-gI mRNA / LNP and gE-gI mRNA / LNP + 10 μg CpG, with each mouse receiving an immunization dose of 10 μg in 100 μL. The control group was immunized with Shingrix vaccine (purchased from GSK, batch number: 4N2AB), and the blank group was immunized with saline. Mice were intramuscularly injected into the tibialis muscle at weeks 0 and 3. Ophthalmic vein blood was collected before injection at weeks 0, 2, and 5, respectively. Blood samples were centrifuged at 13,000 g for 10 minutes. Serum samples were stored at -20°C. Antigen-specific binding antibody IgG titers were measured by ELISA, and cytokines were detected by flow cytometry.

[0241] The ELISA results are shown in Figure 28. The results in Figure 28 showed that the titers of binding antibody IgG induced by immunization with gE-gI mRNA / LNP and immunization with gE-gI mRNA / LNP combined with CpG were comparable to those of the Shingrix vaccine, with no significant difference.

[0242] Figure 29 shows the results of T cell responses stimulated by two different vaccines in this example. The results show that antigen-specific CD8 T cells were induced by immunization with gE-gI mRNA / LNP and immunization with gE-gI mRNA / LNP combined with CpG. + IFN-γ + The percentage of T cells was significantly higher than that induced by the Shingrix vaccine, indicating that the mRNA vaccine corresponding to the gE-gI fusion protein of the present disclosure has the ability to induce robust cellular immunity superior to commercially available vaccines, thereby demonstrating promising application prospects.

[0243] Example 13: Preparation and evaluation of gE-gI fusion proteins from stable cell lines To facilitate subsequent manufacturing and transfection applications, a stable CHO cell line derived gE-gI fusion protein was prepared and subjected to immunological evaluation in this disclosure.

[0244] Preparation of gE-gI fusion protein from stable CHO cell lines: To facilitate subsequent production and transformation, the stable cell line utilized the original signal peptide of gE, so that the sequence of gE was 1 aa to 537 aa of the full-length gE protein (SEQ ID NO: 11), with the original signal peptide being approximately 1 aa to 30 aa. During expression of the fusion protein, the signal peptide 1 aa to 30 aa was removed, resulting in the fusion protein containing the gE extracellular domain from 31 aa to 537 aa (SEQ ID NO: 69) and the gI extracellular domain from 21 aa to 271 aa (SEQ ID NO: 14) after enzymatic digestion. The amino acid sequence of the fusion protein after enzymatic digestion and signal peptide removal is shown in SEQ ID NO: 70.

[0245] The specific preparation method was as follows: The coding sequences for the gE (1–537) and gI (21–271) fusion proteins were also linked using a linker (SEQ ID NO: 15). The gE-gI fusion protein was constructed using a tag-free design in the Canton PMGT vector (Guangzhou Canton Biologics Co., Ltd.), a plasmid for stable transformation. The constructed recombinant plasmid was transfected into CHOZN CHOK1 GSKO (purchased from Merck, USA, catalog number CHOGS) host cells and screened under MSX (Sigma, catalog number GSS-1015-F) stress to obtain cell pools. Target protein expression levels in various cell pools were compared across fed batches, and cell pools with high expression levels were finally selected for monoclonal plating. Using SINGLE CELL PRINTER (Cytena, model F.sight) plating combined with cell imaging, 238 monoclonal clones with good viability were obtained, and the cultures were expanded stepwise. The top 20 clones were obtained by screening according to their expression levels in 24-well and 6-well plates. The target protein expression levels of the top 20 clones were compared by fed batch. The top 5 clones were screened by combining cell proliferation 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 clone was selected for protein expression in EX-CELL CD CHO Fusion Medium at 37°C, 140 rpm, 5% CO2, 85% humidity, and a 25 mm rotation radius (Kuhner, model ISF1-XC). After 6 days of culture, the cell supernatant was collected by centrifugation at 200 g for 8 minutes, purified, and prepared using the tag-free purification method for gE-gI fusion protein described in Example 1.

[0246] Table 6 shows comprehensive data on the stability of the top 5 clones, including cell passage stability and yield change. The best clone was CB4019-clone-16.

[0247] [Table 6]

[0248] FIG. 30 shows the monoclonality confirmation results of the top 1 clone (CB4019-clone-16), which showed that CB4019-clone-16 was derived from the division of a single cell.

[0249] FIG. 31 shows the gE-gI fusion protein obtained by the tag-free purification method of Example 1, with a purity of more than 95%.

[0250] The results in Figure 32 showed that the retention volume of the gE-gI fusion protein derived from the CHO stable cell line was approximately 6.9 mL, and the sample showed a single peak, with both homogeneity and purity exceeding 90%.

[0251] Immunological evaluation of gE-gI fusion proteins derived from stable CHO cell lines: The obtained gE-gI fusion protein was immunologically evaluated according to the method of Example 8, and the results of the immunological evaluation are shown in FIGS.

[0252] As shown in Figure 33, the binding antibody IgG induced by the gE-gI fusion protein derived from the CHO stable cell line in combination with AS01B adjuvant was five times higher than that of the Shingrix vaccine.

[0253] FIG. 34 showed that the titer of neutralizing antibodies induced by the gE-gI fusion protein in combination with AS01B adjuvant was approximately twice that of the Shingrix vaccine.

[0254] Figure 35 shows the CD4 T cells stimulated by two different vaccines in this example. + T cells and CD8 + The results of T cell responses were shown. The results showed that antigen-specific CD4 T cells were induced by gE-gI fusion proteins combined with AS01B-like adjuvants.+ IFN-γ + The levels of T cells were significantly higher than those of Shingrix, ie, approximately 1.4 times the levels of Shingrix.

[0255] Figure 36 shows the levels of cytokines IFN-γ and IL-2 stimulated by two different vaccines in this example, as detected by Elispot assay. The results showed that the levels of IFN-γ and IL-2 induced by the gE-gI fusion protein with the AS01B-like adjuvant were higher than those of Shingrix, with the IFN-γ level being 2.4-fold higher than that of Shingrix.

[0256] Although specific embodiments of the present disclosure have been described in detail, it will be understood that those skilled in the art can make various modifications and changes to the details according to all the teachings disclosed, and these modifications fall within the scope of protection of the present disclosure. The entire present disclosure is intended to be provided by the appended claims and their equivalents.

Claims

1. A fusion protein comprising a first peptide segment and a second peptide segment, wherein the first peptide segment comprises or is composed of the extracellular domain or fragment thereof of the varicella-zoster virus (VZV) gE protein, and the second peptide segment comprises or is composed of the extracellular domain or fragment thereof of the VZV gI protein.

2. The fusion protein according to 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 within the amino acid residues of the gE protein at positions corresponding to positions 150-410 of SEQ ID NO: 11, and / or the second peptide segment comprises at least 50, at least 100, at least 150, or at least 180 consecutive amino acid residues within the amino acid residues of the gI protein at positions corresponding to positions 45-240 of SEQ ID NO:

12.

3. The first peptide segment contains amino acid residues of the gE protein at positions corresponding to positions 150 to 410 of SEQ ID NO: 11, Preferably, the first peptide segment contains amino acid residues of the gE protein at positions corresponding to positions 70 to 485 of SEQ ID NO:

11. Preferably, the first peptide segment contains amino acid residues of the gE protein at positions 63 to 497 of SEQ ID NO: 11, Preferably, the first peptide segment contains amino acid residues of the gE protein at positions 31 to 537 of SEQ ID NO: 11, Preferably, the gE protein has an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with (a) any one of the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 46 to SEQ ID NO: 55, (b) any one of the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 46 to SEQ ID NO: 55, or (c) any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions with (a) any one of the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 46 to SEQ ID NO: 55, Preferably, the first peptide segment 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: 13, SEQ ID NOs: 33 to 42, or SEQ ID NOs: 64 to 69, or includes, or is composed of, a sequence having one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions.

4. The second peptide segment contains amino acid residues of the gI protein at positions corresponding to positions 45-240 of SEQ ID NO: 12, Preferably, the second peptide segment contains amino acid residues of the gI protein at positions 30 to 260 of SEQ ID NO:

12. Preferably, the second peptide segment contains amino acid residues of the gI protein at positions corresponding to positions 21 to 271 of SEQ ID NO:

12. Preferably, the gI protein has an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with (a) the amino acid sequence shown in any one of SEQ ID NOs: 12, SEQ ID NOs: 56 to 58, (b) the amino acid sequence shown in any one of SEQ ID NOs: 12, SEQ ID NOs: 56 to 58, or (c) the amino acid sequence that has one or more (e.g., one, two, three, four, five, six, seven, eight, or nine) amino acid substitutions (preferably conservative substitutions), additions, or deletions with respect to (a) the amino acid sequence shown in any one of SEQ ID NOs: 12, SEQ ID NOs: 56 to 58. Preferably, the fusion protein according to claim 1, wherein the second peptide segment 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, SEQ ID NOs. 43 to SEQ ID NOs. 45, or has a sequence having one or more (for example, one, two, three, four, five, six, seven, eight, or nine) amino acid substitutions (preferably conservative substitutions), additions, or deletions.

5. The second peptide segment is optionally connected to the N-terminus or C-terminus of the first peptide segment via a linker (for example, a peptide linker, for example, the peptide linker shown in sequence number 15). Preferably, the linker is a peptide linker containing one or more (for example, 5 to 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 contains or is composed of the amino acid sequence shown in any one of SEQ ID NOs: 6 to 7, SEQ ID NOs: 20 to 32, SEQ ID NOs: 59 to 63, or SEQ ID NOs: 70, according to claim 1.

6. The fusion protein comprises a signal peptide and / or a detectable label (e.g., a tag protein), Preferably, the fusion protein contains a signal peptide at its N-terminus (for example, the native signal peptide of VZV gE protein, the native signal peptide of VZV gI protein, the gp67 signal peptide, the melittin signal peptide (MSP), or the tissue-type plasminogen activator signal peptide (tPA signal peptide)), Preferably, the fusion protein contains a detectable label at its C-terminus. The fusion protein according to claim 1.

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

8. It further includes one or more selected from 5'UTR, Kosack sequence, start codon, stop codon, 3'UTR, and poly-A tail. Preferably, the RNA molecule according to claim 7 comprises a 5' to 3': 5'UTR, a Kozak sequence, a start codon, a nucleotide sequence encoding a fusion protein, a stop codon, a 3'UTR, and a poly-A tail.

9. The RNA molecule according to claim 7, having one or more of the following characteristics: (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 respect to the nucleotide sequence shown in Sequence ID No. 71, and preferably the nucleotide sequence encoding the fusion protein is shown in Sequence ID No. 71; (2) The nucleotide sequence encoding the fusion protein has one or more stop codons at its 3' end; (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 with respect to the nucleotide sequence shown in Sequence ID No. 17, and preferably the 5'UTR has the nucleotide sequence shown in Sequence 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 with respect to the nucleotide sequence shown in SEQ ID NO: 18, and preferably the 3'UTR has the nucleotide sequence shown in SEQ ID NO: 18; (5) The Kossack sequence is shown in Sequence ID No. 72; (6) The poly-A tail comprises one or more polyadenylic acid sequences, each of which is independently composed of 20 to 120 consecutive adenylic acid sequences, preferably the poly-A tail comprises a plurality of polyadenylic acid sequences, adjacent polyadenylic acid sequences are connected by non-A spacer sequences, preferably the poly-A tail has a nucleotide sequence with 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 respect to 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 (e.g., CAP-0, CAP-1, CAP-2).

10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein according to any one of claims 1 to 6, or an RNA molecule containing the nucleotide sequence encoding the fusion protein.

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

12. A delivery composition comprising a delivery carrier, (i) an RNA molecule containing a nucleotide sequence encoding the fusion protein described in any one of claims 1 to 6, or (ii) an isolated nucleic acid molecule containing the nucleotide sequence encoding the fusion protein or the RNA molecule, or (iii) a vector containing the isolated nucleic acid molecule, Preferably, the delivery carrier is selected from the group consisting of lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, and viral vectors (e.g., replication-deficient retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses). Preferably, the delivery composition comprises the RNA molecule.

13. The delivery composition according to claim 12, wherein the delivery carrier is lipid nanoparticles (LNPs).

14. (i) an RNA molecule comprising a nucleotide sequence encoding the fusion protein according to any one of claims 1 to 6, or (ii) an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein or the RNA molecule, or (iii) a vector comprising the isolated nucleic acid molecule, Preferably, the host cell is selected from the group consisting of prokaryotic cells (e.g., E. coli cells) and eukaryotic cells (e.g., 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 a fusion protein, comprising culturing the host cells described in claim 14 under conditions that enable protein expression, and recovering the fusion protein from the cell culture.

16. An immunogenic composition comprising (i) a fusion protein according to any one of claims 1 to 6, or (ii) an RNA molecule containing a nucleotide sequence encoding the fusion protein, or (iii) an isolated nucleic acid molecule containing a nucleotide sequence encoding the fusion protein or the RNA molecule, or (iv) a vector containing the isolated nucleic acid molecule, or (v) a delivery carrier and any one of (ii) to (iv), and optionally a pharmaceutically acceptable carrier and / or excipient (e.g., an adjuvant), Preferably, the immunogenic composition comprises the fusion protein, or the RNA molecule, or the isolated nucleic acid molecule, or the vector, or the delivery composition, and an adjuvant, wherein the adjuvant is selected from the group consisting of aluminum salt adjuvants, zinc-aluminum hybrid adjuvants (e.g., FH002C), Freund's adjuvants, oil emulsion adjuvants (e.g., MF59 adjuvants), 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 or the delivery composition and the adjuvant. Preferably, the immunogenic composition is a vaccine.

17. A pharmaceutical composition for inducing an immune response to VZV in a subject and / or preventing and / or treating VZV infection or a disease associated with VZV infection in a subject, comprising: (i) a fusion protein according to any one of claims 1 to 6; (ii) an RNA molecule containing a nucleotide sequence encoding the fusion protein; (iii) an isolated nucleic acid molecule containing a nucleotide sequence encoding the fusion protein or the RNA molecule; (iv) a vector containing the isolated nucleic acid molecule; or (v) a delivery composition comprising a delivery carrier and any one of (ii) to (iv); or (vi) a host cell comprising any one of (ii) to (iv); or (vii) an immunogenic composition comprising any one of (i) to (v) and optionally a pharmaceutically acceptable carrier and / or excipient. Preferably, the immunogenic composition is a vaccine. Preferably, the VZV infection is a primary or recurrent VZV infection. Preferably, the disease associated with VZV infection is selected from the group consisting of herpes zoster, varicella, and their complications (e.g., postherpetic neuralgia, pneumonia, encephalomyelitis, conjunctivitis), Preferably, the subject of the test is a mammal such as a human, in the pharmaceutical composition.

18. A method for detecting the presence of an antibody specific to VZV gI protein and / or an antibody specific to VZV gE protein in a sample, comprising using the fusion protein described in any one of claims 1 to 6, Preferably, the method is an immunoassay such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescence immunoassay, fluorescence immunoassay, or radioimmunoassay. Preferably, the method comprises (1) contacting the sample with the fusion protein, and (2) detecting the formation of a fusion protein-antibody immune complex, or detecting the amount of the immune complex, wherein the formation of the immune complex indicates the presence of antibodies specific to VZV gI protein and / or antibodies specific to VZV gE protein in the sample. Preferably, the method further includes detecting the presence of antibodies specific to VZV gI protein and / or antibodies specific to VZV gE protein in the sample using a second antibody having 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), Preferably, the second antibody is specific to a constant region contained in the antibody of the species from which the sample being tested originates (e.g., human). Preferably, the second antibody is an anti-immunoglobulin (e.g., human immunoglobulin) antibody such as an anti-IgG antibody. Preferably, the method is a bodily fluid sample (e.g., whole blood, plasma, serum, salivary excrement, or urine) from a subject (e.g., a mammal, preferably a human).

19. A kit comprising (i) a fusion protein according to any one of claims 1 to 6, or (ii) an RNA molecule containing a nucleotide sequence encoding the fusion protein, or (iii) an isolated nucleic acid molecule containing a nucleotide sequence encoding the fusion protein or the RNA molecule, or (iv) a vector containing the isolated nucleic acid molecule, or (v) a delivery carrier and a delivery composition comprising any one of (ii) to (iv), or (vi) a host cell comprising any one of (ii) to (iv), Preferably, the kit comprises the fusion protein and a second antibody, wherein the second antibody has a detectable label, and optionally the second antibody is specific to a constant region contained in an antibody of the species from which the sample being tested originates.

Images