Herpes zoster mRNA vaccine, its preparation method, and use

Abstract

To provide herpes zoster mRNA vaccine, a preparation method therefor, and use thereof.SOLUTION: Provided is a herpes zoster mRNA vaccine, comprising an RNA encoding a varicella-zoster virus gE glycoprotein or a variant thereof, wherein the vaccine can prevent herpes zoster infection and its complications.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This invention belongs to the technical field of mRNA vaccines, and more particularly to herpes zoster mRNA vaccines, methods for preparing the same, and their use. [Background technology]

[0002] Shingles is an infectious skin disease caused by the reactivation of the varicella-zoster virus (VZV), which has been dormant for a long time in the dorsal root ganglia of the spinal cord or cranial ganglia. Shingles is a common skin disease, often accompanied by neuropathic pain in addition to skin damage. The disease is more likely to occur in the elderly and in immunocompromised or immunodeficient individuals, and is more common in spring and autumn. The incidence increases significantly with age.

[0003] After the body recovers from chickenpox infection, VZV is not completely eliminated but remains dormant in the cranial ganglia, dorsal root ganglia, and autonomic ganglia throughout the nervous system, persisting for life. After infection with VZV and the onset of chickenpox, the body develops specific antibodies against VZV and cellular immunity mediated by T cells (e.g., CD4). + / CD8 + The immune system produces memory T cells (and other immune cells), and these two immune functions play a crucial role in maintaining latent VZV infection and preventing herpes zoster. With increasing age or due to external or internal factors that impair the body's immune system, VZV can spontaneously reactivate and cause herpes zoster (HZ).

[0004] Common activators of shingles include age factors (e.g., aging), cellular immunodeficiency, genetic susceptibility, trauma, systemic diseases (e.g., diabetes, kidney disease), excessive mental stress, and fatigue. Unlike childhood varicella infection, shingles in adults (elderly) is more likely to leave sequelae, the most common of which is postherpetic neuralgia. This is neuralgia (PHN), and the pain can persist for 3 to 12 months even after the shingles has healed.

[0005] Both shingles and postherpetic neuralgia are common and frequently occurring illnesses. The chronic pain caused by shingles (postherpetic neuralgia) and other complications, commonly known as "dragon around the waist," "snake around the waist," or "living snake," seriously impact the patient's quality of life, and older adults and those with weakened immune systems are often affected by these complications. Because the mechanism of pain associated with postherpetic neuralgia is unclear, it remains one of the most difficult types of pain to treat.

[0006] Shingles has two distinctive characteristics: firstly, a high infection rate, and secondly, patients must endure very severe pain. According to survey data from Europe and the United States, the annual incidence of shingles is 2-6‰. The incidence tends to increase with age. Statistical surveys show that the incidence is approximately 4‰ for those aged 10-49, while it reaches as high as 14‰ for those aged 75 and over. Furthermore, women are more susceptible to shingles than men. Currently, the incidence of shingles is on the rise worldwide, and studies from 1994 to 2018 show that the annual incidence of shingles increased by 3.1% each year, with a particularly significant increase in the incidence rate among those aged 20-49.

[0007] China does not include shingles in its list of notifiable infectious diseases under categories A and B, and there are relatively few epidemiological studies covering the entire population. Statistics show that approximately 99.5% of adults over 50 in China carry the VZV virus in their bodies, with about 1.56 million new infections annually. The average annual incidence rate of shingles among those aged 50-60 in China is 2.66‰, and among those over 80... The average annual incidence rate reaches 8.55‰. The incidence of postherpetic neuralgia (PHN) also increases with age; according to studies, the incidence of PHN among elderly shingles patients in China is 18.8%, and among elderly patients over 75 years of age, it reaches 31.7%.

[0008] Vaccination is the most effective way to prevent shingles. Major technological routes for the research and development of shingles vaccines include attenuated live vaccines, recombinant protein vaccines, adenovirus vector vaccines, and mRNA vaccines.

[0009] Currently, there are only four vaccines available worldwide for the prevention of shingles: Merck & Co., Inc.'s live attenuated vaccine Zostavax® (production discontinued), GlaxoSmithKline (GSK)'s recombinant vaccine Shingrix®, SK Chemical Co., Ltd.'s SkyZoster® (sold only in South Korea), and China's recently launched live attenuated vaccine by Changchun BCHT Biotechnology Co., Ltd. Merck & Co., Inc.'s live attenuated vaccine Zostavax® was the world's first approved shingles vaccine, launched in the US and Europe in 2006. However, due to its low efficacy (effectively reducing herpes outbreaks by approximately 70%), it was unsuitable for vaccination in immunocompromised and immunocompromised individuals, severely limiting its applicability, and production was discontinued in 2018. GlaxoSmithKline's (GSK) recombinant protein vaccine, Shingrix®, was launched in the US in 2017, in Europe in 2018, and applied for listing in China in 2019. Global sales of this vaccine reached $2.4 billion in 2021 and $3.2 billion in 2022, placing it among the top 10 best-selling vaccines globally for several consecutive years. However, because this vaccine is a protein subunit vaccine and uses GSK's proprietary adjuvant, it is expensive, difficult to increase production capacity, and, more importantly, has serious side effects, with a significantly higher incidence of side effects compared to attenuated live vaccines.

[0010] mRNA vaccines are the third generation of vaccine technology, following conventional vaccines and protein subunit vaccines. After entering human cells via a specific delivery system, mRNA vaccines use the body's own cells to translate mRNA into protein. This protein is then expressed as a specific antigen protein of the virus, recognized as a foreign antigen by antigen-presenting cells (APCs), promoting the maturation of dendritic cells (DCs), and further activating B and T cells to produce a robust immune response, triggering a dual response of humoral and cellular immunity. mRNA vaccines break away from the conventional immune activation model of vaccines, innovatively using the body's own cells to produce antigens, thereby activating bispecific immunity, forming immunological memory, and providing longer-lasting specific immunity. The greatest advantage of mRNA vaccines is that they can be rapidly developed if the antigen gene sequence of the pathogen is known. mRNA vaccine technology offers several technological advantages, including rapid design and construction, high adaptability to viral mutations, and an efficient, universal, fully synthetic production process platform with standardized ease of production. These advantages result in shorter production links and R&D cycles, a relatively simple process, and the ability to rapidly mass-produce mRNA vaccines.

[0011] Compared to conventional vaccines, the herpes zoster mRNA vaccine has the following advantages: 1) It is non-infectious and non-integrated, eliminating the risk of infection and insertion mutation. 2) The mRNA vaccine activates the body's specific immunity by expressing the antigen in the body, producing more sustained and effective specific immunity. 3) The mRNA vaccine can be stably delivered into cells and efficiently expressed in the body. 4) The preparation of the mRNA vaccine is subject to strict quality control.

[0012] gE is the main target protein in shingles vaccine research and development, and is the highest content in VZV. It is also one of the abundant glycoproteins and plays an important role in viral replication and viral spread between ganglion cells. As a highly glycosylated type I membrane protein, gE is transported between the endoplasmic reticulum (ER), the trans Golgi network (TGN), and the endoplast.

[0013] Given the problems described above, there is an urgent need to provide a novel mRNA vaccine to prevent VZV that can significantly improve the effectiveness of humoral and cellular immunity after vaccination, in order to further reduce the risk of developing shingles in people with weakened immune systems. [Overview of the project]

[0014] The present invention provides a herpes zoster mRNA vaccine that can safely guide the body's cellular mechanisms to produce substantially any protein for any purpose, from natural proteins to antibodies and other entirely novel proteins that may have prophylactic activity both intracellularly and extracellularly. The herpes zoster mRNA vaccine of this disclosure can be used to induce a balanced immune response against VZV, including cellular and humoral immunity, but without many of the risks associated with vaccination with attenuated viruses.

[0015] RNA (e.g., mRNA) vaccines can be used in a variety of situations, depending on the extent or level of the spread of an infectious disease or the unmet medical needs. RNA (e.g., mRNA) vaccines are available to prevent VZV of various genotypes, strains, and isolates. The advantage of RNA (e.g., mRNA) vaccines lies in producing much higher antibody titers and faster response times than commercially available antiviral therapies. While not bound by theory, it is believed that, like mRNA polynucleotides, RNA vaccines are more favorably designed to produce the appropriate protein conformation through translation when the RNA vaccine directs natural cellular mechanisms. Unlike conventional vaccines, which are prepared in vitro and can cause harmful cellular responses, RNA (e.g., mRNA) vaccines are delivered to cell systems in a more natural way.

[0016] Based on the key structure and biological function of the VZV gE protein, this invention innovatively designed a series of mRNA vaccine candidate antigens and successfully screened several herpes zoster mRNA vaccine candidates with novel antigen structures and superior immunogenicity by comparing their immunogenicity with existing mRNA vaccines (e.g., antigen YK-VZV-007, CN108472309A; and the commercially available recombinant subunit vaccine Shingrix®). This formed the basis for the successful development of herpes zoster mRNA vaccines.

[0017] This application provides a novel mRNA vaccine for preventing VZV by combining wild-type VZV gE glycoprotein variants in a specific manner (including sequence shortening, site mutations, and / or sequence deletions) based on the wild-type VZV gE glycoprotein, and accordingly providing a novel mRNA vaccine. Experiments have shown that after vaccination, the effects of humoral and cellular immunity can be significantly improved, and this effect is due to an increase in the content of IgG antibodies against VZV gE protein and immune cells (IFN-γ). + and / or IL-2 + CD4 secretes + and CD8 + An increase in the number of immune cell cytokines (including IFN-γ)+ and IL-2 + We provide a herpes zoster mRNA vaccine that includes the effect of promoting the secretion of (including)

[0018] The mRNA vaccines provided herein may include RNA polynucleotides of at least one VZV glycoprotein provided by the accompanying sequence listing, or fragments, homologs (e.g., having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity), or variants or derivatives thereof.

[0019] In some embodiments, the antigen encodes a VZV gE polypeptide.

[0020] In some embodiments, the VZV gE polypeptide (full length) contains amino acids 1 to 623 (e.g., SEQ ID NOs: 3, 51, 55, 171, 135, 139, 143, 147, 19, 23, 151, 27, 31, 35, 39, 43, 47, 163, 167).

[0021] In some embodiments, the VZV gE polypeptide is a mutant. In some embodiments, the VZV gE mutant is a shortened polypeptide lacking an anchor domain (ER-retaining domain).

[0022] In some embodiments, the abbreviated VZV gE polypeptide contains 1 to 539 amino acids (e.g., SEQ ID NO: 7).

[0023] In some embodiments, the abbreviated VZV gE polypeptide contains 1 to 573 amino acids (e.g., SEQ ID NOs: 15, 59, 63, 67, 71, 75, 79, 83, 87, 91).

[0024] In some embodiments, the truncated VZV gE polypeptide contains 1 to 568 amino acids (e.g., SEQ ID NO: 11).

[0025] In some embodiments, the truncated VZV gE polypeptide comprises amino acids 1-587 (e.g., SEQ ID NO: 95, 99, 103, 107, 111, 115, 119, 123, 127, 131).

[0026] In some embodiments, the truncated VZV gE polypeptide comprises amino acids 1-601 (e.g., SEQ ID NO: 155, 159).

[0027] In some embodiments, the VZV gE variant is a full-length or truncated polypeptide. It also includes mutations in one or more motifs related to targeting to the Golgi apparatus or trans-Golgi network (TGN), and such mutations reduce the VZV gE polypeptide targeted to or localized in the Golgi apparatus or TGN. In particular, the inventors found that the motif related to the localization of the gE protein to the Golgi apparatus or trans-Golgi network is "A" 568 Y 569 R 570 V 571 " motif (the specific mutant antigen sequence includes, for example, YK-VZV-011 (SEQ ID NO: 35), and unexpectedly found that it is particularly the A568D and / or Y5 69K mutations.

[0028] Furthermore, in some embodiments, the VZV gE variant may have a Y582A / G mutation, or a Y569K / A mutation, or a combined mutation of Y582G and Y569A. Other mutation types include phosphorylation acidic motifs such as the S 593 EST 596 DT 598 (SEQ ID NO: 182) site mutation.

[0029] In some embodiments, the mutant VZV gE polypeptide is a full-length or truncated polypeptide. It includes one or more motifs related to VZV gE internal translocation or endocytosis, Y 582 AGL 585This includes the mutation in (SEQ ID NO: 185), which reduces the endocytosis of the VZV gE polypeptide.

[0030] In some embodiments, the VZV gE mutant has the Y569K mutation These are long polypeptides (sequence numbers 27, 31, 35, 39, 43, 47, 143, 147).

[0031] In some embodiments, the VZV gE variant is a full-length polypeptide (SEQ ID NOs: 23, 27, 31, 43, 47, 135, 139, 171) having the Y582A mutation.

[0032] In some embodiments, the VZV gE variant is a full-length polypeptide (SEQ ID NOs: 27, 31, 43, 47) having Y582A and Y569K mutations.

[0033] In some embodiments, the VZV gE mutant is A 593 EA 595 A 596 DA 598 This is a full-length polypeptide having the sequence (SEQ ID NO: 183).

[0034] In some embodiments, the VZV gE mutant is A 593 EA 595 A 596 DA 598 It is a full-length or truncated polypeptide having the sequence (SEQ ID NO: 183) and containing a Y582G mutation, a Y569K mutation, or a combination of Y582G and Y569K mutations.

[0035] In some embodiments, the VZV gE mutant is selected from the group consisting of A568D, Y569K, Y569A, R570E, V571K, Y582A, S593A, S595A, T596A, and T598A and contains one or more site mutations, preferably A568D, Y569K, R570E, and V571K.

[0036] In some embodiments, the site mutation is one of the following groups. 1)A568D, Y569K, R570E, V571K, (YK-VZV-023, YK-VZV-011) 2)A568D, Y569A, R570E, V571K, (YK-VZV-024) 3)Y569K, R570E, V571K, (YK-VZV-025) 4)A568D, Y569K, R570E, V571K, Y582A, (YK-VZV-030, YK-VZV-013) 5)A568D, Y569K, R570E, V571K, Y582G, (YK-VZV-031, YK-VZV-038) 6)A568D, Y582A, (YK-VZV-032) 7)Y569K, Y582A, (YK-VZV-033, YK-VZV-009) 8)R570E, Y582A, (YK-VZV-034) 9)V571K, Y582A, (YK-VZV-035) 10)S593A, S595A, T596A, T598A, (YK-VZV-016, YK-VZV-044) 11)Y582A, S593A, S595A, T596A, T598A, (YK-VZV-037) 12)A568D, Y569K, R570E, V571K, Y582G, S593A, S595A, T596A, T598A, (YK-VZV-039) 13)A568D, Y569K, R570E, V571K, Y582A, S593A, S595A, T596A, T598A, (YK-VZV-014) 14)Y582G, S593A, S595A, T596A, T598A, (YK-VZV-040) 15)Y569K, Y582A, S593A, S595A, T596A, T598A, (YK-VZV-010) 16)A568D, Y569K, R570E, V571K, S593A, S595A, T596A, T598A, (YK-VZV-012) 17) Y582G, (YK-VZV-029) 18) A568D, (YK-VZV-018) 19) Y569K, (YK-VZV-020) 20) R570E, (YK-VZV-021) 21) V571K, (YK-VZV-022) 22)Y582A, (YK-VZV-028, YK-VZV-036)

[0037] In some embodiments, the VZV gE mutant further comprises a sequence deletion, which is selected from one or more of the following groups. 1) A 568 , 2) Y 569 RVDKSPYNQS 579 , 3) Y 569 RVDKSPYNQSMYYAGLPV 587 .

[0038] The VZV RNA antigens of this disclosure have multiple combination types of mutations, the combinations of which include shortening of the C-terminal amino acid of the gE antigen, deletion or substitution of a major amino acid site, and will be further explained below through specific examples.

[0039] The present invention provides a template that can be transcribed to the RNA of the VZV, The present invention further provides a method for preparing the composition, comprising the step of transcribing using the template under conditions suitable for transcription into the RNA.

[0040] In some embodiments, the preparation method is The steps involve synthesizing the DNA sequence of the VZV gE antigen and then homologously recombining it with the PVAX1 vector. Steps to obtain plasmids after transformation and culture of E. coli, The step of obtaining a linearized plasmid after enzymatic digestion of the plasmid. The process includes the step of using T7 RNA polymerase to create a linear plasmid template, adding NTP raw materials, co-transcribe, capping, and then purifying to obtain RNA, preferably mRNA.

[0041] In some embodiments, the purification method includes one or more of lithium chloride precipitation, affinity chromatography, solution exchange by ultrafiltration, and cellulose chromatography.

[0042] In some embodiments, the method for preparing the composition includes the steps of: synthesizing a DNA sequence based on an antigen gene and inserting it into a plasmid DNA construct; cleaving the plasmid DNA with a DNA restriction endonuclease to form a linear template; using the linear plasmid as a template under the catalytic action of T7 RNA polymerase, adding key raw materials such as NTPs and cap analogs, transcribing and preparing to synthesize mRNA, and modifying the RNA; adding a cap to the 5' end; and removing impurities such as contaminants, enzymes, and free nucleotides from the modified mRNA by methods such as lithium chloride precipitation and affinity chromatography.

[0043] In a third aspect, the composition of the present invention is in the form of a vaccine, i.e., a herpes zoster vaccine, the vaccine further comprising a pharmaceutically acceptable carrier in addition to the composition.

[0044] In some preferred embodiments, the pharmaceutically acceptable carrier comprises a lipid mixture, preferably lipid nanoparticles (LNPs).

[0045] In some preferred embodiments, the lipid nanoparticles are, for example, cationic lipids. It includes neutral lipids, structural lipids, and polymer-conjugated lipids.

[0046] In a fourth aspect, the present invention further provides a method for preparing a vaccine composition, comprising the steps of mixing the RNA of the VZV with a pharmaceutically acceptable carrier, for example, encapsulating at least a portion of the RNA within lipid nanoparticles. In some embodiments, the preparation method comprises the steps of mixing the RNA, preferably mRNA, with lipid nanoparticles, encapsulating them within lipid nanoparticles, and purifying to remove unencapsulated components.

[0047] In some more specific embodiments, the preparation method specifically includes the steps of incorporating the mRNA into lipid nanoparticles (LNPs), i.e., mixing a mixture of purified mRNA and lipids in a microfluidic chip and allowing it to self-assemble to form lipid nanoparticles; encapsulating the mRNA within the lipid nanoparticles; dialysis or filtration of the lipid nanoparticle solution to remove unencapsulated mRNA, non-aqueous solvents, and bacteria; storing the filtered lipid nanoparticle solution and packing it into sterile vials to complete the preparation and production of the mRNA vaccine.

[0048] The following is what this application initially discovered:

[0049] This application provides a novel mRNA vaccine for preventing VZV by obtaining several ribonucleic acids encoding VZV gE glycoprotein variants based on wild-type VZV gE glycoprotein, by combining them in a specific manner (including sequence shortening, site mutation, and / or sequence deletion), and experimentally demonstrating that the vaccine can significantly improve the effects of humoral and cellular immunity after vaccination, the effect of which is due to an increase in the content of IgG antibodies against VZV gE protein, and immune cells (IFN-γ + and / or IL-2 + CD4 secretes + and CD8 + An increase in the number of immune cell cytokines (including IFN-γ) + and IL-2 +The present invention provides a herpes zoster mRNA vaccine that includes the effect of promoting the secretion of (including). Specifically, this application includes the following discoveries and results.

[0050] 1. Designed 43 VZV Western blotting detection was performed on gE antigen variants, and the results showed that all 39 antigen sequences had significant protein expression, including YK-VZV-001, YK-VZV-003, YK-VZV-009, YK-VZV-010, YK-VZV-011, YK-VZV-012, YK-VZV-013, YK-VZV-014, YK-VZV-015, YK-VZV-016, YK-VZV-017, YK-VZV-018, YK-VZV-019, YK-VZV-020, YK-VZV-021, YK-VZV-022, YK-VZV-023, YK-VZV-024, YK-VZV-025, and YK-VZV-0 26, YK-VZV-027, YK-VZV-028, YK-VZV-029, YK-VZV-030, YK-VZV-031, YK-VZV-032, Y K-VZV-033, YK-VZV-034, YK-VZV-035, YK-VZV-036, YK-VZV-037, YK-VZV-038, YK-VZ V-039, YK-VZV-040, YK-VZV-044, and YK-VZV-004 (Comparative Example 1), YK-VZV-006 (Comparative Example 2), YK-VZV-045 (Comparative Example 3), and YK-VZV-007 (Comparative Example 4) are included and can be used as effective candidate antigens for immunogenicity screening and evaluation in the next step.

[0051] 2. Based on the 39 sequences listed above, the following 14 variants were identified based on the gE-specific antibody IgG titer results on day 42: YK-VZV-009, YK-VZV-011, YK-VZV-013, YK-VZV-014, YK-VZV-010, YK-VZV-012, YK-VZV-018, YK-VZV-020, YK-VZV-030, YK-VZV-031, YK-VZV-038, YK-VZV-021, YK-VZV-024, YK-VZ V-028 and four control sequences: YK-VZV-004 (Comparative Example 1), YK-VZV-006 (Comparative Example 2), YK-VZV-045 (Comparative Example 3), and YK-VZV-007 (Comparative Example 4) were extracted.

[0052] The above sequences are 3.3 to 5.3 times more potent than the Shingrix® positive control, and simultaneously 1.8 to 2.8 times more potent than the YK-VZV-007 (Comparative Example 4) sequence. Their effectiveness is superior to that of Shingrix®, YK-VZV-007 (Comparative Example 4), YK-VZV-004 (Comparative Example 1), YK-VZV-006 (Comparative Example 2), and YK-VZV-045 (Comparative Example 3).

[0053] CD4 of IFN-γ and IL-2 secreted by the mouse spleen + T cells and CD8 + The following can be understood from the measurement results of the percentage of T cells.

[0054] (1) CD4 + Based on the proportion of T cells, YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020, YK-VZV-012, YK-VZV-018, YK-VZV-028, YK-VZV-038, and YK-VZV-031 were screened, and the above nine sequences were: IFN-γ + CD4 + T cells and IL-2 + CD4 + The total proportion of T cells was more than twice that of the Shingrix®-positive control, up to seven times higher, and approximately 1.8 to 5.3 times higher than that of YK-VZV-007 (Comparative Example 4). IFN-γ + CD4 + The percentage of T cells was 1.00-2.00%, which is more than three times higher than the Shingrix®-positive control, up to 6.7 times higher, and approximately 2.2-4.4 times higher than YK-VZV-007 (Comparative Example 4). IL-2 + CD4 +The percentage of T cells exceeded 1% in all cases, which is more than 3.7 times higher than that of the Shingrix®-positive control vaccine and more than 2.2 times higher than that of YK-VZV-007.

[0055] (2) Furthermore, CD8 + Based on the proportion of T cells, five sequences were screened: YK-VZV-010, YK-VZV-013, YK-VZV-020, YK-VZV-018, and YK-VZV-011. IFN-γ + CD8 + T cells and IL-2 + CD8 + The total percentage of T cells was 4 to 7 times higher than that of the Shingrix® vaccine and approximately 3 to 5 times higher than that of YK-VZV-007 (Comparative Example 4). IFN-γ + CD8 + The T cell ratio was optimal, all above 5.0%, which was 4.5 to 7.0 times higher than the Shingrix® positive control vaccine and approximately 2.5 to 4.5 times higher than YK-VZV-007. IL-2 + CD8 + The T cell ratio was optimal, reaching 0.3-0.5%, which is 2.5-4.0 times higher than the Shingrix® positive control vaccine and approximately 1.5-3.0 times higher than YK-VZV-007. All of these results were significantly higher than those of the comparative examples (YK-VZV-004, YK-VZV-006, YK-VZV-045, YK-VZV-007).

[0056] 4. To verify the above results, the VZV-conjugated-gE antibody IgG titer in the serum of immunized mice on day 56 was further measured and verified based on the measurement results on day 42.

[0057] The results showed that 56 days after immunizing mice with the YK-VZV-009, YK-VZV-010, YK-VZV-011, YK-VZV-013, YK-VZV-020, YK-VZV-014, YK-VZV-012, YK-VZV-018, YK-VZV-021, YK-VZV-024, YK-VZV-028, YK-VZV-030, YK-VZV-031, and YK-VZV-038 vaccines, gE-specific antibody IgG of the above mRNA vaccines was detected. The titers were all superior to those of the Shingrix®-positive control, showing approximately 1.8 to 5.0 times higher than those of the Shingrix® vaccine.

[0058] Among them, the antibody GMT for YK-VZV-011, YK-VZV-013, YK-VZV-020, YK-VZV-018, and YK-VZV-010 was 650 × 10 4 The results were as described above, which were 4.0 to 5.0 times better than the Shingrix® vaccine and more than twice as good as YK-VZV-007 (Comparative Example 4). Furthermore, all of them were significantly better than YK-VZV-004 (Comparative Example 1) and YK-VZV-045 (Comparative Example 3).

[0059] 5. Combining the results of IgG and multi-parameter flow cytometry detection, five optimal antigen sequences (including YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020, and YK-VZV-018) were screened and further validated using the ELISpot method. The results are as follows:

[0060] (1) In all cases, the mRNA vaccine stimulated T cells to secrete the cytokine IFN-γ to over 500 SFU, with the highest reaching nearly 600 SFU, which is approximately 16 to 19 times that of the Shingrix®-positive control, 2.5 to 3.0 times that of Comparative Example 4 (YK-VZV-007), and significantly superior to the cytokine secretion stimulated by the corresponding mRNA vaccines of Comparative Example 1 (YK-VZV-004), Comparative Example 2 (YK-VZV-006), or Comparative Example 3 (YK-VZV-045).

[0061] Among these, the mRNA vaccine prepared with the YK-VZV-010 sequence reached a maximum of 598.8 SFUs, which was 18.7 times higher than the Shingrix®-positive control and nearly 3 times higher than the YK-VZV-007 control.

[0062] IFN-γ secretion levels, from highest to lowest, were YK-VZV-010, YK-VZV-011, YK-VZV-018, YK-VZV-020, and YK-VZV-013. There were no significant differences between the groups, and all showed good and significant immune effects.

[0063] (2) The amount of cytokine IL-2 secreted by T cells stimulated by mRNA vaccines was 400-600 SFU or more in all cases, which is 10-16 times that of the Shingrix®-positive control and 2.0-3.0 times that of the YK-VZV-007 control, and is significantly better than the cytokine secretion stimulated by mRNA vaccines corresponding to Comparative Example 1 (YK-VZV-004), Comparative Example 2 (YK-VZV-006), or Comparative Example 3 (YK-VZV-045).

[0064] Among these, the mRNA vaccine prepared with the YK-VZV-010 sequence reached a maximum of 614.5 SFUs, which was 16.0 times higher than the Shingrix®-positive control and more than 3 times higher than the YK-VZV-007 control.

[0065] The IL-2 secretion levels, from highest to lowest, were YK-VZV-010, YK-VZV-011, YK-VZV-018, YK-VZV-013, and YK-VZV-020. There was no significant difference between the groups, and all showed favorable and significant immune effects.

[0066] 6. Furthermore, the antigen localization results for the five sequences described above indicate that they are localized in either the Golgi apparatus, the cell membrane, or the cytoplasm. [Brief explanation of the drawing]

[0067] [Figure 1]This is a mutant of the VZV gE protein with a major functional domain and sequence deletion. [Figure 2] The YK-VZV-001 to YK-VZV-045 antigens are expressed. [Figure 3] This shows the cellular localization of the VZV gE antigens YK-VZV-010, YK-VZV-011, and YK-VZV-013. [Figure 4] This shows the cellular localization of the VZV gE antigen in YK-VZV-018 and YK-VZV-020. Here, green represents the gE antigen, yellow and red represent GM130 and TGN46, respectively, white arrows represent the cell membrane, and orange arrows represent the Golgi apparatus. [Modes for carrying out the invention]

[0068] The present invention will be further described below with reference to examples. The examples of the present invention are used solely for the purpose of illustrating the present invention and are not intended to limit it. It should be understood that any simple modifications of the present invention based on the technical solutions of the present invention fall within the scope of protection of the present invention.

[0069] In this specification, the term "selected from" means selecting one, two, three or more, or any combination thereof, from the enumerated subjects or elements.

[0070] As used herein, the term “specific compound” may include the said compound, its N-oxide, its solvate, its pharmaceutically acceptable salt, its stereoisomer, and mixtures thereof.

[0071] Design of Antigen Sequences: An antigen is a protein that can induce an immune response (e.g., one that causes the immune system to produce antibodies against the antigen). In this specification, the use of the term “antigen” refers to immunogenic proteins and immunogenic fragments that induce or can induce an immune response to varicella-zoster virus (e.g., VZV), unless otherwise specified. The term “protein” is understood to refer to a peptide, and the term “antigen” to refer to an antigenic fragment. Table 1 provides exemplary sequences of VZV antigens and RNA encoding the VZV antigens of the compositions of this disclosure.

[0072] Nucleic acids: Nucleic acids include polymers of nucleotides (nucleotide monomers). The compositions of this disclosure include RNA having an open reading frame (ORF) encoding a VZV antigen. In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the RNA (e.g., mRNA) further includes a 5'UTR, a 3'UTR, a poly(A) tail, and / or a 5' cap analog.

[0073] Messenger RNA (mRNA): Any RNA that codes for at least one protein (naturally occurring, unnaturally occurring, or modified amino acid polymer) and is translated in vitro, in vivo, in situ, or ex vivo to code for a protein. Unless otherwise stated, nucleic acid sequences described herein may enumerate "T"s in representative DNA sequences, however, it will be understood by those skilled in the art that "T"s are replaced with "U"s when such sequences represent RNA (e.g., mRNA). Accordingly, any DNA disclosed and identified by a specific sequence identification number herein also discloses a corresponding RNA (e.g., mRNA) sequence complementary to the DNA, in which each "T" in the DNA sequence is replaced with a "U".

[0074] An open reading frame (ORF) is a continuous sequence of DNA or RNA that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG). ORFs typically encode proteins. Sequences disclosed herein may also include additional elements such as 5′ and 3′ UTRs.

[0075] Mutant RNA: Antigen variants or other polypeptide variants refer to molecules whose amino acid sequence differs from the wild type, natural, or reference sequence. Antigen / polypeptide variants are different from the natural sequence or Compared to the reference sequence, the mutant may have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence. Typically, the mutant has at least 50% identity with the wild type, natural, or reference sequence. In some embodiments, the mutant has at least 80% or at least 90% identity with the wild type, natural, or reference sequence.

[0076] In some embodiments, the composition comprises RNA or RNA ORF and includes a nucleotide sequence of any of the sequences provided herein (see, for example, the sequence listing and Table 1), or includes a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% identity with any of the nucleotide sequences provided herein.

[0077] The term "identity" refers to the relationship between two or more polypeptide (e.g., antigens) or polynucleotide (nucleic acid) sequences determined by comparing their sequences. Identity also refers to the degree of sequence relevance between or within sequences, determined, for example, by the number of matching amino acid residues or nucleic acid residue sequences between two or more sequences. Identity measures the percentage of identity agreement between the smaller of two or more sequences that have gap alignments (if any) processed by a specific mathematical model or computer program (e.g., "algorithm"). The identity of the relevant antigen or nucleic acid can be readily calculated by known methods. When applied to polypeptide or polynucleotide sequences, the "percentage of identity (%)" is defined as the percentage of residues that are identical (amino acid residues or nucleic acid residues) to the residues (amino acid residues or nucleic acid residues) in the second amino acid sequence or nucleic acid sequence of the candidate amino acid or nucleic acid sequence after the sequences have been aligned and gaps (if necessary) have been introduced to achieve the maximum percentage of identity. Methods and computer programs for comparison are well known in the art. While identity depends on the calculation of the percentage of identity, it is understood that the identity value may differ due to the introduction of gaps and penalties in the calculation. Generally, variants of a particular polynucleotide or polypeptide (e.g., an antigen) have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the particular reference polynucleotide or polypeptide, but less than 100%, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Tools for such comparisons include the BLAST suite (Stephen F. Altschul et al. (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402), etc.

[0078] Accordingly, any polynucleotide encoding a peptide or polypeptide having substitutions, insertions, and / or additions or deletions to a reference sequence, particularly an antigen sequence disclosed herein, is included within the scope of this disclosure. Amino acid residues located in the carboxyl and amino-terminal regions of the amino acid sequence of a peptide or protein may be optionally deleted to provide a truncated sequence. Based on the structure and application of the sequence, the gE protein has several important domains related to its main function (Figure 1). The gE protein has several important domains related to its main function (Figure 3). (1) TM transmembrane domain consisting of amino acids at positions 539-559; (2) A568YRV571: mediates gE transport between the endoplasmic reticulum (ER), trans-Golgi network (TGN), and endoplasty; (3) Y582AGL585: mediates gE transport between the endoplasmic reticulum (ER), trans-Golgi network (TGN), and endoplasty; (4) S593ES595T596DT598: important glycosylation sites that mediate gE transport between the endoplasmic reticulum (ER), trans-Golgi network (TGN), and endoplasty. In some embodiments, important structural sites are removed. By removing and substituting appropriate residues, the transport and expression of the antigen can be altered, thereby enhancing the immunogenicity of the antigen. Such sequences can be readily identified by those skilled in the art. It is also understood that some of the sequences provided herein include, for example, sequence tags or terminal peptide sequences (e.g., N-terminus or C-terminus) that may be removed before use in the preparation of RNA (e.g., mRNA) vaccines.

[0079] As will be recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the range of the target varicella-zoster virus antigen. For example, this specification provides any protein fragment of a reference protein (referring to a polypeptide sequence that is at least one amino acid shorter than the reference antigen sequence, but otherwise identical), provided that the fragment is immunogenic and provides a protective immune response against varicella-zoster virus. In addition to identical but shortened variants of the reference protein, in some embodiments the antigen includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more site mutations, as shown in any sequence provided or referenced herein. The length range of the antigen / antigenic polypeptide may be from about 4, 6, or 8 amino acids to full-length proteins.

[0080] VZV Antigen Variants: The present invention comprises mutant VZV antigenic polypeptides. In some embodiments, the mutant VZV antigen polypeptide is a mutant VZV gE polypeptide. The mutant VZV gE polypeptide is designed to avoid retention of the peptide in the ER / Golgi apparatus, resulting in increased surface expression of the antigen. In some embodiments, the mutant gE polypeptide is shortened to remove the ER-retaining portion or the cytoplasmic tail portion of the polypeptide. In some embodiments, the mutant VZV gE polypeptide is mutated to reduce the localization of the VZV polypeptide to the ER / Golgi apparatus / TGN. The modification inhibits ER capture and thus promotes transport to the cell membrane. Therefore, in some embodiments, the VZV glycoprotein is a mutant gE polypeptide. VZV gE has a TGN-targeting sequence at its C-terminus and is transported from the ER to the TGN in infected and gE-transfected cells. Most of the gE in the TGN is thought to be recovered from the plasma membrane via endocytosis, delivered to the TGN from endosomes, and then recycled back into the plasma membrane. gE accumulates in the TGN along with other VZV proteins (e.g., envelope proteins) involved in the formation of fully enveloped VZV virions. Therefore, mutations that reduce TGN localization and endocytosis are favorable for gE transport to the cell membrane, and mutant VZV gE polypeptides may be any truncated polypeptide lacking an anchor domain (ER-retaining domain). For example, mutant VZV gE polypeptides may be truncated VZV gE polypeptides containing at least amino acids 1-124, e.g., amino acids 1-124, 1-140, 1-160, 1-200, 1-250, 1-300, 1-350, 1-360, 1-400, 1-450, 1-500, 1-511, 1-550 and 1-561, and polypeptide fragments having fragment sizes within the cited size ranges. In some embodiments, mutant VZV gE polypeptides are truncated polypeptides lacking a carboxyl-terminal tail domain. Therefore, in some embodiments, the shortened VZV gE polypeptide contains amino acids 1 to 573 of SEQ ID NO: 59.In some embodiments, the mutant VZV gE polypeptide has at least one mutation in one or more motifs related to ER retention, where the mutation in one or more motifs results in reduced VZV gE polypeptide retention in the ER and / or Golgi apparatus. In some embodiments, the mutant VZV gE polypeptide has at least one mutation in one or more phosphorylated acidic motifs. For example, the mutant VZV gE polypeptide may be a full-length VZV gE polypeptide having a Y582G mutation, a Y569K mutation, or both a Y582G mutation and a Y569K mutation. Alternatively, the mutant VZV gE polypeptide may be antigenic, for example, containing amino acids 1-573 of VZV gE and having a Y569K mutation. It may be a fragment. Alternatively, the mutant VZV gE polypeptide may be an antigenic fragment having a mutation in an acid phosphorylation motif (e.g., the SSTT motif). For example, the mutant VZV gE polypeptide may be an antigenic fragment having the AEAADA sequence (SEQ ID NO: 183).

[0081] Naturally occurring eukaryotic mRNA molecules may contain stabilizing elements, including but not limited to the untranslated regions (UTRs) of their 5′ end (5′UTR) and / or 3′ end (3′UTR), in addition to other structural features such as a 5′-cap structure or a 3′-poly(A) tail. Both the 5′UTR and 3′UTR are generally transcribed from genomic DNA and are elements of immature mRNA. Generally, characteristic structural features of mature mRNA (e.g., a 5′-cap and a 3′-poly(A) tail) are added to the transcribed (immature) mRNA during mRNA processing.

[0082] In some embodiments, the composition comprises an open reading frame encoding at least one antigenic polypeptide having at least one modification, an RNA polynucleotide having at least one 5' terminal cap, and is formulated within lipid nanoparticles. 5'-capping of the polynucleotide can be completed simultaneously during the in vitro transcription reaction using a chemical RNA cap analog to generate a 5'-guanosine cap structure, according to the preparation's protocol. In some cases, 5'-capping of the modified RNA can also be completed using a post-transcriptional vaccinia virus capping enzyme to generate a “cap” structure. Cap 1 structure can be generated using a vaccinia virus capping enzyme and 2'-O methyltransferase to produce m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure can be generated on top of Cap 1 structure, subsequently by 2'-O methylating the third-to-last nucleotide of the 5' end using 2'-O methyltransferase. The cap 3 structure can be generated by 2'-O-methylating the fourth nucleotide from the end of the 5' position on top of the cap 2 structure using a 2'-O methyltransferase. The enzyme can be obtained from a recombinant source. The 3'-poly(A) tail is typically a series of adenine nucleotides attached to the 3' end of the transcribed mRNA. In some cases, it may contain up to approximately 400 adenine nucleotides. In some embodiments, the length of the 3'-poly(A) tail may be a factor necessary for the stability of the individual mRNA.

[0083] Chemical Modification: In some embodiments, the compositions of the Disclosure comprise RNA having an open reading frame encoding a VZV antigen, wherein the nucleic acid comprises a nucleotide and / or nucleoside that is standard (unmodified) or may be modified as known in the Art. In some embodiments, the nucleotides and nucleosides of the Disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides may be naturally occurring modified nucleotides and nucleosides or unnaturally occurring modified nucleotides and nucleosides. Such modifications may include modifications to the sugar, backbone, or nucleic acid base portions of nucleotides and / or nucleosides that are well known in the Art.

[0084] In some embodiments, the naturally occurring modified nucleotides or nucleosides of this disclosure are nucleotides or nucleosides that are commonly known or known in the art. A non-limiting list of such naturally occurring modified nucleotides and nucleosides can be found, among other things, in the widely known MODOMICS database.

[0085] In some embodiments, modified nucleotides or other modified nucleotides present in the non-natural context of this disclosure Nucleosides are nucleotides or nucleosides that are commonly known or known in the Art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be seen, in particular, as published U.S. Patent applications PCT / US2012 / 058519, PCT / US2013 / 075177, PCT / US2014 / 058897, PCT / US2014 / 058891, PCT / US2014 / 070413, PCT / US2015 / 36773, PCT / US2015 / 36759, PCT / US2015 / 36771, or PCT / IB2017 / 051367, all of which are incorporated herein by reference.

[0086] Accordingly, the nucleic acids of this disclosure (e.g., DNA nucleic acids and RNA nucleic acids such as mRNA nucleic acids) may include standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, unnaturally occurring nucleotides and nucleosides, or any combination thereof.

[0087] In some embodiments, the nucleic acids of this disclosure (e.g., DNA nucleic acids and RNA nucleic acids such as mRNA nucleic acids) comprise several (one or more) different types of standard and / or modified nucleotides and nucleosides. In some embodiments, a particular region of the nucleic acid comprises one, two, or more (depending on the case) types of standard and / or modified nucleotides and nucleosides.

[0088] In some embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms exhibit reduced degradation within the cell or organism compared to unmodified nucleic acids containing standard nucleotides and nucleosides.

[0089] In some embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms may exhibit reduced immunogenicity in the cells or organisms compared to unmodified nucleic acids, including standard nucleotides and nucleosides.

[0090] In some embodiments, nucleic acids (e.g., RNA nucleic acids such as mRNA nucleic acids) include unnaturally modified nucleotides introduced during or after the synthesis of the nucleic acid to achieve a desired function or property. The modifications may be present in internucleotide bonds, purine or pyrimidine bases, or sugars. The modifications can be introduced chemically or using polymerases at the ends of the chain or at any other position on the chain. Any region of the nucleic acid may be chemically modified.

[0091] This disclosure provides modified nucleosides and nucleotides of nucleic acids (e.g., RNA nucleic acids such as mRNA nucleic acids). “Nucleoside” refers to a compound obtained by combining a sugar molecule (e.g., pentose or ribose) or a derivative thereof with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred herein as “nucleic acid base”). “Nucleotide” refers to a nucleoside containing a phosphate group. Modified nucleotides can be synthesized by any useful method, such as chemical, enzymatic, or recombinant methods, to contain one or more modified or unnatural nucleosides. The nucleic acid may contain one or more regions of linked nucleosides. Such regions may have variable backbone links. The links may be standard phosphodiester links, in which case the nucleic acid contains nucleotide regions.

[0092] Modified nucleotide base pairs include not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between modified nucleotides containing nucleotides and / or non-standard or modified bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors is between non-standard and standard bases, or between two The bases may be positioned between complementary non-standard base structures, for example, hydrogen bonds of nucleic acids having at least one chemical modification. Examples of such non-standard base pairs are the base pairs between inosine and adenine, cytosine, or uracil in modified nucleotides. Any combination of base / sugar or linker may be incorporated into the nucleic acids of this disclosure.

[0093] In some embodiments, the modified nucleic acid bases in nucleic acids (e.g., RNA nucleic acids such as mRNA nucleic acid) include 1-methyl-psuduridine (m1ψ), 1-ethyl-psuduridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or psuduridine (ψ). In some embodiments, the modified nucleic acid bases in nucleic acids (e.g., RNA nucleic acids such as mRNA nucleic acid) include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpsuduridine, 5-methylcytidine, and / or 5-methoxycytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., two, three, four, or more) of any of the above modified nucleic acid bases, including but not limited to chemical modifications.

[0094] In some embodiments, the mRNA of the Disclosure comprises a 1-methyl-psuduridine (m1ψ) substitution at one or more or all uridine positions of the nucleic acid.

[0095] In some embodiments, the mRNA of the Disclosure comprises 1-methyl-psuduridine (m1ψ) substitutions at one or more or all uridine positions of the nucleic acid, and 5-methylcytidine substitutions at one or more or all cytidine positions of the nucleic acid.

[0096] In some embodiments, the mRNA of the Disclosure comprises pseudouridine (ψ) substitutions at one, more, or all uridine positions of the nucleic acid.

[0097] In some embodiments, for specific modifications, mRNA is modified uniformly (e.g., complete modification, modification throughout the entire sequence). For example, a nucleic acid is uniformly modified with 1-methyl-psuduridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-psuduridine. Similarly, nucleic acids can be uniformly modified for any type of nucleoside residue present in the sequence by substitution with modified residues (e.g., the residues described above).

[0098] The nucleic acid contains approximately 1% to approximately 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., one or more of A, G, U, or C), or any percentage in between (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% This may include %~95%, 10%~100%, 20%~25%, 20%~50%, 20%~60%, 20%~70%, 20%~80%, 20%~90%, 20%~95%, 20%~100%, 50%~60%, 50%~70%, 50%~80%, 50%~90%, 50%~95%, 50%~100%, 70%~80%, 70%~90%, 70%~95%, 70%~100%, 80%~90%, 80%~95%, 80%~100%, 90%~95%, 90%~100%, and 95%~100%). It is understood that the presence of unmodified A, G, U, or C accounts for the remaining proportion.

[0099] The mRNA may contain a minimum of 1% and a maximum of 100% modified nucleotides or any percentage in between, for example, at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the nucleic acid may contain modified nucleotides. The nucleic acid may contain modified pyrimidines such as syl or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracil in the nucleic acid is substituted with modified uracil (e.g., 5-substituted uracil). The modified uracil may be substituted with a compound having a single unique structure, or with multiple compounds having different structures (e.g., two, three, four, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosine in the nucleic acid is substituted with modified cytosine (e.g., 5-substituted cytosine). The modified cytosine may be substituted with a compound having a single unique structure, or with multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0100] Untranslated Regions (UTRs): The mRNAs of this disclosure may contain one or more regions or portions that act or function as untranslated regions. If the mRNA is designed to encode at least one target antigen, the nucleic acid may contain one or more of these untranslated regions (UTRs). The wild-type untranslated regions of nucleic acids are transcribed but not translated. In mRNA, the 5' UTR begins at the transcription start site and continues to the start codon but does not contain the start codon, while the 3' UTR begins immediately after the stop codon and continues to the transcription end signal. Based on various evidence, UTRs have been shown to play a role in regulating the stability and translation of nucleic acid molecules. The regulatory properties of UTRs may be incorporated into the polynucleotides of this disclosure, in particular to enhance molecular stability. Specific features may also be incorporated to ensure controlled downregulation of transcripts in case the transcript is misdirected to an undesirable organ site. Multiple 5'UTR and 3'UTR sequences are known and available in the art.

[0101] In some embodiments of this disclosure, the 5'UTR is a heterologous UTR, i.e., a naturally occurring UTR associated with a different ORF. In another embodiment, the 5'UTR is a synthetic UTR, i.e., one that does not exist in nature. Synthetic UTRs include mutated UTRs to improve properties such as those that increase gene expression, and fully synthetic UTRs. Exemplary 5'UTRs include African clawed frog (Xenopus) or human α- or β-globulin (8278063, 9012219), human cytochrome b-245a polypeptide, and hydroxysteroid (17b) dehydrogenase and tobacco etch virus (US8278063, 9012219).

[0102] In some embodiments, the 5'UTR of the present disclosure includes a sequence selected from sequence numbers 173-174.

[0103] In some preferred embodiments, the 5'UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 174.

[0104] The 3′UTR refers to the region immediately downstream (3′) of the stop codon in mRNA (the codon that signals the termination of translation of the mRNA transcript). The 3′UTR is non-coding. Natural or wild-type 3′UTRs are known to contain embedded adenosine (A) and uridine segment (U) fragments. These AU-rich imprints are particularly common in genes with high metabolic turnover rates.

[0105] The introduction, removal, or modification of AU-rich elements (AREs) in the 3'UTR can be used to modulate the stability of nucleic acids (e.g., RNA) of the Disclosure. When manipulating a particular nucleic acid, one or more copies of AREs can be introduced to further reduce the stability of the nucleic acid of the Disclosure, thereby reducing translation and decreasing the production of the resulting protein. Similarly, Identifying, removing, or mutating AREs can increase intracellular stability, thereby increasing the translation and production of the resulting proteins.

[0106] The 3'UTR may be heterologous or synthetic. With respect to 3'UTRs, globulin UTRs (including African clawed frog β-globulin UTR and human β-globulin UTR) are known in the art (8278063, 9012219, US20110086907). By cloning two consecutive human β-globulin 3'UTRs end-to-end, nucleic acids (e.g., mRNA) encoding modified β-globulins with improved stability in several cell types have been developed, and these are well known in the art (US2012 / 0195936, WO2014 / 071963).

[0107] In some embodiments, the 3'UTR of the present disclosure comprises a sequence selected from sequence number 175.

[0108] Those skilled in the art will understand that heterogeneous or synthetic 5'UTRs can be used with any desired 3'UTR sequence. For example, a heterogeneous 5'UTR can be used with a synthetic 3'UTR or a heterogeneous 3'UTR.

[0109] It should be understood that any UTR from any gene can be incorporated into a region of nucleic acid. Furthermore, multiple wild-type UTRs of any known gene are available. Providing artificial UTRs of variants that are not wild-type regions is also within the scope of this disclosure. These UTRs, or parts thereof, may be oriented in the same direction within a selected transcript, or their orientation or position may be altered. Thus, a 5' or 3' UTR can be inverted, shortened, or lengthened and prepared together with one or more other 5' or 3' UTRs. Where referring to a UTR sequence, the term “modified” as used herein means that the UTR has been modified in any way relative to the reference sequence. For example, a 3' or 5' UTR may be modified compared to a wild-type or native UTR by changes in orientation or position as taught above, or by the incorporation of additional nucleotides, deletion of nucleotides, exchange of nucleotides, or transposition. Any one of these modifications that produces a “modified” UTR (either 3' or 5') includes a variant UTR.

[0110] In vitro transcription of RNA: The polynucleotide-encoding cDNAs described herein can be transcribed using an in vitro transcription (IVT) system. In some embodiments, the RNA transcript is produced in an in vitro transcription reaction using a non-amplified linearized DNA template to produce the RNA transcript. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by enzymatic digestion of circular plasmid DNA. In some embodiments, the plasmid DNA template is used to transfect cells such as bacterial cells (e.g., E. coli such as DH-1 cells). In some embodiments, the transfected cells are cultured to replicate the plasmid DNA, and then the plasmid DNA is isolated and purified. In some embodiments, the DNA template includes an RNA polymerase promoter, such as a T7 promoter, located at 5' of the target gene and operably ligated to it.

[0111] In some embodiments, the in vitro transcription template encodes the 5' untranslated (UTR) region, includes an open reading frame, and encodes the 3' UTR and poly(A) tail. The specific nucleic acid sequence composition and length of the in vitro transcription template depend on the mRNA encoded by the template.

[0112] The "5' untranslated region" (UTR) is the region immediately upstream (i.e., 5′) of the start codon of a non-coding mRNA (i.e., the first codon of the mRNA transcript translated by the ribosome). If an RNA transcript is produced, the 5'UTR may contain a promoter sequence. Such promoter sequences are known in the art. It is understood that such promoter sequences are not present in the vaccines of this disclosure.

[0113] The "3' untranslated region" (UTR) refers to the region immediately downstream (i.e., 3') of a non-coding mRNA stop codon (i.e., the codon that signals the termination of translation of the mRNA transcript).

[0114] An "open reading frame" is a continuous DNA segment that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA), and encodes a polypeptide.

[0115] A "poly(A)tail" is a region of mRNA located downstream of the 3'UTR (i.e., 3') containing multiple consecutive adenosine monophosphates. A poly(A)tail may contain 10 to 300 adenosine monophosphates. For example, a poly(A)tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, a poly(A)tail contains 50 to 250 adenosine monophosphates. In related biological environments (e.g., intracellular and in vivo), poly(A)tails protect mRNA from enzymatic degradation in the cytoplasm and play a role in transcription termination and / or transport and translation of mRNA from the nucleus.

[0116] In some embodiments, nucleic acids contain 200 to 3,000 nucleotides. For example, nucleic acids may contain 200 to 500, 200 to 1,000, 200 to 1,500, 200 to 3,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,500 to 3,000, or 2,000 to 3,000 nucleotides.

[0117] In vitro transcription systems typically include transcription buffer, nucleotide triphosphates (NTPs), RNase inhibitors, inorganic pyrophosphates, and polymerase.

[0118] The aforementioned NTP may be prepared in-house, selected from a supplier, or synthesized as described herein. The aforementioned NTP is selected from, but is not limited to, natural and non-natural (modified) NTPs as described herein.

[0119] Any number of RNA polymerases or variants can be used in the methods of the present disclosure. The polymerase may be selected from bacteriophage RNA polymerases such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or mutant polymerase, but is not limited to these, and is, for example, a polymerase capable of incorporating modified nucleic acids and / or modified nucleotides (including chemically modified nucleic acids and / or nucleotides). In some embodiments, the use of DNase is excluded.

[0120] In some embodiments, RNA transcripts can be capped by enzymatic capping or co-transcription capping reactions. In some embodiments, the RNA includes a 5′ end cap, for example, 7mG(5′)ppp(5′)NlmpNp.

[0121] mRNA purification: The nucleic acid purification described herein includes, but is not limited to, nucleic acid purification, quality assurance, and quality control. Purification can be carried out by methods known in the art, including, but not limited to, lithium chloride precipitation, beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probe (Company: Vedbaek, Denmark), or HPLC-based purification methods such as strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). When used with respect to nucleic acids, the term "purified nucleic acid," etc., means nucleic acid isolated from at least one contaminant. "Contaminant" is any substance that makes another substance unsuitable, impure, or inferior. Thus, purified nucleic acids (e.g., DNA and RNA) exist in a different form or environment than those found in nature, or in a different form or environment than those that existed before the processing or purification method was performed.

[0122] Quality assurance and / or quality control checks may be performed using, but are not limited to, methods such as gel electrophoresis, UV absorbance, or analytical HPLC.

[0123] Quantification: In some embodiments, nucleic acids can be quantified using methods such as ultraviolet-visible spectroscopy (UV / Vis). Non-limiting examples of UV / Vis spectrometers include spectrometers (ThermoFisher, Waltham, MA). Quantified nucleic acids can be analyzed to determine whether they are of appropriate size and to confirm that they have not been degraded. Nucleic acid degradation can be confirmed by HPLC-based purification methods, including but not limited to agarose gel electrophoresis, strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), as well as by methods including but not limited to liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE), and capillary gel electrophoresis (CGE).

[0124] Lipid Nanoparticles (LNPs): In some embodiments, the RNA (e.g., mRNA) of the Disclosure is formulated into lipid nanoparticles (LNPs). Lipid nanoparticles typically comprise ionizable cationic lipids, non-cationic lipids, sterol and PEG lipid fractions, and the target nucleic acid substance. The lipid nanoparticles of this disclosure can be produced by components, compositions and methods commonly known in the art, for example, CN116535381B, TW202340136A, CN116178193B, CN116178193B, CN115784921B, CN115745820B, CN115677518B, CN114957027B, CN114044741B, CN116854754A, CN116785265A, CN116789764A, CN116396178A, CN116375592A, CN116082275A, the aforementioned patents are incorporated herein by reference in their entirety.

[0125] As used herein, the term “cationic lipid” refers to a lipid that is positively charged at a selected pH value. Cationic lipids readily bind to negatively charged nucleic acids, i.e., form lipid nanoparticles (LNPs) through electrostatic interactions with negatively charged phosphate groups present in nucleic acids. LNPs are currently one of the dominant transport carriers.

[0126] In this specification, the term "neutral lipid" refers to an auxiliary lipid that is uncharged or exists in an amphoteric form at a selected pH value. Such neutral lipids may improve the efficiency of nanoparticles by promoting lipid phase transitions, thereby regulating their fluidity to a lipid bilayer structure, and may also influence target organ specificity.

[0127] In this specification, “structural lipids” refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids. Structural lipids are selected from, but are not limited to, cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassisterol, tomatine, ursolic acid, α-tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or any combination thereof.

[0128] In some embodiments, the molar ratio of the cationic lipid to the structural lipid is approximately 1:1 to 5:1, for example, approximately 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, and 2.0:1.

[0129] In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is approximately (1 to 10):1, for example, approximately 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0130] In some embodiments, the molar ratio of the cationic lipid to the structural lipid is approximately (1 to 5):1, for example, approximately 1:1, 2:1, 3:1, 4:1, or 5:1.

[0131] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer-conjugated lipid is (25-65):(5-25):(25-45):(0.5-5). For example, in some embodiments, in terms of the ratio (relative molar number), the cationic lipid is 25 parts, 30 parts, 35 parts, 40 parts, 45 parts, 50 parts, 55 parts, 60 parts or 65 parts, the neutral lipid is 5 parts, 10 parts, 15 parts, 20 parts or 25 parts, the structural lipid is 25 parts, 30 parts, 35 parts, 40 parts or 45 parts, and the polymer-conjugated lipid is 0.5 parts, 1 part, 1.5 parts, 2 parts, 2.5 parts, 3.0 parts, 3.5 parts, 4.0 parts, 4.5 parts or 5.0 parts.

[0132] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer-conjugated lipid is (49-50):10:(38.5-39.5):1. For example, in some embodiments, in terms of the ratio (relative molar number), the cationic lipid is 49 parts or 50 parts, the neutral lipid is 10 parts, the structural lipid is 38.5 parts, 39.0 parts or 39.5 parts, and the polymer-conjugated lipid is 1 part. For example, it is 50:10:38.5:1.5 or 49:10:39.5:1.5.

[0133] As used herein, the term "polymer-conjugated lipid" refers to a lipid modified with polyethylene glycol (PEG). The hydrophilic PEG stabilizes the LNP, adjusts the nanoparticle size by restricting lipid fusion, and extends the half-life of the nanoparticle by reducing non-specific interactions with macrophages. In some embodiments, the polymer-conjugated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. The PEG molecular weight of the PEG modification is usually 350-5000 Da. For example, the polymer-conjugated lipid is distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE (PEG2000), 1,2-dimyristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG PEG2000), and methoxypoly(ethylene glycol) ditetradecylacetamide (ALC0159). It is selected from one or more of them.

[0134] In one embodiment of the composition / carrier of the present disclosure, the polymer-conjugated lipid is DMG-PEG2000.

[0135] In one embodiment of the composition / carrier of the present disclosure, the carrier includes a cationic lipid, a neutral lipid, a structural lipid, and a polymer-conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid substance to the polymer-conjugated lipid is (25-65):(5-25):(25-45):(60.5-5), for example, (45-55):(9-11):(34-43):(0.5-2.5).

[0136] In one embodiment of the composition / carrier of the present disclosure, the carrier includes a cationic lipid, a neutral lipid, a structural lipid, and a polymer-conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid substance to the polymer-conjugated lipid is 50:10:38.5:1.5.

[0137] As used herein, the term "alkyl" means including branched and straight-chain saturated aliphatic monovalent hydrocarbon groups having a specific number of carbon atoms. In the present disclosure, the term "alkylene" means including branched and straight-chain saturated aliphatic divalent hydrocarbon groups having a specific number of carbon atoms. C n~m refers to a group having n to m carbon atoms. For example, C 2~5 alkylene includes C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene. C 2~8 alkylene includes C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, C6 alkylene, C7 alkylene, C8 alkylene. C 1~6Alkylenes include C1 alkylenes, C2 alkylenes, C3 alkylenes, C4 alkylenes, C5 alkylenes, and C6 alkylenes. 1~3 Alkylenes include C1 alkylenes, C2 alkylenes, and C3 alkylenes. 6~15 Linear alkyls include linear alkyls having 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. 12~25 Branched-chain alkyls include branched-chain alkyls having 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms. The alkyl (or alkylene) may be unsubstituted, or it may be substituted, where at least one hydrogen is replaced by another chemical group.

[0138] In this specification, “therapeutic dose” is the amount of a therapeutic agent that, when administered to a patient, can improve a disease or symptom. “Preventive dose” is the amount of a preventive agent that, when administered to a subject, can prevent a disease or symptom. The amount of therapeutic agent constituting the “therapeutic dose” or the amount of preventive agent constituting the “preventive dose” varies depending on the therapeutic / preventive agent, the state and severity of the disease, the age and weight of the patient / subject being treated / prevented, etc. The therapeutic dose and the preventive dose can be typically determined by a person skilled in the art based on their knowledge and this disclosure.

[0139] In this specification, the composition can be used to induce a protective immune response against VZV in a subject requiring it, the protective immune response including, for example, the production of neutralizing antibodies. In some embodiments, the subject is immunocompromised. The subject is 10 years of age or older, for example, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 years of age.

[0140] The vaccines of this disclosure are typically formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticles contain at least one ionizable cationic lipid, at least Each comprises one noncationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG)-modified lipid.

[0141] In some preferred embodiments, the lipid nanoparticles include cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids.

[0142] In some preferred embodiments, the cationic lipid is a compound having the structure of formula I, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G1 is C 1~6 It is alkylene, and G2 is C 2~8 It is alkylene, and G3 is C 1~3 It is alkylene, and L1 is C 6~15 It is a linear alkyl group, and L2 is C 12~25 It is a branched alkyl group. For example, YK-009 with the structure of formula II (see patent CN114044741B).

[0143] [ka]

[0144] In some preferred embodiments, the cationic lipid is a compound having the structure of formula II, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G1 is C 2~8 It is alkylene, and G2 is C 2~8 It is an alkylene, L1 is -C(O)O- or -OC(O)-, L2 is -C(O)O- or -OC(O)-, and R1 is C 6~25 It is a linear or branched alkyl group, and R2 is C 6~25The alkyl group is linear or branched, where G3 is HO(CH2)2- or HO(CH2)3-, G4 is HO(CH2)2- or HO(CH2)3-, and L is (CH2)2- or -(CH2)3- or -(CH2)4-. For example, YK-401 has a II-I structure, and YK-402 has a II-II structure (see patent CN115784921B).

[0145] [ka]

[0146] In some preferred embodiments, the cationic lipid is a compound having the structure of formula III, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G1 is C 2~8 It is alkylene, and G2 is C 2~8 It is alkylene, and R1 is C 6~20 It is a linear or branched alkyl group, and R2 is C 12~25 It is a linear alkyl group, where G3 is HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2)-, (HO(CH2)2)2N(CH2)2)-, CH3O(CH2)2N(CH3)(CH2)2), (CH3)2N(CH2)3SC(O)O(CH2)2)-, (CH3)2N(CH2)3SC(O))-, CH3NH(CH2)2N(CH3)(CH2)2) or CH3CH2NH(CH2)2-. For example, YK-201 has the structure of formula III-I, and YK-202 has the structure of formula III-II (see patent CN115677518B).

[0147] [ka]

[0148] In some preferred embodiments, the cationic lipid is a compound having the structure of formula IV, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G1 is C 1~8 It is alkylene, and G2 is C 2~8is an alkylene, and R1 is C 6~20 is a linear or branched alkyl, and R2 is C 12~25 is a linear or branched alkyl, G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3 or -CH2CH3 or -CH2CH2OH. For example, YK-305 with the structure of formula IV-I and YK-310 with the structure of formula IV-II (see Patent CN115745820B).

[0149] [Chemical formula]

[0150] In some preferred embodiments, the cationic lipid is a compound having the structure of formula V, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, where G 1 and G 2 are each independently an unsubstituted C6-C 10 alkylene, G 3 is an unsubstituted C1-C 12 alkylene, R 1 and R 2 are each independently C6-C 24 alkyl or C6-C 24 alkenyl, R 3 is OR 5 , N, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 where R 4 is C1-C<U+ 12 hydrocarbyl, and R 5 is H or C1-C6 hydrocarbyl. For example, ALC0315 with the structure of formula V-I (see Patent CN108368028B).

[0151] [Chemical formula]

[0152] In some preferred embodiments, the cationic lipid is a compound having the structure of formula VI, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where R4 is -(-(CH2) n Q and -(CH2) n Selected from CHQR, where Q is -OR, -OH, -O(CH2) n Selected from the group consisting of N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R8 and heterorings, where n is 1, 2, or 3, for example, SM102 of the formula VI-I structure (see patent CN110520409A).

[0153] [ka]

[0154] In some preferred embodiments, the cationic lipid is a compound of the formula VII structure, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer.

[0155] [ka]

[0156] In some more preferred embodiments, the cationic lipids include YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

[0157] In some preferred embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:(1~10):1.

[0158] In some preferred embodiments, the molar ratio of the cationic lipid to the structural lipid is (1-5):1.

[0159] In some preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer-conjugated lipid is (25-75):(5-25):(15-65):(0.5-10).

[0160] In some preferred embodiments, the cationic lipid, the neutral lipid, and the structure The molar ratio of lipids to polymer-conjugated lipids is (35-49):(7.5-15):(35-55):(1-5), for example, 49:10:43.5:1.5.

[0161] In some preferred embodiments, the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0162] In some preferred embodiments, the neutral lipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl phospha Selected from one or more of the following: tidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0163] In some more preferred embodiments, the neutral lipid is DOPE and / or DSPC.

[0164] In some more preferred embodiments, the structural lipid is selected from one or more of cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0165] In some more preferred embodiments, the structural lipid is cholesterol.

[0166] In some more preferred embodiments, the polymer-conjugated lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PE Selected from one or more of the following: G-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0167] In some more preferred embodiments, the polymer-conjugated lipid is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimiristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG-PEG2000), and methoxypoly(ethylene glycol) ditetradecylacetamide (ALC-0159).

[0168] Pharmaceutical Formulations: This specification provides compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention or treatment of varicella-zoster virus in humans and other mammals. The compositions provided herein can be used as therapeutic or prophylactic agents. They can be used in pharmaceuticals for the prevention and / or treatment of herpes zoster.

[0169] In some embodiments, the herpes zoster vaccine comprising the RNA described herein can be administered to a subject (e.g., a mammalian subject, e.g., a human subject), and the RNA polynucleotide is translated in vivo to produce an antigenic polypeptide (antigen).

[0170] The “effective amount” of a composition (e.g., containing RNA) is at least partially based on the target tissue, target cell type, method of administration, physical characteristics of the RNA (e.g., length, nucleotide composition and / or degree of modified nucleosides), other components of the vaccine, and other determinants such as the age, weight, height, sex and general health status of the subject. Typically, an effective amount of a composition provides induction or enhancement of an immune response, which is modified by the production of antigens in the target cells. In some embodiments, an effective amount of a composition containing an RNA polynucleotide having at least one chemical modification is more effective than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen. Increased antigen production is demonstrated by increased cell transfection (the percentage of cells transfected with the RNA vaccine), increased protein translation and / or expression from the polynucleotide, decreased nucleolysis (e.g., increased duration of protein translation from modified polynucleotides), or altered antigen-specific immune responses in host cells.

[0171] The term "pharmaceutical composition" refers to a combination of an active agent and an inactive or active carrier, such that the composition is particularly suitable for in vivo or ex vivo diagnostic or therapeutic use. A "pharmaceutically acceptable carrier" does not cause undesirable physiological effects after or when administered to a subject. The carrier of a pharmaceutical composition must be "acceptable" in the sense that it is compatible with the active ingredient and can stabilize the active ingredient. One or more solubilizers may be used as a pharmaceutically acceptable carrier for delivering the active agent.

[0172] In some embodiments, the compositions according to this disclosure (including polynucleotides and polypeptides encoded thereby) can be used to treat or prevent herpes zoster. The compositions may be administered prophylactically or therapeutically to healthy individuals as part of an active immunization regimen, or pre-administered during the incubation period or the early stages of infection during the active infection period after symptom onset. In some embodiments, the amount of RNA provided to cells, tissues, or subjects may be an amount effective for immunoprevention.

[0173] In some embodiments, the composition may be administered by intramuscular injection.

[0174] The composition depends on the extent or level of the spread of infectious diseases or unmet medical needs. They may be used in a variety of environments. As a non-limiting example, RNA vaccines may be used to treat and / or prevent a variety of infectious diseases. Such RNA vaccines have superior properties because they produce much higher antibody titers than commercially available vaccines, produce better neutralizing immunity, produce a more durable immune response, and / or produce a faster response.

[0175] This specification provides pharmaceutical compositions comprising RNA and / or complexes, combined with one or more pharmaceutically acceptable excipients of any choice. In addition to conventional excipients (e.g., any solvent, dispersion medium, diluent or other liquid medium, dispersing or suspension aid, surfactant, isotonic agent, thickener or emulsifier, preservative, etc.), excipients also include, but are not limited to, lipoids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, RNA-transfected cells (e.g., for transplantation into a subject), hyaluronidases, nanoparticle mimics, and combinations thereof.

[0176] The RNA may be prepared or administered alone or in combination with one or more other components. For example, the immunocomposition may include, but is not limited to, an adjuvant, or other components.

[0177] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical compositions of this disclosure will further vary depending on the identity, body type, and / or condition of the subject being treated, and the route through which the composition is administered. For example, the composition may contain between 0.1% and 100%, for example, between 0.5% and 50%, between 1% and 30%, between 5% and 80%, or at least 80% (w / w) of the active ingredient.

[0178] In some embodiments, RNA is prepared using one or more excipients to confer functions such as increasing mRNA stability, increasing cell transfection and protein translation efficiency, and altering the in vivo distribution of mRNA to target specific tissues or cell types.

[0179] Administration / Dosage: This specification provides immunocompositions (e.g., RNA vaccines), methods, kits, and reagents for preventing varicella-zoster virus infection in humans and other mammals. The immunocompositions may be used as therapeutic or prophylactic agents. In some embodiments, the immunocompositions are used to provide prophylactic protection against herpes zoster.

[0180] The subjects may be any mammal, including non-human primates and human subjects.

[0181] In some embodiments, an immune composition (e.g., an mRNA vaccine) is administered to a subject (e.g., a mammalian subject, e.g., a human subject) in an amount effective to induce an antigen-specific immune response. The RNA encoding the VZV gE antigen is expressed and translated in vivo to produce the antigen, which then stimulates the subject's immune response.

[0182] Prophylactic protection against herpes zoster can be achieved after administration of the immunological composition of this disclosure (e.g., mRNA vaccine). The immunological composition may be administered once, twice, three times, four times, or more times, although one dose of the vaccine may be sufficient (in some cases followed by a booster immunization).

[0183] In part with respect to this disclosure, a method is provided for inducing an immune response in a subject to varicella-zoster virus antigen (or multiple antigens). In some embodiments, the method involves administering an immune composition to a subject containing RNA (e.g., mRNA) having an open reading frame encoding the VZV gE glycoprotein, thereby inducing an immune response in the subject to varicella-zoster virus antigen. This involves inducing an immune response specific to the herpes zoster virus antigen response, resulting in increased anti-antigen antibody titers in subjects after vaccination compared to those vaccinated with conventional vaccines (e.g., GSK's recombinant subunit vaccine). "Anti-antigen antibody" refers to a serum antibody specifically bound to an antigen.

[0184] The prophylactic effective dose is the effective dose for preventing viral infection at a clinically acceptable level. In some embodiments, the effective dose is the dosage stated in the vaccine's instructions or accompanying leaflet. As used herein, conventional vaccines refer to vaccines other than the mRNA vaccines of this disclosure. For example, conventional vaccines include, but are not limited to, live microbial vaccines, dead microbial vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, and virus-like particle (VLP) vaccines. In exemplary embodiments, conventional vaccines are vaccines that are approved by a regulatory authority and / or registered with a national drug regulatory agency (e.g., the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA)).

[0185] In other embodiments of this disclosure, a method is provided for inducing an immune response to varicella-zoster virus in a subject. The method comprises administering an immune composition (e.g., an RNA vaccine) comprising an RNA polynucleotide including an open reading frame encoding the VZV gE antigen to a subject, thereby inducing an immune response specific to varicella-zoster virus in the subject.

[0186] In several other embodiments, the immune response is assessed by measuring proteins (antibody titers) in the subject. In other embodiments, serum or antibodies from the immunized subject are tested for their ability to neutralize viral uptake or to mitigate the transformation of human B lymphocytes by varicella-zoster virus. In several other embodiments, the ability to promote a robust T-cell response is measured using techniques well known in the art.

[0187] Furthermore, this specification provides a method for inducing an immune response to varicella-zoster virus in a subject by administering RNA having an open reading frame encoding a first antigen, wherein the RNA does not contain stabilizing elements, and the adjuvant is not prescribed or administered concurrently with the vaccine.

[0188] An immune composition (e.g., an RNA vaccine) may be administered by any route that produces a therapeutically effective outcome. These routes include, but are not limited to, intradermal, intramuscular, or subcutaneous administration. This disclosure provides a method for administering an RNA vaccine to a subject in need of it. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the specific composition, the method of administration, and the method of activity. RNA is usually formulated in unit dosage forms to facilitate administration and ensure uniformity of dose. However, it should be understood that the total daily dose of RNA may be determined by the attending physician within the bounds of reasonable medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for a particular patient will depend on a variety of factors, including the disease being treated and its severity, the activity of the specific compound used, the specific composition used, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the rate of excretion of the specific compound used, the duration of treatment, the composition of the specific compound used or drugs used concurrently, and similar factors well known in the medical field.

[0189] The effective dose of RNA provided herein may be as low as approximately 5 μg, for example, as a single dose or as two 2.5 μg doses. In some embodiments, the effective dose is a total dose of 5 μg to 200 μg. For example, effective doses may be 5 μg, 10 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 6 The total dose may be 5 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, or 200 μg. In some embodiments, the effective dose is a total dose of 5 μg to 200 μg. In some embodiments, the effective dose is a total dose of 10 μg. In some embodiments, the effective dose is a total dose of 20 μg. In some embodiments, the effective dose is a total dose of 75 μg. In some embodiments, the effective dose is a total dose of 150 μg. In some embodiments, the effective dose is a total dose of 200 μg.

[0190] Vaccine effectiveness Some aspects of this disclosure provide formulations of immune compositions (e.g., RNA vaccines) in which RNA is formulated in an effective amount to produce an antigen-specific immune response (e.g., producing antibodies specific to the varicella-zoster virus antigen) in a subject. “Effective amount” is the dose of RNA effective in producing an antigen-specific immune response. This specification further provides methods for inducing an antigen-specific immune response in a subject.

[0191] As used herein, an immune response to a vaccine or LNP of the Disclosure is the generation of a humoral and / or cellular immune response in a subject to one or more varicella-zoster virus proteins present in the vaccine. For the purposes of the Disclosure, “humoral” immune response refers to an immune response mediated by antibody molecules (e.g., including secretory (IgA) or IgG molecules), while “cellular” immune response refers to an immune response mediated by T lymphocytes (e.g., CD4 + Helper cells and / or CD8 +Cellular immunity refers to immune responses mediated by T cells (e.g., CTLs) and / or other leukocytes. A key aspect of cellular immunity is antigen-specific responses induced by cytolytic T cells (CTLs). CTLs are specific to peptide antigens presented in relation to proteins encoded by major histocompatibility complexes (MHC) and expressed on the cell surface. CTLs help induce and promote the destruction of intracellular microorganisms or the lysis of cells infected with such microorganisms. Another aspect of cellular immunity is antigen-specific responses induced by helper T cells. Helper T cells are used to stimulate function and concentrate the activity of nonspecific effector cells on cells that bind to MHC molecules and present peptide antigens on their surface. Cellular immune responses evoke the production of cytokines (e.g., IFN-γ, IL-2, TNF-β), chemokines, and other such molecules produced by activated T cells and / or other leukocytes.

[0192] In some embodiments, the antigen-specific immune response is characterized by measuring the antibody titer against the varicella-zoster virus antigen produced in a subject administered with an immunocomposition provided herein. The antibody titer is the result of measuring the amount of antibody in the subject (e.g., an antibody specific to a particular antigen (e.g., anti-VZV gE glycoprotein) or an antibody specific to the epitope of the antigen). The antibody titer is usually expressed as the reciprocal of the maximum dilution that yields a positive result. For example, enzyme-linked immunosorbent assay (ELISA) is a common measurement used to measure antibody titers.

[0193] In some embodiments, antibody titers are used to determine whether a subject is infected or whether immunity is needed. In some embodiments, antibody titers are used to determine the strength of an autoimmune response, determine the need for booster immunization, determine whether a previous vaccine was effective, and identify recent or previous infections. According to this disclosure, antibody titers may be used to determine the strength of an immune response induced in a subject by an immune composition (e.g., an RNA vaccine).

[0194] In some embodiments, the titer of the anti-VZV gE antigen antibody produced in the subject is against Compared to a control (an unvaccinated subject), the titer increases by at least 1 log. For example, the anti-VZV gE antigen antibody titer produced in a subject may increase by at least 1.5 log, at least 2 log, at least 2.5 log, at least 3 log, at least 3.5 log, or at least 4 log compared to a control. In some embodiments, the anti-VZV gE antigen antibody titer produced in a subject increases by 1, 1.5, 2, 2.5, or 3 log compared to a control. In some embodiments, the anti-VZV gE antigen antibody titer produced in a subject increases by 1 to 4 log compared to a control. For example, the anti-VZV gE antigen antibody titer produced in a subject may increase by 1 to 1.5, 1 to 2, 1 to 2.5, 1 to 3, 1.5 to 2, 1.5 to 2.5, 1.5 to 3, 2 to 2.5, 2 to 3, or 2.5 to 4 log compared to a control.

[0195] In some embodiments, the anti-VZV gE antigen antibody titer produced in the subject is increased by at least twofold compared to the control (vaccinated with the conventional vaccine GSK Shingrix®). For example, the anti-VZV gE antigen antibody titer produced in the subject may be increased by at least one, at least 1.5, at least two, at least 2.5, or at least three times compared to the control GSK.

[0196] In some embodiments, the control is the antibody titer of anti-varicella-zoster virus antigen produced in subjects not administered with an immune composition (e.g., an RNA vaccine). In some embodiments, the control is the antibody titer of anti-varicella-zoster virus antigen produced in subjects administered with the recombinant subunit vaccine Shingrix®.

[0197] In some embodiments, the efficacy of an immune composition (e.g., an RNA vaccine) is measured in a mouse model. For example, the immune composition can be administered to a mouse model, and the induction of neutralizing antibody titers in the mouse model can be determined. Viral attack studies may also be used to evaluate the efficacy of the vaccines of this disclosure. For example, the immune composition can be administered to a mouse model, and the mouse model can be attacked with a virus to measure the survival and / or immune response (e.g., T cell response (e.g., cytokine response)) of the mouse model.

[0198] The present invention further relates to the following embodiments.

[0199] 1. Contains ribonucleic acid (RNA) of varicella-zoster virus (VZV), wherein the RNA encodes wild-type VZV gE glycoprotein or a variant thereof. Here, the sequence of the wild-type VZV gE glycoprotein is shown in SEQ ID NO: 3, and this is an immunogenic composition.

[0200] 2. The composition according to Embodiment 1, wherein the VZV gE glycoprotein mutant comprises one or more mutations among sequence shortening, site mutation, and sequence deletion compared to the wild-type VZV gE glycoprotein.

[0201] 3. The composition according to Embodiment 1 or 2, wherein the sequence shortening includes the absence of a carboxyl-terminal tail domain.

[0202] 4. The sequence shortenings described above belong to the following groups: 1) Shortening of the amino acids from position 540 to 623 from the C-terminus of the gE protein, i.e., retention of the amino acids from position 1 to 539 of the VZVgE protein. 2) Shortening of the amino acids from position 569 to 623 from the C-terminus of the gE protein, i.e., retention of the amino acids from position 1 to 568 of the VZVgE protein. 3) Shortening of the amino acids from position 574 to 623 from the C-terminus of the gE protein, i.e., retention of the amino acids from position 1 to 573 of the VZVgE protein. 4) Shortening of the amino acids from position 588 to 623 from the C-terminus of the gE protein, i.e., retention of the amino acids from position 1 to 587 of the VZVgE protein. 5) Shortening of the amino acids at positions 602-623 from the C-terminus of the gE protein, i.e., retention of the amino acids at positions 1-601 of the VZVgE protein. A composition according to any one of embodiments 1 to 3, selected from any one of the following.

[0203] 5. The VZV gE glycoprotein mutant comprises a site mutation selected from mutations in motifs related to ER retention, endocytosis, and / or localization of the gE protein to at least one motif at at least one site, and mutations in at least one site in the gE C-terminal phosphorylated acid motif. Here, the motif related to ER retention and endocytosis of the gE protein is "Y 582 A 583 G 584 L 585 "Motif (for example, motif Y 582 A 583 G 584 L 585 When a mutation occurs, it can interfere with the endocytosis of the gE protein and reduce the localization of the gE antigen to the trans-Golgi network, for example, including the mutation site Y582A (YK-VZV-013 (SEQ ID NO: 43)). The motifs related to the localization of gE proteins to the Golgi apparatus or trans-Golgi network are "A 568 Y 569 R 570 V 571 " motif (for example, "A 568 Y 569 R 570 V 571 The motif is, and the specific mutant antigen sequence includes, for example, YK-VZV-011 (coordination number 35). The C-terminal oxidative acid motif of the aforementioned gE is "S 593 E 594 S595 T 596 D 597 T 598 " motif (for example, "S 593 E 595 T 596 D 597 T 598 The motif is "A 593 E 594 A 596 D 597 A 598 The composition according to any one of Embodiments 2 to 4, wherein a mutation in the motif can reduce the localization of the gE antigen to the Lance Golgi network, and a specific mutant antigen sequence is, for example, YK-VZV-010 (SEQ ID NO: 31).

[0204] 6. The composition according to any one of Embodiments 2 to 5, wherein the VZV gE glycoprotein mutant comprises a site mutation selected from A568D, Y569K, Y569A, R570E, V571K, Y582A, S593A, S595A, T596A and T598A, for example, selected from A568D, Y569K, R570E and V571K, for example, selected from A568D and Y569K.

[0205] 7. The VZV gE glycoprotein mutant is 1)A568D, Y569K, R570E, V571K, (YK-VZV-023, YK-VZV-011) 2)A568D, Y569A, R570E, V571K, (YK-VZV-024) 3)Y569K, R570E, V571K, (YK-VZV-025) 4)A568D, Y569K, R570E, V571K, Y582A, (YK-VZV-030, YK-VZV-013) 5)A568D, Y569K, R570E, V571K, Y582G, (YK-VZV-031, YK-VZV-038) 6)A568D, Y582A, (YK-VZV-032) 7)Y569K, Y582A, (YK-VZV-033, YK-VZV-009) 8)R570E, Y582A, (YK-VZV-034) 9)V571K, Y582A, (YK-VZV-035) 10)S593A, S595A, T596A, T598A, (YK-VZV-016, YK-VZV-044) 11)Y582A, S593A, S595A, T596A, T598A, (YK-VZV-037) 12)A568D, Y569K, R570E, V571K, Y582G, S593A, S595A, T596A, T598A, (YK-VZV-039) 13)A568D, Y569K, R570E, V571K, Y582A, S593A, S595A, T596A, T598A, (YK-VZV-014) 14)Y582G, S593A, S595A, T596A, T598A, (YK-VZV-040) 15)Y569K, Y582A, S593A, S595A, T596A, T598A, (YK-VZV-010) 16)A568D, Y569K, R570E, V571K, S593A, S595A, T596A, T598A, (YK-VZV-012) 17) Y582G, (YK-VZV-029) 18) A568D, (YK-VZV-018) 19) Y569K, (YK-VZV-020) 20) R570E, (YK-VZV-021) 21) V571K, and (YK-VZV-022) 22)Y582A, (YK-VZV-028, YK-VZV-036) A composition according to any one of embodiments 2 to 6, comprising site mutations selected from.

[0206] 8. The VZV gE glycoprotein mutant is 1)A568D, Y569K, R570E, V571K, (YK-VZV-011) 2)A568D, Y569A, R570E, V571K, (YK-VZV-024) 4)A568D, Y569K, R570E, V571K, Y582A, (YK-VZV-030, YK-VZV-013) 5)A568D, Y569K, R570E, V571K, Y582G, YK-VZV-031, YK-VZV-038) 7)Y569K, Y582A, (YK-VZV-009) 13)A568D, Y569K, R570E, V571K, Y582A, S593A, S595A, T596A, T598A, (YK-VZV-014) 15)Y569K, Y582A, S593A, S595A, T596A, T598A, (YK-VZV-010) 16)A568D, Y569K, R570E, V571K, S593A, S595A, T596A, T598A, (YK-VZV-012) 18) A568D, (YK-VZV-018) 19) Y569K, (YK-VZV-020) 20) R570E, (YK-VZV-021) and 22) Y582A (YK-VZV-028) A composition according to any one of Embodiments 2 to 7, comprising a site mutation selected from.

[0207] 9. The VZV gE glycoprotein mutant is 1) A 568 , 2) Y 569 RVDKSPYNQS 579 , and 3) Y 569 RVDKSPYNQSMYYAGLPV 587 A composition according to any one of embodiments 2 to 8, comprising a sequence deletion selected from.

[0208] 10. The VZV gE glycoprotein variant comprises a combination of site mutations, sequence shortenings, and sequence deletions selected from the following, as described in any one of Embodiments 1 to 9.

[0209] [Table 1] TIFF0007912117000009.tif106169

[0210] 11. The VZV gE glycoprotein variant comprises a combination of site mutations, sequence shortenings and / or sequence deletions selected from the following, as described in any one of Embodiments 1 to 10.

[0211] [Table 2]

[0212] 12. The VZV gE glycoprotein mutants are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the sequence shown in SEQ ID NOs: 27, 31, 35, 39, 43, 47, 63, 71, 75, 87, 103, 111, 115, or 143. A composition according to any one of Embodiments 1 to 11, comprising an array having identity of %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0213] 13. The composition according to any one of Embodiments 1 to 12, wherein the VZV gE glycoprotein variant includes SEQ ID NOs: 31, 35, 43, 63, or 71.

[0214] 14. The composition according to any one of Embodiments 1 to 13, wherein the RNA of VZV has an open reading frame (ORF) encoding VZV gE glycoprotein or a variant thereof, and the sequence of the open reading frame has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 70, 74, 86, 102, 110, 114 or 142 identity.

[0215] 15. The arrangement of the open reading frames is the composition according to Embodiment 14, as shown in Sequence ID No. 30, 34, 42, 62, or 70.

[0216] 16. The composition according to any one of Embodiments 1 to 15, wherein the VZV RNA further comprises a 5' untranslated region (UTR).

[0217] 17. The composition according to Embodiment 16, wherein the 5'UTR has the sequence shown in SEQ ID NOs: 173, 174, 175, 176, or 177.

[0218] 18. The composition according to Embodiment 17, wherein the 5'UTR has the sequence shown in Sequence ID No. 174.

[0219] 19. The composition according to any one of Embodiments 1 to 18, wherein the RNA of the VZV further comprises a 3' untranslated region (UTR).

[0220] 20. The composition according to Embodiment 19, wherein the 3'UTR has the sequence shown in SEQ ID NOs: 178, 179, 180, or 181.

[0221] 21. The composition according to Embodiment 20, wherein the 3'UTR has the sequence shown in Sequence ID No. 178.

[0222] 22. The composition according to any one of Embodiments 1 to 21, wherein the VZV RNA further comprises a poly(A) tail.

[0223] 23. The composition according to Embodiment 22, wherein the poly(A) tail has a length of 50 to 150 nucleotides.

[0224] 24. The composition according to any one of Embodiments 1 to 23, wherein the VZV RNA further comprises a 5' terminal cap.

[0225] 25. The composition according to Embodiment 24, wherein the 5' end cap is 7mG(5′)ppp(5′)NlmpNp.

[0226] 26. The composition according to any one of embodiments 14 to 25, wherein the sequence of the open reading frame is codon-optimized.

[0227] 27. The composition according to Embodiment 26, wherein the sequence of the open reading frame comprises at least one base modification.

[0228] 28. The composition according to Embodiment 27, wherein the base modification is selected from one or more of pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0229] 29. The composition according to Embodiment 28, wherein the base modification comprises replacing uracil with pseudouridine and / or N1-methylpseudridine.

[0230] 30. The composition according to Embodiment 27, 28, or 29, wherein the base modification is a base modification of 1 to 100%, for example, 1% base modification, 2% base modification, 3% base modification, 4% base modification, 5% base modification, 6% base modification, 7% base modification, 8% base modification, 9% base modification, 10% base modification, 15% base modification, 20% base modification, 25% base modification, 30% base modification, 35% base modification, 40% base modification, 45% base modification, 50% base modification, 55% base modification, 60% base modification, 65% base modification, 70% base modification, 75% base modification, 80% base modification, 85% base modification, 90% base modification, 95% base modification, and 100% base modification, and any of the above values ​​as the endpoint.

[0231] 31. The composition according to any one of Embodiments 1 to 30, wherein the RNA sequence of VZV has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 104, 112, 116, or 144 identity.

[0232] 32. The composition according to Embodiment 31, wherein the RNA sequence of the VZV has the sequence shown in SEQ ID NOs. 32, 36, 44, 64, or 72.

[0233] 33. The composition according to any one of Embodiments 1 to 32, wherein the RNA of VZV is mRNA.

[0234] 34. The composition according to any one of Embodiments 1 to 33, wherein the RNA encoding the VZV gE glycoprotein comprises an RNA sequence corresponding to a DNA sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 61, 69, 73, 85, 101, 109, 113, or 141 identity with a DNA sequence of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%61%, 73%, 85%, 101, 109, 113, or 141.

[0235] 35. The composition according to Embodiment 34, wherein the RNA encoding the VZV gE glycoprotein comprises an RNA sequence corresponding to the DNA sequence shown in SEQ ID NOs. 29, 33, 41, 61, or 69.

[0236] 36. A step of providing a template that can be transcribed into RNA of the VZV, The step of transcribing using the template under conditions suitable for transcription into RNA. A method for preparing the composition according to any one of Embodiments 1 to 35, including the following.

[0237] 37. The method according to Embodiment 36, further comprising a purification step selected from lithium chloride precipitation, affinity chromatography, solution exchange by ultrafiltration, and cellulose chromatography.

[0238] 38. A vaccine, comprising a pharmaceutically acceptable carrier, according to any one of Embodiments 1 to 35.

[0239] 39. The composition according to Embodiment 38, wherein the carrier comprises a lipid mixture, the lipid mixture being, for example, lipid nanoparticles (LNPs).

[0240] 40. The composition according to embodiment 38 or 39, wherein the vaccine is an mRNA vaccine.

[0241] 41. The composition according to Embodiment 39 or 40, wherein the lipid mixture is lipid nanoparticles (LNPs), and the lipid nanoparticles include, for example, cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids.

[0242] 42. The cationic lipid is a compound having the structure of formula I, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G1 is C 1~6 It is alkylene, and G2 is C 2~8 It is alkylene, and G3 is C 1~3It is alkylene, and L1 is C 6~15 It is a linear alkyl group, and L2 is C 12~25 The composition according to Embodiment 41, wherein the branched alkyl group is, for example, YK-009 having a structure of formula II.

[0243] [ka]

[0244] 43. The cationic lipid is a compound having the structure of formula II, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G1 is C 2~8 Al It is Kiren, and G2 is C 2~8 It is an alkylene, L1 is -C(O)O- or -OC(O)-, L2 is -C(O)O- or -OC(O)-, and R1 is C 6~25 It is a linear or branched alkyl group, and R2 is C 6~25 The composition according to Embodiment 41, wherein the linear or branched alkyl group is linear, G3 is HO(CH2)2- or HO(CH2)3-, G4 is HO(CH2)2- or HO(CH2)3-, L is (CH2)2- or -(CH2)3- or -(CH2)4-, and for example, the cationic lipid is YK-401 with a formula II-I structure or YK-402 with a formula II-II structure.

[0245] [ka]

[0246] 44. The cationic lipid is a compound having the structure of formula III, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G1 is C 1~6 It is alkylene, and G2 is C 2~8 It is an alkylene, and R1 is C 6~20 It is a linear or branched alkyl group, and R2 is C 12~25The composition according to Embodiment 41, wherein G3 is a branched alkyl group, and G3 is HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2-, or CH3CH2NH(CH2)2-, for example, YK-201 with the formula III-I structure, or YK-202 with the formula III-II structure.

[0247] [ka]

[0248] 45. The cationic lipid is a compound having the structure of formula IV, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G1 is C 1~8 It is alkylene, and G2 is C 2~8 It is an alkylene, and R1 is C 6~25 It is a linear or branched alkyl group, and R2 is C 12~25 The composition according to Embodiment 41, wherein the alkyl group is linear or branched, and G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3, -CH2CH3, or -CH2CH2OH, for example, YK-305 with a structure of formula IV-I, or YK-310 with a structure of formula IV-II.

[0249] [ka]

[0250] 46. ​​The cationic lipid is a compound having the structure of formula V, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where G 1 and G 2 Each of these is an independent, unsubstituted C6~C 10 It is alkylene, G 3 This is unsubstituted C1~C 12It is alkylene, R 1 and R 2 Each is independently C6~C 24 Alkyl or C6-C 24 It is an alkenyl, R 3 is OR 5 , N, -C(=O)OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 And R 4 C1~C 12 It is hydrocarbil, and also R 5 The composition according to Embodiment 41, wherein is H or C1-C6 hydrocarbyl, for example, ALC0315 of the structure of formula VI.

[0251] [ka]

[0252] 47. The cationic lipid is a compound having the structure of formula VI, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where R4 is -(CH2) n Q and -(CH2) n Selected from CHQR, where Q is -OR, -OH, -O(CH2) n The composition according to Embodiment 41, selected from the group consisting of N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R8 and heterocycles, where n is 1, 2 or 3, for example, SM102 of the formula VI-I structure.

[0253] [ka]

[0254] 48. The composition according to Embodiment 41, wherein the cationic lipid is a compound having the structure of formula VII, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer.

[0255] [ka]

[0256] 49. The composition according to Embodiment 41, wherein the cationic lipid is selected from YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

[0257] 50. The composition according to any one of Embodiments 41 to 49, wherein the molar ratio of the cationic lipid to the neutral lipid is (1 to 10):1.

[0258] 51. The composition according to any one of Embodiments 41 to 50, wherein the molar ratio of the cationic lipid to the structural lipid is (1 to 5):1.

[0259] 52. The composition according to any one of Embodiments 41 to 51, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer-conjugated lipid is (25-65):(5-25):(25-45):(0.5-5).

[0260] 53. The molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer-conjugated lipid is (49-50):10:(38.5-39.5):1.5, for example, 5 The composition according to Embodiment 52, wherein the ratio is 0:10:38.5:1.5 or 49:10:39.5:1.5.

[0261] 54. The composition according to any one of Embodiments 41 to 53, wherein the neutral lipid is selected from phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, its derivatives, and any combination thereof.

[0262] 55. The aforementioned neutral lipids are 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distaloyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine A composition according to any one of Embodiments 41 to 54, selected from lysoamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0263] 56. The composition according to any one of Embodiments 41 to 55, wherein the neutral lipid is DOPE and / or DSPC.

[0264] 57. The composition according to any one of Embodiments 41 to 56, wherein the structural lipid is selected from sterols, cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroids, and any combination thereof.

[0265] 58. The composition according to Embodiment 57, wherein the structural lipid includes cholesterol, for example, cholesterol.

[0266] 59. The polymer-conjugated lipid is PEG-modified phosphatidylethanolamine, PEG A composition according to any one of Embodiments 41 to 56, selected from modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and any combination thereof.

[0267] 60. The composition according to Embodiment 59, wherein the polymer-conjugated lipid is selected from distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG-PEG2000), and methoxypoly(ethylene glycol)ditetradecylacetamide (methoxypoly(ALC-0159)).

[0268] 61. The composition according to any one of Embodiments 38 to 60, wherein the effective amount of VZV RNA is 25 μg to 200 μg, preferably 50 μg to 100 μg.

[0269] 62. The composition according to any one of Embodiments 38 to 60, wherein the vaccine is in an injectable dosage form, for example, a liquid formulation or a lyophilized formulation.

[0270] 63. A method for preparing the composition according to any one of Embodiments 38 to 60, comprising the step of mixing the RNA of the VZV with the pharmaceutically acceptable carrier, for example, encapsulating at least a portion of the RNA inside lipid nanoparticles.

[0271] 64. The method according to embodiment 63, further comprising a purification step to remove unencapsulated components, such as dialysis and / or filtration.

[0272] 65. The method according to Embodiment 64, wherein the unencapsulated component is selected from unencapsulated RNA, a water-insoluble solvent, and bacteria.

[0273] 66. Use of a composition according to any one of Embodiments 1 to 62 in the preparation of a pharmaceutical for inducing a protective immune response against VZV in a subject, wherein the protective immune response includes, for example, the production of a neutralizing antibody.

[0274] 66a. A composition according to any one of Embodiments 1 to 62, used to induce a protective immune response against VZV in a subject requiring it, wherein the protective immune response includes, for example, the production of a neutralizing antibody.

[0275] 66b. A method comprising administering to the subject a composition according to any one of Embodiments 1 to 62, wherein the protective immune response includes, for example, the production of a neutralizing antibody, inducing a protective immune response against VZV in the subject to be desired.

[0276] 67. The use, composition, or method according to Embodiment 66, 66a, or 66b, wherein the subject is in an immunodeficient state.

[0277] 68. The use, composition, or method described in 66, 66a, or 66b, wherein the subject is 10 years of age or older, for example, 50, 60, 70, 80 years of age or older.

[0278] 69. The use, composition, or method according to any one of embodiments 66 to 68, wherein the protective immune response further comprises a cellular immune response.

[0279] 70. The use, composition, or method described in any one of Embodiments 66 to 69, wherein the induced protective immune response is used for the prevention of VZV infection.

[0280] 71. The use, composition, or method described in any one of embodiments 66 to 69, wherein the induced protective immune response is used for the prevention of VZV-associated pain.

[0281] Example 1: Design of VZV gE mutant antigen As a highly glycosylated type I membrane protein, gE can be transported between the endoplasmic reticulum (ER), the trans-Golgi network (TGN), and the endoplasmic protoplasm. The gE protein has several important domains related to its main function.

[0282] (1) TM transmembrane domain: Consists of amino acids at positions 539 to 559.

[0283] (2)A 568 YRV 571 Motif: It mediates the transport of gE between the endoplasmic reticulum (ER), the trans-Golgi network (TGN), and the internal protoplasm.

[0284] (3)Y 582 AGL 585 Motif: It mediates the transport of gE between the endoplasmic reticulum (ER), the trans-Golgi network (TGN), and the internal protoplasm.

[0285] (4)S 593 ES 595 T 596 DT 598 Motif: It is an important glycosylation site that mediates the transport of gE between the endoplasmic reticulum (ER), the trans-Golgi network (TGN), and the endoplasmic protoplasm.

[0286] The intracellular carboxyl terminus of gE plays a crucial role in the intracellular transport of gE. Based on the key domains and functions of the gE protein, we designed a series of mRNA antigens targeting mutations in key structural and functional domains of gE, as well as various truncated mutations (see Figure 1).

[0287] Using the wild-type sequence of the gE gene from the VZV virus (see NCBI virus strain sequence number QXN54923.1) as a reference, we designed a series of 43 different gE mutant mRNA sequences.

[0288] Table 1 summarizes mRNAs encoding mutant gE antigens with different C-terminal sequences. YK-VZV-004, YK-VZV-006, YK-VZV-045, and YK-VZV-007 in the table correspond to the corresponding mutations or combinations of mutations in the disclosed patents (US11643441B1, CN114081943A, US20230233671A1, and CN108472309A), respectively.

[0289] [Table 3] TIFF0007912117000019.tif222170TIFF0007912117000020.tif222170TIFF0007912117000021.tif171170

[0290] Example 2: Preparation of VZV gE antigen mRNA vaccine Preparation of transcription template linearization plasmids All antigen gene fragments were synthesized by Genscript Biotech Corporation, cloned into a pVAX vector (purchased from Thermo Fisher Scientific), and successfully constructed a circular plasmid.

[0291] For template preparation, plasmids were linearized using BsaI restriction endonuclease (purchased from Shanghai Beyotime Biotechnology Co., Ltd.). The enzymatic digestion system contained a supercoiled plasmid inserted into the target gene corresponding to Example 1, 10× digestion buffer, BsaI restriction endonuclease, and RNase-free ddH2O. The enzymatic digestion reaction temperature was 37°C, and the digestion time was 3 hours. The enzymatic digestion system is shown in Table 2.

[0292] [Table 4]

[0293] After the enzymatic digestion reaction was complete, the linearization product was recovered using a DNA product purification kit (purchased from Yisheng Biotechnology Co., Ltd.), the product concentration was measured using a micro-ultraviolet spectrophotometer (Denovix), and the length and state of the linearized plasmid template were detected via an agarose gel.

[0294] Preparation of mRNA stock solution In vitro co-transcription capping is a process for preparing mRNA polynucleic acid by modifying or assembling native nucleotide triphosphates (NTPs) according to the base sequence of a template. Adding Clean Cap to this process allows for the generation of mRNA containing the Cap1 structure in a single transcription reaction. The typical reaction process consists of the following system (all purchased from Novoprotein Scientific Inc.).

[0295] [Table 5]

[0296] Once transcription was complete, 1 μl of DNase I (2 U / μl) was added, mixed uniformly, and incubated at 37°C for 20 minutes to remove the template DNA. Subsequently, the transcription product was purified using lithium chloride precipitation.

[0297] 1) 20 μl of the reaction mixture was mixed with 30 μl of RNase-free H2O and 7.5 M lithium chloride (the final concentration of lithium chloride was 2.8 M).

[0298] 2) After homogeneous mixing, the mixture was left at -20°C for 2 hours, then centrifuged at 12000 rpm for 15 minutes, and the supernatant was discarded. 500 μl of 70% ethanol was added to wash the RNA precipitate, and the mixture was centrifuged at 12000 rpm for 5 minutes. This step was repeated once, and the precipitate was collected.

[0299] 3) The mRNA precipitate was dried, dissolved in 100 μl of enzyme-free water, and the purified RNA solution was stored at -80°C. The mRNA concentration and integrity were measured.

[0300] Preparation of mRNA-LNP (mRNA vaccine) Cationic lipids, DSPC, cholesterol, and DMG-PEG2000 were accurately weighed out in 20 mg quantities, dissolved in anhydrous ethanol to prepare a 10 mg / ml solution, and a mixed carrier solution was prepared according to a molar ratio of 49:10:39.5:1.5, which was then used as the organic phase for later use.

[0301] VZV mRNA was dissolved in 50 mM citrate buffer at pH 4.0, diluted to a 0.15 mg / ml solution, and used as the aqueous phase for later use. Using a 3 ml BD (brand name, BiDi in Chinese, same below) syringe, 2 ml of the organic phase was extracted, and using a 10 ml BD syringe, 7 ml of the aqueous phase was extracted. The left pump was used to dispense the organic phase, and the right pump was used to dispense the aqueous phase, and the LNP intermediate solution was prepared in a microfluidic device.

[0302] The preparation volume was 9.0 ml, the organic phase flow rate was 5.0 ml / min, and the aqueous phase flow rate was 15 ml / min. The LNP intermediate solution was collected.

[0303] The above LNP intermediate solution was diluted by adding 9 times its volume of PBS solution, and concentrated by ultrafiltration using a small ultrafiltration apparatus with a pore size of 50 KD. When the volume was concentrated to approximately 10 ml, it was diluted again by adding 4 times its volume of PBS solution and concentrated on the ultrafiltration apparatus until the final concentrated volume was less than 5 ml, at which point ultrafiltration was stopped.

[0304] Example 3: Detection of herpes zoster mRNA vaccine antigen expression by Western blotting. To verify whether the designed antigen mRNA sequence could be expressed in cells and to assess the expression efficiency of various antigen sequences, cells were first transfected with mRNA sequences, and the expression of antigen mRNA in the cells was detected using Western blotting.

[0305] Experimental materials

[0306] [Table 6]

[0307] Experimental Steps Cell culture and transfection: 293T cells were placed in a 12-well plate in a 2.5 × 10⁶ container. 5 Seeds were seeded in one well. The volume of mRNA preparation required for 500 ng of mRNA was calculated, and the corresponding volume of mRNA preparation sample was added directly to the cells and mixed uniformly.

[0308] Experimental group: A mixture of protein samples obtained by transfecting cells with a series of mRNA vaccines prepared in Example 2 and then lysing them. Negative control NC: RIPA lysis solution, Positive control PC: gE purified protein, Internal reference protein loading control: Glyceraldehyde-3-phosphate dehydrogenase GAPDH, Preparation of protein samples: (1) 16 hours after transfection, remove the cells from the incubator, remove all cells from the bottom of the dish by repeated pipetting, transfer to a 1.5 ml centrifuge tube, and centrifuge at 25°C and 1500 rpm for 3 minutes. (2) Discard the supernatant, add 1 ml of PBS, resuspend the cells by pipetting, and centrifuge at 1500 rpm for 3 minutes. (3) Repeat step (2) twice. (4) Add 50 μl of RIPA lysis solution (add 0.5 μl of 100× protein inhibitor to the RIPA lysis solution before use) to each cell tube, place on ice for 30 minutes, and vortex the sample for 30 seconds every 5 minutes during that time. (5) After pre-cooling the centrifuge and completely lysing the cells, centrifuge the sample at 4°C and 12000 rpm for 10 minutes, transfer the supernatant to a new 1.5 ml centrifuge tube, and obtain the test protein sample.

[0309] Development and Analysis: Using a BCA kit, the protein sample concentration was quantitatively detected. 200 μl of BCA working solution and 10 μl of test sample were added. 10 μg of protein sample was pipetteed into a 1.5 ml centrifuge tube, water was added to prepare a 24 μl system, 6 μl of 5× loading buffer was added, and the volume was unified to 30 μl with purified water. The entire protein was boiled to denature it. 3 μg of each sample (9 μl) was aspirated and spotted, 4 μl of marker was spotted, and electrophoresis was performed at 200 V for 30 minutes. The film was transferred using a dry film transfer apparatus, the gel was removed, and a film transfer "sandwich" was created. The negative plate was covered, and voltages of 21V, 23V, and 25V were applied for 1 minute, 4 minutes, and 2 minutes, respectively, followed by blocking at room temperature for 1 hour. Primary antibody culture: The antibody was cultured at room temperature for 1 hour. Antibody preparation (1:2000 dilution): 7.5 μl of antibody was pipetted and 15 ml of diluted primary antibody (anti-VZV gE protein antibody, mouse mAb) was added.

[0310] Film washing: Washed three times for 5 minutes each using 1×TBST buffer. Secondary antibody culture: Cultured at room temperature for 1 hour. Antibody preparation (1:1000 dilution): 30 μl of antibody was pipetted and 30 ml of diluted secondary antibody (goat anti-mouse IgG) was added. Film washing: Washed three times for 5 minutes each using 1×TBST buffer diluted with distilled water. Chromogenic development: Mixed 1 ml each of developer and fixer in a 1:1 ratio, added dropwise to the film to allow chromogenic development, and then photographed.

[0311] Internal reference protein GAPDH: Relative intensity was consistent, indicating the same loading amount for different samples, and based on this, the expression intensities between different samples were compared. Negative control NC: A band corresponding only to the internal reference region was present, and no corresponding band was present in the target protein region, thus meeting the requirements for a negative control. Positive control PC (gE purified protein): A corresponding band was present within the target size, and a corresponding band was present in the internal reference region, thus meeting the requirements for a positive control. Under conditions where the negative and positive settings were reasonable and the bands were clear, it was determined whether or not the experimental samples were expressed and their relative expression levels. Western blotting results of the protein sample mixtures obtained by transfecting cells with a series of mRNA vaccines prepared in Example 2 and then lysing them are shown in Figure 2 of this specification.

[0312] Example 4: Immunization of Balb / c mice Experimental materials Animal: Balb / c mouse, female, 6-8 weeks old. Test materials: 1) mRNA vaccine corresponding to the antigen expressed in Example 3, 2) LNP sample (LNP sample without mRNA prepared in Example 2), 3) GSK recombinant subunit vaccine (Shingrix®), Grouping: Mice were randomly divided into groups based on body weight, with 5 mice in each group.

[0313] Animal immunization: Mice were immunized with the solvent or test substance (liquid mRNA vaccine prepared in Example 2) on day 0 and day 28, respectively. The method was intramuscular injection into the gastrocnemius muscle, and the volume and dosage are as shown in the table below.

[0314] [Table 7] TIFF0007912117000026.tif32169

[0315] [Table 8] TIFF0007912117000028.tif25169

[0316] Sample collection and pretreatment Approximately 0.2 ml of blood was collected from the orbital venous plexus of each animal, the serum was separated (centrifuged at 4000 rpm / min for 10 minutes at 4°C), and stored in a refrigerator at 4°C until processing.

[0317] Health monitoring The measurements are taken twice a day (once in the morning and once in the afternoon) and include, but are not limited to, mortality, disease onset, respiration, secretions, feces, and eating and drinking conditions.

[0318] Humanitarian endpoint According to the IACUC protocol, mice that lost more than 20% of their body weight during the experimental period (using the body weight on day 0 before infection as the baseline, and the body weight on the day of vaccination after infection as the baseline), or mice showing signs of being near death, were euthanized and recorded as deceased animals in the results.

[0319] Example 5: Detection of gE protein-specific IgG antibody titers in Balb / c mouse serum by enzyme immunosorbent assay (ELISA) Main experimental materials and suppliers The varicella-zoster virus (VZV) IgG (human) titer ELISA detection kit (gE protein) was purchased from Acrobiosystems Co., Ltd. The kit contains 1x wash buffer, positive control working solution, negative control working solution, dilution buffer, HRP-goat anti-mouse IgG, substrate solution, stop solution, and an ELISA plate.

[0320] Experimental Steps Preparation of working solution: Preparation of 1× wash buffer: 50 ml of 10× wash buffer was taken and diluted to 500 ml with ultrapure water or deionized water. Positive control working solution and negative control working solution were prepared.

[0321] Pretreatment of test samples: A series of blood samples obtained in Example 4 from day 42 and day 56.

[0322] Antibody titer test: Test samples, positive control PC, and negative control NC were diluted with dilution buffer to a ratio of 1:100 to 1:102400.

[0323] Numbering: Diluted samples corresponding to the wells in the ELISA plate were numbered, and a PC working solution and NC working solution were set for each experiment.

[0324] Sample addition: First, 100 μl of the diluted sample, positive control working solution, and negative control working solution were added to the corresponding wells of the plate. The mixtures were shaken to ensure uniformity and incubated at 37°C for 1.0 hour.

[0325] Plate washing: Discard the liquid in the wells, tap the ELISA plate to dry, wash the plate with 1x wash buffer, immerse in 300 μl / well for 30 seconds, tap the plate, wash the plate a total of three times, and tap to dry.

[0326] Addition of HRP enzyme-labeled substance: HRP-goat anti-mouse IgG was diluted 1000-fold with dilution buffer, 100 μl was added to each well, and the mixture was incubated at 37°C for 1.0 hour.

[0327] Plate cleaning: The plates were cleaned three times by repeating step 6.

[0328] Color development: 100 μl of substrate solution was added to each well and incubated in the dark at 37°C for 20 minutes.

[0329] Stop: Add 50 μl of stop solution to each well and shake the ELISA plate until uniformly mixed.

[0330] Data reading: Using a microplate reader, OD 450 nm and OD 630 The absorbance values ​​of each well were measured at wavelengths of nm, with 630 nm as the background. Then, the data was read within 3 minutes.

[0331] Interpretation of test results: Antibody positive result: OD450 nm-OD 630 nm ≥ 0.1.

[0332] Example 6: Multiparameter flow cytometry (FCM) of CD4 secreting IFN-γ and IL-2 in the spleen of Balb / c mice + T cells and CD8 + Detection of T cell percentage The action of immune checkpoint inhibitors is to prevent T cell depletion. Immune cell function was evaluated by detecting cytokine production in tumor samples. When inflammation or host immune defense occurs, T cells and NK cells carry out an immune response by producing gamma interferon (IFN-γ). Multi-parameter flow cytometry (FCM) was used to identify CD8 cells that produce IFN-γ in mouse spleens. + or CD4 + T cells were quantitatively analyzed.

[0333] γ-interferon (IFN-γ) is a soluble dimeric cytokine and the only member of the type II interferon family. It is primarily secreted by natural killer cells (NK) and natural killer T cells (NKT), plays a role in innate immunity, and is secreted by CD4 Th1 and CD8 cytotoxic T cells during the process of antigen-specific immunity. IFN-γ, or type II interferon, plays an important role in innate and adaptive immunity against viral, certain bacterial, and protozoan infections. IFN-γ is an important activator of macrophages and is also an inducer of major histocompatibility complex type II (MHC II) expression. Interleukin-2 (IL-2) is a cytokine belonging to the chemokine family. This cytokine originates from multiple cells (primarily produced by activated T cells) and has multifaceted effects (primarily promoting lymphocyte growth, proliferation, and differentiation). It plays a crucial role in the body's immune response and antiviral infections, stimulating the proliferation of T cells activated by specific antigens or mitogen factors, activating T cells, promoting cytokine production, stimulating NK cell proliferation, enhancing NK cell killing activity and cytokine production, inducing lymphokine-activated killer cell (LAK) production, promoting B cell proliferation and antibody secretion, and activating macrophages.

[0334] Detection method: FCM method (multi-parameter flow cytometry).

[0335] Detection sample: Spleen removed on day 56 of the immunized mouse model in Example 5.

[0336] Detection objective: CD4 secreting IFN-γ and IL-2 in mouse spleen + T cells and CD8 + This involves measuring the proportion of T cells.

[0337] Detection indicators: CD4 + (IFN-γ + ): All CD4 cells that produce IFN-γ + T cell number, CD4 + (IL-2 + ): All CD4 cells that produce IL-2 + T cell number, CD4 + (IFN-γ + IL-2 + ): CD4 simultaneously produces IFN-γ and IL-2 + T cell number, CD4 + (IFN-γ + or IL-2 + ): All CD4 cells that produce IFN-γ or IL-2 + T cell number, CD8 + (IFN-γ + ): All CD8 cells that produce IFN-γ + T cell number, CD8 + (IL-2 + ): All CD8 cells that produce IL-2 + T cell number, CD8 + (IFN-γ + IL-2 + ): CD8 simultaneously produces IFN-γ and IL-2 + T cell number, CD8 + (IFN-γ + or IL-2 + ): All that produce IFN-γ or IL-2 CD8 + T cell count.

[0338] Experimental materials Main reagents and suppliers

[0339] [Table 9] TIFF0007912117000030.tif48169

[0340] Reagent preparation Each reagent was prepared in the amount required for the experiment, and all reagent preparation ratios were expressed as volume ratios.

[0341] FBS inactivation: FBS stored at -20°C was dissolved by leaving it at 4°C, inactivated by placing it in a 56°C water bath for 30 minutes, filtered through a 0.22 μm filter, stored at 2-8°C, and had an expiration date of 14 days.

[0342] Preparation of 1×RBC lysis buffer: 10×RBC lysis buffer (Mμlti-species) was diluted with sterile water to 1×RBC lysis buffer, homogeneously mixed, and stored.

[0343] Preparation of complete medium containing 1% bivalent antibody: Complete medium was prepared according to RPMI-1640:inactivated FBS=9:1.

[0344] Complete medium containing 1% biantibody was prepared according to the ratio of complete medium to penicillin-streptomycin mixture = 99:1. After homogenization, it was stored at 2-8°C and had a shelf life of 14 days. It was equilibrated to room temperature before use.

[0345] PMA / Ionomycin positive stimulant working solution: 400 μl of PMA working solution and 80 μl of ionomycin (ION) were taken and added to 19.520 ml of complete medium containing 1% biantibody to prepare the positive stimulant working solution, which was used immediately after preparation. The final concentration of PMA was 1 μg / ml, and the final concentration of ionomycin (ION) was 2 μg / ml.

[0346] Negative control: Complete culture medium containing 1% bivalent antibody.

[0347] Preparation of peptide working solution: The protein transport inhibitor working solution and stock solution were diluted 50-fold in complete medium containing 1% bivalent antibody.

[0348] Live / Dead Cell Working Solution: Zombie Violet TM The Fixable Viability solution was diluted 1000-fold with 1×PBS.

[0349] Surface staining antibody: As an example of preparing a dose for one well, prepare the surface staining antibody according to the proportions in the table below, and mix uniformly by pipetting. Store in a dark place at 2°C to 8°C for later use.

[0350] [Table 10]

[0351] 1×Perm / wash buffer was diluted 10-fold with ultrapure water to obtain 10×Perm / wash buffer.

[0352] Intracellular staining antibody: Using the dosage for one well as an example, prepare the intracellular staining antibody according to the proportions in the table below, and mix uniformly by pipetting. Store in a dark place at 2°C to 8°C for later use.

[0353] [Table 11]

[0354] Preparation of cryopreservation solution: Prepared according to FBS:DMSO = 9:1 and used immediately after preparation.

[0355] Operation Steps Isolation of mouse spleen cells A cell strainer was placed in a 6-well plate containing 4 ml of complete medium with 1% bivalent antibody. Then, only the spleen was placed on the cell strainer, and the spleen was gently crushed or pulverized on the cell strainer using the plunger end of a syringe. The resulting cell suspension was passed through the cell strainer and filtered into a 15 ml centrifuge tube. The 6-well plate was washed with 1-2 ml of complete medium with 1% bivalent antibody, and the resulting liquid was filtered and placed in a 15 ml centrifuge tube. The mixture was centrifuged at 20°C and 500 g for 5 minutes. The supernatant was discarded.

[0356] Cells were resuspended in 2 ml of 1 × RBC lysis buffer and lysed for 5 ± 1 minutes. Centrifuged at 20°C and 500 g for 5 minutes. The supernatant was discarded. Cells were washed with 5 ml of complete medium containing 1% bivalent antibody and centrifuged at 20°C and 500 g for 5 minutes. The supernatant was discarded.

[0357] Cells were resuspended in 10 ml of complete medium containing 1% bivalent antibody, and after homogeneous mixing, 100 μl of the cell suspension was immediately transferred to an EP tube. Then, 10 μl of the cell suspension and 10 μl of fluorescent stain were transferred to the EP tube, and after homogeneous mixing, 10 μl was taken and counted with a fluorescence counter to statistically determine the total number of viable cells and cell viability.

[0358] Centrifuge cells in a 15 ml centrifuge tube at 20°C and 400 g for 5 minutes. Discard the supernatant, resuspend the cells in fresh complete medium containing 1% bivalent antibody, and adjust the viable cell concentration of each sample to 1 × 10⁶. 7 The concentration was adjusted to cells / ml.

[0359] After spreading the sample onto the plate, the remaining cells were centrifuged at 20°C and 400g for 5 minutes, and the cell concentration was reduced to (1-2) × 10⁻¹⁶ using cryopreservation solution. 7 The solution was adjusted to cells / ml, dispensed at 1ml / tube into pre-marked cryopreservation tubes, placed in a programmed cooling box, and frozen in a refrigerator at -70°C or below. After 12-24 hours, it was transferred to a liquid nitrogen tank for long-term storage.

[0360] Stimulation of mouse spleen cells Stimulation of mouse spleen cells was performed in a 96-well U-shaped plate, and the experimental date was marked on the plate.

[0361] Positive wells, negative wells, and polypeptide wells were set up according to the experimental procedure. 100 μl / well of the negative control was added to the negative well, 100 μl / well of the positive stimulant working solution was added to the positive well, 100 μl / well of the polypeptide working solution was added to the peptide well, and then 100 μl / well of the cell suspension was added to the positive well, negative well, and peptide well.

[0362] A 96-well plate was placed in a 37°C, 5% CO2 incubator and stimulated for 2 hours ± 5 minutes. Then, 10 μl / well of protein transport inhibitor working solution was added and mixed uniformly. Subsequently, the plate was placed back in the 37°C, 5% CO2 incubator and stimulated for 18 hours ± 10 minutes. The round-bottom 96-well plate was then removed and flow staining was performed.

[0363] Note: The experimental procedures in this section must be performed in a biological safety cabinet, and all reagents used were sterile.

[0364] Cell staining Washing of the sample after stimulation: Remove the round-bottom 96-well plate, centrifuge at 500g at 4°C for 5 minutes, and discard the supernatant. Add 200 μl of PBS to each well, resuspend, mix uniformly, centrifuge at 500g at 4°C for 5 minutes, and discard the supernatant.

[0365] Staining of live / dead cells: Add 50 μl of the prepared live / dead cell working solution to each well, resuspend, mix uniformly, and stain in the dark at 2°C to 8°C for 30 minutes. After staining, add 150 μl of PBS to each well, centrifuge at 500 g at 4°C for 5 minutes, and discard the supernatant.

[0366] Surface staining: Add 50 μl of surface staining antibody to each well, resuspend, mix uniformly, and stain in the dark at 2°C to 8°C for 30 minutes. Add 150 μl of cell staining buffer per well to stop staining, centrifuge at 500 g at 4°C for 5 minutes, and discard the supernatant.

[0367] Cell fixation and membrane disruption: Add 100 μl / well of fixation and permeabilization solution, resuspend, mix homogeneously, and culture in the dark at 4°C for 20 minutes. Add 100 μl / well of 1×Perm / wash buffer to stop the process, centrifuge at 500 g at 4°C for 5 minutes, and discard the supernatant.

[0368] Intracellular staining: Add 50 μl of intracellular staining antibody to each well, resuspend, mix uniformly, and incubate in the dark at 4°C for 50 minutes. Add 150 μl / well of 1× Perm / wash buffer to stop staining, centrifuge at 500 g at 4°C for 5 minutes, and discard the supernatant. Add 200 μl / well of 1× Perm / wash buffer, resuspend, mix uniformly, centrifuge at 500 g at 4°C for 5 minutes, and discard the supernatant.

[0369] Filtration: Add 200 μl / well of cell staining buffer, resuspend, mix uniformly, and filter each cell suspension into a new well plate using a 300-mesh nylon mesh. An example of a loading number is "01-P-1", where "01" is the sample serial number, "P" is the positive well, and the last "1" is the serial number of the well.

[0370] Detection by equipment Instrument name: Flow cytometer (Cytoflex) Instrumental acquisition parameters: The acquisition flow rate was set to high, and the number of recorded cells was 100,000, collected under lymphocyte gauging.

[0371] Data processing Data Analysis: CD3 + Circle the cell, then CD4 + Cells and CD8 + Each cell is circled. CD4 + CD4 + (IFN-γ + ) Subgroup and CD4 + (IL-2 + ) Circle the subgroup, CD8 + CD8 + (IFN-γ + ) Subgroup and CD8 + (IL-2 + The subgroups are circled.

[0372] Data calculation formula: All values ​​in this detection were statistically expressed as percentages, and the valid value was calculated as: Mean value of polypeptide wells - Mean value of negative wells. All numerical values ​​were rounded to two decimal places.

[0373] Example 7: Detection of IFN-γ and IL-2 cytokines secreted by Balb / c mouse spleen cells using enzyme immunoassay spot technology (ELISPOT). Enzyme-Linked Immunospot Assay (ELISPOT) is a highly sensitive detection method in cellular immunology research, capable of detecting antibody-secreting cells (ASCs) and cytokine-secreting cells at the single-cell level. It is more sensitive than ELISA and limiting dilution methods, capable of detecting a single cell secreting a particular protein from 200,000 to 300,000 cells. Furthermore, this method allows for functional detection on live cells after antigen stimulation, and its high specificity, intuitive reliability, and ease of operation have led to its widespread use in the immunology community both domestically and internationally for the detection of secreted CK cells and the measurement of ASCs. After stimulation by an antigen, lymphocytes locally produce cytokines, which are captured by specific monoclonal antibodies on a PVDF membrane (pre-coated) at the bottom of the ELISPOT plate. After cell removal, the captured cytokines bind to biotin-labeled monoclonal antibodies, followed by binding to alkaline phosphatase or horseradish peroxidase-labeled avidin. After adding the substrate and inducing color development, purple or reddish-brown spots appeared on the PVDF membrane, indicating that cells had produced cytokines. The spots were then automatically counted and analyzed using an enzyme-coupled immunosorbent spot analyzer (ELISPOT Reader).

[0374] Using splenic tissue extracted from the mouse model of Example 6, the content of IFN-γ and IL-2 secreted by splenic cells stimulated with a peptide library 56 days post-immunization was detected.

[0375] Sample processing: Process of isolating and cryopreserving mouse spleen cells.

[0376] Elispot method: The content of IFN-γ and IL-2 in the supernatant of spleen cells stimulated with a peptide library was detected.

[0377] Main reagents

[0378] [Table 12]

[0379] Reagent preparation 1) Inactivation of FBS: FBS stored at -20°C was dissolved at 4°C, inactivated in a 56°C water bath for 30 minutes, filtered through a 0.22 μm filter, stored at 2-8°C, and had an expiration date of 30 days.

[0380] 2) Preparation of PMA working solution: Add 1 ml of DMSO to the PMA reagent bottle to dissolve the PMA powder, then transfer to a 15 ml centrifuge tube, rinse the PMA reagent bottle with 1 ml of DMSO, add the rinse solution to the 15 ml centrifuge tube, add 8 ml of DMSO to the 15 ml centrifuge tube, mix uniformly to prepare a 100 μg / ml PMA working solution, and store in a refrigerator at -20°C in the dark.

[0381] 3) Preparation of ION working solution: Add 2 ml of DMSO to the ION reagent bottle and dissolve the ION powder. Transfer this to a 15 ml centrifuge tube and use 1 ml of DMSO to dissolve the ION powder. The N reagent bottle was rinsed, the rinsing step was repeated twice (a total of three times, 1 ml / rinse), the rinse solution was combined in a 15 ml centrifuge tube, 5 ml of DMSO was added to the 15 ml centrifuge tube, and the mixture was thoroughly mixed to prepare a 1 mg / ml ION working solution, which was stored in a refrigerator at -20°C.

[0382] 4) Preparation of complete medium containing 1% biantibody: Complete medium was prepared in accordance with RPMI-1640: inactivated FBS = 9:1. Complete medium containing 1% biantibody was prepared according to complete medium:penicillin-streptomycin mixture = 99:1, and after homogeneous mixing, it was stored at 2-8°C, with a shelf life of 14 days. It was equilibrated to room temperature before use.

[0383] 5) Preparation of positive stimulant working solutions: 400 μl of PMA working solution and 80 μl of ionomycin (ION) were taken and added to 19.520 ml of complete medium to prepare the positive stimulant working solutions, which were used immediately after preparation. The final concentration of PMA was 2 μg / ml and the final concentration of ION was 4 μg / ml.

[0384] 6) Preparation of polypeptide working solution: (1) Preparation of gE peptide library solution: 100 μl of DMSO was added to each tube containing the lyophilized powder, and after dissolution, the polypeptide concentration in each tube was 1 mg / ml / peptide. 900 μl of PBS was added to the dissolved peptide solution to adjust the gE peptide library concentration to 100 μg / ml / peptide.

[0385] (2) Preparation of the gE working solution: The gE peptide library solution was prepared according to a 1:50 ratio in complete medium containing 1% biantibody. After homogeneous mixing, the concentration of the gE working solution was 2 μg / ml / peptide.

[0386] Negative control: The negative control was a complete medium containing 1% bivalent antibody.

[0387] 7) Preparation of 1×PBS solution: Prepare according to 20×PBS:pure water = 1:19, store at room temperature, and use immediately after preparation.

[0388] 8) Preparation of PBS containing FBS: Prepared according to PBS (Biotopped):Inactivated FBS = 200:1 and used immediately after preparation.

[0389] 9) Antibody preparation: (1) Preparation of R4-6A2-biotin: R4-6A2-biotin: PBS containing FBS = 1:1000, prepared and used immediately.

[0390] (2) Preparation of 5H4-biotin: 5H4-biotin: PBS containing FBS = 1:1000, prepared and used immediately.

[0391] Experimental Steps 1) Spleen organs extracted from the mouse model of Example 6 were taken, and on day 56 after immunization, the content of IFN-γ and IL-2 secreted by spleen cells stimulated with the peptide library, as well as CD4 and CD8 T cells producing IFN-γ and IL-2, were detected.

[0392] 2) Test sample: The spleen was removed on day 56 and stored frozen at -80°C.

[0393] Resuscitation of mouse spleen cells Preparation: Before the cells are resuscitated, adjust the temperature of the constant-temperature water bath to 37°C and prepare a complete solution containing 1% biantibody. The entire culture medium was preheated to 37°C.

[0394] Sampling: The sample handler removed mouse spleen cells frozen in a liquid nitrogen tank and transferred them to a box containing liquid nitrogen. The cryopreservation tube was removed from the box, sprayed with 75% alcohol, and placed in the transfer window of the cell culture chamber.

[0395] Resuscitation: The verification team immediately removed the cryopreservation tube through the transfer window and rapidly thawed it in a 37°C water bath. After the cells were fully resuscitated, they were transferred under sterile conditions to a 15 ml centrifuge tube containing 9 ml of complete medium with 1% bivalent antibody.

[0396] Rinsing: Add 1 ml of complete medium containing 1% biantibody to the cryopreservation tube, rinse the cryopreservation tube, and transfer the rinse solution to a 15 ml centrifuge tube.

[0397] Centrifugation: 400g was centrifuged at 18-20°C for 10 minutes.

[0398] Counting: Discard the supernatant, resuspend the cells in 10 ml of complete medium containing 1% bivalent antibody, and after homogeneous mixing, immediately transfer 100 μl of the cell suspension to an EP tube, immediately take 10 μl from the 100 μl cell suspension and add it to an EP tube containing 10 μl of fluorescent staining solution, and after homogeneous mixing, take another 10 μl and count it using a fluorescence counter to statistically determine the total number of viable cells and cell viability (if the activity rate is less than 50%, the counting with the fluorescence counter is repeated once, and the result of the second count is used as the basis).

[0399] Second centrifugation: The remaining cells in the 15 ml centrifugation tube were centrifuged with 400 g at 18-20°C for 5 minutes.

[0400] Resuspension: Discard the supernatant, resuspend the cells in complete medium containing 1% bivalent antibody, and adjust the viable cell concentration to A0:4×10 6 cells / ml, A1:4×10 5 The concentration was adjusted to cells / ml.

[0401] Stimulation of mouse spleen cells Plate washing: Remove the IFN-γ and IL-2 well plates, add 200 μl / well of PBS (the PBS used throughout the experiment is sterile), and wash four times.

[0402] Blocking: Discard the PBS in the wells, tap the plate (to remove as much of the remaining liquid as possible), add 200 μl / well of complete medium containing 1% bivalent antibody, and leave at room temperature for at least 30 minutes (the exact time was honestly recorded).

[0403] Plating: Discard the complete medium containing 1% bivalent antibody in the well plate, tap the plate (to remove as much remaining liquid as possible from the wells), add 100 μl / well of positive stimulant working solution to the positive wells, add 100 μl / well of negative control to the negative control wells, add 100 μl / well of polypeptide working solution to the polypeptide wells, leave the blank wells empty of cells, and add only 200 μl of negative control to each well.

[0404] Cell addition: 100 μl / well of A0 cell suspension was added to the negative and polypeptide wells, and 100 μl / well of A1 cell suspension was added to the positive wells.

[0405] Culture: The plates were wrapped in aluminum foil and placed in a 37°C, 5% CO2 incubator for 21 hours ± 30 minutes.

[0406] Note: The experimental procedures in this section must be performed inside a biological safety cabinet. All of the drugs were sterile reagents.

[0407] Cytokine detection Plate washing: The next day, the original liquid in the well plate was emptied, the plate was tapped (to remove as much remaining liquid as possible from the wells), PBS was added at a rate of 200 μl / well, and the plate was washed five times.

[0408] Secondary antibody addition: Discard the PBS in the wells and tap the plate (remove as much of the remaining liquid as possible from the wells). Add 100 μl / well of IFN-γ:R4-6A2-biotin working solution. Add 100 μl / well of IL-2:5H4-biotin working solution. Incubate at room temperature for 2 hours ± 5 minutes.

[0409] Plate washing: The original liquid in the wells was emptied, the plate was tapped (to remove as much remaining liquid as possible from the wells), 200 μl / well of PBS was added, and the plate was washed 5 times.

[0410] Addition of tertiary antibody: 100 μl / well of streptavidin-HRP working solution was added. After incubation at room temperature for 1 hour ± 2 minutes, the liquid in the well plate was emptied, the plate was tapped (to remove as much remaining liquid as possible from the wells), 200 μl / well of PBS was added, and the plate was washed 5 times.

[0411] Plate washing: The original liquid in the wells was emptied, the plate was tapped (to remove as much remaining liquid as possible from the wells), PBS was added at a rate of 200 μl / well, and the plate was washed 5 times.

[0412] Color development: 100 μl / well of TMB substrate solution, returned to room temperature, was added. After allowing color development at room temperature for 15 ± 1 minutes, the liquid in the well plate was discarded, the well plate was washed with pure water to stop color development, the bottom plate of the well plate was removed, and the bottom surface of the well plate was rinsed.

[0413] Data reading: At room temperature in the dark, after the well plates were completely dry, a photograph was taken using an ELISpot analyzer and the data was read. Camera parameters: Shutter (exposure) (exposure intensity): 400, Gain (gain value): 25. Counting parameters: IFN-γ-mouse-Mab / IL-2-mouse-MabT: Size: 30, Intensity (threshold of optical density): 30, TNTC: 80%.

[0414] Example 8: Distribution of VZV gE antigen in African green monkey kidney cells (Vero) Vero cells are a cell lineage used for cell culture. The "Vero" lineage is isolated from renal epithelial cells extracted from African green monkeys. Vero cells were transfected with various mutant gE antigens (YK-VZV-010, YK-VZV-011, YK-VZV-013, YK-VZV-018, YK-VZV-020) constructed by our company. The transfected cells were stained with gE antigen-labeling antibodies and Golgi marker GM130 / TGN46 antibodies, and the localization of the antigens was observed using a confocal microscope. The results are shown in Figures 3 and 4.

[0415] Preparation of reagents and consumables

[0416] [Table 13] TIFF0007912117000035.tif67169

[0417] Experimental Steps 1) Cell seeding: Vero cells (purchased from Beyotime Biotechnology) were seeded in a confocal dish and observed under a microscope. When the cell seeding density reached approximately 30%, the number of seeded cells was recorded. The cells were cultured overnight.

[0418] 2) Transfection: Transfected with overexpression stock solution (50 ng / well) and cultured for 48 hours.

[0419] 3) Washing: Remove the confocal dish, remove the culture medium directly, add PBS and wash 1 to 3 times, taking care not to shake vigorously during washing.

[0420] 4) Fixation: Cells were fixed by adding 200 μl of 4% paraformaldehyde solution, left at room temperature for 15 minutes, washed three times with 1×PBS, and left at room temperature for 5 minutes each time.

[0421] 5) Permeabilization: Add 200 μl of 0.3% Tritonx-100 (diluted with 1×PBS) and leave the well for 10 minutes. Wash three times with 1×PBS, and let stand at room temperature for 5 minutes each time.

[0422] 6) Blocking: 200 μl of immunohistochemical blocking solution was added to block the sample, and the sample was blocked at room temperature for 2 hours.

[0423] 7) Binding of primary antibody: 100 μl of primary antibody diluted with antibody diluent was added. The specific antibodies and dilutions were VZV gE antibody dilution ratio 1:100, 647-conjμgated GOLGA2 / GM130 antibody dilution ratio 1:100, and CoraLite® 594-conjμgated TGN46 antibody dilution ratio 1:100. The cultures were incubated overnight at 4°C in the dark, and the primary antibody was removed by aspiration (recyclable).

[0424] 8) Washing of primary antibody: Wash with 1×PBS for 5 minutes each time, three times to wash away any unbound primary antibody.

[0425] 9) Secondary antibody culture: 200 μl of fluorescent secondary antibody diluted with antibody diluent was added, resulting in an antibody dilution ratio of 1:300. The culture was performed in the dark at room temperature for 1.5 hours.

[0426] 10) Washing of secondary antibody: To avoid nonspecific staining, the substrate was washed with 1×PBS for 5 minutes each time, three times to wash away any unbound secondary antibody.

[0427] 11) Nucleus staining and sealing: Remove the flame, add one drop of DAPI staining stock solution to each well, seal, and store in a dark place at 4°C.

[0428] 12) Images were taken and observed using a confocal microscope.

[0429] Setting the contrast The comparative settings of this invention are as follows. LNP: Blank LNP that does not contain mRNA vaccine. Shingrix® is a recombinant protein subunit vaccine prepared and currently marketed by GSK Plc. It is the extracellular domain of the VZV gE protein, with amino acids ranging from 1 to 539, and is free of mutations.

[0430] Comparative Example 1 Based on the herpes zoster mRNA vaccine antigen sequence reported in the literature (Morgan A, et al. 2020) and the Moderna patent (US11643441B1), the antigen developed under this patent was found to be gE(1-573aa)Y569A. Based on this, this project designed YK-VZV-004 (SEQ ID NO: 15), which maintained the same mutation site and shortened length as the herpes zoster mRNA vaccine antigen sequence gE(1-573aa)Y569A reported in the Moderna patent (US11643441B1). YK-VZV-004:gE(1-573aa)Y569A was used as one of the antigen screening positive controls, and the immunogenicity of the vaccine was evaluated together with the newly designed mutation and mutation combination sequences of the present invention. For specific results, please refer to the examples.

[0431] Comparative Example 2 Based on the herpes zoster mRNA vaccine antigen sequence reported in patent (CN114081943A), it was found that the full-length VZV gE polypeptide developed in this patent contains a combination of mutations (Y569A, S593A, S595A, T596A, T598A). Based on this, this project designed YK-VZV-006 (SEQ ID NO: 19), which maintained the same combination of mutation sites (Y569A, S593A, S595A, T596A, T598A) as SEQ ID NO: 2 described in patent (CN114081943A). YK-VZV-006:gE(1-623aa), (Y569A, S593A, S595A, T596A, T598A) were also used as one of the antigen screening controls of the present invention to evaluate the immunogenicity of the vaccine together with the newly designed mutants and mutant combination sequences of the present invention. For specific results, please refer to the examples.

[0432] Comparative Example 3 Based on the herpes zoster mRNA vaccine antigen sequence reported in US20230233671A1, it was discovered that this patent discloses the mutant VZV gE protein Y582A. Based on this, this project designed YK-VZV-045 (SEQ ID NO: 171), which maintains the same mutation site combination Y582A as the mRNA vaccine antigen sequence described in US20230233671A1. YK-VZV-045 was also used as one of the antigen screening controls of the present invention, and the immunogenicity of the vaccine was evaluated together with the newly designed mutant and mutant combination sequences of the present invention. For specific results, please refer to the examples.

[0433] Comparative Example 4 Based on the herpes zoster mRNA vaccine antigen sequence reported in CN108472309A, it was discovered that this patent discloses the mutant VZV gE protein Y582A. Based on this, the project designed YK-VZV-007 (SEQ ID NO: 23), which maintained the same combination of mutation sites (Y582A, S593A, S595A, T596A, T598A) as the mRNA vaccine antigen sequence described in CN108472309A. YK-VZV-007 was also used as one of the antigen screening controls of the present invention, and the immunogenicity of the vaccine was evaluated together with the newly designed mutations and mutation combination sequences of the present invention. For specific results, please refer to the examples.

[0434] Summary and Analysis of Results In vitro expression results of VZV gE antigen mutants Western blot detection results for 43 VZV gE antigen variants (Figure 2) showed that the expression of the YK-VZV-002 antigen protein was extremely low or almost nonexistent. The expression results for the YK-VZV-041, YK-VZV-042, and YK-VZV-043 antigen proteins were negative, indicating no expression. Therefore, these four sequences were excluded as they could not be used as candidate antigens for the herpes zoster mRNA vaccine.

[0435] All other antigen variants showed clear protein expression, with the expressed protein size being approximately 63kDa–75kDa, consistent with the theoretical design of the antigen. They could be used as effective candidate antigens for the next step of immunogenicity screening and evaluation.

[0436] Based on the results of the Western blot experiment described above, YK-VZV-001, YK-VZV-003, YK-VZV-004 (Comparative Example 1), YK-VZV-006 (Comparative Example 2), YK-VZV-007 (Comparative Example 4), YK-VZV-009, YK-VZV-010, YK-VZV-011, YK-VZV-012, YK-VZ V-013, YK-VZV-014, YK-VZV-015, YK-VZV-016, YK-VZV-017, YK-VZV-018, YK-VZV-019, YK-VZV-020, YK-VZV-021, YK-VZV-022, YK-VZV-023, YK-VZV-024, YK-VZV-025, YK-VZV All 39 antigen sequences listed above—YK-VZV-026, YK-VZV-027, YK-VZV-028, YK-VZV-029, YK-VZV-030, YK-VZV-031, YK-VZV-032, YK-VZV-033, YK-VZV-034, YK-VZV-035, YK-VZV-036, YK-VZV-037, YK-VZV-038, YK-VZV-039, YK-VZV-040, YK-VZV-044, and YK-VZV-045 (Comparative Example 3)—showed significant protein expression and can be used as effective candidate antigens for immunogenicity screening and evaluation in the next step. Therefore, gE-specific antibody IgG titer detection was performed on these antigens.

[0437] Results of gE protein-specific IgG antibody titers for VZV gE antigen mutants in Balb / c mouse serum (D42) As can be seen from Example 5, the gE protein-specific IgG antibody titer in the serum of mice 42 days after immunization was detected by ELISA, and the detection results are shown in Table 11 below.

[0438] [Table 14] TIFF0007912117000037.tif224168TIFF0007912117000038.tif215168TIFF0007912117000039.tif92168

[0439] Results: Based on the gE-specific antibody IgG titer results on day 42, the titer of the Shingrix®-positive control was 190 × 10⁶. 4 Among these, the mutants that are more effective than Shingrix (registered trademark) are YK-VZV-009, YK-VZV-011, YK-VZV-013, YK-VZV-014, YK-VZV-010, YK-VZV-012, YK-VZV-018, YK-VZV-020, YK-VZV-030, YK-VZV-031, YK-VZV-038, YK-VZV-021, and YK-VZV-024. These were YK-VZV-028, YK-VZV-003, YK-VZV-001, YK-VZV-016, YK-VZV-015, YK-VZV-017, and YK-VZV-004 (Comparative Example 1), YK-VZV-006 (Comparative Example 2), YK-VZV-045 (Comparative Example 3), and YK-VZV-007 (Comparative Example 4), and their effects were shown to be 2.7 to 5.3 times greater than those of the Shingrix® positive control.

[0440] Based on the fact that the gE-specific antibody IgG titer of the above-mentioned YK-VZV-007 sequence (Comparative Example 4) is 1.8 to 2.8 times superior to that of the Shingrix® positive control, the following 14 variants YK-VZV-009, YK-VZV-011, YK-VZV-013, YK-VZV-014, YK-VZV-010, YK-VZV-012, and YK-VZV-018 were further investigated using the Shingrix® positive control as the standard. YK-VZV-020, YK-VZV-030, YK-VZV-031, YK-VZV-038, YK-VZV-021, YK-VZV-024, YK-VZV-028, and four control sequences YK-VZV-004 (Comparative Example 1), YK-VZV-006 (Comparative Example 2), YK-VZV-045 (Comparative Example 3), and YK-VZV-007 (Comparative Example 4) were extracted, and subsequent detection was performed.

[0441] Results of cell-mediated immunization (FCM) with VZV gE antigen variants. Cellular immunity, particularly T-cell immunity, plays a crucial role in the course of VZV infection, and studies have shown that cell-mediated immunity is an important indicator for evaluating the immune efficacy of herpes zoster vaccines. To further evaluate the specific cellular immune response induced by immunizing mice with the above-mentioned antigen mutant mRNA, Example 6 used FCM to evaluate the specific CD4 response induced after immunizing mice with VZV gE antigen mRNA. + T cells and CD8 + We detected the immune response of T cells.

[0442] The above gE-specific antibody IgG titers were used with Shingrix®-positive control as the standard. Based on the fact that its effects are superior to the YK-VZV-007 sequence, the following 14 variants were further developed: YK-VZV-009, YK-VZV-011, YK-VZV-013, YK-VZV-014, YK-VZV-010, YK-VZV-012, YK-VZV-018, YK-VZV-020, YK-VZV-030, YK-VZV-031, YK-VZV-038, YK- The spleens of immunized mice at day 56, containing four control sequences including VZV-021, YK-VZV-024, YK-VZV-028, and YK-VZV-004 (Comparative Example 1), YK-VZV-006 (Comparative Example 2), YK-VZV-045 (Comparative Example 3), and YK-VZV-007 (Comparative Example 4), were removed, and the CD4 secreting IFN-γ and IL-2 in the mouse spleens were detected according to the detection method in Example 6. + T cells and CD8 + The proportion of T cells was detected.

[0443] CD4 + T cells CD4 + The immune effect of T cells plays a crucial role in recovery from VZV infection and the effectiveness of the vaccine, and is one of the most important indicators for evaluating the effectiveness of the vaccine. Splenectomy was performed 56 days after immunization of mice, and the CD4 cells secreting IFN-γ and IL-2 in the mouse spleen were measured. + The percentage of T cells was measured, and the specific results are shown in the table below.

[0444] [Table 15] TIFF0007912117000041.tif224168TIFF0007912117000042.tif57168

[0445] A) Based on the above results, mRNA vaccines (11 in total) of test samples YK-VZV-010, YK-VZV-020, YK-VZV-013, YK-VZV-038, YK-VZV-012, YK-VZV-028, YK-VZV-031, YK-VZV-011, YK-VZV-021, YK-VZV-018, and YK-VZV-009 were screened, and the secreted IFN-γ + CD4 + T cells and IL-2 + CD4 + The total percentage of T cells was more than twice that of the Shingrix®-positive control vaccine, up to seven times higher, and 1.8 to 5.3 times higher than that of YK-VZV-007 (Comparative Example 4). It was also significantly superior to YK-VZV-004 (Comparative Example 1) and YK-VZV-045 (Comparative Example 3).

[0446] Among them, IFN-γ secreted by the mRNA vaccines YK-VZV-010, YK-VZV-020, YK-VZV-013, YK-VZV-011, and YK-VZV-018 + CD4 + T cells and IL-2 + CD4 + The total percentage of T cells was 7.0, 5.2, 3.2, 2.7, and 2.3 times higher than that of the Shingrix®-positive control vaccine, and 5.3, 3.9, 2.4, 2.0, and 1.8 times higher than that of YK-VZV-007 (Comparative Example 4). All of these figures were significantly higher than those of YK-VZV-004 (Comparative Example 1) and YK-VZV-045 (Comparative Example 3).

[0447] The specific details are shown in the table below.

[0448] [Table 16] TIFF0007912117000044.tif21169

[0449] B) However, IFN-γ secreted by the mRNA vaccines YK-VZV-014, YK-VZV-030, and YK-VZV-024 + CD4 + T cells and IL-2 + CD4 + The total percentage of T cells was less than twice that of the Shingrix®-positive control vaccine and less than 1.5 times that of Comparative Example 4 (YK-VZV-007), and was therefore excluded. The specific results are shown in the table below.

[0450] [Table 17]

[0451] IFN-γ + CD4 + Percentage of T cells A) Screen the mRNA vaccines of test samples YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020, YK-VZV-012, YK-VZV-018, YK-VZV-028, YK-VZV-014, YK-VZV-038, and YK-VZV-031, and test for IFN-γ secreted by the mRNA vaccines. + CD4 + The optimal T cell ratio was 1.00–2.00%, which was significantly higher than the comparative examples (YK-VZV-004, YK-VZV-045, YK-VZV-007), more than three times higher than the Shingrix®-positive control vaccine, reaching a maximum of 6.7 times higher, and approximately 2.2–4.4 times higher than comparative example 4 (YK-VZV-007).

[0452] Among them, IFN-γ secreted by mRNA vaccines corresponding to YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020, and YK-VZV-018 + CD4 +The proportion of T cells was 6.7, 5.7, 5.3, 5.3, and 4.0 times higher than that of the Shingrix®-positive control vaccine, and 4.4, 3.8, 3.6, 3.6, and 2.7 times higher than that of Comparative Example 4 (YK-VZV-007), all of which were significantly higher than those of Comparative Example 1 (YK-VZV-004) and Comparative Example 3 (YK-VZV-045).

[0453] The specific results are shown in the table below.

[0454] [Table 18] TIFF0007912117000047.tif101169

[0455] B) IFN-γ secreted by mRNA vaccines YK-VZV-021, YK-VZV-009, YK-VZV-024, and YK-VZV-030 + CD4 + The T cell percentage was less than 1.0%, which is significantly lower than the percentage of cells secreted by the optimal and superior mRNA vaccine (1.0-2.0%), only 2-3 times that of the Shingrix®-positive control vaccine, and about 1-2 times that of Comparative Example 4 (YK-VZV-007). Therefore, YK-VZV-021, YK-VZV-009, YK-VZV-024, and YK-VZV-030 were excluded from the candidate sequences, and the specific results are shown in the table below.

[0456] [Table 19]

[0457] IL-2 + CD4 + T cells A) Screen mRNA vaccines corresponding to test samples YK-VZV-010, YK-VZV-020, YK-VZV-038, YK-VZV-031, YK-VZV-013, YK-VZV-028, YK-VZV-011, YK-VZV-012, YK-VZV-018, YK-VZV-021, and YK-VZV-009, and the IL-2 secreted therefrom. + CD4 + The T cell percentage was optimal, reaching 1.10–3.70%, which was significantly higher than the comparative mRNA vaccines (YK-VZV-004, YK-VZV-045, and YK-VZV-007), more than 3.7 times higher than the Shingrix®-positive control vaccine, more than 2.2 times higher than YK-VZV-007, and significantly superior to comparative examples 1 (YK-VZV-004) and 3 (YK-VZV-045).

[0458] Among them, IL-2 secreted by the mRNA vaccines YK-VZV-010, YK-VZV-020, YK-VZV-013, YK-VZV-011, and YK-VZV-018 + CD4 + The total percentage of T cells was 12.3, 8.3, 4.3, 4.0, and 3.7 times higher than that of the Shingrix®-positive control vaccine, and 7.4, 5.0, 2.6, 2.4, and 2.2 times higher than that of YK-VZV-007 (Comparative Example 4). At the same time, all of these figures were significantly higher than those of YK-VZV-004 (Comparative Example 1) and YK-VZV-045 (Comparative Example 3).

[0459] The specific results are shown in the table below.

[0460] [Table 20] TIFF0007912117000050.tif16169

[0461] B) IL-2 secreted by mRNA vaccines YK-VZV-014, YK-VZV-030, and YK-VZV-024 + CD4 +The percentage of T cells was less than 1.0%, which is significantly lower than the percentage of cells secreted by the optimal and superior mRNA vaccine (greater than 1.0%), less than three times that of the Shingrix®-positive control vaccine, and less than twice that of YK-VZV-007 (Comparative Example 4). Therefore, YK-VZV-014, YK-VZV-030, and YK-VZV-024 were excluded from the candidate sequences, and the specific results are shown in the table below.

[0462] [Table 21]

[0463] (4) Conclusion A) Percentage of immune cells in the mouse spleen after immunizing mice with mRNA vaccines of screened sequences: IFN-γ + CD4 + T cells and IL-2 + CD4 + The total percentage of T cells was more than twice that of the Shingrix®-positive control, reaching up to seven times higher, and 1.8 to 5.3 times higher than that of YK-VZV-007 (Comparative Example 4). FN-γ + CD4 + The percentage of T cells reached 1.00-2.00%, which was more than three times that of the Shingrix®-positive control vaccine, reaching a maximum of 6.7 times, and 2.2-4.4 times that of Comparative Example 4 (YK-VZV-007).

[0464] IL-2 + CD4 + The percentage of T cells exceeded 1% in all cases, which was more than 3.7 times higher than that of the Shingrix® positive control vaccine and more than 2.2 times higher than that of YK-VZV-007. The details are as follows.

[0465] [Table 22] TIFF0007912117000053.tif207169

[0466] In the above sequence, CD4 is induced by YK-VZV-010. + IFN-γ + T cells and CD4 + IL-2 + The proportion of T cells in spleen cells exceeded 2% in all cases, which was approximately 6 to 12 times higher than that of the Shingrix®-positive control vaccine, indicating the most superior immune response.

[0467] B) Secreted by mRNA vaccine of excluded sequences IFN-γ + CD4 + T cells and IL-2 + CD4 + The total percentage of T cells is Shing The rix® positive control vaccine was less than twice as positive, and comparative example 4 (YK-VZV-007) was less than 1.5 times positive. Alternatively, IFN-γ secreted by mRNA vaccines + CD4 + The T cell count was less than 1.0%, which is only 2-3 times higher than the Shingrix®-positive control vaccine and approximately 1-2 times higher than Comparative Example 4 (YK-VZV-007). Alternatively, IL-2 + CD4 + The percentage of T cells was less than 1.0%, which is less than three times that of the Shingrix®-positive control vaccine and less than twice that of YK-VZV-007 (Comparative Example 4). The information is as follows.

[0468] [Table 23] TIFF0007912117000055.tif120170

[0469] CD4 of IFN-γ or IL-2 generated by induction of the above-mentioned mRNA vaccine + T cells make up less than 1% of spleen cells, and IFN-γ is produced simultaneously. + CD4 + T cells and IL-2 + CD4 +The total proportion of T cells was less than 2.0 times that of Shingrix®, indicating poor immune-enhancing effects. Compared to the sequence of portion A mentioned above, it was not suitable as a candidate sequence for a shingles vaccine.

[0470] In summary, IFN-γ is produced by induction of mRNA vaccines of the selected test substances YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020, YK-VZV-012, YK-VZV-018, YK-VZV-028, YK-VZV-038, and YK-VZV-031. + CD4 + T cells and IL-2 + CD4 + Since the T cell effect was good, the above sequence and YK-VZV-004 (Comparative Example 1), YK-VZV-006 (Comparative Example 2), YK-VZV-045 (Comparative Example 3), and YK-VZV-007 (Comparative Example 4) were subsequently used in CD8 + It was used for T-cell immunoanalysis.

[0471] CD8 + T cells CD4 + Based on the T cell results, measurements were performed on test samples YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020, YK-VZV-012, YK-VZV-018, YK-VZV-028, YK-VZV-038, YK-VZV-031, and YK-VZV-004 (Comparative Example 1), YK-VZV-006 (Comparative Example 2), YK-VZV-045 (Comparative Example 3), and YK-VZV-007 (Comparative Example 4), and the CD8 of IL-2 / IFN-γ generated by its induction was measured. + The number of T cells was measured, and the specific results are shown in the table below.

[0472] [Table 24] TIFF0007912117000057.tif191170

[0473] IFN-γ +CD8 + T cells and IL-2 + CD8 + Total T cells A) Based on the above results, IFN-γ secreted by induction of mRNA vaccines of the screened test samples YK-VZV-010, YK-VZV-013, YK-VZV-020, YK-VZV-018, and YK-VZV-011 + CD8 + T cells and IL-2 + CD8 + The total percentage of T cells was 4 to 7 times higher than that of the Shingrix®-positive control vaccine and approximately 3 to 5 times higher than that of YK-VZV-007 (Comparative Example 4), as shown in the table below for specific results.

[0474] [Table 25]

[0475] B) However, IFN-γ secreted by the mRNA vaccines YK-VZV-012, YK-VZV-028, YK-VZV-038, and YK-VZV-031 + CD8 + T cells and IL-2 + CD8 + The total percentage of T cells was less than four times that of the Shingrix®-positive control vaccine and less than three times that of YK-VZV-007 (Comparative Example 4), as shown in the table below for specific results.

[0476] [Table 26]

[0477] IFN-γ + CD8 + T cells A) IFN-γ secreted by mRNA vaccines YK-VZV-010, YK-VZV-013, YK-VZV-020, YK-VZV-018, and YK-VZV-011 + CD8 +The T cell ratio was screened to be optimal, and all were above 5.0%, with YK-VZV-010 having the highest at 8.10%, which was significantly higher than comparative examples 1-4 (YK-VZV-004, YK-VZV-006, YK-VZV-045, YK-VZV-007). The above five sequences were 4.5 to 7.0 times higher than the Shingrix® positive control vaccine and approximately 2.5 to 4.5 times higher than YK-VZV-007. The specific results are shown in the table below.

[0478] [Table 27]

[0479] B) IFN-γ secreted by mRNA vaccines of test samples YK-VZV-012, YK-VZV-028, YK-VZV-038, and YK-VZV-031 + CD8 + The percentage of T cells was less than 5%, which is significantly lower than the percentage of cells secreted by the optimal mRNA vaccine (5.00-8.10%), and only 4.0 times that of the Shingrix® positive control vaccine, and about 1-2 times that of YK-VZV-007. Therefore, it was excluded, and the specific results are shown in the table below.

[0480] [Table 28]

[0481] IL-2 + CD8 + T cells A) IL-2 secreted by mRNA vaccines YK-VZV-010, YK-VZV-013, YK-VZV-018, YK-VZV-011, YK-VZV-028, and YK-VZV-020 + CD8 +The T cell ratio was screened to be optimal, reaching 0.3-0.5%, which was significantly higher than the comparative examples (YK-VZV-004, YK-VZV-006, YK-VZV-045, YK-VZV-007), 2.5-4.0 times higher than the Shingrix®-positive control vaccine, and approximately 1.5-3.0 times higher than YK-VZV-007. The specific results are shown in the table below.

[0482] [Table 29]

[0483] B) IL-2 secreted by mRNA vaccines YK-VZV-038, YK-VZV-012, and YK-VZV-031 + CD8 + The percentage of T cells was less than 0.2%, and its effect was not statistically significant compared to the Shingrix® positive control vaccine and YK-VZV-007, or even inferior. It was only about 0.8 to 1.6 times more effective than the Shingrix® positive control vaccine and about 0.5 to 1.0 times more effective than YK-VZV-007, and the specific results are shown in the table below.

[0484] [Table 30]

[0485] conclusion The percentage of spleen immune cells induced by the screened vaccine candidate, combined with the results of (1) to (3) above: A) IFN-γ + CD8 + T cells and IL-2 + CD8 + The total percentage of T cells was 4 to 7 times higher than that of the Shingrix® vaccine and approximately 3 to 5 times higher than that of YK-VZV-007 (Comparative Example 4). IFN-γ + CD8 +The T cell ratio was optimal, at over 5.0% in all cases, 4.5 to 7.0 times higher than the Shingrix® positive control vaccine, and approximately 2.5 to 4.5 times higher than YK-VZV-007. IL-2 + CD8 + The T cell ratio was optimal, reaching 0.3-0.5%, which is 2.5-4.0 times higher than the Shingrix® positive control vaccine and approximately 1.5-3.0 times higher than YK-VZV-007. All of these results were significantly higher than those of the comparative examples (YK-VZV-004, YK-VZV-006, YK-VZV-045, YK-VZV-007). The details are as follows:

[0486] [Table 31] TIFF0007912117000065.tif183169

[0487] B) Percentage of mouse spleen immune cells induced by the removed test vaccine: IFN-γ + CD8 + T cells and IL-2 + CD8 + The total percentage of T cells was less than four times that of the Shingrix® vaccine and less than three times that of YK-VZV-007 (Comparative Example 4). Alternatively, IFN-γ + CD8 + The percentage of T cells was less than 5%, which is significantly lower than the percentage of cells secreted by the optimal mRNA vaccine (5.20-8.10%), less than 4.0 times that of the Shingrix® positive control vaccine, and about 1-2 times that of YK-VZV-007. Alternatively, IL-2 + CD8 +The percentage of T cells was only 0.2% or less, and its effect was not statistically significant compared to the Shingrix® positive control vaccine and YK-VZV-007, or was even inferior. It was only about 0.8 to 1.6 times more effective than the Shingrix® positive control vaccine and about 0.5 to 1.0 times more effective than YK-VZV-007, and therefore it was excluded. It was done.

[0488] The information is as follows:

[0489] [Table 32] TIFF0007912117000067.tif158169

[0490] IL-2 produced by induction of mRNA vaccines YK-VZV-012 and YK-VZV-031 + CD8 + The percentage of T cells was lower than that of the Shingrix® vaccine and YK-VZV-007 (Comparative Example 4), and therefore it was excluded.

[0491] IFN-γ produced by induction of mRNA vaccines YK-VZV-028 and YK-VZV-038 + CD8 + T cells and IFN-γ + and IL-2 + CD8 + The total percentage of T cells was only about twice that of the Shingrix® vaccine, which was significantly lower. The sequence designed in portion A above (reaching more than 7 times the percentage) had a lower immune effect and was not suitable as a candidate sequence for a shingles vaccine.

[0492] Therefore, based on the above results, the sequences that have a significant advantage in comprehensive immune effects include YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020, and YK-VZV-018. These sequences exhibited good immune effects and were significantly superior to the other sequences, with YK-VZV-010 showing the best effect among them.

[0493] Results of gE protein-specific IgG antibody titers for VZV gE antigen mutants in Balb / c mouse serum (D56) To detect the consistency between the above partial IgG antibody titers and the results of partial VZV gE antigen variant cell immunization, the following partial test samples (YK-VZV-009, YK-VZV-010, YK-VZV-011, YK-VZV-013, YK-VZV-020, YK-VZV-014, YK-VZV-012, YK-VZV-018, YK-VZV-021, YK-VZV-024, YK-VZV-028, YK-VZV) were further tested. Using V-030, YK-VZV-031, YK-VZV-038 and YK-VZV-004 (Comparative Example 1), YK-VZV-045 (Comparative Example 3) and YK-VZV-007 (Comparative Example 4), serum samples were measured 56 days after mouse immunization. The VZV-conjugated-gE antibody IgG titer in the serum was detected referring to the detection method in Example 5, and the results are shown in the table below.

[0494] [Table 33] TIFF0007912117000069.tif193169

[0495] Based on the results above, at day 56, the gE-specific antibody IgG titers of the above mRNA vaccines were all superior to those of the Shingrix®-positive control, being approximately 1.8 to 5.0 times higher than those of the Shingrix® vaccine, and the antibody GMT was 270 to 750 × 10⁻¹⁴. 4 The antibodies are distributed within this range, and among them, the antibody GMT of YK-VZV-010, YK-VZV-011, YK-VZV-018, YK-VZV-020, and YK-VZV-013 is 650 × 10 4 The results were found to be 4.0 to 5.0 times better than the Shingrix® vaccine and more than twice as good as YK-VZV-007 (Comparative Example 4). Furthermore, it was significantly superior to YK-VZV-004 (Comparative Example 1) and YK-VZV-045 (Comparative Example 3).

[0496] The above IgG antibody titers and CD4 + T cells and CD8 + Based on the T cell detection results, five antigens—YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020, and YK-VZV-018—showed the best cellular immune response, and were subsequently used as candidate antigens for further detection and analysis using the ELISpot method.

[0497] Results of VZV gE antigen mutant cell immunofactor (ELISOPT) ELISpot is the gold standard for screening and evaluating the immune effects of candidate vaccine antigen-specific T cells and can be used to distinguish various subunits of activated T cells by cytokines, such as T helper cells (Th)1 cells (producing IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α cytokines), Th2 cells (producing IL-4, IL-5, IL-10, and IL-13 cytokines), and Th17 cells (producing IL-17 cytokines).

[0498] Based on the combined detection results of IgG and cytokines, five optimal antigen sequences (including YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020, and YK-VZV-018) were screened. These were then used in comparative examples 1 (YK-VZV-004), 2 (YK-VZV-006), 3 (YK-VZV-045), and 4 (YK-VZV-007), along with LNP and Shingrix®, as controls. Further detection was performed using the ELISpot method, and the results are as follows.

[0499] 1) The amount of cytokine IFN-γ secreted by stimulating T cells with mRNA vaccines was all above 500 SFU, reaching nearly 600 SFU at its peak. This was approximately 16 to 19 times higher than the Shingrix®-positive control and 2.5 to 3.0 times higher than Comparative Example 4 (YK-VZV-007). It was significantly superior to the cytokines stimulated by the mRNA vaccines corresponding to Comparative Examples 1 (YK-VZV-004), 2 (YK-VZV-006), or 3 (YK-VZV-045).

[0500] Among these, the mRNA vaccine prepared using the YK-VZV-010 sequence reached a maximum of 598.8 SFUs, which was 18.7 times higher than the Shingrix®-positive control and nearly 3 times higher than the YK-VZV-007 control.

[0501] IFN-γ secretion levels, from highest to lowest, were YK-VZV-010, YK-VZV-018, YK-VZV-011, YK-VZV-020, and YK-VZV-013. There was no significant difference between the groups, and all groups showed favorable and significant immune effects.

[0502] [Table 34]

[0503] 2) The amount of cytokine IL-2 secreted by T cells stimulated by the mRNA vaccine was 400-600 SFU or higher in all cases, which was 10-16 times higher than the Shingrix®-positive control and 2.0-3.0 times higher than the YK-VZV-007 control. This was superior to the cytokine secretion stimulated by the mRNA vaccines corresponding to Comparative Example 1 (YK-VZV-004), Comparative Example 2 (YK-VZV-006), or Comparative Example 3 (YK-VZV-045).

[0504] Among these, the mRNA vaccine prepared with the YK-VZV-010 sequence reached a maximum of 614.5 SFUs, which was 16.00 times higher than the Shingrix®-positive control and more than 3 times higher than the YK-VZV-007 control.

[0505] The IL-2 secretion levels, from highest to lowest, were YK-VZV-010, YK-VZV-011, YK-VZV-018, YK-VZV-013, and YK-VZV-020. There were no significant differences between the groups, and all showed significant and favorable immune effects.

[0506] [Table 35]

[0507] Cell localization results of VZV gE antigen variants C-terminal shortening and "A 568 Y 569 R 570 V 571 The motif mutation resulted in reduced localization of the gE antigen to the transgolster network.

[0508] "A 568 Y 569 R 570 V 571 The "motif" (SEQ ID NO: 184) is a transport motif that targets the gE polypeptide to the transgolster network, and YK-VZV-018 (SEQ ID NO: 63) and YK-VZV-020 (SEQ ID NO: 71) encode a shortened polypeptide (retaining 1-573aa) in which the last 50 amino acids from the C-terminal region have been deleted, and A 568 Y 569 R 570 V 571 Mutations are introduced at any one position within the sequence (e.g., A568D and Y569K site mutations). The results of Example 8 showed that both the YK-VZV-018 and YK-VZV-020 mutants reduced the localization of gE polypeptide to the trans-Golgi network and enhanced cell membrane expression.

[0509] "A 568 Y 569 R 570 V 571 " or "Y 582 A 583 G 584 L 585Any combination of mutant motifs reduced the localization of gE antigens to the trans-Golgi network.

[0510] YK-VZV-011 (SEQ ID NO: 35) and YK-VZV-013 (SEQ ID NO: 43) encode full-length gE polypeptides, and "A 568 Y 569 R 570 V 571 It has mutations in each part of the "motif" (sequence number 184), and furthermore, YK-VZV-013's "Y 582 A 583 G 584 L 585 The endocytosis motif (coordination number 185) also underwent a mutation (e.g., Y582A). The results of Example 8 are "A 568 Y 569 R 570 V 571 " or "Y 582 A 583 G 584 L 585 Any combination of mutant motifs showed that all gE antigens were expressed on the cell membrane.

[0511] 3) "A 593 E 594 A 595 A 596 D 597 A 598 Full-length gE mutants with the mutation motifs Y569K and Y582A mutations showed reduced localization to the trans-Golgi network.

[0512] YK-VZV-010 (SEQ ID NO: 31) codes for a full-length gE polypeptide, and its A 593 E 594 A 595 A 596 D 597 A 598 (Sequence code 183) The array is "S 593 E 594 S 595 T 596 D 597 T 598The "" motif (SEQ ID NO: 182) was substituted. YK-VZV-010 substituted the Ser / Thr-rich "SSTT" acidic cluster with an Ala-rich sequence, and in addition, the YK-VZV-010 mRNA vaccine also had Y569K and Y582A mutations. The results of Example 8 showed that YK-VZV-010 enhances the cell membrane expression of the gE polypeptide.

[0513] Each of the above mutants had modifications that reduced the localization of the encoded gE protein to the trans-Golgi network and enhanced its transport to the serosa membrane.

[0514] [Table 36]

[0515] Summary: In summary, the present invention designs 43 antigen sequences encoding VZV gE protein variants, screens for several sequences with superior efficacy, and includes several antigen sequences that are more effective than at least the vaccine Shingrix® and YK-VZV-004 (Comparative Example 1), YK-VZV-006 (Comparative Example 2), YK-VZV-045 (Comparative Example 3), and YK-VZV-007 (Comparative Example 4), and such results were surprising. Among these sequences, the sequences with mutation combinations of YK-VZV-010 (full-length gE, Y569K, Y582A, S593A, S595A, T596A, T598A), YK-VZV-011 (full-length gE, A568D, Y569K, R570E, V571K), YK-VZV-013 (full-length gE, A568D, Y569K, R570E, V571K, Y582A), YK-VZV-018 (gE1-573aa, A568D), and YK-VZV-020 (gE1-573aa, Y569K) were more preferred. The table below shows the complete sequence information and its number for this application, taking the antigen sequence of YK-VZV-001 as an example. In this table, SEQ ID NOs. 2 and 3 are the nucleic acid sequence ("ORF-NT") and amino acid sequence ("ORF-AA") of the open reading frame of the antigen YK-VZV-001, respectively, and SEQ ID NOs. 1 and 4 are the DNA sequence ("DNA") and mRNA sequence ("mRNA") of the antigen YK-VZV-001, respectively.

[0516] [Table 37] TIFF0007912117000074.tif226170TIFF0007912117000075.tif226170TIFF0007912117000076.tif229170TIFF00079121170 00077.tif228170TIFF0007912117000078.tif228170TIFF0007912117000079.tif227170TIFF0007912117000080.tif100170

[0517] Sequence ID 1: YK-VZV-001-DNA TCGACCTGCTGCTGGAATGGCTGTACGTGCCCATCGATCCTACCTGCCAGCCTATGCGGCTGTACTCCACCTGTCTGTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCTACAGAGTGGACAAGAGCCCTTACAACCAGAGCATGTACTACGCCGGCCTGCCTGTGGACGACTTCGAGGATAGCGAGAGCACCGACACCGAGGAAGAGTTCGGCAACGCCATTGGAGGATCTCACGGCGGCAGCAGCTATACCGTGTACATCGACAAGACCCGGTGAgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0518] sequence number 2 YK-VZV-001-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCA CAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCAGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGT ACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGGATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACACCACCTGTTTCCAGGACGTG GTGGTGGATGTGGACTGCGCGAGAACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGGGAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACACCAGCACACTGTTCGACGAGCTGGAACTGGACCCTCCTGAGATTGAACCCGGGGTGCTGAAGGTGCTGAGAACCGAGAAGCAGTACCTGGGAGTGTACATCTGGAACATGAGAGGCAGCGA CGGCACCTCTACCTACGCCACCTTTCTGGTCACATGGAAGGGCGACGAGAAAACACGGAACCCCACACCAGCTGTGACCCCTCAACCTAGAGGCGCCGAGTTTCACATGTGGAATTACCACAGCCACGTGTTCAGCGTGGGCGATACCTTTAGCCTGGCCATGCATCTGCAGTACAAGATCCACGAGGCCCCTTTCGACCTGCTGCTGGAATGGCTGTACGTGCCCATCGATCCTACCTGCCAGCCTATGCGGCTGTACTCCACCTGTCTGTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCTACAGAGTGGACAAGAGCCCTTACAACCAGAGCATGTACTACGCCGGCCTGCCTGTGGACGACTTCGAGGATAGCGAGAGCACCGACACCGAGGAAGAGTTCGGCAACGCCATTGGAGGATCTCACGGCGGCAGCAGCTATACCGTGTACATCGACAAGACCCGGTGA

[0519] Accession number 3 YK-VZV-001-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRVDKSPYNQSMYYAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYIDKTR

[0520] Accession number 4 YK-VZV-001-mRNA AGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGU

[0521] Sequence ID 5 YK-VZV-002-DNA

[0522] Sequence ID 6: YK-VZV-002-ORF-NT

[0523] sequence number 7 YK-VZV-002-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNG DDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPE IEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHM NSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYA

[0524] sequence number 8 YK-VZV-002 mRNA AAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUCAGAUAUGCCUGAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0525] Sequence ID 9 YK-VZV-003-DNA AATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAAGCCTGAgctggagcctcggtggccatgcttcttgccccttgggcctcc ccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0526] Sequence ID 10: YK-VZV-003-ORF-NT AAGCCTAAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAAGCCTGA

[0527] sequence number 11 YK-VZV-003-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDD RHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPG VLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVAS TVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKA

[0528] sequence number 12 YK-VZV-003 mRNA GGCGCCGAGUUUCACAUGUGGAAUUACCACAGCCACGUGUUCAGCGUGGGCGAUACCUUUAGCCUGGCCAUGCAUCUGCAGUACAAGAUCCACGAGGCCCCUUUCGACCUGCUGCUGGAAUGGCUGUACGUGCCCAUCGAUCCUACCUGCCAGCCUAUGCGGCUGUACUCCACCUGUCUGUAUCACCCCAACGCUCCCCAGUGCCUGAGCCACAUGAAUAGCGGCUGCACCUUCACAAGCCCUCACCUGGCUCAGCGAGUGGCCAGCACAGUGUACCAGAAUUGCGAGCACGCCGACAAUUACACCGCCUACUGUCUGGGCAUCAGCCACAUGGAACCUAGCUUCGGCCUGAUCCUGCACGAUGGCGGCACAACCCUGAAGUUCGUGGAUACCCCUGAGAGCCUGAGCGGCCUGUAUGUGUUCGUGGUGUACUUCAACGGCCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAAGCCUGAgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0529] SEQ ID NO: 13 YK-VZV-004-DNA AGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGGATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACACCACCTGTTTCCAGGACGTGGTGGTGGATGTGGACTGCGCCGAGAACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGGGAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACACCAGCACACTGTTCGACGAGCTGGAACTGGACCCTCCTGAGATTGAACCCGGGGTGCTGAAGGTGCTGAGAACCGAGAAGCAGTACCTGGGAGTGTACATCTGGAACATGAGAGGCAGCGACGGCACCTCTACCTACGCCACCTTTCTGGTCACA。 TGGAAGGGCGACGAGAAAACACGGAACCCCACACCAGCTGTGACCCCTCAACCTAGAGGCGCCGAGTTTCACATGTGGAATTACCACAGCCACGTGTTCAGCGTGGGCGATACCTTTAGCCTGGCCATGCATCTGCAGTACAAGATCCACGAGGCCCCTTTCGACCTGCTGCTGGAATGGCTGTACGTGCCCATCGATCCTACCTGCCAGCCTATGCGGCTGTACTCCACCTGTCTGTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAAGCCGCCAGAGTGGACAAGTGAgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0530] sequence number 14 YK-VZV-004-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCA CAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCAGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGT ACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGGATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACACCACCTGTTTCCAGGACGTG GTGGTGGATGTGGACTGCGCGAGAACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGGGAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACACCAGCACACTGTTCGACGAGCTGGAACTGGACCCTCCTGAGATTGAACCCGGGGTGCTGAAGGTGCTGAGAACCGAGAAGCAGTACCTGGGAGTGTACATCTGGAACATGAGAGGCAGCGA CGGCACCTCTACCTACGCCACCTTTCTGGTCCACATGGAAGGGCGACGAGAAAACCGGAACCCCACACCAGCTGTGACCCCTCAACCTAGAGGCGCGAGTTTCACATGTGGAATTACCACAGCCACGTGTTCAGCGTGGCGATACCTTTAGCCTGGCCATGCATCTGCAGTACAAGATCCACGAGGCCCTTTCGACC TGCTGCTGGAATGGCTGTACGTGCCCATCGATCCTACCTGCCAGCCTATGCGGCTGTACTCCACCTGTCTGTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCT ACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGGCGGCCTGTATGTGTTCGTGGTGTACTTACAACGGCCACGTGGAAGCCGTCTACACCGTGGTGTCTACCGTGGACACTTCGTGAACGCCATCGAGGAAAGA GGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAAGCCGCCAGAGTGGACAAGTGA

[0531] sequence no. 15 YK-VZV-004-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAARVDK

[0532] SEQ ID NO: 16 YK-VZV-004-mRNA AGAAAUAGAGAGAAAAGAGUAAGUAAGAAAUAUAAAGACCACCAUGGGCACCGUGAACAAGCCUUGUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAUUCCUGUGCGGGCUAGCGUGCUGAGAAUACGACGACGACUUCCACAUCGACGAGGACAAGCGUGUACGAGCCCUACUACCACAGCGUCGAUAGCGAUCGCCGAGUCUACUAGCCUAGCUGGCAACACAGAGGCGAGGAGACAGAAGGCCUACGAACCACAAGGCCUACCACAACAGCCCCUACAUCUGGCCCCCGGAACGACUACGACGAUGGCUUCCUGGAAAAUUGCCCACGAGCACCACGGCGUGGUACAAUCAAGGCAGGCAUCGACACGCGGCGAGAGACUGAUGCAGCCUA

[0533] Array number 17 YK-VZV-006-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCC

[0534] Sequence ID 18: YK-VZV-006-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGG

[0535] sequence number 19 YK-VZV-006-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSP YIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPA IQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVF SVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNGSCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVE AVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITVPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAARVDKSPYNQSMYYAGLPVDDFEDAEAADAEEEFGNAIGGSHGGSSYTVYIDKTR

[0536] SEQ ID NO:20 YK-VZV-006 mRNA AUUGCGAGCACGCCGACAAUUACACCGCCUACUGUCUGGGCAUCAGCCACAUGGAACCUAGCUUCGGCCUGAUCCUGCACGAUGGCGGCACAACCCUGAAGUUCGUGGAUACCCCUGAGAGCCUGAGCGGCCUGUAUGUGUUCGUGGUGUACUUCAACGGCCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAGGCCGCUAGAGUGGACAAGAGCCCUUACAACCAGAGCAUGUACUACGCCGGCCUGCCUGUGGACGAUUUCGAGGAUGCUGAAGCCGCCGAUGCCGAGGAAGAGUUUGGCAACGCCAUUGGCGGAUCUCACGGCGGCAGCAGCUAUACCGUGUACAUCGACAAGACCCGGUGAgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0537] SEQ ID NO: 21 YK-VZV-007-DNA

[0538] sequence number 22 YK-VZV-007-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAG AGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACG TGATCCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGG TCCTTTCTGCCCAGCCTGACATGTACCGGGGATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACACCACCTGTTTCCAGGACGTGGTGGATGTGGACTGCGCGAGAACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGGGAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACAC

[0539] sequence number 23 YK-VZV-007-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYG DVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGGTST YATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVD TPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRWDKSPYNQSMYAAGLPVDDFEDAEAADAEEEFGNAIGGSHGGSSYTVYIDKTR

[0540] SEQ ID NO:24 YK-VZV-007 mRNA AGACUCCUCCCAUCCUCUCCCCUGUCCCUGUCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUC AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0541] Sequence ID 25 YK-VZV-009-DNA AGGATAGCGAGAGCACCGACACCGAGGAAGAGTTCGGCAACGCCATTGGAGGATCTCACGGCGGCAGCAGCTATACCGTGTACATCGACAAGACCCGGTGAgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA [[ID=③]]

[0542] [[ID=④]] [[ID=⑤]]Sequence No. 26 YK-VZV-009-ORF-NT[[ID=⑥]] AAGCCTAAAAGAAATCACACCCGGAATCCCGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCAAGAGAGTGGACAAGAGCCCTTACAACCAGTCTATGTATGCCGCCGGACTGCCCGTGGACGATTTCGAGGATAGCGAGAGACACCGACACCGAGAGAGAGTTCGGCAACGCCATTGGAGGATCTCACGGCGGCAGCAGCTATACCGTGTACATCGACAAGACCCGGTGA

[0543] sequence number 27 YK-VZV-009-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYG DVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTST YATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRWASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVD TPESLSGLYVFFVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAAWTGGLAAVVLLCLVIFLICTAKRMRVKAKRVDKSPYNQSMYAAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYIDKTR

[0544] sequence number 28 YK-VZV-009-mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGU ACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGAUGGCUUCCUGGAAAUGCCCACGAGCACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCUACACAGAUGAGCGCCCAAGA GGACCUGGGAGAUGAUACCGGCAUCCACGUGAUCCCCACACUGAACGGCGACGACAGACACAAGAUCGUGAACGUGGACCAGCGGCAGUACGGCGACGUGUUCAAGGGCGACCUGAAUCCUAAGCCUCAGGGCCAGCGCCUGAUCGAGGUGUCCGUGGAAGAGAAUCACCCCUUCACACUGAGAGCCCCUAUCCAGAGAAUCUACGGCGUGCGCUAUACCGAGACAUGGUCCUUUCUGCCCAGCCUGACAUGUACCGGGGGAUGCCGCUCCUGCCAUCCAGCACAUUUGCCUGAAGCACACCACCUGUUUCCAGGACGUGGUGGUGGAUGUGGACUGCGCCGAGAACACCAAAGAGGAUCAGCUGGCCGAGAUCAGCUACCGGUUCCAGGGAAAGAAAGAAGGGCCGACCAGCCUUGGAUCGUGGUCAACACCAGCACACUGU

[0545] Accession No. 29 YK-VZV-010-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAA

[0546] Sequence number 30 YK-VZV-010-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGG

[0547] sequence number 31 YK-VZV-010-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENH PFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLA MHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNGSCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAY TVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAKRVDKSPYNQSMYAAGLPVDDFEDAEAADAEEEFGNAIGGSHGGSSYTVYIDKTR

[0548] SEQ ID NO:32 YK-VZV-010 mRNA AAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAGGCCAAGAGAGUGGACAAGAGCCCUUACAACCAGUCUAUGUAUGCCGCCGGACUGCCCGUGGACGAUUUUGAGGAUGCUGAAGCCGCCGACGCCGAGGAAGAAUUUGGCAACGCCAUUGGCGGAAGCCACGGCGGCAGUAGCUAUACCGUGUACAUCGACAAGACCCGGUGAgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0549] SEQ ID NO: 33 YK-VZV-011-DNA ATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGCGCGTGAAGGACAAAGAGAAGGACAAGTCCCCTTACAACCAGAGCATGTACTACGCCGGCCTGCCTGTGGACGACTTCGAGGATAGCGAGAGCACCGACACCGAGGAAGAGTTCGGCAACGCCATTGGAGGATCTCACGGCGGCAGCAGCTATACCGTGTACATCGACAAGACCCGGTGAgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0550] SEQ ID NO: 34 YK-VZV-011-ORF-NT ATGCATCTGCAGTACAAGATCCACGAGGCCCTTTCGACCTGCTGCTGGAATGGCTGTACGTGCCCATCGATCCTACCTGCCAGCCTATGCGGCTGTACTCCACCTGTCTGTTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGC ACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGGCGGCCTGTATGTGTTCGTGGTGTACTTCACGGCCACGTGGAAGCCGTGGCCTACACC GTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTTGCCTGGTCATCTTCCTGATC TGCACCGCCAAGCGGATGCGCGTGAAGGACAAAGAGAAGGACAAGTCCCCTTACAACCAGAGCATGTACTACGCCGGCCTGCCTGTGGACGACTTCGAGGATAGCGAGAGCACCGACACCGAGGAAGAGTTCGGCAACGCCATTGGAGGATCTCACGGCGGCAGCAGCTATACCGTGTACATCGACAAGACCCGGTGA

[0551] sequence number 35 YK-VZV-011-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYG DVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTST YATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRWASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVD TPESLSGLYVFFVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAAWTGGLAAVVLLCLVIFLICTAKRMRVKDKEKDKSPYNQSMYYAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYIDKTR

[0552] sequence number 36 YK-VZV-011-mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGAUGGCUUCCUGGAAAAUGCCCACGAGCACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCG

[0553] SEQ ID NO: 37 YK-VZV-012-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATC AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0554] Sequence ID 38 YK-VZV-012-ORF-NT GCTATACCGTGTACATCGACAAGACCCGGTGA

[0555] sequence number 39 YK-VZV-012-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYG DVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGGTST YATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVD TPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITVPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKDKEKDKSPYNQSMYYAGLPVDDFEDAEAADAEEEFGNAIGGSHGGSSYTVYIDKTR

[0556] SEQ ID NO:40 YK-VZV-012 mRNA

[0557] Array number 41 YK-VZV-013-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGGATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACACCACCTGTTTCCAGGACGTGGTGGTGGATGTGGACTGCGCCGAGAACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGGGAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACACCAGCACACTGT

[0558] sequence number 42 YK-VZV-013-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGCTTCCTGGGAAAATGCCCACGAGCACCACGG GTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGG

[0559] sequence number 43 YK-VZV-013-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYG DVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGGTST YATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVD TPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITVPNGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKDKEKDKSPYNQSMYAAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYIDKTR

[0560] SEQ ID NO:44 YK-VZV-013 mRNA UACAUCGACAAGACCCGGUGAgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcU gagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0561] Sequence ID 45 YK-VZV-014-DNA AATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGCGCGTGAAGGACAAAGAGAAGGACAAGTCCCCTTACAACCAGTCTATGTATGCCGCCGGACTGCCCGTGGACGATTTTGAGGATGCTGAAGCCGCCGACGCCGAGGAAGAATTTGGCAACGCCATTGGCGGAAGCCACGGCGGCAGTAGCTATACCGTGTACATCGACAAGACCCGGTGAgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0562] SEQ ID NO: 46 YK-VZV-014-ORF-NT TGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGGCGGCCTGTATGTGTTCGTGGTGTACTTCCAACGGCCACGTGGAAGCGTGGCCTACACCGTGGTGTCTACCGTGGACACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGC CGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGCGCGTGAAGGACAAAGAGAAGGACAAGTCCCCTTACAACCAGTCTATGTATGCCGCCGGACTGCCCGTGGACGATTTTGAGGATGCTGAAGCCGCCGACGCCGAGGAAGAATTGGCAACGCCATTGGCGGAAGCCACGGCGGCAGTAGCTATACCGTGTACATCGACAAGACCCGGTGA

[0563] sequence number 47 YK-VZV-014-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYG DVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTST YATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRWASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVD TPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITVPNPGTSPLLRYAAAWTGGLAAVVLLCLVIFLICTAKRMRVKDKEKDKSPYNQSMYAAGLPVDDFEDAEAADAEEEFGNAIGGSHGGSSYTVYIDKTR

[0564] sequence number 48 YK-VZV-014-mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAG AUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGAUGGCU UCCUGGAAAAUGCCCACGAGCACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCUACACAGAUGAGCGCCCAAGAGGACCUGGGAGAUGAUACCGGCAUCCACGUGAUCCCCACACUGAACGGCGACGACAGACACAAGAUCGUGAACGUGGACCAGCGGCAGUACGGCGACGUGUUCAAGGGCGACCUGAAUCCUAAGCCUCAGGGCCAGCGCCUGAUCGAGGUGUCCGUGGAAGAGAAUCACCCCUUCACACUGAGAGAGCCCCUAUCCAGAGAAUCUACGGCGCGUGCGCUAUACCGAGACAUGGUCCUUUCUGCC

[0565] Array number 49 YK-VZV-015-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCG

[0566] sequence number 50 YK-VZV-015-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCT

[0567] sequence number 51 YK-VZV-015-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENT KEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVAS TVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAMYYAGLPVDDFEDSESTDTEEFGNAIGGSHGGSSYTVYIDKTR

[0568] SEQ ID NO:52 YK-VZV-015 mRNA CCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAGGCCAUGUACUAUGCCGGCCUGCCUGUGGACGACUUCGAGGAUAGCGAGAGCACCGACACCGAGGAAGAGUUCGGCAACGCCAUUGGAGGAUCUCACGGCGGCAGCAGCUAUACCGUGUACAUCGACAAGACCCGGUGAgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0569] SEQ ID NO: 53 YK-VZV-016-DNA GTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCATGTACTATGCCGGCCTGCCTGTGGACGATTTCGAAGATGCTGAAGCCGCCGACGCCGAGGAAGAGTTTGGAAACGCCATTGGCGGAAGCCACGGCGGCAGCAGCTACACTGTGTACATCGACAAGACCCGGTGAgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0570] SEQ ID NO: 54 YK-VZV-016-ORF-NT CAGCCACGTGTTCAGCGTGGGCGATACCTTTAGCCTGGCCATGCATCTGCAGTACAAGATCCACGAGGCCCTTTCGACCTGCTGCTGGAATGGCTGTACGTGCCATCGATCCTACCTGCCAGCCTATGCGGCTGTACTCCACCTGTCTGTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGC ACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGGCGGCCTGTATGTGTTCGTGGT GTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGGAATCCCGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTG CTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCATGTACTATGCCGGCCTGCCTGTGGACGATTTCGAAGATGCTGAAGCCGCCGACGCCGAGGAAGAGTTTGGAAACGCCATTGGCGGAAGCCACGGCGGCAGCACTACACTGTGTACATCGACAAGACCCGGTGA

[0571] sequence number 55 YK-VZV-016-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQ YGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGS DGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDG GTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITVPNPGTSPLLRYAAAWTGGLAAVVLLCLVIFLICTAKRMRVKAMYYAGLVPDDFEDAEAADAEEEFGNAIGGSHGGSSYTVYIDKTR

[0572] sequence number 56 YK-VZV-016-mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGAUGGCUUCCUGGAAAAUGCCCACGAGCACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCG

[0573] Array number 57 YK-VZV-017-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTA

[0574] Sequence ID 58 YK-VZV-017-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGG

[0575] sequence number 59 YK-VZV-017-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETW SFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPT CQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRVDK

[0576] SEQ ID NO:60 YK-VZV-017 mRNA CCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAGGCCUACAGAGUGGACAAGUGAUAAUAGgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0577] SEQ ID NO: 61 YK-VZV-018-DNA CATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGACTACAGAGTGGACAAGTGATAATAGgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0578] Accession No. 62 YK-VZV-018-ORF-NT AGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGGCGGCCTGTATGTGTTCGTGGTGACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACACTTCG TGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGGACTACAGAGTGGACAAGTGA

[0579] sequence number 63 YK-VZV-018-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDR HKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVL KVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVY QNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKDYRVDK

[0580] SEQ ID NO:64 YK-VZV-018 mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCG ACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGAUGGCUUCCUGGAAAAUGCCCACGAGCACCACGGCGUGUACAAUCA AGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCUACACAGAUGAGCGCCCAAGAGGACCUGGGAGAUGAUACCGGCAUCCACGUGAUCCCCACACUGAACGGCGACGACAGACACAAGAUCGUGAACGUGGACCAGCGGCAGUACGGCGACGUGUUCAAGGGCGACCUGAAUCCUAAGCCUCAGGGCCAGCGCCUGAUCGAGGUGUCCGUGGAAGAGAAUCACCCCUUCACACUGAGAGCCCCUAUCCAGAGAAUCUACGGCGUGCGCUAUACCGAGACAUGGUCCUUUCUGCCCAGCCUGACAUGUACCGGGGGAUGCCGCUCCUGCCAUCCAGCACAUUUGCCUGAAGCACACCACCUGUUUCCAGGACGUGG

[0581] Accession No. 65 YK-VZV-019-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAA

[0582] Accession number 66 YK-VZV-019-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGG

[0583] sequence number 67 YK-VZV-019-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDR HKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVL KVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVY QNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKYRVDK

[0584] SEQ ID NO:68 YK-VZV-019 mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUC

[0585] Sequence ID 69 YK-VZV-020-DNA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0586] Sequence ID 70: YK-VZV-020-ORF-NT

[0587] sequence number 71 YK-VZV-020-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDR HKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVL KVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVY QNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAKRVDK

[0588] SEQ ID NO:72 YK-VZV-020 mRNA GUGCCUGAGCCACAUGAAUAGCGGCUGCACCUUCACAAGCCCUCACCUGGCUCAGCGAGUGGCCAGCACAGUGUACCAGAAUUGCGAGCACGCCGACAAUUACACCGCCUACUGUCUGGGCAUCAGCCACAUGGAACCUAGCUUCGGCCUGAUCCUGCACGAUGGCGGCACAACCCUGAAGUUCGUGGAUACCCCUGAGAGCCUGAGCGGCCUGUAUGUGUUCGUGGUGUACUUCAACGGCCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAGGCCAAGAGAGUGGACAAGUGAUAAUAGgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0589] SEQ ID NO: 73 YK-VZV-021-DNA GTACAAGATCCACGAGGCCCCTTTCGACCTGCTGCTGGAATGGCTGTACGTGCCCATCGATCCTACCTGCCAGCCTATGCGGCTGTACTCCACCTGTCTGTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCTATGAGGTGGACAAGTGATAATAGgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0590] SEQ ID NO: 74 YK-VZV-021-ORF-NT CAGCCACGTGTTCAGCGTGGGCGATACCTTTAGCCTGGCCATGCATCTGCAGTACAAGATCCACGAGGCCCTTTCGACCTGCTGCTGGAATGGCTGTACGTGCCCATCGATCCTACCTGCCAGCCTATGCGGCTGACTCCACCTGTCTGTATCACCCCAACGCTCCCC AGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCA CAACCCTGAAGTTCGTGGATACCCCTGGAGAGCCTGAGCGGCCTGTATGGTTCGTGGTGACTTCCAACGGCCACGTGGAAGCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCT GCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCTATGAGGTGGACAAGTGA

[0591] sequence number 75 YK-VZV-021-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDR HKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVL KVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVY QNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYEVDK

[0592] SEQ ID NO:76 YK-VZV-021 mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGAUGGCUUCCUGGAAAAUGCCCACGAGCACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCUACACAGAUGAGCGCCCAAGAGGACCUGGGAGAUGAUACCGGCAUCCACGUGAUCCCCACACUGAACGGCGACGACAGACACAAGAUCGUGAACGUGGACC

[0593] Array number 77 YK-VZV-022-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCG

[0594] Array number 78 YK-VZV-022-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAA

[0595] sequence number 79 YK-VZV-022-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHIC LKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNGSCTFTSPHLAVASTVYQNCEHADNYTA YCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRKDK

[0596] SEQ ID NO:80 YK-VZV-022 mRNA GAGAGUGAAGGCCUACCGGAAGGACAAGUGAUAAAUAGgcUggagccUcggUggccaUgcUUcUUgccccUUugggccUccccccagccccUccUccUccUUccUgcacccgUaccccgUggUcUUUgaaUaaagUcUgagUgggcggAA

[0597] sequence number81 YK-VZV-023-DNA AATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGCGCGTGAAGGACAAAGAGAAGGACAAGTGATAATAGgctggagcctcggtggccatgcttcttgccc cttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0598] Sequence ID 82: YK-VZV-023-ORF-NT AGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGCGCGTGAAGGACAAAGAGAAGGACAAGTGA

[0599] sequence number 83 YK-VZV-023-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDR HKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVL KVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVY QNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKDKEKDK

[0600] SEQ ID NO:84 YK-VZV-023 mRNA 。

[0601] Array number 85 YK-VZV-024-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGGATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACACCACCTGTTTCCAGGACGTGGTGGTGGATGTGGACTGCGCCGAGAACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGGGAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACACCAGCACACTGT

[0602] sequence number 86 YK-VZV-024-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCT GGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCAGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCCCCAAGAGGACCTGGGG AGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCCGCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGA ATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACACCACTGTTTCCAGGACGTGGTGGATGTGGACTGCGCGCGAGAACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGG GAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACACCAGCACACTGTTCGACGAGCTGGAACTGGACCCTCCTGAGATTGAACCCGGGGTGCTGAAGGTGCTGAGAACCGAGAAGCAGTACCTGGGAGTGTACATCTGGAACATGAGAGGCAGCGACGGCACCTCTACCTACGCCACCTTTCTGGTCACATGGAAGGGCGACGAGAAAACACGGAACCCCACACCAGCTGTGACCCCTCAACCTAGAGGCGCCGAGTTTCACATGTGGAATTACCACAGCCACGTGTTCAGCGTGGGCGATACCTTTAGCCTGGCCATGCATCTGCAGTACAAGATCCACGAGGCCCCTTTCGACCTGCTGCTGGAATGGCTGTACGTGCCCATCGATCCTACCTGCCAGCCTATGCGGCTGTACTCCACCTGTCTGTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGACGCCGAGAAGGACAAGTGA

[0603] Accession number 87 YK-VZV-024-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKDAEKDK

[0604] Accession number 88 YK-VZV-024-mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGC

[0605] Array number 89 YK-VZV-025-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATC

[0606] Sequence ID 90 YK-VZV-025-ORF-NT

[0607] sequence number 91 YK-VZV-025-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETW SFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPT CQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITVPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKKEKDK

[0608] SEQ ID NO:92 YK-VZV-025 mRNA CCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGCGCGUGAAGAAAGAGAAGGACAAGUGAUAAUAGgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0609] SEQ ID NO: 93 YK-VZV-026-DNA CATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCTACAGAGTGGACAAGAGCCCTTACAACCAGAGCATGTACTACGCCGGCCTGCCTGTTTGATAATAGgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0610] Accession No. 94 YK-VZV-026-ORF-NT CCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAAATTACACCGCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGGCGGCCTGTATGTGTTCGTGGTACTTTCAACGGCCACGTGGAAGCGTGGCCTACACCGTGGTGTCTACCGTGGACACTTCGT GAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCTACAGAGTGGACAAGAGCCCTTACAACCAGAGCATGTACTACGCCGGCCTGCCTGTTGA

[0611] sequence number 95 YK-VZV-026-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRVDKSPYNQSMYYAGLPV

[0612] [[ID=*4*]]Sequence number 96 YK-VZV-026-mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAG AUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGAUGGCU UCCUGGAAAAUGCCCACGAGCACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCUACACAGAUGAGCGCCCAAGAGGACCUGGGAGAUGAUACCGGCAUCCACGUGAUCCCCACACUGAACGGCGACGACAGACACAAGAUCGUGAACGUGGACCAGCGGCAGUACGGCGACGUGUUCAAGGGCGACCUGAAUCCUAAGCCUCAGGGCCAGCGCCUGAUCGAGGUGUCCGUGGAAGAGAAUCACCCCUUCACACUGAGAGAGCCCCUAUCCAGAGAAUCUACGGCGCGUGCGCUAUACCGAGACAUGGUCCUUUCUGCC

[0613] Array number 97 YK-VZV-027-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACC

[0614] Accession number 98 YK-VZV-027-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACG

[0615] sequence number 99 YK-VZV-027-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNG DDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPE IEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHL AQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICT AKRMRVKAMYYAGLPV

[0616] SEQ ID NO:100 YK-VZV-027 mRNA cccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0617] Sequence ID 101 YK-VZV-028-DNA GAGAGTGAAGGCTATGTATGCCGCCGGACTGCCTGTTTGATAATAGgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctt tgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0618] Sequence ID 102: YK-VZV-028-ORF-NT TGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCTATGTATGCCGCCGGACTGCCTGTTTGA

[0619] sequence number 103 YK-VZV-028-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRH KIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKV LRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNC EHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAMYAAGLPV

[0620] SEQ ID NO:104 YK-VZV-028 mRNA UCAGCGUGGGCGAUACCUUUAGCCUGGCCAUGCAUCUGCAGUACAAGAUCCACGAGGCCCCUUUCGACCUGCUGCUGGAAUGGCUGUACGUGCCCAUCGAUCCUACCUGCCAGCCUAUGCGGCUGUACUCCACCUGUCUGUAUCACCCCAACGCUCCCCAGUGCCUGAGCCACAUGAAUAGCGGCUGCACCUUCACAAGCCCUCACCUGGCUCAGCGAGUGGCCAGCACAGUGUACCAGAAUUGCGAGCACGCCGACAAUUACACCGCCUACUGUCUGGGCAUCAGCCACAUGGAACCUAGCUUCGGCCUGAUCCUGCACGAUGGCGGCACAACCCUGAAGUUCGUGGAUACCCCUGAGAGCCUGAGCGGCCUGUAUGUGUUCGUGGUGUACUUCAACGGCCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAGGCUAUGUAUGCCGCCGGACUGCCUGUUUGAUAAUAGgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0621] SEQ ID NO: 105 YK-VZV-029-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGGATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACACCACCTGTTTCCAGGACGTGGTGGTGGATGTGGACTGCGCCGAGAACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGGGAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACACCAGCACACTGTTCGACGAGCTGGAACTGGACCCTCCTGAGATTGAACCCGGGGTGCTGAAGGTGCTGAGAACCGAGAAGCAGTACCTGGGAGTGTACATCTGGAACATGAGAGGCAGCGACGGCACCTCTA。

[0622] sequence number 106 YK-VZV-029-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGG CCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCAGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCACACTGAACGGCGACGACAGACACAAGATCGT GAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGATGCCGCCTGCCATCCAGCACATTTGC CTGAAGCACACCACCTGTTTCCAGGACGTGGTGGTGGATGTGGACTGCGCGAGAACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGGGAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACACCAGCACACTGTTCGACGAGCTGGAACTGGACCCTCCTGAGATTGAACCCGGGGTGCTGAAGGTGCTGGAACCGAGAAGC AGTACCTGGGAGTGTACATCTGGAACATGAGAGGCAGCGACGGCACCTCTACCTACGCCACCTTTCTGGTCACATGGAAGGGCGACGAGAAAACAGGAACCCCACACCAGCTGTGACCCCTCAACCTAGAGGCGCGAGTTTCACATGTGGAATTACCACAGCCACGTGTTCAGCGTGGGCGATACCTTTAGCCTGGCCATGCATCTGCAG TACAAGATCCACGAGGCCCTTTCGACCTGCTGCTGGAATGGCTGTACGTGCCCATCGATCCTACCTGCCAGCCTATGCGGCGTACTCCACCTGTCTGTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCC GACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGGCGGCCTGTATGTGTTCGTGGTGTACTTACAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAA AGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCTATGTATGGCCCGGACTGCCTGTTTGA

[0623] sequence number 107 YK-VZV-029-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRH KIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKV LRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNC EHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAMYGAGLPV

[0624] SEQ ID NO:108 YK-VZV-029 mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGAUGGCUUCCUGGAAAAUGCCCACGAG

[0625] Array number 109 YK-VZV-030-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGC

[0626] Accession number 110 YK-VZV-030-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCC

[0627] sequence number 111 YK-VZV-030-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETW SFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLY STCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKDKKDKSPYNQSMYAAGLPV

[0628] SEQ ID NO:112 YK-VZV-030 mRNA CCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGCGCGUGAAGGACAAAGAGAAGGACAAGUCCCCUUACAACCAGUCUAUGUAUGCCGCCGGACUGCCCGUUUGAUAAUAGgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0629] SEQ ID NO: 113 YK-VZV-031-DNA ATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGCGCGTGAAGGACAAAGAGAAGGACAAGTCCCCTTACAACCAGTCTATGTATGGCGCCGGACTGCCCGTTTGATAATAGgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0630] SEQ ID NO: 114 YK-VZV-031-ORF-NT AACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACCGCCGACAATTACACCGCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGGAGAGCCTGAGCGGCCTGTATGTGTTCGTGTGTACTTCCAACGGCCACGTGGAAGCGTGGCCTACACCGTGGTG TCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGCGCGTGAAGGACAAAGAGAAGGACAAGTCCCCTTACAACCAGTCTATGTATGGCCCGGACTGCCCGTTTGA

[0631] sequence number 115 YK-VZV-031-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKI VNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEK QYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTA YCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKDKEKDKSPYNQSMYGAGLPV

[0632] SEQ ID NO:116 YK-VZV-031 mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUA GCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCC CCGGAACGACUACGAUGGCUUCCUGGAAAAUGCCCACGAGCACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCUACACAGAUGAGCGCCCAAGAGGACCUGGGAGAUGAUACCGGCAUCCACGUGAUCCCCACACUGAACGGCGACGACAGACACAAGAUCGUGAACGUGGACCAGCGGCAGUACGGCGACGUGUUCAAGGGCGACCUGAAUCCUAAGCCUCAGGGCCAGCGCCUGAUCGAGGUGUCCGUGGAAGAGAAUCACCCCUUCACACUGAGAGAGCCCCUAUCCAGAGAA

[0633] Array No. 117 YK-VZV-032-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACA

[0634] Accession number 118 YK-VZV-032-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGG

[0635] sequence number 119 YK-VZV-032-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHIC LKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNGSCTFTSPHLAVASTVYQNCEHADNYTA YCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKDYRVDKSPYNQSMYAAGLPV

[0636] SEQ ID NO:120 YK-VZV-032 mRNA GAGAGUGAAGGACUACAGAGUGGACAAGAGCCCUUACAACCAGUCUAUGUAUGCCGCGGACUGCCCGUUUGAUAAUAGgcUggagccUcggUggccaUgcUUcUUgccccUugggccUccccccagccccUccUccccUUccUUccUgcacccgUaccccgUggUcUUUgaaUaaagUcUgagUgggcggcgAA

[0637] sequence number 121 YK-VZV-033-DNA CAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCAAGAGAGTGGACAAGAGCCCTTACAACCAGTCTATGTATGCCGCCGGACTGCCCGT TTGATAATAGgctggagcctcggtggccatgcttcttgcccttgggcctcccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0638] sequence number 122 YK-VZV-033-ORF-NT TACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTCTCTGCTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCAAGAGAGTGGACAAGAGCCCTTACAACCAGTCTATGTATGCCGCCGGACTGCCGTTTGA

[0639] sequence number 123 YK-VZV-033-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKI VNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEK QYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTA YCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITVPNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAKRVDKSPYNQSMYAAGLPV

[0640] Accession No. 124 YK-VZV-033-mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGAUGGCUUCCUGGAAAAUGCCCACGAGCACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCUACACAGAUGAGCGCCCAAGAGGACCUGGGAGAUGAUACCGGCAUCCACGUGAUCCCCACACUGAACGGCGACGACAGACACAAGAUCGUGAACGUGGACCAGCGGCAGUACGGCGACGUGUUCAAGGGCGACCUGAAUCCUAAGCCUCAGGGCCAGCGCCUGAUCGAGGUGUCCGUGGAAGAGAAUCACCCCUUCACACUGAGAGCCCCUAUCCAGAGAAUCUACGGCGUGCGCUAUACCGAGACAUGGUCCUUUCUGCCCAGCCUGACAUGUACCGGGGAUGCCGCUCCUGCCAUCCAGCACAUUUGCCUGAAGCACACCACCUGUUUCCAGGACGUGGUGGUGGAUGUGGACUGCGCCGAGAACACCAAAGAGGAUCAGCUGGCCGAGAUCAGCUACCGGUUCCAGGGAAAGAAAGAGGCCGACCAGCCUUGGAUCGUGGUCAACACCAGCACACUGU

[0641] Array number 125 YK-VZV-034-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGGATGCCGCTCCTGCCATCCAG

[0642] Array No. 126 YK-VZV-034-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCC

[0643] sequence number 127 YK-VZV-034-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKI VNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEK QYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTA YCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYEVDKSPYNQSMYAAGLPV

[0644] SEQ ID NO:128 YK-VZV-034 mRNA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0645] Sequence ID 129 YK-VZV-035-DNA ctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0646] Sequence ID 130 YK-VZV-035-ORF-NT GCCAAGCGGATGAGAGTGAAGGCCTACCGGAAGGACAAGAGCCCTTTACAACCAGTCTATGTATGCCGCCGGACTGCCCGTTTGA

[0647] sequence number 131 YK-VZV-035-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKI VNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEK QYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTA YCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRKDKSPYNQSMYAAGLPV

[0648] SEQ ID NO:132 YK-VZV-035 mRNA UCAGCGUGGGCGAUACCUUUAGCCUGGCCAUGCAUCUGCAGUACAAGAUCCACGAGGCCCCUUUCGACCUGCUGCUGGAAUGGCUGUACGUGCCCAUCGAUCCUACCUGCCAGCCUAUGCGGCUGUACUCCACCUGUCUGUAUCACCCCAACGCUCCCCAGUGCCUGAGCCACAUGAAUAGCGGCUGCACCUUCACAAGCCCUCACCUGGCUCAGCGAGUGGCCAGCACAGUGUACCAGAAUUGCGAGCACGCCGACAAUUACACCGCCUACUGUCUGGGCAUCAGCCACAUGGAACCUAGCUUCGGCCUGAUCCUGCACGAUGGCGGCACAACCCUGAAGUUCGUGGAUACCCCUGAGAGCCUGAGCGGCCUGUAUGUGUUCGUGGUGUACUUCAACGGCCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAGGCCUACCGGAAGGACAAGAGCCCUUACAACCAGUCUAUGUAUGCCGCCGGACUGCCCGUUUGAUAAUAGgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0649] Array No. 133 YK-VZV-036-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGGATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACACCACCTGTTTCCAGGACGTGGTGGTGGATGTGGACTGCGCCGAGAACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGGGAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACACCAGCACACTGTTCGACGAGCTGGAACTGGACCCTCCTGAGATTGAACCCGGGGTGCTGAAGGTGCTGAGAACCGAGAAGCAGTACCTGGGA

[0650] sequence number 134 YK-VZV-036-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCC GAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATG AGGCGCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCAC CCCTTCACACTGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACACCACCTGTTTCCAGGACGTGGTGGTGGATGGACTGCGCCGAGAACACC

[0651] sequence number 135 YK-VZV-036-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQ YGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGS DGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDG GTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAAWTGGLAAVVLLCLVIFLICTAKRMRVKAMYAAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYIDKTR

[0652] SEQ ID NO:136 YK-VZV-036 mRNA AGACUCCUCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGU AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0653] Sequence ID 137 YK-VZV-037-DNA TTGGCAACGCCATTGGCGGAAGCCACGGCGGCAGTAGCTATACCGTGTACATCGACAAGACCCGGTGATAATAGgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgt accccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0654] Sequence ID 138 YK-VZV-037-ORF-NT CACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCTATGTATGCCGCCGGACTGCCCGTGGACGATTTTGAGGATGCTGAAGCCGCCGACGCCGAGGAAGAATTGGCAACGCCATTGGCGGAAGCCACGGCGGCAGTAGCTATACCGTGTACATCGACAAGACCCGGTGA

[0655] sequence number 139 YK-VZV-037-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQ YGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPVIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGS DGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDG GTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAAWTGGLAAVVLLCLVIFLICTAKRMRVKAMYAAGLPVDDFEDAEAADAEEEFGNAIGGSHGGSSYTVYIDKTR

[0656] Array number 140 YK-VZV-037-mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGAUGGCUUCCUGGAAAAUGCCCACGAGCACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCUACACAGAUGAGCGCCCAAGAGGACCUGGGAGAUGAUACCGGCAUCCACGUGAUCCCCACACUGAACGGCGACGACAGACACAAGAUCGUGAACGUGGACCAGCGGCAGUACGGCGACGUGUUCAAGGGCGACCUGAAUCCUAAGCCUCAGGGCCAGCGCCUGAUCGAGGUGUCCGUGGAAGAGAAUCACCCCUUCACACUGAGAGCCCCUAUCCAGAGAAUCUACGGCGUGCGCUAUACCGAGACAUGGUCCUUUCUGCCCAGCCUGACAUGUACCGGGGAUGCCGCUCCUGCCAUCCAGCACAUUUGCCUGAAGCACACCACCUGUUUCCAGGACGUGGUGGUGGAUGUGGACUGCGCCGAGAACACCAAAGAGGAUCAGCUGGCCGAGAUCAGCUACCGGUUCCAGGGAAAGAAAGAGGCCGACCAGCCUUGGAUCGUGGUCAACACCAGCACACUGUUCGACGAGCUGGAACUGGACCCUCCUGAGAUUGAACCCGGGGUGCUGAAGGUGCUGAGAACCGAGAAGCAGUACCUGGGA

[0657] Array number 141 YK-VZV-038-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGGATGCCGCTCCTGCCATCCAG

[0658] Array number 142 YK-VZV-038-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGT

[0659] sequence number 143 YK-VZV-038-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENT KEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVV YFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKDKEKDKSPYNQSMYGAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYIDKTR

[0660] SEQ ID NO:144 YK-VZV-038 mRNA GCGCGUGAAGGACAAAGAGAAGGACAAGUCCCCUUACAACCAGUCUAUGUAUGGCGCCGGACUGCCCUGGACGAUUUCGAGGAUAGCGAGAGCACCGACACCGAGGAAGAGUUCGGCAACGCCAUUGGAGGAUCUCACGGCGGCAGCAGCUAUACCGUGUACAUCGACAAGACCCGGUGAUAAUAGgcUggagccUcggUgg ccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0661] Sequence ID 145 YK-VZV-039-DNA GCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGCGCGTGAAGGACAAAGAGAAGGACAAGTCCCCTTACAACCAGTCTATGTATGGCGCCGGACTGCCCGTGGACGATTTTGAGGATGCTGAAGCCGCCGACGCCGAGGAAGAATTTGGCAACGCCATTGGCGGAAGCCACGGCGGCAGTAGCTATACCGTGTACATCGACAAGACCCGGTGATAATAGgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0662] SEQ ID NO: 146 YK-VZV-039-ORF-NT AACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTG CACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGGCGGCCTGTATGTGTTCGTGGTGTACTTCACGGCCACGTGGAAGCGTGGCCTACACCGTGGTGTCTACCGTGGACACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACT GCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGCGCGTGAAGGAC AAAGAGAAGGACAAGTCCCCTTACAACCAGTCTATGTATGGCGCGGACTGCCCGTGGACGATTTTGAGGATGCTGAAGCCGCCGACGCCGAGGAAGAATTGGCAACGCCATTGGCGGAAGCCACGGCGCAGTAGCTATACCGTGTACATCGACAAGACCCGGTGA

[0663] sequence number 147 YK-VZV-039-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYG DVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTST YATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRWASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVD TPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITVPNPGTSPLLRYAAAWTGGLAAVVLLCLVIFLICTAKRMRVKDKEKDKSPYNQSMYGAGLPVDDFEDAEAADAEEEFGNAIGGSHGGSSYTVYIDKTR

[0664] sequence number 148 YK-VZV-039-mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGAUGGCUUCCUGGAAAAUGCCCACGAGCACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCUACACAGAUGAGCGCCCAAGAGGACCUGGGAGAUGAUACCGGCAUCCACGUGAUCCCCACACUGAACGGCGACGACAGACACAAGAUCGUGAACGUGGACC

[0665] Array number 149 YK-VZV-040-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGC

[0666] Sequence ID 150 YK-VZV-040-ORF-NT

[0667] sequence number 151 YK-VZV-040-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYG DVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGGTST YATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVD TPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITVPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRWDKSPYNQSMYGAGLPVDDFEDAEAADAEEEFGNAIGGSHGGSSYTVYIDKTR

[0668] SEQ ID NO:152 YK-VZV-040 mRNA GUGCCUGAGCCACAUGAAUAGCGGCUGCACCUUCACAAGCCCUCACCUGGCUCAGCGAGUGGCCAGCACAGUGUACCAGAAUUGCGAGCACGCCGACAAUUACACCGCCUACUGUCUGGGCAUCAGCCACAUGGAACCUAGCUUCGGCCUGAUCCUGCACGAUGGCGGCACAACCCUGAAGUUCGUGGAUACCCCUGAGAGCCUGAGCGGCCUGUAUGUGUUCGUGGUGUACUUCAACGGCCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAGGCCUACAGAGUGGACAAGAGCCCUUACAACCAGUCUAUGUAUGGCGCCGGACUGCCCGUGGACGAUUUUGAGGAUGCUGAAGCCGCCGACGCCGAGGAAGAAUUUGGCAACGCCAUUGGCGGAAGCCACGGCGGCAGUAGCUAUACCGUGUACAUCGACAAGACCCGGUGAUAAUAGgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0669] SEQ ID NO: 153 YK-VZV-041-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGGATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACACCACCTGTTTCCAGGACGTGGTGGTGGATGTGGACTGCGCCGAGAACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGGGAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACACCAGCACACTGTTCGACGAGCTGGAACTGGACCCTCCTGAGATTGAACCCGGGGTGCTGAAGGTGCTGAGAACCGAGAAGCAGTACCTGGGAGTGTACATCTGGAACATGAGAGGCAGCGACGGCACCTCTA。

[0670] sequence number 154 YK-VZV-041-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGC AGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCACACTGAACGGC GACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGG GATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACCACCTGTTTCCAGGACGTGGTGGTGGATGGACTGCGCCGAGACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGGGAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACACCAGCACACTGTTCGACGAGCTGGAACTGGACCCTCCTGAG ATTGAACCCGGGGTGCTGAAGGTGCTGAGAACCGAGAAGCAGTACCTGGGAGTGTACATCTGGAACATGAGAGGCAGCGACGGCACCTCTACCTACGCCACCTTTCTGGTCCACATGGAAGGGCGACGAGAAAACAGGAACCCCACACCAGCTGTGACCCCTCAACCTAGAGGCGCGAGTTTCACATGTGGAATTACCACAGCCACGTGTTCAGCGTGGGCGATAC CTTTAGCCTGGCCATGCATCTGCAGTACAAGATCCACGAGGCCCTTTCGACCTGCTGCTGGAATGGCTGTACGTGCCATCGATCCTACCTGCCAGCTATGCGGCTGTACTCCACCTGTCTGTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATT GCGAGCACGCCGACAAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGGCGGCCTGTATGTGTTCGTGGTGTACTTCACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACACTTCGTGAACGCCATCGAGGAAAGA GGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCGACGATTTCGAGACAGCGAGAGCACCGATACCGAGGAAGAAT

[0671] sequence number 155 YK-VZV-041-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKADDFEDSESTDTEEE

[0672] SEQ ID NO: 156 YK-VZV-041-mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCC

[0673] Accession No. 157 YK-VZV-042-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTA

[0674] Sequence ID 158 YK-VZV-042-ORF-NT

[0675] sequence number 159 YK-VZV-042-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLDGDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKG DLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAP FDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKADDFEDAEAADAEEE

[0676] SEQ ID NO:160 YK-VZV-042 mRNA GCCUGAGCGGCCUGUAUGUGUUCGUGGUGUACUUCAACGGCCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAGGCCGACGAUUUCGAGGAUGCCGAGGCCGCCGAUGCUGAGGAAGAAUGAUAAUAGgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0677] SEQ ID NO: 161 YK-VZV-043-DNA CCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCGACGATTTCGAGGACAGCGAGAGCACCGATACCGAGGAAGAGTTCGGCAACGCCATTGGCGGATCTCACGGCGGCAGCAGCTATACCGTGTACATCGACAAGACCCGGTGATAATAGgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0678] SEQ ID NO: 162 YK-VZV-043-ORF-NT TGCTGCTGGAATGGCTGTACGTGCCCATCGATCCTACCTGCCAGCCTATGCGGCTGTACTCCACCTGTCTGTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAAT TGCGAGCACGCCGACAAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGGCGGCCTGTATGTGTTCGTGGTGTACTTCACGGCCACGTGGAAGCCGTG GCCTACACCGTGGTGTCTACCGTGGACACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCT GTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCGACGATTTCGAGGACAGCGAGAGCACCGATACCGAGGAAGAGTTCGGCAACGCCATTGGCGGATCTCACGGCGGCAGCAGCTATACCGTGTACATCGACAAGACCCGGTGA

[0679] sequence number 163 YK-VZV-043-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQ RQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWN MRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNGSCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFG LILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKADDFEDSESTDTEEFGNAIGGSHGGSSYTVYIDKTR

[0680] SEQ ID NO:164 YK-VZV-043 mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGAUGGCUUCCUGGAAAAUGCCCACGAGCACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCUACACAGAUGAGCGCCCAAGAGGACCUGGGAGAUGAUACCGGCAUCCACGUGAUCCCCACA

[0681] Array number 165 YK-VZV-044-DNA AGACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACCGCCACCATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCA

[0682] Accession No. 166 YK-VZV-044-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCC

[0683] Array number 167 YK-VZV-044-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKG DLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKADDFEDAEAADAEEEFGNAIGGSHGGSSYTVYIDKTR[[ID= eight]] \n[[ID= nine]]

[0684] \n[[ID= ten]] \n[[ID= eleven]]Array number 168 YK-VZV-044-mRNA\n[[ID= twelve]]<0zzzzzz> It should be noted that there seems to be an error in the original text where the tag <0zzzzzz> is used instead of . The translation is based on the provided text with this correction assumed. GAUGGCGGCACAACCCUGAAGUUCGUGGAUACCCCUGAGAGCCUGAGCGGCCUGUAUGUGUUCGUGGUGUACUUCAACGGCCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACAAGCCCACUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAGGCCGACGAUUUCGAGGAUGCCGAGGCCGCCGAUGCUGAGGAAGAAUUUGGCAACGCCAUUGGCGGCUCUCACGGCGGCAGUAGCUAUACCGUGUACAUCGACAAGACCCGGUGAUAAUAGgcUggagccUcggUggccaUgcUUcUUgccccUUgggccUccccccagccccUccUccccUUccUgcacccgUacccccgUggUcUUUgaaUaaagUcUgagUgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0685] SEQ ID NO: 169 YK-VZV-045-DNA GGCTGTACTCCACCTGTCTGTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTGAGCGGCCTGTATGTGTTCGTGGTGTACTTCAACGGCCACGTGGAAGCCGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTTGTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCTACAGAGTGGACAAGAGCCCTTACAACCAGAGCATGTACGCTGCCGGCCTGCCTGTGGACGACTTCGAGGATAGCGAGAGCACCGACACCGAGGAAGAGTTCGGCAACGCCATTGGAGGATCTCACGGCGGCAGCAGCTATACCGTGTACATCGACAAGACCCGGTGAgctggagcctcggtggccatgcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0686] SEQ ID NO: 170 YK-VZV-045-ORF-NT ATGGGCACCGTGAACAAGCCTGTTGTGGGCGTGCTGATGGGCTTCGGCATCATCACAGGCACCCTGCGGATCACCAATCCTGTGCGGGCTAGCGTGCTGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACCAACAGCGTGTACGAGCCCTACTACCACAGCGATCACGCCGAGTCTAGCTGGGTCAACAGAGGCGAGAGCAGCAGAAAGGCCTACGACCACAACAGCCCCTACATCTGGCCCCGGAACGACTACGATGGCTTCCTGGAAAATGCCCACGAGCACCACGGCGTGTACAATCAAGGCAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGAGCGCCCAAGAGGACCTGGGAGATGATACCGGCATCCACGTGATCCCCACACTGAACGGCGACGACAGACACAAGATCGTGAACGTGGACCAGCGGCAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCTAAGCCTCAGGGCCAGCGCCTGATCGAGGTGTCCGTGGAAGAGAATCACCCCTTCACACTGAGAGCCCCTATCCAGAGAATCTACGGCGTGCGCTATACCGAGACATGGTCCTTTCTGCCCAGCCTGACATGTACCGGGGATGCCGCTCCTGCCATCCAGCACATTTGCCTGAAGCACACCACCTGTTTCCAGGACGTGGTGGTGGATGTGGACTGCGCCGAGAACACCAAAGAGGATCAGCTGGCCGAGATCAGCTACCGGTTCCAGGGAAAGAAAGAGGCCGACCAGCCTTGGATCGTGGTCAACACCAGCACACTGTTCGACGAGCTGGAACTGGACCCTCCTGAGATTGAACCCGGGGTGCTGAAGGTGCTGAGAACCGAGAAGCAGTACCTGGGAGTGTACATCTGGAACATGAGAGGCAGCGACGGCACCTCTACCTACGCCACCTTTCTGGTCACATGGAAG GGCGACGAGAAAACAGGAACCCCACACCAGCTGTGACCCTCAACCTAGAGGCGCGAGTTTCACATGTGGAATTACCACAGCCACGTGTTCAGCGTGGGCGATACCTTTAGCCTGGCCATGCATCTGCAGTACAAGATCCACGAGGCCCTTTCGACCTGCTGCTGGAATGGCTGTACGTGCCATCGATCCTACCTGCCAGCCTAGCGGCTGTACTCCACCTGT CTGTATCACCCCAACGCTCCCCAGTGCCTGAGCCACATGAATAGCGGCTGCACCTTCACAAGCCCTCACCTGGCTCAGCGAGTGGCCAGCACAGTGTACCAGAATTGCGAGCACGCCGACAATTACACCGCTACTGTCTGGGCATCAGCCACATGGAACCTAGCTTCGGCCTGATCCTGCACGATGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGAGCCTG AGCGGCCTGTATGGTTCGTGGTGTACTTCCAACGGCCACGTGGAAGCGTGGCCTACACCGTGGTGTCTACCGTGGACACTTCGTGAACGCCATCGAGGAAAGAGGCTTCCCTCCAACTGCTGGACAGGCCTCCTGCCACCACCAAGCCTAAAGAAATCACACCCGTGAATCCCGGCACAAGCCCACTGCTTAGATACGCCGCTTGGACAGGCGGACTGGCTGCTGTT GTTCTGCTGTGCCTGGTCATCTTCCTGATCTGCACCGCCAAGCGGATGAGAGTGAAGGCCTACAGAGTGGACAAGAGCCCTTACAACCAGAGCATGTACGCTGCCGGCCTGCCTGTGGACGACTTCGAGGATAGCGAGAGCACCGACCACGAGGAAGAGTTCGGCAACGCCATTGGAGGATCTCACGGCGGCAGCAGCTATACCGTGTACATCGACAAGACCCGGTGA

[0687] sequence number 171 YK-VZV-045-ORF-AA MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRVDKSPYNQSMYAAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYIDKTR

[0688] Accession number 172 YK-VZV-045-mRNA AGACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACCGCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCA

[0689] Sequence ID 173 5′ UTR 5′AGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC3′

[0690] Sequence ID 174 5′ UTR 5′AGAC...

Claims

1. It contains ribonucleic acid (RNA) of the varicella-zoster virus (VZV), wherein the RNA encodes a variant of the wild-type VZV gE glycoprotein. Here, the sequence of the wild-type VZV gE glycoprotein is sequence number 3, Here, the variant of the wild-type VZV gE glycoprotein includes a mutation relative to SEQ ID NO: 3, and the mutation is one of the following: a) Shortening of SEQ ID NO: 3 to a VZV gE polypeptide having amino acids at positions 1-573, and substitution of Y569K. b) Shortening of SEQ ID NO: 3 to a VZV gE polypeptide having amino acids at positions 1-587, and substitution of A568D, Y569K, R570E, V571K, and Y582A. c) Shortening of SEQ ID NO: 3 to a VZV gE polypeptide having amino acids at positions 1-587, and substitution of A568D, Y569K, R570E, V571K, Y582G d) Replacement of Y569K and Y582A e) Replacement of Y569K, Y582A, S593A, S595A, T596A and T598A f) Replacement of A568D, Y569K, R570E and V571K g) Replacement of A568D, Y569K, R570E, V571K, S593A, S595A, T596A and T598A h) Replacement of A568D, Y569K, R570E, V571K and Y582A i) Replacement of A568D, Y569K, R570E, V571K, Y582A, S593A, S595A, T596A and T598A An immunogenic composition selected from the following.

2. The aforementioned mutations are any of the following: a) Shortening of SEQ ID NO: 3 to a VZV gE polypeptide having amino acids at positions 1-573, and substitution of Y569K. e) Replacement of Y569K, Y582A, S593AS595A, T596A and T598A f) Replacement of A568D, Y569K, R570E and V571K h) Replacement of A568D, Y569K, R570E, V571K and Y582A Selected from, Or, The amino acid sequences of the VZV gE glycoprotein variants are as shown in SEQ ID NOs: 27, 31, 35, 39, 43, 47, 71, 111, or 115, or, The RNA sequence of the aforementioned VZV is as shown in SEQ ID NOs. 28, 32, 36, 40, 44, 48, 72, 112, or 116, or, The RNA of the aforementioned VZV is mRNA, or, The composition according to claim 1, wherein the RNA sequence encoding the VZV gE glycoprotein corresponds to the DNA sequence shown in SEQ ID NOs. 25, 29, 33, 37, 41, 45, 69, 109, or 113.

3. The composition according to claim 1, wherein the RNA of VZV has an open reading frame (ORF) encoding the VZV gE glycoprotein, and the sequence of the open reading frame is shown in SEQ ID NOs. 26, 30, 34, 38, 42, 46, 70, 110, or 114.

4. The composition according to claim 1, wherein the VZV RNA further comprises a 5' untranslated region (UTR).

5. The composition according to claim 4, wherein the sequence of the 5'UTR is shown in sequence number 173, 174, 175, 176, or 177.

6. The composition according to claim 1, wherein the VZV RNA further comprises a 3' untranslated region (UTR).

7. The composition according to claim 6, wherein the sequence of the 3'UTR is shown in sequence numbers 178, 179, 180, or 181.

8. The composition according to claim 1, wherein the VZV RNA further comprises a poly(A) tail.

9. The composition according to claim 8, wherein the poly(A) tail has a length of 50 to 150 nucleotides.

10. The composition according to claim 1, wherein the VZV RNA further comprises a 5' terminal cap.

11. The composition according to claim 10, wherein the 5' end cap is 7mG(5')ppp(5')NlmpNp.

12. The composition according to claim 3, wherein the sequence of the open reading frames is codon-optimized.

13. The composition according to claim 12, wherein the sequence of the open reading frame comprises at least one base modification.

14. The composition according to claim 13, wherein the base modification is selected from one or more of pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine.

15. The composition according to claim 14, wherein the base modification is to replace uracil with pseudouridine and / or N1-methylpseudridine.

16. The composition according to claim 13, 14, or 15, wherein the base modification is a base modification of 1 to 100%.

17. The composition according to claim 16, wherein the base modification is 100% base modification.

18. A step of providing a template that can be transcribed into the RNA of the VZV, The step of transcribing using the template under conditions suitable for transcription into RNA. A method for preparing the composition according to any one of claims 1 to 15, including the following:

19. The method according to claim 18, further comprising a purification step selected from lithium chloride precipitation, affinity chromatography, solution exchange by ultrafiltration, and cellulose chromatography.

20. The composition according to claim 1, wherein the composition is a vaccine and further comprises a pharmaceutically acceptable carrier.

21. The composition according to claim 20, wherein the carrier comprises a lipid mixture.

22. The composition according to claim 21, wherein the lipid mixture is lipid nanoparticles (LNPs).

23. The composition according to claim 20, 21, or 22, wherein the vaccine is an mRNA vaccine.

24. The composition according to claim 22, wherein the lipid nanoparticles include cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids.

25. The cationic lipid is selected from YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA. 【Chemistry 1】 【change】 Or, The molar ratio of the cationic lipid to the neutral lipid is (1 to 10):

1. Or, The molar ratio of the cationic lipid to the structural lipid is (1 to 5):

1. Or, The aforementioned neutral lipids are selected from phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, their derivatives, and any combination thereof. Or, The aforementioned neutral lipids are 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Dieter PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,Selected from 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof, Or, The composition according to claim 24, wherein the neutral lipid is DOPE and / or DSPC.

26. The composition according to claim 24, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-75):(5-25):(15-65):(0.5-10).

27. The composition according to claim 26, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (35-49):(7.5-15):(35-55):(1-5).

28. The composition according to claim 27, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 49:10:43.5:1.

5.

29. The composition according to claim 24, wherein the structural lipid is selected from sterols, cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroids, and any combination thereof.

30. The composition according to claim 29, wherein the structural lipid comprises cholesterol.

31. The composition according to claim 30, wherein the structural lipid is cholesterol.

32. The composition according to claim 24, wherein the polymer-conjugated lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and any combination thereof.

33. The composition according to claim 32, wherein the polymer-conjugated lipid is selected from distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG-PEG2000), methoxypoly(ethylene glycol) ditetradecylacetamide (ALC-0159), and combinations thereof.

34. The composition according to claim 20, wherein the effective amount of VZV RNA is 25 μg to 200 μg.

35. The composition according to claim 34, wherein the effective amount of VZV RNA is 50 μg to 100 μg.

36. The composition according to claim 20, wherein the vaccine is in the form of an injectable dosage.

37. The composition according to claim 36, wherein the vaccine is a liquid formulation or a lyophilized formulation.

38. A method for preparing the composition according to any one of claims 20 to 22, 24 to 37, comprising the step of mixing the RNA of the VZV with the pharmaceutically acceptable carrier.

39. A method for preparing the composition according to claim 38, comprising the step of encapsulating at least a portion of the RNA inside lipid nanoparticles.

40. A composition according to claim 1, for use in inducing a protective immune response against VZV in a subject requiring it.

41. The composition according to claim 40, wherein the protective immune response includes the production of a neutralizing antibody.

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