Herpes zoster mRNA vaccine, method of preparation and use.

Description

1 / 121 Herpes zoster mRNA vaccine, method of preparation and use. TECHNICAL FIELD

[0001] This disclosure pertains to the technical field of mRNA vaccines and relates specifically to a herpes zoster mRNA vaccine, a method of preparation thereof and a use thereof. FUNDAMENTALS

[0002] Herpes zoster is an infectious skin disease caused by the reactivation of the varicella-zoster virus (VZV), which has been latent in the dorsal root ganglia or cranial ganglia for a prolonged period. Herpes zoster is a common dermatological disease that is often accompanied by neuropathic pain, in addition to skin damage. The disease occurs mainly in elderly people, immunosuppressed or immunodeficient groups, and is more common in spring and autumn. The incidence shows a significant increase with age.

[0003] VZV is not completely eliminated from the human body after recovery from varicella infection, but remains latent in cranial ganglia, dorsal root ganglia, and autonomic ganglia throughout the neuroaxis and persists throughout life. After VZV infection resulting in varicella, the body produces specific antibodies against VZV and cellular immunity mediated by T cells (e.g., CD4+ / CD8+ / memory T cells, etc.). These two immunological functions play an important role in maintaining latent VZV infection and preventing herpes zoster. VZV can reactivate spontaneously with age or when certain external or internal factors cause suppression of the body's immunity, leading to herpes zoster (HZ).

[0004] Common triggering factors for herpes zoster include age factors (e.g., old age), cellular immunodeficiency, genetic susceptibility, trauma, systemic diseases (e.g., diabetes, kidney disease, etc.), stress, fatigue, etc. In contrast to chickenpox infection in children, herpes zoster in adults (elderly) can easily lead to complications, with postherpetic neuralgia (PHN) being the most common complication, which can cause pain lasting 3 to 12 months after the herpes zoster has healed.

[0005] Both herpes zoster and postherpetic neuralgia are common and prevalent diseases. Chronic pain (postherpetic neuralgia) and other complications caused by Petition 870260035339, dated 04 / 15 / 2026, pp. 129 / 249 2 / 121 Herpes zoster, commonly known as shingles, seriously affects the quality of life of patients, particularly affecting the elderly and those with weak immune systems. Due to the unknown mechanism of the pain, post-herpetic neuralgia remains one of the most intractable pains.

[0006] Herpes zoster has two notable characteristics: a high infection rate; and significant suffering for patients. Research data from Europe and the United States show that the annual incidence of herpes zoster is generally 2 to 6%. The incidence tends to increase with age. Statistics indicate that the incidence among individuals aged 10 to 49 is approximately 4%, while the incidence among those over 75 is as high as 14%. Furthermore, women are slightly more likely to develop herpes zoster than men. The global incidence of herpes zoster is increasing. Research data from 1994 to 2018 show that the annual incidence of herpes zoster increases by 3.1% per year, with a more pronounced increase in incidence, especially among individuals aged 20 to 49.

[0007] Herpes zoster has not been included in the category of Class A and B notifiable infectious diseases in China, and there have been relatively few relevant epidemiological studies across the entire population. According to statistics, in China, approximately 99.5% of adults aged 50 years or older are carriers of latent VZV, with about 1.56 million new cases reported each year. In China, the average annual incidence of herpes zoster among individuals aged 50 to 60 years is 2.66%, while the average annual incidence among those aged 80 years or older is 8.55%. The incidence of postherpetic neuralgia (PHN) also increases with age, with studies showing that in China, the incidence of PHN among elderly patients with herpes zoster is 18.8%, and the incidence of PHN among those over 75 years of age is 31.7%.

[0008] Vaccines are the most effective way to prevent shingles. The main technological routes involved in the development of shingles vaccines include live attenuated vaccines, recombinant protein vaccines, adenovirus vector vaccines, and mRNA vaccines.

[0009] There are only four vaccines available worldwide to prevent shingles: Zostavax® (discontinued), a live attenuated vaccine from Merck; Shingrix®, a recombinant protein vaccine from GlaxoSmithKline (GSK); SkyZoster® (sold only in Korea). Petition 870260035339, dated 04 / 15 / 2026, pp. 130 / 249 3 / 121 South) from SK Chemicals; and a recently launched live attenuated vaccine from BCHT Biotechnology in China. Merck's Zostavax® live attenuated vaccine was the first shingles vaccine approved for marketing worldwide, launched in the United States and Europe, respectively, in 2006. However, due to its low protective efficacy (with a protective efficiency of approximately 70% effective in reducing the development of shingles) and inadequacy for individuals with immunosuppression or immune system disorders, its scope of application was very limited, and the vaccine was discontinued in 2018. GSK's Shingrix® recombinant protein vaccine was launched in the United States in 2017, then in Europe in 2018, and sought approval in China in 2019. With global sales reaching US$2.4 billion in 2021 and US$3.2 billion in 2022, the vaccine has been among the top ten best-selling vaccines in the world for several consecutive years.However, the vaccine is a protein subunit vaccine, which is expensive and has difficulty scaling up production capacity due to the use of a special adjuvant from GSK. More importantly, the vaccine has serious side effects, with a significantly higher incidence of adverse reactions than that of live attenuated vaccines.

[0010] mRNA vaccines represent the third generation of vaccine technology, following traditional vaccines and protein subunit vaccines. The mRNA vaccine enters human cells through a specific delivery system and uses the body's own cells to translate the mRNA into protein. The expressed protein becomes a specific antigenic protein possessed by the virus and is recognized as a foreign antigen by antigen-presenting cells (APCs), which drives the maturation of dendritic cells (DCs) and subsequently activates B cells and T cells to generate a robust immune response, triggering humoral and cellular immune responses. The mRNA vaccine breaks the pattern of immune activation of traditional vaccines by innovatively using the body's own cells to produce antigens, thus activating dual specific immunity, establishing immune memory, and providing longer-lasting specific immunity.The greatest advantage of mRNA vaccines lies in their ability to be rapidly developed once the antigenic gene sequences of the pathogen are known. Other technical advantages include rapid design and construction, high adaptability to viral mutations, an efficient and universal platform for fully synthetic production processes, ease of standardized production, and more. Petition 870260035339, dated 04 / 15 / 2026, pp. 131 / 249 4 / 121 of mRNA vaccines result in short production chains and development cycles, relatively simple processes, and rapid large-scale production capabilities.

[0011] The herpes zoster mRNA vaccine has the following advantages over traditional vaccines: 1) it is a non-infectious and non-integrating agent with no risk of infection or insertion of mutations; 2) the mRNA vaccine activates specific immunity in humans through in vivo expression of antigens, allowing for more durable and effective specific immunity; 3) the mRNA vaccine allows for stable distribution in cells for efficient in vivo expression; 4) the production of mRNA vaccines can undergo rigorous quality control.

[0012] gE, an important target protein for the development of herpes zoster vaccines, is one of the most abundant glycoproteins in VZV and plays a key role in viral replication and viral transmission between ganglion cells. As a highly glycosylated type I membrane protein, gE can be transported between the endoplasmic reticulum (ER), the trans-Golgi network (TGN), and endosomes.

[0013] Based on the above problems, in order to further reduce the risk of herpes zoster in individuals with low immunity, there is an urgent need to provide a new mRNA vaccine to prevent VZV, which can significantly increase the effect of humoral and cellular immunity after vaccination. SUMMARY

[0014] This disclosure provides a herpes zoster mRNA vaccine that can safely target the body's cellular mechanism to produce virtually any protein of interest, ranging from natural proteins to antibodies and other entirely novel proteins that may have preventive activity inside and outside cells. The herpes zoster mRNA vaccine of this disclosure can be used to elicit a balanced immune response against VZV, including cellular and humoral immune responses, without many of the risks associated with attenuated viral vaccination.

[0015] RNA (e.g., mRNA) vaccines can be used in a variety of settings, depending on the prevalence of infection or the degree or level of unmet medical need. RNA (e.g., mRNA) vaccines can be used to prevent VZV in a variety of genotypes, strains, and isolates. RNA vaccines (e.g., mRNA) Petition 870260035339, dated 04 / 15 / 2026, pp. 132 / 249 5 / 121 example, mRNA) are superior because they generate much greater antibody potency and earlier responses than commercially available antiviral therapeutic treatments. While not wishing to be limited by theory, it is believed that, like mRNA polynucleotides, RNA vaccines are better engineered to produce an appropriate protein conformation through translation when the RNA vaccine attaches to a natural cellular mechanism. Unlike traditional vaccines, which are manufactured ex vivo and can trigger adverse cellular responses, RNA (e.g., mRNA) vaccines are delivered to cellular systems in a more natural way.

[0016] This disclosure describes the innovative design of a series of mRNA vaccine candidate antigens based on the important biological structure and function of the VZV gE protein, compares their immunogenicity with that of prior art mRNA vaccines (e.g., YK-VZV-007 antigen, see CN108472309A; and the commercially available recombinant subunit vaccine Shingrix®), and successfully screens a series of herpes zoster mRNA vaccine candidates with novel antigenic structures and superior immunogenicity, providing the basis for the successful development of herpes zoster mRNA vaccines.

[0017] Based on the wild-type VZV gE glycoprotein, this disclosure obtains a variety of ribonucleic acids encoding VZV gE glycoprotein variants through a specific combination (including sequence truncation, site mutation, and / or sequence deletion) and consequently provides a novel mRNA vaccine for VZV prevention. Experiments have demonstrated that the vaccine can significantly enhance the effect of humoral and cellular immunity after vaccination, which includes: improving IgG antibody content against the VZV gE protein, increasing the number of immune cells (including CD4+ and CD8+ cells secreting IFN-γ and / or IL-2+), and promoting cytokine secretion (including IFN-γ and IL-2+) by immune cells in the herpes zoster mRNA vaccine.

[0018] The mRNA vaccine, as provided in this document, may comprise an RNA polynucleotide of at least one VZV glycoprotein provided in the attached Sequence Listing, or a fragment, homologous (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), variant or derivative thereof. Petition 870260035339, dated 04 / 15 / 2026, pp. 133 / 249 6 / 121

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

[0020] In some embodiments, the VZV gE polypeptide (total length) comprises amino acids 1 to 623 (e.g., SEQ ID NO: 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 variant. In some embodiments, the VZV gE variant is a truncated VZV gE polypeptide lacking an anchor domain (ER retention domain).

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

[0023] In some embodiments, the truncated VZV gE polypeptide comprises amino acids 1 to 573 (e.g., SEQ ID NO: 15, 59, 63, 67, 71, 75, 79, 83, 87, 91).

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

[0025] In some embodiments, the truncated VZV gE polypeptide comprises amino acids 1 to 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 to 601 (e.g., SEQ ID NO: 155, 159).

[0027] In some embodiments, the VZV gE variant is a full-length or truncated polypeptide. In this document, the VZV gE variant further comprises a mutation in one or more motifs associated with Golgi or trans-Golgi network (TGN) targeting, resulting in decreased targeting or localization of the VZV gE polypeptide to Golgi or TGN. In particular, the present inventors have unexpectedly discovered that motifs associated with gE protein targeting to Golgi or trans-Golgi network comprise an A568Y569R570V571 motif (with a specific mutant antigen sequence, such as YK-VZV-011 (SEQ ID NO: 35)), especially having an A568D mutation and / or a Y569K mutation.

[0028] In addition, in some forms, the gE variant of VZV may have a Y582A / G mutation, or a Y569K / A mutation, or a combination of Y582G and Y569A mutations. Other mutation types also include a site mutation in an acid motif. Petition 870260035339, dated 04 / 15 / 2026, pp. 134 / 249 7 / 121 phosphorylated, as S593EST596DT598 (SEQ ID NO: 182).

[0029] In some embodiments, the VZV gE variant polypeptide is a full-length or truncated polypeptide. In this document, the VZV gE variant polypeptide further comprises a mutation in one or more Y582AGL585 motifs (SEQ ID NO: 185) associated with VZV gE internalization or endocytosis, resulting in reduced endocytosis of the VZV gE polypeptide.

[0030] In some embodiments, the gE variant of VZV is a full-length polypeptide having a Y569K mutation (SEQ ID NO: 27, 31, 35, 39, 43, 47, 143, 147).

[0031] In some embodiments, the gE variant of VZV is a full-length polypeptide having a Y582A mutation (SEQ ID NO: 23, 27, 31, 43, 47, 135, 139, 171).

[0032] In some embodiments, the gE variant of VZV is a full-length polypeptide having a Y582A mutation and a Y569K mutation (SEQ ID NO: 27, 31, 43, 47).

[0033] In some embodiments, the gE variant of VZV is a full-length polypeptide having a sequence A593EA595A596DA598 (SEQ ID NO: 183).

[0034] In some embodiments, the gE variant of VZV is a full-length or truncated polypeptide having an A593EA595A596DA598 sequence (SEQ ID NO: 183) and having a Y582G mutation, or a Y569K mutation, or a combination of Y582G and Y569K mutations.

[0035] In some embodiments, the gE variant of VZV comprises any one or more site mutations selected from the group consisting of: A568D, Y569K, Y569A, R570E, V571K, Y582A, S593A, S595A, T596A, T598A; preferably A568D, Y569K, R570E, V571K.

[0036] In some embodiments, the site mutation is any of the following combinations: 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) Petition 870260035339, de 15 / 04 / 2026, pág. 135 / 249 8 / 121 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; (YKVZV-039) 13) A568D; Y569K; R570E; V571K; Y582A; S593A; S595A; T596A; T598A; (YKVZV-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-VZV012) 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 gE variant of VZV further comprises a sequence deletion, wherein the sequence deletion is selected from any one or more of the group consisting of: 1)A568, 2) Y569RVDKSPYNQS579 (SEQ ID NO: 186), 3) Y569RVDKSPYNQSMYYAGLPV587 (SEQ ID NO: 187).

[0038] The VZV gE antigen of this disclosure has several mutation combinations, comprising amino acid truncations at the C-terminus of the gE antigen and deletions or substitutions at key amino acid sites, as further described below by means of specific examples.

[0039] This disclosure also provides a method of preparation for composition, comprising: Petition 870260035339, dated 04 / 15 / 2026, pp. 136 / 249 9 / 121 provide a template that can transcribe VZV RNA; Transcribe the RNA using the template under conditions suitable for transcription.

[0040] In some embodiments, the preparation method comprises the following steps: synthesizing a DNA sequence of the VZV gE antigen and then recombining it into the PVAX1 vector by homologous recombination; to obtain a plasmid after transformation and culture of E. coli; to obtain a linearized plasmid after plasmid digestion; Using T7 RNA polymerase with the linearized plasmid as a template, add NTP starting materials, followed by co-transcriptional capping, and then purify to obtain RNA, preferably mRNA.

[0041] In some preferred embodiments, the purification method comprises one or a combination of the following: lithium chloride precipitation, affinity chromatography, ultrafiltration exchange and cellulose chromatography.

[0042] In some embodiments, the preparation method for the composition comprises synthesizing a DNA sequence according to an antigen gene and inserting it into a plasmid DNA construct. The plasmid DNA is cleaved using DNA restriction endonuclease to obtain a linearized template. Under the catalysis of T7 RNA polymerase, with the linearized plasmid as a template, key starting materials such as NTP analogs and caps are added for transcription and mRNA synthesis, followed by RNA modification where a cap is added at the 5' end. The modified mRNA is then purified by methods such as lithium chloride precipitation and affinity chromatography to remove contaminants, enzymes, free nucleotides, and other impurities.

[0043] In a third aspect, the composition of the present disclosure is in the form of a vaccine, that is, a vaccine against herpes zoster, comprising a pharmaceutically acceptable carrier in addition to the composition.

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

[0045] In some preferred embodiments, the lipid nanoparticle comprises, for example, a cationic lipid, a neutral lipid, a structural lipid, and a lipid conjugated with a polymer. Petition 870260035339, dated 04 / 15 / 2026, pp. 137 / 249 10 / 121

[0046] In a fourth aspect, the present disclosure also provides a method for preparing the vaccine composition, comprising mixing VZV RNA with a pharmaceutically acceptable carrier, for example, to encapsulate at least a portion of the RNA in lipid nanoparticles. In some embodiments, the preparation method comprises the following steps: mixing RNA, preferably mRNA, with lipid nanoparticles to encapsulate it in lipid nanoparticles, followed by purification to remove an unencapsulated component.

[0047] In some more specific embodiments, the preparation method comprises the following specific steps: formulating the mRNA into lipid nanoparticles (LNPs), i.e., mixing the purified mRNA with a lipid mixture on a microfluidic chip, allowing the self-assembly of lipid nanoparticles, with the mRNA encapsulated in liposomes; the lipid nanoparticle solution is subjected to dialysis or filtration to remove unencapsulated mRNA, non-aqueous solvent, and bacteria; the filtered lipid nanoparticle solution is packaged in sterile vials for storage to complete the preparation and production of an mRNA vaccine.

[0048] This publication discovered for the first time: Based on the wild-type VZV gE glycoprotein, this disclosure obtains a series of ribonucleic acids encoding VZV gE glycoprotein variants through a specific combination (including sequence truncation, site mutation, and / or sequence deletion) and provides a novel mRNA vaccine for VZV prevention. Experiments have demonstrated that the vaccine can significantly enhance the effect of humoral and cellular immunity after vaccination, including: improving IgG antibody content against the VZV gE protein, increasing the number of immune cells (including CD4+ and CD8+ cells secreting IFN-γ and / or IL-2+), and promoting cytokine secretion (including IFN-γ and IL-2+) by immune cells in the herpes zoster mRNA vaccine. Specifically, this disclosure includes the following findings and results: 1. The Western Blot detection results of 43 projected VZV gE antigen variants showed that 39 antigen sequences exhibited significant protein expression, including YK-VZV-001, YK-VZV-003, YK-VZV-009, YK-VZV-001, YK-VZV-011, YKVZV-012, YK-VZV-013, YK-VZV-014, YK-VZV-015, YK-VZV-016, YK-VZV-017, YK- Petition 870260035339, of 15 / 04 / 2026, p. 138 / 249 11 / 121 VZV-018, YK-VZV-019, YK-VZV-020, YK-VZV-021, YK-VZV-022, YK-VZV-023, YKVZV-024, YK-VZV-025, YK-VZV-026, YK-VZV-020, YK-VZV-028, YK-VZV-028 YK-VZV-029, YKVZV-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-038 YK-VZV-039, YK-VZV-040, YK-VZV-044, as well as YK-VZV-004 (Comparative Example 1), YK-VZV-006 (Comparative Example 2), YK-VZV-045 (Comparative Example 3), and YK-VZV-040 (Comparative Example 4), can serve as effective candidate antigens for subsequent immunogenicity screening and evaluation.

[0049] 2. Based on the 39 sequences above and the gE-specific antibody IgG titer results on day 42, a total of 14 variants were extracted, including 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-VZV-028, as well as 4 control sequences including 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).

[0050] The effect of these sequences was 3.3 to 5.3 times that of the positive control Shingrix® and 1.8 to 2.8 times that of the YK-VZV-007 sequence (Comparative Example 4). The effect of these sequences was superior to 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).

[0051] 3. As shown by the results of measuring the percentage of CD4+ T cells and CD8+ cells that secrete IFN-γ and IL-2 in the spleens of mice, (i) a total of 9 sequences were screened based on the percentage of CD4+ T cells, including YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020, YK-VZV-012, YKVZV-018, YK-VZV-028, YK-VZV-038 and YK-VZV-031; The total percentage of TIFN-γ4CD4+ cells and IL-2+ CD4+ T cells was 2 times or more and up to 7 times that of the Shingrix® positive control and approximately 1.8 to 5.3 times that of YK-VZV-007 (Comparative Example 4); The percentage of IFN-γ4CD4+ T cells was 1.00 to 2.00%, which was 3 times or more and up to 6.7 times that of the positive control vaccine Shingrix® and approximately 2.2 to 4.4 times Petition 870260035339, dated 04 / 15 / 2026, pp. 139 / 249 12 / 121 a of YK-VZV-007 (Comparative Example 4); The percentage of IL-2+ CD4+ T cells was greater than 1%, which was 3.7 times or more than that of the positive control vaccine Shingrix® and 2.2 times or more than that of YK-VZV-007; (ii) a total of 5 sequences were further screened based on the percentage of CD8+ T cells, including YK-VZV-010, YK-VZV-013, YK-VZV-020, YK-VZV-018 and YK-VZV-011; The total percentage of IFN-γ+CD8+ T cells and IL-2+CD8+ T cells was 4 to 7 times or more that of the Shingrix® vaccine and approximately 3 to 5 times that of YK-VZV-007 (Comparative Example 4); The percentage of IFN-γ+CD8+ T cells was ideal at 5.0% or more, which was 4.5 to 7 times or more than that of the Shingrix® positive control vaccine and approximately 2.5 to 4.5 times that of YK-VZV-007; The percentage of IL-2+ CD8+ T cells was ideal at 0.3 to 0.5%, which was 2.5 to 4 times or more than that of the Shingrix® positive control vaccine and approximately 1.5 to 3 times that of YK-VZV-007; The results were significantly superior to those of the comparative examples (YK-VZV-004, YK-VZV-006, YK-VZV-045 and YK-VZV-007).

[0052] 4. To validate the above results, the titer of VZV gE-bound IgG antibodies in the serum of mice immunized on day 56 was measured again for validation based on the day 42 test results.

[0053] The results showed that on day 56 after mice were immunized with mRNA vaccines including 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, the gE-specific IgG antibody titers of these vaccines were higher than the Shingrix® positive control, being approximately 1.8 to 5.0 times that of the Shingrix® vaccine; Specifically, the GMT antibody of YK-VZV-011, YK-VZV-013, YK-VZV-020, YK-VZV-018, and YK-VZV-010 can reach 650 x 10⁴ or more, being 4.0 to 5.0 times or more than that of the Shingrix® vaccine and 2 times or more than that of YK-VZV-007 (Comparative Example 4); furthermore, the results were significantly better than those of YK-VZV-004 (Comparative Example 1) and YK-VZV-045 (Comparative Example 3). Petition 870260035339, dated 04 / 15 / 2026, pp. 140 / 249 13 / 121

[0054] 5. Through comprehensive analysis of IgG results and multiparameter flow cytometry, a total of 5 ideal antigen sequences (including YK-VZV-010, YKVZV-013, YK-VZV-011, YK-VZV-020 and YK-VZV-018) were screened and subsequently validated by ELISPOT, and the results are as follows: (i) The amount of IFN-γ cytokine secreted by T cells stimulated by the mRNA vaccine was 500 SFU or more, with a maximum close to 600 SFU, which was approximately 16 to 19 times that of the Shingrix® positive control and 2.5 to 3.0 times that of Comparative Example 4 (YK-VZV-007); and which was significantly better than that of the secreted and stimulated mRNA vaccine cytokine corresponding to Comparative Example 1 (YK-VZV-004), Comparative Example 2 (YK-VZV-006) or Example Comparative 3 (YK-VZV-045).

[0055] Specifically, the mRNA vaccine prepared from the YK-VZV-010 sequence reached a maximum of 598.8 SFU, which was 18.7 times that of the Shingrix® positive control and almost 3 times that of the YK-VZV-007 control.

[0056] The IFN-γ secretion levels from high to low were YK-VZV-010, YK-VZV-011, YK-VZV-018, YK-VZV-020 and YK-VZV-013, with no significant difference between the groups, indicating a good and significant immunological effect.

[0057] (ii) The amount of IL-2 cytokine secreted by T cells stimulated by the mRNA vaccine was 400 to 600 SFU or more, which was 10 to 16 times that of the Shingrix® positive control and 2.0 to 3.0 times that of the YK-VZV-007 control; and which was significantly better than that of the cytokine secreted and stimulated by the mRNA vaccine corresponding to Comparative Example 1 (YK-VZV-004), Comparative Example 2 (YK-VZV-006) or Example Comparative 3 (YK-VZV-045).

[0058] Specifically, the mRNA vaccine prepared from the YK-VZV-010 sequence achieved a maximum of 614.5 SFU, which was 16.0 times that of the Shingrix® positive control and 3 times or more that of the YK-VZV-007 control.

[0059] The IL-2 secretion levels from high to low were YK-VZV-010, YK-VZV-011, YK-VZV-018, YK-VZV-013 and YK-VZV-020, with no significant difference between the groups, indicating a good and significant immunological effect.

[0060] 6. In addition, the antigen localization results of the five sequences above Petition 870260035339, dated 04 / 15 / 2026, pp. 141 / 249 14 / 121 indicated its location in the Golgi apparatus, cell membrane, or cytoplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. 1 shows the main functional domains of the VZV gE protein and a series of deletion mutants; FIG. 2 shows the antigen expression of YK-VZV-001 for YK-VZV-045; FIG. 3 shows the cellular localization of VZV gE antigens from YK-VZV-010, YKVZV-011 and YK-VZV-013; FIG. 4 shows the cellular localization of VZV gE antigens from YK-VZV-018 and YKVZV-020, where green represents gE antigen; yellow and red represent GM130 and TGN46, respectively; white arrow represents cell membrane; and orange arrow represents Golgi apparatus. DETAILED DESCRIPTION OF THE PREFERRED MODALITY

[0062] The present disclosure is further described below by way of example. It should be understood that the examples in this disclosure are given only to illustrate the present disclosure and are not intended to limit the present disclosure, and any simple improvements to the present disclosure under the premise of the technical solution of the present disclosure are within the scope of protection of the present disclosure.

[0063] As used in this document, the term selected from means selecting any one or any combination of two or three or more of the objects or elements listed.

[0064] It should be understood that the term a compound, as used herein, may include the compound, an N-oxide thereof, a solvate thereof, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, and a mixture thereof.

[0065] Antigen sequence design: Antigens are proteins capable of inducing an immune response (e.g., causing an immune system to produce antibodies against the antigens). As used herein, the term antigen is used to encompass immunogenic proteins and immunogenic fragments (an immunogenic fragment that induces or is capable of inducing an immune response) to a varicella-zoster virus (e.g., VZV), unless otherwise indicated. It should be understood that the term protein encompasses peptides and the term Petition 870260035339, dated 04 / 15 / 2026, pp. 142 / 249 15 / 121 antigen comprises antigenic fragments. Exemplary sequences of the VZV antigens and the RNA encoding the VZV antigens of the composition of this disclosure are provided in Table 1.

[0066] Nucleic acids: Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). The composition of the present disclosure comprises an 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 comprises a 5' UTR, a 3' UTR, a poly(A) tail and / or a 5' cap analog.

[0067] Messenger RNA (mRNA) is any RNA that encodes (at least one) protein (a polymer of naturally occurring, unnatural, or modified amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. Those skilled in the art will appreciate that, unless otherwise indicated, the nucleic acid sequences set forth in this disclosure may recite a T in a representative DNA sequence, but where the sequence represents RNA (e.g., mRNA), the T will be replaced by a U. Consequently, any DNA disclosed and identified in this document by a particular sequence identification number also discloses a corresponding RNA (e.g., mRNA) sequence that is complementary to the DNA, in which each T in the DNA sequence is replaced by a U.

[0068] An open reading frame (ORF) is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG)) and ending with a stop codon (e.g., TAA, TAG, or TGA, or UAA or UAG). An ORF typically encodes a protein. It should be understood that the sequences disclosed in this document may still include additional elements, such as 5' and 3' UTRs.

[0069] RNA variant: Antigen variants or other polypeptide variants refer to molecules that differ in their amino acid sequence from a wild-type, native, or reference sequence. Antigen / polypeptide variants may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence, compared to a native or reference sequence. Typically, variants have at least 50% identity with a wild-type, native, or reference sequence. In Petition 870260035339, dated 04 / 15 / 2026, pp. 143 / 249 16 / 121 In some forms, the variants share at least 80% or at least 90% identity with a wild-type, native, or reference sequence.

[0070] In some embodiments, a composition comprises an RNA or an RNA ORF comprising a nucleotide sequence of any of the sequences provided in this document (see, for example, Sequence Listing and Table 1), or comprising 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% identity with a nucleotide sequence of any of the sequences provided in this document.

[0071] The term identity refers to a relationship between the sequences of two or more polypeptides (e.g., antigens) or polynucleotides (nucleic acids), as determined by sequence comparison. Identity also refers to the degree of sequence relatedness between or between sequences, as determined by the number of matches between chains of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identical matches between the smallest of two or more sequences with gap alignments (if any) addressed by a specific mathematical model or computer program (e.g., algorithm). The identity of related antigens or nucleic acids can be easily calculated by known methods.Percent identity (%), when applied to a polypeptide or polynucleotide sequence, is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid or nucleic acid sequence of a second sequence after alignment of the sequences and introduction of gaps (if necessary), to achieve maximum percent identity. Methods and computer programs for alignment are well known in the art. It should be understood that identity depends on a percent identity calculation, but may differ in value due to gaps and penalties introduced in the calculation.Generally, variants of a specific polynucleotide or polypeptide (e.g., antigen) have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity with the polynucleotide or polypeptide of origin. Petition 870260035339, dated 04 / 15 / 2026, pp. 144 / 249 17 / 121 specific reference, as determined by the sequence alignment programs and parameters described in this document and known to those skilled in the art. Such alignment tools include those of the BLAST suite (Stephen F. Altsch, et al. (1997), Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25: 3389-3402).

[0072] As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions, and / or additions and deletions relative to the reference sequences, particularly the antigen sequences disclosed in this document, are included within the scope of this disclosure. Amino acid residues located in the carboxy- and amino-terminal regions of a peptide or protein's amino acid sequence may optionally be deleted to provide a truncated sequence. Depending on the structure and use of the sequence, gE proteins have a number of important domains related to their primary functions (see FIG. 1). gE proteins have a number of important domains related to their primary functions (see FIG. 3).(1) TM transmembrane domain, consisting of amino acids 539 to 559; (2) A568YRV571, which mediates gE transport between the endoplasmic reticulum (ER), transGolgi network (TGN), and endosomes; (3) Y582AGL585, which mediates gE transport between the endoplasmic reticulum (ER), transGolgi network (TGN), and endosomes; (4) S593ES595T590DT598, which is an important glycosylation site and mediates gE transport between the endoplasmic reticulum (ER), transGolgi network (TGN), and endosomes. In some embodiments, important structural sites may be removed and replaced with appropriate residues, thereby altering antigen transport and expression to increase antigen immunogenicity. Such sequences can be easily identified by those skilled in the art.It should also be understood that some of the sequences provided in this document contain sequence markers or terminal peptide sequences (e.g., at the N-terminus or C-terminus) that may be deleted, for example, before use in the preparation of an RNA vaccine (e.g., mRNA).

[0073] As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of varicella-zoster virus antigens of interest. For example, any protein fragment of a reference protein (meaning a Petition 870260035339, dated 04 / 15 / 2026, pages 145 / 249 18 / 121 polypeptide sequence that is at least one amino acid residue shorter than a reference antigen sequence, but otherwise identical), provided that the fragment is immunogenic and confers a protective immune response against varicella-zoster virus. In addition to variants that are identical to a reference protein but are truncated, in some embodiments, the antigen comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more site mutations, as shown in any of the sequences provided or referenced in this document. Antigenic antigens / polypeptides can vary in length from approximately 4, 6, or 8 amino acids to full-length proteins.

[0074] VZV Antigen Variants: This disclosure includes a variant VZV antigenic polypeptide. In some embodiments, the variant VZV antigenic polypeptide is a variant VZV gE polypeptide. The variant VZV gE polypeptide is designed to avoid ER / Golgi retention of the polypeptide, leading to increased surface expression of the antigen. In some embodiments, the variant gE polypeptide is truncated to remove an ER-retaining portion or a cytoplasmic tail portion of the polypeptide. In some embodiments, the variant VZV gE polypeptide is mutated to reduce the localization of the VZV polypeptide to the ER / Golgi / TGN. Such modifications inhibit ER trapping and therefore accelerate transport to the cell membrane. Thus, in some embodiments, the VZV glycoprotein is a variant gE polypeptide.VZV gE has a TGN targeting sequence at its C-terminus and is transported from the ER to the TGN in gE-infected and transfected cells. Most gE in the TGN appears to be retrieved from the plasma membrane by endocytosis and distributed from endosomes to the TGN, followed by recycling back to the plasma membrane. gE accumulates in the TGN along with other VZV proteins (e.g., tegument proteins) associated with the production of fully enveloped VZV virions. Thus, mutations to reduce TGN localization and endocytosis contribute to gE transport to the cell membrane. The VZV variant gE polypeptide can be any truncated polypeptide lacking an anchor domain (ER retention domain).For example, the VZV variant gE polypeptide may be a truncated VZV gE polypeptide comprising at least amino acids 1 to 124, such as amino acids 1 to 124, 1 to 140, 1 to 160, 1 to 200, 1 to 250, 1 to 300, 1 to 350, 1 to 360, 1 to 400, 1 to 450, 1 to 500, 1 to 511, 1 to 550 and 1 to 561, as well as a polypeptide fragment with fragment sizes. Petition 870260035339, dated 04 / 15 / 2026, pp. 146 / 249 19 / 121 within the cited size ranges. In some embodiments, the VZV variant gE polypeptide is a truncated polypeptide lacking a carboxy-terminal tail domain. Thus, in some embodiments, the truncated VZV gE polypeptide comprises amino acids 1 to 573 of SEQ ID NO: 59. In some embodiments, the variant VZV gE polypeptide has at least one mutation in one or more ER-associated retention motifs, wherein the mutation in one or more motifs results in decreased retention of the VZV gE polypeptide in the ER and / or Golgi. In some embodiments, the variant VZV gE polypeptide has at least one mutation in one or more phosphorylated acid motifs. For example, the variant VZV gE polypeptide may be a full-length VZV gE polypeptide with a Y582G mutation, a Y569K mutation, or a Y582G mutation and a Y569K mutation.Alternatively, the VZV variant gE polypeptide may be an antigenic fragment comprising, for example, amino acids 1 to 573 of VZV gE and having a Y569K mutation. Alternatively, the VZV variant gE polypeptide may be an antigenic fragment with a mutation in a phosphorylated acid motif (e.g., SSTT motif, SEQ ID NO: 188). For example, the VZV variant gE polypeptide may be an antigenic fragment with an AEAADA sequence (SEQ ID NO: 183).

[0075] Naturally occurring eukaryotic mRNA molecules may contain stabilizing elements, including but not limited to an untranslated region (UTR) at their 5' end (5' UTR) and / or at their 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 the 3' UTR are typically transcribed from genomic DNA and are elements of early mRNA. Structural features typical of mature mRNA (e.g., the 5' cap and the 3' poly(A) tail) are typically added to the transcribed (early) mRNA during mRNA processing.

[0076] In some embodiments, the composition comprises an RNA polynucleotide with an open reading frame encoding at least one antigenic polypeptide with at least one modification and at least one 5' terminal cap and is formulated within a lipid nanoparticle. 5' capping of polynucleotides can be performed simultaneously during an in vitro transcription reaction using an RNA cap chemical analog to generate a 5' guanosine cap structure according to the manufacturer's protocols. In some Petition 870260035339, dated 04 / 15 / 2026, pp. 147 / 249 In cases 20 / 121, 5' capping of the modified RNA can also be performed post-transcriptionally using a vaccinia virus capping enzyme to generate a cap structure. Cap structure 1 can be generated using a vaccinia virus capping enzyme and a 2'-O methyltransferase to generate: m7G(5')ppp(5')G-2'-O-methyl. Cap structure 2 can be generated from Cap structure 1 followed by 2'-O-methylation of the antepenultimate 5' nucleotide using a 2'-O methyltransferase. Cap structure 3 can be generated from Cap structure 2 followed by 2'-O-methylation of the preantepenultimate 5' nucleotide using a 2'-O methyltransferase. The enzymes can be derived from recombinant sources. The 3' poly(A) tail is typically a stretch of adenine nucleotides added to the 3' end of the transcribed mRNA. In some cases, it can comprise up to approximately 400 adenine nucleotides.In some modalities, the length of the 3' poly(A) tail may be an essential element in relation to the stability of the individual mRNA.

[0077] Chemical Modification: In some embodiments, the composition of the present disclosure comprises an RNA having an open reading frame encoding a VZV antigen, wherein the nucleic acid comprises nucleotides and / or nucleosides that may be standard (unmodified) or modified as known in the art. In some embodiments, the nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides may be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications may include those in the sugar, backbone, or nucleobase portions of the nucleotides and / or nucleosides, as recognized in the art.

[0078] In some embodiments, the modified naturally occurring nucleotides or nucleosides of the present disclosure are nucleotides or nucleosides as generally known or recognized in the art. Non-limiting examples of such modified naturally occurring nucleotides and nucleosides can be found particularly in the widely recognized MODOMICS database.

[0079] In some embodiments, the modified, non-naturally occurring nucleotides or nucleosides of the present disclosure are nucleotides or nucleosides as generally known or recognized in the art. Non-limiting examples of such nucleotides and Petition 870260035339, dated 04 / 15 / 2026, pages 148 / 249 21 / 121 modified nucleosides of non-natural occurrence can be found particularly in the published US Applications No. 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.

[0080] Thus, the nucleic acids of disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) may comprise standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, unnaturally occurring nucleotides and nucleosides, or any combination thereof.

[0081] In some embodiments, the nucleic acids of disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) comprise a plurality (more than one) of different types of standard and / or modified nucleotides and nucleosides. In some embodiments, a particular region of the nucleic acid contains one, two, or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0082] In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced into a cell or organism, exhibits reduced degradation in the cell or organism, respectively, compared to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

[0083] In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

[0084] In some embodiments, nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise unnaturally unmodified nucleotides that are introduced during the synthesis or post-synthesis of nucleic acids to achieve desired functions or properties. The modification may be present in internucleotide bonds, purine or pyrimidine bases, or sugars. The modification may be chemically synthesized or introduced by a polymerase at the terminus of a chain or elsewhere in the chain. Any region of a nucleic acid may be chemically modified. Petition 870260035339, dated 04 / 15 / 2026, pp. 149 / 249 22 / 121 modified.

[0085] This disclosure provides modified nucleosides and nucleotides of a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids). A nucleoside refers to a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to in this document as a nucleobase). A nucleotide refers to a nucleoside that includes a phosphate group. Modified nucleotides may be synthesized by any useful method, such as chemical, enzymatic, or recombinant methods, to include one or more modified or non-natural nucleosides. The nucleic acid may comprise one or more regions of linked nucleosides. Such regions may have variable backbone linkages. The linkage may be a standard phosphodiester bond, in which case the nucleic acid would comprise a region of nucleotides.

[0086] Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, for example, in those nucleic acids having at least one chemical modification. An example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any base / sugar or ligand combination may be incorporated into the nucleic acids of the present disclosure.

[0087] In some embodiments, the modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxyuridine (mo5U), 5-methylcytidine (m5C), and / or pseudouridine (ψ). In some embodiments, the modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine, and / or 5-methoxycytidine. In some embodiments, the polyribonucleotides Petition 870260035339, dated 04 / 15 / 2026, pages 150 / 249 23 / 121 comprise a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.

[0088] In some embodiments, the mRNA of the present disclosure comprises a 1-methyl-pseudouridine (m1ψ) substitution at one or more or all uridine positions of the nucleic acid.

[0089] In some embodiments, the mRNA of the present disclosure comprises a 1-methyl-pseudouridine (m1ψ) substitution at one or more or all of the uridine positions of the nucleic acid and a 5-methylcytidine substitution at one or more or all of the cytidine positions of the nucleic acid.

[0090] In some embodiments, the mRNA of the present disclosure comprises a pseudouridine (ψ) substitution at one or more or all uridine positions of the nucleic acid.

[0091] In some embodiments, mRNA is uniformly modified (e.g., fully modified, modified along the sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with 1-methylpseudouridine, meaning that all uridine residues in the mRNA sequence are replaced by 1-methylpseudouridine. Similarly, a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by substitution with a modified residue, such as those presented above.

[0092] Nucleic acid may contain from approximately 1% to approximately 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C) or any intermediate percentage (for example, 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% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100% and 95% to 100%). It should be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C. Petition 870260035339, dated 04 / 15 / 2026, pp. 151 / 249 24 / 121

[0093] The mRNA may contain a minimum of 1% and a maximum of 100% modified nucleotides or any intermediate percentage, such as 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 a modified pyrimidine, such as a modified uracil 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 replaced by a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure or it can be replaced by a plurality of compounds having different structures (for example, 2, 3, 4 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 replaced by a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine may be replaced by a compound with a single unique structure or may be replaced by a plurality of compounds with different structures (e.g., 2, 3, 4, or more unique structures).

[0094] Untranslated Region (UTR): The mRNA of the present disclosure may comprise one or more regions or portions that act or function as an untranslated region. When mRNA is designed to encode at least one antigen of interest, the nucleic acid may comprise one or more of these untranslated regions (UTRs). A wild-type untranslated region of nucleic acid is transcribed but not translated. In mRNA, the 5' UTR begins at the transcription start site and continues through, but not including, the start codon; while the 3' UTR begins immediately after the stop codon and continues through to the transcription termination signal. There is growing evidence for the regulatory role of the UTR in the stability and translation of nucleic acid molecules. The regulatory features of the UTR can be incorporated into the polynucleotides of the present disclosure to particularly enhance the stability of the molecule.Specific features can also be incorporated to ensure controlled downregulation of transcripts in case they are misdirected to undesirable organ sites. A variety of UTR 5' and UTR 3' sequences are known and are... Petition 870260035339, dated 04 / 15 / 2026, pp. 152 / 249 25 / 121 available in the technique.

[0095] In some embodiments of the present disclosure, the 5' UTR is a heterologous UTR, that is, a UTR found in nature associated with a different ORF. In another embodiment, the 5' UTR is a synthetic UTR, that is, it does not occur in nature. Synthetic UTRs include UTRs that have been mutated to enhance their properties, such as UTRs that increase gene expression, as well as fully synthetic UTRs. Exemplary 5' UTRs include Xenopus or human α-globin or β-globin (8278063; 9012219), human cytochrome b-245a polypeptide, hydroxysteroid (17b) dehydrogenase, and smoke mosaic virus (US8278063, 9012219).

[0096] In some embodiments, the UTR 5' of this disclosure comprises a selected sequence of SEQ ID NO: 173 to 174.

[0097] In some preferred embodiments, the UTR 5' of this disclosure comprises a selected sequence of SEQ ID NO: 174.

[0098] A 3' UTR is a region of an mRNA that is directly downstream (3') of the stop codon (the codon in an mRNA transcript that signals a termination of translation). The 3' UTR does not encode a protein (it is non-coding). Native or wild-type 3' UTRs are known to have stretches of adenosine (A) and uridine (U) incorporated into them. These AU-rich elements are particularly prevalent in genes with high turnover rates.

[0099] The introduction, removal, or modification of AU-rich elements (AREs) in the 3' UTR region can be used to modulate the stability of nucleic acids (e.g., RNA) of the present disclosure. By manipulating a specific nucleic acid, one or more copies of an ARE can be introduced to make the nucleic acids of the present disclosure less stable, thereby reducing translation and decreasing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability, thereby increasing translation and the production of the resulting protein.

[0100] The 3' UTR can be heterologous or synthetic. With respect to 3' UTRs, globin UTRs (including Xenopus β-globin UTRs and human β-globin UTRs) are known in the art (8278063, 9012219, US20110086907). A nucleic acid (e.g., mRNA) encoding a modified β-globin with enhanced stability in some types Petition 870260035339, dated 04 / 15 / 2026, pp. 153 / 249 The 26 / 121 cell model was developed by cloning two sequential 3' human β-globin UTRs from head to tail and is well known in the art (US2012 / 0195936, WO2014 / 071963).

[0101] In some embodiments, UTR 3' of this disclosure comprises a selected sequence of SEQ ID NO: 175.

[0102] Those versed in the art will understand that 5' UTRs that are heterologous or synthetic can be used with any desired 3' UTR sequence. For example, a heterologous 5' UTR can be used with a synthetic 3' UTR or a heterologous 3' UTR.

[0103] It should be understood that any UTR of any gene can be incorporated into a region of a nucleic acid. Furthermore, multiple wild-type UTRs of any known gene can be used. It is also within the scope of this disclosure to provide artificial UTRs that are not variants of the wild-type region. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they are selected, or they may be altered in orientation or location. Thus, a 5' or 3' UTR may be inverted, shortened, lengthened, or prepared with one or more other 5' or 3' UTRs. As used herein, the term altered, as it refers to a UTR sequence, means that the UTR has been altered in some way relative to a reference sequence.For example, a 3' or 5' UTR can be altered relative to a wild-type or native UTR by changing its orientation or location as taught above, or it can be altered by adding additional nucleotides, deleting nucleotides, exchanging or transposing nucleotides. Any of these alterations producing an altered UTR (whether 3' or 5') comprises a variant UTR.

[0104] In vitro RNA transcription: cDNA encoding the polynucleotides described in this document can be transcribed using an in vitro transcription (IVT) system. In some embodiments, the RNA transcript is generated in an in vitro transcription reaction using an unamplified linearized DNA template to generate the RNA transcript. In some embodiments, the DNA template is isolated DNA. In some embodiments, the DNA template is cDNA. In some embodiments, the cDNA is formed by digestion of circular plasmid DNA. In some embodiments, cells, such as bacterial cells, such as E. coli cells, such as DH-1 cells, are transfected with a plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the Petition 870260035339, dated 04 / 15 / 2026, pp. 154 / 249 27 / 121 The plasmid DNA is then isolated and purified. In some embodiments, the DNA template comprises an RNA polymerase promoter, such as a 5' T7 promoter, operationally linked to the target gene.

[0105] In some embodiments, an in vitro transcription template encodes a 5' untranslated region (UTR), contains an open reading frame, and encodes a 3' UTR and a poly(A) tail. The specific nucleic acid sequence composition and length of the in vitro transcription template will depend on the mRNA encoded by the template.

[0106] The 5' untranslated region (UTR) refers to a region of an mRNA that is directly upstream (i.e., 5') of the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not code for a polypeptide. When generating RNA transcripts, the 5' UTR may comprise a promoter sequence. Such promoter sequences are known in the art. It should be understood that such promoter sequences will not be present in the vaccine of this disclosure.

[0107] Untranslated 3' region (UTR) refers to a region of an mRNA that is directly downstream (i.e., 3') of the stop codon (i.e., the codon in an mRNA transcript that signals a termination of translation) that does not code for a polypeptide.

[0108] An open reading frame is a continuous stretch of DNA that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA) and codes for a polypeptide.

[0109] A poly(A) tail is a region of an mRNA that is downstream, such as directly downstream (i.e., 3') of the 3' UTR, containing multiple consecutive adenosine monophosphates. The poly(A) tail may contain 10 to 300 adenosine monophosphates. For example, the 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, the poly(A) tail contains 50 to 250 adenosine monophosphates. In a relevant biological context (e.g., in cells, in vivo), the poly(A) tail functions to protect an mRNA from enzymatic degradation, for example, in the cytoplasm, and assists in the termination of transcription and / or export of mRNA from the nucleus and translation.

[0110] In some forms, nucleic acid comprises 200 to 3000 nucleotides. By Petition 870260035339, dated 04 / 15 / 2026, pages 155 / 249 28 / 121 For example, nucleic acid can comprise 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

[0111] The in vitro transcription system typically comprises a transcription buffer, a nucleotide triphosphate (NTP), an RNase inhibitor, an inorganic pyrophosphate, and a polymerase.

[0112] NTP can be manufactured in-house, selected from a supplier, or synthesized as described herein. NTP can be selected from, but is not limited to, those described in this document, including natural and non-natural (modified) NTPs.

[0113] Any number of RNA polymerases or variants may be used in the method of the present disclosure. The polymerase may be selected from, but is not limited to, a phage RNA polymerase, such as a T7 RNA polymerase, a T3 RNA polymerase, an SP6 RNA polymerase and / or a mutant polymerase, such as, but not limited to, polymerases capable of incorporating modified nucleic acids and / or modified nucleotides (including chemically modified nucleic acids and / or nucleotides). Some embodiments exclude the use of DNase.

[0114] In some embodiments, the RNA transcript may be capped by enzymatic capping or co-transcriptional capping reactions. In some embodiments, the RNA comprises a 5' terminal cap, such as 7mG(5')ppp(5')NlmpNp.

[0115] mRNA Purification: Purification of the nucleic acids described in this document may include, but is not limited to, nucleic acid purification, quality assurance, and quality control. Purification may be performed by methods known in the art, such as, but not limited to, lithium chloride precipitation, beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNA™ oligo-T capture probes (Corporation, Vedbaek, Denmark), or HPLC-based purification methods, such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term purified, when used in relation to a nucleic acid, such as a purified nucleic acid, refers to one that is separated from at least one contaminant. A contaminant is any substance Petition 870260035339, dated 04 / 15 / 2026, pages 156 / 249 29 / 121 that renders another substance unsuitable, impure, or inferior. Thus, purified nucleic acid (e.g., DNA and RNA) is present in a form or environment different from that in which it is found in nature, or a form or environment different from that in which it was present before being subjected to a processing or purification method.

[0116] Quality assurance and / or quality control verification may be performed using methods such as, but not limited to, gel electrophoresis, UV absorption or analytical HPLC.

[0117] Quantification: In some embodiments, nucleic acid can be quantified using methods such as, but not limited to, ultraviolet-visible (UV / Vis) spectroscopy. A non-limiting example of a UV / Vis spectrometer is a spectrometer (Thermo Fisher, Waltham, MA). The quantified nucleic acid can be analyzed to determine if the nucleic acid is of appropriate size and to verify that no nucleic acid degradation has occurred. Nucleic acid degradation can be verified by methods such as, but not limited to, agarose gel electrophoresis; HPLC-based purification methods, such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC); liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE), and capillary gel electrophoresis (CGE).

[0118] Lipid nanoparticle (LNP): In some embodiments, the RNA (e.g., mRNA) of the present disclosure is formulated into a lipid nanoparticle (LNP). The lipid nanoparticle typically comprises an ionizable cationic lipid, a non-cationic lipid, a sterol, a PEG lipid component, and a nucleic acid payload of interest. The lipid nanoparticles of the present disclosure can be generated using components, compositions and methods commonly known in the art, see, for example, CN116535381B, TW202340136A, CN116178193B, CN116178193B, CN115784921B, CN115745820B, CN115677518B, CN114957027B, CN114044741B, CN116854754A, CN116785265A, CN116789764A, CN116396178A, CN116375592A and CN116082275A, all of which are incorporated herein by reference in their entirety.

[0119] As used herein, the term cationic lipid refers to a lipid that is positively charged at a selected pH value. Cationic lipids are prone to Petition 870260035339, dated 04 / 15 / 2026, pp. 157 / 249 30 / 121 bind to negatively charged nucleic acids, that is, to form lipid nanoparticles (LNPs) by interacting with negatively charged phosphate groups present in nucleic acids through electrostatic forces. LNPs are currently one of the main carriers of distribution.

[0120] As used herein, the term neutral lipid refers to an auxiliary lipid that is discharged or exists in a zwitterionic form at a selected pH value. The neutral lipid can regulate the flow of nanoparticles in a lipid bilayer structure and improve efficiency by promoting lipid phase transition and can also affect target organ specificity.

[0121] As used herein, a structural lipid refers to a lipid that enhances the stability of nanoparticles by filling the gaps between lipids. The structural lipid may be selected from, but is not limited to, the group consisting of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassinosterol, 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, corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or any combination thereof.

[0122] In some embodiments, the molar ratio of the cationic lipid to the structural lipid is about 1:1 to 5:1, for example, about 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 or 2.0:1.

[0123] In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is about (1 to 10):1, for example, about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0124] In some embodiments, the molar ratio of the cationic lipid to the structural lipid is about (1 to 5):1, for example, about 1:1, 2:1, 3:1, 4:1 or 5:1.

[0125] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25 to 65):(5 to 25):(25 to 45):(0.5 to 5). For example, in some embodiments, by proportion (relative number of moles), 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; the polymer-conjugated lipid Petition 870260035339, dated 04 / 15 / 2026, pages 158 / 249 31 / 121 polymer is 0.5 part, 1 part, 1.5 part, 2 parts, 2.5 parts, 3.0 parts, 3.5 parts, 4.0 parts, 4.5 parts or 5.0 parts.

[0126] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (49 to 50):10:(38.5 to 39.5):1. For example, in some embodiments, by proportion (relative number of moles), 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; the polymer-conjugated lipid is 1 part; such as 50:10:38.5:1.5 or 49:10:39.5:1.5.

[0127] As used herein, the term polymer-conjugated lipid refers to a lipid modified with polyethylene glycol (PEG). Hydrophilic PEG stabilizes LNPs, regulates nanoparticle size by limiting lipid fusion, and increases nanoparticle half-life by reducing nonspecific 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, or PEG-modified dialkylglycerol. The molecular weight of the PEG for the PEG modification is generally from 350 to 5000 Da. For example, the lipid conjugated with the polymer is selected from one or more of distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), or methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[0128] In one embodiment of the compositions / carriers of the present disclosure, the lipid conjugated with polymer is DMG-PEG2000.

[0129] In one embodiment of the compositions / carriers of the present disclosure, the carrier comprises a neutral lipid, a structural lipid and a polymer-conjugated lipid, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer-conjugated lipid is (25 to 65):(5 to 25):(25 to 45):(0.5 to 5), such as (45 to 55):(9 to 11):(34 to 43):(0.5 to 2.5).

[0130] In one embodiment of the compositions / carriers of the present disclosure, the carrier comprises a neutral lipid, a structural lipid, and a polymer-conjugated lipid, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the lipid Petition 870260035339, dated 04 / 15 / 2026, pages 159 / 249 32 / 121 combined with polymer is 50:10:38.5:1.5.

[0131] As used herein, thermoalkyl is intended to include linear branched saturated aliphatic monovalent hydrocarbon groups with a specified number of carbon atoms. As used herein, thermoalkylene is intended to include linear branched saturated aliphatic divalent hydrocarbon groups with a specified number of carbon atoms. Cn-m refers to a group with carbon atoms. For example, C2-5 alkylene included C2 alkylene, C3 alkylene, C4 alkylene and C5 alkylene. C28 alkylene includes C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, Cô alkylene, C7 alkylene and C8 alkylene. C1-6 alkylene includes C1 alkylene, C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene and C1-3 alkylene. C1-3 alkylene includes C1 alkylene, C2 alkylene and C3 alkylene. C1-15 linear alkyl includes a linear alkyl group with 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbon atoms.C12-25 branched alkyl includes a branched alkyl group with 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms. An alkyl (or alkylene) group may be unsubstituted, or an alkyl (or alkylene) group may be substituted in which at least one hydrogen is replaced by another chemical group.

[0132] As used herein, a therapeutically effective amount is an amount of a therapeutic agent that improves a disease or condition when administered to a patient. A prophylactically effective amount is an amount of a prophylactic agent that prevents a disease or condition when administered to an individual. The therapeutically effective amount for a therapeutic agent or the prophylactically effective amount for a prophylactic agent varies with the therapeutic / prophylactic agent, the disease state and its severity, the age and weight of a patient / individual to be treated / prevented, etc. The therapeutically effective amount and the prophylactically effective amount can be routinely determined by those skilled in the art based on their knowledge and the present disclosure.

[0133] As used in this document, the composition can be used to induce a protective immune response against VZV in an individual in need thereof, wherein the protective immune response comprises, for example, the production of a neutralizing antibody. In some embodiments, the individual is immunocompromised. In some embodiments, the individual is 10 years of age or older, such as 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, Petition 870260035339, dated 04 / 15 / 2026, pp. 160 / 249 33 / 121 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.

[0134] The vaccines of this disclosure are typically formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticle comprises at least one ionizable cationic lipid, at least one non-cationic lipid, at least one sterol and / or at least one polyethylene glycol (PEG) modified lipid.

[0135] In some preferred embodiments, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a structural lipid and a lipid conjugated with a polymer.

[0136] In some preferred embodiments, the cationic lipid is a compound having the structure of formula I, or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein G1 is C1-6 alkylene; G2 is C2-8 alkylene; G3 is C1-3 alkylene; L1 is C1-5 linear alkyl; L2 is C12-25 branched alkyl. For example, YK-009 with the structure of formula II (see patent CN114044741B).

[0138] In some preferred embodiments, the cationic lipid is a compound having the structure of formula II, or an N-oxide, a solvate, a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein Gi is C2-8 alkylene; G2 is C2-8 alkylene; Li is -C(O)O- or OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C6-25 linear or branched alkyl; R2 is C6-25 linear or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is Petition 870260035339, dated 04 / 15 / 2026, pp. 161 / 249 34 / 121 (CH2)2-, -(CH2)3-, or -(CH2)4-. For example, YK-401 with the formula structure II-I or YK402 with the formula structure II-II (see patent CN115784921B). G3N-G,—L,—R, LZ N Gp Lp Rp

[0139] formula II YK-402 formula II-II

[0140] In some preferred embodiments, the cationic lipid is a compound having the structure of formula III, or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein Gi is C1-6 alkylene; G2 is C2-8 alkylene; Ri is C6-20 linear or branched alkyl; R2 is C12-25 branched alkyl; 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 structure of formula III-I or YK-202 with the structure of formula III-II (see patent CN115677518B). Petition 870260035339, dated 04 / 15 / 2026, pp. 162 / 249 35 / 121

[0141] Formula III formula IϠ-I THE YK-202 formula III-II

[0142] In some preferred embodiments, the cationic lipid is a compound having the structure of formula IV, or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein Gi is C1-8 alkylene; G2 is C2-8 alkylene; Ri is C6-25 linear or branched alkyl; R2 is C12-25 linear or branched alkyl; G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3, -CH2CH3 or -CH2CH2OH; For example, YK-305 with the structure of formula IV-I or YK-310 with the structure of formula IV-II (see patent CN115745820B).

[0143] formula IV Petition 870260035339, dated 04 / 15 / 2026, pp. 163 / 249 36 / 121 IV-I formula formula IV-II

[0144] In some preferred embodiments, the cationic lipid is a compound having the structure of formula V, or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein G1 and G2 are each independently unsubstituted C₁-C₂O alkylene; G3 is unsubstituted C₁-C₁₂ alkylene; R1 and R2 are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl; R3 is OR₅, N, -C(=O)OR₄, -OC(=O)R₄, or -NR₅C(=O)R₄; R4 is C₁-C₁₂ hydrocarbyl; and R5 is H or C₁-C₂O hydrocarbyl; for example, ALC₃15 having the structure of formula VI (see patent CN108368028B). Formula V, see (CN108368028B, ALC0315) Petition 870260035339, dated 04 / 15 / 2026, pp. 164 / 249 37 / 121 Formula VI

[0145] In some preferred embodiments, the cationic lipid is a compound with the structure of formula VI, or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein R4 is selected from -(CH2)nQ and -(CH2)nCHQR; Q is selected from the group consisting of -OR, -OH, -O(CH2)nN(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(H)(R), -N(R)S(O)2R8 and heterocycle; né 1, 2 or 3; for example, SM 102 with the structure of formula VI-I (see patent CN1105209A).

[0146] formula VI, see (CN 110520409A, SM102) formula VI-I

[0147] In some preferred embodiments, the cationic lipid is a compound having the structure of formula VII, or an N-oxide, a solvate, a pharmaceutically acceptable salt, or a stereoisomer thereof. Petition 870260035339, dated 04 / 15 / 2026, pp. 165 / 249 38 / 121 formula VII, see (CN102625696B, DLIN-MC3-DMA)

[0148] In some preferred embodiments, the cationic lipid comprises YK-009, YK-401, YK-305, ALC0315, SM102 and DLIN-MC3-DMA.

[0149] In some preferred embodiments, the molar ratio of the cationic lipid to the neutral lipid is (1 to 10):1.

[0150] In some preferred embodiments, the molar ratio of the cationic lipid to the structural lipid is (1 to 5): 1.

[0151] In some preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the lipid conjugated with polymer is (25 to 75):(5 to 25):(15 to 65):(0.5 to 10).

[0152] In some preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the lipid conjugated with polymer is (35 to 49):(7.5 to 15):(35 to 55):(1 to 5), such as 49:10:43.5:1.5.

[0153] In some preferred embodiments, the neutral lipid includes one or more phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and derivatives thereof.

[0154] In some more preferred embodiments, the neutral lipid is selected from one or more of the following: l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-snglycero-phosphocholine (DMPC), l,2-dioleoyl-glycero-phosphocholine-3 (DOPC), 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleo-glycero-3-phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2cholesteryl-hemisuccinoyl-sn-glycero-3 -phosphocholine (OChemsPC), 1 -hexadecyl-sn-glycero-3 phosphocholine (LCL6), Lyso l,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-snglycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-snglycero-3-phospholamine (DOPE), l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3 Petition 870260035339, dated 04 / 15 / 2026, pp. 166 / 249 39 / 121 phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, sodium salt of 1,2-dioleoyl-sn-glycero-3-phosphorac-(1-glycerol) (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or mixtures thereof.

[0155] In some of the more preferred modalities, the neutral lipid is DOPE and / or DSPC.

[0156] In some preferred embodiments, the structural lipid is selected from one or more of the following: cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassinosterol, tomatine, ursolic acid, α-tocopherol, or corticosteroid.

[0157] In some of the more preferred forms, the structural lipid is cholesterol.

[0158] In some preferred embodiments, the lipid conjugated with polymer is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol.

[0159] In some more preferred embodiments, the lipid conjugated with polymer is selected from one or more of distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPEPEG2000), dimyristoylglycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) or methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[0160] Pharmaceutical preparation: This document provides compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention or treatment of varicella-zoster virus in humans and other mammals, for example. The compositions provided in this document can be used as therapeutic or prophylactic agents. They can be used in medications to prevent and / or treat herpes zoster.

[0161] In some embodiments, a herpes zoster vaccine containing RNA, as described in this document, can be administered to an individual (e.g., a mammalian individual, such as a human individual), and the RNA polynucleotide is translated in vivo into Petition 870260035339, dated 04 / 15 / 2026, pp. 167 / 249 40 / 121 produce an antigenic polypeptide (antigen).

[0162] An effective amount of a composition (e.g., comprising RNA) is based at least in part on the target tissue, target cell type, mode of administration, physical characteristics of the RNA (e.g., length, nucleotide composition, and / or degree of nucleoside modification), other vaccine components, and other determinants such as age, body weight, height, sex, and general health of the individual. Typically, an effective amount of a composition provides an induced or enhanced immune response based on antigen production in the individual's cells. In some embodiments, an effective amount of a composition containing an RNA polynucleotide having at least one chemical modification is more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen.Increased antigen production can be evidenced by increased cell transfection (the percentage of cells transfected with the RNA vaccine), increased translation and / or expression of polynucleotide proteins, decreased nucleic acid degradation (e.g., as evidenced by increased translation time of proteins from a modified polynucleotide), or altered host cell antigen-specific immune response.

[0163] The term pharmaceutical composition refers to the combination of an active agent with an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo. A pharmaceutically acceptable carrier does not cause undesirable physiological effects after or during administration to an individual. The carrier in the pharmaceutical composition must also be acceptable in the sense that it is compatible with and capable of stabilizing the active ingredient. One or more solubilizers may be used as pharmaceutical carriers for the distribution of the active agent.

[0164] In some embodiments, the composition (comprising a polynucleotide and the polypeptide it encodes) according to the present disclosure can be used to treat or prevent herpes zoster. The composition can be administered prophylactically or therapeutically to healthy individuals as part of an active immunization schedule or at the onset of infection during the incubation phase or during active infection after the onset of symptoms. In some embodiments, the amount of RNA delivered to the cell, tissue or Petition 870260035339, dated 04 / 15 / 2026, pp. 168 / 249 41 / 121 per individual may be an effective amount for immunoprophylaxis.

[0165] In some modalities, the composition may be administered intramuscularly.

[0166] The composition may be used in a variety of settings, depending on the prevalence of infection or the degree or level of unmet medical need. As a non-limiting example, RNA vaccines may be used to treat and / or prevent a variety of infectious diseases. RNA vaccines have superior properties in that they produce much higher antibody titers, better neutralizing immunity, produce more durable immune responses, and / or produce earlier responses than commercially available vaccines.

[0167] Pharmaceutical compositions comprising RNA and / or complexes are provided in this document, optionally in combination with one or more pharmaceutically acceptable excipients. In addition to traditional excipients (e.g., any and all solvents, dispersing media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives), excipients may include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, RNA-transfected cells (e.g., for transplantation into an individual), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0168] RNA can be formulated or administered alone or in combination with one or more other components. For example, an immunizing composition may comprise other components including, but not limited to, adjuvants.

[0169] The relative amounts of the active ingredient, the pharmaceutically acceptable excipient and / or any additional ingredients in the pharmaceutical composition according to this disclosure will vary depending on the identity, size and / or condition of the individual being treated and also depending on the route by which the composition is to be administered. By way of example, the composition may comprise 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.

[0170] In some embodiments, an RNA is formulated using one or more excipients to increase mRNA stability, enhance cell transfection efficiency, and improve efficiency. Petition 870260035339, dated 04 / 15 / 2026, pp. 169 / 249 42 / 121 of protein translation, altering mRNA biodistribution to target specific tissues or cell types, etc.

[0171] Dosage / Administration: Immunizing compositions (e.g., RNA vaccines), methods, kits, and reagents for the prevention of varicella-zoster virus in humans and other mammals are provided in this document. The immunizing compositions may be used as therapeutic or prophylactic agents. In some embodiments, the immunizing compositions are used to provide prophylactic protection against herpes zoster.

[0172] An individual can be any mammal, including non-human primates and human individuals.

[0173] In some embodiments, an immunizing composition (e.g., RNA vaccine) is administered to an individual (e.g., a mammalian individual, such as a human individual) 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 an immune response in the individual.

[0174] Prophylactic protection against herpes zoster can be achieved after administration of the immunizing composition (e.g., RNA vaccine) of this disclosure. The immunizing composition can be administered once, twice, three times, four times or more, but a single administration of the vaccine may be sufficient (optionally followed by a single booster).

[0175] A method for inducing an immune response against a varicella-zoster virus antigen (or multiple antigens) in an individual is provided in one aspect of this disclosure. In some embodiments, the method comprises administering to an individual an immunizing composition comprising an RNA (e.g., mRNA) with an open reading frame encoding a VZV gE glycoprotein, thereby inducing a specific immune response to the varicella-zoster virus antigen in the individual, wherein the anti-antigen antibody titer in the individual is increased after vaccination relative to the anti-antigen antibody titer in an individual vaccinated with a traditional vaccine (e.g., GSK recombinant subunit vaccine). An anti-antigen antibody is a serum antibody that binds specifically to an antigen.

[0176] A prophylactically effective dose is an effective dose that prevents viral infection in Petition 870260035339, dated 04 / 15 / 2026, pp. 170 / 249 43 / 121 a clinically acceptable level. In some embodiments, the effective dose is a dose listed in a package insert for the vaccine. As used herein, traditional vaccine refers to a vaccine other than the mRNA vaccines of the present disclosure. For example, traditional vaccine includes, but is not limited to, live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, virus-like particle (VLP) vaccines, etc. In exemplary embodiments, traditional vaccine is a vaccine that has achieved regulatory approval and / or is registered by a national drug regulatory body, such as the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA).

[0177] A method for inducing an immune response against a varicella-zoster virus in an individual is provided in other aspects of this disclosure. The method comprises administering to an individual an immunizing composition (e.g., RNA vaccine) comprising an RNA polynucleotide comprising an open reading frame encoding a VZV gE antigen, thereby inducing a specific immune response to the varicella-zoster virus in the individual.

[0178] In other embodiments, the immune response is assessed by determining the protein (antibody titer) in the individual. In other embodiments, the ability of the serum or antibody of an immunized individual to neutralize viral uptake or reduce the transformation of human B lymphocytes by varicella-zoster virus is tested. In other embodiments, the ability to promote a robust T-cell response is measured using techniques recognized in the art.

[0179] Also provided in this document is a method for inducing an immune response against a varicella-zoster virus in an individual by administering to the individual an RNA with an open reading frame encoding a first antigen, wherein the RNA does not comprise a stabilizing element and wherein an adjuvant is not co-formulated or co-administered with the vaccine.

[0180] The immunizing composition (e.g., RNA vaccine) may be administered by any route that results in a therapeutically effective outcome. These routes include, but are not limited to, intradermal, intramuscular, or subcutaneous administration. The present disclosure provides a method comprising administering an RNA vaccine to an individual in need thereof. The exact amount required will vary from individual to individual. Petition 870260035339, dated 04 / 15 / 2026, pp. 171 / 249 44 / 121 individual, depending on the species, age and general condition of the individual, the severity of the disease, the particular composition, its mode of administration, its mode of activity, etc. RNA is typically formulated in the form of dosage units for ease of administration and uniformity of dosage. However, it should be understood that the total daily dosage of RNA may be determined by the attending physician within the scope of good medical judgment.The specific therapeutically effective, prophylactically effective, or imaging dose level appropriate for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the patient's age, body weight, general health, sex, and diet; the timing of administration, route of administration, and rate of excretion of the specific compound employed; the duration of treatment; medications used in combination with or coinciding with the specific compound employed; and similar factors well known in the medical art.

[0181] The effective amount of RNA, as provided in this document, may be as low as 5 μg, administered, for example, as a single dose or as two doses of 2.5 μg. In some embodiments, the effective amount is a total dose of 5 μg to 200 μg. For example, the effective amount may be a total dose of 5 μg, 10 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 55 pg, 60 pg, 65 pg, 70 pg, 75 pg, 80 pg, 85 pg, 90 pg, 95 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, 150 pg, 160 pg, 170 pg, 180 pg, 190 pg, or 200 pg. In some embodiments, the effective amount is a total dose of 5 μg to 200 μg. In some embodiments, the effective amount is a total dose of 10 μg. In some embodiments, the effective amount is a total dose of 20 μg. In some embodiments, the effective amount is a total dose of 75 μg. In some embodiments, the effective amount is a total dose of 150 μg. In some embodiments, the effective amount is a total dose of 200 μg.

[0182] Vaccine efficacy Some aspects of this disclosure provide a preparation of the immunizing composition (e.g., RNA vaccine), in which RNA is formulated in an amount effective to produce an antigen-specific immune response in an individual (e.g., to produce an antibody specific to a varicella-zoster virus antigen). An effective amount is a dose of RNA effective to produce a specific immune response. Petition 870260035339, dated 04 / 15 / 2026, pp. 172 / 249 45 / 121 of the antigen. A method for inducing an antigen-specific immune response in an individual is also provided in this document.

[0183] As used in this document, the immune response to the vaccine or LNP of this disclosure is the generation of a humoral and / or cellular immune response in an individual to (one or more) varicella-zoster virus proteins present in the vaccine. For the purposes of this disclosure, humoral immune response refers to an immune response mediated by antibody molecules, including, for example, secretory (IgA) or IgG molecules, while cellular immune response refers to an immune response mediated by T lymphocytes (e.g., CD4+ helper T cells and / or CD8+ T cells, such as CTLs) and / or other leukocytes. An important aspect of cellular immunity involves an antigen-specific response by cytotoxic T lymphocytes (CTLs). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (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 involves a specific antigen response by helper T cells. Helper T cells serve to stimulate the function and focus the activity of non-specific effector cells on cells displaying peptide antigens in association with MHC molecules on their surface. The cellular immune response also leads to the production of cytokines (e.g., IFN-γ, IL-2, TNF-β), chemokines, and other molecules produced by activated T cells and / or other leukocytes.

[0184] In some embodiments, the antigen-specific immune response is characterized by measuring an antibody titer of varicella-zoster virus antigen produced in an individual administered with the immunizing composition, as provided in this document. Antibody titer is a measurement of the amount of antibodies within an individual, for example, antibodies that are specific to a particular antigen (e.g., an anti-gE glycoprotein of VZV) or an epitope of the antigen. Antibody titer is typically expressed as the reciprocal of the maximum dilution that gives a positive result. For example, enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers.

[0185] In some modalities, antibody titers are used to assess whether a Petition 870260035339, dated 04 / 15 / 2026, pp. 173 / 249 46 / 121 individuals have been infected or to determine if immunization is necessary. In some embodiments, antibody titers are used to determine the strength of an autoimmune response, to determine if a booster immunisation is needed, to determine if a prior vaccine is effective, and to identify any recent or past infections. According to the present disclosure, antibody titers can be used to determine the strength of an immune response induced in an individual by an immunising composition (e.g., RNA vaccine).

[0186] In some embodiments, the antibody titer of VZV anti-gE antigen produced in the individual is increased by at least 1 log relative to a control (unvaccinated individual). For example, the antibody titer of VZV anti-gE antigen produced in the individual may be increased by at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, or at least 4 log relative to a control. In some embodiments, the antibody titer of VZV anti-gE antigen produced in the individual is increased by 1, 1.5, 2, 2.5, or 3 log relative to a control. In some embodiments, the antibody titer of VZV anti-gE antigen produced in the individual is increased by 1 to 4 log relative to a control. For example, the antibody titer of anti-gE VZV antigen produced in an individual may be increased 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 relative to a control.

[0187] In some embodiments, the antibody titer of VZV anti-gE antigen produced in the individual is increased by at least 2 times compared to a control (vaccinated with the traditional GSK Shingrix® vaccine). For example, the antibody titer of VZV anti-gE antigen produced in the individual may be increased by at least 1 time, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, or at least 3.5 times compared to the GSK control.

[0188] In some embodiments, the control is a varicella-zoster virus antigen antibody titer produced in an individual not administered with an immunizing composition (e.g., RNA vaccine). In some embodiments, the control is a varicella-zoster virus antigen antibody titer produced in an individual administered with the Shingrix® recombinant subunit vaccine.

[0189] In some forms, the ability of an immunizing composition (e.g., Petition 870260035339, dated 04 / 15 / 2026, pp. 174 / 249 The effectiveness of the 47 / 121 RNA vaccine is measured in a murine model. For example, the immunizing composition can be administered to a murine model and the murine model can be assayed for induction of neutralizing antibody titers. Viral challenge studies can also be used to evaluate the efficacy of the vaccines of this disclosure. For example, the immunizing composition can be administered to a murine model, the murine model can be challenged with virus, and the murine model can be tested for survival and / or immune response (e.g., T-cell response, cytokine response).

[0190] This disclosure also refers to the following modalities: 1. An immunogenic composition comprising a varicella-zoster virus (VZV) ribonucleic acid (RNA) encoding a wild-type VZV gE glycoprotein or a variant thereof; where the wild-type VZV glycoprotein gE sequence is SEQ ID NO: 3.

[0191] 2. The composition, according to embodiment 1, in which the VZV gE glycoprotein variant comprises one or more of the following mutations relative to the wild-type VZV gE glycoprotein: a sequence truncation, a site mutation, and a sequence deletion.

[0192] 3. The composition, according to embodiment 2, in which the sequence truncation comprises a lack of a carboxy-terminal tail domain.

[0193] 4. The composition, according to any of the modalities 1 to 3, in which the sequence truncation is selected from any of the group consisting of: 1) truncation of amino acids 540 to 623 at the C-terminal of the gE protein; and retention of amino acids 1 to 539 of the VZV gE protein; 2) truncation of amino acids 569 to 623 at the C-terminal of the gE protein; that is, retention of amino acids 1 to 568 of the VZV gE protein; 3) truncation of amino acids 574 to 623 at the C-terminal of the gE protein; that is, retention of amino acids 1 to 573 of the VZV gE protein; 4) truncation of amino acids 588 to 623 at the C-terminal of the gE protein; that is, retention of amino acids 1 to 587 of the VZV gE protein; 5) truncation of amino acids 602 to 623 at the C-terminal of the gE protein; that is, retention of amino acids 1 to 601 of the VZV gE protein. Petition 870260035339, dated 04 / 15 / 2026, pp. 175 / 249 48 / 121

[0194] 5. The composition, according to any of embodiments 2 to 4, in which the VZV gE glycoprotein variant comprises a site mutation selected from: a mutation in a motif associated with ER retention and endocytosis of the gE protein and / or at least one site mutation in at least one motif associated with Golgi or trans-Golgi network localization and at least one site mutation in a phosphorylated acid motif at the C-terminal of gE; where the motif associated with ER retention and gE protein endocytosis includes a Y582A583G584L585 motif (e.g., a mutation in the Y582A583G584L585 motif may prevent gE protein endocytosis and reduce gE antigen localization in the trans-Golgi network, such as the Y582A mutation site, YK-VZV-013 (SEQ ID NO: 43)); The motif associated with the localization of the gE protein to Golgi or trans-Golgi network includes an A568Y569R570V571 motif (for example, the A568Y569R570V571 motif is a specific mutant antigen sequence, such as YK-VZV-011 (SEQ ID NO: 35)); The phosphorylated acid motif at the C-terminus of gE includes an S593E594S595T596D597T598 motif (e.g., a mutation of the motif). The sequence S593E594S595T596D597T598 for a motif A593E594A595A596D597A598 may reduce the localization of the gE antigen in the trans-Golgi network, with a specific mutant antigen sequence, such as YK-VZV-010 (SEQ ID NO: 31)).

[0195] 6. The composition, according to any of embodiments 2 to 5, in which the VZV gE glycoprotein variant 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.

[0196] 7. The composition, according to any of the embodiments from 2 to 6, in which the VZV gE glycoprotein variant comprises a site mutation selected from: 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) Petition 870260035339, dated 04 / 15 / 2026, pp. 176 / 249 49 / 121 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; (YKVZV-039) 13) A568D; Y569K; R570E; V571K; Y582A; S593A; S595A; T596A; T598A; (YKVZV-014) 14) Y582G; S593A; S595A; T596A; T598A; (YK-VZV-040) 15) Y569K; Y582A; S593A; S595A; T596A; T598A; (YK-VZV-010) 13) A568D; Y569K; R570E; V571K; Y582A; S593A; S595A; T596A; T598A; (YKVZV-014) 17) Y582G; (YK-VZV-029) 18) A568D; (YK-VZV-018) 19) Y569K; (YK-VZV-020) 20) R570E; (YK-VZV-021) 21) V571K; e (YK-VZV-022) 22) Y582A; (YK-VZV-028, YK-VZV-036).

[0197] 8. The composition, according to any of the embodiments from 2 to 7, in which the VZV gE glycoprotein variant comprises a site mutation selected from: 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; (YKVZV-014); 15) Y569K; Y582A; S593A; S595A; T596A; T598A; (YK-VZV-010) Petition 870260035339, dated 04 / 15 / 2026, pp. 177 / 249 50 / 121 16) A568D; Y569K; R570E; V571K; S593A; S595A; T596A; T598A; (YK-VZV012) 18) A568D; (YK-VZV-018) 19) Y569K; (YK-VZV-020) 20) R570E; (YK-VZV-021); and 22) Y582A; (YK-VZV-028).

[0198] 9. The composition, according to any of embodiments 2 to 8, in which the VZV gE glycoprotein variant comprises a sequence deletion selected from: 1) A568; 2) Y569RVDKSPYNQS579; and 3) Y569RVDKSPYNQSMYYAGLPV587.

[0199] 10. The composition, according to any of the embodiments from 1 to 9, in which the VZV gE glycoprotein variant comprises a combination of a site mutation, a sequence truncation, and a sequence deletion selected from: Petition 870260035339, dated 04 / 15 / 2026, pp. 178 / 249 51 / 121 Table 1: VZV gE glycoprotein variants comprising combinations of sequence truncation, site mutations, and sequence deletions. Name Sequence truncation Site mutation Sequence deletion YK-VZV-018 gE (1 to 573) A568D No sequence deletion YK-VZV-020 gE (1 to 573) Y569K No sequence deletion YK-VZV-021 gE (1 to 573) R570E No sequence deletion YK-VZV-024 gE (1 to 573) A568D; Y569A; R570E; V571K No sequence deletion YK-VZV-028 gE (1 to 587) Y582A -Y569RVDKSPYNQS579- YK-VZV-030 gE (1 to 587) A568D; Y569K; R570E; V571K; Y582A No sequence deletion YK-VZV-031 gE (1 to 587) A568D; Y569K; R570E; V571K; Y582G No sequence deletion YK-VZV-038 No sequence truncation A568D; Y569K; R570E; V571K; Y582G No sequence deletion YK-VZV-009 No sequence truncation Y569K; Y582A No sequence deletion YK-VZV-010 No sequence truncation Y569K; Y582A; S593A; S595A; T596A; T598A No sequence deletion YK-VZV-011 No sequence truncation A568D; Y569K; R570E; V571K No sequence deletion YK-VZV-012 No sequence truncation A568D; Y569K; R570E; V571K; S593A; S595A; T596A;T598A No sequence deletion YK-VZV-013 No sequence truncation A568D; Y569K; R570E; V571K; Y582A No sequence deletion YK-VZV-014 No sequence truncation A568D; Y569K; R570E; V571K; Y582A; S593A; S595A; T596A; T598A No sequence deletion;

[0200]

[0201] 11. The composition, according to any of the embodiments from 1 to 10, in which the VZV gE glycoprotein variant comprises a combination of a site mutation, a sequence truncation and / or a sequence deletion selected from: Petition 870260035339, dated 04 / 15 / 2026, pp. 179 / 249 52 / 121 Table 2: VZV gE glycoprotein variants comprising site mutations and, optionally, sequence truncation and / or sequence deletion. Name Sequence truncation Site mutation Sequence deletion YK-VZV-018 gE (1 to 573) A568D No sequence deletion YK-VZV-020 gE (1 to 573) Y569K No sequence deletion YK-VZV-010 No sequence truncation Y569K; Y582A; S593A; S595A; T596A; T598A No sequence deletion YK-VZV-011 No sequence truncation A568D; Y569K; R570E; V571K No sequence deletion YK-VZV-013 No sequence truncation A568D; Y569K; R570E; V571K; Y582A No sequence deletion

[0202] 12. The composition according to any of embodiments 1 to 11, wherein the VZV gE glycoprotein variant comprises a sequence with 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 with the sequence of SEQ ID NO: 27, 31, 35, 39, 43, 47, 63, 71, 75, 87, 103, 111, 115 or 143.

[0203] 13. The composition, according to any of the embodiments from 1 to 12, in which the VZV gE glycoprotein variant comprises SEQ ID NO: 31, 35, 43, 63 or 71.

[0204] 14. The composition according to any of the modalities 1 to 13, in which the VZV RNA has an open reading frame (ORF) encoding a VZV gE glycoprotein or a variant thereof, wherein the open reading frame comprises a sequence with 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 with SEQ ID NO: 26, 30, 34, 38, 42, 46, 62, 70, 74, 86, 102, 110, 114 or 142.

[0205] 15. Composition, according to embodiment 14, in which the open read phase comprises a sequence of SEQ ID NO: 30, 34, 42, 62 or 70.

[0206] 16. The composition, according to any of the modalities from 1 to 15, in which the Petition 870260035339, dated 04 / 15 / 2026, pp. 180 / 249 53 / 121 VZV RNA also includes a 5' untranslated region (UTR).

[0207] 17. Composition, according to embodiment 16, wherein UTR 5' comprises a sequence as shown in SEQ ID NO: 173, 174, 175, 176 or 177.

[0208] 18. Composition, according to embodiment 17, wherein UTR 5' comprises a sequence as shown in SEQ ID NO: 174.

[0209] 19. The composition, according to any of the modalities from 1 to 18, in which the VZV RNA also includes a 3' untranslated region (UTR).

[0210] 20. Composition, according to embodiment 19, wherein UTR 3' comprises a sequence as shown in SEQ ID NO: 178, 179, 180 or 181.

[0211] 21. Composition, according to embodiment 20, wherein UTR 3' comprises a sequence as shown in SEQ ID NO: 178.

[0212] 22. The composition, according to any of the modalities from 1 to 21, in which the VZV RNA also includes a poly(A) tail.

[0213] 23. Composition, according to embodiment 22, in which the poly(A) tail has a length of 50 to 150 nucleotides.

[0214] 24. The composition, according to any of the modalities from 1 to 23, in which the VZV RNA also includes a 5' terminal cap.

[0215] 25. The composition, according to modality 24, in which the terminal cap 5' is 7mG(5')ppp(5')NlmpNp.

[0216] 26. The composition, according to any of the modalities from 14 to 25, in which the sequence of the open reading frame is optimized by codons.

[0217] 27. Composition, according to modality 26, in which the open reading phase sequence comprises at least one base modification.

[0218] 28. The composition, according to embodiment 27, in which the base modification is selected from one or more of the following: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxypseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine. Petition 870260035339, dated 04 / 15 / 2026, pp. 181 / 249 54 / 121

[0219] 29. The composition, according to embodiment 28, in which the base modification comprises the substitution of uracil for pseudouridine and / or N1-methylpseudouridine.

[0220] 30. The composition, according to embodiment 27, 28 or 29, in which the base modification is from 1 to 100% base modification, such as 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 a range with any of the above values ​​as endpoints.

[0221] 31. The composition according to any of the modalities 1 to 30, in which the VZV RNA comprises a sequence with 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 with SEQ ID NO: 28, 32, 36, 40, 44, 48, 64, 72, 76, 88, 104, 112, 116, or 144.

[0222] 32. The composition, according to embodiment 31, in which the VZV RNA comprises a sequence as shown in SEQ ID NO: 32, 36, 44, 64 or 72.

[0223] 33. The composition, according to any of the modalities 1 to 32, in which the VZV RNA is mRNA.

[0224] 34. The composition, according to any of the modalities 1 to 33, in which 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%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 25, 29, 33, 37, 41, 45, 61, 69, 73, 85, 101, 109, 113 or 141.

[0225] 35. The composition, according to embodiment 34, in which the RNA encoding the VZV gE glycoprotein comprises an RNA sequence corresponding to the DNA sequence, as shown in SEQ ID NO: 29, 33, 41, 61 or 69. Petition 870260035339, dated 04 / 15 / 2026, pp. 182 / 249 55 / 121

[0226] 36. A method for preparing the composition according to any of the embodiments from 1 to 35, comprising: to provide a template that can transcribe VZV RNA; Transcribe RNA using the template under conditions suitable for transcription.

[0227] 37. The method, according to embodiment 36, further comprising a purification step selected from: lithium chloride precipitation, affinity chromatography, ultrafiltration exchange and cellulose chromatography.

[0228] 38. The composition, according to any of the embodiments from 1 to 35, wherein the composition is a vaccine and further comprises a pharmaceutically acceptable carrier.

[0229] 39. The composition, according to embodiment 38, in which the carrier comprises a lipid mixture, such as a lipid nanoparticle (LNP).

[0230] 40. The composition, according to modality 38 or 39, in which the vaccine is an mRNA vaccine.

[0231] 41. The composition, according to embodiment 39 or 40, in which the lipid mixture is a lipid nanoparticle (LNP), for example, comprising a cationic lipid, a neutral lipid, a structural lipid and a lipid conjugated with a polymer.

[0232] 42. The composition, according to embodiment 41, wherein the cationic lipid is a compound having the structure of formula I, or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein Gi is C1-6 alkylene; G2 is C2-8 alkylene; G3 is C1-3 alkylene; Li is C1-15 linear alkyl; L2 is C12-25 branched alkyl; for example, the cationic lipid is YK-009 having the structure of formula II.

[0233] OH I formula I Petition 870260035339, dated 04 / 15 / 2026, pp. 183 / 249 56 / 121 VK-009 formula II

[0234] 43. The composition, according to embodiment 41, wherein the cationic lipid is a compound having the structure of formula II, or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein Gi is C2-5-alkylene; G2 is C2-8-alkylene; Li is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C6-25 linear or branched alkyl; R2 is C6-25 linear or branched alkyl; G3 is H0(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2-, -(CH2)3-, or -(CH2)4-; For example, the cationic lipid is YK-401 with the formula II-I structure or YK-402 with the formula II-II structure.

[0235] G3 NG!—b— l / N Gp L2 R2 G4 formula II-I Petition 870260035339, dated 04 / 15 / 2026, pp. 184 / 249 57 / 121 formula II-II

[0236] 44. The composition, according to embodiment 41, wherein the cationic lipid is a compound having the structure of formula III, or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein Gi is C1-6 alkylene; G2 is C2-8 alkylene; Ri is C6-20 linear or branched alkyl; R2 is C12-25 branched alkyl; 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.

[0237] Formula III formula III-I Petition 870260035339, dated 04 / 15 / 2026, pp. 185 / 249 58 / 121 O ΥK-202 formula IP-II

[0238] 45. The composition, according to embodiment 41, wherein the cationic lipid is a compound having the structure of formula IV, or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein Gi is C1-8 alkylene; G2 is C2-8 alkylene; Ri is C6-25 linear or branched alkyl; R2 is C12-25 linear or branched alkyl; G3 is HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3, -CH2CH3 or -CH2CH2OH; for example, YK-305 with the structure of formula IV-I or YK-310 with the structure of formula IV-II.

[0239] YK-305 formula IV-I Petition 870260035339, dated 04 / 15 / 2026, pp. 186 / 249 59 / 121 OH HO'' formula IV-II

[0240] 46. The composition, according to embodiment 41, wherein the cationic lipid is a compound having the structure of formula V, or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein G1 and G2 are each independently unsubstituted C₁-C₂O alkylene; G3 is unsubstituted C₁-C₁₂ alkylene; R1 and R2 are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl; R3 is OR₅, N, -C(=O)OR₄, -OC(=O)R₄, or -NR₅C(=O)R₄; R4 is C₁-C₁₂ hydrocarbyl; and R5 is H or C₁-C₂O hydrocarbyl; for example, ALC₃15 having the structure of formula VI.

[0241] HO R1 G1G2 R2formula V formula VI

[0242] 47. The composition, according to embodiment 41, in which the cationic lipid is a compound with the structure of formula VI, or an N-oxide, a solvate, a salt Petition 870260035339, dated 04 / 15 / 2026, pp. 187 / 249 60 / 121 pharmaceutically acceptable or a stereoisomer thereof, wherein R4 is selected from (CH2)nQ and -(C1hjnCHQR); Q is selected from the group consisting of -OR, -OH, O(CH2)nN(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 heterocycle; n is 1, 2 or 3; formula VI formula VI-I

[0243] 48. The composition, according to embodiment 41, wherein the cationic lipid is a compound having the structure of formula VII, or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof. Formula VII

[0244] 49. The composition, according to embodiment 41, in which the cationic lipid is selected from YK-009, YK-401, YK-305, ALC0315, SM102 and DLIN-MC3-DMA.

[0245] 50. The composition, according to any of the embodiments from 41 to 49, in which the molar ratio of the cationic lipid to the neutral lipid is (1 to 10): 1. Petition 870260035339, dated 04 / 15 / 2026, pp. 188 / 249 61 / 121

[0246] 51. The composition, according to any of the embodiments from 41 to 50, in which the molar ratio of the cationic lipid to the structural lipid is (1 to 5):1.

[0247] 52. The composition, according to any of the embodiments from 41 to 51, in which the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the lipid conjugated with polymer is (25 to 65):(5 to 25):(25 to 45):(0.5 to 5).

[0248] 53. The composition, according to embodiment 52, in which the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the lipid conjugated with polymer is (49 to 50):10:(38.5 to 39.5):1.5, such as 50:10:38.5:1.5 or 49:10:39.5:1.5.

[0249] 54. The composition, according to any of the embodiments from 41 to 53, in which the neutral lipid is selected from phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, derivatives thereof and any combination thereof.

[0250] 55. The composition, according to any of the modalities 41 to 54, whereby the neutral lipid is selected within: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2dimyristoyl-sn-glycero-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-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-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-snglycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-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, sodium salt of 1,2-dioleoyl-sn-glycero-3-phosphorac-(1-glycerol) (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl phosphatidyl ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine. Petition 870260035339, dated 04 / 15 / 2026, pp. 189 / 249 62 / 121 (LPE) and mixtures thereof.

[0251] 56. The composition, according to any of the modalities from 41 to 55, in which the neutral lipid is DOPE and / or DSPC.

[0252] 57. The composition, according to any of the embodiments from 41 to 56, in which the structural lipid is selected from: sterol, cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassinosterol, tomatine, ursolic acid, α-tocopherol, corticosteroid and any combination thereof.

[0253] 58. The composition, according to embodiment 57, in which the structural lipid comprises cholesterol; for example, the structural lipid is cholesterol.

[0254] 59. The composition, according to any of the embodiments from 41 to 56, in which the lipid conjugated with polymer 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.

[0255] 60. The composition, according to embodiment 59, in which the lipid conjugated with polymer is selected from: distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPEPEG2000), dimyristoylglycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), methoxypolyethylene glycol ditetradecylacetamide (ALC-0159) and any combination thereof.

[0256] 61. The composition, according to any of the modalities 38 to 60, in which the VZV RNA has an effective dose of 25 μg to 200 μg; preferably 50 μg to 100 μg.

[0257] 62. The composition, according to any of the embodiments 38 to 60, in which the vaccine is an injection, such as a liquid preparation or a lyophilized preparation.

[0258] 63. A method for preparing the composition according to any of embodiments 38 to 60, comprising: mixing the VZV RNA with a pharmaceutically acceptable carrier, for example, to encapsulate at least a portion of the RNA in lipid nanoparticles.

[0259] 64. The method according to embodiment 63, further comprising a purification step to remove a non-encapsulated component, such as dialysis and / or filtration.

[0260] 65. The method, according to embodiment 64, in which the non-encapsulated component Petition 870260035339, dated 04 / 15 / 2026, pp. 190 / 249 63 / 121 is selected from among: non-encapsulated RNA, a non-aqueous solvent, and bacteria.

[0261] 66. A use of the composition, according to any of the embodiments from 1 to 62, in the preparation of a medicament to induce a protective immune response against VZV in an individual, wherein the protective immune response comprises, for example, the production of a neutralizing antibody.

[0262] 66a. The composition, according to any of embodiments 1 to 62, for use in inducing a protective immune response against VZV in an individual in need thereof, wherein the protective immune response comprises, for example, the production of a neutralizing antibody.

[0263] 66b. A method for inducing a protective immune response against VZV in an individual in need thereof, comprising administering to the individual the composition according to any of the embodiments 1 to 62, wherein the protective immune response comprises, for example, the production of a neutralizing antibody.

[0264] 67. Use, composition or method, according to modality 66, 66a or 66b, in which the individual is immunocompromised.

[0265] 68. Use, composition or method, according to modality 66, 66a, 66b or 67, where the individual is 10 years of age or older, such as 50, 60, 70, 80 years of age or older.

[0266] 69. Use, composition or method, according to any of the modalities 66a 68, in which the protective immune response also includes a cellular immune response.

[0267] 70. Use, composition or method, according to any of the modalities from 66 to 69, in which the induction of a protective immune response is for the prevention of VZV infection.

[0268] 71. Use, composition or method, according to any of the modalities from 66 to 69, in which the induction of a protective immune response is for the prevention of VZV-related pain.

[0269] Example 1: VZV gE mutant antigen design As a highly glycosylated type I membrane protein, gE can be transported between the endoplasmic reticulum (ER), the trans-Golgi network (TGN), and endosomes. gE proteins have several important domains related to their primary functions.

[0270] (1) TM transmembrane domain, which consists of amino acids 539 to 559; Petition 870260035339, dated 04 / 15 / 2026, pp. 191 / 249 64 / 121 (2) motif A568YRV571, which mediates the transport of gE between the endoplasmic reticulum (ER), the trans-Golgi network (TGN) and endosomes; (3) motif Y582AGL585, which mediates the transport of gE between the endoplasmic reticulum (ER), the trans-Golgi network (TGN) and endosomes; (4) Motif S593ES595T596DT598, which is an important glycosylation site and mediates gE transport between the endoplasmic reticulum (ER), the trans-Golgi network (TGN) and endosomes.

[0271] The intracellular carboxyl terminal of gE plays a decisive role in the intracellular transport of gE. Based on the important domains of gE proteins and their functions, a series of mRNA antigens targeting mutations in the main structural / functional domains of gE and mutations in different truncated forms were designed (as shown in FIG. 1).

[0272] Using the wild-type sequence of the VZV gE gene (with reference to NCBI strain sequence number QXN54923.1), a series of 43 different variant gE mRNA sequences was designed: Table 3 summarizes the mRNAs encoding variant gE antigens with different C-terminal sequences, in which YK-VZV-004, YK-VZV-006, YK-VZV-045, and YK-VZV-007 correspond to the mutation sequences or combinations of mutations in the disclosed patents (US11643441B1, CN114081943A, US20230233671A1, and CN108472309A), respectively. Table 3: Herpes zoster mRNA vaccine antigens SEQ ID NO: Name Sequence truncation: (truncated to) Site mutation Sequence deletion 7 YK-VZV-002 gE (1 to 539) Wild type (WT) / 11 YK-VZV-003 gE (1 to 568) WT / 59 YK-VZV-017 gE (1 to 573) WT / 15 YK-VZV-004 Y569A (Comparative Example 1) / 63 YK-VZV-018 A568D / 67 YK-VZV-019 A568 71 YK-VZV-020 Y569K / 75 YK-VZV-021 R570E / 79 YK-VZV-022 V571K / 83 YK-VZV-023 A568D; Y569K; R570E; V571K / Petition 870260035339, dated 04 / 15 / 2026, pp. 192 / 249 65 / 121 87 YK-VZV-024 A568D; Y569A; R570E; V571K / 91 YK-VZV-025 Y569K; R570E; V571K -A568- 95 YK-VZV-026 gE (1 a 587) WT 99 YK-VZV-027 Y569RVDKSPYNQS579 103 YK-VZV-028 Y582A Y569RVDKSPYNQS579 107 YK-VZV-029 Y582G Y569RVDKSPYNQS579 111 YK-VZV-030 A568D; Y569K; R570E; V571K; Y582A / 115 YK-VZV-031 A568D; Y569K; R570E; V571K; Y582G / 119 YK-VZV-032 A568D; Y582A / 123 YK-VZV-033 Y569K; Y582A / 127 YK-VZV-034 R570E; Y582A / 131 YK-VZV-035 V571K; Y582A / 171 YK-VZV-045 gE (1 a 623) comprimento total Y582A (Exemplo Comparativo 3) / 3 YK-VZV-001 WT Comprimento total / 51 YK-VZV-015 Y569RVDKSPYNQS579 55 YK-VZV-016 S593A; S595A; T596A; T598A Y569RVDKSPYNQS579 135 YK-VZV-036 Y582A Y569RVDKSPYNQS579 139 YK-VZV-037 Y582A; S593A; S595A; T596A; T598A Y569RVDKSPYNQS579 143 YK-VZV-038 A568D; Y569K; R570E; V571K; Y582G / 147 YK-VZV-039 A568D; Y569K; R570E; V571K; Y582G; S593A; S595A; T596A; T598A / 19 YK-VZV-006 Y569A; S593A; S595A; T596A; T598A (Exemplo Comparativo 2) / 23 YK-VZV-007 Y582A; S593A; S595A;T596A; T598A (Comparative Example 4) / 151 YK-VZV-040 Y582G; S593A; S595A; T596A; T598A / 27 YK-VZV-009 Y569K; Y582A / 31 YK-VZV-010 Y569K; Y582A; S593A; S595A; T596A; T598A / 35 YK-VZV-011 A568D; Y569K; R570E; V571K / 39 YK-VZV-012 A568D; Y569K; R570E; V571K; S593A; S595A; T596A; T598A / 43 YK-VZV-013 A568D; Y569K; R570E; V571K; Y582A / 47 YK-VZV-014 A568D; Y569K; R570E; V571K; Y582A; S593A; S595A; T596A; T598A / ; Petition 870260035339, dated 04 / 15 / 2026, pp. 193 / 249 66 / 121 163 YK-VZV-043 / Y569RVDKSPYNQSM YYAGLPV587 167 YK-VZV-044 S593A; S595A; T596A; T598A Y569RVDKSPYNQSM YYAGLPV587 155 YK-VZV-041 gE (1 to 601) / Y569RVDKSPYNQSM YYAGLPV587 159 YK-VZV-042 / Y569RVDKSPYNQSM YYAGLPV587

[0273] Example 2: Preparation of the VZV gE antigen mRNA vaccine Preparation of the linearized plasmid of the transcription template. All fragments of the antigen gene were artificially synthesized and cloned into the pVAX vector (acquired from Thermo Fisher Scientific) by GenScript Biotech Corporation, resulting in a successfully constructed circular plasmid.

[0274] The model was prepared by linearizing the plasmid using BsaI restriction endonuclease (acquired from Shanghai Beyotime Biotechnology Co., Ltd.). The digestion system included the supercoiled plasmid inserted into the target gene corresponding to Example 1, 10x digestion buffer, BsaI restriction endonuclease, and RNase-free ddH2U. The digestion reaction was conducted at 37°C for 3 hours, and the digestion system is shown in Table 4. Table 4: Digestion reaction system Component Quantity pDNA 1 to 2 μg 10x digestion buffer 5.0 μL BsaI (10 U / μL) 1.0 μL RNase-free DdH2U Up to 50 μL

[0275] After completion of the digestion reaction, the linearized product was recovered using a DNA product purification kit (purchased from Yeasen Biotechnology (Shanghai) Co., Ltd.). The product concentration was measured using an ultra-micro UV-Vis spectrophotometer (Denovix), and the length and state of the linearized plasmid template were detected by agarose gel electrophoresis.

[0276] Preparation of mRNA stock solution The in vitro co-transcriptional capping reaction is a process that assembles modified or natural nucleoside triphosphates (NTPs) according to the template base sequence to prepare an mRNA polynucleotide, during which Clean Cap is added to generate an mRNA with a Cap1 structure through a one-step transcription reaction. Petition 870260035339, dated 04 / 15 / 2026, pp. 194 / 249 The 67 / 121 conventional reaction system consists of the following components (all acquired from...). Suzhou Novoprotein Scientific Co., Ltd.): Table 5: In vitro co-transcriptional capping reaction system Component Quantity cDNA Model 1.0 μg 10x Transcription Buffer 2.0 μL NTP (100 mM each) 2.0 μL each Cap1-GAG (100 mM) 2.0 μL RNase Inhibitor 0.5 μL T7 RNA Polymerase 2.0 μL RNase-Free DdH2O Up to 20 μL Incubation at 37°C for 2 hours

[0277] After transcription was completed, 1 μL of DNase I (2 U / μL) was added, well mixed, and incubated at 37°C for 20 minutes to remove the DNA template. The transcription product was then purified by lithium chloride precipitation.

[0278] 1) 30 μL of RNase-free H2O and 7.5 M lithium chloride (with a final concentration of 2.8 M) were added to 20 μL of reaction mixture.

[0279] 2) After mixing well, the mixture was left to stand at -20°C for 2 hours, followed by centrifugation at 12,000 rpm for 15 minutes, and the supernatant was discarded. The RNA pellet was washed with 500 μL of 70% ethanol, followed by centrifugation at 12,000 rpm for 5 minutes, which was repeated once to collect the precipitate.

[0280] 3) After drying, the mRNA precipitate was dissolved in 100 μL of enzyme-free water and the purified RNA solution was stored at -80°C. The concentration and integrity of the mRNA were determined.

[0281] Preparation of mRNA-LNP (mRNA vaccine) mg each of cationic lipid, DSPC, cholesterol and DMG-PEG2000 were accurately weighed and dissolved in anhydrous ethanol to prepare a solution with a concentration of 10 mg / mL, which was formulated into a carrier mixture at a molar ratio of 49:10:39.5:1.5 as the organic phase for further use.

[0282] VZV mRNA was dissolved in 50 mM citrate buffer (pH 4.0) and diluted to prepare a solution with a concentration of 0.15 mg / mL as the aqueous phase for further use. The LNP intermediate solution was prepared in a microfluidic device by Petition 870260035339, dated 04 / 15 / 2026, pp. 195 / 249 68 / 121 extraction of 2 mL of the organic phase using a BD syringe (brand, Bidi in Chinese, same as below) of 3 mL and 7 mL of the aqueous phase using a BD syringe of 10 mL, with the organic phase in the left pump and the aqueous phase in the right pump.

[0283] The preparation volume was 9.0 mL; the organic phase flow rate was 5.0 mL / min; the aqueous phase flow rate was 15 mL / min; the LNP intermediate solution was collected.

[0284] The intermediate LNP solution was diluted by adding 9 times the volume of PBS and concentrated by ultrafiltration in a small-scale ultrafiltration device with an ultrafiltration membrane pore size of 50 kD. The mixture was further diluted by adding 4 times the volume of PBS and concentrated in the ultrafiltration device when it was concentrated to approximately 10 mL. Ultrafiltration was stopped when the final concentrated volume was less than 5 mL.

[0285] Example 3: Detection of herpes zoster mRNA vaccine antigen expression by Western Blot To verify whether the designed antigen mRNA sequence can be expressed in cells and to evaluate the expression efficiency of different antigen sequences, cells were first transfected with the mRNA sequence and antigen mRNA expression in cells was detected using Western blot.

[0286] Experimental materials Table 6: Main experimental materials and sources Material Name Source 293T Cells Purchased from ATCC mRNA Vaccine Series Prepared in Example 2 RIPA Lysis Buffer Thermo Fisher Scientific BCA Kit Thermo Fisher Scientific Primary Antibody (anti-VZV gE protein antibody, mouse mAb) Abcam Secondary Antibody (anti-mouse goat IgG) ACRO

[0287] Experimental steps Cell culture and transfection: 293T cells were inoculated into a 12-well plate at a density of 2.5 x 10⁵ cells / well. The mRNA preparation volume required for 500 ng of mRNA was calculated, and the corresponding sample volume was determined. Petition 870260035339, dated 04 / 15 / 2026, pp. 196 / 249 The 69 / 121 mRNA preparation was added directly to the cells and mixed well.

[0288] Experimental group: a mixture of protein samples obtained by lysis of cells transfected with a series of mRNA vaccines prepared in Example 2; Negative control (NC): RIPA lysis buffer; Positive control (PC): purified gE protein; Internal control of reference protein load: glyceraldehyde 3-phosphate dehydrogenase (GAPDH); Protein sample preparation: (i) After 16 hours of transfection, cells were removed from the incubator and repeatedly pipetted to separate them from the bottom of the plate, then transferred to a 1.5 mL centrifuge tube and centrifuged at 1500 rpm for 3 minutes at 25°C. (ii) The supernatant was discarded. Cells were pipetted and resuspended in 1 mL of PBS and centrifuged at 1500 rpm for 3 minutes at 25°C. (iii) The previous step was repeated twice. (iv) 50 μL of ripa lysis buffer were added to each cell tube (with 0.5 μL of 100* protease inhibitor added before using the ripa lysis buffer) and placed on ice for 30 minutes, during which time the samples were vortexed for 30 seconds every 5 minutes. (v) The centrifuge was pre-cooled. After the cells were completely lysed, the samples were centrifuged at 12000 rpm for 10 minutes at 4°C.The supernatant was pipetted and transferred to a new 1.5 mL centrifuge tube to obtain protein samples for testing.

[0289] Development and analysis: The concentration of protein samples was quantified using a BCA kit, and 200 μL of BCA working solution and 10 μL of the sample to be tested were added. 10 μg of protein samples were pipetted into a 1.5 mL centrifuge tube and water was added to prepare a 24 μL system. 6 μE of 5* loading buffer were added and the volume was completed to 30 μE with purified water. Total proteins were boiled for denaturation. 3 μg of sample (9 μE) were pipetted for spotting, along with 4 μE of marker, and subjected to electrophoresis at 200 V for 30 minutes. Membrane transfer was performed using a dry transmembrane instrument. The gel was removed and a membrane transfer sandwich was prepared. The negative plate was covered and membrane transfer was performed at 21 V, 23 V, and 25 V. Petition 870260035339, dated 04 / 15 / 2026, pp. 197 / 249 70 / 121 1, 4, and 2 minutes, respectively. The membrane was then blocked at room temperature for 1 hour. Primary antibody incubation: The membrane was incubated at room temperature for 1 hour. Antibody preparation (diluted 1:2000): 7.5 μL of antibody were pipetted and 15 mL of primary antibody dilution buffer (anti-VZV gE protein antibody, mouse mAb) were added.

[0290] Membrane washing: The membrane was washed 3 times for 5 minutes each using 1x TBST buffer. Secondary antibody incubation: The membrane was incubated at room temperature for 1 hour. Antibody preparation (diluted 1:1000): 30 μL of antibody were pipetted and 30 mL of secondary antibody dilution buffer (anti-mouse goat IgG) were added. Membrane washing: The membrane was washed 3 times for 5 minutes each using 1x TBST buffer diluted with distilled water. Color development: 1 mL of each developing solution and fixation solution were mixed in a 1:1 ratio. The mixture was added dropwise to the membrane for color development and photographed.

[0291] Internal reference protein GAPDH: The relative intensity was consistent, indicating an equal amount of charge in all samples, based on which expression levels were compared between different samples. Negative control (NC): Only the internal reference had matching bands, and there was no matching band in the target protein region, which met the negative control requirements. Positive control (PC) (purified gE protein): There were matching bands within the target size range, and the internal reference had matching bands, which met the positive control requirements. Under conditions where the positive and negative controls were adequately defined and the bands were clearly defined, the expression of the experimental samples and the relative expression level were determined.The Western blot results of the mixture of protein samples obtained by lysis of cells transfected with a series of mRNA vaccines prepared in Example 2 are shown in FIG. 2.

[0292] Example 4: Immunization of Balb / c mice Experimental materials Animals: Balb / c mice, females, 6 to 8 weeks old. Test substances: 1) mRNA vaccine corresponding to the antigen expressed in Petition 870260035339, dated 04 / 15 / 2026, pp. 198 / 249 71 / 121 Example 3; 2) LNP sample (non-mRNA LNP sample prepared in Example 2); 3) recombinant subunit vaccine (Shingrix®) from GSK. Grouping: The mice were randomly grouped according to body weight, with 5 mice per group.

[0293] Animal immunization: On day 0 and day 28, mice were immunized with vehicle or test substance (liquid mRNA vaccine prepared in Example 2) via intramuscular injection into the gastrocnemius muscle. Volumes and doses are shown in the table below. Table 7: Immunization protocol in mice Group Number of animals Immunization Blood collection time Euthanasia time Vaccine to be tested Inoculation dose (pg / long-haired mouse) Inoculation volume (pL / long-haired mouse) Route Frequency 1 5 Vehicle 0 100 Intramuscular injection (gastrocnemius) Days 0 and 28 Blood collection from the orbital vein on days 42 and 56 Spleen removal on day 56 2 5 YK-VZV-001 10 100 3 5 YK-VZV-003 10 100 4 5 YK-VZV-004 10 100 5 5 YK-VZV-006 10 100 6 5 YK-VZV-007 10 100 7 5 YK-VZV-009 10 100 8 5 YK-VZV-010 10 100 9 5 YK-VZV-011 10 100 10 5 YK-VZV-012 10 100 11 5 YK-VZV-013 10 100 12 5 YK-VZV-014 10 100 13 5 YK-VZV-015 10 100 14 5 YK-VZV-016 10 100 15 5 YK-VZV-017 10 100 16 5 YK-VZV-018 10 100 Petition 870260035339, dated 04 / 15 / 2026, pp. 199 / 249 72 / 121 17 5 YK-VZV-019 10 100 18 5 YK-VZV-020 10 100 19 5 YK-VZV-021 10 100 20 5 YK-VZV-022 10 100 21 5 YK-VZV-023 10 100 22 5 YK-VZV-024 10 100 Table 7 (continued) Group Number of animals Immunization Blood collection time Euthanasia time Vaccine to be tested Inoculation dose (pg / long-haired mouse) Inoculation volume (pL / long-haired mouse) Route Frequency 23 5 YK-VZV-025 10 100 Intramuscular injection (gastrocnemius) Days 0 and 28 Blood collection from the orbital vein on days 42 and 56 Spleen removal on day 56 24 5 YK-VZV-026 10 100 25 5 YK-VZV-027 10 100 26 5 YK-VZV-028 10 100 27 5 YK-VZV-029 10 100 28 5 YK-VZV-030 10 100 29 5 YK-VZV-031 10 100 30 5 YK-VZV-032 10 100 31 5 YK-VZV-033 10 100 32 5 YK-VZV-034 10 100 33 5 YK-VZV-035 10 100 34 5 YK-VZV-036 10 100 35 5 YK-VZV-037 10 100 36 5 YK-VZV-038 10 100 37 5 YK-VZV-039 10 100 38 5 YK-VZV-040 10 100 Petition 870260035339, dated 04 / 15 / 2026, pp. 200 / 249 73 / 121 39 5 YK-VZV-044 10 100 40 5 YK-VZV-045 10 100 41 5 LNP 10 100 42 5 Shingrix® 10 100

[0294] Sample collection and pre-treatment Approximately 0.2 mL of blood was collected from the orbital venous plexus of each animal. The serum was separated (by centrifugation at 4000 rpm for 10 minutes at 4°C) and stored in a refrigerator at 4°C for further processing.

[0295] Health Monitoring Twice a day (once in the morning and once in the afternoon), including but not limited to observations of death, illness, respiration, secretions, feces, as well as food and water intake.

[0296] Human endpoint According to the IACUC protocol, any mouse experiencing more than 20% body weight loss during the experiment (body weight on day 0 is used as baseline body weight before infection, and body weight on the day of inoculation is used as baseline body weight after infection) and / or showing signs of impending death will be sacrificed and recorded as a dead animal in the results.

[0297] Example 5: Detection of specific IgG antibody titer for gE protein in the serum of Balb / c mice by enzyme-linked immunosorbent assay (ELISA) Main experimental materials and sources The varicella-zoster virus (VZV) IgG titer (gE protein) ELISA assay kit (human) was purchased from Beijing ACROBiosystems Co., Ltd., which includes 1x wash buffer, positive control working solution, negative control working solution, dilution buffer, goat anti-mouse IgG HRP, substrate solution, stop solution, and ELISA plate.

[0298] Experimental steps Preparation of the working solution: Preparation of 1x washing buffer: 50 mL of 10x washing buffer were diluted with ultrapure water or deionized water and the volume was Petition 870260035339, dated 04 / 15 / 2026, pp. 201 / 249 74 / 121 fixed in 500 mL. The positive control working solution and the negative control working solution were prepared.

[0299] Pre-treatment of samples to be tested: A series of blood samples collected on day 42 and day 56 in Example 4.

[0300] Antibody titer assay: The samples to be tested, positive control (PC) and negative control (NC) were diluted with dilution buffer from 1:100 to 1:102400.

[0301] Numbering: The diluted samples were numbered corresponding to the wells of the ELISA plate. PC working solution and NC working solution were defined for each experiment.

[0302] Sample addition: 100 μL of diluted sample, positive control working solution, and negative control working solution were first added to the well of the corresponding plate. The plate was shaken to mix thoroughly and incubated at 37°C for 1.0 hour.

[0303] Plate washing: The liquid in the wells was discarded and the ELISA plate was tapped dry. The plate was washed with 1x washing buffer (300 μL / well, soaked for 30 seconds) a total of 3 times and tapped dry.

[0304] Addition of HRP enzyme conjugate: HRP anti-mouse goat IgG was diluted 1000 times with dilution buffer, then added to the plate at 100 μL per well and incubated at 37°C for 1.0 hour.

[0305] Plate washing: The washing step (step 6) was repeated 3 times.

[0306] Color development: 100 μL of substrate solution were added to each well and incubated in the dark at 37°C for 20 minutes.

[0307] Termination: 50 μL of stopping solution were added to each well and the plate was shaken to mix thoroughly.

[0308] Reading: The absorbance of each well at OD450 nm and OD630 nm (with 630 nm as background) was measured using a microplate reader and read within 3 minutes after completion.

[0309] Interpretation of test results: Determination of positive antibodies: OD450 nm - OD630 nm > 0.1.

[0310] Example 6: Detection of the percentage of CD4+ T cells and CD8+ T cells secreting IFN-γ and IL-2 in the spleen of Balb / c mice by multiparameter flow cytometry (FCM) Petition 870260035339, dated 04 / 15 / 2026, pp. 202 / 249 75 / 121 The role of immune checkpoint inhibitors is to prevent T cell exhaustion. Immune cell function is assessed by detecting cytokine production in tumor samples. T cells and NK cells produce interferon gamma (IFN-γ) to mount an immune response under inflammatory conditions or host immune defense. CD8+ or CD4+ T cells producing IFN-γ in mouse spleens were quantified using multiparameter flow cytometry (FCM).

[0311] Interferon gamma (IFN-γ) is a soluble dimeric cytokine and the only member of the type II interferons. It is secreted primarily by natural killer (NK) cells and natural killer (NKT) T cells and plays a role in innate immunity; in the process of antigen-specific immunity, it is secreted by CD4 Th1 and CD8 cytotoxic T cells. Interferon IFN-γ or type II plays an important role in innate and adaptive immunity against viral, certain bacterial, and protozoan infections. IFN-γ serves as an important activator of macrophages and an inducer of the expression of major histocompatibility complex class II (MHC II). Interleukin-2 (IL-2) is a cytokine of the chemokine family. It is a cytokine of multicellular origin (produced primarily by activated T cells) with pleiotropic effects (primarily promoting lymphocyte growth, proliferation, and differentiation). It plays an important role in the body's immune response and in antiviral infection.It can stimulate the proliferation of T cells that have been activated by specific antigens or mitogenic factors; activate T cells and promote cytokine production; stimulate NK cell proliferation, increase NK killing activity and cytokine production, and induce the production of lymphokine-activated killer (LAK) cells; promote B cell proliferation and antibody secretion; and activate macrophages.

[0312] Detection method: FCM (multiparameter flow cytometry).

[0313] Sample for detection: Spleen removed on day 56 from the immunized mouse model in Example 5.

[0314] Detection objective: To determine the percentage of CD4+ T cells and CD8+ T cells secreting IFN-γ and IL-2 in the spleens of mice. Detection indicators: CD4+ (IFN-γ+): total number of CD4+ T cells that produce IFN-γ; CD4+(IL-2+): total number of CD4+ T cells that produce IL-2; Petition 870260035339, dated 04 / 15 / 2026, pp. 203 / 249 76 / 121 CD4+ (IFN-γ+ IL-2+): total number of CD4+ T cells that produce IFN-γ and IL-2; CD4+ (IFN-γ+ or IL-2+): total number of CD4+ T cells that produce IFN-γ or IL-2; CD8+ (IFN-γ+): total number of CD8+ T cells that produce IFN-γ; CD8+ (IL-2+): total number of CD8+ T cells that produce IL-2; CD8+ (IFN-γ+IL-2+): total number of CD8+ T cells that produce IFN-γ and IL-2; CD8+ (IFN-γ+ or IL-2+): total number of CD8+ T cells that produce IFN-γ or IL-2.

[0315] Experimental materials Main sources of reagents Table 8: Reagents for sample detection Manufacturer Name Fetal Bovine Serum (FBS) ExCell Bio RPMI 1640 Medium GIBCO Penicillin-Streptomycin Mixture GIBCO Phosphate Buffered Saline (PBS) Biotopped 10x RBC Lysis Buffer (Multi-Species) Thermo Fisher Zombie Violet™ Fixable Viability Kit Biolegend Protein Transport Inhibitor (Brefeldin A Solution) Biolegend Fixation and Permeabilization Solution BD Perm / Wash Buffer BD Ionomycin / PMA Sigma Mouse Anti-CD3 PerCP / Cyanine 5.5 Biolegend Mouse Anti-CD4 FITC Biolegend Mouse Anti-CD8a Alexa Fluor® 700 Biolegend Mouse Anti-IFN-γ APC Biolegend Mouse Anti-IL-2 PE Biolegend Cell Staining Buffer Biolegend

[0316] Test solution preparation Each reagent was prepared according to the quantity required for the Petition 870260035339, dated 04 / 15 / 2026, pp. 204 / 249 77 / 121 experiment and all reagents were prepared in the volume ratio.

[0317] FBS Inactivation: FBS stored at -20°C was thawed at 4°C, then inactivated in a water bath at 56°C for 30 minutes, filtered through a 0.22 μm filter and stored at 2 to 8°C with an effective period of 14 days.

[0318] Preparation of 1x RBC Lysis Buffer: 10x RBC Lysis Buffer (Multiple species) were diluted in 1x RBC Lysis Buffer with sterile water and thoroughly mixed for later use.

[0319] Preparation of complete medium containing 1% double antibiotics: The complete medium was prepared at a ratio of RPMI-1640: inactivated FBS = 9:1.

[0320] Complete medium containing 1% dual antibiotics was prepared at a ratio of complete medium: penicillin-streptomycin mixture = 99:1, well mixed and stored at 2 to 8°C with an effective period of 14 days. The complete medium was equilibrated at room temperature before use.

[0321] Positive stimulating working solution for PMA / ionomycin: 400 μE of PMA working solution and 80 μL of ionomycin (ION) were added to 19.520 mL of complete medium containing 1% double antibiotics to prepare a positive stimulating working solution, which was ready for use. The final concentration of PMA was 1 μg / mL and the final concentration of ionomycin (ION) was 2 μg / mE.

[0322] Negative control: complete medium containing 1% double antibiotics.

[0323] Preparation of the peptide working solution: The Protein Transport Inhibitor working solution was diluted 50 times with complete medium containing 1% double antibiotics.

[0324] Live / Dead Working Solution: Zombie Violet™ Fixable Viability Solution was diluted 1000 times with 1x PBS.

[0325] Surface staining antibody: Taking the amount for one well as an example, the surface staining antibody was prepared according to the proportions in the table below, well mixed by pipetting and stored in the dark at 2°C to 8°C for later use. Petition 870260035339, dated 04 / 15 / 2026, pages 205 / 249 78 / 121 Table 9: Preparation of the surface staining antibody Antibody Name | Antibody Volume / Well | Cell Staining Buffer | Mouse anti-CD3 PerCP / Cyanin 5.5 | 2 μL / test | 45.2 μL | Mouse anti-CD4 FITC | 2 μL / test | Mouse anti-CD8a Alexa Fluor® | 0.8 μL / test

[0326] 1* Perm / Wash Buffer: 10* Perm / Wash Buffer were diluted 10 times with ultrapure water.

[0327] Intracellular staining antibody: Taking the amount for one well as an example, the intracellular staining antibody was prepared according to the proportions in the table below, well mixed by pipetting and stored in the dark at 2°C to 8°C for later use. Table 10: Preparation of the intracellular staining antibody Antibody Name | Antibody Volume / Well | Perm Buffer / Wash | Mouse Anti-IFN-γ | APC | 2 μL / test | 44 μL | Mouse Anti-IL-2 | PE | 2 μL / test

[0328] Preparation of cryopreservation solution: prepared at a ratio of FBS: DMSO = 9:1, ready for use.

[0329] Procedure Isolation of mouse spleen cells A cell filter was placed in a 6-well plate containing 4 mL of complete medium containing 1% double antibiotics. The spleen was then placed separately in the cell filter and gently crushed or ground with the plunger end of a syringe. The resulting cell suspension was filtered through the cell filter and transferred to a 15 mL centrifuge tube. The 6-well plate was rinsed with 1 to 2 mL of complete medium containing 1% double antibiotics, and the resulting liquid was filtered and transferred to the 15 mL centrifuge tube, which was centrifuged at 500 g for 5 minutes at 20 °C. The supernatant was discarded.

[0330] The cells were resuspended in 2 mL of 1x RBC Lysis Buffer, lysed for 5 ± 1 minutes, and centrifuged at 500 g for 5 minutes at 20°C. The supernatant was discarded. The Petition 870260035339, dated 04 / 15 / 2026, pages 206 / 249 79 / 121 cells were washed with 5 mL of complete medium containing 1% double antibiotics and centrifuged at 500 g for 5 minutes at 20 °C. The supernatant was discarded.

[0331] The cells were resuspended in 10 mL of complete medium containing 1% double antibiotics, well mixed, and 100 μL of cell suspension were immediately added to an EP tube. 10 μL of cell suspension and 10 μL of fluorescent staining solution were then added to the EP tube, well mixed, and 10 μL of the mixture were taken for counting with a fluorescence counter. The total number of viable cells and cell viability were counted.

[0332] The cells in the 15 mL centrifuge tube were centrifuged at 400 g for 5 minutes at 20°C and the supernatant was discarded. The cells were resuspended in fresh complete medium containing 1% double antibiotics and the concentration of viable cells in each sample was adjusted to 1 *107 cells / mL.

[0333] After the samples were plated, the remaining cells were centrifuged at 400 g for 5 minutes at 20°C and the cell concentration was adjusted to (1 to 2) *107 cells / mL with cryopreservation solution. The mixture was divided into aliquots in pre-labeled cryotubes at 1 mL per vial. The cryotubes were placed in a programmed cooling box, cryopreserved in a refrigerator below -70°C for 12 to 24 hours, and transferred to a liquid nitrogen tank for long-term storage.

[0334] Stimulation of mouse spleen cells Stimulation of mouse spleen cells was performed in a 96-well U-shaped plate labeled with the date of the experiment.

[0335] Positive wells, negative wells, and peptide wells were set up based on experimental requirements. 100 μL / well of negative control was added to the negative wells, 100 μL / well of positive stimulant working solution was added to the positive wells, and 100 μL / well of peptide working solution was added to the peptide wells. 100 μL / well of cell suspension was then added to the positive wells, negative wells, and peptide wells.

[0336] The 96-well plate was stimulated in a 5% CO2 incubator at 37°C for 2 hours ± 5 minutes, then supplemented with 10 μL / well of Protein Transport Inhibitor working solution, mixed well, and continuously stimulated in the CO2 incubator. Petition 870260035339, dated 04 / 15 / 2026, pp. 207 / 249 80 / 121 5% at 37°C for an additional 18 hours ± 10 minutes. The 96-well round-bottom plate was then removed for flow cytometry staining.

[0337] Note: All experimental procedures in this section must be conducted in a biosafety cabinet, using sterile reagents.

[0338] Cell staining Stimulated washing samples: The 96-well round-bottom plate was removed and centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. 200 μL of PBS were added to each well, resuspended, and thoroughly mixed, and the plate was centrifuged at 500 g for 5 minutes at 4°C. The supernatant was discarded.

[0339] Live / Dead Staining: 50 μL of prepared live / dead working solution were added to each well, resuspended, thoroughly mixed, and stained in the dark at 2°C to 8°C for 30 minutes. After staining, 150 μL of PBS were added to each well, and the plate was centrifuged at 500 g for 5 minutes at 4°C. The supernatant was discarded.

[0340] Surface staining: 50 μL of surface staining antibody were added to each well, resuspended and thoroughly mixed, and stained in the dark at 2°C to 8°C for 30 minutes. 150 μL of Cell Staining Buffer were added to each well to complete the staining, and the plate was centrifuged at 500 g for 5 minutes at 4°C. The supernatant was discarded.

[0341] Cell fixation and permeabilization: 100 pL of Fixation and Permeabilization Solution were added to each well, resuspended and mixed, and incubated in the dark at 4°C for 20 minutes. 100 pL of 1* Perm / Wash Buffer were added to each well for termination, and the plate was centrifuged at 500 g for 5 minutes at 4°C. The supernatant was discarded.

[0342] Intracellular staining: 50 pL of intracellular staining antibody were added to each well, resuspended and thoroughly mixed, and incubated in the dark at 4°C for 50 minutes. 150 pL of 1* Perm / Wash Buffer were added to each well to complete the staining, and the plate was centrifuged at 500 g for 5 minutes at 4°C. The supernatant was discarded. 200 pL of 1* Perm / Wash Buffer were added to each well, resuspended and thoroughly mixed, and the plate was centrifuged at 500 g for 5 minutes at 4°C. The supernatant was discarded. Petition 870260035339, dated 04 / 15 / 2026, pages 208 / 249 81 / 121

[0343] Filtration: 200 μL of Cell Staining Buffer were added to each well, resuspended and thoroughly mixed, and the cell suspension was filtered through a 300 mesh nylon screen and transferred to a new well plate. Example of sample numbering: “01-P-1”, where “01” represents the sample number, “P” represents the positive well, and the final “1” represents the well number.

[0344] Instrument detection Instrument name: Flow cytometer (Cytoflex); Instrument acquisition parameters: Set the acquisition flow rate to high speed and record a total of 100,000 cells collected from the lymphocyte gate. Data processing Data analysis: CD3+ cells were identified, and then CD4+ and CD8+ cells were identified separately. The CD4+ (IFN-γ+) and CD4+ (IL-2+) subpopulations were identified from the CD4+ cells; the CD8+ (IFN-γ+) and CD8+ (IL-2+) subpopulations were identified from the CD8+ cells.

[0345] Data calculation formula: All values ​​in this assay were calculated as percentages; effective value = mean of peptide wells - mean of negative wells. All values ​​were retained with one decimal place.

[0346] Example 7: Detection of IFN-γ and IL-2 cytokine secretion from Balb / c mouse spleen cells by enzyme-linked immunospot assay (ELISPOT) Enzyme-linked immunosuppressant assay (ELISPOT) is a highly sensitive detection method in cellular immunology research that can detect antibody-secreting cells (ASCs) and cytokine-secreting cells (CKs) at the single-cell level. It offers greater sensitivity than ELISA and limited dilution methods and can detect a protein-secreting cell from 200,000 to 300,000 cells. Furthermore, the method allows for the functional assessment of viable cells stimulated by antigens, demonstrating high specificity, high intuitive reliability, and operability, making it widely used in the detection of CK-secreting cells or ASC assays in the field of immunology both domestically and internationally. After antigen stimulation, lymphocytes locally produce cytokines, which are captured by specific monoclonal antibodies (pre-coated) on the PVDF membrane at the bottom of the ELISPOT plate. After cell removal, the captured cytokines bind... Petition 870260035339, dated 04 / 15 / 2026, pages 209 / 249 82 / 121 to biotin-labeled monoclonal antibodies and subsequently to avidin labeled with alkaline phosphatase or horseradish peroxidase. After adding substrate for color development, purple or reddish-brown spots appear on the PVDF membrane, indicating cytokine production by the cells. The spots are automatically counted and analyzed by an ELISPOT reader.

[0347] The spleen removed from the mouse model in Example 6 was used to detect the levels of IFN-γ and IL-2 secreted by spleen cells after stimulation of the peptide library on day 56 post-immunization.

[0348] Sample processing: Follow the procedure for isolation and cryopreservation of mouse spleen cells.

[0349] ELISPOT Method: Detect IFN-γ and IL-2 levels in spleen cell supernatant after peptide library stimulation.

[0350] Main reagents Table 11: Reagents for sample detection Name Source Mouse spleen cells Medleader FBS ExCell Bio RPMI-1640 medium GIBCO DMSO Sigma Phosphate buffered saline (1* PBS) Biotopped Phosphate buffered saline (20* PBS) Solarbio Forbol 12-myristate 13-acetate (PMA) Sigma Inomycin (ION) Sigma Penicillin-streptomycin mixture Gibco ELISpot Plus: Mouse IFN-γ (HRP) MabTech ELISpot Plus: Mouse IL-2 (HRP) MabTech Preparation of the test solution 1) FBS Inactivation: FBS stored at -20°C was thawed at 4°C, then inactivated in a water bath at 56°C for 30 minutes, filtered through a 0.22 μm filter, and stored at 2 to 8°C with an effective period of 30 days. Petition 870260035339, dated 04 / 15 / 2026, pp. 210 / 249 83 / 121

[0351] 2) Preparation of the PMA working solution: 1 mL of DMSO was added to a PMA reagent bottle to dissolve the PMA powder, and the mixture was transferred to a 15 mL centrifuge tube. The PMA reagent bottle was rinsed with 1 mL of DMSO, and the rinsing solution was transferred to the 15 mL centrifuge tube. 8 mL of DMSO were then added to the 15 mL centrifuge tube and mixed well to prepare a PMA working solution with a concentration of 100 μg / mL, which was stored in the dark in a refrigerator at -20°C.

[0352] 3) Preparation of the ION working solution: 2 mL of DMSO were added to an ION reagent bottle to dissolve the ION powder, and the mixture was transferred to a 15 mL centrifuge tube. The ION reagent bottle was rinsed with 1 mL of DMSO, then the rinsing step was repeated two more times (a total of 3 times, each with 1 mL), and the rinsing solution was transferred to the 15 mL centrifuge tube. 5 mL of DMSO were then added to the 15 mL centrifuge tube and mixed well to prepare an ION working solution with a concentration of 1 mg / mL, which was stored in a refrigerator at -20°C.

[0353] 4) Preparation of complete medium containing 1% double antibiotics: The complete medium was prepared at a ratio of RPMI-1640: inactivated FBS = 9:1. Complete medium containing 1% double antibiotics was prepared at a ratio of complete medium: penicillin-streptomycin mixture = 99:1, well mixed and stored at 2 to 8°C with an effective period of 14 days. The complete medium was equilibrated at room temperature before use.

[0354] 5) Preparation of positive stimulant working solution: 400 g / L of PMA working solution and 80 g / L of ionomycin (ION) were added to 19.520 mL of complete medium to prepare a positive stimulant working solution, which was ready for use. The final concentration of PMA was 2 g / mL and the final concentration of ION was 4 g / mL.

[0355] 6) Preparation of peptide working solution: (i) Preparation of the gE peptide library solution 100 g / L of DMSO were added to each tube of lyophilized powder. After dissolution, the peptide concentration in each tube was 1 mg / mL per peptide. 900 g / L of PBS were added to the dissolved peptide solution to adjust the concentration of the gE peptide library to 100 mg / mL per peptide. Petition 870260035339, dated 04 / 15 / 2026, pp. 211 / 249 84 / 121

[0356] (ii) Preparation of the gE working solution The gE working solution was prepared at a ratio of gE peptide library solution: complete medium containing 1% double antibiotics = 1:50, resulting in a concentration of 2 μg / mL per peptide.

[0357] Negative control: complete medium containing 1% double antibiotics.

[0358] 7) Preparation of 1* PBS solution: prepared in a ratio of 20* PBS: pure water = 1:19, stored at room temperature, ready for use.

[0359] 8) Preparation of PBS containing FBS: prepared at a PBS ratio (Biotopped): FBS deactivated = 200:1, ready to use.

[0360] 9) Antibody preparation: (i) R4-6A2-biotin preparation: prepared at a ratio of R4-6A2-biotin: PBS containing FBS = 1:1000, ready for use.

[0361] (ii) 5H4-biotin preparation: prepared at a ratio of 5H4-biotin: PBS containing FBS = 1:1000, ready for use.

[0362] Experimental steps 1) The spleen removed from the mouse model in Example 6 was used to detect the levels of IFN-γ and IL-2 secreted by spleen cells after stimulation of the peptide library on day 56 post-immunization, as well as CD4+ and CD8+ T cells producing IFN-γ and IL-2.

[0363] 2) Sample for detection: Spleen removed on day 56, cryopreserved at -80°C.

[0364] Resuscitation of mouse spleen cells Preparation: Before cell resuscitation, the temperature of the thermostatic water bath was adjusted to 37°C and the complete medium containing 1% double antibiotics was pre-warmed to 37°C.

[0365] Sampling: Mouse spleen cells cryopreserved in the liquid nitrogen tank were removed by a sample administrator and transferred to a box containing liquid nitrogen. The cryotubes were removed from the box, sprayed with 75% ethanol, and then placed in a transfer window of the cell culture room. Petition 870260035339, dated 04 / 15 / 2026, pp. 212 / 249 85 / 121

[0366] Resuscitation: The cryotubes were immediately removed from the transfer window by a validator and rapidly thawed in a thermostatic water bath at 37°C. Once fully thawed, the cells were aseptically transferred to a 15 mL centrifuge tube containing 9 mL of complete medium containing 1% double antibiotics.

[0367] Rinse: 1 mL of complete medium containing 1% double antibiotics was added to a cryogenic flask, then the cryogenic flask was rinsed and the rinse solution was transferred back to the 15 mL centrifuge tube.

[0368] Centrifugation: Cells were centrifuged at 400 g for 10 minutes at 18 to 20°C.

[0369] Counting: The supernatant was discarded. Cells were resuspended in 10 mL of complete medium containing 1% double antibiotics, well mixed, and 100 μL of cell suspension were added to an EP tube. 10 μL of 100 μL of cell suspension were then added to an EP tube containing 10 μL of fluorescent staining solution, well mixed, and 10 μL of the mixture were taken for counting with a fluorescence counter. The total number of viable cells and cell viability were counted (if viability is less than 50%, repeat the count with the fluorescence counter once and the results of the second count will prevail).

[0370] Re-centrifugation: The remaining cells in the 15 mL centrifuge tube were centrifuged at 400 g for 5 minutes at 18 to 20°C.

[0371] Resuspension: The supernatant was discarded. The cells were resuspended in complete medium containing 1% double antibiotics and the viable cell concentration was adjusted to A0: 4 x 10⁶ cells / mL and A1: 4 x 10⁵ cells / mL.

[0372] Stimulation of mouse spleen cells Plate washing: IFN-γ and IL-2 plates were washed with 200 μL / well of PBS (PBS used throughout the experiment was sterile) a total of 4 times.

[0373] Blocking: The PBS in the wells was discarded and the plate was tapped (to the greatest extent, remove residual liquid in the wells). 200 μL / well of complete medium containing 1% double antibiotics was added and the plate was left at room temperature for at least 30 minutes (record the exact time).

[0374] Plating: The complete medium containing 1% double antibiotics in the well plate was discarded and the plate was tapped (to the greatest extent, remove residual liquid in the wells). Petition 870260035339, dated 04 / 15 / 2026, pp. 213 / 249 86 / 121 wells); 100 μL / well of positive stimulant working solution was added to the positive wells; 100 μL / well of negative control was added to the negative wells; 100 μL / well of peptide working solution was added to the peptide wells; 200 μL / well of negative control was added to the cell-free blank wells.

[0375] Cell addition: 100 μL / well of A0 cell suspension were added to the negative wells and peptide wells, and 100 μL / well of A1 cell suspension were added to the positive wells.

[0376] Incubation: The plate was covered with aluminum foil and stimulated in a 5% CO2 incubator at 37°C for 21 hours ± 30 minutes.

[0377] Note: All experimental procedures in this section must be conducted in a biosafety cabinet, using sterile reagents.

[0378] Cytokine detection Plate washing: The following day, the original liquid in the well plate was discarded. The plate was shaken (to the greatest extent, to remove residual liquid in the wells) and washed with 200 μL / well of PBS a total of 5 times.

[0379] Secondary antibody addition: PBS in the wells was discarded and the plate was tapped (to the greatest extent, remove residual liquid in the wells). IFN-γ: R4-6A2-biotin working solution was added at 100 μL / well. IL-2: 5H4-biotin working solution was added at 100 μL / well. The plate was incubated at room temperature for 2 hours ± 5 minutes.

[0380] Plate washing: The original liquid in the well plate was discarded. The plate was tapped (to the greatest extent, to remove residual liquid in the wells) and washed with 200 μL / well of PBS a total of 5 times.

[0381] Tertiary antibody addition: Streptavidin-HRP working solution was added at 100 μL / well. The plate was incubated at room temperature for 1 hour ± 2 minutes and the liquid in the well plate was discarded. The plate was tapped (to the greatest extent, remove residual liquid in the wells) and washed with 200 μL / well of PBS a total of 5 times.

[0382] Plate washing: The original liquid in the well plate was discarded. The plate was tapped (to the greatest extent, to remove residual liquid in the wells) and washed with 200 μL / well of PBS a total of 5 times. Petition 870260035339, dated 04 / 15 / 2026, pp. 214 / 249 87 / 121

[0383] Color development: TMB substrate solution, which had been brought to room temperature, was added at 100 μL / well. After color development at room temperature for 15 ± 1 minutes, the liquid in the well plate was discarded and the plate was washed with pure water to complete color development. The bottom plate was removed from the well plate and the bottom surface of the well plate was rinsed.

[0384] Reading: After the well plate was completely dried at room temperature in the dark, an ELISPOT analyzer was used to photograph and read. Camera parameters: Shutter (Exposure): 400; Gain: 25. Counting parameters: IFN-γ-Mouse-Mab / IL-2Mouse-MabT: Size: 30; Intensity (threshold for optical density): 30; TNTC: 80%.

[0385] Example 8: Distribution of VZV gE antigen in Chlorocebus sabaeus (Vero) kidney cells Vero cells are a cell line used in cell cultures. The Vero cell line was isolated from renal epithelial cells extracted from Chlorocebus sabaeus. Vero cells were transfected with different gE antigen variants constructed in this document (YK-VZV-010, YK-VZV-011, YK-VZV-013, YK-VZV-018, and YK-VZV-020). Transfected cells were stained with gE antigen-labeling antibodies and with Golgi marker antibodies GM130 / TGN46, and antigen localization was observed using confocal microscopy. The results are shown in FIGS 3 and 4.

[0386] Preparation of reagents and consumables Table 12: Reagents and consumables for sample detection Name Source μ-Slide 8 Light-shielded well incubation box ibidi VZV gE protein anti-antibody Abcam Donkey anti-mouse secondary antibody 488 Yeasen Biotechnology (Shanghai) Co., Ltd. Fluorescence quenching sealing agent (containing DAPI) Beyotime Biotechnology Co., Ltd. Phosphate buffered saline (1* PBS) Beyotime Biotechnology Co., Ltd. Phosphate buffered saline (20* PBS) Beyotime Biotechnology Co., Ltd. Immunostaining permeabilization buffer (Triton X-100) Beyotime Biotechnology Co., Ltd. 4% paraformaldehyde fixing solution Beyotime Biotechnology Co., Ltd. QuickBlock™ primary antibody dilution buffer for immunostaining Beyotime Biotechnology Co., Ltd. Petition 870260035339, dated 04 / 15 / 2026, pp. 215 / 249 88 / 121 QuickBlock™ blocking buffer for immunostaining Beyotime Biotechnology Co., Ltd. QuickBlock™ secondary antibody dilution buffer for immunofluorescence Beyotime Biotechnology Co., Ltd. GOLGA2 / GM130 antibody conjugated with 647 Proteintech TGN46 antibody conjugated with CoraLite® 594 Proteintech Experimental stages 1) Cell inoculation: Vero cells (acquired from Beyotime Biotechnology Co., Ltd.) were inoculated into a confocal petri dish, and the number of inoculated cells was recorded when the cell inoculation density was observed to be around 30% under a microscope. The cells were incubated overnight.

[0387] 2) Transfection: Cells were transfected with overexpression stock solution (50 ng / well) and incubated for 48 hours.

[0388] 3) Washing: The confocal petri dish was removed and the internal medium was discarded. The petri dish was washed with PBS 1 to 3 times, avoiding vigorous shaking.

[0389] 4) Fixation: The cells were fixed with 200 μL of paraformaldehyde solution at 4%, left at room temperature for 15 minutes and washed with 1x PBS three times, leaving at room temperature for 5 minutes each time.

[0390] 5) Permeabilization: The cells were permeabilized with 200 μL of 0.3% Triton X-100 (diluted with 1x PBS) for 10 minutes and washed with 1x PBS three times, leaving at room temperature for 5 minutes each time.

[0391] 6) Blocking: The cells were blocked with 200 μL of immunostaining blocking buffer and incubated at room temperature for 2 hours.

[0392] 7) Primary antibody binding: 100 qL of primary antibody diluted with antibody dilution buffer were added, with specific antibodies and dilution ratios as follows. VZV gE antibody was diluted 1:100, GOLGA2 / GM130 antibody conjugated with 647 was diluted 1:100, and TGN46 antibody conjugated with CoraLite® 594 was diluted 1:100. The mixture was incubated in the dark at 4°C overnight and the primary antibody was aspirated (it can be reused).

[0393] 8) Washing of the primary antibody: The mixture was washed with 1x PBS three times for 5 minutes each time to remove unbound primary antibody.

[0394] 9) Secondary antibody incubation: 200 qL of fluorescent secondary antibody Petition 870260035339, dated 04 / 15 / 2026, pp. 216 / 249 89 / 121 diluted with antibody dilution buffer at a ratio of 1:300 were added. The mixture was incubated in the dark at room temperature for 1.5 hours.

[0395] 10) Washing of secondary antibodies: The mixture was washed with 1x PBS three times for 5 minutes each time to remove unbound secondary antibodies, preventing non-specific staining.

[0396] 11) Nuclear staining and sealing: The structure was removed and a drop of DAPI staining solution was added to each well. The Petri dish was covered with a lid and stored in the dark at 4°C.

[0397] 12) A confocal microscope was used for photographing and observation.

[0398] Control configuration This disclosure defines the following controls: LNP: Blank LNP without mRNA vaccine; Shingrix®: Shingrix® recombinant protein subunit vaccine produced and marketed by GSK, consisting of the extracellular region of the VZV gE protein, amino acids 1 to 539, without mutations.

[0399] Comparative Example 1 Based on the herpes zoster mRNA vaccine antigen sequence reported in the literature (Morgan A, et al. 2020) and Moderna (US11643441B1), the antigen developed by the patent was found to be gE(1-573aa)Y569A, based on which YK-VZV-004 (SEQ ID NO: 15) was designed in the present disclosure, which retains the same mutation site and truncation length as the herpes zoster mRNA vaccine antigen sequence gE(1-573aa)Y569A reported in the Moderna patent (US11643441B1). YK-VZV-004:gE(1573aa)Y569A was used as one of the positive controls for antigen screening, along with the newly designed mutation sequences or mutation combinations in the present disclosure for vaccine immunogenicity assessment. Specific results are shown in the examples.

[0400] Comparative Example 2 Based on the antigen sequence of the herpes zoster mRNA vaccine reported in the patent (CN114081943A), it was found that the full-length VZV gE polypeptide developed by the patent comprises a combination of mutations (Y569A, S593A, S595A, Petition 870260035339, dated 04 / 15 / 2026, pp. 217 / 249 90 / 121 Based on the T596A and T598A mutations, YK-VZV-006 (SEQ ID NO: 19) was designed in this disclosure, retaining the same combination of mutation sites (Y569A, S593A, S595A, T596A, and T598A) as SEQ ID NO: 2 mentioned in the patent (CN114081943A). YK-VZV006: gE(1-623aa) (Y569A, S593A, S595A, T596A, and T598A) was also used as one of the antigen screening controls in this disclosure, along with the newly designed mutation sequences or mutation combinations in this disclosure for vaccine immunogenicity assessment. Specific results are shown in the examples.

[0401] Comparative Example 3 Based on the herpes zoster mRNA vaccine antigen sequence reported in US20230233671A1, the patent discloses the VZV Y582A mutant gE protein, based on which YK-VZV-045 (SEQ ID NO: 171) was designed in the present disclosure, which retains the same mutation site combination (Y582A) as the mRNA vaccine antigen sequence mentioned in US20230233671A1. YK-VZV-045 was also used as one of the antigen screening controls in the present disclosure, along with the newly designed mutation sequences or mutation combinations in the present disclosure for vaccine immunogenicity assessment. Specific results are shown in the examples.

[0402] Comparative Example 4 Based on the herpes zoster mRNA vaccine antigen sequence reported in CN108472309A, the patent discloses the VZV Y582A mutant gE protein, based on which YK-VZV-007 (SEQ ID NO: 23) was designed in the present disclosure, which retains the same combination of mutation sites (Y582A, S593A, S595A, T596A, and T598A) as the mRNA vaccine antigen sequence mentioned in CN108472309A. YK-VZV007 was also used as one of the controls for antigen screening in the present disclosure, along with the newly designed mutation sequences or mutation combinations in the present disclosure for vaccine immunogenicity assessment. Specific results are shown in the examples.

[0403] Summary and analysis of results Results of in vitro expression of VZV gE antigen variants Petition 870260035339, dated 04 / 15 / 2026, pp. 218 / 249 91 / 121 Western blot detection results for 43 VZV gE antigen variants (FIG. 2) show that YK-VZV-002 exhibits extremely low or almost no antigenic protein expression; YK-VZV-041, YK-VZV-042, and YK-VZV-043 show negative results with no antigenic protein expression. Consequently, these four sequences could not be used as candidate antigens for the herpes zoster mRNA vaccine and were therefore excluded.

[0404] All other antigen variants exhibit significant protein expression, with the size of the expressed protein ranging from approximately 63 kDa to 75 kDa, which is consistent with the theoretical design of the antigen. These antigen variants can be used as effective candidate antigens for screening and subsequent immunogenicity assessment.

[0405] As shown by the results of the Western blot experiments above, all 39 antigen sequences of YK-VZV-001, YK-VZV-003, YK-VZV-004 (Example Comparative 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-VZV-013, YK-VZV-04, YK-VZV-01 YK-VZV-015, YK-VZV-016, YK-VZV-017, YK-VZV-018, YK-VZV-019 YKVZV-020, YK-VZV-021, YK-VZV-022, YK-VZV-023, YK-VZV-024, YK-VZV-023 YK-VZV-025, YKVZV-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, YKVZV-038, YK-VZV-039, YK-VZV-040, YK-VZV-044, and YK-VZV-045 (Example 3) can be used as gene expression candidates subsequent evaluation. Therefore, the gE-specific antibody IgG titer assay was conducted against these antigens.

[0406] Results of gE protein-specific IgG antibody titers of VZV gE antigen variants in Balb / c camundo sister (D42) As shown in Example 5, specific IgG antibody titers for the gE protein in mouse serum were measured on day 42 post-immunization by ELISA. The results are shown in Table 13. Petition 870260035339, dated 04 / 15 / 2026, pp. 219 / 249 92 / 121 Table 13: Specific IgG antibody titers for VZV gE protein in mouse serum (day 42) Substância de teste Anticorpo GMT (x 104) Anticorpo GMT da sustancia de teste / anticorpo GMT de Shingrix® (vezes) Anticorpo de teste de sustancia de teste / anticorpo GMT de YK-VZV-007 (vezes) YK-VZV-009 1000 5.3 2.8 YK-VZV-010 1000 5.3 2.8 YK-VZV-013 1000 5.3 2.8 YK-VZV-014 1000 5.3 2.8 YK-VZV-011 950 5.0 2.6 YK-VZV-012 944 5.0 2.6 YK-VZV-018 870 4.6 2.4 YK-VZV-020 800 4.2 2.2 YK-VZV-030 662 3.5 1.8 YK-VZV-031 659 3.5 1.8 YK-VZV-038 654 3.4 1.8 YK-VZV-021 650 3.4 1.8 YK-VZV-024 640 3.4 1.8 YK-VZV-028 630 3.3 1.8 YK-VZV-003 560 2.9 1.6 YK-VZV-001 540 2.8 1.5 YK-VZV-016 530 2.8 1.5 YK-VZV-015 520 2.7 1.4 YK-VZV-017 510 2.7 1.4 YK-VZV-023 190 1.0 0.5 YK-VZV-022 163 0.9 0.5 YK-VZV-026 163 0.9 0.5 YK-VZV-027 163 0.9 0.5 YK-VZV-033 163 0.9 0.5 YK-VZV-037 163 0.9 0.5 YK-VZV-039 163 0.9 0.5 YK-VZV-044 163 0.9 0.5 YK-VZV-040 162 0.9 0.5 YK-VZV-032 160 0.8 0.4 YK-VZV-034 154 0.8 0.4 YK-VZV-025 151 0.8 0.4 YK-VZV-035 150 0.8 0.4 YK-VZV-036 136 0.7 0.4 YK-VZV-029 102 0.5 0.3 LNP 0 0.0 0,0 Shingrix® 190 1.0 0.5 Petition 870260035339, dated 04 / 15 / 2026, pages 220 / 249 93 / 121 YK-VZV-004 (Comparative Example 1) 400 2.1 1.1 YK-VZV-006 (Comparative Example 2) 360 1.9 1.0 YK-VZV-045 (Comparative Example 3) 352 1.9 1.0 YK-VZV-007 (Comparative Example 4) 360 1.9 1.0 As shown by the gE-specific antibody IgG titer results on day 42, the Shingrix® positive control had a titer result of 190 x 10⁴, while the following variants outperformed Shingrix®, including YK-VZV-009, YK-VZV-011, YK-VZV013, YK-VZV-014, YK-VZV-010, YK-VZV-012, YK-VZV-018, YK-VZV-020, YK-VZV030, YK-VZV-031, YK-VZV-038, YK-VZV-021, YK-VZV-024, YK-VZV-028, YK-VZV003, YK-VZV-001, YK-VZV-016, YK-VZV-015, YK-VZV-017, 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), whose titer was 2.7 to 5.3 times that of the Shingrix® positive control.

[0407] Using the Shingrix® positive control as a standard, and based on the fact that the IgG titer result of the gE-specific antibody above was also 1.8 to 2.8 times better than that of the YK-VZV-007 sequence (Comparative Example 4), a total of 14 variants were additionally extracted, including YK-VZV-009, YK-VZV-011, YK-VZV013, YK-VZV-014, YK-VZV-010, YK-VZV-012, YK-VZV-018, YK-VZV-020, YK-VZV030, YK-VZV-031, YK-VZV-038, YK-VZV-021, YK-VZV-024, YK-VZV-028, as well as 4 control sequences including 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) for subsequent testing.

[0408] Results of cellular immunity to VZV gE antigen variants (FCM) Cellular immunity, particularly T-cell immunity, plays a crucial role in the VZV infection process. Studies have demonstrated that the cell-mediated immune response is an important indicator for evaluating the immunological effect of the herpes zoster vaccine. To further evaluate the specific cellular immune response induced by immunizing mice with the above-mentioned antigen variant mRNA, the Petition 870260035339, dated 04 / 15 / 2026, pages 221 / 249 94 / 121 specific CD4+ and CD8+ T cell immune responses induced by immunization of mice with VZV gE antigen mRNA were detected by FCM in Example 6.

[0409] Using the Shingrix® positive control as a standard, and based on the fact that the IgG titer result of the gE-specific antibody above was also better than that of the YK-VZV-007 sequence, a total of 14 variants were additionally extracted, including the spleens of mice immunized on day 56 of YK-VZV-009, YK-VZV-011, YK-VZV013, YK-VZV-014, YK-VZV-010, YK-VZV-012, YK-VZV-018, YK-VZV-020, YK-VZV030, YK-VZV-031, YK-VZV-038, YK-VZV-021, YK-VZV-024, YK-VZV-028, as well as 4 sequences of Control cells including 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 used, and the percentage of CD4+ T cells and CD8+ T cells secreting IFN-γ and IL-2 in mouse spleens was measured according to the detection method in Example 6.

[0410] CD4+ T cells The immunological effect of CD4+ T cells plays a crucial role in recovery from VZV infection and in the immunological effect of the vaccine. It is one of the most important indicators for evaluating the immunological effect of the vaccine. The spleens of immunized mice were removed on day 56, and the percentage of CD4+ T cells secreting IFN-γ and IL-2 in the mouse spleens was measured. The specific results are shown in the table below. Table 14: Summary of CD4+ T cell assay results Test substance CD4+ (IFN-γ') CD4+ (IL-2+) CD4+ (IFN-γ' or IL-2+) CD4+ (IFN-γ+) Compared to Shingrix® (times) Compared to YK-VZV007 (times) CD4+ (IL-2+) Compared to Shingrix® (times) Compared to YK-VZV007 (times) CD4+ (IFN-γ' or IL-2+) Compared to Shingrix® (times) Compared to YK-VZV007 (times) YK-VZV-010 2.00% 6.7 4.4 3.70% 12.3 7.4 4.20% 7.0 5.3 YK-VZV-013 1.70% 5.7 3.8 1.30% 4.3 2.6 1.90% 3.2 2.4 YK-VZV-011 1.60% 5.3 3.6 1.20% 4.0 2.4 1.60% 2.7 2.0 YK-VZV-020 1.60% 5.3 3.6 2.50% 8.3 5.0 3.10% 5.2 3.9 YK-VZV-012 1.40% 4.7 3.1 1.20% 4.0 2.4 1.70% 2.8 2.1 Petition 870260035339, dated 04 / 15 / 2026, pp. 222 / 249 95 / 121 YK-VZV-018 1.20% 4.0 2.7 1.10% 3.7 2.2 1.40% 2.3 1.8 YK-VZV-028 1.20% 4.0 2.7 1.30% 4.3 2.6 1.70% YK-VZV-012. 1.10% 3.7 2.4 0.80% 2.7 1.6 1.10% 1.8 1.4 YK-VZV-038 1.10% 3.7 2.4 1.40% 4.7 2.8 1.90% 3.2 2.4 YK-VZ-VZV 2.30% 2.30% 1.40% 4.7 2.8 1.70% 2.8 2.1 YK-VZV-021 0.90% 3.0 2.0 1.10% 3.7 2.2 1.50% 2.5 1.9 YK-VZV-009 0.80% 2.70% 2.18 .2.17 1.40% 2.3 1.8 YK-VZV-024 0.70% 2.3 1.6 0.60% 2.0 1.2 0.90% 1.5 1.1 YK-VZV-030 0.60% 2.0 1.3 0.80% 2.71% 1.1.1.8% lnp 0.10% 0.3 0.2 0.10% 0.3 0.2 0.20% 0.3 0.3 Shingrix® 0.30% 1.0 0.7 0.30% 1.0 0.6 0.60% 1.0 0.8 YK-VZV-0.040.5% Comparative 1.8 1.2 0.55% 1.8 1.1 0.95% 1.6 1.2 YK-VZV-045 (Comparative Sample 3) 0.50% 1.7 1.1 0.30% 1.0 0.6 0.50% 0.8 0.6 YK-VZV-045 (Comparative Sample) 0.45% 1.5 1.0 0.50% 1.7 1.0 0.80% 1.3 1.0 Note: Shingrix® is a recombinant protein vaccine from GSK.

[0411] Percentage of total IFN-y+CD4+ T cells and IL-2+ CD4+ T cells A) Based on the above results, mRNA vaccines prepared from the test substances 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 (11 in total) were excluded. The total percentage of IFN-γ+CD4+ T cells and IL-2+CD4+ T cells secreted by these mRNA vaccines was 2 times or more and up to 7 times that of the positive control vaccine Shingrix® and 1.8 to 5.3 times that of YK-VZV-007 (Comparative Example 4). Furthermore, the results were significantly better than those of YK-VZV-004 (Comparative Example 1) and YK-VZV-045 (Comparative Example 3).

[0412] Specifically, the total percentage of IFN-γ+CD4+ T cells and IL-2+ CD4+ T cells secreted by mRNA vaccines prepared from YK-VZV-010, YK-VZV-020, YK-VZV-013, YK-VZV-011, and YK-VZV-018 was 7.0, 5.2, 3.2, 2.7, and 2.3 times that of the positive control vaccine Shingrix®, respectively; 5.3, 3.9, 2.4, 2.0, and 1.8 times that of YK-VZV-007 (Comparative Example 4), respectively; and significantly higher than that of YK-VZV004 (Comparative Example 1) and YK-VZV-045 (Comparative Example 3). Petition 870260035339, dated 04 / 15 / 2026, pp. 223 / 249 96 / 121

[0413] Details are shown in the table below. Table 15: Total percentage of IFN-γ+ CD4+ T cells and IL-2+ CD4+ T cells induced by mRNA vaccines - 1 CD4+ test substance (IFN-y+ or IL2+) Compared to Shingrix® (times) Compared to YK-VZV-007 (times) YK-VZV-010 4.20% 7.0 5.3 YK-VZV-020 3.10% 5.23 YK-VZV-007 1.90% 3.2 2.4 YK-VZV-038 1.90% 3.2 2.4 YK-VZV-012 1.70% 2.8 2.1 YK-VZV-028 1.70% 2.8 2.1 YK-VZV-031 1.70% 2.8 YK-VZV-011 1.60% 2.7 2.0 YK-VZV-021 1.50% 2.5 1.9 YK-VZV-018 1.40% 2.3 1.8 YK-VZV-009 1.40% 2.3 1.8 LNP ® 30.30x 0.60% 1.0 0.8 YK-VZV-004 (Comparative Sample 1) 0.95% 1.6 1.2 YK-VZV-045 (Comparative Sample 3) 0.50% 0.8 0.6 YK-VZV-007 (Comparative Sample 4) 1.80.80%

[0414] B) However, the total percentage of IFN-γ+CD4+ T cells and IL-2+CD4+ T cells secreted by mRNA vaccines prepared from the test substances YK-VZV-014, YK-VZV-030 and YK-VZV-024 was less than 2 times that of the Shingrix® positive control vaccine and less than 1.5 times that of Comparative Example 4 (YK-VZV-007), so these sequences were excluded. The specific results are shown in the table below.

[0415] Table 16: Total percentage of IFN-γ+CD4+ T cells and IL-2+CD4+ T cells induced by mRNA vaccines - 2 Test substance CD4+ (IFN-γ+ or IL2+) Compared to Shingrix® (times) Compared to YK-VZV-007 (times) YK-VZV-014 1.10% 1.8 1.4 YK-VZV-030 1.10% 1.8 1.4 YK-VZV-024 0.90% 1.5 1.1 LNP 0.20% 0.3 0.3 Shingrix® 0.60% 1.0 0.8 Petition 870260035339, dated 04 / 15 / 2026, pp. 224 / 249 97 / 121 YK-VZV-004 (Comparative Example 1) 0.95% 1.6 1.2 YK-VZV-045 (Comparative Example 3) 0.50% 0.8 0.6 YK-VZV-007 (Comparative Example 4) 0.80% 1.3 1.0

[0416] Percentage of IFN-γ+ CD4+ T cells A) The mRNA vaccines prepared from the 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-014, YK-VZV-038 and YK-VZV-031 were screened. The percentage of IFNγ+CD4+ T cells secreted by these mRNA vaccines was ideally 1.00 to 2.00%, which was significantly higher than that of the comparative examples (YK-VZV-004, YK-VZV-045 and YK-VZV-007); which was 3 times or more and up to 6.7 times that of the positive control vaccine Shingrix®; and this was approximately 2.2 to 4.4 times that of Comparative Example 4 (YK-VZV-007).

[0417] Specifically, the percentage of IFN-γ' CD4+ T cells secreted by the mRNA vaccines corresponding to YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020 and YKVZV-018 was 6.7, 5.7, 5.3, 5.3 and 4.0 times that of the positive control vaccine Shingrix®, respectively; 4.4, 3.8, 3.6, 3.6 and 2.7 times that of Comparative Example 4 (YK-VZV-007), respectively; and significantly higher than that of Comparative Example 1 (YK-VZV004) and Comparative Example 3 (YK-VZV-045).

[0418] The specific results are shown in the table below. Petition 870260035339, dated 04 / 15 / 2026, pp. 225 / 249 98 / 121 Table 17: IFN-γ+ CD4+ T cells induced by mRNA vaccines - 1 Test substance CD4+ IFN-y+ Compared to Shingrix® (times) Compared to YKVZV-007 (times) YK-VZV-010 2.00% 6.7 4.4 YK-VZV-013 1.70% 5.7 3.8 YK-VZV-011 1.60% 5.3 3.6 YK-VZV-020 1.60% 5.3 3.6 YK-VZV-012 1.40% 4.7 3.1 YK-VZV-018 1.20% 4.0 2.7 YK-VZV-028 1.20% 4.0 2.7 YK-VZV-014 1.10% 3.7 2.4 YK-VZV-038 1.10% 3.7 2.4 YK-VZV-031 1.00% 3.3 2.2 LNP 0.10% 0.3 0.2 Shingrix® 0.30% 1.0 0.7 YK-VZV-004 (Comparative Example 1) 0.55% 1.8 1.2 YK-VZV-045 (Comparative Example 3) 0.50% 1.7 1.1 YK-VZV-007 (Comparative Example 4) 0.45% 1.5 1.0

[0419] B) Since the percentage of IFN-γ+ CD4+ T cells secreted by mRNA vaccines prepared from the test substances YK-VZV-021, YK-VZV-009, YK-VZV-024, and YK-VZV-030 was less than 1.0%, which was significantly lower than the ideal and relatively ideal percentage of cells secreted by mRNA vaccines (1.0 to 2.0%), only 2 to 3 times that of the Shingrix® positive control vaccine and approximately 1 to 2 times that of Comparative Example 4 (YK-VZV-007), YK-VZV-021, YK-VZV-009, YK-VZV-024, and YK-VZV-030 were excluded from the candidate sequences. The specific results are shown in the table below. Table 18: IFN-γ+ CD4+ T cells induced by mRNA vaccines - 2 Test substance CD4+ IFN-γ+ Compared to Shingrix® (times) Compared to YKVZV-007 (times) YK-VZV-021 0.90% 3.0 2.0 YK-VZV-009 0.80% 2.7 1.8 YK-VZV-024 0.70% 2.3 1.6 YK-VZV-030 0.60% 2.0 1.3 LNP 0.10% 0.3 0.2 Shingrix® 0.30% 1.0 0.7 Petition 870260035339, dated 04 / 15 / 2026, pages 226 / 249 99 / 121 YK-VZV-004 (Comparative Example 1) 0.55% 1.8 1.2 YK-VZV-045 (Comparative Example 3) 0.50% 1.7 1.1 YK-VZV-007 (Comparative Example 4) 0.45% 1.5 1.0

[0420] IL-2+ CD4+ T cells A) The mRNA vaccines corresponding to the test substances YK-VZV-010, YKVZV-020, YK-VZV-038, YK-VZV-031, YK-VZV-013, YK-VZV-028, YK-VZV-011, YKVZV-012, YK-VZV-018, YK-VZV-021, and YK-VZV-009 were screened. The percentage of IL-2+ CD4+ T cells secreted by these mRNA vaccines was ideally 1.10 to 3.70%, which was significantly higher than that of the comparative sample mRNA vaccines (YK-VZV004, YK-VZV-045, and YK-VZV-007); which was 3.7 times or more than the positive control vaccine Shingrix® and 2.2 times or more than that of YK-VZV-007; and which was significantly higher than that of Comparative Example 1 (YK-VZV-004) and Comparative Example 3 (YK-VZV-045).

[0421] Specifically, the percentage of IL-2+ CD4+ T cells secreted by mRNA vaccines prepared from YK-VZV-010, YK-VZV-020, YK-VZV-013, YK-VZV-011, and YK-VZV-018 was 12.3, 8.3, 4.3, 4.0, and 3.7 times that of the positive control vaccine Shingrix®, respectively; 7.4, 5.0, 2.6, 2.4, and 2.2 times that of YK-VZV-007 (Comparative Example 4), respectively; and significantly higher than that of YK-VZV-004 (Comparative Example 1) and YK-VZV-045 (Comparative Example 3).

[0422] The specific results are shown in the table below. Table 19: IL-2+ CD4+ T cells induced by mRNA vaccines - 1 Test substance CD4+ IL-2+ Compared to Shingrix® (times) Compared to YKVZV-007 (times) YK-VZV-010 3.70% 12.3 7.4 YK-VZV-020 2.50% 8.3 5.0 YK-VZV-038 1.40% 4.7 2.8 YK-VZV-031 1.40% 4.7 2.8 YK-VZV-013 1.30% 4.3 2.6 YK-VZV-028 1.30% 4.3 2.6 YK-VZV-011 1.20% 4.0 2.4 YK-VZV-012 1.20% 4.0 2.4 YK-VZV-018 1.10% 3.7 2.2 YK-VZV-021 1.10% 3.7 2.2 YK-VZV-009 1.10% 3.7 2.2 Petition 870260035339, dated 04 / 15 / 2026, pages 227 / 249 100 / 121 LNP 0.10% 0.3 0.2 Shingrix® 0.30% 1.0 0.6 YK-VZV-004 (Comparative Example 1) 0.55% 1.8 1.1 YK-VZV-045 (Comparative Example 3) 0.30% 1.0 0.6 YK-VZV-007 (Comparative Example 4) 0.50% 1.7 1.0 B) Since the percentage of IL-2+ CD4+ T cells secreted by the mRNA vaccines prepared from the test substances YK-VZV-014, YK-VZV-030, and YK-VZV-024 was less than 1.0%, which was significantly lower than the ideal and relatively ideal percentage of cells secreted by mRNA vaccines (more than 1.0%), 3 times or less that of the positive control vaccine Shingrix®, and less than twice that of YK-VZV-007 (Comparative Example 4), YK-VZV-014, YK-VZV-030, and YK-VZV-024 were excluded from the candidate sequences. The specific results are shown in the table below. Table 20: IL-2+ CD4+ T cells induced by mRNA vaccines - 2 Test substance CD4+ IL-2+ Compared to Shingrix® (times) Compared to YKVZV-007 (times) YK-VZV-014 0.80% 2.7 1.6 YK-VZV-030 0.80% 2.7 1.6 YK-VZV-024 0.60% 2.0 1.2 LNP 0.10% 0.3 0.2 Shingrix® 0.30% 1.0 0.6 YK-VZV-004 (Comparative Example 1) 0.55% 1.8 1.1 YK-VZV-045 (Comparative Example 3) 0.30% 1.0 0.6 YK-VZV-007 (Comparative Example 4) 0.50% 1.7 1.0

[0423] IV) Conclusion A) After immunizing mice with mRNA vaccines prepared from the selected sequences, the percentage of immune cells in the spleens of the mice is as follows: The total percentage of IFN-γ+ CD4+ T cells and IL-2+ CD4+ T cells was 2 times or more and up to 7 times that of the Shingrix® positive control and 1.8 to 5.3 times that of YK-VZV-007 (Comparative Example 4); Petition 870260035339, dated 04 / 15 / 2026, pp. 228 / 249 101 / 121 the percentage of IFN-Y+ CD4+ T cells was 1.00 to 2.00%, which was 3 times or more and up to 6.7 times that of the Shingrix® positive control vaccine and approximately 2.2 to 4.4 times that of Comparative Example 4 (YK-VZV-007); The percentage of IL-2+ CD4+ T cells was greater than 1%, which was 3.7 times or more that of the positive control vaccine Shingrix® and 2.2 times or more that of YK-VZV-007; the details are as follows: Table 21: CD4+ T cells producing IFN-γ+ or IL-2+ induced by mRNA vaccines Test substance CD4+ (IFN-y+) CD4+ (IL-2+) CD4+ (IFN-y+ or IL-2+) CD4+ (IFN-y+) Compared with Shingrix® (times) Compared with yk-vzv007 (times) CD4+ (IL-2+) Compared with Shingrix® (times) compared to yk-vzv007 (sometimes) CD4+ (IFN-y+ or IL-2+) Compared to Shingrix® (sometimes) Compared to yk-vzv007 (sometimes) YK-VZV-010 2.00% 6.7 4.4 3.70% 7.0 5.3 YK-VZV-013 1.70% 5.7 3.8 1.30% 4.3 2.6 1.90% 3.2 2.4 YK-VZV-011 1.60% 5.3 3.6 1.20% 4.0 2.4 1.60% 2.07 YK-VZV-020 1.60% 5.3 3.6 2.50% 8.3 5.0 3.10% 5.2 3.9 YK-VZV-012 1.40% 4.7 3.1 1.20% 4.0 2.4 1.70% 2.8 YK-VZV-018 1.20% 4.0 2.7 1.10% 3.7 2.2 1.40% 2.3 1.8 YK-VZV-028 1.20% 4.0 2.7 1.30% 4.3 2.6 1.70% 2.8 2.1 YK-VZ-VZV-038 1.70% 1.40% 4.7 2.8 1.90% 3.2 2.4 YK-VZV-031 1.00% 3.3 2.2 1.40% 4.7 2.8 1.70% 2.8 2.1 LNP 0.10% 0.3 0.2 0.10% 0.00.20% 0.3 0.3 Shingrix® 0.30% 1.0 0.7 0.30% 1.0 0.6 0.60% 1.0 0.8 YK-VZV-004 (Comparative Sample 1) 0.55% 1.8 1.2 0.55% 1.81 1.1.1.65%2 YK-VZV-045 (Comparative Example 3) 0.50% 1.7 1.1 0.30% 1.0 0.6 0.50% 0.8 0.6 YK-VZV-007 (Comparative Example 4) 0.45% 1.5 1.0 0.50% 1.7 1.0 0.80% 1.3 1.0

[0424] Among the sequences above, the percentage of YK-VZV-010-induced IFN-γ+CD4+ T cells and IL-2+ CD4+ T cells in spleen cells was greater than 2%, which was approximately 6 to 12 times that of the Shingrix® positive control vaccine, indicating the best Petition 870260035339, dated 04 / 15 / 2026, pages 229 / 249 102 / 121 immune effect.

[0425] B) The percentage of immune cells secreted by mRNA vaccines prepared from the deleted sequences is as follows: The total percentage of IFN-γ+CD4+ T cells and IL-2+CD4+ T cells was less than 2 times that of the Shingrix® positive control vaccine and less than 1.5 times that of Comparative Example 4 (YK-VZV-007); or the percentage of IFN-γ+CD4+ T cells secreted by the mRNA vaccines was less than 1.0%, which was only 2 to 3 times that of the Shingrix® positive control vaccine and approximately 1 to 2 times that of Comparative Example 4 (YK-VZV-007); or the percentage of IL-2+CD4+ T cells was less than 1.0%, which was 3 times or less that of the Shingrix® positive control vaccine and less than 2 times that of YK-VZV-007 (Example Comparative 4); the details are as follows: Table 22: CD4+ T cells producing IFN-γ' or IL-2+ induced by mRNA vaccines Test substance CD4+ (IFN-γ·) CD4· (IL-2·) CD4· (IFN-γ· or IL-2·) CD4+ (IFN-γ·) Compared to Shingrix® (times) Compared to YK-VZV007 (times) CD4· (IL-2·) Compared to Shingrix® (times) Compared to YK-VZV007 (times) CD4· (IFN-γ· or IL-2·) Compared to Shingrix® (times) Compared to YK-VZV007 (times) YK-VZV-014 1.10% 3.7 2.4 0.80% 2.7 1.6 1.10% 1.8 1.4 YK-VZV-021 0.90% 3.0 2.0 1.10% 3.7 2.2 1.50% 2.5 1.9 YK-VZV-009 0.80% 2.7 1.8 1.10% 3.7 2.2 1.40% 2.3 1.8 YK-VZV-024 0.70% 2.3 1.6 0.60% 2.0 1.2 0.90% 1.5 1.1 YK-VZV-030 0.60% 2.0 1.3 0.80% 2.7 1.6 1.10% 1.8 1.4 LNP 0.10% 0.3 0.2 0.10% 0.3 0.2 0.20% 0.3 0.3 Shingrix® 0.30% 1.0 0.7 0.30% 1.0 0.6 0.60% 1.0 0.8 YK-VZV-004 (Comparative Example 1) 0.55% 1.8 1.2 0.55% 1.8 1.1 0.95% 1.6 1.2 YK-VZV-045 (Comparative Example 3) 0.50% 1.7 1.1 0.30% 1.0 0.6 0.50% 0.8 0.6 YK-VZV-007 (Comparative Example 4) 0.45% 1.5 1.0 0.50% 1.7 1.0 0.80% 1.3 1.0

[0426] The percentage of CD4+ T cells producing IFN-γ or IL-2 induced by vaccines Petition 870260035339, dated 04 / 15 / 2026, pages 230 / 249 The percentage of mRNA in spleen cells (103 / 121) was less than 1%, and the total percentage of IFNγ+ CD4+ T cells and IL-2+ CD4+ T cells was less than 2.0 times that of Shingrix®, indicating a weak immune-boosting effect. Therefore, these sequences were not suitable as candidate sequences for the herpes zoster vaccine compared to the sequences in Part A above.

[0427] In summary, mRNA vaccines prepared from the test substances YKVZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020, YK-VZV-012, YK-VZV-018, YKVZV-028, YK-VZV-038 and YK-VZV-031 were selected. IFN-γ+CD4+ T cells and IL-2+CD4+ T cells induced by these mRNA vaccines had an excellent effect. Therefore, the above sequences, as well as YK-VZV-004 (Comparative Example 1), YK-VZV006 (Comparative Example 2), YK-VZV-045 (Comparative Example 3) and YK-VZV-007 (Comparative Example 4) were used for subsequent CD8+ T cell immunoassays.

[0428] CD8+ T cells Based on the results for CD4+ T cells, the 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, YK-VZV-031, 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 tested, and the number of CD8+ T cells producing IL-2 / IFN-γ induced by these sequences was measured. The specific results are shown in the table below. Table 23: Summary of CD8+ T cell assay results Test substance CD8+ IFN-γ+ CD8+ IL-2+ CD8+ (IFN-γ+ or IL-2+) CD8+ (IFN-γ+) Compared to Shingrix® (times) Compared to yk-vzv007 (times) CD8+ (IL-2+) Compared to Shingrix® (times) Compared to yk-vzv007 (times) CD8+ (IFN-γ+ or IL-2+) Compared to Shingrix® (times) Compared to yk-vzv007 (times) YK-VZV-010 8.10% 7.36 4.26 0.50% 4.17 2.78 7.90% 7.18 4.94 YK-VZV-013 6.90% 6.27 3.63 0.40% 3.33 2.22 7.10% 6.45 4.44 YK-VZV-020 5.60% 5.09 2.95 0.30% 2.50 1.67 5.60% 5.09 3.50 YK-VZV-018 5.20% 4.73 2.74 0.40% 3.33 2.22 5.20% 4.73 3.25 YK-VZV-011 5.00% 4.55 2.63 0.40% 3.33 2.22 5.10% 4.64 3.19 YK-VZV-012 4.20% 3.82 2.21 0.10% 0.83 0.56 4.20% 3.82 2.63 YK-VZV-028 3.10% 2.82 1.63 0.40% 3.33 2.22 3.10% 2.82 1.94 YK-VZV-038 2.40% 2.18 1.26 0.20% 1.67 1.11 2.30% 2.09 1.44 Petition 870260035339, dated 04 / 15 / 2026, pages 231 / 249 104 / 121 YK-VZV-031 1.80% 1.64 0.95 0.10% 0.83 0.56 1.90% 1.73 1.19 lnp 0.00% - - 0.00% - - 0.00% - - Shingrix® 1.10% 1.00 0.58 0.12% 1.00 0.67 1.10% 1.00 0.69 YK-VZV-004 (Comparative Example 1) 2.80% 2.55 1.47 0.21% 1.75 1.17 2.70% 2.45 1.69 YK-VZV-006 (Comparative Example 2) 1.90% 1.73 1.00 0.20% 1.67 1.11 1.50% 1.36 0.94 YK-VZV-045 (Comparative Example 3) 1.50% 1.36 0.79 0.16% 1.33 0.89 1.40% 1.27 0.88 YK-VZV-007 (Comparative Example 4) 1.90% 1.73 1.00 0.18% 1.50 1.00 1.60% 1.45 1.00 Note: Shingrix® is a recombinant protein vaccine from GSK.

[0429] Total percentage of IFN-γ+CD8+ T cells and IL-2+ CD8+ T cells A) Based on the above results, mRNA vaccines prepared from the test substances YK-VZV-010, YK-VZV-013, YK-VZV-020, YK-VZV-018, and YK-VZV011 were screened. The total percentage of IFN-γ+CD8+ T cells and IL-2+CD8+ T cells induced by these mRNA vaccines was 4 to 7 times or more that of the Shingrix® positive control vaccine and approximately 3 to 5 times that of YK-VZV-007 (Comparative Example 4). The specific results are shown in the table below. Table 24: Total percentage of IFN-γ+CD8+ T cells and IL-2+CD8+ T cells induced by mRNA vaccines - 1 Test substance CD8+ (IFN-γ+ or IL-2+) Compared to Shingrix® (times) Compared to YK-VZV-007 (times) YK-VZV-010 7.90% 7.18 4.94 YK-VZV-013 7.10% 6.45 4.44 YK-VZV-020 5.60% 5.09 3.50 YK-VZV-018 5.20% 4.73 3.25 YK-VZV-011 5.10% 4.64 3.19 lnp 0.00% - - Shingrix® 1.10% 1.00 0.69 YK-VZV-004 (Comparative Example 1) 2.70% 2.45 1.69 YK-VZV-006 (Comparative Example 2) 1.50% 1.36 0.94 Petition 870260035339, dated 04 / 15 / 2026, pages 232 / 249 105 / 121 YK-VZV-045 (Comparative Example 3) 1.40% 1.27 0.88 YK-VZV-007 (Comparative Example 4) 1.60% 1.45 1.00

[0430] B) However, the total percentage of IFN-γ+ CD8+ T cells and IL-2+ CD8+ T cells secreted by mRNA vaccines prepared from the test substances YK-VZV-012, YK-VZV-028, YK-VZV-038 and YK-VZV-031 was less than 4 times that of the positive control vaccine Shingrix® and 3 times or less that of YK-VZV-007 (Comparative Example 4). The specific results are shown in the table below. Table 25: Total percentage of IFN-γ+ CD8+ T cells and IL-2+ CD8+ T cells induced by mRNA vaccines - 2 Test substance CD8+ (IFN-γ+ or IL-2+) Compared to Shingrix® (times) Compared to YK-VZV-007 (times) YK-VZV-012 4.20% 3.82 2.63 YK-VZV-028 3.10% 2.82 1.94 YK-VZV-038 2.30% 2.09 1.44 YK-VZV-031 1.90% 1.73 1.19 LNP 0.00% - - Shingrix® 1.10% 1.00 0.69 YK-VZV-004 (Comparative Example 1) 2.70% 2.45 1.69 YK-VZV-006 (Comparative Example 2) 1.50% 1.36 0.94 YK-VZV-045 (Comparative Example 3) 1.40% 1.27 0.88 YK-VZV-007 (Comparative Example 4) 1.60% 1.45 1.00

[0431] T cells IFN-γ+CD8+ A) The mRNA vaccines prepared from the test substances YK-VZV-010, YK-VZV-013, YK-VZV-020, YK-VZV-018, and YK-VZV-011 were classified. The percentage of IFN-γ+CD8+ T cells secreted by these mRNA vaccines was ideal at 5.0% or more, with the percentage for YK-VZV-010 being the highest at 8.10%, which was significantly higher than that of Comparative Examples 1 to 4 (YK-VZV-004, YK-VZV-006, YK-VZV-045, and YK-VZV-007). The percentage of these five sequences was 4.5 to 7.0 times or more that of the Shingrix® positive control vaccine and approximately 2.5 to 4.5 times that of YK-VZV-007. You Petition 870260035339, dated 04 / 15 / 2026, pp. 233 / 249 106 / 121 specific results are shown in the table below. Table 26: IFN-γ+ CD8+ T cells induced by mRNA vaccines - 1 Test substance CD8+ (IFN-γ+) Compared to Shingrix® (times) Compared to YK-VZV-007 (times) YK-VZV-010 8.10% 7.36 4.26 YK-VZV-013 6.90% 6.27 3.63 YK-VZV-020 5.60% 5.09 2.95 YK-VZV-018 5.20% 4.73 2.74 YK-VZV-011 5.00% 4.55 2.63 LNP 0.00% - - Shingrix® 1.10% 1.00 0.58 YK-VZV-004 (Comparative Example 1) 2.80% 2.55 1.47 YK-VZV-006 (Comparative Example 2) 1.90% 1.73 1.00 YK-VZV-045 (Comparative Example 3) 1.50% 1.36 0.79 YK-VZV-007 (Comparative Example 4) 1.90% 1.73 1.00

[0432] B) Since the percentage of IFN-γ+CD8+ T cells secreted by mRNA vaccines prepared from the test substances YK-VZV-012, YK-VZV-028, YK-VZV-038 and YK-VZV-031 was less than 5%, which was significantly lower than the ideal percentage of cells secreted by mRNA vaccines (5.00 to 8.10%), only 4 times or less than the Shingrix® positive control vaccine and approximately 1 to 2 times that of YK-VZV-007, these sequences were excluded. The specific results are shown in the table below. Table 27: IFN-γ+CD8+ T cells induced by mRNA vaccines - 2 Test substance CD8+ (IFN-γ+) Compared to Shingrix® (times) Compared to YK-VZV-007 (times) YK-VZV-012 4.20% 3.82 2.21 YK-VZV-028 3.10% 2.82 1.63 YK-VZV-038 2.40% 2.18 1.26 YK-VZV-031 1.80% 1.64 0.95 LNP 0.00% - - Shingrix® 1.10% 1.00 0.58 YK-VZV-004 (Comparative Example 1) 2.80% 2.55 1.47 YK-VZV-006 (Comparative Example 2) 1.90% 1.73 1.00 YK-VZV-045 (Example 1.50% 1.36 0.79 Petition 870260035339, dated 04 / 15 / 2026, pages 234 / 249 107 / 121 Comparative Example 3) YK-VZV-007 (Comparative Example 4) 1.90% 1.73% 1.00%

[0433] IL-2+ CD8+ T cells A) The mRNA vaccines prepared from the test substances YK-VZV-010, YK-VZV-013, YK-VZV-018, YK-VZV-011, YK-VZV-028, and YK-VZV-020 were classified. The percentage of IL-2+ CD8+ T cells secreted by these mRNA vaccines was ideally 0.3 to 0.5%, which was significantly higher than that of the comparative examples (YK-VZV-004, YK-VZV-006, YK-VZV-045, and YK-VZV-007); and which was 2.5 to 4.0 times or more that of the positive control vaccine Shingrix® and approximately 1.5 to 3.0 times that of YK-VZV-007. The specific results are shown in the table below. Table 28: IL-2+ CD8+ T cells induced by mRNA vaccines - 1 Test substance CD8+ (IL-2+) Compared to Shingrix® (times) Compared to YKVZV-007 (times) YK-VZV-010 0.50% 4.17 2.78 YK-VZV-013 0.40% 3.33 2.22 YK-VZV-018 0.40% 3.33 2.22 YK-VZV-011 0.40% 3.33 2.22 YK-VZV-028 0.40% 3.33 2.22 YK-VZV-020 0.30% 2.50 1.67 LNP 0.00% - - Shingrix® 0.12% 1.00 0.67 YK-VZV-004 (Comparative Example) 1) 0.21% 1.75 1.17 YK-VZV-006 (Comparative Example 2) 0.20% 1.67 1.11 YK-VZV-045 (Comparative Example 3) 0.16% 1.33 0.89 YK-VZV-007 (Comparative Example 4) 0.18% 1.50 1.00

[0434] B) Since the percentage of IL-2+ CD8+ T cells secreted by mRNA vaccines prepared from the test substances YK-VZV-038, YK-VZV-012, and YK-VZV031 was only 0.2% or less, whose effect was not significantly different or worse than that of the positive control vaccine Shingrix® and YK-VZV-007, and which was only approximately 0.8 to 1.6 times that of the positive control vaccine Shingrix® and approximately 0.5 to 1.0 times that of YK-VZV-007, these sequences were excluded. The Petition 870260035339, dated 04 / 15 / 2026, pp. 235 / 249 108 / 121 specific results are shown in the table below. Table 29: IL-2+ CD8+ T cells induced by mRNA vaccines - 2 Test substance CD8+ (IL-2+) Compared to Shingrix® (times) Compared to YKVZV-007 (times) YK-VZV-038 0.20% 1.67 1.11 YK-VZV-012 0.10% 0.83 0.56 YK-VZV-031 0.10% 0.83 0.56 LNP 0.00% - - Shingrix® 0.12% 1.00 0.67 YK-VZV-004 (Comparative Example 1) 0.21% 1.75 1.17 YK-VZV-006 (Comparative Example 2) 0.20% 1.67 1.11 YK-VZV-045 (Comparative Example 3) 0.16% 1.33 0.89 YK-VZV-007 (Comparative Example 4) 0.18% 1.50 1.00 Conclusion:

[0435] Based on the results from (i) to (iii) above, the candidate vaccines were screened and the percentage of immune cells in the spleens of mice induced by these vaccines is as follows: A) The total percentage of IFN-γ+CD8+ T cells and IL-2+CD8+ T cells was 4 to 7 times or more that of the Shingrix® vaccine and approximately 3 to 5 times that of YK-VZV-007 (Comparative Example 4); The percentage of IFN-γ+CD8+ T cells was ideal at 5.0% or more, which was 4.5 to 7 times or more than that of the Shingrix® positive control vaccine and approximately 2.5 to 4.5 times that of YK-VZV-007; The percentage of IL-2+ CD8+ T cells was ideal at 0.3 to 0.5%, which was 2.5 to 4 times or more than that of the Shingrix® positive control vaccine and approximately 1.5 to 3 times that of YK-VZV-007; The results were significantly superior to those of the comparative examples (YK-VZV-004, YK-VZV-006, YK-VZV-045 and YK-VZV-007); the details are as follows: Petition 870260035339, dated 04 / 15 / 2026, pp. 236 / 249 109 / 121 Table 30: CD8+ T cells producing IFN-γ or IL-2+ induced by mRNA-1 vaccines Test substance CD8+ IFN-γ' CD8+ IL-2+ CD8+ (IFN-γ' or IL-2+) CD8+ (IFN-γ') Compared to Shingrix® (times) Compared to YK-VZV007 (times) CD8+ (IL-2+) Compared to Shingrix® (times) Compared to YK-VZV007 (times) CD8+ (IFN-γ' or IL-2+) Compared to Shingrix® (times) Compared to YK-VZV007 (times) YK-VZV-010 8.10% 7.36 4.26 0.50% 4.17 2.78 7.90% 7.18 4.94 YK-VZV-013 6.90% 6.27 3.63 0.40% 3.33 2.22 7.10% 6.45 4.44 YK-VZV-020 5.60% 5.09 2.95 0.30% 2.50 1.67 5.60% 5.09 3.50 YK-VZV-018 - - 0.00% - - 0.00% - - Shingrix® 1.10% 1.00 0.58 0.12% 1.00 0.67 1.10% 1.00 0.69 YK-VZV-004 (Comparative Example 1) 2.80% 2.55 1.47 0.21% 1.75 1.17 2.70% 2.45 1.69 YK-VZV-006 (Comparative Example 2) 1.90% 1.73 1.00 0.20% 1.67 1.11 1.50% 1.36 0.94 YK-VZV-045 (Comparative Example 3) 1.50% 1.36 0.79 0.16% 1.33 0.89 1.40% 1.27 0,88 YK-VZV-007 (Comparative Example 4) 1.90% 1.73 1.00 0.18% 1.50 1.00 1.60% 1.45 1.00

[0436] B) The percentage of immune cells in the spleens of mice induced by vaccines prepared from the excluded test substances is as follows: The total percentage of IFN-γ+CD8+ T cells and IL-2+CD8+ T cells was less than 4 times that of the Shingrix® vaccine and 3 times or less that of YK-VZV-007 (Comparative Example 4); Or, the percentage of IFN-γ+CD8+ T cells was less than 5%, which was significantly lower than the ideal percentage of cells secreted by mRNA vaccines (5.20 to 8.10%), only 4 times or less than the Shingrix® positive control vaccine and approximately 1 to 2 times that of YK-VZV-007; Alternatively, the percentage of IL-2+ CD8+ T cells was only 0.2% or less, the effect of which was not significantly different from or worse than that of the positive control vaccine Shingrix® and YK-VZV-007, and which was only approximately 0.8 to 1.6 times that of the control vaccine. Petition 870260035339, dated 04 / 15 / 2026, pages 237 / 249 110 / 121 positive Shingrix® and approximately 0.5 to 1.0 times that of YK-VZV-007, so these sequences were excluded; The details are as follows: Table 31: CD8+ T cells producing IFN-γ or IL-2+ induced by mRNA-2 vaccines Test substance CD8+ IFN-γ+ CD8+ IL-2+ CD8+ (IFN-γ+ or IL-2+) CD8+ (IFN-γ') Compared to Shingrix® (times) Compared to yk-vzv007 (times) CD8+ (IL-2+) Compared to Shingrix® (times) Compared to yk-vzv007 (times) CD8+ (IFN-γ+ or IL-2+) Compared to Shingrix® (times) Compared to yk-vzv007 (times) YK-VZV-012 4.20% 3.82 2.21 0.10% 0.83 0.56 4.20% 3.82 2.63 YK-VZV-028 3.10% 2.82 1.63 0.40% 3.33 2.22 3.10% 2.82 1.94 YK-VZV-038 2.40% 2.18 1.26 0.20% 1.67 1.11 2.30% 2.09 1.44 YK-VZV-031 1.80% 1.64 0.95 0.10% 0.83 0.56 1.90% 1.73 1.19 LNP 0.00% - - 0.00% - - 0.00% - - Shingrix® 1.10% 1.00 0.58 0.12% 1.00 0.67 1.10% 1.00 0.69 YK-VZV-004 (Comparative Example 1) 2.80% 2.55 1.47 0.21% 1.75 1.17 2.70% 2.45 1.69 YK-VZV-006 (Comparative Example 2) 1.90% 1.73 1.00 0.20% 1.67 1.11 1.50% 1.36 0.94 YK-VZV-045 (Comparative Example 3) 1.50% 1.36 0.79 0.16% 1.33 0.89 1.40% 1.27 0.88 YK-VZV-007 (Comparative Example 4) 1.90% 1.73 1.00 0.18% 1.50 1.00 1.60% 1.45 1.00

[0437] The percentage of CD8+ T cells producing IL-2+ induced by mRNA vaccines prepared from YK-VZV-012 and YK-VZV-031 was lower than that of the Shingrix® vaccine and YK-VZV-007 (Comparative Example 4). Therefore, these sequences were excluded.

[0438] The total percentage of IFN-γ-producing CD8+ T cells and IFN-γ+ and IL-2+-producing CD8+ T cells induced by mRNA vaccines prepared from YK-VZV028 and YK-VZV-038 was only approximately 2 times that of the Shingrix® vaccine, which was significantly lower than that of the sequences designed in Part A above (up to 7 times or Petition 870260035339, dated 04 / 15 / 2026, pp. 238 / 249 111 / 121 plus), indicating a weak immunological effect. Therefore, these sequences were not suitable as candidate sequences for the herpes zoster vaccine.

[0439] Therefore, based on the results above, the sequences with significant advantages in overall immune effect include: YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV020 and YK-VZV-018, with YK-VZV-010 showing the best effect.

[0440] Results of specific IgG antibody titers for gE protein of VZV gE antigen variants in serum from Balb / c (D56) mice To verify the consistency of some of the IgG antibody titers above and some of the results of the VZV gE antigen variant cell immunoassay, some of the following test substances (including 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, YK-VZV-038, YK-VZV-004 (Comparative Example 1), YK-VZV-045 (Comparative Example 3) and YK-VZV-007) were used. (Comparative Example 4)) were also used. Serum samples from mice immunized on day 56 were determined and VZV gE-bound IgG antibody titers in serum were measured according to the detection method in Example 5. The results are shown in the table below: Table 32: VZV gE-bound IgG antibody titers in serum (day 56) Test substance GMT antibody (x 104) GMT antibody from test substance / GMT antibody of Shingrix® GMT antibody from test substance / GMT antibody of YK-VZV-007 YK-VZV-010 750 5.00 2.68-101010 4.73 2.54 YK-VZV-018 698 4.65 2.49 YK-VZV-020 690 4.60 2.46 YK-VZV-013 669 4.46 2.39 YK-VZV-031 480 3.27 1.17 YK-VZV-030 454 3.03 1.62 YK-VZV-038 445 2.97 1.59 YK-VZV-009 420 2.80 1.50 YK-VZV-014 410 2.73 1.46 YK-VZV-012 40 5.40 1.50 YK-VZV-021 373 2.49 1.33 YK-VZV-028 357 2.38 1.28 Petition 870260035339, of 15 / 04 / 2026, p. 239 / 249 112 / 121 YK-VZV-024 274 1.83 0.98 LNP 0 0.00 0.00 Shingrix® 150 1.00 0.54 YK-VZV-004 (Comparative Example 1) 330 2.20 1.18 YK-VZV-045 (Comparative Example 170) 1.13 0.61 YK-VZV-007 (Comparative Sample 4) 280 1.87 1.00

[0441] Based on the above results, on day 56, the gE-specific IgG antibody titers of the above mRNA vaccines were all higher than the Shingrix® positive control, being approximately 1.8 to 5.0 times that of the Shingrix® vaccine, with the antibody GMT ranging from 270 to 750 x 104. Specifically, the antibody GMT of YK-VZV-010, YK-VZV011, YK-VZV-018, YK-VZV-020 and YK-VZV-013 can reach 650 x 104 or more, being 4.0 to 5.0 times or more that of the Shingrix® vaccine and 2 times or more that of YK-VZV-007 (Comparative Example 4). Furthermore, the results were significantly better than those of YK-VZV-004 (Comparative Example 1) and YK-VZV-045 (Comparative Example 3).

[0442] Based on IgG antibody titers, as well as CD4+ T cell and CD8+ T cell assay results, the five antigens YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020 and YK-VZV-018 exhibited the best cellular immunity effect, making them suitable candidate antigens for further detection and analysis by ELISPOT.

[0443] Results of cellular immunological factors of VZV gE antigen variants (ELISOPT) ELISOPT is the gold standard for screening and evaluating the immunological effects of antigen-specific T cells from vaccine candidates and can be used to differentiate between cytokine-activated T cell subpopulations, such as type 1 helper T (Th) 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).

[0444] Through comprehensive analysis of IgG and cytokine assay results, a total of 5 ideal antigen sequences (including YK-VZV-010, YK-VZV-013, YK-VZV-011, YK-VZV-020 and YK-VZV-018) were classified and further detected by ELISPOT along with Comparative Example 1 (YK-VZV-004), Comparative Example 2 (YK-VZV-006), Example Petition 870260035339, dated 04 / 15 / 2026, pp. 240 / 249 113 / 121 Comparative Example 3 (YK-VZV-045) and Comparative Example 4 (YK-VZV-007), using LNP and Shingrix® as controls. The result is as follows: 1) The amount of IFN-γ cytokine secreted by T cells stimulated by the mRNA vaccine was 500 SFU or more, with a maximum close to 600 SFU, which was approximately 16 to 19 times that of the Shingrix® positive control and 2.5 to 3.0 times that of Comparative Example 4 (YK-VZV-007); and which was significantly better than that of the cytokine secreted and stimulated by the mRNA vaccine corresponding to Comparative Example 1 (YK-VZV-004), Comparative Example 2 (YK-VZV-006) or Example Comparative 3 (YK-VZV-045).

[0445] Specifically, the mRNA vaccine prepared from the YK-VZV-010 sequence reached a maximum of 598.8 SFU, which was 18.7 times that of the Shingrix® positive control and almost 3 times that of the YK-VZV-007 control.

[0446] The IFN-γ secretion levels from high to low were YK-VZV-010, YK-VZV-018, YK-VZV-011, YK-VZV-020 and YK-VZV-013, with no significant difference between the groups, indicating a good and significant immunological effect. Petition 870260035339, dated 04 / 15 / 2026, pages 241 / 249 114 / 121 Table 33: Secretion of IFN-γ+ from spleen cells induced by mRNA vaccines Test substance IFN-γ+ (SFU) Compared to Shingrix® (times) Compared to YK-VZV-007 (times) YK-VZV-010 598.8 18.71 2.99 YK-VZV-018 565 17.66 2.82 YK-VZV-011 561.7 17.55 2.80 YK-VZV-020 522.4 16.33 2.61 YK-VZV-013 511 15.97 2.55 LNP 1.2 0.04 0.01 Shingrix® 32.0 1.00 0.16 YK-VZV-004 (Comparative Example 1) 280.3 8.76 1.40 YK-VZV-006 (Comparative Example 2) 220.3 6.88 1.10 YK-VZV-045 (Comparative Example 3) 200.1 6.25 1.00 YK-VZV-007 (Comparative Example 4) 200.3 6.26 1.00

[0447] 2) The amount of IL-2 cytokine secreted by T cells stimulated by the mRNA vaccine was 400 to 600 SFU or more, which was 10 to 16 times that of the Shingrix® positive control and 2.0 to 3.0 times that of the YK-VZV-007 control; and which was significantly better than that of the cytokine secreted and stimulated by the mRNA vaccine corresponding to Comparative Example 1 (YK-VZV-004), Comparative Example 2 (YK-VZV-006) or Example Comparative 3 (YK-VZV-045).

[0448] Specifically, the mRNA vaccine prepared from the YK-VZV-010 sequence achieved a maximum of 614.5 SFU, which was 16.00 times that of the Shingrix® positive control and 3 times or more that of the YK-VZV-007 control.

[0449] The IL-2 secretion levels from high to low were YK-VZV-010, YK-VZV-011, YK-VZV-018, YK-VZV-013 and YK-VZV-020, with no significant difference between the groups, indicating a good and significant immunological effect. Table 34: IL-2+ secretion from spleen cells induced by mRNA vaccines Test substance IL-2+ (SFU) Compared to Shingrix® (times) Compared to YK-VZV-007 (times) YK-VZV-010 614.5 16.00 3.07 YK-VZV-011 490.3 12.77 2.45 Petition 870260035339, dated 04 / 15 / 2026, pages 242 / 249 115 / 121 YK-VZV-018 452.3 11.78 2.26 YK-VZV-013 441.6 11.50 2.20 YK-VZV-020 418.9 10.91 2.09 LNP 16.1 0.42 0.08 Shingrix® 38.4 1.00 0.19 YK-VZV-004 (Comparative Example 1) 274.7 7.15 1.37 (Comparative Example 4) 200.4 5.22 1.00

[0450] Results of cellular localization of VZV gE antigen variants 1) Reduction of the gE antigen localization in the trans-Golgi network through C-terminal truncation and mutations in the A1568Y569R570V571 motif. The A568Y569R570V571 motif (SEQ ID NO: 184) is a transport motif that targets the gE polypeptide to the trans-Golgi network. YK-VZV-018 (SEQ ID NO: 63) and YKVZV-020 (SEQ ID NO: 71) encode a truncated polypeptide with 50 terminal amino acids deleted from the C-terminal region (retaining 1-573aa) and mutations at any site of the A568Y569R570V571 sequence (e.g., mutations at A568D and Y569K). The results from Example 8 showed that both YK-VZV-018 and YK-VZV-020 variants led to reduced localization of the gE polypeptide to the trans-Golgi network and increased cell membrane expression.

[0451] 2) Reduction of gE antigen localization to the trans-Golgi network by any combination of mutated motifs A568Y569R570V571 or Y582A583G584L585 YK-VZV-011 (SEQ ID NO: 35) and YK-VZV-013 (SEQ ID NO: 43) encode a full-length gE polypeptide with mutations at each site of the motif A568Y569R570V571 (SEQ ID NO: 184). In addition, YK-VZV-013 also has mutations (e.g., Y582A) in the endocytosis motif Y582A583G584L585 (SEQ ID NO: 185). The results from Example 8 showed that any combination of mutated motifs A568Y569R570V571 or Y582A583G584L585 resulted in the expression of the gE antigen on the cell membrane. Petition 870260035339, dated 04 / 15 / 2026, pp. 243 / 249 116 / 121

[0452] 3) Reduced localization of full-length gE variants with mutated motif A593E594A595A596D597A598 and Y569K and Y582A mutations in the trans-Golgi network YK-VZV-010 (SEQ ID NO: 31) encodes a full-length gE polypeptide, with the sequence A593E594A595A596D597A598 (SEQ ID NO: 183) replacing the motif S593E594S595T596D597T598 (SEQ ID NO: 182). YK-VZV-010 replaces the Ser / Thr-rich SSTT acid cluster with an Ala-rich sequence. In addition, the YKVZV-010 mRNA vaccine also has Y569K and Y582A mutations. The results from Example 8 showed that the YK-VZV-010 gE polypeptide showed increased cell membrane expression.

[0453] These variants are each modified to reduce the localization of the encoded gE protein to the trans-Golgi network and increase transport to the plasma membrane. Table 35: Antigen localization results of different VZV antigen sequences in Vero cells Mutation site Antigen sequence SEQ ID NO Expression Cellular location Full-length gE; Y569K; Y582A; S593A; S595A; T596A; T598A YK-VZV-010 31 +++ Showing location in Golgi and cell membrane Full-length gE; A568D; Y569K; R570E; V571K YK-VZV-011 35 ++ Showing location in Golgi and cell membrane Full-length gE; A568D; Y569K; R570E; V571K; Y582A YK-VZV-013 43 ++ Showing location in Golgi, cytoplasm and cell membrane gE (1-573aa); A568D YK-VZV-018 63 +++ Showing localization in Golgi, cytoplasm and cell membrane gE (1-573aa); Y569K YK-VZV-020 71 +++ Showing localization in Golgi, cytoplasm and cell membrane Summary In conclusion, this disclosure projected 43 antigen sequences encoding variants of the VZV gE protein and selected several sequences with excellent performance, including those that at least outperformed the Shingrix® vaccine, such as YKVZV-004 (Comparative Example 1), YK-VZV-006 (Comparative Example 2), YK-VZV-045 (Comparative Example 3), and YK-VZV-007 (Comparative Example 4), which is quite remarkable. Petition 870260035339, dated 04 / 15 / 2026, pp. 244 / 249 117 / 121 surprising. Among these sequences, the 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 (gE 1-573aa, A568D) and YK-VZV020 (gE 1-573aa, Y569K) were preferred. The following table shows the complete sequence information and numbering of this disclosure. For example, in the YK-VZV-001 antigen sequences, SEQ ID NO: 2 and 3 represent the nucleic acid sequence (ORF-NT) and the amino acid sequence (ORF-AA) of the open reading frame for the YK-VZV001 antigen, respectively; SEQ ID NO: 1 and 4 represent the DNA sequence (DNA) and the mRNA sequence (mRNA) for the YK-VZV-001 antigen, respectively. Table 36: List of sequences SEQ ID NO: Name Sequence Type 1 YK-VZV-001-DNA DNA 2 YK-VZV-001-ORF-NT ORF-NT 3 YK-VZV-001-ORF-AA ORF-AA 4 YK-VZV-001-mRNA mRNA 5 YK-VZV-002-DNA DNA 6 YK-VZV-002-ORF-NT ORF-NT 7 YK-VZV-002-ORF-AA ORF-AA 8 YK-VZV-002-mRNA mRNA 9 YK-VZV-003-DNA DNA 10 YK-VZV-003-ORF-NT ORF-NT 11 YK-VZV-003-ORF-AA ORF-AA 12 YK-VZV-003-mRNA mRNA 13 YK-VZV-004-DNA DNA 14 YK-VZV-004-ORF-NT ORF-NT 15 YK-VZV-004-ORF-AA ORF-AA 16 YK-VZV-004-mRNA mRNA 17 YK-VZV-006-DNA DNA 18 YK-VZV-006-ORF-NT ORF-NT 19 YK-VZV-006-ORF-AA ORF-AA 20 YK-VZV-006-mRNA mRNA 21 YK-VZV-007-DNA DNA 22 YK-VZV-007-ORF-NT ORF-NT 23 YK-VZV-007-ORF-AA ORF-AA 24 YK-VZV-007-mRNA mRNA 25 YK-VZV-009-DNA DNA 26 YK-VZV-009-ORF-NT ORF-NT Petition 870260035339, de 15 / 04 / 2026, pág. 245 / 249 118 / 121 27 YK-VZV-009-ORF-AA ORF-AA 28 YK-VZV-009-mRNA mRNA 29 YK-VZV-010-DNA DNA 30 YK-VZV-010-ORF-NT ORF-NT 31 YK-VZV-010-ORF-AA ORF-AA 32 YK-VZV-010-mRNA mRNA 33 YK-VZV-011-DNA DNA 34 YK-VZV-011-ORF-NT ORF-NT 35 YK-VZV-011-ORF-AA ORF-AA 36 YK-VZV-011-mRNA mRNA 37 YK-VZV-012-DNA DNA 38 YK-VZV-012-ORF-NT ORF-NT 39 YK-VZV-012-ORF-AA ORF-AA 40 YK-VZV-012-mRNA mRNA 41 YK-VZV-013-DNA DNA 42 YK-VZV-013-ORF-NT ORF-NT 43 YK-VZV-013-ORF-AA ORF-AA 44 YK-VZV-013-mRNA mRNA 45 YK-VZV-014-DNA DNA 46 YK-VZV-014-ORF-NT ORF-NT 47 YK-VZV-014-ORF-AA ORF-AA 48 YK-VZV-014-mRNA mRNA 49 YK-VZV-015-DNA DNA 50 YK-VZV-015-ORF-NT ORF-NT 51 YK-VZV-015-ORF-AA ORF-AA 52 YK-VZV-015-mRNA mRNA 53 YK-VZV-016-DNA DNA 54 YK-VZV-016-ORF-NT ORF-NT 55 YK-VZV-016-ORF-AA ORF-AA 56 YK-VZV-016-mRNA mRNA 57 YK-VZV-017-DNA DNA 58 YK-VZV-017-ORF-NT ORF-NT 59 YK-VZV-017-ORF-AA ORF-AA 60 YK-VZV-017-mRNA mRNA 61 YK-VZV-018-DNA DNA 62 YK-VZV-018-ORF-NT ORF-NT 63 YK-VZV-018-ORF-AA ORF-AA 64 YK-VZV-018-mRNA mRNA 65 YK-VZV-019-DNA DNA 66YK-VZV-019-ORF-NT ORF-NT 67 YK-VZV-019-ORF-AA ORF-AA 68 YK-VZV-019-mRNA mRNA 69 YK-VZV-020-DNA DNA Petition 870260035339, of 15 / 04 / 2026, p. 246 / 249 119 / 121 70 YK-VZV-020-ORF-NT ORF-NT 71 YK-VZV-020-ORF-AA ORF-AA 72 YK-VZV-020-mRNA mRNA 73 YK-VZV-021-DNA DNA 74 YK-VZV-021-ORF-NT ORF-NT 75 YK-VZV-021-ORF-AA ORF-AA 76 YK-VZV-021-mRNA mRNA 77 YK-VZV-022-DNA DNA 78 YK-VZV-022-ORF-NT ORF-NT 79 YK-VZV-022-ORF-AA ORF-AA 80 YK-VZV-022-mRNA mRNA 81 YK-VZV-023-DNA DNA 82 YK-VZV-023-ORF-NT ORF-NT 83 YK-VZV-023-ORF-AA ORF-AA 84 YK-VZV-023-mRNA mRNA 85 YK-VZV-024-DNA DNA 86 YK-VZV-024-ORF-NT ORF-NT 87 YK-VZV-024-ORF-AA ORF-AA 88 YK-VZV-024-mRNA mRNA 89 YK-VZV-025-DNA DNA 90 YK-VZV-025-ORF-NT ORF-NT 91 YK-VZV-025-ORF-AA ORF-AA 92 YK-VZV-025-mRNA mRNA 93 YK-VZV-026-DNA DNA 94 YK-VZV-026-ORF-NT ORF-NT 95 YK-VZV-026-ORF-AA ORF-AA 96 YK-VZV-026-mRNA mRNA 97 YK-VZV-027-DNA DNA 98 YK-VZV-027-ORF-NT ORF-NT 99 YK-VZV-027-ORF-AA ORF-AA 100 YK-VZV-027-mRNA mRNA 101 YK-VZV-028-DNA DNA 102 YK-VZV-028-ORF-NT ORF-NT 103 YK-VZV-028-ORF-AA ORF-AA 104 YK-VZV-028-mRNA mRNA 105 YK-VZV-029-DNA DNA 106 YK-VZV-029-ORF-NT ORF-NT 107 YK-VZV-029-ORF-AA ORF-AA 108YK-VZV-029-mRNA mRNA 109 YK-VZV-030-DNA DNA 110 YK-VZV-030-ORF-NT ORF-NT 111 YK-VZV-030-ORF-AA ORF-AA 112 YK-VZV-030-mRNA mRNA Petition 870260035339, of 15 / 04 / 2026, p. 247 / 249 120 / 121 113 YK-VZV-031-DNA DNA 114 YK-VZV-031-ORF-NT ORF-NT 115 YK-VZV-031-ORF-AA ORF-AA 116 YK-VZV-031-mRNA mRNA 117 YK-VZV-032-DNA DNA 118 YK-VZV-032-ORF-NT ORF-NT 119 YK-VZV-032-ORF-AA ORF-AA 120 YK-VZV-032-mRNA mRNA 121 YK-VZV-033-DNA DNA 122 YK-VZV-033-ORF-NT ORF-NT 123 YK-VZV-033-ORF-AA ORF-AA 124 YK-VZV-033-mRNA mRNA 125 YK-VZV-034-DNA DNA 126 YK-VZV-034-ORF-NT ORF-NT 127 YK-VZV-034-ORF-AA ORF-AA 128 YK-VZV-034-mRNA mRNA 129 YK-VZV-035-DNA DNA 130 YK-VZV-035-ORF-NT ORF-NT 131 YK-VZV-035-ORF-AA ORF-AA 132 YK-VZV-035-mRNA mRNA 133 YK-VZV-036-DNA DNA 134 YK-VZV-036-ORF-NT ORF-NT 135 YK-VZV-036-ORF-AA ORF-AA 136 YK-VZV-036-mRNA mRNA 137 YK-VZV-037-DNA DNA 138 YK-VZV-037-ORF-NT ORF-NT 139 YK-VZV-037-ORF-AA ORF-AA 140 YK-VZV-037-mRNA mRNA 141 YK-VZV-038-DNA DNA 142 YK-VZV-038-ORF-NT ORF-NT 143 YK-VZV-038-ORF-AA ORF-AA 144 YK-VZV-038-mRNA mRNA 145 YK-VZV-039-DNA DNA 146 YK-VZV-039-ORF-NT ORF-NT 147 YK-VZV-039-ORF-AA ORF-AA 148 YK-VZV-039-mRNA mRNA 149 YK-VZV-040-DNA DNA 150 YK-VZV-040-ORF-NTORF-NT 151 YK-VZV-040-ORF-AA ORF-AA 152 YK-VZV-040-mRNA mRNA 153 YK-VZV-041-DNA DNA 154 YK-VZV-041-ORF-NT ORF-NT 155 YK-VZV-04-ORF-AAF-AAF-AA Petition 870260035339, of 15 / 04 / 2026, p. 248 / 249 121 / 121 156 YK-VZV-041-mRNA mRNA 157 YK-VZV-042-DNA DNA 158 YK-VZV-042-ORF-NT ORF-NT 159 YK-VZV-042-ORF-AA ORF-AA 160 YK-VZV-042-mRNA 161 mRNA YK-VZV-043-DNA DNA 162 YK-VZV-043-ORF-NT ORF-NT 163 YK-VZV-043-ORF-AA ORF-AA 164 YK-VZV-043-mRNA mRNA 165 YK-VZV-044-DNA 165 YK-VZV-04-VZV-NT-4-VZOR-NT ORF-NT 167 YK-VZV-044-ORF-AA ORF-AA 168 YK-VZV-044-mRNA mRNA 169 YK-VZV-045-DNA DNA 170 YK-VZV-045-ORF-NT ORF-NT 171 YK-VZV-04-AARF-AAF-127 YK-VZV-045-mRNA mRNA 173 UTR 5' Artificially Synthesized 174 UTR 5' C3 (complement component 3) 175 UTR 5' Artificially Synthesized 176 UTR 5' Artificially Synthesized 177 UTR 5' Synthesized 178 UTR 3' from Geneglobin-β to Homo sapiens hemoglobin (TA) 179 UTR 3' Homo sapiens β-globin hemoglobin gene 180 UTR 3' PCBP4 gene 181 UTR 3' CYBA_1.5x 182 Motif S593ES595T596DT598 Amino acid sequence 183 Motif A593EA595A596DA598 Amino acid sequence 184 Motif A568YRV571 Amino acid sequence 185 Motif Y582AGL585 Amino acid sequence 186 Motif Y569RVDKSPYNQS579 Amino acid sequence 187 Motif Y569RVDKSPYNQSMYYAGLP V587 Amino acid sequence 188 Motif SSTT Amino acid sequence 189 AYRVYAGLSSTT Amino acid sequence 190 YAGLSSTT Amino acid sequence 191 AYRVYAGL Amino acid sequence. Petition 870260035339, dated 04 / 15 / 2026, p. 249 / 249

Claims

1 / 8 CLAIMS 1. Immunogenic composition, characterized in that it comprises a ribonucleic acid (RNA) of a varicella-zoster virus (VZV) encoding a wild-type VZV gE glycoprotein or a variant thereof; wherein the sequence of the wild-type VZV gE glycoprotein is SEQ ID NO: 3; wherein the variant of the VZV gE glycoprotein is the variant selected from: YK-VZV020, YK-VZV-030, YK-VZV-031, YK-VZV-009, YK-VZV-010, YK-VZV-011, YK-VZV012, YK-VZV-013 and YK-VZV-014.Table 1 Name Sequence truncation Site mutation Sequence deletion YK-VZV-020 gE (1 to 573) Y569K No sequence deletion YK-VZV-030 gE (1 to 587) A568D; Y569K; R570E; V571K; Y582A No sequence deletion YK-VZV-031 gE (1 to 587) A568D; Y569K; R570E; V571K; Y582G No sequence deletion YK-VZV-009 No sequence truncation Y569K; Y582A No sequence deletion YK-VZV-010 No sequence truncation Y569K; Y582A; S593A; S595A; T596A; T598A No sequence deletion YK-VZV-011 No sequence truncation A568D; Y569K; R570E; V571K No sequence deletion YK-VZV-012 No sequence truncation A568D; Y569K; R570E; V571K; S593A; S595A; T596A; T598A No sequence deletion YK-VZV-013 No sequence truncation A568D; Y569K; R570E; No sequence truncation Petition 870260004046, dated 01 / 15 / 2026, page 311 / 318 2 / 8 Petition 870260004046, dated 01 / 15 / 2026, page 311 / 318 2 / 8 V571K; Y582A YK-VZV-014 No sequence truncation A568D; Y569K; R570E; V571K; Y582A; S593A; S595A; T596A; T598A No sequence deletion.

2. Composition according to claim 1, characterized in that the VZV glycoprotein gE variant is the variant selected from: YK-VZV-020, YK-VZV010, YK-VZV-011 and YK-VZV-013; Table 2 Name Sequence truncation Site mutation Sequence deletion YK-VZV-020 gE (1 to 573) Y569K No sequence deletion YK-VZV-010 No sequence truncation Y569K; Y582A; S593A; S595A; T596A; T598A No sequence deletion YK-VZV-011 No sequence truncation A568D; Y569K; R570E; V571K No sequence deletion YK-VZV-013 No sequence truncation A568D; Y569K; R570E; V571K; Y582A Without sequence deletion, or the amino acid sequence of the VZV glycoprotein gE variant is as shown in SEQ ID NO: 27, 31, 35, 39, 43, 47, 71, 111 or 115; or the VZV RNA sequence is as shown in SEQ ID NO: 28, 32, 36, 40, 44, 48, 72, 112 or 116; or the VZV RNA is mRNA;or, the RNA sequence encoding the VZV gE glycoprotein corresponding to a DNA sequence shown in SEQ ID NO: 25, 29, 33, 37, 41, 45, 69, 109 or 113. Petition 870260004046, dated 15 / 01 / 2026, pp. 312 / 318 3 / 8; 3. Composition according to claim 1, characterized in that the VZV RNA has an open reading frame (ORF) encoding a VZV gE glycoprotein, the sequence of the open reading frame being as shown in SEQ ID NO: 26, 30, 34, 38, 42, 46, 70, 110 or 114.

4. Composition according to claim 1, characterized in that the VZV RNA further comprises a 5' untranslated region (UTR).

5. Composition according to claim 4, characterized in that the sequence of UTR 5' is as shown in SEQ ID NO: 173, 174, 175, 176 or 177.

6. Composition according to claim 1, characterized in that the VZV RNA further comprises a 3' untranslated region (UTR).

7. Composition according to claim 6, characterized in that the sequence of UTR 3' is as shown in SEQ ID NO: 178, 179, 180 or 181.

8. Composition according to claim 1, characterized in that the VZV RNA further comprises a poly(A) tail.

9. Composition according to claim 8, characterized in that the poly(A) tail has a length of 50 to 150 nucleotides.

10. Composition according to claim 1, characterized in that the VZV RNA further comprises a 5' terminal cap.

11. Composition according to claim 10, characterized in that the terminal cap 5' is 7mG(5')ppp(5')NlmpNp.

12. Composition according to claim 3, characterized in that the sequence of the open reading frame is codon-optimized.

13. Composition according to claim 12, characterized in that the open read phase sequence comprises at least one base modification.

14. Composition according to claim 13, characterized in that the base modification is selected from one or more of the following: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, Petition 870260004046, of 01 / 15 / 2026, p. 313 / 318 4 / 8 hydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

15. Composition according to claim 14, characterized in that the base modification is the substitution of uracil for pseudouridine and / or N1-methylpseudouridine.

16. Composition, according to claim 13, 14 or 15, characterized in that the base modification is from 1 to 100% base modification.

17. Composition according to claim 16, characterized in that the base modification is 100% base modification.

18. Method for preparing the composition, as defined in any one of claims 1 to 17, characterized in that it comprises: providing a template that can transcribe VZV RNA; transcribing the RNA using the template under conditions suitable for transcription.

19. Method according to claim 18, characterized in that it further comprises a purification step selected from: lithium chloride precipitation, affinity chromatography, ultrafiltration exchange and cellulose chromatography.

20. Composition according to claim 1, characterized in that the composition is a vaccine and further comprises a pharmaceutically acceptable carrier.

21. Composition according to claim 20, characterized in that the carrier comprises a lipid mixture.

22. Composition according to claim 21, characterized in that the lipid mixture is a lipid nanoparticle (LNP).

23. Composition according to claim 20, 21 or 22, characterized in that the vaccine is an mRNA vaccine.

24. Composition according to claim 23, characterized in that the lipid nanoparticle (LNP) comprises a cationic lipid, a neutral lipid, a structural lipid, and a polymer-conjugated lipid.

25. Composition according to claim 24, characterized in that the cationic lipid is selected from: YK-009, YK-401, YK-305, ALC0315, SM102 and DLIN-MC3-DMA: Petition 870260004046, dated 15 / 01 / 2026, pp. 314 / 318 5 / 8 YK-305 Petition 870260004046, dated 15 / 01 / 2026, pp. 315 / 318 6 / 8 DLIN-MC3; 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 neutral lipid is selected from phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, derivatives thereof and any combination thereof; or, the neutral lipid is selected from: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-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-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-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-snglycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-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-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-estearoyl-2-oleoyl-estearoylethanolamine (SOPE),1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof; or, the neutral lipid is DOPE and / or DSPC.

26. Composition according to claim 24, characterized in that the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the lipid conjugated with polymer is (25 to 75):(5 to 25):(15 to 65):(0.5 to 10).

27. Composition, according to claim 26, characterized in that the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the lipid conjugated with polymer is (35 to 49):(7.5 to 15):(35 to 55):(1 to 5).

28. Composition according to claim 27, characterized in that the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the lipid conjugated with the polymer is 49:10:43.5:1.

5.

29. Composition according to claim 24, characterized in that the structural lipid is selected from: sterol, cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassinosterol, tomatine, ursolic acid, α-tocopherol, corticosteroid and any combination thereof.

30. Composition according to claim 29, characterized in that the structural lipid comprises cholesterol.

31. Composition according to claim 30, characterized in that the structural lipid is cholesterol.

32. Composition according to claim 24, characterized in that the lipid conjugated with polymer 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. Composition according to claim 32, characterized in that the lipid conjugated with polymer is selected from: distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), methoxypolyethylene glycol ditetradecylacetamide (ALC-0159) and any combination thereof.

34. Composition according to claim 20, characterized in that the VZV RNA has an effective dose of 25 μg 200 μg.

35. Composition according to claim 34, characterized in that VZV has an effective dose of 50 μg to 100 μg.

36. Composition according to claim 20, characterized in that the vaccine is an injection.

37. Composition according to claim 36, characterized in that the vaccine is a liquid preparation or a lyophilized preparation.

38. Method for preparing the composition, as defined in any of claims 20 to 37, characterized in that it comprises: mixing VZV RNA with a pharmaceutically acceptable carrier.

39. Method according to claim 38, characterized in that the method comprises encapsulating at least one portion of RNA in lipid nanoparticles.

40. Use of the composition, as defined in any one of claims 1 to 17, 20 to 37, characterized in that it is in the preparation of a medicament to induce a protective immune response against VZV in a subject.

41. Use, according to claim 40, characterized in that the protective immune response comprises the production of a neutralizing antibody. Petition 870260004046, dated 15 / 01 / 2026, pp. 318 / 318

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