RNA vaccine and related product thereof

Abstract

Provided are an RNA vaccine and a related product thereof, relating to the field of biomedicine. The RNA vaccine is a therapeutic vaccine. An antigen contained in the RNA vaccine comprises a combination of HPV16E6721 and HPV18E6721 or a combination of HPV16E672, HPV18E672 and HPV16 / 18E1. Compared with existing vaccines, the provided vaccine can induce viral clearance during a low-copy replication phase of the virus, and has a better immune effect.

Description

An RNA vaccine and related products

[0001] Cross-references to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 2025100389910, filed on January 10, 2025, entitled "An RNA Vaccine and Related Products Thereof," the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of biomedicine, and more specifically, to an RNA vaccine and related products. Background Technology

[0004] Human papillomavirus (HPV) is a group of DNA viruses with high host and tissue specificity, capable of infecting human skin and mucous membrane cells. The HPV family contains over 200 known types, some of which are closely associated with human health problems. Based on the type of lesions they cause, HPV can be classified into low-risk and high-risk types. Low-risk HPV is usually associated with benign skin lesions such as warts, while high-risk HPV is associated with the development of malignant tumors, especially cervical cancer. The harm of HPV is mainly reflected in its association with various cancers. High-risk HPV types, such as HPV 16 and 18, are the leading cause of cervical cancer; it is estimated that approximately 70% of cervical cancer cases are related to infection with these types of HPV. Currently, marketed HPV-based vaccines are primarily preventative vaccines. For those already infected, there is currently no drug that can directly clear HPV. Conventional treatment options mainly include (1) relying on the body's own immune system and inflammation control to promote the body's natural clearance of HPV, which is a relatively long process (generally 6-12 months); (2) using surgery to treat or remove the lesions. Therefore, the market lacks a highly effective HPV treatment drug with better treatment adherence.

[0005] In recent years, mRNA drugs have attracted widespread attention due to their short preparation cycle and flexible design. Notably, compared to recombinant protein drugs, mRNA can activate T-cell immunity after entering the body, thereby enhancing the killing of infected cells. Currently, the market still lacks safe and effective therapeutic mRNA vaccines against HPV.

[0006] In view of this, this disclosure is hereby made. Summary of the Invention

[0007] This disclosure provides an RNA vaccine and related products.

[0008] The embodiments of this disclosure are implemented as follows:

[0009] In a first aspect, embodiments of this disclosure provide a nucleic acid molecule or nucleic acid composition, comprising: any one or a combination of nucleic acid molecule 1 and nucleic acid molecule 2; wherein the nucleic acid molecule 1 comprises: interconnected HPV16 E6, HPV16 E7, HPV16 E2 and HPV16 E1; and the nucleic acid molecule 2 comprises: interconnected HPV18 E6, HPV18 E7, HPV18 E2 and HPV18 E1.

[0010] Secondly, embodiments of this disclosure provide a nucleic acid molecule or nucleic acid composition, comprising: any one or more combinations of nucleic acid molecule 3, nucleic acid molecule 4, and nucleic acid molecule 5; wherein the nucleic acid molecule 3 comprises: interconnected HPV16 E6, HPV16 E7, and HPV16 E2; wherein the nucleic acid molecule 4 comprises: interconnected HPV18 E6, HPV18 E7, and HPV18 E2; and wherein the nucleic acid molecule 5 comprises: a fusion of HPV16 and HPV18 E1.

[0011] Thirdly, this disclosure provides a biological material selected from any one of the following: (1) an expression cassette containing the nucleic acid molecule or nucleic acid composition described in the foregoing embodiments; (2) a carrier containing the nucleic acid molecule or nucleic acid composition or the expression cassette; (3) a recombinant microorganism containing the nucleic acid molecule or nucleic acid composition or the expression cassette or the carrier; and (4) a cell line containing the nucleic acid molecule or nucleic acid composition or the expression cassette or the carrier.

[0012] Fourthly, this disclosure provides an RNA vaccine comprising: any one or a combination of vaccine 1 and vaccine 2; or any one or a combination of vaccine 3, vaccine 4 and vaccine 5; wherein the antigen of vaccine 1 comprises nucleic acid molecule 1 as described in the foregoing embodiments; the antigen of vaccine 2 comprises nucleic acid molecule 2 as described in the foregoing embodiments; the antigen of vaccine 3 comprises nucleic acid molecule 3 as described in the foregoing embodiments; the antigen of vaccine 4 comprises nucleic acid molecule 4 as described in the foregoing embodiments; and the antigen of vaccine 5 comprises nucleic acid molecule 5 as described in the foregoing embodiments.

[0013] Fifthly, this disclosure provides the application of nucleic acid molecules or nucleic acid compositions or biological materials as described in the foregoing embodiments in the preparation of RNA vaccines.

[0014] In a sixth aspect, embodiments of this disclosure provide a composition comprising: the nucleic acid molecule or nucleic acid composition described in the foregoing embodiments, the biological material described in the foregoing embodiments, or the RNA vaccine described in the foregoing embodiments.

[0015] In a seventh aspect, embodiments of this disclosure provide a drug comprising: a drug delivery carrier and a nucleic acid drug; the nucleic acid drug comprising: the nucleic acid molecule or nucleic acid composition described in the foregoing embodiments, or the biological material described in the foregoing embodiments, or the RNA vaccine described in the foregoing embodiments.

[0016] Eighthly, this disclosure provides a kit comprising: the nucleic acid molecule or nucleic acid composition described in the foregoing embodiments, the biological material described in the foregoing embodiments, the RNA vaccine described in the foregoing embodiments, the composition described in the foregoing embodiments, or the drug described in the foregoing embodiments.

[0017] Ninthly, embodiments of this disclosure provide a treatment method comprising administering the drug described in the foregoing embodiments to a subject to be treated.

[0018] This disclosure has the following beneficial effects:

[0019] (1) By optimizing HPV antigens, a therapeutic RNA vaccine for HPV is provided. Compared with the combination of antigens that only select E6 and E7, E2 and E1 are added, which can enable the final RNA vaccine to act on the entire process of early HPV infection and lesions, that is, to induce viral clearance during the stage of low viral replication.

[0020] (2) This disclosure optimizes the combination and order of E6, E7, E1 and E2, which improves the potency of the vaccine compared with other antigen combinations and orders;

[0021] (3) Based on optimized antigens, different types of RNA vaccines are provided, such as samRNA vaccines and nrmRNA vaccines; compared with non-replicating RNA vaccines, samRNA can better stimulate cytotoxic T cell immune responses. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this disclosure and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 shows a schematic diagram of the structures of nr-mRNA and samRNA;

[0024] Figure 2 shows the intracellular expression of eGFP mRNA with different backbones;

[0025] Figure 3 shows the expression of different backbone saRNAs and nrmRNAs observed under a fluorescence microscope;

[0026] Figure 4 shows the intracellular expression of eGFP mRNA with different backbones; where A. relative expression intensity of eGFP saRNA with different backbones; B. percentage of eGFP-positive cells;

[0027] Figure 5 shows the expression of different backbone saRNAs observed under a fluorescence microscope;

[0028] Figure 6 shows the immunization effects of different mRNA vaccines; where A. tumor growth curves after immunization with different mRNA vaccines; B. antigen-specific CD4+ T cell immunity after immunization with different mRNA vaccines; C. antigen-specific CD8+ T cell immunity after immunization with different mRNA vaccines.

[0029] Figure 7 shows the expression of AI-optimized mRNA-LNPs detected by Western blotting; where A. expression of the AI-optimized mRNA sequence of HPV16; B. expression of the AI-optimized mRNA sequence of HPV18.

[0030] Figure 8 shows cell-mediated immunization experiments with different optimized antigen sequences of HPV16; where A is antigen-specific CD4+ T cell immunization and B is antigen-specific CD8+ T cell immunization.

[0031] Figure 9 shows cell-mediated immunization experiments with different optimized antigen sequences of HPV18; where A is antigen-specific CD4+ T cell immunization and B is antigen-specific CD8+ T cell immunization.

[0032] Figure 10 shows the Western blot analysis of mRNA-LNP expression in cells at different HPV16-E1 sites;

[0033] Figure 11 shows the Western blot analysis of mRNA-LNP expression in cells at different HPV18-E1 sites;

[0034] Figure 12 shows the immunization results of samRNA-LNP vaccines at different E1 positions; where A. antigen-specific CD4+ T cell immunization against HPV16; B. antigen-specific CD8+ T cell immunization against HPV16; C. antigen-specific CD4+ T cell immunization against HPV18; D. antigen-specific CD8+ T cell immunization against HPV18.

[0035] Figure 13 shows the immunization results of nrmRNA-LNP vaccines at different E1 positions; where A. antigen-specific CD4+ T cell immunization against HPV16; B. antigen-specific CD8+ T cell immunization against HPV16; C. antigen-specific CD4+ T cell immunization against HPV18; D. antigen-specific CD8+ T cell immunization against HPV18.

[0036] Figure 14 shows the relative expression levels of different signal peptides in the cell supernatant as detected by dot blot.

[0037] Figure 15 shows saRNAs with different signal peptides. Immunological analysis of HPV vaccine; including A. antigen-specific CD4+ T cell immunity; B. antigen-specific CD8+ T cell immunity;

[0038] Figure 16 is a schematic diagram of the linker construction between 16E67 and 16E2;

[0039] Figure 17 shows the results of different linker saRNA vaccines detected by Western blot (WB) and cellular immunity; where A. WB detection of cell expression; B. Antigen-specific CD4+ T cell immunity; C. Antigen-specific CD8+ T cell immunity.

[0040] Figure 18 shows the four groups of saRNA. Immunological analysis of HPV covalent vaccines; including: A. Antigen-specific CD4+ T cell immunity against HPV16; B. Antigen-specific CD8+ T cell immunity against HPV16; C. Antigen-specific CD4+ T cell immunity against HPV18; D. Antigen-specific CD8+ T cell immunity against HPV18.

[0041] Figure 19 shows the two groups of saRNAs. Immunological analysis of HPV covalent vaccines; including A. antigen-specific CD4+ T cell immunization against HPV16 and HPV18; B. antigen-specific CD8+ T cell immunization against HPV16 and HPV18;

[0042] Figure 20 shows nrmRNA. Immunological analysis of HPV covalent vaccines; including: A. Antigen-specific CD4+ T cell immunity against HPV16; B. Antigen-specific CD8+ T cell immunity against HPV16; C. Antigen-specific CD4+ T cell immunity against HPV18; D. Antigen-specific CD8+ T cell immunity against HPV18. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions in the embodiments of this disclosure will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0044] On one hand, embodiments of this disclosure provide a nucleic acid molecule or nucleic acid composition, comprising: any one or a combination of nucleic acid molecule 1 and nucleic acid molecule 2.

[0045] The nucleic acid molecule 1 includes: interconnected HPV16 E6, HPV16 E7, HPV16 E2 and HPV16 E1;

[0046] The nucleic acid molecule 2 includes interconnected HPV18 E6, HPV18 E7, HPV18 E2 and HPV18 E1.

[0047] In some embodiments, the interconnection is a sequential connection.

[0048] In some embodiments, the connection is made either directly or via a linker.

[0049] This disclosure does not specifically limit the "linker" used in the embodiments, and flexible or rigid linkers conventional in the art can be used for sequence connection. In some embodiments, the linker can be any one or more of (GGGS)n, (GGGGS)m, (GGS)n, (GS)n, (G)n, G(P)nG, (GSPAG)n, GG(ASPA)nGG, (EAAAK)n, G(TPT)nG, and (A)n, where n and m are positive integers selected from 1 to 5, specifically any one or any two of 1, 2, 3, 4, and 5.

[0050] In some embodiments, the amino acid sequence of HPV16 E6 has at least 80% identity with the sequence shown in SEQ ID NO:32.

[0051] The phrase "having at least 80% identity" in this article can specifically refer to any one or any two of the following: 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, and 100%. The same applies below and will not be repeated hereafter.

[0052] In some embodiments, the amino acid sequence of the HPV16 E7 has at least 80% identity with the sequence shown in SEQ ID NO:33.

[0053] In some embodiments, the amino acid sequence of the HPV16 E2 has at least 80% identity with the sequence shown in SEQ ID NO:35.

[0054] In some embodiments, the amino acid sequence of the HPV16 E1 has 80% identity with the sequence shown in SEQ ID NO:34.

[0055] In some embodiments, the amino acid sequence of the HPV18 E6 has at least 80% identity with the sequence shown in SEQ ID NO:36.

[0056] In some embodiments, the amino acid sequence of the HPV18 E7 has at least 80% identity with the sequence shown in SEQ ID NO:37.

[0057] In some embodiments, the amino acid sequence of the HPV18 E2 has at least 80% identity with the sequence shown in SEQ ID NO:39.

[0058] In some embodiments, the amino acid sequence of the HPV18 E1 has 80% identity with the sequence shown in SEQ ID NO:38.

[0059] In some embodiments, the nucleotide sequence of the nucleic acid molecule 1 includes a sequence having at least 80% identity with the sequence shown in SEQ ID NO:50, 58 or 64.

[0060] In some embodiments, the nucleotide sequence of the nucleic acid molecule 2 includes a sequence having at least 80% identity with the sequence shown in SEQ ID NO:51.

[0061] On the other hand, embodiments of this disclosure provide a nucleic acid molecule or nucleic acid composition, comprising: any one or more combinations of nucleic acid molecule 3, nucleic acid molecule 4, and nucleic acid molecule 5;

[0062] The nucleic acid molecule 3 includes: interconnected HPV16 E6, HPV16 E7 and HPV16 E2;

[0063] The nucleic acid molecule 4 includes: interconnected HPV18 E6, HPV18 E7 and HPV18 E2;

[0064] The nucleic acid molecule 5 includes: HPV16 and 18 fusion E1, HPV16 / 18E1 fusion, HPV16 / 18E1.

[0065] In some embodiments, the connection is made either directly or via a linker.

[0066] In some embodiments, the amino acid sequence of HPV16 E6 has at least 80% identity with the sequence shown in SEQ ID NO:32.

[0067] In some embodiments, the amino acid sequence of the HPV16 E7 has at least 80% identity with the sequence shown in SEQ ID NO:33.

[0068] In some embodiments, the amino acid sequence of the HPV16 E2 has at least 80% identity with the sequence shown in SEQ ID NO:35.

[0069] In some embodiments, the amino acid sequence of the HPV18 E6 has at least 80% identity with the sequence shown in SEQ ID NO:36.

[0070] In some embodiments, the amino acid sequence of the HPV18 E7 has at least 80% identity with the sequence shown in SEQ ID NO:37.

[0071] In some embodiments, the amino acid sequence of the HPV18 E2 has at least 80% identity with the sequence shown in SEQ ID NO:39.

[0072] In some embodiments, the nucleotide sequence of the nucleic acid molecule 3 includes a sequence having at least 80% identity with the sequence shown in SEQ ID NO:31.

[0073] In some embodiments, the nucleotide sequence of the nucleic acid molecule 4 includes a sequence having at least 80% identity with the sequence shown in SEQ ID NO:47.

[0074] In some embodiments, the nucleotide sequence of the HPV16 and 18 fusion E1 has 80% identity with the sequence shown in SEQ ID NO:52.

[0075] On the other hand, this disclosure provides a biological material selected from any of the following: (1) an expression cassette containing the nucleic acid molecule or nucleic acid composition described in any of the foregoing embodiments; (2) a carrier containing the nucleic acid molecule or nucleic acid composition or the expression cassette; (3) a recombinant microorganism containing the nucleic acid molecule or nucleic acid composition or the expression cassette or the carrier; and (4) a cell line containing the nucleic acid molecule or nucleic acid composition or the expression cassette or the carrier.

[0076] On the other hand, this disclosure provides an RNA vaccine, which includes: any one or a combination of vaccine 1 and vaccine 2; or any one or a combination of vaccine 3, vaccine 4 and vaccine 5;

[0077] The antigen of vaccine 1 includes nucleic acid molecule 1 as described in any of the foregoing embodiments; the antigen of vaccine 2 includes nucleic acid molecule 2 as described in any of the foregoing embodiments; the antigen of vaccine 3 includes nucleic acid molecule 3 as described in any of the foregoing embodiments; the antigen of vaccine 4 includes nucleic acid molecule 4 as described in any of the foregoing embodiments; and the antigen of vaccine 5 includes nucleic acid molecule 5 as described in any of the foregoing embodiments.

[0078] In some embodiments, vaccine 1, vaccine 2, vaccine 3, vaccine 4, and vaccine 5 are either samRNA vaccines or nrmRNA vaccines. samRNA is a self-amplifying mRNA, similar to saRNA or sa-mRNA. nrmRNA is a non-replicating mRNA, similar to nr-mRNA.

[0079] In some embodiments, the samRNA vaccine comprises: an antigen region and a non-structural protein region of the virus.

[0080] In some embodiments, the non-structural protein region of the virus includes polynucleotides encoding any one or more of nsp1, nsp2, nsp3, and nsp4.

[0081] In some embodiments, the amino acid sequence of nsp1 is as shown in SEQ ID NO:3; the amino acid sequence of nsp2 is as shown in any one of SEQ ID NO:4, 18-20; the amino acid sequence of nsp3 is as shown in SEQ ID NO:5; and the amino acid sequence of nsp4 is as shown in SEQ ID NO:6.

[0082] In some embodiments, the polynucleotide encoding nsp1 is shown in SEQ ID NO:21; the polynucleotide encoding nsp2 is shown in any one of SEQ ID NO:22, 25-27; the polynucleotide encoding nsp3 is shown in SEQ ID NO:23; and the polynucleotide encoding nsp4 is shown in SEQ ID NO:24.

[0083] In some embodiments, the samRNA vaccine further includes any one or more of a promoter region, a 5'UTR region, a subgenomic promoter region, a 3'UTR region, and a polyA structural region. In some embodiments, the samRNA vaccine comprises, from its sequence 5' to 3', a promoter region, a 5'UTR region, a subgenomic promoter region, an antigen region, a 3'UTR region, and a polyA structural region connected in sequence.

[0084] In some embodiments, the nucleotide sequence of the promoter region is shown in SEQ ID NO:1.

[0085] In some embodiments, the nucleotide sequence of the 5'UTR region is shown in SEQ ID NO:2.

[0086] In some embodiments, the subgenomic promoter region is shown as SEQ ID NO:7.

[0087] In some embodiments, the nucleotide sequence of the 3'UTR region is shown in SEQ ID NO:8.

[0088] In some embodiments, the nucleotide sequence of the polyA structural region is shown in any one of SEQ ID NO: 9 to 17.

[0089] In some embodiments, the nrmRNA includes any one or more of an antigen region and a promoter region, a 5'UTR region, a 3'UTR region, and a polyA structural region. In some embodiments, the nrmRNA vaccine includes, from its sequence 5' to 3', a promoter region, a 5'UTR region, an antigen region, a 3'UTR region, and a polyA structural region connected in sequence.

[0090] In some embodiments, the samRNA vaccine and / or the nrmRNA vaccine further includes an encoding signal peptide region. The encoding signal peptide region is located at the 5' end of the antigen region.

[0091] In some embodiments, the nucleotide sequence of the signal peptide is shown in any one of SEQ ID NO:46, 65-73.

[0092] On the other hand, this disclosure provides the application of nucleic acid molecules or nucleic acid compositions or biological materials as described in any of the foregoing embodiments in the preparation of RNA vaccines.

[0093] On the other hand, this disclosure provides a composition comprising: the nucleic acid molecule or nucleic acid composition described in any of the foregoing embodiments, the biological material described in any of the foregoing embodiments, or the RNA vaccine described in any of the foregoing embodiments.

[0094] In some embodiments, the composition further includes a drug delivery carrier.

[0095] In some embodiments, the drug delivery carrier includes any one or more of nanoparticles and RNA-targeting ligands.

[0096] In some embodiments, the nanoparticles include any one or more of lipid nanoparticles, polymer nanoparticles, lipid polymer complexes, and inorganic nanoparticles.

[0097] On the other hand, this disclosure provides a drug comprising: a drug delivery carrier and a nucleic acid drug; the nucleic acid drug comprising: the nucleic acid molecule or nucleic acid composition described in any of the foregoing embodiments, or the biological material described in any of the foregoing embodiments, or the RNA vaccine described in any of the foregoing embodiments.

[0098] In some embodiments, the nucleic acid drug is encapsulated in or connected to the drug delivery carrier. The drug delivery carrier is the same as described in any of the foregoing embodiments and will not be repeated here.

[0099] On the other hand, this disclosure provides a kit comprising: the nucleic acid molecule or nucleic acid composition described in any of the foregoing embodiments, the biological material described in any of the foregoing embodiments, the RNA vaccine described in any of the foregoing embodiments, the composition described in any of the foregoing embodiments, or the drug described in any of the foregoing embodiments.

[0100] Furthermore, embodiments of this disclosure provide a treatment method comprising administering the drug described in any of the foregoing embodiments to a subject to be treated.

[0101] In some embodiments, the treatment method is a method for treating HPV.

[0102] In some embodiments, the treatment of HPV includes treating early, middle, or late-stage HPV.

[0103] The features and performance of this disclosure will be further described in detail below with reference to embodiments.

[0104] Example: Validating the effects of different saRNA and nrmRNA backbones on vaccines.

[0105] The basic structure of saRNA includes: a T7 promoter region (SEQ ID NO:1), a 5'UTR region (SEQ ID NO:2), viral non-structural protein regions (nsp1-4) (SEQ ID NO:3-6, 18-20), a subgenomic promoter region (SEQ ID NO:7), an antigen region, a 3'UTR region (SEQ ID NO:8), and a polyA structural region (SEQ ID NO:9-17), as shown in Figure 1. The nucleotide sequences of the amino acid sequences (nsp1-4) described in SEQ ID NO:3-6 are shown sequentially in SEQ ID NO:21-24. The nucleotide sequences of the amino acid sequences (nsp2) described in SEQ ID NO:18-20 are shown sequentially in SEQ ID NO:25-27.

[0106] This embodiment uses eGFP as a reporter system to verify the effects of different backbones through cellular-level expression. The sequence of Venezuelan equine encephalomyelitis virus (TC-83Venezuelan Equine Encephalitis Virus, VEEV) was selected as the backbone basis in this embodiment, and the specific procedures are as follows.

[0107] (1) Different skeletons

[0108] This embodiment provides different saRNA backbones and uses the nrmRNA backbone disclosed by BioNTech (refer to US20240075165A1) as a control group. The different saRNA backbones are shown in Table 1.

[0109] Table 1 Different skeletons

[0110] Note: "NO:1" is an abbreviation of SEQ ID NO:1, "NO:2" is an abbreviation of SEQ ID NO:2, and so on.

[0111] (2) Construction of saRNA-eGFP expression vector

[0112] Based on the different saRNA backbones designed in Table 1, the eGFP antigen sequence (NO:28) was cloned into the above backbones using homologous recombination to obtain the corresponding saRNA expression vectors: saRNA.V1-eGFP, saRNA.V2-eGFP, saRNA.V3-eGFP, saRNA.V4-eGFP, saRNA.V5-eGFP, saRNA.V6-eGFP, saRNA.V7-eGFP, saRNA.V8-eGFP, saRNA.V9-eGFP, saRNA.V10-eGFP, saRNA.V11-eGFP, saRNA.V12-eGFP, saRNA.V13-eGFP, saRNA.V14-eGFP, and saRNA.V15-eGFP. Simultaneously, nrmRNA-eGFP (NO:29) was constructed by replacing the antigen sequence with an eGFP sequence based on BNT162b2.

[0113] (3) Plasmid preparation and saRNA synthesis and purification

[0114] The strains containing the target plasmids saRNA-V1-eGFP to saRNA-V15-eGFP obtained above were fermented and amplified, and then the fermented cells were collected for plasmid preparation. In this example, the QIAGEN Endofree Plasmid Maxi Kit (catalog number 12362, QIAGEN) was used for plasmid extraction, and the recovered plasmids were calibrated using Nanodrop.

[0115] The prepared plasmids were linearized using restriction endonucleases (BspQI or AsisI), and the linearized DNA was recovered using ethanol precipitation. The concentration of the linearized plasmids was determined using Nanodrop.

[0116] (4) Preparation of saRNA

[0117] Based on the template prepared above, an in vitro transcription reaction was prepared (as shown in Table 2, referring to the HanHai Bio IVT transcription kit), and then reacted at 37°C for 2 h. After the IVT reaction was completed, 1 μl of DNase I was added to the reaction solution, and the reaction was carried out again at 37°C for 0.5 h.

[0118] Table 2 Reaction System

[0119] The reaction solution prepared by the above reaction was purified using magnetic beads (VAHTS mRNA). The mRNA stock solution was obtained using the Capture Beads (N412-01-AA, Nanjing Novizan) strategy for further research.

[0120] (5) Cell expression and detection

[0121] Frozen K562 cells were passaged and cultured until a sufficient number of cells were obtained. After treatment, the cells were plated into 24-well plates and incubated overnight at 37°C with CO2. Subsequently, mRNA transfection was performed using Lipofectamine mRNA transfection reagent. TM MessengerMAX TM The mRNA was transfected into K562 cells using a transfection reagent (LMRNA001, Thermofisher Scientific). Samples were then taken on days 1, 4, 7, 8, 9, 12, 14, and 20 for fluorescence detection and fluorescent cell proportion assays. The total fluorescence intensity was used to characterize the expression intensity of the target protein, and the fluorescent cell proportion was used to characterize the persistence and stability of saRNA expression.

[0122] The experimental results are shown in Figure 2. Different backbones mediated significant differences in the expression intensity and stability of the target gene. As shown in Figure 3, under a fluorescence microscope, the optimized saRNA backbone significantly affected the expression of the target protein eGFP. The preferred saRNA backbones (saRNA.V6, saRNA.V5, and saRNA.V8) showed non-inferiority to the control group nrmRNA at the cellular level. As shown in Figure 4, the target gene expression intensity and stability mediated by the saRNA.V10-eGFP, saRNA.V11-eGFP, saRNA.V12-eGFP, saRNA.V13-eGFP, saRNA.V14-eGFP, and saRNA.V15-eGFP experimental groups were all non-inferiority to saRNA.V6-eGFP. The preferred saRNA backbones (saRNA.V10, saRNA.V11, and saRNA.V14) showed slightly better expression at the cellular level than saRNA.V6-eGFP.

[0123] As shown in Figure 5, under a fluorescence microscope, saRNA.V10, saRNA.V11, and saRNA.V14 showed comparable or better expression of the target protein eGFP compared to the preferred backbone saRNA.V6-eGFP.

[0124] Examples show that mRNA vaccines with different backbones have different immunogenic effects.

[0125] (1) Select mRNA backbone vectors with different expression intensities and forms.

[0126] HPV16 antigens 16E6, 16E7, and 16E2 were designed and linked together using a flexible linker to form the fusion antigen 16E672 (NO:30). Codon optimization was performed on the amino acid sequence of the fusion antigen to obtain the corresponding nucleic acid sequence NO:31. NO:31 was cloned into nrmRNA, saRNA.V1, saRNA.V5, and saRNA.V6, respectively, to obtain the expression vectors nrmRNA.16E672, saRNA.V1-16E672, saRNA.V5-16E672, and saRNA.V6-16E672.

[0127] (2) Plasmid preparation and linearization: Referring to the previous examples, this example uses the QIAGEN Endofree Plasmid Maxi Kit (catalog number 12362, QIAGEN) for plasmid extraction. The recovered plasmid concentration was determined using Nanodrop. The prepared plasmids were linearized using the restriction endonuclease BspQI, and the linearized DNA was recovered using ethanol precipitation. The recovered plasmid concentration was determined using Nanodrop.

[0128] (3) Preparation of mRNA: nrmRNA.16E672, saRNA.V1-16E672, saRNA.V5-16E672 and saRNA.V6-16E672 were prepared according to the technical method described in step (4) of the previous embodiment.

[0129] (4) Preparation of mRNA-LNP: mRNA and lipids (the lipids consisted of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), cholesterol, distearate phosphatidylcholine (DSPC), and cationic lipids dissolved in alcohol) were rapidly mixed using a microfluidic system, resulting in lipid precipitation and the encapsulation of mRNA into LNPs by charge interaction. The mRNA-LNP complex was then concentrated and the solution was changed to form four mRNA-LNP formulations, which were adjusted to a concentration of 150 μg / ml.

[0130] (5) Animal immunization and testing:

[0131] The four groups of mRNA-LNP vaccines prepared above were used to immunize mice. The specific protocol was as follows: 6-7 week old female C57BL / 6J mice were used. On day 6 (D6) after inoculation with TC-1 cells, the subcutaneous tumor volume of the C57BL / 6J mice was measured, and experimental animals with uniform tumor size were selected. Intramuscular injections were administered into the bilateral lateral thighs of the mice. Immunization was carried out on days 6, 13, and 20. Eight days after the third immunization (D28), blood samples were collected for humoral immunity testing. On day 28, the mouse spleen was isolated, and splenic lymphocytes were prepared. Antigen-specific CD4+ T and CD8+ T cell immunity was detected using ICS after peptide stimulation.

[0132] As shown in Figure 6, the tumor suppression effects of saRNA.V5-16E672 and saRNA.V6-16E672 were better than those of saRNA.V1-16E672 and nrmRNA.16E672.

[0133] Examples and verification of the effects of different antigen combinations

[0134] Based on saRNA and nrmRNA backbones, antigens of HPV16E6 (NO:32), HPV16E7 (NO:33), HPV16E1 (NO:34), HPV16E2 (NO:35), HPV18E6 (NO:36), HPV18E7 (NO:37), HPV18E1 (NO:38), and HPV18E2 (NO:39) were selected for fusion. Different sequences of E1, E6, E7, and E2 were linked by a flexible linker to obtain the final HPV antigens, specifically including: HPV16E672 (NO:30), HPV18E672 (NO:40), HPV16E1672 (NO:41), HPV18E1672 (NO:42), HPV16E6721 (NO:43), HPV18E6721 (NO:44), and HPV16 / 18E1. fusion(NO:45).

[0135] The antigen was codon optimized to obtain its nucleic acid sequence, as follows.

[0136] Table 3 HPV antigens

[0137] Preparation of saRNA-LNP vaccine: Sequences NO.31, 47, 50, 51, 53–57, 59–60, and 62–64 were synthesized and cloned into the saRNA vector saRNA.V6 to obtain the corresponding saRNA expression plasmid. The corresponding mRNA-LNP vaccine was prepared according to the description in the preceding examples (preparation of mRNA-LNP).

[0138] Western blot analysis was used to verify the in vitro expression of saRNA-LNP: 293T cells were passaged until a sufficient number of cells were obtained. The cells were then trypsinized into 6-well plates and incubated overnight at 37°C with CO2. The next day, liposome-encapsulated mRNA-HPV was transfected into the plated 293T cells. After 24 hours of culture, the cells were lysed and protein samples were collected.

[0139] The experimental results are shown in Figure 7. The protein expression levels of saRNAs corresponding to sequences NO.53, NO.54, NO.55, and NO.57 were better than those of the control group NO.50.

[0140] Immunization study in healthy mice: The 14 groups of mRNA-LNP vaccines prepared above were used to immunize mice. Specific protocol: 6-7 week old female C57BL / 6J mice were used. Immunization was performed via intramuscular injection into the bilateral lateral thighs on day 1 (D1). Twenty days post-immunization (D21), the spleens of the mice were isolated for splenic lymphocyte preparation. Antigen-specific CD4 counts were detected using ICS after peptide stimulation. + T and CD8+ T-cell immunity.

[0141] As shown in Figure 8, for HPV16, the vaccine group without the 16E1 protein (NO:31) was significantly superior to the vaccine group containing the 16E1 protein (NO:50, NO:53 to NO:57) in inducing CD8+ T cell immune responses. As shown in Figure 9, for all experimental groups containing HPV16E1, the preferred sequence was NO:50. A similar trend was observed for HPV18, where the vaccine group without the 18E1 antigen (NO:47) showed significantly better CD8+ T cell immune responses. + The T-cell immune response was superior to that of the vaccine group containing 18E1 (NO:51, NO:59 to NO:63); for all optimized experimental groups, the preferred antigen sequences were NO:64 and NO:59.

[0142] Based on the above immunization results, in the saRNA vaccine, the CD8+ of the experimental group in which E1 was introduced... + Cellular immunity was significantly reduced.

[0143] Example: Verification of the effect of E1 placement order on cellular immunity

[0144] Based on NO:50 and NO:51, with completely identical nucleic acids, the nucleic acid sequence was adjusted to obtain sequences NO:48 and NO:49. Then, NO:48 and NO:49 were cloned into the saRNA vectors saRNA.V6 and nrmRNA.eGFP, respectively, to obtain the corresponding expression plasmids saRNA.V6-16E1672, saRNA.V6-18E1672, nrmRNA-16E1672, and nrmRNA-18E1672. Referring to the description in the previous embodiment (preparation of mRNA-LNP), the corresponding vaccine was prepared. In this embodiment, cell expression detection and mouse immunization were performed on saRNA-LNPs with different sequences. In the immunization experiment of this embodiment, the immunization procedure for the HPV16 experimental group was: immunization on D1 and D21, and detection on D35; for the HPV18 experimental group, immunization on D1 and detection on D21.

[0145] As shown in Figures 10 and 11, the intracellular expression results revealed that 16E1672 was significantly more expressed than 16E6721 in both saRNA and nrmRNA. A similar trend was observed for HPV18, with 18E1672 being significantly more expressed than 18E6721.

[0146] As shown in Figure 12, for the saRNA vaccine, 16E6721 (NO:50) was slightly better than the 16E1672 vaccine group (NO:48) in inducing CD8+ T cell immune responses, and 18E6721 (NO:51) was slightly better than the 18E1672 vaccine group (NO:49) in inducing CD8+ T cell immune responses.

[0147] As shown in Figure 13, for mRNA vaccines, 16E6721 (NO:50) was significantly better than the 16E1672 vaccine group (NO:48) in inducing CD8+ T cell immune responses, and 18E6721 (NO:51) was significantly better than the 18E1672 vaccine group (NO:49) in inducing CD8+ T cell immune responses.

[0148] Example: Verifying the effect of signal peptides on vaccines

[0149] Signal peptides SP1–SP10 (nucleic acid sequences shown in SEQ ID NO:46, 65–73, respectively) were obtained and then cloned into saRNA.V6-16E672 to obtain saRNA.V6-16E672-SP2 / SP3 / SP4 / SP5 / SP6 / SP7 / SP8 / SP9 / SP10. The corresponding vaccines were prepared according to the description in the previous embodiment (preparation of mRNA-LNP). In this embodiment, the cell expression of saRNA-LNPs of different signal peptides was detected, and the three experimental groups with the best expression in the supernatant were selected for mouse immunization. In this immunization experiment, the immunization procedure was: immunization on D1 and D21, and detection on D35.

[0150] As shown in Figure 14, different signal peptides affect the secretion of the target antigen. Based on the relative expression levels in cells, SP3, SP5, and SP8 were selected for mouse immunization experiments. As shown in Figure 15, selecting different signal peptides can enhance cellular immune responses. Signal peptide SP8 exhibits a better CD8+ T cell immune response.

[0151] Example: Verifying the impact of different linkers on vaccines

[0152] In the aforementioned embodiments, the linker of the originally constructed vector saRNA.V6-16E1672 between 16E67 and 16E2 was GL1 (3*GGGGS, schematic diagram as shown in Figure 16). In this embodiment, the linker sequences GL2 and GL3 (amino acid sequences are 3*EAAAK and GSGATNFSLLKQAGDVEENPGP, respectively) were used to replace the GL1 sequence in saRNA.V6-16E672 to obtain saRNA.V6-16E672-GL2 / GL3. Following the description in the aforementioned embodiments (preparation of mRNA-LNP), the corresponding vaccine was prepared. In this embodiment, cell expression detection and mouse immunization were performed on HPV16E672 saRNA-LNP with different linkers. In this immunization experiment, the immunization procedure was: immunization on D1 and D21, and detection on D35.

[0153] As shown in Figure 17, there was no significant difference in intracellular expression mediated by GL1 and GL2, while GL3 showed protein cleavage due to the presence of P2A. Also as shown in Figure 17, the GL2 and GL3 experimental groups mediated slightly better CD4+ T cell immunity in mice than GL1, while GL1 mediated slightly better CD8+ T cell immunity than both GL2 and GL3 experimental groups.

[0154] Example, bivalent vaccine

[0155] This embodiment provides six groups of saRNA vaccines and four groups of nrmRNA vaccines.

[0156] (1) Four groups of saRNA vaccines: The antigens of the four groups of saRNA were cloned into saRNA.V6 (see the above examples) to obtain saRNA.V6-antigen, and the signal peptide SP1 was added to the 5' end of the saRNA.V6-antigen to obtain saRNA.V6-SP1-antigen. The antigen sequences of each group are shown in Table 4.

[0157] Table 4 saRNA vaccine

[0158] The prepared vaccine was used to immunize mice, and the results were measured on day 1 and day 21.

[0159] As shown in Figure 18, vac3 did not show a significant decrease in cellular immunity compared to vac1 when E1 antigen was present. In contrast, vac2 and vac4 showed inferior cellular immunity compared to vac3 and vac1.

[0160] (2) Two groups of saRNA vaccines: Selected antigen sequences were cloned into saRNA.V6 and saRNA.V11 respectively to obtain saRNA.V6-antigen and saRNA.V11-antigen, and signal peptide SP1 was added to obtain saRNA.V6-SP1-antigen (Vac5) and saRNA.V11-SP1-antigen (Vac6). The antigen sequences of each group are shown in Table 5.

[0161] Table 5 saRNA vaccine

[0162] The prepared vaccine was used to immunize mice on days 1 and 21, and the results were measured on day 35.

[0163] As shown in Figure 19, the cell-mediated immunity generated by Vac6 stimulation is slightly better than that of Vac5, meaning that saRNA.V11 (using continuous polyA) and saRNA.V6 (containing linker polyA) have comparable or better effects.

[0164] (3) Four groups of nrmRNAs: The antigens of the four groups of nrmRNAs were cloned into the nrmRNA backbone (NO:29) to obtain nrmRNA-antigens, and the signal peptide SP1 was added to the 5' end of the nrmRNA-antigens to obtain nrmRNAs. -SP1- antigen, the antigen sequences of each group are shown in Table 6.

[0165] Table 6 nrmRNA vaccines

[0166] The prepared vaccine was used to immunize mice, and the results were measured on day 1 and day 21.

[0167] As shown in Figure 20, nrmRNA HPV vaccine and saRNA HPV vaccines elicit different immune responses. In mouse immunization results with the bivalent nrmRNA vaccine, vac8 and vac9, when the E1 antigen was present, did not show a significant decrease in cellular immunity compared to vac7. Comparing vac8 and vac9, there was no significant difference in cellular immunity between them.

[0168] The following are some of the sequences involved in the embodiments of this disclosure:

[0169] The above description is merely a preferred embodiment of this disclosure and is not intended to limit this disclosure. Various modifications and variations can be made to this disclosure by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure. Industrial applicability

[0170] In summary, this disclosure provides a therapeutic RNA vaccine for HPV by optimizing HPV antigens. Compared to antigen combinations that only select E6 and E7, the addition of E2 and E1 allows the final RNA vaccine to target the entire process of early HPV infection and lesions, i.e., it can induce viral clearance during the low-copy replication stage. Furthermore, the optimized combination and order of E6, E7, E1, and E2 improves vaccine potency compared to other antigen combinations and orders. Based on the optimized antigens, different types of RNA vaccines are provided, such as samRNA vaccines and nrmRNA vaccines. Compared to non-replicating RNA vaccines, samRNA can better stimulate cytotoxic T-cell immune responses.

Claims

1. A nucleic acid molecule or nucleic acid composition, characterized in that, It includes: Any one or a combination of nucleic acid molecule 1 and nucleic acid molecule 2: The nucleic acid molecule 1 includes: interconnected HPV16 E6, HPV16 E7, HPV16 E2 and HPV16 E1; The nucleic acid molecule 2 includes interconnected HPV18 E6, HPV18 E7, HPV18 E2 and HPV18 E1.

2. The nucleic acid molecule or nucleic acid composition according to claim 1, characterized in that, The connection method is either a direct connection or a connection via a linker; Optionally, the linker includes a flexible or rigid linker; Optionally, the amino acid sequence of the HPV16 E6 has at least 80% identity with the sequence shown in SEQ ID NO:32; Optionally, the amino acid sequence of the HPV16 E7 has at least 80% identity with the sequence shown in SEQ ID NO:33; Optionally, the amino acid sequence of the HPV16 E2 has at least 80% identity with the sequence shown in SEQ ID NO:35; Optionally, the amino acid sequence of the HPV16 E1 has 80% identity with the sequence shown in SEQ ID NO:34; Optionally, the amino acid sequence of the HPV18 E6 has at least 80% identity with the sequence shown in SEQ ID NO:36; Optionally, the amino acid sequence of the HPV18 E7 has at least 80% identity with the sequence shown in SEQ ID NO:37; Optionally, the amino acid sequence of the HPV18 E2 has at least 80% identity with the sequence shown in SEQ ID NO:39; Optionally, the amino acid sequence of the HPV18 E1 has 80% identity with the sequence shown in SEQ ID NO:38; Optionally, the nucleotide sequence of the nucleic acid molecule 1 includes a sequence having at least 80% identity with the sequence shown in SEQ ID NO:50, 58 or 64; Optionally, the nucleotide sequence of the nucleic acid molecule 2 includes a sequence having at least 80% identity with the sequence shown in SEQ ID NO:

51.

3. A nucleic acid molecule or nucleic acid composition, characterized in that, It includes any one or more combinations of nucleic acid molecules 3, 4, and 5; The nucleic acid molecule 3 includes: interconnected HPV16 E6, HPV16 E7 and HPV16 E2; The nucleic acid molecule 4 includes: interconnected HPV18 E6, HPV18 E7 and HPV18 E2; The nucleic acid molecule 5 includes: HPV16 and 18 fusion E1.

4. The nucleic acid molecule or nucleic acid composition according to claim 3, characterized in that, The connection method is either a direct connection or a connection via a linker; Optionally, the linker includes a flexible linker; Optionally, the amino acid sequence of the HPV16 E6 has at least 80% identity with the sequence shown in SEQ ID NO:32; Optionally, the amino acid sequence of the HPV16 E7 has at least 80% identity with the sequence shown in SEQ ID NO:33; Optionally, the amino acid sequence of the HPV16 E2 has at least 80% identity with the sequence shown in SEQ ID NO:35; Optionally, the amino acid sequence of the HPV18 E6 has at least 80% identity with the sequence shown in SEQ ID NO:36; Optionally, the amino acid sequence of the HPV18 E7 has at least 80% identity with the sequence shown in SEQ ID NO:37; Optionally, the amino acid sequence of the HPV18 E2 has at least 80% identity with the sequence shown in SEQ ID NO:39; Optionally, the nucleotide sequence of the nucleic acid molecule 3 includes a sequence having at least 80% identity with the sequence shown in SEQ ID NO:31; Optionally, the nucleotide sequence of the nucleic acid molecule 4 includes a sequence having at least 80% identity with the sequence shown in SEQ ID NO:47; Optionally, the nucleotide sequence of the HPV16 and 18 fusion E1 has 80% identity with the sequence shown in SEQ ID NO:

52.

5. A biomaterial, characterized in that, The biological material is selected from any one of the following: (1) an expression cassette containing the nucleic acid molecule or nucleic acid composition of any one of claims 1 to 4; (2) a carrier containing the nucleic acid molecule or nucleic acid composition or the expression cassette; (3) a recombinant microorganism containing the nucleic acid molecule or nucleic acid composition or the expression cassette or the carrier; (4) a cell line containing the nucleic acid molecule or nucleic acid composition or the expression cassette or the carrier.

6. An RNA vaccine, characterized in that, This includes: vaccines Any one or a combination of vaccine 1 and vaccine 2; Or any combination of one or more of vaccines 3, 4 and 5; The antigen of vaccine 1 includes the nucleic acid molecule 1 as described in claim 1 or 2; The antigen of the vaccine 2 includes the nucleic acid molecule 2 as described in claim 1 or 2; The antigen of the vaccine 3 includes the nucleic acid molecule 3 as described in claim 3 or 4; The antigen of the vaccine 4 includes the nucleic acid molecule 4 as described in claim 3 or 4; The antigen of the vaccine 5 includes the nucleic acid molecule 5 as described in claim 3 or 4.

7. The RNA vaccine according to claim 6, characterized in that, The types of vaccines 1, 2, 3, 4 and 5 are samRNA vaccines or nrmRNA vaccines; Optionally, the samRNA vaccine comprises: an antigen region and a non-structural protein region of the virus; Optionally, the non-structural protein region of the virus includes polynucleotides encoding any one or more of nsp1, nsp2, nsp3, and nsp4; Optionally, the amino acid sequence of nsp1 is as shown in SEQ ID NO:3; the amino acid sequence of nsp2 is as shown in any one of SEQ ID NO:4, 18 to 20; the amino acid sequence of nsp3 is as shown in SEQ ID NO:5; and the amino acid sequence of nsp4 is as shown in SEQ ID NO:

6. Optionally, the polynucleotide encoding nsp1 is shown in SEQ ID NO:21; the polynucleotide encoding nsp2 is shown in any one of SEQ ID NO:22, 25-27; the polynucleotide encoding nsp3 is shown in SEQ ID NO:23; and the polynucleotide encoding nsp4 is shown in SEQ ID NO:

24. Optionally, the samRNA vaccine further includes any one or more of the following: promoter region, 5'UTR region, subgenomic promoter region, 3'UTR region, and polyA structural region; Optionally, the nucleotide sequence of the promoter region is shown in SEQ ID NO:1; Optionally, the nucleotide sequence of the 5'UTR region is as shown in SEQ ID NO:2; Optionally, the subgenomic promoter region is shown in SEQ ID NO:7; Optionally, the nucleotide sequence of the 3'UTR region is shown in SEQ ID NO:8; Optionally, the nucleotide sequence of the polyA structural region is shown in any one of SEQ ID NO: 9 to 17; Optionally, the nrmRNA includes any one or more of the following: antigen region and promoter region, 5'UTR region, 3'UTR region and polyA structural region; Optionally, the samRNA vaccine and / or the nrmRNA vaccine further include: a region encoding a signal peptide; Optionally, the region encoding the signal peptide is located at the 5' end of the antigen region; Optionally, the nucleotide sequence of the signal peptide is as shown in any one of SEQ ID NO:46, 65-73.

8. The use of the nucleic acid molecule or nucleic acid composition as described in any one of claims 1 to 4 or the biological material as described in claim 5 in the preparation of RNA vaccines.

9. A composition, characterized in that, It includes: The nucleic acid molecule or nucleic acid composition according to any one of claims 1 to 4, the biological material according to claim 5, or the RNA vaccine according to claim 6 or 7.

10. The composition according to claim 9, characterized in that, The composition further includes: a drug delivery carrier; Optionally, the drug delivery carrier includes any one or more of nanoparticles and RNA-targeting ligands; Optionally, the nanoparticles include any one or more of the following: lipid nanoparticles, polymer nanoparticles, lipid polymer complexes, and inorganic nanoparticles.

11. A drug, characterized in that, It includes: a drug delivery carrier and a nucleic acid drug; the nucleic acid drug includes: a nucleic acid molecule or nucleic acid composition according to any one of claims 1 to 4, or a biological material according to claim 5, or an RNA vaccine according to claim 6 or 7.

12. A reagent kit, characterized in that, It includes: The nucleic acid molecule or nucleic acid composition according to any one of claims 1 to 4, the biological material according to claim 5, the RNA vaccine according to claim 6 or 7, the composition according to claim 9 or 10, or the drug according to claim 11.

13. A treatment method, characterized in that, It includes administering the drug of claim 11 to the subject to be treated.

14. The treatment method according to claim 13, characterized in that, The treatment method described is a method for treating HPV.

15. The treatment method according to claim 14, characterized in that, The treatment of HPV includes treatment of early, middle or late stages of HPV.

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