Monoantigen or multi-antigen mRNA vaccine for preventing or treating hepatitis B virus infection and application thereof

Abstract

The invention is applicable to the technical field of biological medicines, and provides a single-antigen or multi-antigen mRNA vaccine for preventing or treating hepatitis B virus infection and application thereof. The protein coded by mRNA comprises a fusion protein (Pan-HLA-DR-epitope-preS1-Fc) formed by fusing a hepatitis B virus surface antigen and / or a hepatitis B virus front surface antigen 1 with a pan-HLA-DR binding epitope and a human IgG1 Fc segment. After mRNA is wrapped into a delivery system through lipid nanoparticles, a single-antigen or multi-antigen mRNA vaccine is prepared. The mRNA vaccine preparation can specifically enhance humoral immune response and cellular immune response aiming at hepatitis B virus surface antigen and / or hepatitis B virus front surface antigen 1, and can be used for preventing and treating hepatitis B; all the components in the mRNA vaccine preparation can be widely obtained, the vaccine cost is effectively reduced, the vaccine yield is increased, and good practical application value is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to monoantigen or polyantigen mRNA vaccines for the prevention or treatment of hepatitis B virus infection and their applications. Background Technology

[0002] Hepatitis B virus (HBV) belongs to the Hepatotropic DNA Viridae family. HBV is a hepatotropic DNA virus whose genome includes four open reading frames (ORFs): S, C, P, and X, which encode the viral capsid protein, nucleocapsid, polymerase, and X protein, respectively. The capsid protein's ORF contains multiple synchronous translation start codons that determine three overlapping polypeptides at the C-terminus. These three polypeptides constitute three protein forms: large (L), medium (M), and small (S). The L protein comprises the preS1, preS2, and S regions, with the preS2 region exhibiting the strongest immunogenicity. The preS1 region, located at the outermost end of the viral particle, exists only on intact, infectious HBV particles. It interacts with the NTCP binding domain on hepatocytes, participating in the direct binding of the virus to hepatocytes and playing a crucial role in HBV assembly and release.

[0003] There are currently nearly 300 million people living with HBV worldwide. Without effective intervention, chronic hepatitis B infection can progress to serious liver diseases such as cirrhosis and liver cancer. The liver's immune tolerance is a fundamental characteristic for maintaining liver homeostasis and preventing excessive immune inflammation and damage, but it is also a key reason why various pathogens can persist and are difficult for the immune system to clear. Although hepatitis B virus can be effectively prevented through vaccination, currently used interferon and nucleic acid drugs still have certain limitations. Therefore, there is an urgent need to develop new, safe, and effective anti-HBV treatments and strategies.

[0004] Therapeutic hepatitis B vaccines present hepatitis B virus-related antigens through various pathways, activating the patient's own immune system to clear HBV. This represents a novel and effective treatment strategy for achieving virological control, functional cure, and even complete cure of chronic HBV. Currently, marketed or clinically developed therapeutic hepatitis B vaccines mainly include recombinant protein adjuvant vaccines, DNA vaccines, and viral vector vaccines. However, these vaccines lack significant immune activation capabilities and struggle to effectively overcome immune depletion caused by chronic HBV infection, posing a significant challenge to the development of therapeutic HBV vaccines. Therefore, this invention proposes a single-antigen or multi-antigen mRNA vaccine for the prevention or treatment of hepatitis B virus infection and its application. Summary of the Invention

[0005] The purpose of this invention is to provide a single-antigen or multi-antigen mRNA vaccine for the prevention or treatment of hepatitis B virus infection and its application, aiming to solve the problem that existing vaccines have insufficient immune activation capacity and are difficult to break the immune depletion caused by chronic HBV infection.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a variety of HBV antigen-encoded mRNA molecules, wherein the proteins encoded by the mRNA molecules include hepatitis B surface antigen (HBsAg) and / or hepatitis B pre-surface antigen 1 fused with a pan-HLA-DR binding epitope and a human IgG1 Fc fragment (Pan-HLA-DR-epitope-preS1-Fc).

[0008] Furthermore, the mRNA molecule includes any one of (a1)-(a5):

[0009] (a1) An mRNA molecule having one or more of the nucleotide sequences shown in SEQ ID NO: 1-SEQ ID NO: 26 (corresponding to NCY01-NCY26 respectively), wherein the nucleotide sequence includes a 5' untranslated region (5'UTR), a coding region (CDS) and a 3' untranslated region (3'UTR);

[0010] (a2) A nucleic acid in which one or more nucleotides have been substituted, deleted or added in the nucleotide sequence defined in (a1);

[0011] (a3) The coding region has at least 70% homology with the nucleotide sequence of (a1) and encodes HBsAg or hepatitis B virus pre-surface antigen 1 (preS1) or Pan-HLA-DR-epitope-preS1-Fc; the 5' untranslated region and the 3' untranslated region have at least 50% homology.

[0012] (a4) Nucleic acids that are partially or completely complementary to any one of (a1)-(a3);

[0013] (a5) An mRNA molecule that hybridizes under strict conditions to the mRNA molecules defined by (a1), (a2) or (a3) ​​and has the function of encoding HBsAg, preS1-Fc (a fusion protein of hepatitis B virus pre-surface antigen 1 and human IgG1 Fc segment) and / or Pan-HLA-DR-epitope-preS1-Fc.

[0014] Furthermore, in order to achieve the best immune effect of mRNA vaccines, mRNA molecules include one or more modifications, including untranslated region modifications and nucleotide modifications;

[0015] Among them, untranslated region modification includes at least one of 5' cap structure modification, 5' end untranslated region sequence introduction, 3' end untranslated region sequence introduction, and 3' end polyadenylation introduction;

[0016] Nucleotide modifications include at least one of the following: pseudouridine, 5-methoxyuridine, 5-methylcytidine, 2-thiouridine, N6-methyladenosine (m6A), N1-methyladenosine (m1A), 2'-O-methylation, 5-methylcytosine (m5C), 5-hydroxymethylcytosine (5hmC), N4-methylcytidine (m4C), 7-methylguanosine (m7G), N2-methylguanosine (m2G), N1-methylpseudouridine, m2,7G, m2,2,7G, and Nm.

[0017] The pseudouridine is selected from at least one of the following: 4-thiopseudouridine, 2-thiopseudouridine, 1-carboxymethylpseudouridine, 1-propynylpseudouridine, 1-tauronic acid methylpseudouridine, N1-methylpseudouridine, 4-thio-1-methylpseudouridine, 2-thio-1-methylpseudouridine, 1-methyl-1-denitropseudouridine, 2-thio-1-methyl-1-denitropseudouridine, dihydropseudouridine, 2-thio-dihydropseudouridine, 4-methoxypseudouridine, and 4-methoxy-2-thiopseudouridine; preferably N1-methylpseudouridine.

[0018] Furthermore, 0%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the nucleotides in the mRNA molecule are modified.

[0019] Furthermore, the nucleotide sequence of the mRNA molecule includes the same or different nucleotide modifications.

[0020] A second aspect of the invention provides a pharmaceutical composition comprising mRNA and a delivery vector encapsulating the mRNA;

[0021] The mRNA is the aforementioned mRNA molecule or a mixture of two different aforementioned mRNA molecules; the delivery carrier includes, but is not limited to, one or more of lipid nanoparticles (LNPs), liposomes, polymers, micelles, and viruses; preferably, lipid nanoparticles. The lipid nanoparticles include cationic lipids, helper phospholipids, sterol lipids, and polyethylene glycol (PEG) modified lipids.

[0022] Furthermore, the cationic lipid is an ionizable cationic lipid, which may be selected from any one or more of the following: methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester (DLin-MC3-DMA), N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethylamine (DLin-KC2-DMA), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butyryl)oxy)heptadecanoic acid ester (L319), heptadecanoic acid 9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid ester (SM102); preferably SM102.

[0023] Furthermore, the auxiliary phospholipid is preferably one or more of DSPC, DOPC, DPPG, DOPS and DOPE, more preferably DSPC and / or DOPS, and most preferably DSPC.

[0024] Furthermore, the sterol lipids are selected from one or more of cholesterol, cholesterol esters, sterol hormones, sterol vitamins and phytosterols, more preferably from one or more of cholesterol, cholesterol esters and phytosterols, and most preferably cholesterol.

[0025] The PEG-modified lipid is preferably one or more of DAG-PEG, DAA-PEG, DMG-PEG, Cer-PEG, and DSPE-PEG, more preferably PEG-DMG. Preferably, the relative molecular mass of the PEG is 2000-5000, for example, 2000, 3000, 4000, or 5000; most preferably, the PEG-modified lipid is mPEG2000-DMG.

[0026] Furthermore, the lipid nanoparticles are prepared by combining SM102, DSPC, cholesterol, and mPEG2000-DMG; the molar ratio of SM102, DSPC, cholesterol, and mPEG2000-DMG in the lipid nanoparticles is 30-70:1-20:20-50:0.1-5, preferably 50:10:38.5:1.5. These liposome nanoparticles exhibit good antigen-presenting properties.

[0027] A third aspect of the invention provides an mRNA vaccine comprising the pharmaceutical composition of claim 4 or 5.

[0028] Furthermore, mRNA vaccines can be administered in various formulations, such as lyophilized or injectable forms. No specific limitations are specified here.

[0029] Furthermore, mRNA vaccines can be administered via intravenous, intramuscular, subcutaneous, or local routes.

[0030] Furthermore, the mRNA vaccine is an injectable vaccine, administered via intramuscular injection. The dosage for intramuscular injection is 10μg-20μg per dose; a course of treatment consists of 2-4 doses, with each dose spaced 14 days apart.

[0031] In a fourth aspect, the present invention provides a DNA molecule that can be transcribed to the above-mentioned mRNA molecule.

[0032] A fifth aspect of the invention provides an expression vector, expression cassette, or host cell containing the aforementioned mRNA molecule or DNA molecule.

[0033] A sixth aspect of the present invention provides a method for preparing the above-mentioned mRNA molecule, comprising transcribing the above-mentioned DNA molecule.

[0034] A seventh aspect of the present invention provides the use of the above-described mRNA molecule, pharmaceutical composition, mRNA vaccine, DNA molecule, expression vector, expression cassette or host cell in the preparation of a drug having any one or more of the following effects;

[0035] 1) Prevention or treatment of liver diseases mediated by hepatitis B virus;

[0036] 2) Prevention, treatment or testing for hepatitis B virus.

[0037] Furthermore, the liver diseases mediated by hepatitis B virus include hepatitis, cirrhosis, liver failure, and liver cancer mediated by hepatitis B virus; preferably hepatitis, which includes acute hepatitis and chronic hepatitis.

[0038] Compared with the prior art, the beneficial effects of the present invention are:

[0039] 1. Compared with other traditional vaccine production methods, the hepatitis B virus mRNA vaccine preparation of this invention can be prepared by in vitro transcription of mRNA, without relying on cell amplification. It can easily achieve monitoring and quality control of all production processes, and saves processes such as cell culture, antigen extraction and purification, further saving time and economic costs.

[0040] 2. The hepatitis B virus mRNA vaccine preparation of this invention contains endogenously expressed protein antigens that, compared to heterologously prepared antigens, have high fidelity in terms of spatial structure and post-translational modifications such as glycosylation when translated into proteins after entering the recipient's body. This can elicit an effective immune response against the corresponding pathogen and enhance the protective effect of the vaccine.

[0041] 3. The hepatitis B virus mRNA vaccine prepared in this invention contains nucleic acid sequences encoding HLA-DR helper epitopes and human IgG1 Fc fragments, which can increase antigen presentation efficacy, improve mRNA molecule stability, prolong in vivo half-life, and help the vaccine enhance the patient's own immune system to break immune tolerance and induce strong cellular and humoral immune responses.

[0042] 4. Compared with DNA vaccines, the hepatitis B virus mRNA vaccine prepared in this invention does not require the translation protein antigen to enter the cell nucleus, thus enhancing antigen production while avoiding the risk of genome integration. Furthermore, as a common transient component in the body, mRNA is easily cleared by the body's physiological metabolic pathways, without adding extra metabolic burden to the recipient.

[0043] 5. The mRNA of the hepatitis B virus mRNA vaccine preparation prepared in this invention can activate multiple intracellular pathogen-related receptors (PRRs), has its own adjuvant effect, does not require the addition of additional adjuvants, can induce an acquired immune response against pathogens by promoting different innate immune responses, and does not pose a risk of potential hepatitis B infection due to attenuated live vaccine vaccination.

[0044] 6. The hepatitis B virus mRNA vaccine formulation prepared in this invention incorporates untranslated region (UTR) and nucleotide modifications to achieve optimal immunization efficacy. These modifications include 5' capping via co-transcription, introduction of 5' UTR sequences, introduction of 3' UTR sequences, introduction of 3' polyadenylate sequences, and nucleotide modifications such as replacing uridine with pseudouridine. These modifications further enhance mRNA stability, improve protein translation efficiency, and strengthen vaccine immunization. Therefore, it has significant practical application value. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the plasmid structure in Example 2.

[0046] Figure 2 Electrophoresis gel images of different mRNA molecules in Example 2: A represents NCY01-NCY16; B represents NCY17-NCY26.

[0047] Figure 3 The graph shows the protein expression levels of different mRNA molecules in HEK 293T cells in Example 3: A represents NCY01-NCY16; B represents NCY17-NCY26.

[0048] Figure 4The anti-HBs antibody level or anti-preS1 IgG antibody level induced by the single antigen mRNA vaccine in Example 5 in healthy mice is shown in Figure 5. A represents the anti-HBs antibody level induced by 16 single antigen mRNA vaccines encoding HBsAg (NCY01-NCY16) in healthy mice; B represents the anti-preS1 IgG antibody level induced by 10 single antigen mRNA vaccine candidates encoding Pan-HLA-DR-epitope-preS1-Fc (NCY17-NCY26) in healthy mice.

[0049] Figure 5 The levels of anti-HBs antibodies and anti-preS1 IgG antibodies induced in healthy mice by the dual-antigen combination mRNA vaccine (a total of 24 combination regimens) and the marketed subunit vaccine in Example 5.

[0050] Figure 6 The levels of HBsAg-specific or preS1-specific IFN-γ-secreting T cells induced in healthy mice by the dual-antigen combination mRNA vaccine (24 combination schemes in total) and the marketed subunit vaccine in Example 5.

[0051] Figure 7 The inhibitory effects of the single antigen mRNA vaccine and entecavir (ETV) in Example 7 on serum HBsAg and HBV DNA in HBV mice were investigated.

[0052] Figure 8 The inhibitory effects of the dual-antigen combination mRNA vaccine (a total of 24 combination regimens) and entecavir (ETV) in Example 7 on serum HBsAg and HBV DNA in HBV mice were investigated.

[0053] Figure 9 Serum HBsAb and anti-preS1 IgG levels induced in HBV mice by the dual-antigen combination mRNA vaccine of Example 7. Detailed Implementation

[0054] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. Unless otherwise specified, the methods used in this invention are conventional methods in this technical field. In this invention, materials, reagents, or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0055] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0056] Example 1: Design of hepatitis B virus mRNA vaccine sequence;

[0057] mRNA candidate sequence design:

[0058] HBV is an enveloped DNA virus belonging to the Hepadnaviridae family. Its genome includes four open reading frames (ORFs): S, C, P, and X, which encode the viral capsid protein, nucleocapsid, polymerase, and X protein, respectively. The capsid protein's open reading frame contains multiple synchronous translation start codons, which determine three overlapping polypeptides at the C-terminus. These three polypeptides constitute three protein forms: large (L), medium (M), and small (S). The L protein includes the preS1, preS2, and S regions. HBV-associated antigen load is a major factor leading to T cell exhaustion in the liver. Studies have confirmed that HBsAg levels in HBV-infected patients can reach as high as 400 ng / mL (accounting for 0.4% of total serum protein), playing a crucial role in suppressing the HBV-specific immune response. HBsAg can directly act on dendritic cells, inhibiting their cytokine production. Traditional vaccines commonly use HBsAg to achieve anti-HBV effects.

[0059] The PreS1 region, located at the outermost end of the viral particle, exists only on intact, infectious hepatitis B virus particles. It interacts with the NTCP binding domain of the hepatocyte receptor, participating in the direct binding of the virus to hepatocytes and playing a crucial role in HBV assembly and release. Furthermore, the addition of the Fc fusion protein increases molecular stability, prolongs the in vivo half-life, and allows for Fc-mediated effector functions. In this invention, other HBV antigens (preS1) will be used as the antigen design basis for the vaccine, based on a redesigned HBs antigen.

[0060] HBV is genotyped based on its entire genome sequence, with 10 recognized genotypes (AJ). It can also be classified into serotypes and subtypes based on B-cell antigen epitopes corresponding to envelope proteins, namely adr, adw, ayr, or ayw, and further subdivided into 10 serological subtypes. Currently, most prophylactic hepatitis B vaccines used clinically are derived from the A2-adw strain, while type C is predominant among infected individuals in China. Therefore, the mRNA sequences encoding HBsAg and preS1 antigens in this invention are designed based on the genome sequence of the C-adr strain (GenBank: AY123041.1).

[0061] Because viral proteins are expressed at low levels in mammals, codon optimization is employed to increase their expression. In organisms, any amino acid corresponds to multiple different codons, but during translation, one codon often dominates in frequency of use. Utilizing preferred codons maximizes translation speed and accelerates protein synthesis. The more stable the secondary structure of the mRNA molecule, the longer its half-life, thus improving the in vivo stability of the mRNA molecule and ensuring relatively long-term sustained expression of the antigen. In this invention, 26 sequences were obtained through sequence optimization.

[0062] Example 2: mRNA preparation;

[0063] 2.1 Full Gene Synthesis of Template Plasmid: The ORF (open reading frame) sequence encoding the antigen or interleukin is concatenated with the T7 promoter sequence, 5'UTR sequence, 3'UTR sequence, and polyA sequence, and then the full gene is synthesized using pUC57 as a vector to obtain the template plasmid (e.g., Figure 1 (As shown).

[0064] 2.2 Obtaining the transcribed template DNA sequence by PCR: Using the linearized template plasmid as a template, polyT long primers, high-fidelity DNA polymerase, dNTPs and other raw materials (all purchased from Nanjing Novizan Biotechnology Co., Ltd.) were used to obtain the transcribed template linear DNA on a PCR instrument according to the procedure recommended in the instruction manual.

[0065] The PCR reaction system is as follows:

[0066] Table 1 PCR reaction system

[0067]

[0068] The PCR reaction procedure is as follows:

[0069] Table 2 PCR reaction procedure

[0070]

[0071] 2.3 In vitro transcription reaction for mRNA preparation (using a 40 μL reaction system as an example): The prepared linear DNA template was mixed with T7 RNA polymerase, rNTPs mononucleotides, capping enzyme, and other raw materials (purchased from Jiangsu Shenji Biotechnology Co., Ltd.) according to the manufacturer's instructions. The co-transcription and capping method was used, and the transcription reaction was carried out at 37°C for 2 hours. After the transcription reaction, the template linear DNA was digested using DNase (purchased from Novizan) to reduce the risk of residual template DNA.

[0072] The in vitro transcription reaction system (40 μL system) is as follows:

[0073] Table 3 In vitro transcription reaction system

[0074]

[0075] 2.4 Purification: The mRNA from the in vitro transcription reaction was purified using an RNA purification kit (NEB). The purified mRNA was dissolved in TE buffer and ready for subsequent coating with the formulation. Electrophoresis results ( Figure 2 Figures A and B show that the mRNA is of good purity. The specific nucleotide sequences of the mRNA molecules are shown as NCY01-NCY26 (i.e., SEQ ID NO: 1-SEQ ID NO: 26).

[0076] Example 3: mRNA sequence transfection into HEK 293T cells to verify protein expression levels;

[0077] HEK 293T cells were seeded in six-well plates, with 5 × 10⁶ cells per well. 5 Cells were incubated at 37°C for 24 hours. 2 μg of each mRNA sequence was transfected using jetMESSENGER transfection reagent. The specific procedure was as follows: 2 μg of mRNA was added to 200 μL of mRNA buffer, mixed well, then 4 μL of jetMESSENGER reagent was added and mixed again. After incubation at room temperature for 15 minutes, the mixture was added dropwise to the cells. Untransfected cells served as a negative control and were incubated at 37°C. Forty-eight hours after transfection, a portion of the culture supernatant was collected. Cells were collected, an appropriate amount of lysis buffer was added, and the mixture was gently pipetted to mix. The cells were incubated on ice for 30 minutes, vortexing every 5 minutes. Then, the cells were centrifuged at 12000 rpm for 15 minutes at 4°C, and the cell culture supernatant was collected. The expression levels of HBsAg and preS1 in the cell lysate were detected by enzyme-linked immunosorbent assay (ELISA). The experimental results are as follows: Figure 3 As shown, the designed mRNA sequence can efficiently express the corresponding protein in HEK 293T cells.

[0078] Example 4: Preparation and characterization of hepatitis B mRNA vaccine formulation;

[0079] Preparation of lipid solution: SM102, DSPC, cholesterol and mPEG2000-DMG were dissolved in ethanol solution at a molar ratio of 50:10:38.5:1.5; the molar concentration of SM102 in ethanol solution was controlled to be 2.112 mmol / L.

[0080] mRNA solution preparation: Dissolve the mRNA in a 10mM citrate buffer solution at pH=4.0 to prepare a 50μg / mL solution; In the dual-antigen combination mRNA vaccine, the mRNA encoding HBsAg (NCY01-NCY16) and the mRNA encoding Pan-HLA-DR-epitope-preS1-Fc (NCY17-NCY26) are mixed at a molar ratio of 2:1.

[0081] Preparation of lipid nanoparticles: The Inano L+ nanomedicine preparation system (purchased from Myanna Instruments Technology Co., Ltd.) was used. mRNA and lipid solutions were respectively drawn up using a syringe at a volume ratio of 3:1 and inserted into a microfluidic chip (R-SDM chip purchased from Myanna Instruments Technology Co., Ltd.). The parameters were set as follows: Flowrate ratio: 3:1, Total flow rate: 12 mL / min, and the lipid nanoparticle solution was obtained by mixing.

[0082] Solution replacement: The lipid nanoparticle solution was added to an ultrafiltration tube and centrifuged for ultrafiltration. The solution was replaced multiple times with phosphate buffer to obtain the final product.

[0083] Example 5: Immunogenicity assessment in animals;

[0084] 5.1 Animal immunization with single-antigen mRNA vaccines;

[0085] Immunogenicity of single-antigen mRNA vaccines was tested using C57BL / 6 mice (male, 6 weeks primiparous, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). The immunogenicity of 16 single-antigen mRNA vaccines encoding HBsAg (NCY01-NCY16) and 10 single-antigen mRNA vaccines encoding Pan-HLA-DR-epitope-preS1-Fc (NCY17-NCY26) was evaluated under two-dose immunization (days 0 and 14) and single-dose 10 μg doses. The experiment consisted of 28 groups of 5 mice each. The negative control group received PBS, while the control group received a 1 / 10th dose of the marketed subunit vaccine (Engerix-B, GlaxoSmithKline). After dilution with PBS to the specified concentration, the vaccines were administered intramuscularly via the thigh, unilaterally. Seven days after two immunizations, 100-150 μL of blood was collected, incubated at 4°C for at least 2 hours, centrifuged at 3000 rpm for 15 minutes to separate the serum, and the sample was stored at -80°C for subsequent immunological analysis.

[0086] 5.2 Animal immunization with a dual-antigen combination mRNA vaccine;

[0087] Immunogenicity of dual-antigen combination mRNA vaccines was tested using C57BL / 6 mice (male, 6 weeks primordial age, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). The study evaluated antibody production from 24 dual-antigen mRNA vaccines administered via double-dose immunization (days 0 and 14) or a single 20 μg dose. The experiment consisted of 26 groups, with 5 mice in each group. The negative control group received PBS, while the control group received a 1 / 10th dose of a marketed subunit vaccine (Engerix-B, GlaxoSmithKline). The vaccine was diluted with PBS to the specified concentration and then administered intramuscularly via the thigh, unilaterally. Seven days after two immunizations, 100-150 μL of blood was collected, incubated at 4°C for at least 2 hours, centrifuged at 3000 rpm for 15 minutes, and the serum was separated. The sample was stored at -80°C for subsequent immunological analysis. Mice were euthanized, and the spleen was removed and ground in a 70 μm cell filter. Red blood cell lysis buffer (purchased from Solarbio) was added and incubated at room temperature for 5 minutes. Then, 10 mL of PBS was added, and the mixture was centrifuged at 1600 rpm for 5 minutes. The cells were then resuspended in cell culture medium to prepare a single-cell suspension.

[0088] 5.3 Antibody titer detection;

[0089] The hepatitis B virus surface antibody quantitative assay kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) was used to quantify the hepatitis B virus surface antibody in mouse serum according to the instructions.

[0090] Hepatitis B virus preS1 antibody was detected using an ELISA method: PreS1 was diluted to 2 μg / mL with coating buffer and added to each well (50 μL) in an ELISA plate (Corning 9018). Coating was performed overnight at 4°C. The plate was washed once with PBS (260 μL per well). Blocking was performed at 37°C for two hours with 5% blocking buffer (5% FBS). Serum samples were diluted to an appropriate dilution with PBS, and 50 μL was added to each well of the blocked ELISA plate. The plate was incubated at 37°C for one hour. The plate was washed five times with PBST (260 μL each time), and 50 μL of enzyme-conjugated anti-mouse IgG-HRP (1:10000 diluted by PBS) was added to each well. The plate was incubated at 37°C for half an hour. The plate was washed five times with PBST (260 μL each time), and 100 μL of substrate TMB was added to each well. The plate was incubated at room temperature in the dark until substrate color development was observed. Add 50 μL of stop solution (2 N H2SO4) to each well to stop the color development. Read the plate using a microplate reader and the readings are OD 450-OD 630.

[0091] Experimental results are as follows Figure 4As shown, the monoantigen mRNA vaccine group induced high levels of antigen-specific antibodies (anti-HBs antibodies and anti-preS1 IgG antibodies), with titers significantly higher than those in the marketed subunit vaccine group. This result indicates that the monoantigen mRNA vaccine can effectively induce an HBV antigen-specific humoral immune response.

[0092] Experimental results are as follows Figure 5 As shown, mice in the dual-antigen combination mRNA vaccine group (a total of 24 combinations) all produced high levels of anti-HBs and anti-preS1 IgG antibodies, and the titers were significantly higher than those in the marketed subunit vaccine group. Since the marketed subunit vaccine does not contain the preS1 component, mice in this vaccine group did not produce anti-preS1 IgG antibodies.

[0093] 5.4 Enzyme-linked immunospot assay;

[0094] To assess the level of vaccine-induced antigen-specific IFN-γ secretory T cells, an IFN-γ ELISpot assay kit (purchased from MABTECH) was used, and the procedure was followed according to the instructions. The specific steps were as follows: Dilute the coating antibody with PBS to a concentration of 15 μg / mL, and prepare a volume of 100 μL per well. Remove the PVDF plate and pre-wet it with 35% ethanol (15 μL / well, maximum 1 min). Wash the plate 5 times with sterile water (200 μL / well). Add 100 μL of coating antibody per well and incubate overnight at 4-8°C. Remove the coating solution, wash 4 times with sterile PBS (200 μL / well), and block with 1640 complete medium containing 10% FBS (200 μL / well). Incubate in a cell culture incubator for at least 30 min. Dilute the protein concentration to 2 μg / mL with 1640 complete medium. Remove the ELISpot plate, discard the culture medium, and add 100 μL of antigen per well (final concentration 1 μg / mL). For the negative control, add 100 μL of culture medium. Add 100 μL of the isolated single-cell suspension per well (cell count 2 × 10⁻⁶). 5Place the plate in a cell culture incubator for 18-24 hours. Remove the ELISpot plate, discard the liquid, wash 5 times with PBS (200 μL / well), and blot dry. Dilute the primary antibody (detection antibody-coupled biotin) 1:1000 with antibody dilution buffer (final concentration: 1 μg / mL), add 100 μL to each well, and incubate at room temperature for 2 hours. Discard the primary antibody, wash 5 times with PBS (200 μL / well), and blot dry. Dilute the secondary antibody (Streptavidin-ALP) 1:1000 with antibody dilution buffer, add 100 μL to each well, and incubate at room temperature for 1 hour. Discard the secondary antibody, wash 5 times with PBS (200 μL / well), and blot dry. Add 100 μL of filtered chromogenic buffer BCIP / NBT-plus to each well, protect from light, and observe the color development status in real time. Rinse the plate thoroughly under running water, air dry, and store at room temperature in the dark. Read the plate using an ELISpot reader.

[0095] Experimental results are as follows Figure 6 As shown, mice immunized with the dual-antigen combination mRNA vaccine group (a total of 24 combinations) produced high levels of HBsAg-specific or preS1-specific IFN-γ-secreting T cells, with significantly better results than the marketed subunit vaccine group.

[0096] Example 6: Construction of the HBV-carrier mouse model;

[0097] Construction of the rAAV / HBV 1.3 mouse model: Normal adult male C57BL / 6J mice, aged 5-6 weeks, were injected with 3 × 10⁻⁶ recombinant rAAV / HBV 1.3 virus via tail vein injection into each mouse. 10 GC (purchased from Paizhen Biotechnology Co., Ltd.). Serum was collected from mice 8 weeks later. Mice with serum HBsAg concentrations greater than 1000 IU / mL were designated as HBV-carrier mice.

[0098] Example 7: In vivo pharmacodynamic evaluation in animals;

[0099] 7.1 Administration to animals;

[0100] The therapeutic efficacy of the vaccine was evaluated using rAAV-HBV1.3 mice.

[0101] Evaluation of the single-component mRNA vaccine (NCY01-NCY26): A four-dose administration regimen was used, with each dose administered two weeks apart, at a dose of 10 μg / dose. Untreated mice received PBS injections under identical conditions. The vaccine, diluted to the specified concentration with PBS, was administered intramuscularly via the thigh, unilaterally. Entecavir (Entecavir, ETV, purchased from Selleck Chemicals) was used as a control, diluted to an appropriate concentration and administered via gavage at 50 μg / kg once daily. The experiment consisted of 28 groups, with 6 mice in each group.

[0102] Evaluation of two-component mRNA vaccines (24 combinations in total): A four-dose administration regimen was used, with each dose given two weeks apart, at a dose of 20 μg / dose. Untreated mice received PBS injections under identical conditions. The vaccine, diluted to the specified concentration with PBS, was administered intramuscularly via the thigh, unilaterally. Entecavir (ETV, purchased from Selleck Chemicals) was used as a control, diluted to an appropriate concentration and administered by gavage at 50 μg / kg once daily. The experiment consisted of 26 groups, with 6 mice in each group.

[0103] Two weeks after the last administration, 100-150 μL of blood was collected, incubated at 4°C for at least 2 hours, centrifuged at 3000 rpm for 15 minutes to separate the serum, and the sample was stored at -80°C for subsequent immunological analysis.

[0104] 7.2 Detection of HBsAg and HBV DNA in mouse serum;

[0105] Use the Hepatitis B virus surface antigen detection kit (purchased from Antu Biotechnology Co., Ltd.) and follow the instructions; use the Hepatitis B virus nucleic acid detection kit (PCR-fluorescent probe method, purchased from Sansure Biotech) and follow the instructions.

[0106] Experimental results are as follows Figure 7 As shown, compared with the untreated PBS group and the ETV group, the single-antigen mRNA vaccine (NCY01-NCY26) significantly reduced the serum HBsAg and HBV DNA levels in HBV mice. Among them, the mRNA vaccine encoding HBsAg (NCY01-NCY16) was relatively more effective. In contrast, the ETV treatment only reduced serum HBV DNA levels and did not reduce serum HBsAg levels.

[0107] Experimental results are as follows Figure 8As shown, compared with the untreated PBS group and the ETV group, the dual-antigen combination mRNA vaccine (a total of 24 combinations) can significantly reduce the serum HBsAg and HBV DNA levels in HBV mice, achieving near-zero or complete zeroing. In contrast, the ETV treatment can only reduce serum HBV DNA levels, but cannot reduce serum HBsAg levels.

[0108] 7.3 Antibody titer detection;

[0109] The hepatitis B virus surface antibody quantification kit (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) was used to quantify the hepatitis B virus surface antibody in mouse serum according to the instructions.

[0110] Hepatitis B PreS1 antibody was detected using an ELISA method: PreS1 was diluted to 2 μg / mL with coating buffer and added to each well (50 μL) in an ELISA plate (Corning 9018). Coating was performed overnight at 4°C. The plate was washed once with PBS (260 μL per well). Blocking was performed at 37°C for two hours with 5% blocking buffer (5% FBS). Serum samples were diluted to an appropriate dilution with PBS, and 50 μL was added to each well of the blocked ELISA plate. The plate was incubated at 37°C for one hour. The plate was washed five times with PBST (260 μL each time), and 50 μL of enzyme-conjugated anti-mouse IgG-HRP (1:10000 diluted by PBS) was added to each well. The plate was incubated at 37°C for half an hour. The plate was washed five times with PBST (260 μL each time), and 100 μL of substrate TMB was added to each well. The plate was incubated at room temperature in the dark until substrate development was observed. Add 50 μL of stop solution (2 N H2SO4) to each well to stop the color development. Read the plate using a microplate reader and the readings are OD 450-OD 630.

[0111] Experimental results are as follows Figure 9 As shown, rAAV-HBV1.3 mice treated with dual-antigen combination mRNA vaccines (a total of 24 combinations) all produced high levels of anti-HBs and anti-preS1 IgG antibodies. However, the ETV group failed to induce specific antibodies in the mice. The combined results indicate that both single-antigen mRNA vaccines and dual-antigen combination mRNA vaccines can clear the virus, reverse the immune tolerance state caused by chronic HBV infection, effectively induce HBV antigen-specific humoral immunity, and achieve seroconversion.

[0112] In summary, both the monoantigen mRNA vaccine and the dual-antigen combined mRNA vaccine of this invention can: 1) significantly reduce serum HBsAg levels in chronically HBV-infected mice; 2) significantly reduce peripheral blood HBV DNA levels; 3) induce significant antigen-specific antibody seroconversion; and 4) induce significant antigen-specific T cell responses. Among these, the dual-antigen combined mRNA vaccine exhibits a synergistic effect, effectively reducing serum HBsAg levels in HBV-infected mice while simultaneously inducing the production of anti-HBsAg and anti-preS1 IgG, thus preventing HBV reinfection and achieving effective prevention and / or treatment of hepatitis B virus.

[0113] The sequence information involved in this application is as follows:

[0114] In SEQ ID NO: 1: 5'UTR: nucleotides 1-53 of SEQ ID NO: 1; CDS: nucleotides 54-737 of SEQ ID NO: 1; 3'UTR: nucleotides 738-847 of SEQ ID NO: 1.

[0115] In SEQ ID NO: 2: 5'UTR: nucleotides 1-52 of SEQ ID NO: 2; CDS: nucleotides 53-736 of SEQ ID NO: 2; 3'UTR: nucleotides 737-846 of SEQ ID NO: 2.

[0116] In SEQ ID NO: 3: 5'UTR: nucleotides 1-240 of SEQ ID NO: 3; CDS: nucleotides 241-924 of SEQ ID NO: 3; 3'UTR: nucleotides 925-1034 of SEQ ID NO: 3.

[0117] In SEQ ID NO: 4: 5'UTR: nucleotides 1-58 of SEQ ID NO: 4; CDS: nucleotides 59-742 of SEQ ID NO: 4; 3'UTR: nucleotides 743-852 of SEQ ID NO: 4.

[0118] In SEQ ID NO: 5: 5'UTR: nucleotides 1-58 of SEQ ID NO: 5; CDS: nucleotides 59-742 of SEQ ID NO: 5; 3'UTR: nucleotides 743-852 of SEQ ID NO: 5.

[0119] In SEQ ID NO: 6: 5'UTR: nucleotides 1-58 of SEQ ID NO: 6; CDS: nucleotides 59-742 of SEQ ID NO: 6; 3'UTR: nucleotides 743-852 of SEQ ID NO: 6.

[0120] In SEQ ID NO: 7: 5'UTR: nucleotides 1-240 of SEQ ID NO: 7; CDS: nucleotides 241-924 of SEQ ID NO: 7; 3'UTR: nucleotides 925-1034 of SEQ ID NO: 7.

[0121] In SEQ ID NO: 8: 5'UTR: nucleotides 1-58 of SEQ ID NO: 8; CDS: nucleotides 59-742 of SEQ ID NO: 8; 3'UTR: nucleotides 743-852 of SEQ ID NO: 8.

[0122] In SEQ ID NO: 9: 5'UTR: nucleotides 1-58 of SEQ ID NO: 9; CDS: nucleotides 59-742 of SEQ ID NO: 9; 3'UTR: nucleotides 743-852 of SEQ ID NO: 9.

[0123] In SEQ ID NO: 10: 5'UTR: nucleotides 1-58 of SEQ ID NO: 10; CDS: nucleotides 59-742 of SEQ ID NO: 10; 3'UTR: nucleotides 743-852 of SEQ ID NO: 10.

[0124] In SEQ ID NO: 11: 5'UTR: nucleotides 1-58 of SEQ ID NO: 11; CDS: nucleotides 59-742 of SEQ ID NO: 11; 3'UTR: nucleotides 743-852 of SEQ ID NO: 11.

[0125] In SEQ ID NO: 12: 5'UTR: nucleotides 1-53 of SEQ ID NO: 12; CDS: nucleotides 54-737 of SEQ ID NO: 12; 3'UTR: nucleotides 738-847 of SEQ ID NO: 12.

[0126] In SEQ ID NO: 13: 5'UTR: nucleotides 1-240 of SEQ ID NO: 13; CDS: nucleotides 241-924 of SEQ ID NO: 13; 3'UTR: nucleotides 925-1034 of SEQ ID NO: 13.

[0127] In SEQ ID NO: 14: 5'UTR: nucleotides 1-52 of SEQ ID NO: 14; CDS: nucleotides 53-736 of SEQ ID NO: 14; 3'UTR: nucleotides 737-846 of SEQ ID NO: 14.

[0128] In SEQ ID NO: 15: 5'UTR: nucleotides 1-58 of SEQ ID NO: 15; CDS: nucleotides 59-742 of SEQ ID NO: 15; 3'UTR: nucleotides 743-852 of SEQ ID NO: 15.

[0129] In SEQ ID NO: 16: 5'UTR: nucleotides 1-58 of SEQ ID NO: 16; CDS: nucleotides 59-742 of SEQ ID NO: 16; 3'UTR: nucleotides 743-852 of SEQ ID NO: 16.

[0130] In SEQ ID NO: 17: 5'UTR: nucleotides 1-53 of SEQ ID NO: 17; CDS: nucleotides 54-1163 of SEQ ID NO: 17; 3'UTR: nucleotides 1164-1273 of SEQ ID NO: 17.

[0131] In SEQ ID NO: 18: 5'UTR: nucleotides 1-240 in SEQ ID NO: 18; CDS: nucleotides 241-1350 in SEQ ID NO: 18; 3'UTR: nucleotides 1351-1460 in SEQ ID NO: 18.

[0132] In SEQ ID NO: 19: 5'UTR: nucleotides 1-52 of SEQ ID NO: 19; CDS: nucleotides 53-1162 of SEQ ID NO: 19; 3'UTR: nucleotides 1163-1272 of SEQ ID NO: 19.

[0133] In SEQ ID NO: 20: 5'UTR: nucleotides 1-58 of SEQ ID NO: 20; CDS: nucleotides 59-1168 of SEQ ID NO: 20; 3'UTR: nucleotides 1169-1278 of SEQ ID NO: 20.

[0134] In SEQ ID NO: 21: 5'UTR: nucleotides 1-58 of SEQ ID NO: 21; CDS: nucleotides 59-1168 of SEQ ID NO: 21; 3'UTR: nucleotides 1169-1278 of SEQ ID NO: 21.

[0135] In SEQ ID NO: 22: 5'UTR: nucleotides 1-53 of SEQ ID NO: 22; CDS: nucleotides 54-1163 of SEQ ID NO: 22; 3'UTR: nucleotides 1164-1273 of SEQ ID NO: 22.

[0136] In SEQ ID NO: 23: 5'UTR: nucleotides 1-240 in SEQ ID NO: 18; CDS: nucleotides 241-1350 in SEQ ID NO: 23; 3'UTR: nucleotides 1351-1460 in SEQ ID NO: 23.

[0137] In SEQ ID NO: 24: 5'UTR: nucleotides 1-52 of SEQ ID NO: 24; CDS: nucleotides 53-1162 of SEQ ID NO: 24; 3'UTR: nucleotides 1163-1272 of SEQ ID NO: 24.

[0138] In SEQ ID NO: 25: 5'UTR: nucleotides 1-58 of SEQ ID NO: 25; CDS: nucleotides 59-1168 of SEQ ID NO: 25; 3'UTR: nucleotides 1169-1278 of SEQ ID NO: 25.

[0139] In SEQ ID NO: 26: 5'UTR: nucleotides 1-58 of SEQ ID NO: 26; CDS: nucleotides 59-1168 of SEQ ID NO: 26; 3'UTR: nucleotides 1169-1278 of SEQ ID NO: 26.

[0140] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention.

Claims

1. An mRNA molecule, characterized in that, The proteins encoded by the mRNA molecule include HBsAg and / or Pan-HLA-DR-epitope-preS1-Fc; Wherein, HBsAg is hepatitis B virus surface antigen, and Pan-HLA-DR-epitope-preS1-Fc is a fusion protein of hepatitis B virus pre-surface antigen 1 fused with pan-HLA-DR binding epitope and human IgG1 Fc segment.

2. The mRNA molecule as described in claim 1, characterized in that, The mRNA molecule includes any one of (a1)-(a5): (a1) An mRNA molecule having one or more of the nucleotide sequences shown in SEQ ID NO: 1-SEQ ID NO: 26, said nucleotide sequences including a 5' untranslated region, a coding region and a 3' untranslated region; (a2) A nucleic acid in which one or more nucleotides have been substituted, deleted or added in the nucleotide sequence defined in (a1); (a3) The nucleotide sequence described in (a1) has at least 70% homology in the coding region and encodes HBsAg or preS1 or Pan-HLA-DR-epitope-preS1-Fc; the 5' untranslated region and the 3' untranslated region have at least 50% homology. (a4) Nucleic acids that are partially or completely complementary to any one of (a1)-(a3); (a5) An mRNA molecule that hybridizes under strict conditions to the mRNA molecules defined by (a1), (a2) or (a3) ​​and has the function of encoding HBsAg, preS1-Fc and / or Pan-HLA-DR-epitope-preS1-Fc; Wherein, preS1 is hepatitis B virus pre-surface antigen 1, and preS1-Fc is a fusion protein of hepatitis B virus pre-surface antigen 1 and human IgG1 Fc segment.

3. The mRNA molecule as described in claim 1, characterized in that, The mRNA molecule includes one or more modifications, including untranslated region modifications and nucleotide modifications; The untranslated region modification includes at least one of the following: 5' cap structure modification, 5' end untranslated region sequence introduction, 3' end untranslated region sequence introduction, and 3' end polyadenylate introduction.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes mRNA and a delivery vector encapsulating the mRNA; Wherein, the mRNA is the mRNA molecule of any one of claims 1-3 or a mixture of two different mRNA molecules of any one of claims 1-3; the delivery carrier is selected from one or more of lipid nanoparticles, liposomes, polymers, micelles and viruses; the lipid nanoparticles include cationic lipids, helper phospholipids, sterol lipids and polyethylene glycol modified lipids.

5. The pharmaceutical composition according to claim 4, characterized in that, The lipid nanoparticles were prepared by combining SM102, DSPC, cholesterol, and mPEG2000-DMG. The molar ratio of SM102, DSPC, cholesterol and mPEG2000-DMG in the lipid nanoparticles is 30-70:1-20:20-50:0.1-5.

6. mRNA vaccine, characterized in that, The mRNA vaccine comprises the pharmaceutical composition of claim 4 or 5.

7. A DNA molecule, characterized by, The DNA molecule can be transcribed to obtain the mRNA molecule according to any one of claims 1-3.

8. An expression vector, expression cassette, or host cell, characterized in that, The expression vector, expression cassette, or host cell contains the mRNA molecule of any one of claims 1-3 or the DNA molecule of claim 7.

9. The use of the mRNA molecule of any one of claims 1-3, the pharmaceutical composition of any one of claims 4-5, the mRNA vaccine of claim 6, the DNA molecule of claim 7, the expression vector, expression cassette or host cell of claim 8 in the preparation of a drug having any one or more of the following effects; 1) Prevention or treatment of liver diseases mediated by hepatitis B virus; 2) Prevention, treatment or testing for hepatitis B virus.

10. The application as described in claim 9, characterized in that, The liver diseases mediated by hepatitis B virus include hepatitis, cirrhosis, liver failure, and liver cancer mediated by hepatitis B virus. The hepatitis mentioned includes acute hepatitis and chronic hepatitis.

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