Preparation of new mRNA vaccine, and use thereof in pharmaceutical combination for treating hepatitis b virus disease

Abstract

Provided in the present invention are a preparation of a new mRNA vaccine, and the use thereof in a pharmaceutical combination for treating the hepatitis B virus disease. Specifically, provided in the present invention is a therapeutic mRNA vaccine against chronic hepatitis B infection, which vaccine contains an mRNA encoding a large protein (L protein) region of a hepatitis B virus surface antigen, and also contains two mRNAs respectively encoding a middle protein (M protein) region of the hepatitis B virus surface antigen and encoding a small protein (S protein) region thereof, or contains an mRNA encoding the small protein (S protein) region. The therapeutic mRNA vaccine of the present invention can break the immune tolerance state in chronic hepatitis B infection, and has the potential to functionally cure chronic hepatitis B infection clinically.

Description

Preparation of a novel mRNA vaccine and its application in drug combinations for the treatment of hepatitis B virus disease Technical Field

[0001] The present invention relates to the technical field of mRNA vaccines, and in particular to the preparation of a novel mRNA vaccine and its application in a drug combination for treating hepatitis B virus disease. Background Art

[0002] According to statistics, there are approximately 300 million patients with chronic hepatitis B infection (CHB) worldwide. It is a global disease that seriously endangers human health. Currently, clinical drugs for the treatment of CHB include interferon and nucleoside (acid) analogues, which work by activating innate immunity and inhibiting RNA in the HBV replication cycle, respectively. These two drugs are only effective in a small number of patients when used alone. Their combined use can improve the functional cure rate, but the efficacy varies greatly among hepatitis B patients with different clinical characteristics. In CHB patients, a large amount of HBV surface antigen - HBsAg continuously stimulates the immune system, forming immune tolerance, and no longer produces antibodies HBsAb against the surface antigen. At the same time, the T cell immune response is also suppressed, making HBV extremely difficult to eliminate. Reactivating the production of HBsAb, breaking immune tolerance, and then reactivating the antiviral immune response are the key and prerequisite for the treatment of CHB.

[0003] Breaking immune tolerance is marked by the sustained production of high levels of HBsAb, requiring the reactivation of B cells and maintenance of antibody production. Controlling viral infection and replication, suppressing high HBsAg levels, and preventing the persistent depletion of HBsAb and B cells are crucial. The N-terminal Pre-S1 region of the HBV surface antigen L protein mediates viral binding to and invasion of the hepatocyte surface receptor NTCP. Antibody levels against Pre-S1 in chronic hepatitis B patients are positively correlated with cure rates and negatively correlated with cccDNA content. Therefore, specific activation of antibodies against Pre-S1 is a prerequisite for breaking immune tolerance. Pre-S1 or Pre-S peptides have failed to effectively inhibit persistent viral infection and replication. The L protein possesses a natural Pre-S antigenic structure, but it cannot be stably expressed on the cell membrane or secreted alone. It is present only in the endoplasmic reticulum or Golgi apparatus and is cytotoxic. HBV virus-like particles (VLPs) contain S and M proteins, as well as membrane components, which effectively support L protein. LMS VLPs, expressed and secreted by mammalian cells and combined with adjuvants, can activate the production of antibodies against both Pre-S and S proteins, but HBsAg clearance is low. LMS VLPs contain the highest S protein content, followed by M protein, with L protein content being the lowest. Therefore, insufficient L protein antigen activation may contribute to these results. Furthermore, LMS VLP preparation involves gene transduction, monoclonal screening, fermentation, and protein purification, resulting in a long production cycle and complex production costs and quality control.

[0004] mRNA vaccines have a short design and preparation cycle and are low-cost, with minimal complexity in the production and purification processes. More importantly, mRNA modification technology and LNP delivery overcome the barriers of immunotoxicity and expression stability, offering broad application prospects. Unlike recombinant protein or inactivated viral vaccines, mRNA vaccines do not require adjuvants, resulting in no side effects. They can also activate a certain degree of innate immune response, enhancing immune efficacy. Unlike methods expressing antigens via viral vectors or DNA, mRNA does not risk integration into host cell DNA. It is gradually degraded after translation in the body and is no longer expressed after sufficient antibodies and immune memory have been generated through multiple immunizations. Studies have been conducted to generate mRNA encoding the L protein or to fuse mRNA encoding pre-S1 with other HBV antigens, soluble sPD-1, and soluble sCD40L sequences for the treatment of HBV in mice. Results have shown that L mRNA must be used in combination with antiviral drugs to be effective, while pre-S1 fusion mRNA has not demonstrated key antibody data. Treatment with mRNA encoding full-length HBsAg showed increased HBsAb and decreased surface antigen, but this study was not attempted in HBV transgenic Tg mice in a fully immune-tolerant state.

[0005] Therefore, there is a need in this field to develop a therapeutic hepatitis B vaccine that can produce sufficient antibodies and break through immune tolerance. Summary of the Invention

[0006] The purpose of the present invention is to provide a therapeutic hepatitis B vaccine that can produce sufficient antibodies and break through immune tolerance.

[0007] In a first aspect of the present invention, a vaccine composition is provided, comprising:

[0008] The first polynucleotide N L , which encodes the hepatitis B virus surface antigen L protein; and

[0009] The second polynucleotide N S , which encodes the hepatitis B virus surface antigen S protein.

[0010] In another preferred embodiment, the vaccine composition further comprises a third polynucleotide N M , which encodes the hepatitis B virus surface antigen M protein.

[0011] In another preferred embodiment, the L protein includes an N-terminal Pre-S region and a C-terminal S protein region.

[0012] In another preferred embodiment, the M protein includes the N-terminal Pre-S2 region and the C-terminal S protein region.

[0013] In another preferred embodiment, the amino acid sequence of the L protein is shown in SEQ ID NO: 1.

[0014] In another preferred embodiment, the amino acid sequence of the M protein is shown in SEQ ID NO: 2.

[0015] In another preferred embodiment, the amino acid sequence of the S protein is shown in SEQ ID NO: 3.

[0016] In another preferred embodiment, in the vaccine composition, the polynucleotide encoding the immunogen consists only of N L and N S Composed of, or consisting only of N L 、N M and N s composition.

[0017] In another preferred embodiment, in the vaccine composition, N L and N S The molar ratio is 1:10-10:1, preferably 1:5-5:1, more preferably 1:2-2:1, for example 1:1.

[0018] In another preferred embodiment, in the vaccine composition, N L and N M The molar ratio is 1:10-10:1, preferably 1:5-5:1, more preferably 1:2-2:1, for example 1:1.

[0019] In another preferred embodiment, the polynucleotide N L 、N M and N S Each independently is DNA or mRNA.

[0020] In another preferred embodiment, the nucleic acid sequences encoding the L protein, M protein and / or S protein are derived from nucleic acid sequences corresponding to hepatitis B virus surface antigens of the same or different genotypes.

[0021] In another preferred embodiment, N L 、N M and / or N S The nucleic acid sequence is codon-optimized.

[0022] In another preferred embodiment, the N L 、N M and / or N S The encoding nucleic acid sequence is optimized or modified, and the optimization or modification does not change the antigenic epitope that the Pre-S region binds to the receptor.

[0023] In another preferred embodiment, the transcription start sites of the M protein and the S protein in the L protein coding sequence are mutated so that the L protein can only be translated.

[0024] In another preferred embodiment, the transcription start site of the S protein in the M protein coding sequence is mutated so that it can only be translated into the M protein.

[0025] In another preferred embodiment, the polynucleotide N L 、N M and N S For mRNA.

[0026] In another preferred embodiment, N L The mRNA sequence encoding the L protein is shown in SEQ ID NO: 4.

[0027] In another preferred embodiment, N M The mRNA sequence encoding the M protein is shown in SEQ ID NO: 5.

[0028] In another preferred embodiment, N S The mRNA sequence encoding the S protein is shown in SEQ ID NO:6.

[0029] In another preferred embodiment, N L and N S fused into the same mRNA.

[0030] In another preferred embodiment, N L 、N M and N S fused into the same mRNA.

[0031] In another preferred embodiment, the mRNA has a structure of Formula I: Z1-Z2-Z3-Z4-Z5-Z6 (I)

[0032] Where,

[0033] Z1 is none or 5' capping element;

[0034] Z2 is the 5′-UTR element;

[0035] Z3 is none or a signal peptide coding sequence;

[0036] Z4 is a coding sequence for a hepatitis B virus surface antigen protein, wherein the hepatitis B virus surface antigen protein is selected from the group consisting of L protein, M protein, S protein, or a combination thereof;

[0037] Z5 is the 3′-UTR element;

[0038] Z6 is the polyA tail element.

[0039] In another preferred embodiment, the 5' capping element is cap0 or cap1.

[0040] In another preferred embodiment, Z4 comprises a polynucleotide sequence selected from SEQ ID NO: 4, 5 or 6 or a combination thereof.

[0041] In another preferred embodiment, the Z4 sequence is shown in any one of SEQ ID NO: 4, 5 or 6.

[0042] In another preferred embodiment, the Z4 has a structure as shown in the following formula (II): N1-L1-N2-L2-N3 (II)

[0043] Where,

[0044] N1, N2 and N3 are each independently none, or N L Sequence, or N M Sequence, or N S sequence;

[0045] L1 and L2 are each independently a self-cleaving junction fragment encoding sequence;

[0046] Furthermore, at least one of N1, N2, and N3 is N L Sequence, one for N S sequence.

[0047] In another preferred embodiment, the self-cleaving linker fragment is selected from the following group: IRES, P2A, or a combination thereof.

[0048] In another preferred embodiment, the mRNA is a modified mRNA, preferably, the modification includes pseudouracil modification.

[0049] In another preferred embodiment, the polynucleotide is loaded in a vector.

[0050] In another preferred embodiment, the N L and N S Loaded in the same carrier or different carriers.

[0051] In another preferred embodiment, the N L 、N M and N S Loaded in the same carrier or different carriers.

[0052] In another preferred embodiment, the vector is selected from the group consisting of liposome nanoparticles (LNP), polymer particles, bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, or combinations thereof.

[0053] In another preferred embodiment, the vector is a plasmid.

[0054] In another preferred embodiment, the vector is an adeno-associated virus vector.

[0055] In another preferred embodiment, the carrier is liposome nanoparticles (LNP).

[0056] In another preferred embodiment, the vaccine composition is a liposome nanoparticle, which includes a liposome layer located on the outside and the polynucleotide encapsulated inside, preferably the polynucleotide is mRNA.

[0057] In another preferred embodiment, mRNAs encoding different hepatitis B virus surface antigen proteins are encapsulated in the same LNP or different LNPs.

[0058] In another preferred embodiment, the components of the liposome layer are selected from the following groups: cationic lipids (such as SM-102), auxiliary lipids (such as DSPC), cholesterol, PEG-modified lipids (such as PEG2000-DMG), or a combination thereof.

[0059] In another preferred embodiment, the liposome layer comprises or consists of the following components: cationic lipid SM-102, auxiliary lipid DSPC, cholesterol and PEG2000-DMG lipid.

[0060] In another preferred embodiment, the liposome layer comprises or consists of the following components in molar ratio:

[0061] 40-60% (preferably 50-55%) cationic lipid SM-102;

[0062] 5-15% (preferably 10-12%) auxiliary lipid DSPC;

[0063] 30-45% (preferably 35%-38.5%) cholesterol; and

[0064] 0.5-2% (preferably 1-1.5%) PEG2000-DMG lipid.

[0065] In another preferred embodiment, the average particle size of the liposome nanoparticles is 50-250 nm.

[0066] In another preferred embodiment, the polydispersity index (PDI) of the liposome nanoparticles is 0.01-0.3.

[0067] In another preferred embodiment, the vaccine composition further contains an immunostimulatory factor or a polynucleotide encoding an immunostimulatory factor.

[0068] In another preferred embodiment, the immune stimulatory factor is selected from the following group: CD40L protein, mitochondrial antiviral signaling protein MAVS protein, CD79a, CD79b, CD19, CD21 and CD81 protein.

[0069] In another preferred embodiment, the immunostimulatory factor is used as a B cell activation auxiliary signal or a B cell activation second signal or a B cell activation co-stimulatory signal to enhance the immune response intensity and antiviral effect of the mRNA vaccine.

[0070] In another preferred embodiment, the immunostimulatory factor is a protein or a polypeptide.

[0071] In another preferred embodiment, the polynucleotide encoding the immunostimulatory factor is mRNA or DNA.

[0072] In another preferred embodiment, the polynucleotide encoding the immunostimulatory factor is loaded in a vector, preferably in a plasmid or virus.

[0073] In another preferred embodiment, after the vaccine composition is transfected into cells, the hepatitis B virus surface antigen L protein, S protein and optional M protein are stably expressed on the cell surface.

[0074] In another preferred embodiment, the vaccine composition further comprises a vaccinologically acceptable diluent, excipient, adjuvant, or a combination thereof.

[0075] In another preferred embodiment, the vaccine composition does not contain an adjuvant.

[0076] In another preferred embodiment, the vaccine composition is in liquid dosage form, solid dosage form, or gel dosage form.

[0077] In another preferred embodiment, the vaccine composition is administered by a method selected from the group consisting of subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, oral and nasal spraying, and aerosol inhalation.

[0078] In a second aspect of the present invention, an engineered host cell is provided, wherein the engineered host cell expresses membrane-bound hepatitis B virus surface antigen L protein and S protein.

[0079] In another preferred embodiment, the host cell is obtained by transfecting the vaccine composition described in the first aspect of the present invention.

[0080] In another preferred embodiment, the host cell further expresses membrane-bound hepatitis B virus surface antigen M protein.

[0081] In another preferred embodiment, the host cell further expresses an immunostimulatory factor.

[0082] In another preferred embodiment, the immune stimulatory factor is selected from the following group: CD40L protein, mitochondrial antiviral signaling protein MAVS protein, CD79a, CD79b, CD19, CD21 and CD81 protein.

[0083] In another preferred embodiment, the hepatitis B virus surface antigen expressed by the host cell activates B cell immunity.

[0084] In another preferred embodiment, the host cell is selected from the group consisting of muscle cells, macrophages, dendritic cells, fibroblasts, endothelial cells, epithelial cells, adipocytes, neutrophils, and lymphocytes.

[0085] In a third aspect of the present invention, a method for preparing the engineered host cell according to the second aspect of the present invention is provided, comprising the steps of:

[0086] The vaccine composition described in the first aspect of the present invention is transduced into immune cells to obtain the engineered host cells.

[0087] In a fourth aspect of the present invention, there is provided use of the vaccine composition of the first aspect of the present invention or the engineered host cell of the second aspect of the present invention for preparing a medicament for use in a subject:

[0088] (a) Prevention of hepatitis B virus infection;

[0089] (b) inducing the production of specific antibodies against hepatitis B virus; and / or

[0090] (c) Treatment of chronic hepatitis B infection.

[0091] In another preferred embodiment, the drug is a vaccine.

[0092] In another preferred embodiment, the engineered host cell is a homologous or heterologous cell of the subject.

[0093] In another preferred embodiment, the chronic hepatitis B is immune-tolerant chronic hepatitis B.

[0094] In the fifth aspect of the present invention, a drug for treating chronic hepatitis B infection is provided, the drug comprising the vaccine composition described in the first aspect of the present invention or the host cell described in the second aspect of the present invention.

[0095] In another preferred embodiment, the drug is a vaccine.

[0096] In another preferred embodiment, the vaccine induces the production of antibodies against natural Pre-S antigen epitopes in chronic hepatitis B infection.

[0097] In another preferred embodiment, the chronic hepatitis B is immune-tolerant chronic hepatitis B.

[0098] In a sixth aspect of the present invention, a method for preventing and / or treating hepatitis B is provided, comprising the steps of:

[0099] The vaccine composition of the first aspect of the present invention or the engineered immune cells of the second aspect of the present invention are administered to a subject in need.

[0100] In another preferred embodiment, the subject includes humans or non-human mammals.

[0101] In another preferred embodiment, the non-human mammals include rodents and non-human primates.

[0102] In a seventh aspect of the present invention, a method for producing anti-hepatitis B virus antibodies is provided, comprising the steps of:

[0103] The vaccine composition described in the first aspect of the present invention or the host cell described in the second aspect of the present invention is used to immunize an animal, thereby producing the anti-hepatitis B virus antibody.

[0104] In another preferred embodiment, the animals include humans and non-human mammals (such as rodents, eg, mice and rats).

[0105] In another preferred embodiment, the antibody is an antibody against the Pre-S antigen epitope.

[0106] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0108] Figure 1 shows the proportion of cells positive for Pre-S2 antigen on the surface of monoclonal cells detected by flow cytometry.

[0109] FIG2 shows the expression levels of L protein and S protein in the total protein, cell membrane fraction and cytoplasm fraction of monoclonal cells detected by western blot.

[0110] FIG3 shows the distribution of membrane raft structures on the surface of monoclonal cells observed using a transmission electron microscope.

[0111] FIG4 shows the integrity of L mRNA, M mRNA, and S mRNA detected by capillary electrophoresis.

[0112] FIG5 shows the particle sizes of L mRNA, M mRNA, and S mRNA complexes encapsulated by LNPs detected by dynamic light scattering.

[0113] Figure 6 shows the surface potential of LNP-encapsulated L mRNA, M mRNA, and S mRNA complexes detected using phase analysis light scattering.

[0114] Figure 7 shows the physicochemical properties, encapsulation efficiency, and endotoxin detection results of the L mRNA, M mRNA, and S mRNA complexes after LNP encapsulation.

[0115] Figure 8 shows the proportion of cells positive for Pre-S2 and S antigens on the cell surface detected by flow cytometry after 293T cells were transfected with L mRNA, M mRNA and S mRNA in vitro.

[0116] FIG9 shows the expression levels of target proteins in cells and culture supernatants detected by Western blotting after L mRNA, M mRNA and S mRNA were transfected into 293T cells in vitro.

[0117] FIG10 shows the protein feature analysis and VLP characterization in the purified LMS VLP.

[0118] Figure 11A shows the HBsAb levels in HBV Tg mice after immunization with mRNA vaccine.

[0119] Figure 11B shows the trend of anti-S antigen antibody titers after immunization of HBV Tg mice with mRNA vaccine.

[0120] Figure 11C shows the trend of anti-preS antigen antibody titers after immunization of HBV Tg mice with mRNA vaccine.

[0121] Figure 12 shows the individual HBsAg levels in HBV Tg mice after immunization with mRNA vaccine.

[0122] Figure 13 is a correlation analysis between the HBsAg reduction ratio and HBsAb levels after immunization of HBV Tg mice with mRNA vaccine.

[0123] Figure 14 shows the levels of HBsAg, HBsAb, and HBeAg after immunization of the pAAV HBV1.2 mouse model with mRNA vaccine.

[0124] Figure 15 shows the results of indirect ELISA detection of the binding of purified IgG in the serum of HBV Tg mice immunized with mRNA vaccine to Pre-S peptide, HBsAg recombinant protein and LMS VLP particles.

[0125] FIG16( a ) shows the selection of 293 monoclonal cells positive for surface Pre-S2 antigen using flow cytometry.

[0126] FIG16( b ) shows the VLPs produced by L+S-expressing 293 monoclonal cells observed using a scanning electron microscope.

[0127] Figure 17(a) is a schematic diagram of the plasmids used to transduce 293 cells.

[0128] Figures 17(b) and 17(c) show flow cytometry detection of hepatitis B antigen expression on the surface of 293 cells.

[0129] FIG18( a ) shows that the 293 stable cell line transduced with L or L+S plasmid expresses L antigen on its surface.

[0130] FIG18( b ) shows the flow cytometry detection of L antigen expression in 293 stable cell lines transduced with L+S plasmid.

[0131] FIG18( c ) shows the Western blot analysis of the L antigen expressed in the 293 stable cell line transduced with the L+S plasmid.

[0132] FIG19( a ) shows the expression results of preS antigen and S antigen detected by flow cytometry after co-transduction of L, M, and S plasmids into mouse embryonic fibroblast Balb / 3T3 cells.

[0133] FIG19( b ) shows the ratio of IFNγ-positive cells in CD3-positive cells detected by flow cytometry after T cells were co-incubated with Balb / 3T3 cells expressing LMS antigen.

[0134] FIG20( a ) is a schematic diagram of the experimental scheme in which rAAV8 HBV1.3 (1×10 1 vg / mouse) mice were immunized with different doses of LS, LMS mRNA, S mRNA or GFP mRNA.

[0135] Figure 20(b) shows the changes in HBsAg antigen after rAAV8 HBV1.3 (1×101 vg / mouse) was injected with different immune mRNAs.

[0136] Figure 20(c) shows the changes in anti-HBsAb antibodies after rAAV8 HBV1.3 (1×101 vg / mouse) was injected with different immune mRNAs.

[0137] Figure 20(d) shows the changes in HBeAg antigen after rAAV8 HBV1.3 (1×101 vg / mouse) was injected with different immunization mRNAs.

[0138] Figure 20(e) shows the changes in HBV DNA levels after rAAV8 HBV1.3 (1×101 vg / mouse) was administered with different immune mRNAs.

[0139] FIG21( a ) is a schematic diagram of the experimental scheme in which rAAV8 HBV1.3 (5×10 1 vg / mouse) mice were immunized with different doses of LMS mRNA, S mRNA, or GFP mRNA.

[0140] Figure 21(b) shows the changes in serum HBsAg antigen in the mice in Figure 20a after immunization with mRNA.

[0141] Figure 21(c) is a comparison of HBsAg antigen in the serum of the mice in Figure 20a at week 0 and week 9 after immunization with mRNA.

[0142] Figure 21(d) shows the changes in serum anti-HBsAg antibodies in the mice in Figure 20a after immunization with mRNA.

[0143] Figure 21(e) shows the correlation analysis between serum HBsAb activation levels and HBsAg reduction levels at week 0 and week 6.

[0144] Figure 21(f) shows the changes in HBeAg in mouse serum from week 0 to week 9.

[0145] FIG22a shows the effect of the positive control HBIg in inhibiting HBeAg expression in an in vitro HBV infection HepG2-hNTCP cell line model.

[0146] FIG22 b shows the effect of negative control serum in inhibiting HBeAg expression in an in vitro HBV infection HepG2-hNTCP cell line model.

[0147] FIG22c shows the effect of LMS mRNA immune serum in inhibiting HBeAg expression in an in vitro HBV infection HepG2-hNTCP cell line model.

[0148] FIG22d shows the effect of LS mRNA immune serum in inhibiting HBeAg expression in an in vitro HBV infection HepG2-hNTCP cell line model.

[0149] FIG22e shows the effect of S mRNA immune serum in inhibiting HBeAg expression in an in vitro HBV infection HepG2-hNTCP cell line model.

[0150] Figure 22f shows the effective neutralizing antibody concentration in the serum sample solution calculated by comparison with the specific concentration value of the HBIg positive control antibody IC50.

[0151] Figure 23(a) shows the changes in body weight of mice after immunization with different doses of LMS mRNA or PBS in the acute toxicity test of LMS mRNA vaccine in mice.

[0152] Figure 23(b) shows the weights of the heart, liver, spleen, lung, and kidney of mice at the end of the acute toxicity experiment. Representative results are presented as mean ± standard deviation (SD).

[0153] Figure 24(a) shows the liver and kidney function analysis in the acute toxicity test of the LMS mRNA vaccine, including the levels of liver function indicators aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at the end of the acute toxicity test.

[0154] Figure 24(b) shows renal function indicators at the end of the acute toxicity study, including urea, uric acid (UA), and creatinine (CR) levels. Representative results are presented as mean ± standard deviation (SD).

[0155] FIG25 shows H&E staining and pathological analysis of the main organs (heart, liver, spleen, lung and kidney) of mice inoculated with different doses of LMS mRNA or PBS at the end of the acute toxicity test. DETAILED DESCRIPTION

[0156] After extensive and in-depth research, the inventors have developed for the first time the preparation of a new type of mRNA vaccine and its application in a drug combination for treating hepatitis B virus disease. Specifically, the present invention provides a therapeutic mRNA vaccine for chronic hepatitis B infection, which comprises an mRNA encoding the large protein (L protein) region of the hepatitis B virus surface antigen, and also comprises two mRNAs encoding the middle protein (M protein) region of the hepatitis B virus surface antigen and the small protein (S protein) region, or an mRNA encoding the small protein (S protein) region. The mRNA vaccine of the present invention can be added with one or more mRNA molecules encoding different immunostimulatory factors to further activate B cells and enhance humoral immune responses for the treatment of chronic hepatitis B infection. The mRNA therapeutic vaccine of the present invention can activate a strong antiviral immune response in mice. It is the first mRNA vaccine prepared that can break the immune tolerance state in chronic hepatitis B infection without the need for combined administration and the addition of adjuvants. The vaccine of the present invention can significantly improve the viral serological indicators of HBV transgenic mice and pAAV HBV1.2 mice, and has the potential to functionally cure chronic hepatitis B infection in clinical practice. On this basis, the present invention has been completed.

[0157] the term

[0158] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0159] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0160] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0161] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reaction), ie, has a reasonable benefit / risk ratio.

[0162] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is tolerated by them. Those skilled in the art will appreciate that the "therapeutically effective amount" may vary depending on the form of the vaccine composition, route of administration, excipients used, severity of the disease, and combination therapy with other drugs.

[0163] HBV surface antigen

[0164] HBV has three surface antigens: the large surface antigen protein region (L protein), the middle surface antigen protein region (M protein), and the small surface antigen protein region (S protein). All three are encoded by the same variable reading frame. Structurally, with the S protein at the C-terminus, the N-terminus is extended with the Pre-S2 sequence to form the M protein. Further extension of the N-terminus with the Pre-S1 sequence forms the L protein. Pre-S1 and Pre-S2 together constitute Pre-S.

[0165] During viral infection, three surface antigens are synthesized in the endoplasmic reticulum and mature in the Golgi apparatus. The S protein and Pre-S2 can be glycosylated, and both contain transmembrane domains at their C-termini. Functionally, the S protein is the primary component of the surface antigens of viruses and virus-like particles (VLPs), providing structural support and assisting in nucleocapsid assembly and viral egress. The S protein binds to heparan sulfate on the surface of hepatocytes via its α-antigenic determinant, inducing amidation of the Pre-S1 domain of the L protein. The amidated Pre-S1 then binds to the sodium taurocholate cotransporting polypeptide (NTCP) receptor, promoting viral invasion.

[0166] Inhibiting the binding of Pre-S1 on the surface of viral particles to NTCP receptors can effectively block viral infection and invasion. Antibody levels against Pre-S1 are positively correlated with high CHB cure rates and negatively correlated with cccDNA (covalently closed circular DNA) content. Therefore, designing Pre-S with natural antigen epitopes and activating a specific humoral immune response against the Pre-S structure is the key to mRNA vaccine design.

[0167] mRNA vaccines

[0168] The term "mRNA" as used herein refers to a type of single-stranded ribonucleic acid that is transcribed from one strand of DNA as a template and carries genetic information to guide protein synthesis.

[0169] The term "vaccine" as used herein refers to a composition suitable for use in animals (including humans) that, upon administration, induces an immune response strong enough to minimally help prevent, ameliorate or cure clinical disease caused by viral infection.

[0170] The terms "vaccine composition of the present invention" and "mRNA vaccine of the present invention" used herein are used interchangeably and both refer to the vaccine composition described in the first aspect of this application.

[0171] The present invention also provides a vaccine composition containing the mRNA vaccine of the present invention. In a preferred embodiment, the vaccine composition of the present invention is provided in the form of an mRNA-LNP vaccine.

[0172] The term "lipid nanoparticles" (Lipid nanoparticles, LNP) as used herein refers to particles with at least one nanometer size, which comprise at least one lipid. In a preferred embodiment, the lipid includes but is not limited to neutral phospholipids and polyethylene glycol-lipids. The term "neutral phospholipids" as used herein refers to uncharged, non-phosphoglyceride phospholipid molecules. The term "polyethylene glycol-lipid" or "PEG-modified lipid" as used herein refers to a molecule comprising a lipid portion and a polyethylene glycol portion.

[0173] Research has confirmed that L protein can only be stably expressed on the cell membrane surface and present a Pre-S structure when supported by M and S proteins (at least the S protein). Therefore, the combination of L, M, and S proteins is used as the active ingredient in a vaccine to stimulate the production of neutralizing antibodies.

[0174] mRNA vaccines offer the advantages of rapid preparation, low production costs, and the ability to activate innate immune responses. The target antigen is endogenously expressed and processed for presentation on the cell membrane, eliminating the risk of genomic integration. After a period of expression and immune response, mRNA gradually degrades and is not permanently expressed in cells. After multiple mRNA immunizations, the body can generate a sufficiently strong memory response to a specific antigen.

[0175] The present invention designs mRNA molecules expressing L protein, M protein and S protein, which are encapsulated using LNPs respectively, and transfected with a mixture of L mRNA, M mRNA and S mRNA or a mixture of L mRNA and S mRNA. With the support of M protein and S protein or only S protein, a high proportion of L protein is presented on the cell membrane surface, which can activate B cells by directly presenting or presenting Pre-S natural antigen epitopes through MHC molecules. In HBV Tg mice with complete immune tolerance, the production of antibodies against natural Pre-S antigen epitopes and HBsAb antibodies is significantly activated, effectively breaking immune tolerance and clearing surface antigens. At the same time, immunostimulatory factor mRNA represented by CD40L is designed and prepared. After co-transfection and expression, it can bind to CD40 on the surface of B cells and can serve as a second signal to enhance B cell activation, further enhancing the humoral immune response.

[0176] The vaccine composition provided herein may be in any suitable form, depending on the method of administration desired by the patient. It may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such kits typically (but not necessarily) include instructions for use. It may include a plurality of such unit dosage forms.

[0177] The vaccine composition is suitable for any appropriate route of administration, such as injection (including subcutaneous, intradermal, intramuscular, intraperitoneal, microneedle or intravenous injection), inhalation or oral, nasal, or anal administration. The composition can be prepared by any method known in the pharmaceutical art, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions.

[0178] HBV mRNA vaccine

[0179] The presentation of natural antigen epitopes and the stable expression of antigens are crucial for HBV mRNA vaccines. At the same time, the mRNA sequence significantly affects its stability and translation efficiency. Therefore, the present invention optimizes its gene expression sequence (CDS sequence) while maintaining the natural amino acid sequence of L protein, M protein and S protein. Non-coding region 5'-UTR and 3'-UTR are added before and after the CDS sequence, as well as polyadenylic acid (polyA). On this basis, a plasmid template is constructed and in vitro transcription is performed, significantly improving mRNA stability and translation efficiency. Functional elements such as T7 promoter and prokaryotic expression resistance gene are also included in the transcription template. 5' capping of mRNA and incorporation of modified nucleosides are performed during in vitro transcription. Cationic LNP packaging is performed after mRNA purification and quality control, and finally LNP / mRNA is subjected to quality testing.

[0180] The specific amino acid sequence is shown below:

[0181] L protein (SEQ ID NO: 1)

[0182] M protein (SEQ ID NO: 2)

[0183] S protein (SEQ ID NO: 3)

[0184] The present invention provides an mRNA vaccine encoding the large hepatitis B surface antigen (HBsAg) protein (L), the middle hepatitis B surface antigen (HBsAg) protein (M), and the small hepatitis B surface antigen (S), or encoding both the L and S proteins, for use as a therapeutic vaccine in HBV infection or chronic hepatitis B infection. The vaccine of the present invention may comprise the mRNA of the present invention, or a complex formed by the mRNA and liposome nanoparticles (LNPs). The vaccine of the present invention can induce the production of large amounts of antibodies against the S protein in a chronic hepatitis B infection model, break through the immune tolerance in chronic hepatitis B infection, and significantly reduce HBsAg serum levels. The vaccine of the present invention can achieve a partial functional cure in a chronic hepatitis B infection model.

[0185] The present invention designs, optimizes and prepares mRNA encoding L protein, M protein and S protein respectively as components of the above mRNA vaccines, and the coding sequences are shown in SEQ ID NO:4-6.

[0186] In one embodiment, a therapeutic mRNA vaccine for chronic hepatitis B infection is provided, which comprises L mRNA, M mRNA and S mRNA, or comprises L mRNA and S mRNA, and the different mRNAs are encapsulated in LNPs and then mixed for use.

[0187] In one embodiment, mammalian cells are transfected with a combination of L mRNA, M mRNA, and S mRNA, or a combination of L mRNA and S mRNA, and the expression of L protein, M protein, and S protein can be detected in the cell membrane and cell culture supernatant.

[0188] Using the mRNA vaccine of the present invention to immunize HBV Tg mice in a state of complete immune tolerance can activate the production of high levels of HBsAb, break the immune tolerance in CHB, significantly reduce the HBsAg levels of some mice, and improve viral serum indicators, indicating that the mRNA vaccine has the potential to functionally cure CHB.

[0189] HBV Tg mice were immunized with the mRNA vaccine of the present invention. After purifying the antibody IgG in the mouse serum and removing the anti-S IgG antibody, the remaining IgG could bind to HBV virus particles, indicating that the vaccine can activate the production of specific antibodies against the natural Pre-S antigen epitope.

[0190] After immunizing HBV Tg mice with the mRNA vaccine of the present invention and completing the treatment, spleen T cells were isolated and stimulated with mouse cells co-expressing L protein, M protein and S protein to activate the expression of IFN-γ, indicating that the mRNA vaccine specifically activated the T cell immune response against the Pre-S antigen epitope.

[0191] Immunization of the pAAV HBV1.2 mouse model with the mRNA vaccine of the present invention activated the production of HBsAb, significantly reduced the HBsAg and HBeAg levels of some mice, and improved viral serum indicators.

[0192] Immunization of the rAAV HBV1.3 mouse model with the mRNA vaccine of the present invention activated the production of HBsAb, significantly reduced the HBsAg and HBeAg levels of some mice, and improved viral serum indicators.

[0193] In one embodiment, the mRNA vaccine of the present invention can be co-administered with an immune stimulatory factor (eg, CD40L).

[0194] The invention designs and prepares mRNA encoding CD40L, and after transfecting mammalian cells, incubates with PBMC or B cells separated from mouse peripheral blood, significantly activates B cells.

[0195] The present invention designs and prepares mRNA encoding CD40L, and immunizes HBV Tg mice together with mRNA vaccine, which can enhance the humoral immune response of mRNA vaccine, increase the level of anti-water production and significantly reduce the level of surface antigen.

[0196] The present invention also provides a method for inducing the production of antibodies against natural Pre-S antigen epitopes in a chronic hepatitis B infection model and maintaining the production for a long time, which comprises administering the mRNA vaccine of the present invention to a subject in need.

[0197] The main advantages of the present invention include:

[0198] 1) After the mRNA vaccine of the present invention is transfected into cells, it can stably express hepatitis B surface antigen and present the Pre-S epitope on the cell surface by co-expressing L protein, S protein and optional M protein, thus solving the problem that L protein alone cannot be stably expressed on the cell surface.

[0199] 2) The mRNA vaccine of the present invention has a simple preparation process, a short production cycle, low cost, good immune effect, and can significantly activate the production of high-level antibodies on the cell surface.

[0200] 3) The mRNA vaccine of the present invention can break immune tolerance in the HBV Tg model, activate the production of specific antibodies against the natural Pre-S antigen structure and Pre-S-dependent T cell immune response.

[0201] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0202] Example 1 Expression and Detection of Cell Membrane L Protein

[0203] First, L protein, M protein, and S protein expression plasmids (all pD2531 plasmids) were used to stably transform glutamine synthetase-deficient (Gln-) 293 cells. The main steps were:

[0204] (1) The three plasmids were linearized using PvuI-HF enzyme (NEB);

[0205] (2) PEIPrime (Serochem) was mixed with the linearized plasmid and transfected into 293(Gln-) cells in shake flasks. The groups included pD2531:L+pD2531:S and pD2531:L+pD2531:M+pD2531:S. Untransfected 293(Gln-) cells were used as controls.

[0206] (3) One week after transfection, when cell viability and proliferation were good, the transfected cells were cultured in glutamine-deficient 293 medium (Gibco). Two weeks later, samples were taken for flow cytometry (FACS) analysis to detect antigen expression.

[0207] (4) The limiting dilution method was used to select and culture monoclonal cells, and finally the monoclonal cells 293:LS(4C6) and 293:LS(4G2) expressing L protein and S protein, and the monoclonal cell 293:LMS(A4) expressing L protein, M protein and S protein were obtained.

[0208] After multiple passages, FACS analysis revealed a 45-55% positive rate for the Pre-S2 antigen on the surface of these monoclonal cells (Figure 1). A membrane protein extraction kit (Beyotime) was used to extract the membrane protein (MP) and cytoplasmic protein (CyP) of 293, 293:LS(4C6), and 293:LS(4G2). The main steps involved hypotonic treatment of the cells, 2-3 cycles of liquid nitrogen freeze-thaw, low-speed centrifugation to pellet the nuclei, high-speed centrifugation to pellet the membrane fraction, and membrane protein solubilization. Whole cell protein lysed with NP-40 lysis buffer was used as a control. Western blotting (WB) was used to detect the S antigen region in each fraction. As shown in Figure 2, the cell membrane fraction contained a certain proportion of S protein and L protein (at a molecular weight of approximately 40 kDa).

[0209] The above results all indicate that, with the support of S protein or M protein + S protein, L protein can be stably presented on the cell membrane surface ( Figures 1 , 2 , 16 , 17 , 18 ).

[0210] At the same time, similar to other studies, when the pD2531:L plasmid was stably transfected into 293 cells alone, it was found that most cells died due to cytotoxicity during the screening process and single clones could not be selected.

[0211] Example 2 Observation of cell membrane raft structure

[0212] The membrane raft structure is a dynamic lipid raft structure that protrudes from the cell membrane surface and contains lipids, proteins and sugar molecules. The membrane raft plays a certain role in the invasion, assembly and budding of the virus. The expression of viral surface antigens can sometimes lead to the formation of this structure on the membrane surface and the presentation of a certain density of regularly arranged antigen molecules, which has a strong promoting effect on enhancing the immunogenicity of the antigen. Therefore, in this embodiment, a transmission electron microscope (TEM) was used to observe the distribution of membrane rafts on the surface of 293:LS (4G2) cells and 293:LMS (A4) cells, and 293 (Gln-) cells were used as a control. The main steps include: centrifugation after cell shake flask culture for a period of time, fixation with 2.5% glutaraldehyde, and sending the structure to a third party for TEM sample preparation and photography. The results are shown in Figure 3. Compared with the control group cells, the surfaces of these two monoclonal cells contained a larger number of membrane raft structures, indicating that the co-expression of S protein and L protein, or S protein, M protein and L protein can form membrane raft structures (Figure 3), and the L protein presented by the cell membrane may have stronger immunogenicity.

[0213] Example 3 mRNA vaccine preparation

[0214] Based on the results of Example 1, L protein is stably presented on the cell membrane only when L and S proteins are expressed simultaneously, or when L, M, and S proteins are expressed simultaneously. mRNAs expressing these three surface antigen proteins were designed, optimized, and prepared, respectively. Liposome nanoparticles (LNPs) were used as delivery vehicles, and intramuscular injection of the mRNA vaccine was used as the immunization method. In vitro validation demonstrated that these three proteins could be efficiently expressed in mammalian cells.

[0215] The preparation method of mRNA vaccine mainly includes: synthesizing the optimized L protein, M protein and S protein CDS sequences respectively, cloning the DNA fragments into the template plasmid respectively, preparing mRNA and purification by in vitro transcription, and performing LNP packaging and quality control testing.

[0216] CDS sequence optimization involves adjusting codon preference for expression in humans, increasing the G / C ratio, adjusting the frequency of commonly used codons, and avoiding specific restriction sites to maintain high mRNA structure stability and enhance translation efficiency in humans and mammals. The transcription start sites of the M and S proteins in the sequence are mutated to enable transcription and translation into the L protein only. Similarly, the transcription start site of the S protein in the M protein sequence is mutated to enable translation into the M protein only.

[0217] The mRNA sequence is shown below:

[0218] L mRNA (SEQ ID NO: 4):

[0219] M mRNA (SEQ ID NO: 5)

[0220] S mRNA (SEQ ID NO: 6)

[0221] The transcription template plasmid includes T7 promoter, 5'-UTR, coding region (ORF), 3'-UTR and polyA, kanamycin KanR resistance sequence and other elements.

[0222] In vitro transcription can be performed using a three-step, two-step, or one-step method to produce mRNA molecules with Cap1 or Cap0 5' capping, 3' tailing, and incorporation of pseudouracil and / or other modified nucleosides. DNA template removal is performed using DNase I, and mRNA is purified using lithium chloride. After reconstitution in RNase-free water, mRNA concentration and purity are measured using a Nanodrop UV spectrophotometer, length is determined by agarose gel electrophoresis, capping efficiency and tailing are determined by LC-MS, and mRNA integrity is assessed by capillary electrophoresis (Figure 4).

[0223] The present invention uses liposome nanoparticles LNP to encapsulate mRNA. LNP contains four components, namely cationic lipid SM-102 (50%), auxiliary lipid DSPC (10%), cholesterol Chol (38.5%), and PEG2000-DMG lipid (1.5%). These four components are dissolved in anhydrous ethanol in a molar ratio to form an organic phase. The mRNA and organic phase in the aqueous phase are injected into a microfluidic device in proportion, and the LNP / mRNA mixture is mixed to obtain a final LNP / mRNA mixture after buffer exchange, filtration sterilization and concentration. Nanosight is used to detect the particle size (results shown in Figure 5) and the polydispersity coefficient PDI, and phase analysis light scattering is used to detect the surface potential (results shown in Figure 6). RiboGreen is used to detect the encapsulation efficiency and mRNA concentration, and Limulus amebocyte lysate is used to detect the bacterial endotoxin content of LNP / mRNA. The results are summarized as shown in Figure 7. Store at 4°C until use.

[0224] All LNP / mRNA injections of the present invention are used within one month after encapsulation.

[0225] Example 4 Verification of in vitro expression of mRNA vaccine

[0226] 293T cells were plated into 6-well plates, with 6×10^5 cells per well. After overnight, each well was treated with 5μg of LNP-encapsulated L mRNA, M mRNA, and S mRNA, 5μg of L mRNA + 5μg of S mRNA, or 5μg of L mRNA + 5μg of M mRNA + 5μg of S mRNA. 48 hours after transfection, the cells were collected and divided into two parts: one for FACS analysis and the other for membrane protein extraction and WB analysis together with the culture supernatant. The same transfection method was used, and FACS analysis was performed 96 hours after transfection. Specifically:

[0227] (1) FACS detection: Wash each group of cells once with pre-cooled PBS, centrifuge, add 3% BSA and block on ice for 30 minutes. Divide the cells into two parts, centrifuge, add diluted anti-Pre-S2 and anti-S antibodies respectively, incubate on ice for 1 hour, wash three times with PBS, add FITC anti-mouse and FITC anti-rabbit fluorescent secondary antibodies respectively, incubate on ice for 0.5 hours, wash three times with PBS and detect on the machine.

[0228] (2) Membrane protein extraction and WB detection: The membrane protein extraction method is the same as that in Example 1, mainly including cell hypotonic treatment, liquid nitrogen freeze-thaw 2-3 times, low-speed centrifugation to precipitate cell nuclei, high-speed centrifugation to precipitate membrane components, and membrane protein solubilization. 5× loading buffer was added to the cell membrane lysate and cell culture supernatant, and then the cells were placed in a boiling water bath for 5 minutes. Polyacrylamide gel electrophoresis was then performed. After the electrophoresis, protein immunoblotting was performed. The protein was transferred to a PVDF membrane, blocked with 5% skim milk powder at room temperature for 1 hour, incubated with anti-Pre-S2 and anti-S antibodies at 4°C overnight, washed 3 times with TBST, incubated with HRP anti-mouse antibody at room temperature for 1 hour, washed 3 times with TBST, and the PVDF membrane was developed and exposed using an enhanced chemiluminescence reagent, and photographed.

[0229] The FACS results, shown in Figure 8, show that, while the Pre-S2 signal was highest in the M mRNA transfection group, the Pre-S2 and S protein signals were highest when the three mRNAs were co-transduced compared to the other groups, indicating a higher proportion of the corresponding antigens presented on the membrane surface. The WB results, shown in Figure 9, show that when the three mRNAs were co-transduced, the cell membrane presented all three proteins, with the L protein expression level and proportion significantly increased compared to L mRNA transfection alone. Furthermore, S and M proteins were detectable in the cell culture supernatant during co-transduction.

[0230] Example 5 Study on the effect of mRNA vaccine on breaking immune tolerance in HBV transgenic mice

[0231] In 1.3-copy HBV transgenic Tg Balb / c mice, the activation of hepatitis B surface antibodies by an mRNA vaccine in a state of chronic hepatitis B immune tolerance was investigated, and the correlation between antigen levels and antibody production levels was studied. After breeding with 1.3-copy HBV Tg mice, the HBV carrier-positive rate in the offspring was 75%, and HBV viral particles were expressed from birth and carried throughout life. Using the Abbott reagent chemiluminescence method to detect hepatitis B surface antigen (HBsAg) in the serum of transgenic mice, the level reached 15517.07±9376.81 IU / mL; using the Shengxiang Hepatitis B Virus Nucleic Acid Quantification Kit (PCR-fluorescent probe method) to detect serum HBV DNA, the level reached 103-105 IU / ml. HBsAg and HBV DNA levels did not differ significantly by mouse sex; the hepatitis B surface antibody (HBsAb) levels in all transgenic mice were 0. This transgenic mouse is primarily used to study the immune pathogenesis of hepatitis B and evaluate the efficacy of anti-hepatitis B virus drugs. It is currently the most widely used HBV transgenic mouse in China.

[0232] Fifty-five HBV Tg mice with different HBsAg levels were divided into 11 groups, with 5 mice in each group. Each group contained mice with high HBsAg levels (>10,000 IU / mL) and mice with low HBsAg levels (<10,000 IU / mL). They were injected with different mRNA vaccines, proteins, or PBS. The groups included:

[0233] PBS;

[0234] Luciferase Luc mRNA (10 μg);

[0235] L+M+S mRNA(5+5+5μg / 10+10+10μg);

[0236] L+S mRNA (5+5μg / 10+10μg);

[0237] L mRNA (5 μg / 10 μg);

[0238] S mRNA (10 μg);

[0239] Virus-like particles LMS VLP (5μg) + 125μg aluminum adjuvant adjuvant (InvivoGen);

[0240] Pre-S peptide (5 μg) + 125 μg adjuvant.

[0241] Mice were immunized by intramuscular injection on days 0, 8, 15, 29, and 113 (16 weeks). Blood was collected from the eye sockets on day 0 and every two weeks thereafter for serum. Serum HBsAg and HBsAb were measured using the Abbott chemiluminescence assay, and serum HBV DNA was measured using the Shengxiang Hepatitis B Virus Nucleic Acid Quantitative Detection Kit.

[0242] Luc mRNA was obtained commercially. Pre-S protein was obtained by prokaryotic expression and purification. The specific method includes the following steps:

[0243] (1) The construction of the prokaryotic expression system includes synthesizing the Pre-S gene and cloning it into the pET:28a prokaryotic expression plasmid after the 6×His tag. The plasmid carries the KanR resistance gene and isopropyl-β-D-thiogalactopyranoside IPTG. The constructed plasmid is transformed into BL21 (DE3) competent cells, shaken and plated, colonies are selected after overnight growth, colony PCR is performed, and the target band is confirmed according to the molecular weight after agarose gel electrophoresis. The positive colonies are selected and sent to Jinweizhi for sequencing. After verifying the correct sequence, the bacterial solution is amplified and the bacteria are preserved;

[0244] (2) The prokaryotic expression and purification process includes bacterial recovery, amplification using LB medium, IPTG-induced expression, bacterial liquid collection and lysis, inclusion body protein extraction and renaturation, and Ni column purification to obtain the target protein;

[0245] (3) Finally, endotoxins were removed using Thermo Fisher Pierce High-Capacity Endotoxin Removal Resin, and endotoxins were detected using the GenScript Endotoxin Detection Kit.

[0246] In this example, LMS VLPs were obtained by fermentation and purification of the 293:LMS(A4) monoclonal cells secreting LMS VLP particles constructed and screened in Example 1. The specific steps included amplifying and fermenting the 293 monoclonal cells from a 500 mL shake flask to a 2.5 L shake flask, centrifuging the cells at 3000 rpm / min, removing cell debris by centrifugation at 8000 rpm / min, concentrating 50-70 times with a 300K ultrafiltration membrane and replacing with D-PBS, and then purifying the VLPs using hydroxyapatite CHT adsorption, size exclusion chromatography SEC, and virus / biomacromolecule resin chromatography column core400. The purity of the LMS VLPs and the ratio of each protein were detected by silver staining, the L protein, M protein, and S protein were further confirmed by western blot, and the VLP morphology and size were detected by TEM. The results are shown in Figure 10.

[0247] The results of HBsAb detection are shown in Figure 11. Two weeks after the first immunization (week 2), all mice in the L+M+S mRNA high-dose group and 80% of the mice in the low-dose group produced surface antibodies. 80%, 60%, and 60% of the mice in the L+S mRNA high- and low-dose groups and the S mRNA group produced antibodies, respectively. The overall antibody level in the L+S mRNA group was greater than that in the S mRNA group. In comparison, 60% of the mice in the LMS VLP group produced antibodies. Two weeks after the second immunization (week 4), all mice in the L+M+S mRNA, L+S mRNA, and S mRNA groups produced antibodies and all broke immune tolerance (surface antibody levels greater than 10 IU / L). Although all mice in the LMS VLP group produced antibodies at week 4, immune tolerance was not completely broken until week 6. The L mRNA group never produced antibodies. At the same time, starting from the 4th week, the surface antibodies of most mice in the L+M+S mRNA, L+S mRNA, S mRNA and LMS VLP groups always maintained a high level, especially the first two groups. Starting from the 14th week, antibodies greater than 40,000 IU / L were produced and continued to rise. Two weeks after the last immunization in week 16 (week 18), the antibody level continued to rise. The HBsAg test results are shown in Figure 12. From week 0 to week 18, the surface antigen of one mouse with low antigen level in each of the L+M+S mRNA, S mRNA and LMS VLP groups was significantly reduced. A correlation analysis was performed on the antigen reduction and antibody production levels in mice that produced antibodies from week 0 to week 6. The results showed that there was no significant correlation, indicating that the effect of mRNA vaccine on activating antibodies and breaking immune tolerance is not affected by antigen level (Figure 13).

[0248] Example 6 Study on the therapeutic effect of mRNA vaccine in pAAV HBV1.2 mouse model

[0249] The construction of the pAAV HBV1.2 copy mouse model is specifically as follows: 10 μg of pAAV HBV1.2 plasmid is injected into C57 / BL6 mice through the tail vein at 8% of the mouse body weight, causing transient cardiac congestion, resulting in increased pressure in the lower layer, and the plasmid solution flows to the hepatic vein and renal vein. The pressure opens the barrier between endothelial cells, causing a transient defect in the liver cell membrane. After 12h-72h, the mice return to normal, and pAAV HBV1.2 is transferred to the liver cells to replicate and express HBV virus particles. Three days after modeling, HBsAg and HBV DNA generally reach the highest levels, and surface antigen positivity can be maintained for about 1 month.

[0250] On the fourth day of modeling, 40 mice with successful modeling were divided into 8 groups and injected with different mRNA vaccines, proteins or PBS. The groups included: PBS, Luc mRNA (10 μg), L+M+S mRNA (5+5+5 μg), L+S mRNA (5+5 μg), L mRNA (5 μg), S mRNA (5 μg), LMS VLP (5 μg) + 125 μg adjuvant, Pre-S peptide(5μg)+125μg Mice were immunized by intramuscular injection on days 0 and 15. Blood was collected from the orbital cavity on day 0 and weekly thereafter for serum collection until the end of the study. Serum HBsAg and HBsAb were measured using the Abbott chemiluminescence assay, and serum HBV DNA was measured using the Shengxiang Hepatitis B Virus Nucleic Acid Quantitative Detection Kit.

[0251] The results are shown in Figure 14. Within the 21-day window period after the successful construction of the pAAV HBV1.2 mouse model, the combination of three or two mRNAs significantly inhibited the production of HBV surface antigens and envelope antigens after immunization. Unlike the HBV Tg model, L mRNA immunization alone also had an inhibitory effect, which may be related to the relatively low level of hepatitis B antigen in this model and the significant decrease in antigen expression over time in the short term. The FASC and WB results of in vitro mRNA transfection in Example 4 showed that L mRNA also had a certain expression in the early stage after transfection, so it was able to activate the production of certain antibodies in this model. In addition, the activation of antibodies was slightly different from that of antigens. 14 days after the first immunization, only the combination of three or two mRNAs significantly activated the production of high-level antibodies. High-level antibodies were not produced until the 21st day after immunization with L mRNA or S mRNA alone.

[0252] Example 7 Study on the therapeutic effect of mRNA vaccine in rAAV HBV1.3 mouse model

[0253] A recombinant AAV8 virus carrying 1.3 copies of the full-length HBV genome was injected into the blood at 1×10 10 or 5×10 10 vg / mL was injected intravenously into mice, and a stable HBV continuous replication model could be established after 4 weeks, and a high level of serum and viral indicators could generally be maintained for 3 months.

[0254] The rAAV HBV1.3 mouse model was constructed as follows: 1×10 10 or 5×10 10 vg / mL of rAAV HBV1.3 virus liquid was added, and a stable HBV replication model was established 4 weeks after modeling (see Figure 21a for the modeling process).

[0255] After the model was successfully established, 30 mice with successful modeling were divided into 6 groups and injected with different mRNA vaccines or proteins or PBS, including PBS, L+M+S mRNA (5+5+5μg), L+S mRNA (5+5μg), L mRNA (5μg), S mRNA (5μg), LMS VLP (5μg) + 125μg adjuvant. The specific injection time is shown in Figure 21a. Mice were immunized by intramuscular injection on day 0 and day 15 of immunization. Blood was collected from the orbits on day 0 and every week thereafter, and serum was obtained until the end of the experiment after 3 months. Serum HBsAg and HBsAb were detected using the Abbott reagent chemiluminescence method. Serum HBV DNA was detected using the Shengxiang Hepatitis B Virus Nucleic Acid Quantitative Detection Kit. Figure 20 is 1×10 10 vg / mL model immunization experiment, HBsAg, anti-HBsAg antibody, HBeAg antigen, and HBV DNA level changes, Figure 21 is 5×10 10 vg / mL model immunization experiment, and changes in HBsAg, anti-HBsAg antibodies, and HBeAg antigen levels.

[0256] Example 8 mRNA vaccine activates antigen-specific T cell immune response in HBV Tg mice

[0257] In this example, the spleen T cells isolated from the animal model of Example 5 were stimulated by LMS co-expressing mouse cells to examine the mRNA vaccine's activation of Pre-S natural antigen-dependent T cell immune responses.

[0258] The specific process is as follows: (1) constructing pCDNA3.1(+)-:L protein, pCDNA3.1(+):M protein and pCDNA3.1(+):S protein with different resistance genes Protein expression plasmid, plasmid linearization, then use Lipo3000 to co-transduce these three plasmids into mouse embryonic fibroblast Balb / 3T3, 96h after transfection, resistance gene screening, enrichment of antigen expression positive cells, screening for two weeks and FACS detection (Figure 19a), ready for incubation with T cells; (2) Take the mice with better treatment effect in Example 5 and kill them, take out the spleen and grind it, use Percoll density gradient centrifugation to separate lymphocytes, then use CD3 magnetic beads to separate T cells, count and dilute with cell culture medium; (3) Each group of T cells is added to the antigen-expressing Balb / 3T3 cells at a ratio of 1:5, incubate for 12 hours, use pre-cooled PBS to wash to obtain each group of cells, treat the cells with fixative, wash with PBS, incubate with anti-CD3e antibody, wash with PBS, and use True-PhosTM The membrane was permeabilized with Perm Buffer (BioLegend), incubated with anti-IFNγ antibody, washed with PBS, and the proportion of IFNγ-positive cells in CD3-positive cells was detected by flow cytometry ( FIG19 b ).

[0259] Example 9 Detection of specific antibodies against the Pre-S natural antigen epitope in HBV Tg mice immunized with mRNA vaccine

[0260] In this example, IgG in the serum of HBV Tg mice immunized with mRNA vaccine was purified, and anti-S antibodies were removed by S antigen binding screening to eliminate the interference of anti-S IgG antibodies. The binding of IgG to HBV virus particles after screening was detected to determine whether mRNA vaccine immunization activated the production of specific antibodies against the re-S natural antigen epitope in HBV Tg mice.

[0261] The specific process is as follows: (1) In Example 5, after immunization with mRNA vaccine for 3 times until the 14th week, PBS, Luc mRNA, L+M+S mRNA, L+S mRNA, L mRNA, S mRNA, LMS VLP, and Pre-S group mouse serum were extracted every two weeks. After the serum of each group accumulated to 1 mL, IgG was purified using a Protein A column; (2) The diluted Pre-S polypeptide, HBsAg recombinant protein, and LMS VLP particles were taken respectively, coated on an ELISA plate at 4°C overnight, washed with PBS, blocked with goat serum, and 1 μg of IgG sample was added to each plate. The plates were incubated at 4°C overnight, washed with PBST, and HRP anti-mouse was added. After incubation at 37°C for 2 h, the plates were washed with PBST, TMB colorimetric solution was added for reaction, and 2M H2SO4 stop solution was added. The CD450-OD603 value was detected by an ELISA reader. (3) Streptavidin magnetic beads were used to bind to biotin-labeled HBsAg recombinant protein to remove anti-S IgG; (4) Diluted serum of hepatitis B patients (containing HBV particles), Pre-S peptide and HBsAg recombinant protein were taken separately, coated on the ELISA plate overnight at 4°C, washed with PBS, blocked with diluted normal human serum, added with 1 μg of IgG sample screened by HBsAg-magnetic beads, incubated overnight at 4°C, washed with PBST, added with HRP anti-mouse, incubated at 37°C for 2h, washed with PBST, added with TMB colorimetric solution, and then added with 2M H2SO4 stop solution, and the CD450-OD603 value was detected by enzyme marker.

[0262] The experimental results are shown in Figure 15. Compared with the control group, HBV Tg mice immunized with L+M+S mRNA, L+S mRNA and LMS VLP all produced IgG antibodies against Pre-S polypeptide and HBsAg. SmRNA and Pre-S activated the production of antibodies against HBsAg and Pre-S polypeptide, respectively (Figure 11c, Figure 15).

[0263] Example 10 Analysis of serum antibodies inhibiting in vitro cell line infection after different mRNA vaccines immunized mice

[0264] In this example, C57 mice were immunized with mRNA and serum samples were extracted (the specific effective neutralizing antibody concentration is unknown). An in vitro model of HBV-infected HepG2-hNTCP cell line was used to detect and compare the in vitro inhibitory effects of immune serum on HBV-infected HepG2-hNTCP cell line at different concentration gradients. By detecting HBeAg levels, the HBeAg inhibition rate of different serum samples under different concentration conditions was determined, and the IC50 corresponding dilution factor and concentration used when the serum sample inhibition rate on HBV infection reached 50% were calculated. The experimental steps include: (1) preparing HepG2-hNTCP cell line; (2) infecting HepG2-hNTCP cell line with HBV that has infection and replication ability under the conditions of adding HBIg positive control antibody (Figure 22a) or serum samples with different concentration gradients (Figure 22c, Figure 22d, Figure 22e) or without adding serum samples (Figure 22b), with three replicates set for each group. Serum groups 1-4 consisted of PBS, LMS mRNA, LS mRNA, and S mRNA-immunized mouse serum, respectively. (3) On the seventh day after HBV infection, culture supernatants were collected. (4) HBeAg levels in the supernatants were measured. The calculated results were compared with the IC50 concentration of the HBeAg positive control antibody to determine the specific concentration of effective neutralizing antibodies in the serum sample stock solution (Figure 22f). The experimental results were used to evaluate the neutralizing ability of the serum samples and their effective antibody concentrations.

[0265] Example 11 Toxicological analysis of mice after immunization with different doses of mRNA vaccine

[0266] Male ICR mice aged 5 to 6 weeks were selected and injected intramuscularly on days 0 and 3 with 1.5 μg, 7.5 μg, or 37.5 μg of LMS mRNA (mass ratio 1:1:1), respectively. An equal volume of PBS was used as a control (n=8). During the experiment, mice were observed every 3-4 days for mortality, appearance, and behavioral activity, and body weight changes were recorded (Figure 23a) until 21 days after the second dose.

[0267] At the end of the experiment, the mouse serum was separated and the weights of the main organs (including heart, liver, spleen, lungs and kidneys) were weighed (Figure 23b). At the same time, liver function (Figure 24a) and renal function indicators (Figure 24b) were detected in the mouse serum. Liver function indicators include alanine aminotransferase (ALT) and aspartate aminotransferase (AST); renal function indicators include urea (UREA), uric acid (UA) and creatinine (CR). After rinsing the organs with PBS, they were fixed with 4% paraformaldehyde for 2 days and then stained with H&E for pathological analysis (Figure 25).

[0268] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A vaccine composition, characterized in that The vaccine composition comprises: The first polynucleotide N L , which encodes the hepatitis B virus surface antigen L protein; and The second polynucleotide N S , which encodes the hepatitis B virus surface antigen S protein.

2. The vaccine composition according to claim 1, wherein N L and N S The molar ratio is 1:10-10:1, preferably 1:5-5:1, and more preferably 1:2-2:

1.

3. The vaccine composition according to claim 1, wherein The vaccine composition further comprises a third polynucleotide N M , which encodes the hepatitis B virus surface antigen M protein.

4. The vaccine composition according to claim 1, wherein The transcription start sites of the M protein and the S protein in the L protein coding sequence are mutated so that the L protein can only be translated.

5. The vaccine composition according to claim 1 or 3, wherein The polynucleotide is mRNA.

6. The vaccine composition according to claim 5, wherein N L The mRNA sequence encoding the L protein is shown in SEQ ID NO: 4; M The coding mRNA sequence of the M protein is shown in SEQ ID NO: 5; S The mRNA sequence encoding the S protein is shown in SEQ ID NO:

6.

7. The vaccine composition according to claim 1, wherein The vaccine composition is a liposome nanoparticle, which includes a liposome layer located on the outside and the polynucleotide encapsulated inside.

8. An engineered host cell, characterized in that The engineered host cell expresses membrane-bound hepatitis B virus surface antigen L protein and S protein; or expresses membrane-bound hepatitis B virus surface antigen L protein, S protein and M protein.

9. The host cell according to claim 8, wherein The host cell is obtained by transfecting the vaccine composition according to claim 1 or 3.

10. The method for preparing an engineered host cell according to claim 8, wherein: Including steps: The vaccine composition according to claim 1 is transduced into immune cells to obtain the engineered host cells.

11. Use of the vaccine composition of claim 1 or the engineered host cell of claim 8 for the preparation of a medicament for use in a subject: (a) Prevention of hepatitis B virus infection; (b) inducing the production of specific antibodies against hepatitis B virus; and / or (c) Treatment of chronic hepatitis B infection.

12. The use according to claim 11, characterized in that The chronic hepatitis B is immune-tolerant chronic hepatitis B.

13. A drug for treating chronic hepatitis B infection, characterized in that: The medicament comprises the vaccine composition according to claim 1 or the host cell according to claim 8.

14. A method for preventing and / or treating hepatitis B, comprising the steps of: Administering the vaccine composition of claim 1 or the engineered host cell of claim 8 to a subject in need thereof.

15. A method for producing anti-hepatitis B virus antibodies, comprising the steps of: An animal is immunized using the vaccine composition according to claim 1 or the engineered host cell according to claim 8 to produce the anti-hepatitis B virus antibody.

Images