Lentiviral vector, lentiviral particle for treating hepatitis B, and preparation method thereof and use thereof

Abstract

The present invention discloses a lentiviral vector particle and a lentiviral vector particle for treating hepatitis B infection. The lentiviral vector and particle of the present invention comprise a nucleotide sequence encoding a hepatitis B virus antigen. The large S antigen of hepatitis B virus is particularly selected as a candidate and can be applied in pharmaceutical compositions or vaccines for treating and / or preventing hepatitis B virus infection and for treating and / or preventing diseases caused by hepatitis B virus infection, thereby providing excellent therapeutic and prophylactic effects in patients in need thereof.

Description

[Technical field]

[0001] The present application relates to the biomedical field, in particular to lentiviral vectors and lentiviral particles for treating and / or preventing Hepatitis B, as well as preparation methods and uses thereof. [Background technology]

[0002] Hepatitis B virus (HBV) infection is one of the serious public health problems worldwide. HBV infection is an important cause of chronic hepatitis B, liver cirrhosis, and hepatocellular carcinoma. Drugs commonly used for clinical treatment of chronic HBV infection mainly include nucleoside analogs and interferon. Nucleoside analogs cannot completely eliminate HBV DNA in liver cells, and long-term use can easily lead to the emergence of drug-resistant mutant strains and rebound after drug withdrawal. After a course of interferon treatment (usually 48 weeks), the incidence of HBeAg seroconversion is only 33%, and the HBsAg conversion rate is less than 10%. Interferon monotherapy has a negative conversion rate of 3% to 7% for HBsAg, which is only slightly higher than that of nucleoside analogs, and the many side effects of interferon also limit its use.

[0003] Currently, the widely used hepatitis B protein vaccine induces humoral immunity and produces protective antibodies to achieve the purpose of prevention. Numerous studies have found that protective antibodies can only eliminate extracellular virus particles, but cannot eliminate intracellular viruses, making it difficult to treat infected patients. After antiviral treatment, the incidence of hepatocellular carcinoma in HBsAg-positive people is significantly higher than that in HBsAg-negative people. HBsAg-negative conversion is associated with improved liver function, improved histopathology, and improved long-term prognosis. This is currently recommended by the latest domestic and international chronic hepatitis B prevention guidelines. The ideal treatment goal is functional cure, i.e., HBsAg negativity, i.e., functional cure or clinical cure is the main goal of current chronic hepatitis B treatment. The art still needs drugs that can be used to treat and / or prevent HBV infection or treat and / or prevent diseases caused by HBV.

[0004] The HBV sequences inserted into the lentiviral vector in CN109923212A are surface antigens of genotypes A and C, Pol, HBx, MHC I and MHC II epitope sequences, and surface B cell epitopes of genotypes A and C. Combined with VLPs, the results shown are only immunogenicity results, and the therapeutic effect is unknown. The antigen sequences disclosed by CN1209340A are assigned to HBV genotype A sequences, which are mainly distributed in Europe and Central Africa. Summary of the Invention [Means for solving the problem]

[0005] Contents of the invention The present invention provides a lentiviral vector for the treatment of hepatitis B. The lentiviral vector comprises a nucleotide sequence encoding at least one hepatitis B virus antigen, the hepatitis B virus antigen being selected from the group consisting of core antigen (HBcAg), PreS1 antigen (PreS1) and large S antigen (LargeS).Preferably, the encoded hepatitis B virus antigen is the large S antigen (LargeS).

[0006] The amino acid sequence of the core antigen (HBcAg) may be the sequence set forth in SEQ ID NO:1; and / or the amino acid sequence of the PreS1 antigen (PreS1) may be the sequence set forth in SEQ ID NO:4 or SEQ ID NO:7; and / or the amino acid sequence of the large S antigen (LargeS) may be the sequence set forth in SEQ ID NO:10 or SEQ ID NO:13.

[0007] The coding nucleotide sequence of the core antigen (HBcAg) may be the sequence shown in SEQ ID NO:3; and / or the coding nucleotide sequence of the PreS1 antigen (PreS1) may be the sequence shown in SEQ ID NO:6 or SEQ ID NO:9; and / or the coding nucleotide sequence of the large S antigen (LargeS) may be the sequence shown in SEQ ID NO:12 or SEQ ID NO:15.

[0008] The present invention also provides a method for preparing lentiviral particles for treating hepatitis B, comprising: a) co-transfecting a host cell with a lentiviral vector according to the invention, a packaging vector expressing Gag, Rev and / or Pol proteins and an envelope vector expressing an envelope protein, or transfecting a lentiviral vector according to the invention into a host cell capable of expressing an envelope protein and one or more of Gag, Rev and Pol proteins; b) culturing the transfected host cells to package the lentiviral vector into lentiviral vector particles; and c) harvesting the lentiviral vector particles produced in step b). The above method is characterized by comprising the steps of:

[0009] The present invention also provides a preparation of lentiviral particles for the treatment of hepatitis B. The preparation of lentiviral particles comprises a lentiviral vector according to the present invention, or the lentiviral particles are prepared by the preparation method according to the present invention.

[0010] The present invention also provides a lentiviral vector according to the invention or a preparation of a lentiviral particle according to the invention for the treatment and / or prevention of a hepatitis B virus infection in a subject in need thereof or for the treatment and / or prevention of a disease caused by a hepatitis B virus infection in a subject in need thereof. A lentiviral vector according to the invention or a preparation of a lentiviral particle according to the invention may be implemented in a pharmaceutical composition, such as a vaccine, in particular together with a pharma- ceutically acceptable carrier.

[0011] The subject according to the present invention may in particular be a mammal, such as a human.

[0012] The present invention also provides a pharmaceutical composition for treating and / or preventing a Hepatitis B virus infection in a subject in need thereof, or for treating and / or preventing a disease caused by a Hepatitis B virus infection in a subject in need thereof, said pharmaceutical composition comprising a lentiviral vector according to the invention, or a preparation of lentiviral particles according to the invention, and a pharma- ceutical acceptable carrier.

[0013] The beneficial effects of the present invention include the following: The immunogen of the present invention can cover the sequences of most epidemic strains in China, and the non-integrating lentiviral vector according to the present invention can optimize antigen presentation and break immune tolerance under the premise of ensuring the safety of the non-integrating lentiviral vector. The animal experimental data contained herein demonstrates the good immunogenicity of the non-integrating lentiviral vector and can induce strong immune responses in wild mice. In a mouse model of chronic HBV infection, significant therapeutic effects have also been confirmed, including the elimination of HBV antigens and viral DNA, the conversion of antibodies to positive, and the ability to activate strong T cell immune responses, with the potential for significant clinical conversion. [Brief description of the drawings]

[0014] [Figure 1a] FIG. 1a is a plasmid map. [Figure 1b] FIG. 1b is a plasmid map. [Diagram 2] FIG. 2 shows the experimental timeline of the studies presented herein. [Diagram 3] Figure 3 shows the detection of HBsAg in mice. Figures 3A to 3E show AAV-HBV-infected mice intramuscularly injected with lentiviruses JW27, JW28, JW27+JW28, JW29, and JW30 (horizontal axis: time (weeks); vertical axis: HBsAg (IU / mL)). [Figure 4] Figure 4 shows detection of HBV-DNA in mice. Figures 4A to 4E show AAV-HBV-infected mice intramuscularly injected with lentiviruses JW27, JW28, JW27+JW28, JW29, and JW30 (horizontal axis: time (weeks); vertical axis: HBV-DNA Log10 (IU / mL)). [Diagram 5]Figures 5A-5E are graphs showing the results of using a mouse IFN-γ ELISPOT assay to analyze induced T cell responses, where Figures 5A-5E are, respectively, the JW27 group, the JW28 group, the JW27 group + JW28 group, the JW29 group, and the JW30 group (vertical axis: from left to right: HBsAg pool; PreS1-GT B pool; PreS1-GT C pool; PreS2 pool, and core pool; vertical axis: SFC / 106 splenocytes). [Figure 6] FIG. 6 shows the results of HBcAg immunohistochemistry in liver tissues of various tested mice in the following order from left to right and top to bottom: JW27-259, JW27-273, JW27-269, JW28-225, JW28-264, JW28-266, JW27+JW28-234, JW27+JW28-236, JW27+JW28-201, JW29-211; JW29-249, JW29-268, JW30-226, JW30-227 and JW30-260. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Specific implementation The present invention will be further explained and described below in conjunction with the embodiments, but the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. Based on the present invention and the embodiments, all other inventions and embodiments obtained by those skilled in the art without creative work should be within the protection scope of the present invention.

[0016] Materials and reagents used in the following examples may be obtained from commercial sources unless otherwise specified.

[0017] Unless otherwise indicated or defined, all terms used have their normal meaning in the art as would be understood by a person skilled in the art. Reference is made, for example, to standard manuals, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual; 2000"; Lewin, "Genes VIII"; and Roitt et al., "Immunology" (8th edition), as well as to the general existing techniques. In addition, unless otherwise specified, all methods, steps, techniques and operations not specifically detailed can and have been performed in a manner known per se, which would be understood by a person skilled in the art in the field of the present invention. For example, reference is made to the standard manuals, the general prior art mentioned above, and other references cited therein.

[0018] As used herein, the word "and / or" includes all combinations of the items connected by the word and is to be treated as if each combination were individually listed herein. For example, "A and / or B" includes "A," "A and B," and "B." For example, "A, B, and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0019] When the words "contains" or "comprises" are used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the sequence or may have additional amino acids or cores at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to the skilled artisan that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained under certain practical conditions (e.g., when expressed in a specific expression system), but does not substantially affect the function of the polypeptide. Therefore, when a specific amino acid sequence of a polypeptide is described in the text of the specification and claims, it also covers a polypeptide that does not contain the methionine encoded by the start codon at the N-terminus, but does contain the methionine. The coding nucleotide sequence may also contain a start codon, and vice versa.

[0020] A "polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and are single- or double-stranded RNA or DNA polymers that optionally contain synthetic, non-natural or altered nucleotide bases. Nucleotides are referred to by their one-letter designation as follows: "A" is adenosine or deoxyadenosine (for RNA or DNA, respectively), "C" is cytidine or deoxycytidine, "G" is guanosine or deoxyguanosine, "U" means uridine, "T" means deoxythymidine, "R" means purine (A or G), "Y" means pyrimidine (C or T), "K" means G or T, "H" means A or C or T, "D" means A, T or G, "I" means inosine, and "N" means any nucleotide.

[0021] "Polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues correspond to an artificial chemical analogue of a naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence", and "protein" can also include modified forms, including, but not limited to, glycosylation, lipid linkage, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation, and ADP ribosylation.

[0022] "Regulatory sequence" and "regulatory element" are used interchangeably and refer to sequences upstream (5' non-coding sequences), in the middle, or downstream (3' non-coding sequences) of a coding sequence that influence the transcription, RNA processing, processing of the associated coding sequence, or stability of the translated nucleotide sequence. Regulatory sequences can include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0023] As used herein, the term "operably linked" refers to the connection of regulatory elements (such as, but not limited to, promoter sequences, transcription termination sequences, etc.) to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleotide sequence is controlled and regulated by the transcriptional control elements. Techniques for operably linking regulatory element regions to nucleic acid molecules are known.

[0024] The term "treatment" or "treating" refers to the administration of an active agent for the purpose of curing, relieving, alleviating, altering, correcting, ameliorating, improving, or affecting the condition (e.g., disease) under consideration, the symptoms of the condition, or preventing or delaying the onset of the symptoms, complications, or biochemical signs of the disease, or otherwise halting or inhibiting further development of the disease, condition, or disorder in a statistically significant manner.

[0025] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of acquiring a considered disease and / or one or all of the symptoms of the disease.

[0026] 1. Lentiviral vectors expressing hepatitis B virus (HBV) antigens, lentiviral particles, and methods for preparing the same The present invention provides a lentiviral vector comprising a nucleotide sequence encoding a Hepatitis B virus antigen.

[0027] As used herein, "lentiviral vector" refers to a nucleic acid construct derived from a lentivirus that is used to transduce a transgene containing a cis-acting lentiviral RNA or DNA sequence into a host cell. The lentiviral vector can be replication-defective, e.g., the lentiviral vector lacks the coding sequence of a functional lentiviral protein, such as Gag, Pol, Rev, and / or Env protein. When the replication-defective lentiviral vector is packaged into a lentiviral particle, the lentiviral protein (e.g., Gag, Pol, Rev, and / or Env) needs to be provided in trans.

[0028] Lentiviral vector can be in the form of RNA or RNA.For example, said lentiviral vector can be in the form of recombinant DNA molecule, such as plasmid (also called lentiviral transfer vector).Lentiviral vector can also refer to the genomic nucleic acid molecule contained in the complete lentiviral particle, which is a dimer of single-stranded RNA molecule.Lentiviral vector can also refer to the DNA sequence that is integrated into host cell.

[0029] Lentiviral vectors can be derived from, for example, human immunodeficiency virus (HIV-1 or HIV-2), monkey immunodeficiency virus (SIV), equine infectious encephalitis virus (EIAV), goat arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV), and feline immunodeficiency virus (FIV), and have been modified to remove genetic determinants responsible for pathogenicity and to introduce foreign expression cassettes.

[0030] In some preferred embodiments, the lentiviral vector is a non-integrating lentiviral vector. Non-integrating lentiviral vectors can effectively avoid the potential risk of viral DNA integration into the human genome.

[0031] "Lentiviral particle" or "lentiviral vector particle," as used interchangeably herein, refers to a packaged viral particle (e.g., a lentiviral vector described herein) that contains lentiviral proteins and its associated lentiviral genome, which packaged viral particle is capable of infecting a host cell and expressing in the host the proteins encoded by the viral genome.

[0032] The hepatitis B virus antigens according to the present invention may be antigens from different genotypes or different serotypes of hepatitis B virus. For example, the HBV may be selected from genotypes A, B, C, D, E, F, G, and H. Preferably, the HBV is genotype C. Alternatively, the HBV may be selected from serotypes ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq+, and adrq-. Preferably, the HBV is serotype adw or adr. In some preferred embodiments, the HBV is genotype C, serotype adr.

[0033] The Hepatitis B virus antigen encoded in the lentiviral vector of the present invention is selected from the group consisting of core antigen (HBcAg), PreS1 antigen (PreS1) and large S antigen (LargeS), or a combination thereof. In some preferred embodiments, the encoded Hepatitis B virus antigen is large S antigen (LargeS).

[0034] In some embodiments, the Hepatitis B virus core antigen (HBcAg) comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the coding nucleotide sequence of the Hepatitis B virus core antigen (HBcAg) is codon optimized for expression in humans. In some embodiments, the nucleotide sequence encoding the Hepatitis B virus core antigen (HBcAg) is set forth in SEQ ID NO: 2 or 3.

[0035] In some embodiments, the encoded Hepatitis B virus PreS1 antigen (PreS1) comprises the amino acid sequence set forth in SEQ ID NO: 4 or 7. In some embodiments, the coding nucleotide sequence of the Hepatitis B virus PreS1 antigen (PreS1) is codon-optimized for expression in humans. In some embodiments, the coding nucleotide sequence of the Hepatitis B virus PreS1 antigen (PreS1) is set forth in SEQ ID NO: 5, 6, 8 or 9, particularly SEQ ID NO: 6 or 9.

[0036] In some embodiments, the encoded Hepatitis B virus large S antigen (LargeS) comprises the amino acid sequence shown in SEQ ID NO: 10 or 13. In some specific embodiments, the coding nucleotide sequence of the Hepatitis B virus large S antigen (LargeS) is codon-optimized for expression in humans. In some embodiments, the coding nucleotide sequence of the Hepatitis B virus large S antigen (LargeS) is shown in SEQ ID NO: 11, 12, 14 or 15, particularly SEQ ID NO: 12 or 15.

[0037] In some preferred embodiments, the encoded Hepatitis B virus antigen is Hepatitis B virus large S antigen (Large S). In some preferred embodiments, the encoded Hepatitis B virus large S antigen (Large S) comprises the amino acid sequence set forth in SEQ ID NO: 13. In some preferred embodiments, the encoded nucleotide sequence of the Hepatitis B virus large S antigen (Large S) is set forth in SEQ ID NO: 15.

[0038] In some embodiments, the nucleotide sequence encoding the Hepatitis B virus is operably linked to a regulatory element. In some embodiments, the regulatory element is a promoter. A suitable promoter can be, for example, the β2 microglobulin promoter (β2m). An example of the β2m promoter can be found in International Patent Application Publication WO2013 / 174630.

[0039] In some embodiments, the lentiviral vector further comprises one or more or all of the following operably linked elements selected from the following: 5'LTR, ψ, RRE, cPPT / CTS, WPRE, and 3'LTR, In some embodiments, the 3'LTR lacks the U3 region (ΔU3).

[0040] In another aspect, the present invention provides a method for preparing a lentiviral vector particle comprising a nucleotide sequence encoding a Hepatitis B virus antigen, the method comprising the steps of: a) co-transfecting a suitable host cell with a lentiviral vector of the invention and one or more packaging vectors expressing Gag, Rev and / or Pol and an envelope vector expressing an envelope protein, or transfecting a lentiviral vector of the invention into a suitable host cell capable of expressing Gag, Rev, Pol and / or envelope protein; b) culturing the transfected host cells to package the lentiviral vector into lentiviral vector particles; and c) harvesting the lentiviral vector particles produced in step b). The above method further comprises the steps of:

[0041] In some embodiments, the suitable host cells capable of expressing Gag, Rev, Pol and / or envelope proteins are used to express Gag, Rev and / or Pol prior to transfection with the lentiviral vector of the invention. Transfection of various packaging vectors and envelope vectors expressing envelope proteins can also be performed.

[0042] Any other viral or non-viral envelope protein can be used for pseudotyped packaging, as long as the envelope protein is suitable for packaging and for entering target cells. In some embodiments, the envelope protein is the envelope glycoprotein of vesicular stomatitis virus (VSV-G). For example, the envelope protein can be the vesicular stomatitis virus envelope protein of Indiana serotype (GenBank Accession No. J02428) or New Jersey serotype (GenBank Accession No. P04882).

[0043] It is known in the art and to those skilled in the art that one or more suitable packaging vectors expressing Gag, Rev and / or Pol, as well as envelope vectors expressing suitable envelope proteins for packaging lentiviruses, can be readily obtained. In some embodiments, the lentiviral vector is a plasmid.

[0044] Suitable host cells for preparing lentiviral vector particles include, but are not limited to, 293 cells, such as 293T cells.

[0045] General methods for constructing lentiviral vectors and packaging viral particles suitable for the present invention can be found in Chinese Patents or Chinese Patent Application Publications No. 101291688A, No. 102083462B, No. 104039968B, No. 102083462B, etc.

[0046] In another aspect, the present invention provides a lentiviral vector particle (also referred to as a preparation of lentiviral particles) comprising a nucleotide sequence encoding a Hepatitis B virus antigen, said lentiviral vector particle comprising a lentiviral vector of the invention or prepared by the method of the invention referred to above.

[0047] 2. Treatment and / or prevention of diseases In another aspect, the present invention provides that the lentiviral vectors and / or lentiviral vector particles of the present invention (also referred to as lentiviral particle preparations) are prepared for the treatment and / or prevention of Hepatitis B virus infection in a subject in need thereof, or for the treatment and / or prevention of a disease caused by Hepatitis B virus infection in a subject in need thereof.

[0048] In another aspect, the present invention provides a pharmaceutical composition for treating and / or preventing a hepatitis B virus infection in a subject in need thereof, or for treating and / or preventing a disease caused by a hepatitis B virus infection in a subject in need thereof, comprising at least one lentiviral vector particle of the present invention and a pharma- ceutical acceptable carrier. In some embodiments, the pharmaceutical composition is a hepatitis B therapeutic vaccine.

[0049] As used herein, a "pharmaceutical acceptable carrier" is a substance that can be added to an active pharmaceutical ingredient to aid in formulation or to stabilize the formulation without causing significant adverse toxicological effects to the patient, including, but not limited to, a disintegrant, adhesive, filler, buffer, isotonicity agent, stabilizer, antioxidant, surfactant, or lubricant.

[0050] "Diseases caused by hepatitis B virus infection" include, but are not limited to, hepatitis caused by hepatitis B virus infection; cirrhosis; complications associated with cirrhosis, including ascites, esophageal varices, spontaneous peritonitis, and hepatic encephalopathy; liver cancer; liver failure; kidney disease; inflammation or swelling of blood vessels; anemia; and the like.

[0051] In some embodiments, the pharmaceutical composition comprises two or more lentiviral vectors of the present invention, each of which comprises a nucleotide sequence encoding different hepatitis B virus antigens.For example, the pharmaceutical composition can comprise two kinds of lentiviral vector particles of the present invention, where a first lentiviral vector particle comprises a nucleotide sequence encoding hepatitis B virus core antigen (HBcAg), and a second lentiviral vector particle comprises a nucleotide sequence encoding hepatitis B virus PreS1 antigen (PreS1).

[0052] As used herein, the term "subject" refers to a mammal, for example a human. In some embodiments, the subject is infected with hepatitis B virus. In some embodiments, the subject is not infected with hepatitis B virus. In some embodiments, the subject is infected with hepatitis B virus and shows symptoms of disease caused by hepatitis B virus infection. In some embodiments, the subject is infected with hepatitis B virus but does not show symptoms of disease caused by hepatitis B virus infection.

[0053] In some embodiments, the Hepatitis B virus may be selected from genotypes A, B, C, D, E, F, G, and H. Preferably, the Hepatitis B virus is genotype C. In some embodiments, the Hepatitis B virus may be selected from serotypes ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq+, and adrq-. Preferably, the Hepatitis B virus is serotype adw or adr. In some preferred embodiments, the Hepatitis B virus is genotype C, serotype adr.

[0054] A "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition containing the compound that is at least sufficient to produce a therapeutic effect following administration to a subject. Thus, a "therapeutically effective amount" or "therapeutically effective dose" is the amount necessary to treat, prevent, cure, ameliorate, block, or partially block a symptom of the disease or disorder.

[0055] The "therapeutically effective amount" of the lentiviral vector particle of the present invention preferably results in a reduction in the hepatitis B viral load, a reduction in the severity of hepatitis B symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of suffering, injury or disability caused by the disease. For example, for the treatment of hepatitis B infection, a "therapeutically effective amount" preferably reduces the hepatitis B viral load by at least about 10%, preferably at least about 20%, more preferably at least about 30%, compared to a subject not receiving treatment. More preferably, it reduces the hepatitis B viral load by at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, more preferably at least about 100%. Alternatively, the "therapeutically effective amount" preferably reduces HBsAg levels in a subject by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95%, more preferably at least about 100%.

[0056] In some specific embodiments, the administered dose of the lentiviral vector particles of the invention is about 1 x 10 per dose. 7 TU / ml ~ approx. 1 x 10 8 TU, for example, about 1 x 10 7 TU, approx. 2×10 7 TU, approx. 3×10 7 TU, approx. 4×10 7 The lentiviral vector particles may comprise approximately 5×10 7 TU, approx. 6×10 7 TU, approx. 7×10 7 TU, approx. 8×10 7 TU, approx. 9×10 7 TU, approx. 10×10 7 TU, preferably about 5 x 10 7TU. TU (transducing unit) refers to the number of biologically active (e.g., capable of infecting and entering a target cell) viral particles.

[0057] The lentiviral vector particles or pharmaceutical compositions of the present invention can be administered through one or more routes of administration using one or more methods known in the art. Those skilled in the art will appreciate that the route and / or mode of administration will vary according to the desired results. Suitable routes of administration include, but are not limited to, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, etc. In some embodiments, the lentiviral vector particles or pharmaceutical compositions of the present invention are administered by intramuscular and / or intravenous injection.

[0058] Exemplary treatment regimens of the lentiviral vector particles (also referred to as preparations of lentiviral vector particles) or pharmaceutical compositions of the present invention can be once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, once every three months to once every six months, or the initial dosing interval is slightly shorter (e.g., once a week to once every three weeks), and the subsequent dosing interval is increased (e.g., once a month to once every three to six months). The lentiviral vector or pharmaceutical composition of the present invention can be administered once, twice, or more times. In some specific embodiments, the lentiviral vector particles or pharmaceutical compositions of the present invention are administered twice, for example, once in the first week and once again in the second week.

[0059] 3. Methods for constructing and generating LV For the construction of recombinant lentiviral vectors of the present invention expressing core antigen (HBcAg), PreS1 antigen (PreS1), and / or large S antigen (LargeS), the sequences of HBcAg, PreS1, and large S derived from Hepatitis B virus strain FMC#97 were downloaded from GenBank and codon-optimized. The mammalian codon-optimized sequences encoding HBcAg, PreS1, and large S were cloned into the BamHI and XhoI restriction enzyme sites of the FLAP-SP1b2m-WPREm plasmid to generate pFLAP-SP1b2m-transgene-WPREm. The plasmids were generated using the maxiprep kit (Macherey-Nagel, Duren, Germany). The sequences were confirmed by double-stranded sequencing.

[0060] Lentiviral particles were generated by transient calcium phosphate co-transfection of HEK293T cells. Briefly, HEK293T cells were co-transfected with plasmid vector pFLAP-SP1b2m-transgene-WPREm, VSV-G envelope of Indiana serotype or New Jersey plasmid, and packaged pSD-GP-niNDK(D64V) plasmid. The viral particles were harvested 48 hours after transfection and stored at -80°C. Viral titers were determined by transduction of HEK293T cells with aphidicolin followed by qPCR.

[0061] In another aspect, the invention provides a virus production method for producing lentiviral vector particles comprising a lentiviral vector of the invention, a suitable packaging vector, a suitable packaging vector, and / or a suitable host cell, such as a 293 cell. Reagents may also include cell transfection reagents.

[0062] Working Example

[0063] Example 1. Construction of a candidate hepatitis B therapeutic lentiviral vaccine 1.1. Genotyping and serotyping of hepatitis B During reproduction, hepatitis B virus can undergo mutations in the genome nucleotide sequence of the hepatitis B virus, which can sometimes lead to changes in the biological characteristics of the virus. The nucleotide differences between different genotypes of HBV can be used to trace the route of HBV transmission, identify the source of infection, and determine the relationship of transmission and pathogenesis. At present, there are 10 genotypes of HBV identified, and the geographic distribution of the 10 genotypes has been revealed. Among the genotypes, genotype A is mainly distributed in Northwest Europe and Central Africa, genotypes B and C are common in Asia, genotype D is the dominant genotype in the Mediterranean region and the Middle East to India, and genotype E is mainly found in West Africa. Types F and H are more common in South America, and genotype G may originate from Central America. In most parts of China, patients infected with HBV are mainly genotypes B and C, accounting for more than 95%, while reports of genotypes A and D are only found in some ethnic minority areas. The geographical distribution characteristics of HBV genotypes in China are also quite different. The C genotype is more than 90% in most regions, and is mainly prevalent in the north, while the B genotype is prevalent in the south. Research has shown that compared with genotype B, genotype C infection has a higher risk of causing more serious liver diseases, such as cirrhosis and liver cancer.

[0064] HBV can be divided into 9 serotypes: ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq+ and adrq-. The serotypes only reflect some amino acid differences in the envelope protein, and do not truly reflect the phylogenetic relationship of the virus; a single nucleic acid change in gene sequence can change the serotype, and thus the serotypes do not truly reflect the differences of HBV in gene sequence. Research has found that there is no strict correspondence between serotypes and genotypes. Different serotypes can belong to the same genotype, and the same serotype can be distributed into different genotypes (see Table 1). In China, the serotypes are mostly adw and adr subtypes.

[0065] [Table 1]

[0066] 1.2. Antigen design and codon optimization Three immunogens were selected as research targets: core antigen (HBcAg), PreS1 antigen (PreS1 antigen) and large S antigen (large S antigen, including PreS1, PreS2 and surface antigen HBsAg). Since the main genotypes of HBV in China are B and C types, HBV strain 536207 (GenBank: AY220698.1) of genotype B, serotype adw, and HBV strain FMU#14 (GenBank: AF411408.1) of genotype C, serotype adr, their full-length sequences are: preS1 coding gene at position 2848-3205, a total of 3215 bases; large S coding gene at position 2848-835, a total of 360 bases; and core coding gene at position 1901-2452, a total of 1203 bases. According to the HBsAg amino acid sequence alignment of the genotype C HBV strains, it was found that the 204th amino acid is mostly Ser, and the 204th amino acid of this virus strain is Arg, so that the regulated codon AGA is AGC (highlighted in the sequence). In addition, due to the high homology of the core gene, there are only four amino acid differences between different virus strains. After comparing the amino acid sequences of the core protein encoded by the B and C virus strains, the 5th, 83rd, 87th, and 97th amino acids were selected. The most frequently occurring amino acid coding sequences. The specific coding sequences of each antigen obtained are as follows:

[0067] HBV genotype B preS1 coding sequence (SEQ ID NO:5) ATGGGAGGTTGGTCTTCCAAACCTCGAAAAGGCATGGGGACAAATCTTTCTGTCCCCAATCCCCTGGGATTCTTCCCCGATCATCAGTTGGACCCTGCATTCAAAGCCAACTCAGAAAATCCAGATTGGGACCTCAACCCGCACAAGGACAACTGGCCGGACGCCAACAAGGTGGGAGTGGGAGCATTCGGGCCAGGGTTCACCCCTCCCCATGGGGGACTGTTGGGGTGGAGCCCTCAGGCTCAGGGCCTACTCACAACTGTGCCAGCAGCTCCTCCTCCTGCCTCCACCAATCGGCAGTTAGGAAGGCAGCCTACTCCCTTATCTCCACCTCTAAGGGACACTCATCCTCAGGCCTGA

[0068] HBV genotype B large S (large S) coding sequence (SEQ ID NO: 11)

[0069] HBV genotype B preS1 coding sequence (SEQ ID NO:8) ATGGGAGGTTGGTCTTCCAAACCTCGAAAAGGCATGGGGACGAATCTTTCTGTTCCCAATCCTTGGGATTCTTTCCCGATCACCAGTTGGACCCTGCGTTCGGAGCCAACTCAAACAATCCAGATTGGGACTTCAACCCCAACAAGGATCACTGGCCAGAGGCAAATCAGGTAGGAGCG GGAGCATTCGGGCCAGGGTTCACCCCACCACACGGCGGTCTTTTGGGGTGGAGCCCTCAGGCTCAAGGCATATTGACAACAGTGCCAGTAGCACCTCCTCTGCCTCCACCAATCGGCAGTCAGGGAGACAGCCTACTCCCATCTCTCCACCTCTAAGAGACAGTCATCCTCAGGCCTGA

[0070] HBV genotype C large S coding sequence (SEQ ID NO: 14)

[0071] HBV Core coding sequence (SEQ ID NO:2) ATGGACATTGACCCGTATAAAGAATTTGGAGCTTCTGTGGAGTTACTCTCTTTTTGCCTTCTGACTTCTTTCCTTCTATTCGAGATCTCCTCGACACCGCCTCTGCTCTGTATCGGGAGGCCTTAGAGTCTCCGGAA CATTGTTCACCTCACCATACGGCACTCAGGCAAGCTATTCTGTGTTGGGGTGAGTTAATGAATCTAGCCACCTGGGTGGGAAGTAATTTGGAAGATCCAGCATCCAGGGAATTAGTAGTCAGCTATGTCAACGTTTAAT ATGGGCCTAAAAAATCAGACAACTATTGTGGTTTCACATTTCCTGTCTTACTTTTGGGAGAGAAACTGTTCTTGAATATTTGGTGTCTTTTGGAGTGTGGATTCGCACTCCTCCCGCATATAGACCGCCAAATGCCCCT ATCTTATCAACACTTCCGGAAACTACTGTTGTTAGACGAAGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAATCTCAATGTTAG

[0072] The above coding sequence has been provided to Suzhou GENEWIZ Company for gene synthesis. Prior to synthesis, the GC content, high level structure, and elimination of restriction enzyme sites in each host are optimized according to human codons according to priority. The optimized sequence is as follows:

[0073] HBV genotype B preS1 codon-optimized sequence (adw-preS1) (SEQ ID NO:6) ATGGGCGGCTGGAGCAGCAAGCCCAGAAAGGGCATGGGCACCAACCTGAGCGTGCCCAACCCCCTGGGCTTCTTCCCCGACCACCAGCTGGACCCCGCCTTCAAGGCCAACAGCGAGAACCCCGACTGGGACCTGAACCCCCACAAGGACAACTGGCCTGACGCCAACAAGGTGGGCGTGGGAGCTTTCGGCCCTGGCTTCACCCCTCCCCATGGAGGACTGCTGGGCTGGAGCCCTCAGGCTCAGGGACTGCTGACCACAGTGCCCGCTGCTCCTCCTCCTGCCAGCACCAACAGGCAGCTGGGCAGACAGCCCACACCCCTGAGCCCTCCTCTGAGAGACACCCACCCCCAGGCCTGA

[0074] Sequence optimized for codons of HBV genotype B large S (adw-LargeS) (SEQ ID NO: 12)

[0075] HBV genotype C preS1 codon-optimized sequence (adr-preS1) (SEQ ID NO: 9) ATGGGCGGATGGTCCTCCAAGCCTAGAAAGGGCATGGGCACCAATCTGTCCGTGCCCAACCCCTGGGCTTTTTCCCCGATCACCAGTTAGATCCTGCCTTCGGCGCCAACAGCAACAACCCTGACTGGGACTTCAACCCCAACAAGGATCACTGGCCCGAGGCCAATCAAGTGGGCGCT GGAGCTTTCGGCCCTGGCTTCACACCTCCCCATGGAGGACTGCTGGGCTGGTCCCCTCAGGCCCAGGGAATTCTCACAACAGTGCCCGTGGCCCCTCCTCCCGCTAGCACCAACAGGCAGAGCGGCAGGCAGCCCACACCTATCAGCCCCCCTCTGAGAGATTCCCCCCCAGGCCTGA

[0076] HBV genotype C large S codon-optimized sequence (adr-LargeS) (SEQ ID NO: 15)

[0077] HBV core codon-optimized sequence (SEQ ID NO:3) ATGGACATCGACCCTTACAAGGAGTTCGGCGCCAGCGTGGAGCTCCTGAGCTTCCTGCCCAGCGACTTCTTTCCCAGCATCAGAGACCTGCTGGACACCGCCACGCCCTGTACAGGGAAGCCCTGGAGAGCCCCGAG CACTGTAGCCCTCACCACACCGCCCTCAGACAGGCCATCCTGTGCTGGGGCGAGCTGATGAACCTGGCCACCTGGGTGGGAAGCAACCTGGAAGACCCCGCCTCCAGGGAGCTGGTGGTGTCCTACGTGAACGTGAAC ATGGGCCTGAAGATCAGGCAGCTGCTGTGGTTCCACATCAGCTGCCTGACCTTCGGCAGAGAACCGTGCTGGAGTACCTGGTGAGCTTCGGCGTCTGGATCAGAACCCCTCCTGCCTACAGACCCCCTAACGCCCCC ATCCTGTCCACCCTGCCCGAGACCACAGTGGTGAGGAGGAGAGGCAGGAGCCCCAGAAGGAGGACCCCTAGCCCCAGGAGGAGGAGAAGCCAGTCCCCCAGGAGAAGAAGGTCCCAGTCCAGGGAGAGCCATGCTGA

[0078] The 5' of each sequence was inserted into the BamH I restriction enzyme site GGATCC, and the 3' was inserted into the Xhol restriction enzyme site CTCGAG. After synthesis, the target gene sequences were assembled into the eukaryotic expression vector pcDNA3.1(+). Sequencing verified that the immunogen genes were full-length and correct. The amino acid sequences of the selected antigens are as follows:

[0079] HBV core amino acid sequence (SEQ ID NO:1) MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

[0080] HBV type B preS1 amino acid sequence (SEQ ID NO:4) MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFKANSENPDWDLNPHKDNWPDANKVGVGAFGPGFTPPHGGLLGWSPQAQGLLTTVPAAPPPASTNRQLGRQPTPLSPPLRDTHPQA

[0081] HBV type C preS1 amino acid sequence (SEQ ID NO:7) MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVGAGAFGPGFTPPHGGLLGWSPQAQGILTTVPVAPPPASTNRQSGRQPTTPISPPLRDSHPQA

[0082] HBV type B large S amino acid sequence (SEQ ID NO: 10) MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFKANSENPDWDLNPHKDNWPDANKVGVGAFGPGFTPPHGGLLGWSPQAQGLLTTVPAAPPPASTNRQLGRQPTPLSPPLRDTHPQAMQWNSTTFHQTLQDPRVRALYFPAGGSSSGTVSPAQNTVSAISSILSKTGDPVPNMENIASGLLGPLLVLQAGFFLLTKIL TIPS

[0083] HBV type C large S amino acid sequence (SEQ ID NO: 13) MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVGAGAFGPGFTPPHGGLLGWSPQAQGILTTVPVAPPPASTNRQSGRQPTPISPPLRDSHPQAIQWNSTTFHQALLDPRVRGLYFPAGGSSSGTVNPVPTTASPTSSIFSRTGDPAPNMENTTSGFLGPLLVLQAGFFLLTRIL TIPS

[0084] 1.3. Lentivirus Construction The recombinant plasmid containing HBV type C preS1, large S and core antigen was co-transfected with packaging vector and envelope vector to carry out lentivirus packaging. The plasmid vector structure used is shown in Figure 1. The corresponding serial numbers of the synthesized lentiviruses are JW27 (containing the codon-optimized nucleic acid sequence of HBV core shown in SEQ ID NO:3, which expresses the amino acid sequence shown in SEQ ID NO:1), JW28 (containing the codon-optimized nucleic acid sequence of HBV genotype C preS1 shown in SEQ ID NO:9, which expresses the amino acid sequence shown in SEQ ID NO:7) and JW29 (containing the codon-optimized nucleic acid sequence of HBV genotype C large S shown in SEQ ID NO:15, which expresses the amino acid sequence shown in SEQ ID NO:13). In addition, the control group is a lentivirus with inserted green fluorescent protein GFP, named JW30, and the inserted GFP sequence is as follows:

[0085] GFP coding sequence (SEQ ID NO: 16) ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACT TGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCTGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTG TTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCA GACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAA

[0086] Example 2. Evaluation of efficacy of lentiviral vaccine for the treatment of hepatitis B in the AAV8-1.3HBV mouse model 2.1 Experimental Method Establishment of a mouse HBV infection model C57bl / 6j mice aged 6-8 weeks or weighing about 20 g were used. The mice were purchased from Beijing Weitonglihua Company. After purchase, the mice were adapted to the environment of the animal room for about one week. AAV8-1.3HBV 5.10 10GC (genome copy) / mouse was injected into the tail vein, and the injection volume was 200 μL. The AAV8-1.3HBV used (purchased from Guangzhou Paizhen Biotechnology Co., Ltd.) is genotype D HBV, and the genome sequence of the genotype D HBV is shown in SEQ ID NO:17.

[0087] Immunization with lentiviral vaccine for the treatment of hepatitis B Mice were injected with AAV8-1.3HBV via the tail vein for 4 weeks to establish a persistent infection model. The mice were injected with JW27, JW28, JW29 and JW30 lentiviruses via the hind leg muscles. The injection volume was 5.10 7 TU and the injection volume was 50 μL. JW27 is a lentivirus expressing HBV genotype C core antigen, JW28 is a lentivirus expressing HBV genotype C preS1 antigen, JW29 is a lentivirus expressing HBV genotype C large S antigen (preS1+preS2+HBsAg) antigen, and JW30 is a control lentivirus expressing GFP.

[0088] HBsAg detection 3μL of serum is added to 297μL of PBS to prepare a 100-fold dilution, and the dilution is sent to Jinyu Medical for HBsAg detection. The detection kit is Abbott Hepatitis B Virus Surface Antigen Quantitative Determination Kit (chemiluminescence microparticle immunoassay method), and the detection limit is 5IU / mL.

[0089] Mouse serum HBV-DNA detection The viral nucleic acid quantitative determination kit (one-step method) from Hunan Shengxiang Biomedical is used, and the operation steps are performed according to the instructions.

[0090] Anti-HBs detection 3μL of serum is added to 297μL of PBS to prepare 100-fold dilution, and the dilution is sent to Jinyu Medical for anti-HBs detection. The detection kit is Abbott Hepatitis B Virus Surface Antibody Quantitative Determination Kit (chemiluminescence microparticle immunoassay), and the detection limit is 10IU / mL.

[0091] HE and IHC detection of liver tissue Mice were anesthetized by intraperitoneal injection of chloral hydrate. After the mice were deeply anesthetized, the mouse thoracic cavity was opened with scissors, and 5ml of PBS was injected from the apex of the heart, and the color change of the liver was observed until the liver turned white. A part of the liver tissue was taken in formalin fixative and transferred to Ribiology (Shanghai Ruibaohe Biotechnology Co., Ltd.) for paraffin embedding, and then HE staining and HBcAg+ staining were performed.

[0092] Mouse IFN-gamma Elispot detection Single cell acquisition and stimulation: After cervical dislocation of the mouse, immerse the mouse in 75% alcohol for 5 minutes; open the abdominal cavity of the mouse, remove the spleen, and place the spleen in 5mL of 1640 medium; crush the spleen with gauze, transfer the crushed material to a 15mL centrifuge tube, centrifuge at 600g for 5 minutes, discard the supernatant; resuspend with 3mL of lysis solution and mix thoroughly; after lysis for 3 minutes, add 5mL of 1640 medium containing 10% FCS; centrifuge at 600g for 5 minutes, discard the supernatant; resuspend and resuspend with 1mL of complete medium count; use peptide libraries for stimulation of core, PreS1, PreS2 and HBsAg respectively, the final working concentration of each peptide is 2ug / mL, and PMA+ionomycin stimulation is used as a positive control.

[0093] IFN-gamma Elispot assay of cell fractions: (1) Cells were plated in a 96-well plate at 1 × 10 6Cells are added at a concentration of 100 μL / 100 μL, 100 μL of IL-2 stimulatory peptide is added as needed, the final concentration of each peptide is 2 μg / mL, and incubated at 37° C., 5% carbon dioxide for 6 days, on the third day half of the medium is replaced and the same concentrations of IL-2 and stimulatory peptide are added. (2) Antibody overnight coating of Elispot plates: Dilute anti-IFN-gamma antibody 1:200 in sterile PBS according to the number of wells required for the experiment (as recommended by the instructions) and add 100 μL / well to the Elispot plate and incubate at 4° C. overnight. (3) Remove the Elispot plate that was incubated overnight, discard the liquid in the wells, and add 200 μL of complete medium (wash once, leave for 3 minutes, discard). Add 200 μL of complete medium and seal at room temperature for 2 hours. (4) After closure, discard the culture medium and resuspend the cells of the control group and treatment group in addition to the positive and negative well controls to a final volume of 100-150 μL. After gentle mixing, place in a humidified box and incubate at 37°C in a 5% carbon dioxide incubator for approximately 24 hours. (5) Discard the culture. Add 200 μL of distilled water / well and wash twice, leaving each time for 3-5 minutes, then discard it. Tap the remaining liquid dry on absorbent paper. Add 200 μL / well of PBS + 0.05% Tween and wash three times. (6) Dilute detection antibody conjugated to biotin at 1:250 in PBS+10% NCS, add 100 μL / well, and incubate at room temperature for 2 hours. (7) Discard the liquid and add 200 μL / well of PBS + 0.05% Tween and wash three times, discarding after 1-2 minutes each time. (8) Dilute streptavidin-HRP in PBS+10% NCS at a 1:100 ratio, add 100 μL / well, and incubate at room temperature for 1 hour. (9) Discard the reaction solution, add 200 μL / well of PBS + 0.05% Tween, and wash 4 times. Discard after 1-2 minutes each time. Add 200 μL / well of PBS, wash twice, and discard. (10) Add 1 drop of color-developing substrate per 1 ml of color-developing buffer. After mixing, add 100 μL of it to the well. React at room temperature and avoid light for 5 to 60 minutes. When a clear red spot appears, gently rinse the board with tap water for 5 minutes to stop the reaction. (11) Allow the board to air dry.

[0094] Experimental design The experimental timeline is shown in Figure 2. The experimental schedule is shown in Table 2 below. JW27, JW28 and JW30 were immunized with lentivirus coated with New Jersey VSV-G for the third injection at week 10. Serum was collected weekly for HBsAg, HBeAg and HBV-DNA detection.

[0095] [Table 2] JPEG2024516115000003.jpg200170

[0096] 2.2 Experimental Results HBV mouse model In this experiment, 5E+10 GC (genome copies) of AAV8-1.3HBV virus was injected into 100 mice, 50 males and 50 females, numbered 174-273 (174-223 for males and 224-273 for females) via tail vein. After 2 weeks, 10 mice and 10 females were randomly selected for HBsAg and HBV DNA testing to verify whether the mice were infected with HBV. The results are shown in Table 3 and Table 4 below.

[0097] [Table 3]

[0098] [Table 4]

[0099] The results show that AAV8-1.3HBV successfully caused infection in the selected mice. At 4 weeks, 100 mice were tested for HBsAg and HBV DNA. According to the data of HBsAg (Table 5 below) and HBV DNA (Table 6 below), it can be seen that all 100 mice were successfully infected with HBV.

[0100] [Table 5] JPEG2024516115000007.jpg218170

[0101] [Table 6] JPEG2024516115000009.jpg255162JPEG2024516115000010.jpg128162

[0102] Grouping Due to individual differences, the levels of HBsAg and HBV DNA in mice are not uniform. To reduce the differences between individuals, 25 mice with similar surface antigens and viral parameters were selected from male and female mice and randomly divided into 5 groups, 5 males and 5 females each grouped as shown in Table 7 below.

[0103] [Table 7] JPEG2024516115000012.jpg86170

[0104] Immunization with therapeutic lentiviral vaccines After confirming the establishment of persistent HBV infection model in mice, the five groups were immunized with therapeutic vaccines, and the five groups were intramuscularly injected with LV-JW27, LV-JW28, LV-JW27+28, LV-JW29 and LV-JW30. The vaccine injection dose was 5E+7 TU / bottle, and the volume was 50 μl. Among them, LV-JW27+28 was a combined immunization, and LV-JW27 and LV-JW28 were injected into the left and right leg muscles, respectively. The second immunization boost was performed one week later. Five weeks after the second immunization, the third immunization was performed. The third immunization boost was replaced with lentivirus enveloped with New Jersey VSV-G. The dose was 5E+7 TU / mouse, and the volume was 50 μl. Due to the lack of JW29 New Jersey VSV-G enveloped lentivirus, only four groups, LV-JW27, LV-JW28, LV-JW27+28, and LV-JW30, received the third immunization boost.

[0105] Efficacy of a therapeutic lentiviral vaccine against hepatitis B infection in mice Changes in HBsAg levels in peripheral blood From 4 weeks after the first immunization, peripheral blood was collected weekly for detection of HBsAg and HBV DNA in serum. The most obvious effect was in the JW29 group (Figure 3D). Five male mice showed a sharp decline in HBsAg after the first immunization, and three female mice continued to have a decline in HBsAg after 9 weeks. There were three mice HBsAg that became negative by 19 weeks, five HBsAg that became negative at 27 weeks, and six HBsAg that became negative at 38 weeks, with a negative conversion rate of 60% (Table 8 below). In the JW27 group (Figure 3A), one male mouse died at 7 weeks. After the first immunization, HBsAg for the five male mice decreased and then returned. After 15 weeks, the HBsAg of the 9 mice in the group continued to decline, and at 38 weeks, 4 of the 9 mice achieved HBsAg negativity (Table 8). In the JW28 group (Figure 3B), HBsAg remained fluctuating and relatively stable from the first immunization until 15 weeks. After 15 weeks, HBsAg decreased until the end of the observation period. One mouse became HBsAg negative (Table 8). There is no significant effect overall in the treatment. In the JW27+JW28 combination immunization group (Figure 3C), the HBsAg of the mice in the group remained stable from the first immunization until the third immunization at 10 weeks. After 10 weeks of immunization, the HBsAg of 4 mice began to decline continuously until they became negative. At 27 weeks, 5 mice achieved antigen negativity, and by the end of the 38-week observation, a total of 6 mice achieved HBsAg negativity in the group (Table 8). The JW30 group was the control group (Figure 3E). Except for one mouse that shed HBV by itself, the HBsAg of the remaining 9 mice remained stable during the observation period.

[0106] [Table 8]

[0107] Changes in HBV DNA levels in peripheral blood HBV DNA in peripheral blood of mice in the JW27 group (Figure 4A) gradually increased until 15 weeks after the first immunization and maintained a high level. HBV DNA in four male mice remained at high levels but did not decrease significantly until 38 weeks, while five female mice began to have a decline in HBV DNA after 15 weeks, and three female mice achieved HBV DNA clearance at 38 weeks (Table 9 below). HBV DNA in peripheral blood of mice in the JW27 group (Figure 4B) also gradually increased until 15 weeks after the first immunization and then generally maintained a high level. By 38 weeks, one mouse became negative (Table 9), and the remaining nine mice continued to maintain a high level with no apparent effect. HBV DNA in peripheral blood of the JW27+JW28 combination immunization group (Figure 4C) also remained stable or tended to increase until 15 weeks after the first immunization. After 15 weeks, 8 mice had a significant decrease in HBV DNA, and 2 mice achieved HBV DNA negativity at 38 weeks (Table 9). The peripheral blood HBV DNA of mice in the JW29 group (Figure 4D) fluctuated from the first immunization until 13 weeks, and was generally maintained at a relatively stable level. After 15 weeks, 6 mice had a significant decrease in HBV DNA, which was still present at 23 weeks. Five mice achieved HBV DNA negativity, and 6 mice, including 4 female mice and 2 male mice, had HBV DNA negativity by 38 weeks (Table 9), and these 6 mice also achieved HBsAg negativity and reached a cured state. In the JW30 control group (Figure 4E), except for one mouse that became negative by itself, the HBV DNA of the remaining 9 mice remained stable during the observation period.

[0108] [Table 9]

[0109] Detection of HBV DNA in peripheral blood that excludes anti-HBs Surface antibodies are protective antibodies produced by the body against Hepatitis B virus. The appearance of surface antibodies indicates that the mice have specific immunity against HBV infection and the virus becomes undetectable in serum. Anti-HBs was detected in the serum of peripheral blood of mice in JW27, JW28, JW27+JW28, JW29, and JW30 groups at different time points, and the results are shown in the following table (Table 10 below): the time points of antibody conversion in JW27, JW28, and JW29 groups all correspond to the time points when HBsAg becomes negative.

[0110] [Table 10] JPEG2024516115000016.jpg255156JPEG2024516115000017.jpg19156

[0111] Mouse spleen cell IFN-γ ELISPOT detection Spleen cells from groups JW27, JW28, JW27+JW28, JW29 and JW30 were isolated and added to test wells for overnight culture, and specific antigens genotype B preS1 polypeptide, genotype C preS1 polypeptide, preS2 polypeptide, core polypeptide and HBsAg were added simultaneously.

[0112] The results showed that the JW29 group was the strongest, and the JW27 group, JW28 group, JW27+JW28 group and JW29 group all had immune responses against specific antigens (Figure 5).

[0113] HBcAg immunohistochemistry of liver tissue The HBcAg immunohistochemistry results of individual liver tissues from each group are shown in Figure 6 under a microscope at 200x magnification. Three fields were taken for each to calculate the number of HBcAg positive cells, and the average was taken to obtain the positive rate. The results are shown in Table 11 below.

[0114] [Table 11]

[0115] 2.3 Conclusion In summary, the hepatitis B therapeutic lentiviral vaccine of the present invention, in particular the therapeutic vaccine capable of expressing Large S antigen, has good therapeutic effects on hepatitis B and can achieve HBsAg clearance and HBV DNA clearance.

Claims

1. A lentiviral vector for the treatment of hepatitis B, characterized in that the lentiviral vector comprises a nucleotide sequence encoding at least one hepatitis B viral antigen, the hepatitis B viral antigen being selected from the group consisting of core antigen (HBcAg), PreS1 antigen (PreS1) and large S antigen (LargeS).

2. The lentiviral vector for treating hepatitis B according to claim 1, wherein the encoded hepatitis B viral antigen is large S antigen (LargeS).

3. A lentiviral vector for treating hepatitis B according to claim 1 or 2, wherein the amino acid sequence of the core antigen (HBcAg) is the sequence shown in SEQ ID NO: 1; and / or the amino acid sequence of the PreS1 antigen (PreS1) is the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 7; and / or the amino acid sequence of the large S antigen (LargeS) is the sequence shown in SEQ ID NO: 10 or SEQ ID NO:

13.

4. A lentiviral vector for treating hepatitis B according to claim 1 or 2, wherein the coding nucleotide sequence of the core antigen (HBcAg) is the sequence shown in SEQ ID NO: 3; and / or the coding nucleotide sequence of the PreS1 antigen (PreS1) is the sequence shown in SEQ ID NO: 6 or SEQ ID NO: 9; and / or the coding nucleotide sequence of the large S antigen (LargeS) is the sequence shown in SEQ ID NO: 12 or SEQ ID NO:

15.

5. A method for preparing lentiviral particles for treating hepatitis B, comprising: a) co-transfecting a host cell with the lentiviral vector according to claim 1 or 2, a packaging vector expressing Gag, Rev and / or Pol proteins and an envelope vector expressing an envelope protein, or transfecting a lentiviral vector according to claim 1 or 2 into a host cell capable of expressing an envelope protein and one or more of Gag, Rev and Pol proteins; b) culturing the transfected host cells to package the lentiviral vector into lentiviral vector particles; and c) harvesting the lentiviral vector particles produced in step b). The method comprising the steps of:

6. A preparation of lentiviral particles for the treatment of hepatitis B, the preparation comprising a lentiviral vector according to claim 1 or 2.

7. A preparation of lentiviral particles for the treatment of hepatitis B, wherein the lentiviral particles are prepared by the method of claim 5.

8. A lentiviral vector as described in claim 1 or 2 for the treatment and / or prevention of hepatitis B virus infection or for the treatment and / or prevention of a disease caused by hepatitis B virus infection in a subject in need of such treatment and / or prevention.

9. A preparation of lentiviral particles as described in claim 6 or 7 for the treatment and / or prevention of hepatitis B virus infection or for the treatment and / or prevention of a disease caused by hepatitis B virus infection in a subject in need of such treatment and / or prevention.

10. The lentiviral vector of claim 8, wherein the subject is a mammal.

11. A preparation of lentiviral particles as described in claim 9, wherein the subject is a mammal.

12. A pharmaceutical composition for the treatment and / or prevention of hepatitis B virus infection in a subject in need thereof, or for the treatment and / or prevention of a disease caused by hepatitis B virus infection in a subject in need thereof, the pharmaceutical composition comprising a lentiviral vector according to claim 1 or 2 and a pharma- ceutical acceptable carrier.

13. A pharmaceutical composition for the treatment and / or prevention of hepatitis B virus infection in a subject in need thereof, or for the treatment and / or prevention of a disease caused by hepatitis B virus infection in a subject in need thereof, the pharmaceutical composition comprising a preparation of lentiviral particles as described in claim 6 and a pharma- ceutical acceptable carrier.

14. A pharmaceutical composition for the treatment and / or prevention of hepatitis B virus infection in a subject in need thereof, or for the treatment and / or prevention of a disease caused by hepatitis B virus infection in a subject in need thereof, the pharmaceutical composition comprising a preparation of lentiviral particles as described in claim 7 and a pharma- ceutical acceptable carrier.

15. The pharmaceutical composition of claim 12, wherein the subject is a mammal.

16. The pharmaceutical composition of claim 13, wherein the subject is a mammal.

17. The pharmaceutical composition of claim 14, wherein the subject is a mammal.

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