WO2022133230A1 - Combination therapy for treating hepatitis b virus infection - Google Patents

Combination therapy for treating hepatitis b virus infection Download PDF

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Publication number
WO2022133230A1
WO2022133230A1 PCT/US2021/064060 US2021064060W WO2022133230A1 WO 2022133230 A1 WO2022133230 A1 WO 2022133230A1 US 2021064060 W US2021064060 W US 2021064060W WO 2022133230 A1 WO2022133230 A1 WO 2022133230A1
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seq
hbv
polynucleotide sequence
amino acid
antigen
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PCT/US2021/064060
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French (fr)
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Jason Lee DEHART
Christian James MAINE
Craig Stuart PACE
Heather Lynn Davis
Farah Riad ITANI
George Kukolj
Brett Steven MARRO
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Janssen Pharmaceuticals, Inc.
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Publication of WO2022133230A1 publication Critical patent/WO2022133230A1/en

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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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    • C12N2730/00—Reverse transcribing DNA viruses
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    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present disclosure relates generally to compositions and kits comprising an RNA interference (RNAi) component and an RNA replicon vaccine and their uses for treating hepatitis B virus infection or inhibiting the expression of at least one hepatitis B virus gene.
  • RNAi RNA interference
  • the hepatitis B virus is a hepatotrophic, double-stranded DNA containing virus. Although DNA is the genetic material, the replication cycle involves a reverse transcription step to copy a pregenomic RNA into DNA.
  • Hepatitis B virus is classified as one member of the Hepadnaviruses and belongs to the family of Hepadnaviridae.
  • the primary infection of adult humans with hepatitis B virus causes an acute hepatitis with symptoms of organ inflammation, fever, jaundice and increased liver transaminases in blood. Those patients that are not able to overcome the virus infection suffer a chronic disease progression over many years with increased risk of developing cirrhotic liver or liver cancer. Perinatal transmission from hepatitis B virus-infected mothers to newborns also leads to chronic hepatitis.
  • cccDNA serves as a template for transcription of five major viral mRNAs, which are 3.5, 3.5, 2.4, 2.1 and 0.7 kb long. All mRNAs are 5'-capped and polyadenylated at the 3'-end. There is sequence overlap at the 3'- end between all five mRNAs.
  • One 3.5 kb mRNA serves as template for core protein and polymerase production.
  • the same transcript serves as a pre-genomic replication intermediate and allows the viral polymerase to initiate the reverse transcription into DNA.
  • Core protein is needed for nucleocapsid formation.
  • the other 3.5 kb mRNA encodes pre-core, the secretable e-antigen (HBeAg).
  • HBeAg secretable e-antigen
  • the 2.4 and 2.1 kb mRNAs carry the open reading frames (“ORF”) pre-Sl, pre-S2 and S for expression of viral large, medium and small surface antigen.
  • ORF open reading frames
  • the s-antigen is associated with infectious, complete particles.
  • blood of infected patients also contains non-infectious particles derived from s-antigen alone, free of genomic DNA or polymerase. The function of these particles is not fully understood.
  • the complete and lasting depletion of detectable s-antigen in blood is considered as a reli able indicator for hepatitis B virus clearance.
  • the 0.7 kb mRNA encodes the X protein. This gene product is important for efficient transcription of viral genes and also acts as a transactivator on host gene expression. The latter activity seems to be important for hepatocyte transformation during development of liver cancer.
  • Chronic HBV infection can be classified into five phases: (I) HBeAg-positive chronic infection, (II) HBeAg-positive chronic hepatitis, (III) HBeAg-negative chronic infection, (IV) HBeAg- negative chronic hepatitis and (V) HBsAg-negative phase.
  • HCC cirrhosis and hepatocellular carcinoma
  • Nucleoside analogs as inhibitors of reverse transcriptase activity are typically the first treatment option for many patients.
  • Long term administration of lamivudine, tenofovir, and/or entecavir has been shown to suppress hepatitis B virus replication, sometimes to undetectable levels, with improvement of liver function and reduction of liver inflammation typically seen as the most important benefits.
  • only few patients achieve complete and lasting remission after the end of treatment.
  • the hepatitis B virus develops drug resistance with increasing duration of treatment. This is especially difficult for patients co-infected with hepatitis B and human immunodeficiency virus (HIV). Both viruses are susceptible to nucleoside analogue drugs and may co-develop resistance.
  • HIV human immunodeficiency virus
  • Pegylated interferon-alpha has been used to treat mild to moderate chronic hepatitis B patients.
  • current treatment of chronic hepatitis B has limited efficacy (Erha et al., Gut. 2005 Jul; 54(7): 1009-1013).
  • the Asian genotype B gives very poor response rates.
  • Co-infection with hepatitis D virus (HDV) or human immunodeficiency virus has been shown to render interferon-alpha therapy completely ineffective. Patients with strong liver damage and heavy fibrotic conditions are not qualified for interferon-alpha therapy.
  • hepatitis B virus-specific RNA interference (RN Ai) agents have been previously shown to inhibit expression of HBV gene expression.
  • RN Ai hepatitis B virus-specific RNA interference
  • U.S. Patent Application Publication No. 2013/0005793, to Chin et al., which is incorporated herein by reference in its entirety discloses certain double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of hepatitis B virus gene.
  • dsRNA double-stranded ribonucleic acid
  • HBV Hepatitis B viral
  • a method of treating a Hepatitis B viral (HBV) infection in a subject enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection, decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection, decreasing expression of one or more Hepatitis B Virus (HBV) polypeptide(s), more particularly of one or more polypeptide(s) from HBsAg and HBeAg, in a subject in need thereof, and/or increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with Hepatitis B viral (HBV) infection, wherein the method comprises administering to the subject:
  • RNAi component having:
  • a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
  • a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111;
  • nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
  • IVS internal ribosome entry sequence
  • first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
  • the method further comprises administering to the subject another agent for treating infection caused by hepatitis B virus HBV.
  • the other agent can be a nucleoside analog.
  • the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof.
  • the other agent is a nucleic acid polymer (NAP).
  • NAP nucleic acid polymer
  • the NAP can, for example, be selected from REP2139 or REP2165.
  • REP2139 has a sequence of (A,5'MeC) 20 with each linkage being phosphorothioated and every ribose being 2’0 methylated (which is disclosed as SEQ ID NOTO in WO2016/04525, the content of which is incorporated herein by reference in its entirety).
  • REP2165 has a sequence of (A,5'MeC) 20 with each linkage being phosphorothioated, every rbose being 2’0 methylated except adenosines at positions 11, 21, and 31, where riboses are 2’OH (which is disclosed as SEQ ID NO: 13 in WO2016/04525).
  • the NAP can also be other exemplary nucleic acid polymers, which include, but are not limited to, REP2006, REP2031, REP2055, STOPSTM (S-antigen transport-inhibiting oligonucleotide polymers), and those disclosed in Patent Application Publication Nos. WO200424919; WO201221985; and WO202097342 and U.S. Patent Nos. 7,358,068;
  • the subject has chronic HBV infection.
  • Another general aspect of the application relates to a combination or a kit for use in treating a HBV infection, such as a chronic HBV infection (CHB), with or without viral co-infection, e.g., with or without co-infection with HDV and/or HCV and/or HIV, more particularly with or without co-infection with at least HDV, and/or for treating chronic HDV infection (CHD) in a subject in need thereof, comprising:
  • RNAi component having:
  • a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
  • a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111;
  • a pharmaceutical composition comprising an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3’-end:
  • IVS internal ribosome entry sequence
  • first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
  • the combination or kit is for use in enhancing an immune response, decreasing viral replication, and/or decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptides, more particularly of one or more polypeptide(s) selected from HBsAg and HBeAg, in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection.
  • HBV Hepatitis B Virus
  • the combination or kit further comprises another agent for treating infection caused by hepatitis B virus HBV.
  • the other agent can be a nucleoside analog.
  • the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof.
  • the other agent is a nucleic acid polymer (NAP).
  • NAP nucleic acid polymer
  • the NAP can, for example, be selected from REP2139 or REP2165.
  • REP2139 has a sequence of (A,5'MeC) 20 with each linkage being phosphorothioated and every ribose being 2’0 methylated (which is disclosed as SEQ ID NOTO in WO2016/04525, the content of which is incorporated herein by reference in its entirety).
  • REP2165 has a sequence of (A,5'MeC) 20 with each linkage being phosphorothioated, every rbose being 2’0 methylated except adenosines at positions 11, 21, and 31, where riboses are 2’OH (which is disclosed as SEQ ID NO: 13 in WO2016/04525).
  • the NAP can also be other exemplary nucleic acid polymers, which include, but are not limited to, REP2006, REP2031, REP2055, STOPSTM (S-antigen transport-inhibiting oligonucleotide polymers), and those disclosed in Patent Application Publication Nos. WO200424919; WO201221985; and WO202097342 and U.S. Patent Nos. 7,358,068;
  • RNAi component and a nucleic acid molecule comprising a non-naturally occurring polynucleotide, optionally a nucleoside analog, in the manufacture of a medicament for treating a Hepatitis B viral (HBV) infection in a subject, enhancing an immune response in a subject with a HBV infection, decreasing viral replication in a subject with a HBV infection, decreasing the expression of one or more HBV polypeptide(s), more particularly of one or more polypeptide(s) from HBsAg and HBeAg, in a subject in need thereof, modulating Hepatitis B viral (HBV) capsid assembly or disassembly in a subject with a HBV infection, and/or increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with
  • HBV Hepatitis B viral
  • RNAi component comprises:
  • a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
  • a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111 ; and
  • nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3’-end:
  • IVS internal ribosome entry sequence
  • first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen; and
  • the nucleoside analog is preferably entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof.
  • the treatment comprises a first phase conducted before a second phase, and a) the first phase comprises administering the RNAi component to the subject to thereby decrease the HBsAg to a level low enough to allow recovery of T cell function, preferably to a serum HBsAg level of less than 1000, 100, 10, or 1 lU/mL; and b) the second phase comprises administering the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence to the subject.
  • the second phase does not comprise administering the RNAi component to the subject.
  • the second phase further comprises administering the RNAi component to the subject.
  • the first phase of the treatment can last about 1-24 months, such as 1-12 months, 1-3 months, 4-6 months, 7-9 months, 10-12 months, or any period of time in between.
  • the second phase of the treatment can last about 1-24 months, such as 1-12 months, 4-6 months, 7-9 months, 10-12 months, 13-18 months, 19-24 months, or any period of time in between.
  • the second phase comprises administering the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence and administering the RNAi component to the subject over the same treatment period.
  • the second phase comprises administering the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence and administering the RNAi component to the subject over different treatment periods, wherein the administration of the RNAi component stops first, and the administration of the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence continues, for example, for an additional 1 month, 2-3 months, 4-5 months, 6 or more months, or any period of time in between, until the end of the second phase.
  • the RNAi component is administered subcutaneously or intravenously, preferably subcutaneously, at an amount of about 40-1000 mg per dose, more particularly about 40-250 mg per dose, such as about 100-200 mg per dose, more particularly about 200 mg per dose, and it is administered weekly, every two weeks, every 4 weeks, monthly, every 2 months, or every 3 months, preferably every 4 weeks or monthly.
  • a subject has achieved at least one of the following features a)-e), more particularly more than one of the following features a)-e), more particularly at least features a), b) and c), more particularly all of features a)-d), during or after the treatment with a combination according to an embodiment of the application: a) decreased HBV replication as measured by serum HBV DNA level, preferably undetectable serum HBV DNA level; b) decreased expression of one or more HBV polypeptide(s), preferably decreased expression of HBsAg as measured by serum HBsAg level, preferably undetectable serum HBsAg level; c) enhanced HBV-specific T cell responses; d) loss of HBeAg or serocoversion for HBeAg, if the subject is HBeAg positive before the treatment; and e) seroconversion for HBsAg, preferably, the subject has achieved functional cure after the treatment; more preferably, when administered to a cohort of CHB patients, a greater
  • a combination according to an embodiment of the application is for use in decreasing viral replication, as measured by serum HBV DNA, in the subject with CHB, wherein the subject is not already suppressed by nucleoside/nucleotide analogue therapy.
  • the combination can also be used for decreasing the expression of one or more HBV polypeptides in the subject with CHB, such as HBsAg in serum of the subject.
  • the combination can further be used for bringing about an enhanced HBV-specific T cell response, which can be enhanced in a quantitative and/or qualitative manner, in the subject with CHB.
  • the enhanced T cell response in the subject with CHB can result in one or more of the following: a) greater number of HBV-specific T cells; b) greater breadth of HBV-specific T cells in that more HBV antigens are recognized by the T cells; c) greater function of HBV-specific T cells in that when re-stimulated with HBV antigens (such as HBV peptide pools), the T cells secrete greater number of cytokines, e.g., polyfunctional T cells; d) enhanced clearance of HBV-infected cells, such as cells containing cccDNA, or of non-infected cells that have integrated HBV DNA; and e) functional cure in the subject with CHB through enhancing immune responses, especially HBV-specific T cell responses.
  • a combination according to an embodiment of the application is for use in treating a subject co-infected with CHB and another chronic infection with at least one of: hepatitis D virus (HDV); hepatitis C virus (HCV); or human immunodeficiency virus (HIV).
  • the combination can be used in a method of decreasing the serum levels of HDV RNA in a subject chronically co-infected with both HBV and HDV; a method of normalizing alanine aminotransferase (ALT) level in a subject chronically coinfected with HBV and HDV; or a method of eradicating HDV infection in a subject chronically co-infected with HBV and HDV.
  • the first or the second RNAi agent comprises at least one modified nucleotide or at least one modified intemucleoside linkage. In another variation, substantially all of the nucleotides in the first and the second RNAi agents are modified nucleotides.
  • the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent. In one aspect, the targeting ligand comprises N-acetyl-galactosamine.
  • the targeting ligand is selected from the group consisting of (NAGI 3), (NAG13)s, (NAGI 8), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), and (NAG39)s.
  • NAGI 3 NAG13)s, (NAGI 8), (NAG18)s, (
  • the targeting ligand is (NAG25), (NAG25)s, (NAG31), (NAG31)s, (NAG37), or (NAG37)s.
  • the targeting ligand is conjugated to the sense strand of the first or the second RNAi agent.
  • the targeting ligand is conjugated to the 5’ terminus of the sense stand of the first or the second RNAi agent.
  • the first and the second RNAi agents further comprise a targeting ligand that is conjugated to each of the first and the second RNAi agents.
  • the targeting ligand comprises N-acetyl-galactosamine.
  • the targeting ligand is selected from the group consisting of (NAGI 3), (NAG13)s, (NAGI 8), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), and (NAG39)s.
  • NAGI 3 NAG13)s, (NAGI 8), (NAG18)s, (
  • the targeting ligand is (NAG25), (NAG25)s, (NAG31), (NAG31)s, (NAG37), or (NAG37)s.
  • the targeting ligand is conjugated to the sense strand of each of the first and second RNAi agents.
  • the targeting ligand is conjugated to the 5’ terminus of the sense stand of each of the first and the second RNAi agents.
  • the first and the second RNAi agents independently comprise a duplex selected from the group consisting of: an antisense strand comprising SEQ ID NO:93 and a sense strand comprising SEQ ID NO: 102; an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 103; an antisense strand comprising SEQ ID NO: 95 and a sense strand comprising SEQ ID NO: 103; an antisense strand comprising SEQ ID NO:96 and a sense strand comprising SEQ ID NO: 104; an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 108; an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 109; an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 105; and an antisense strand comprising SEQ ID NO: 100 and
  • the first and the second RNAi agents are each independently conjugated to a targeting ligand comprising N-acetyl- galactosamine, and the first and the second RNAi agents independently comprise a duplex selected from the group consisting of: an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 103; an antisense strand comprising SEQ ID NO:96 and a sense strand comprising SEQ ID NO: 104; an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 108; an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 105; and an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 110.
  • the ratio of the first RNAi agent to the second RNAi agent by weight is in the range of about 1 :2 to about 5: 1. In another variation, the ratio of the first RNAi agent to the second RNAi agent by weight is about 2: 1. In still another variation, the molar ratio of the first RNAi agent to the second RNAi agent is in the range of about 1 :2 to about 5: 1. In another variation, the molar ratio of the first RNAi agent to the second RNAi agent is about 2: 1.
  • the first and the second RNAi agents are each independently conjugated to (NAG37)s, the first RNAi agent comprises an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 103, and the second RNAi agent comprises an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 108.
  • one of the first or second HBV antigens is an HBV core or an HBV pol antigen.
  • the non-naturally occurring polynucleotide sequence further comprises, ordered from the 5’ - to 3 ’-end:
  • the non-naturally occurring polynucleotide sequence further comprises, ordered from the 5’ - to 3 ’-end:
  • each of the first, second, third and fourth HBV antigens is different from each other.
  • each of the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
  • a first HBV Pre-S 1 antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: i;
  • a second HBV Pre-S 1 antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 3;
  • an HBV PreS2.S antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 5;
  • an HBV core antigen comprising, preferably consisting of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7; and
  • an HBV polymerase antigen comprising, preferably consisting of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 9, preferably, each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a signal peptide, and the HBV PreS2.S antigen comprises an internal signal peptide.
  • the HBV core antigen comprises, preferably consists of, an amino acid sequence that is at least 98% identical to at least one of SEQ ID NOs: 84, 85, or 86, such as at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 84, 85, or 86.
  • the last five C-terminal amino acids of the HBV core antigen comprise a VVR amino acid sequence, more particularly a VVRR (SEQ ID NO: 91) amino acid sequence, more particularly a VVRRR (SEQ ID NO: 92) amino acid sequence.
  • each of the HBV surface antigen, the HBV core antigen and the HBV pol antigen comprises:
  • each of the HBV surface antigens, the HBV core antigen and the HBV pol antigen comprises one or more epitopes for HLA- A*ll:01.
  • each of the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
  • the HBV core antigen consists of the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86;
  • the HBV pol antigen consisting of the amino acid sequence of SEQ ID NO: 9, preferably, each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a signal peptide, such as the signal peptide comprising the amino acid sequence of SEQ ID NO: 77.
  • each of the polynucleotide sequences encoding the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
  • a polynucleotide sequence encoding the first HBV Pre-S 1 antigen having a sequence that is at least 90% identical to SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2;
  • a polynucleotide sequence encoding the second HBV Pre-S 1 antigen having a sequence that is at least 90% identical to SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4;
  • a polynucleotide sequence encoding the HBV PreS2.S antigen having a sequence that is at least 90% identical to SEQ ID NO: 6, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 6;
  • a polynucleotide sequence encoding the HBV core antigen having a sequence that is at least 90% identical to SEQ ID NO: 8, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 8; and
  • the polynucleotide sequence encoding the HBV pol antigen having a sequence that is at least 90% identical to SEQ ID NO: 10, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 10, preferably, the polynucleotide sequence encoding each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a polynucleotide sequence encoding a signal peptide, and the HBV PreS2.S antigen comprises an internal signal peptide.
  • each of the polynucleotide sequences encoding the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
  • each of the first, second and third autoprotease peptides independently comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2 A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof.
  • each of the first, second and third autoprotease peptides comprises the peptide sequence of P2A, such as a P2A sequence of SEQ ID NO: 11.
  • each of the first, second and third IRES is derived from encephalomyocarditis virus (EMCV) or Enterovirus 71 (EV71).
  • EMCV encephalomyocarditis virus
  • EV71 Enterovirus 71
  • each of the first, second and third IRES comprises the polynucleotide sequence of SEQ ID NO: 13 or 14.
  • the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence comprises, ordered from the 5’- to 3’-end:
  • polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3 a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5;
  • (6) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
  • the nucleic acid molecule comprises the non-naturally occurring polynucleotide sequence of any one of SEQ ID NOs: 15 to 54.
  • the application relates to a vector comprising a non-naturally occurring polynucleotide sequence of the application.
  • the vector is a DNA plasmid.
  • the vector is a DNA viral vector or an RNA viral vector.
  • RNA replicon comprising, ordered from the 5’- to 3’-end:
  • RNA virus (1) a 5’ untranslated region (5’-UTR) required for nonstructural protein-mediated amplification of an RNA virus;
  • RNA virus a 3’ untranslated region (3’-UTR) required for nonstructural protein-mediated amplification of the RNA virus.
  • RNA replicon comprising, ordered from the 5’- to 3’-end,
  • (9) optionally, a poly adenosine sequence.
  • the DLP motif is from a virus species selected from the group consisting of Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MTDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (U AV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus
  • EEEV Eastern equine encephalitis virus
  • the fourth autoprotease peptide is selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof.
  • the fourth autoprotease peptide comprises the peptide sequence of P2A.
  • RNA replicon comprising, ordered from the 5’- to 3’-end,
  • polynucleotide sequences encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4 having the nucleic acid sequences of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively,
  • the polynucleotide sequence encoding the P2A sequence comprises SEQ ID NO: 12
  • the non-naturally occurring polynucleotide sequence comprises the polynucleotide sequence of any one of SEQ ID NOs: 15 to 54
  • the RNA replicon further comprises a poly adenosine sequence at the 3 ’-end of the replicon.
  • the poly adenosine sequence has the sequence of SEQ ID NO: 64.
  • the application relates to an RNA replicon comprising the polynucleotide sequence of any one of SEQ ID NOs: 65 to 72.
  • the application relates to a nucleic acid molecule comprising a DNA sequence encoding an RNA replicon of the application.
  • the nucleic acid further comprises a T7 promoter operably linked to the 5 ’-end of the DNA sequence. More preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO: 73.
  • the application relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a nucleic acid molecule, a vector, or an RNA replicon of the application, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises: (1) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9; or (2) a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleo
  • the application relates to a method for vaccinating a subject against HBV, the method comprising administering to the subject a combination of pharmaceutical compositions of the application.
  • the method comprises administering to the subject a first composition comprising an RNAi component, a second composition comprising a nucleic acid molecule comprising a non- naturrally occurring polynucleotide sequence encoding one or more HBV antigens, and a third composition comprising a nucleic acid molecule encoding at least one identical HBV antigen, wherein the second and third composition comprise a prime-boost regimen.
  • the prime-boost regimen comprises a priming composition comprising an RNA replicon of the application and a boosting composition comprising a vector which is not an RNA replicon and which encodes at least one identical HBV epitope, preferably, at least one identical HBV antigen, as the priming composition.
  • the boosting composition comprises a Modified Vaccinia Ankara (MV A) vector, an adenovirus vector, or a plasmid vector.
  • MV A Modified Vaccinia Ankara
  • the prime-boost regimen comprises a boosting composition comprising an RNA replicon of the application and a priming composition comprising a vector which is not an RNA replicon and which encodes at least one identical HBV epitope, such as at least one identical HLA epitope, preferably, at least one identical HBV antigen, as the boosting composition.
  • the priming composition comprises a Modified Vaccinia Ankara (MV A) vector, an adenovirus vector, or a plasmid vector.
  • MV A Modified Vaccinia Ankara
  • the application relates to an isolated host cell comprising a nucleic acid molecule, a vector, or an RNA replicon of the application.
  • the application relates to a method of producing an RNA replicon, comprising transcribing a nucleic acid of the application, in vivo or in vitro.
  • FIGs. 1A-1C show the experimental design for the RNAi and RNA replicon combination therapy.
  • FIG. 1 A shows the groups to be tested with the combination therapy.
  • FIG. IB shows provides subject and readout information for the combination therapy experiment.
  • FIG. 1C shows a schematic of the experimental design.
  • FIGs. 2A-2B show schematics of antigen designs (FIG. 2 A) and bicistronic and tetracistronic vaccine designs (FIG. 2B).
  • the antigen designs (FIG. 2A) include Pol with inactivating point mutations, truncated Core with C terminal deletion (SEQ ID NO:76), PreS2.S, and PreSl showing the cystatin S Signal peptide (SEQ ID NO:77).
  • FIGS. 3A-3F show graphs of HBsAg serum concentrations (geometric mean ⁇ 95% confidence intervals) in mice that were administered HBV siRNA + HBV tetra-3 replicon (Group 1) compared to mice that received HBV siRNA + control replicon (Group 4) at wk 0 (immediately prior to first siRNA dose; FIG. 3A & 3D); wk 6 (immediately prior to the first replicon dose; FIG. 3B & 3E) and wk 14 (2 wks after the last replicon dose; FIG. 3C & 3F).
  • FIGs. 3A-3C show serum HBsAg concentrations for mice from the efficacy and immunogenicity cohorts combined.
  • FIGs. 3D-3F show HBsAg serum concentrations in mice in the efficacy (E) and immunogenicity (I) cohorts separately.
  • FIGs. 4A-4B show graphs demonstrating HBsAg serum concentration in mice that were administered HBV siRNA + HBV tetra-3 replicon compared to HBV siRNA + control replicon over time.
  • FIG. 4A shows absolute HBsAg concentrations.
  • FIG. 4B shows delta HBsAg concentrations normalized to wk 0 HBsAg concentrations.
  • Asterisks indicate statistically significant difference (P ⁇ 0.05) calculated by Mann- Whitney test comparing HBV siRNA + HBV replicon and HBV siRNA + rFF replicon.
  • FIG. 5 shows a graph of HBV-specific IFNg T cell responses in mice treated with HBV siRNA + HBV tetra-3 replicon or AAT siRNA + HBV tetra-3 -replicon as compared to mice that received the siRNA vehicle + replicon. Statistical significance was determined by Mann- Whitney test).
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”
  • any numerical value such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL.
  • a concentration range of 1 mg/mL to 10 mg/mL includes 0.9 mg/mL to 11 mg/mL.
  • the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
  • An “epitope” as used herein is a set of amino acid residues that form a site recognized by an immunoglobulin, T cell receptor or human leukocyte antigen (HLA) molecule.
  • the HLA proteins are encoded by clusters of genes that form a region located on chromosome 6 known as the Major Histocompatibility Complex (MHC), in recognition of the important role of the proteins encoded by the MHC loci in graft rejection. Accordingly, the HLA proteins are also referred to as MHC proteins.
  • HLA or MHC proteins are cell surface glycoproteins that bind peptides at intracellular locations and deliver them to the cell surface, where the combined ligand is recognized by a T cell. Class I MHC proteins are found on virtually all of the nucleated cells of the body.
  • the class I MHC proteins bind peptides present in the cytosol and form peptide-MHC protein complexes that are presented at the cell surface, where they are recognized by cytotoxic CD8+ T cells.
  • Class II MHC proteins are usually found only on antigen-presenting cells such as B lymphocytes, macrophages, and dendritic cells.
  • Each MHC Class I receptor consists of a variable a chain and a relatively conserved P2-microglobulin chain.
  • Three different, highly polymorphic class I a chain genes have been identified. These are called HLA- A, HLA-B, and HLA-C. Variations in the a chain accounts for all of the different class I MHC genes in the population.
  • the half maximal effective concentration (EC 50 ) is intended in accordance with its general meaning in the field. It may more particularly refer to the concentration of a compound which induces a response halfway between the baseline and maximum, typically after a specified exposure time.
  • the EC 50 value is commonly used as a measure of a compound’s potency, with a lower value generally indicating a higher potency.
  • a “non-naturally occurring” nucleic acid or polypeptide refers to a nucleic acid or polypeptide that does not occur in nature.
  • a “non-naturally occurring” nucleic acid or polypeptide can be synthesized, treated, fabricated, and/or otherwise manipulated in a laboratory and/or manufacturing setting.
  • a non-naturally occurring nucleic acid or polypeptide can comprise a naturally-occurring nucleic acid or polypeptide that is treated, processed, or manipulated to exhibit properties that were not present in the naturally-occurring nucleic acid or polypeptide, prior to treatment.
  • a “non-naturally occurring” nucleic acid or polypeptide can be a nucleic acid or polypeptide isolated or separated from the natural source in which it was discovered, and it lacks covalent bonds to sequences with which it was associated in the natural source.
  • a “non-naturally occurring” nucleic acid or polypeptide can be made recombinantly or via other methods, such as chemical synthesis.
  • the term “priming composition,” “priming immunization,” or “prime immunization” refers to primary antigen stimulation by using a first composition of the invention.
  • the term “priming” or “potentiating” an immune response refers to a first immunization using an antigen which induces an immune response to the desired antigen and recalls a higher level of immune response to the desired antigen upon subsequent re-immunization with the same antigen.
  • the term “boosting composition,” “boosting immunization,” or “boost immunization” refers to an additional immunization administered to, or effective in, a mammal after the primary immunization.
  • the term “boosting” an immune response refers to the administration of a composition delivering the same antigen as encoded in the priming immunization.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions.
  • pharmaceutically acceptable salt refers to a salt of any of the compounds or compositions herein which are known to be non-toxic and are commonly used in the pharmaceutical literature.
  • the pharmaceutically acceptable salt of a compound retains the biological effectiveness of the compounds described herein and are not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts can be found in Berge et al., Pharmaceutical Salts, J. Pharmaceutical Sciences, January 1977, 66(1), 1-19.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 2 -hydroxy ethylsulfonic acid, p- toluenesulfonic acid, stearic acid and salicylic acid.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines; substituted amines including naturally occurring substituted amines; cyclic amines; and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, tri ethyl amine, tripropylamine, and ethanolamine.
  • the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts.
  • the pharmaceutically acceptable salts according to the invention may be prepared from the parent compound containing acidic or basic group through conventional chemical procedures.
  • such salts can be prepared through the reaction of the compounds in the form of free acid or base with stoichiometric appropriate base or acid in water, organic solvent or the mixture thereof.
  • nonaqueous medium like ether, ethyl acetate, ethanol, isopropanol, acetonitrile etc. are preferable.
  • the terms “patient” and “subject” refer to a human or a great ape (e.g., chimpanzee, orangutan, bonobo, gorilla).
  • the patient or subject is a human that has been or will be the object of treatment, observation or experiment.
  • the combinations, compositions, and methods described herein can be useful in both human therapy and veterinary applications for species that can be chronically infected by HBV.
  • the subject has an HBV infection, more particularly a chronic HBV infection (CHB).
  • CHB chronic HBV infection
  • the subject can have a CHB, with or without viral co-infection.
  • a “viral co-infection” refers to an infection with at least two types of virus.
  • a “viral co-infection” can be an infection with at least two types of virus simultaneously.
  • a “viral coinfection” can also be a superinfection, wherein an infection with one or more types of virus is in addition to a pre-existing infection with one or more other types of virus.
  • the subject has an HBV-HDV co-infection, an HBV -HIV co-infection, or an HBV-HCV co-infection.
  • the subject has a CHB-HDV co-infection, a CHB-HIV co-infection, or a CHB-HCV co-infection. More preferably, the subject has a viral coinfection with CHB and chronic HDV.
  • hepatitis B virus or “HBV” refers to a specific virus of the hepadnaviridae family.
  • HBV is a small (e.g., 3.2 kb) hepatotropic DNA virus that encodes four open reading frames and seven proteins.
  • the seven proteins encoded by HBV include small (S), middle (M), and large (L) surface antigens (HBsAg) or envelope (Env) proteins, pre-core protein, core protein, viral polymerase (Pol), and HBx protein.
  • HBV expresses three surface antigens, or envelope proteins, L, M, and S, with S being the smallest and L being the largest, and M in the middle.
  • the L protein is encoded by the Pre-S 1-Pre-S2-S domains, the M protein the Pre-S2-S domains and the S protein only the S domain.
  • Core protein is the subunit of the viral nucleocapsid. Pol is needed for synthesis of viral DNA (reverse transcriptase, RNaseH, and primer), which takes place in nucleocapsids localized to the cytoplasm of infected hepatocytes.
  • PreCore is the core protein with an N-terminal signal peptide and is proteolytically processed at its N and C termini before secretion from infected cells, as the so-called hepatitis B e-antigen (HBeAg).
  • HBx protein is required for efficient transcription of covalently closed circular DNA (cccDNA).
  • HBx is not a viral structural protein. All viral proteins of HBV have their own mRNA except for core and polymerase, which share an mRNA. With the exception of the protein pre-Core, none of the HBV viral proteins are subject to post-translational proteolytic processing.
  • the HBV virion contains a viral envelope, nucleocapsid, and single copy of the partially double-stranded DNA genome.
  • the nucleocapsid comprises 120 dimers of core protein and is covered by a lipid membrane embedded with the S, M, and L viral envelope or surface antigen proteins.
  • the virus is uncoated and the capsidcontaining relaxed circular DNA (rcDNA) with covalently bound viral polymerase migrates to the nucleus.
  • rcDNA relaxed circular DNA
  • phosphorylation of the core protein induces structural changes, exposing a nuclear localization signal enabling interaction of the capsid with so- called importins.
  • rcDNA covalently closed circular DNA genome (further converted into a minichromosome with the addition of histones and other host factors) from which overlapping transcripts encode for HBeAg, HBsAg, Core protein, viral polymerase and HBx protein.
  • HBV pre-genomic RNA
  • pgRNA pre-genomic RNA
  • HBV HBV is divided into four main serotypes (adr, adw, ayr, ayw) based on antigenic epitopes present on the envelope proteins, and into eight genotypes (A, B, C, D, E, F, G, and H) based on the sequence of the viral genome.
  • genotypes are distributed over different geographic regions. For example, the most prevalent genotypes in Asia are genotypes B and C. Genotype D is dominant in Africa, the Middle East, and India, whereas genotype A is widespread in Northern Europe, sub-Saharan Africa, and West Africa.
  • a patient or subject having a “chronic hepatitis B virus (CHB) infection,” “CHB,” “CHB infection,” or “CHB virus infection” refers to the ordinary meaning in the art, more particularly to a patient or subject chronically infected with HBV and having detectable HBsAg (with or without HBeAg) in the blood for six or more months after HBV detection.
  • CHB chronic hepatitis B virus
  • CHB infection can be classified into four phases which typically, but not always, progress from one to the next: (I) HBeAg-positive chronic infection (previously known as immune tolerant), (II) HBeAg-positive chronic hepatitis (previously known as immune active), (III) HBeAg-negative chronic infection (previously known as inactive carrier), and (IV) HBeAg-negative chronic hepatitis (previously known as reactivation).
  • the different phases of chronic HBV infection can also be characterized by differences in viral load, liver enzyme levels (necroinflammatory activity), HBeAg, or HBsAg load or presence of antibodies to these antigens.
  • cccDNA levels in untreated subjects stay relatively constant at approximately 10 to 50 copies per cell but may be as low as 1 to 2 copies per cell when suppressed by nucleos(t)ide analogue therapy, even though viremia can vary considerably.
  • the persistence of the cccDNA species leads to chronicity.
  • a chronic HBV infection can be characterized the laboratory criteria published by the Centers for Disease Control and Prevention (CDC), such as: (i) negative for IgM antibodies to hepatitis B core antigen (IgM anti-HBc) and positive for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), or nucleic acid test for hepatitis B virus DNA, or (ii) positive for HBsAg or nucleic acid test for HBV DNA, or positive for HBeAg two times at least 6 months apart.
  • CDC Centers for Disease Control and Prevention
  • therapeutically effective amount refers to that amount of each component of the combination disclosed and/or described herein that is sufficient to affect treatment, as defined herein, when administered to a subject in need of such treatment.
  • the therapeutically effective amount will vary depending upon, for example, the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the particular combination and/or composition, the dosing regimen to be followed, timing of administration, the manner of administration, all of which can readily be determined by one of ordinary skill in the art.
  • the therapeutically effective amount can be ascertained experimentally, for example by assaying blood concentration of each component of the combination, or theoretically, by calculating bioavailability by one of ordinary skill in the art in view of the present disclosure.
  • a therapeutically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an HBV infection or a symptom associated therewith; (ii) reduce the duration of an HBV infection or symptom associated therewith; (iii) prevent the progression of an HBV infection or symptom associated therewith; (iv) cause regression of an HBV infection or symptom associated therewith; (v) prevent the development or onset of an HBV infection, or symptom associated therewith; (vi) treat or retreat a chronic HBV infection that recurs due to relapse after functional cure is achieved or symptom associated therewith; (vii) prevent the recurrence of an HBV infection or symptom associated therewith; (viii) reduce hospitalization of a subject having an HBV infection; (ix) reduce hospitalization length of a subject having an HBV infection; (x) increase the survival of a subject with an HBV
  • a therapeutically effective amount can also be an amount of the combination and/or composition sufficient to reduce HBsAg levels consistent with evolution to clinical seroconversion; achieve sustained HBsAg clearance associated with reduction of infected hepatocytes by a subject’s immune system; induce HBV-antigen specific activated T-cell populations; and/or achieve persistent loss of HBsAg during or after treatment that then preferably persists at 6 months or more after the end of treatment, most preferably for life.
  • Examples of a target index include a serum HBsAg level that is below a threshold of, e.g., 100 lU/mL of HBsAg and/or HBV-specific CD8 T cells responses of greater numbers or of greater polyfunctionality than, e.g., at the beginning of the treatment.
  • target indexes include, but are not limited to, serum HBV DNA levels lower than the lower limit of quantification (LLoQ) or lower than 20 lU/mL, more particularly lower than 15 lU/mL, more particularly lower than 10 lU/mL; serum ALT concentration lower than 3 times the upper normal limit, or lower than 129 U/L if the subject is a male subject, or lower than 108 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 120 U/L if the subject is a male subject or lower than 105 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 90 U/L if the subject is a male subject or lower than 57 U/L if the subject is a female subject; HBeAg-negative serum; serum HBsAg level of 1000 lU/ml or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly of 10 l
  • inducing an immune response when used with reference to the methods described herein encompasses providing a therapeutic immunity for treating against a pathogenic agent, e.g., HBV.
  • inducing an immune response means producing an immunity in a subject in need thereof, e.g., to provide a therapeutic effect against a disease, such as HBV infection.
  • inducing an immune response refers to causing or improving cellular immunity, e.g., HBV-specific CD4+ and CD8+ T cell responses. In certain embodiments, this T cell response can bring about functional cure for the treated patient who has CHB.
  • inducing an immune response refers to causing or improving a humoral immune response against HBV infection. In certain embodiments, “inducing an immune response” refers to causing or improving a cellular and a humoral immune response against HBV infection.
  • the term “protective immunity” or “protective immune response” means that the vaccinated subject can control an infection with the pathogenic agent against which the vaccination was done. Usually, the subject having developed a “protective immune response” develops only mild to moderate clinical symptoms or no symptoms at all. Usually, a subject having a “protective immune response” or “protective immunity” against a certain agent will not die as a result of the infection with said agent.
  • the term “functional cure” or “FC” refers to a state of a subject who had CHB where the serum of the subject remains free of detectable HBV DNA and HBsAg after the subject is off all HBV treatment(s) for at least 6 months.
  • the serum of the subject with FC has undetectable HBV DNA and is HBsAg-negative 6 months after the end of HBV treatment.
  • the HBsAg loss in a subject with FC can be with or without HBsAg seroconversion.
  • the FC lasts at least 1 year, preferably at least 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years. Most preferably, the FC lasts for life.
  • the term “recovery of T cell function” refers to the re-activation of exhausted and otherwise non-functional HBV-specific T cells such that they are able to perform their normal effector functions.
  • Examples of recovered HBV-specific T cell response include, but are not limited to, killing or non-cytolytic control of HBV-infected hepatocytes, killing of hepatocytes with integrated HBV DNA expressing HBsAg and, once FC is achieved, performing surveillance and killing infected cells that reactivate over time. The net effect is to keep the serum free of virus products, such as HBV DNA, HBsAg and other HBV proteins.
  • the terms and phrases “in combination,” “in combination with,” “co-delivery,” and “administered together with” in the context of the administration of two or more therapies or components to a subject refers to simultaneous administration or subsequent administration of two or more therapies or components, such as two vectors, e.g., DNA plasmids, peptides, or a therapeutic combination and an adjuvant. “Simultaneous administration” can be administration of the two or more therapies or components at least within the same day.
  • two components are “administered together with” or “administered in combination with,” they can be administered in separate compositions sequentially within a short time period, such as 24, 20, 16, 12, 8 or 4 hours, or within 1 hour, or they can be administered in a single composition at the same time. “Subsequent administration” can be administration of the two or more therapies or components in the same day or on separate days. The use of the term “in combination with” does not restrict the order in which therapies or components are administered to a subject.
  • a first therapy or component e.g., an RNAi component
  • a second therapy or component e.g., a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence
  • a first therapy or component e.g., an RNAi component
  • a second therapy or component e.g., a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence
  • a first therapy or component e.g., an RNAi component
  • a second therapy or component e.g., a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence
  • a first therapy or component e.g., an RNAi component
  • a second therapy or component e.g., a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence
  • the application relates to a method for treating an HBV infection in an individual in need thereof, comprising administering to the individual an RNAi component, as described herein under 1), and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence as described herein under 2).
  • the application relates to an RNAi component, as described herein under 1), for use in treating an HBV infection in a subject in need thereof, in combination with a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence as described herein under 2).
  • the application relates to the use of an RNAi component, as described herein under 1), in the manufacture of a medicament for the treatment of hepatitis B Virus (HBV) infection, wherein the medicament is for administration, or to be administered, in combination with a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence, as described herein under 2).
  • HBV hepatitis B Virus
  • RNAi component described herein may contain particular components arranged in a particular order, those having ordinary skill in the art will appreciate that the concepts disclosed herein may equally apply to other components arranged in other orders that can be used in RNAi of the application.
  • the application contemplates use of any of the applicable components in any combination that can be used the application, whether or not a particular combination is expressly described.
  • the invention generally relates to a therapeutic combination comprising one or more HBV RNAi and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence.
  • RNAi component an effective amount of an RNAi component and an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens or a pharmaceutically acceptable salt thereof.
  • the combination of an effective amount of an RNAi component and an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens can be for use in treating a HBV infection, such as a chronic HBV infection (CHB), with or without viral co-infection, e.g., with or without co-infection with HDV and/or HCV and/or HIV, more particularly with or without co-infection with at least HDV, and/or for treating chronic HDV infection (CHD) in a subject in need thereof.
  • CHB chronic HBV infection
  • CHD chronic HDV infection
  • the combination is for use in enhancing an immune response, decreasing viral replication, and/or decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptides, more particularly of one or more polypeptide(s) selected from HBsAg and HBeAg, in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection.
  • HBV Hepatitis B Virus
  • an RNAi component for use in combination with a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens in the treatment of a Hepatitis B Virus infection, more particularly a chomic HBV infection (CHB) with or without a viral co-infection, and/or in the treatment of a chronic Hepatitis D Virus (HDV) infection, wherein the RNAi component and the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens are as defined herein, and wherein the RNAi component optionally is indicated for simultaneous, sequential, or separate administration with the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens.
  • CHB chomic HBV infection
  • HDV chronic Hepatitis D Virus
  • nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence comprising one or more HBV antigens for use in combination with an RNAi component in the treatment of a Hepatitis B Virus infection, more particularly a chomic HBV infection (CHB) with or without a viral co-infection, and/or in the treatment of a chronic Hepatitis D Virus (HDV) infection, wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens and the RNAi component are as defined herein, and wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens optionally is indicated for simultaneous, sequential, or separate administration with the RNAi component.
  • CHB chomic HBV infection
  • HDV chronic Hepatitis D Virus
  • HBV Hepatitis B Virus
  • hepatitis B virus or “HBV” refers to a virus of the hepadnaviridae family.
  • HBV is a small (e.g., 3.2 kb) hepatotropic DNA virus that encodes four open reading frames and seven proteins.
  • the seven proteins encoded by HBV include small (S), medium (M), and large (L) surface antigen (HBsAg) or envelope (Env) proteins, pre-Core protein, core protein, viral polymerase (Pol), and HBx protein.
  • HBV expresses three surface antigens, or envelope proteins, L, M, and S, with S being the smallest and L being the largest.
  • Core protein is the subunit of the viral nucleocapsid. Pol is needed for synthesis of viral DNA (reverse transcriptase, RNaseH, and primer), which takes place in nucleocapsids localized to the cytoplasm of infected hepatocytes.
  • PreCore is the core protein with an N-terminal signal peptide and is proteolytically processed at its N and C termini before secretion form infected cells, as the so-called hepatitis B e-antigen (HBeAg). HBx protein is required for efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a viral structural protein. All viral proteins of HBV have their own mRNA except for core and polymerase, which share an mRNA. With the exception of the protein pre-Core, none of the HBV viral proteins are subject to post-translational proteolytic processing.
  • the HBV virion contains a viral envelope, nucleocapsid, and single copy of the partially double-stranded DNA genome.
  • the nucleocapsid comprises 120 dimers of core protein and is covered by a capsid membrane embedded with the S, M, and L viral envelope or surface antigen proteins.
  • the virus is uncoated and the capsidcontaining relaxed circular DNA (rcDNA) with covalently bound viral polymerase migrates to the nucleus.
  • rcDNA relaxed circular DNA
  • phosphorylation of the Core protein induces structural changes, exposing a nuclear localization signal enabling interaction of the capsid with so- called importins.
  • rccDNA covalently closed circular DNA genome from which overlapping transcripts encode for HBeAg, HBsAg, Core protein, viral polymerase and HBx protein.
  • Core protein, viral polymerase, and pre- genomic RNA (pgRNA) associate in the cytoplasm and self-assemble into immature pgRNA- containing capsid particles, which further convert into mature rcDNA-capsids and function as a common intermediate that is either enveloped and secreted as infections virus particles or transported back to the nucleus to replenish and maintain a stable cccDNA pool.
  • pgRNA pre- genomic RNA
  • HBV is divided into four serotypes (adr, adw, ayr, ayw) based on antigenic epitopes present on the envelope proteins, and into eight genotypes (A, B, C, D, E, F, G, and H) based on the sequence of the viral genome.
  • the HBV genotypes are distributed over different geographic regions. For example, the most prevalent genotypes in Asia are genotypes B and C. Genotype D is dominant in Africa, the Middle East, and India, whereas genotype A is widespread in Northern Europe, sub-Saharan Africa, and West Africa.
  • HBV Antigens are distributed over different geographic regions. For example, the most prevalent genotypes in Asia. Genotype D is dominant in Africa, the Middle East, and India, whereas genotype A is widespread in Northern Europe, sub-Saharan Africa, and West Africa.
  • HBV antigen As used herein, the terms “HBV antigen,” “antigenic polypeptide of HBV,” “HBV antigenic polypeptide,” “HBV antigenic protein,” “HBV immunogenic polypeptide,” and “HBV immunogen” all refer to a polypeptide capable of inducing an immune response against an HBV in a subject. The induced response can be a humoral and/or cellular mediated response.
  • the HBV antigen can be a polypeptide of HBV, a fragment or epitope thereof, or a combination of multiple HBV polypeptides, portions or derivatives thereof.
  • an HBV antigen is capable of raising in a host a protective immune response, e.g., inducing an immune response against a viral disease or infection, and/or producing an immunity (i.e., vaccinates) a subject against a viral disease or infection, that protects the subject against the viral disease or infection.
  • an HBV antigen can comprise a polypeptide or immunogenic fragment(s) thereof from any HBV protein, such as HBeAg, pre-core protein, HBsAg (S, M, or L proteins), core protein, viral polymerase, or HBx protein derived from any HBV genotype, e.g., genotype A, B, C, D, E, F, G, and/or H, or combination thereof.
  • each of the terms “HBV core antigen,” “HBeAg” and “core antigen” refers to an HBV antigen capable of inducing an immune response against an HBV core protein in a subject.
  • the induced immune response can be a humoral and/or cellular mediated response.
  • Each of the terms “core,” “core polypeptide,” and “core protein” refers to the HBV viral core protein.
  • Full-length core antigen is typically 183 amino acids in length and includes an assembly domain (amino acids 1 to 149) and a nucleic acid binding domain (amino acids 150 to 183).
  • the 34-residue nucleic acid binding domain is required for pre- genomic RNA encapsidation.
  • HBV core protein is dimeric in solution, with the dimers self-assembling into icosahedral capsids. Each dimer of core protein has four a-helix bundles flanked by an a-helix domain on either side. Truncated HBV core proteins lacking the nucleic acid binding domain are also capable of forming capsids.
  • an HBV antigen is a truncated HBV core antigen.
  • a “truncated HBV core antigen,” refers to an HBV antigen that does not contain the entire length of an HBV core protein but is capable of inducing an immune response against the HBV core protein in a subject.
  • an HBV core antigen can be modified to delete one or more amino acids of the highly positively charged (arginine rich) C-terminal nucleic acid binding domain of the core antigen, which typically contains seventeen arginine (R) residues.
  • a truncated HBV core antigen of the application is preferably a C-terminally truncated HBV core protein which does not comprise the HBV core nuclear import signal and/or a truncated HBV core protein from which the C-terminal HBV core nuclear import signal has been deleted.
  • a truncated HBV core antigen comprises a deletion in the C-terminal nucleic acid binding domain, such as a deletion of 1 to 34 amino acid residues of the C-terminal nucleic acid binding domain, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acid residues, preferably a deletion of 31-34 C-terminal amino acid residues of the C-terminal nucleic acid binding domain.
  • a truncated HBV core antigen comprises a deletion in the C-terminal nucleic acid binding domain, preferably a deletion of 31 C-terminal amino acid residues of the C-terminal nucleic acid binding domain.
  • an HBV core antigen amino acid sequence is operably linked to a signal peptide for secretion. Any suitable signal peptide can be used.
  • an HBV core antigen is operably linked at its N-terminus to a Cystatin S precursor signal peptide, to enhance secretion.
  • the Cystatin S precursor signal peptide has the amino acid sequence of SEQ ID NO: 77.
  • a coding sequence of an HBV core antigen is operably linked to a coding sequence of Cystatin S precursor signal peptide having the polynucleotide sequence of SEQ ID NO: 90.
  • An HBV core antigen of the application can be a consensus sequence derived from multiple HBV genotypes (e.g., genotypes A, B, C, D, E, F, G, and H).
  • Consensus sequence means an artificial sequence of amino acids based on an alignment of amino acid sequences of homologous proteins as determined by an alignment of amino acid sequences of homologous proteins. The alignment can be conducted using methods or algorithm known in the art, such as using Clustal Omega. It can be the calculated order of most frequent amino acid residues, found at each position in a sequence alignment, based upon sequences of HBV antigens (e.g., core, pol, etc.) from at least 100 natural HBV isolates.
  • HBV antigens e.g., core, pol, etc.
  • a consensus sequence can be non-naturally occurring and different from the native viral sequences.
  • Consensus sequences can be designed by aligning multiple HBV antigen sequences from different sources using a multiple sequence alignment tool, and at variable alignment positions, selecting the most frequent amino acid.
  • a consensus sequence of an HBV antigen is derived from HBV genotypes A, B, C, and D.
  • the term “consensus antigen” is used to refer to an antigen having consensus sequence.
  • An exemplary truncated HBV core antigen lacks the nucleic acid binding function and is capable of inducing an immune response in a mammal against at least two HBV genotypes.
  • a truncated HBV core antigen is capable of inducing a T cell response in a mammal against at least HBV genotypes A, B, C and D.
  • a truncated HBV core antigen is capable of inducing a CD8 T cell response in a human subject against at least HBV genotypes A, B, C and D.
  • an HBV core antigen of the application comprises one or more T cell epitopes for MHC class I HLA alleles.
  • an HBV core antigen comprises one or more epitopes selected from the group consisting of HLA- A* 11:01 epitopes, HLA-A*02:01 epitopes, HLA-A*A0101 epitopes, and HLA-B*40:01 epitopes.
  • an HBV core antigen comprises two or more, such as 2, 3, or 4, of T cell epitopes selected from the group consisting of HLA-A*11:01 epitopes, HLA-A*02:01 epitopes, HLA- A*A0101 epitopes, and HL A-B* 40: 01 epitopes. More preferably, an HBV core antigen comprises HLA-A*ll:01, HLA-A*02:01, HLA-A*A0101, and HLA-B*40:01 T cell epitopes. More preferably, an HBV core antigen comprises one or more HLA-A* 11:01 epitope(s).
  • an HBV core antigen of the application is a consensus antigen, preferably a consensus antigen derived from at least two, preferably all, of HBV genotypes A, B, C, and D, more preferably a truncated consensus antigen derived from HBV genotypes A, B, C, and D.
  • An exemplary truncated HBV core consensus antigen consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 86, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 86.
  • the HBV core antigen comprises, preferably consists of, an amino acid sequence that is at least 98% identical to SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86.
  • SEQ ID NO: 86 is a core consensus antigen derived from HBV genotypes A, B, C, and D.
  • SEQ ID NO: 7 contains a 34-amino acid C-terminal deletion of the highly positively charged (arginine rich) nucleic acid binding domain of the native core antigen.
  • an HBV core antigen of the application retains one or more of the C-terminal arginine residues and has the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, to thereby restore the HLA-A* 11:01 epitope in the HBV core antigen.
  • the last five C-terminal amino acids of an HBV core antigen comprise a VVR amino acid sequence, more particularly a VVRR (SEQ ID NO: 91) amino acid sequence, more particularly a VVRRR (SEQ ID NO: 92) amino acid sequence.
  • an HBV core antigen is a truncated HBV antigen consisting of the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86.
  • an HBV core antigen is encoded by a polynucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89, respectively.
  • the HBV core antigen consists of the amino acid sequence of SEQ ID NO: 86 and is encoded by the polynucleotide sequence of SEQ ID NO: 89.
  • an HBV core antigen consists of an amino acid sequence, or is encoded by a polynucleotide sequence, as described in U.S. Patent Application Publication No. US2019/0185828, the content of which is herein incorporated by reference in its entirety.
  • HBV polymerase antigen As used herein, the term “HBV polymerase antigen,” “HBV Pol antigen,” or “HBV pol antigen” refers to an HBV antigen capable of inducing an immune response against an HBV polymerase in a subject.
  • the immune response can be a humoral and/or cellular mediated response.
  • polymerase polymerase polypeptide
  • Poly polymerase polypeptide
  • Poly polypeptide
  • an HBV antigen comprises an HBV Pol antigen, or any immunogenic fragment or combination thereof.
  • An HBV Pol antigen can contain further modifications to improve immunogenicity of the antigen, such as by introducing mutations into the active sites of the polymerase and/or RNase domains to decrease or substantially eliminate certain enzymatic activities.
  • an HBV Pol antigen of the invention does not have reverse transcriptase activity and RNase H activity and is capable of inducing an immune response in a mammal against at least two HBV genotypes.
  • an HBV Pol antigen is capable of inducing a T cell response in a mammal against at least two, preferably all, of HBV genotypes A, B, C and D. More preferably, a HBV Pol antigen is capable of inducing a CD8 T cell response in a human subject against at least HBV genotypes A, B, C and D.
  • an HBV Pol antigen is an inactivated Pol antigen.
  • an inactivated HBV Pol antigen comprises one or more amino acid mutations in the active site of the polymerase domain.
  • an inactivated HBV Pol antigen comprises one or more amino acid mutations in the active site of the RNaseH domain.
  • an inactivated HBV pol antigen comprises one or more amino acid mutations in the active site of both the polymerase domain and the RNaseH domain.
  • the “YXDD” motif (SEQ ID NO: 74) in the polymerase domain of an HBV pol antigen that can be required for nucleotide/metal ion binding can be mutated, e.g., by replacing one or more of the aspartate residues (D) with asparagine residues (N), eliminating or reducing metal coordination function, thereby decreasing or substantially eliminating reverse transcriptase function.
  • the “DEDD” motif (SEQ ID NO: 75) in the RNaseH domain of an HBV pol antigen required for Mg 2+ coordination can be mutated, e.g., by replacing one or more aspartate residues (D) with asparagine residues (N) and/or replacing the glutamate residue (E) with glutamine (Q), thereby decreasing or substantially eliminating RNaseH function.
  • an HBV pol antigen is modified by (1) mutating the aspartate residues (D) to asparagine residues (N) in the “YXDD” motif (SEQ ID NO: 74) of the polymerase domain; and (2) mutating the first aspartate residue (D) to an asparagine residue (N) and the first glutamate residue (E) to a glutamine residue (N) in the “DEDD” motif (SEQ ID NO: 75) of the RNaseH domain, thereby decreasing or substantially eliminating both the reverse transcriptase and RNaseH functions of the pol antigen.
  • an HBV pol antigen amino acid sequence is operably linked to a signal peptide for secretion. Any suitable signal peptides can be used.
  • an HBV pol antigen is operably linked at its N-terminus to a Cystatin S precursor signal peptide, to enhance secretion.
  • the Cystatin S precursor signal peptide has the amino acid sequence of SEQ ID NO: 77.
  • a coding sequence of an HBV pol antigen is operably linked to a coding sequence of Cystatin S precursor signal peptide having the polynucleotide sequence of SEQ ID NO: 90.
  • an HBV pol antigen of the application comprises one or more T cell epitopes for MHC class I HLA alleles.
  • an HBV pol antigen comprises one or more epitopes selected from the group consisting of HLA- A* 11:01 epitopes, HLA-A*24:02 epitopes, HLA-A*02:01 epitopes, and HLA-A*A0101 epitopes.
  • an HBV pol antigen comprises two or more, such as 2, 3, or 4, T cell epitopes selected from the group consisting of HLA-A*11:01 epitopes, HLA-A*24:02 epitopes, HLA- A*02:01 epitopes, and HLA-A*A0101. More preferably, an HBV pol antigen comprises HLA-A*ll:01, HLA-A*24:02, HLA-A*02:01, and HLA- A* A0101 T cell epitopes. More preferably, an HBV pol antigen comprises one or more HLA-A*11:01 epitope(s).
  • an HBV pol antigen is a consensus antigen, preferably a consensus antigen derived from at least two, preferably all, of HBV genotypes A, B, C, and D, more preferably an inactivated consensus antigen derived from HBV genotypes A, B, C, and D.
  • An exemplary HBV pol consensus antigen according to the application comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 9, preferably at least 98% identical to SEQ ID NO: 9, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 9.
  • SEQ ID NO: 9 is a pol consensus antigen derived from HBV genotypes A, B, C, and D comprising four mutations located in the active sites of the polymerase and RNaseH domains.
  • the four mutations include mutation of the aspartic acid residues (D) to asparagine residues (N) in the “YXDD” motif (SEQ ID NO: 74) of the polymerase domain; and mutation of the first aspartate residue (D) to an asparagine residue (N) and mutation of the glutamate residue (E) to a glutamine residue (Q) in the “DEDD” (SEQ ID NO: 75) motif of the RNaseH domain.
  • an HBV pol antigen comprises the amino acid sequence of SEQ ID NO: 9.
  • an HBV pol antigen consists of the amino acid sequence of SEQ ID NO: 9.
  • an HBV pol antigen is encoded by a polynucleotide sequence of SEQ ID NO: 10.
  • an HBV pol antigen contains or consists of an amino acid sequence, or is encoded by a polynucleotide sequence as described in U.S. Patent Application Publication No.
  • each of the terms “HBV surface antigen,” “surface antigen,” “HBV envelope antigen,” “envelope antigen,” and “env antigen” refers to an HBV antigen capable of inducing or eliciting an immune response against one or more HBV surface antigens or envelope proteins in a subject.
  • the immune response can be a humoral and/or cellular mediated response.
  • Each of the terms “HBV surface protein,” “surface protein,” “HBV envelope protein,” and “envelope protein” refers to HBV viral surface or envelope proteins.
  • HBV expresses three surface antigens, or envelope proteins.
  • Gene S is the gene of the HBV genome that encodes the surface antigens.
  • the surface antigen gene is one long open reading frame but contains three in frame “start” (ATG) codons that divide the gene into three sections, pre-Sl, pre-S2, and S. Because of the multiple start codons, polypeptides of three different sizes called large (L) or L-surface antigen, middle (M) or M-surface antigen, and small (S) or S-surface antigen are produced, also named the HBV L, M and S envelope proteins. Two different promoters (PreSl and PreS2) drive transcription of the L, M, and S- surface antigen coding sequences resulting in three different translated proteins, the L, M and S envelope proteins.
  • the PreS2 promoter is sometimes referred to as the PreS2/S promoter since it is driving M-surface antigen and S-surface antigen transcription separately.
  • the amino acid sequence of the L-surface antigen is in-frame with the M and S-surface antigen sequences.
  • the L-surface antigen contains the M- and S-surface antigen domains and the M-surface antigen includes the S-surface antigen domain.
  • the L-, M- and S-surface antigen are co-C-terminal and share the entire S domain. Relative to S, M has an additional domain, pre-S2, at its N terminus, and relative to M, L has a pre-Sl domain.
  • an HBV antigen is an HBV PreSl antigen, which is encoded by a pre-Sl gene section and contains only the Pre-Sl domain of the L antigen.
  • the PreSl antigen can have various lengths, such as having 99 to 109 amino acids.
  • An HBV PreSl antigen of the application can contain the sequence of any naturally occurring PreSl domain, and variants or derivatives thereof.
  • an HBV antigen is an HBV PreS2.S antigen, which is encoded by the pre-S2 and S gene sections and contains the PreS2 domain and the S domain.
  • the PreS2 domain can be about 55 amino acids long and the S-domain can contain about 226 amino acids.
  • An HBV PreS2.S antigen of the application can contain the sequences of any of the naturally occurring PreS2 and S domains, and variants or derivatives thereof.
  • an internal signal peptide of PreS2.S is left intact to facilitate secretion PreS2.S protein products of the HBV M and HBV S antigens.
  • an HBV PreS2.S antigen is an HBV M surface antigen.
  • an HBV PreS2.S antigen is an HBV S surface antigen.
  • an HBV PreS2.S antigen encompasses an HBV M surface antigen and an HBV S surface antigen.
  • an HBV surface antigen amino acid sequence is operably linked to or contains a signal peptide for secretion. Any suitable signal peptides can be used.
  • an HBV Pre-S 1 antigen amino acid sequence is operably linked at its N- terminus to a Cystatin S precursor signal peptide to enhance secretion.
  • the Cystatin S precursor signal peptide has the amino acid sequence of SEQ ID NO: 77.
  • a coding sequence of an HBV Pre-S 1 antigen is operably linked to a coding sequence of Cystatin S precursor signal peptide having the polynucleotide sequence of SEQ ID NO: 90.
  • an HBV antigen comprises an HBV surface antigen, or any immunogenic fragment or combination thereof.
  • An HBV surface antigen is capable of inducing an immune response in a subject against at least one of L-surface antigen, M-surface antigen, and S-surface antigen proteins.
  • an HBV surface antigen such as a Pre-S 1 or PreS2.S antigen, is a consensus antigen, preferably a consensus antigen derived from at least two HBV genotypes A, B, C, and D, and more preferably a consensus antigen derived from HBV genotypes A, B, C, and D.
  • an HBV surface antigen of the application comprises one or more T cell epitopes for MHC class I HLA alleles.
  • an HBV surface antigen comprises one or more T cell epitopes selected from the group consisting of HLA- A* 11:01 epitopes, HLA-A*24:02 epitopes, and HLA-A*A2402 epitopes.
  • an HBV Pre-S 1 antigen comprises one or more T cell epitopes selected from the group consisting of HLA-A*11 :01 epitopes and HLA-A*24:02 epitopes.
  • an HBV Pre-Sl antigen comprises HLA-A*ll:01 and HLA-A*24:02 T cell epitopes. More preferably, an HBV Pre-Sl antigen comprises one or more HLA- A*11:01 epitope(s).
  • an HBV PreS2.S antigen comprises one or more T cell epitopes selected from the group consisting of HLA-A*l l:01 epitopes, HLA-A*24:02 epitopes, and HLA-A*A2402 epitopes. More preferably, an HBV PreS2.S antigen comprises HLA-A*ll:01, HLA-A*24:02, and HLA-A*A2402 T cell epitopes. More preferably, an HBV PreS2.S antigen comprises one or more HLA-A* 11:01 epitope(s).
  • an HBV surface antigen is a Pre-S 1 antigen.
  • An exemplary Pre-S 1 antigen according to the application comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3.
  • an HBV surface antigen is a Pre-S2.S antigen.
  • An exemplary Pre-S2.S antigen according to the application comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 5.
  • an HBV surface antigen is a Pre-S 1 antigen consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
  • an HBV surface antigen is encoded by a polynucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • an HBV surface antigen is a Pre-S2.S antigen consisting of the amino acid sequence of SEQ ID NO: 5.
  • an HBV surface antigen is encoded by a polynucleotide sequence of SEQ ID NO: 6.
  • an HBV surface antigen is an S-surface antigen.
  • An exemplary S-surface antigen according to the application consists of an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 79, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 79.
  • SEQ ID NO: 81 is an HBV consensus S-surface antigen derived from HBV genotypes A, B, C, and D.
  • an S-surface antigen consists of the amino acid sequence of SEQ ID NO: 79.
  • an HBV surface antigen is encoded by a polynucleotide sequence of SEQ ID NO: 78.
  • an HBV surface antigen is M-surface antigen, or any immunogenic fragment or combination thereof.
  • the M-surface antigen is a consensus antigen, preferably a consensus antigen derived from at least two, preferably all, of HBV genotypes A, B, C, and D, and more preferably a consensus antigen derived from HBV genotypes A, B, C, and D.
  • the M- surface antigen is capable of inducing or eliciting an immune response against M-surface antigen in a subject.
  • An exemplary M-surface antigen according to the application comprises or consists of an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 82, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO:
  • SEQ ID NO: 82 is an HBV consensus M-surface antigen derived from HBV genotypes A, B, C, and D.
  • an M-surface antigen consists of the amino acid sequence of SEQ ID NO: 82.
  • an HBV surface antigen is an L-surface antigen, or any immunogenic fragment or combination thereof.
  • the L-surface antigen is a consensus antigen, preferably a consensus antigen derived from at least two, preferably all, of HBV genotypes A, B, C, and D, and more preferably a consensus antigen derived from HBV genotypes A, B, C, and D.
  • the L-surface antigen is capable of inducing or eliciting an immune response against L-surface antigen in a subject.
  • An exemplary L-surface antigen according to the application comprises or consists of an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 83, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO:
  • SEQ ID NO: 83 is an HBV consensus L-surface antigen derived from HBV genotypes A, B, C, and D.
  • an M-surface antigen consists of the amino acid sequence of SEQ ID NO: 83.
  • an HBV surface antigen comprises a portion of any one of the L-, M-, and S-surface antigens, or any combination thereof.
  • an HBV surface antigen can comprise or consist of the N-terminal L-surface antigen domain.
  • An HBV surface antigen can also comprise or consist of the M-surface antigen domain.
  • An HBV surface antigen can also comprise or consist of the N-terminal L-surface antigen domain and the M- surface antigen domain.
  • An HBV surface antigen can also comprise or consist of the N- terminal L-surface antigen domain, the M-surface antigen domain, and a portion of the S- surface antigen domain.
  • An exemplary example of such a surface antigen according to the application consists of an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 81, such as at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 81, preferably at least 98% identical to SEQ ID NO: 81, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 81.
  • SEQ ID NO: 81 is a consensus antigen derived from HBV genotypes A, B, C, and D, containing the N-terminal L-surface antigen domain, the entire M- surface antigen domain, and a 15 -amino acid C-terminal tail from the S-surface antigen domain.
  • an HBV surface antigen consists of the amino acid sequence of SEQ ID NO: 81.
  • an HBV surface antigen is encoded by a polynucleotide sequence of SEQ ID NO: 80 that encodes an HBV surface antigen.
  • an HBV surface antigen consists of an amino acid sequence, or is encoded by a polynucleotide sequence, described in European Patent Application No. 19180926, the content of which is herein incorporated by reference in its entirety.
  • the application provides a nucleic acid molecule comprising a non-naturally polynucleotide sequence encoding an HBV antigen according to the application, and a vector comprising the non-naturally occurring nucleic acid.
  • a nucleic acid molecule can comprise any non-naturally occurring polynucleotide sequence encoding an HBV antigen of the application, which can be made using methods known in the art in view of the present disclosure.
  • a non-naturally occurring polynucleotide encodes at least one of a truncated HBV core antigen, an HBV polymerase antigen, an HBV Pre-S 1 antigen, and an HBV Pre-S2.S antigen of the application.
  • a polynucleotide can be in the form of RNA or in the form of DNA obtained by recombinant techniques (e.g., cloning) or produced synthetically (e.g., chemical synthesis).
  • the DNA can be single-stranded or double-stranded or can contain portions of both double-stranded and single-stranded sequence.
  • the DNA can, for example, comprise genomic DNA, cDNA, or combinations thereof.
  • the polynucleotide can also be a DNA/RNA hybrid.
  • the polynucleotides and vectors of the application can be used for recombinant protein production, expression of the protein in host cell, or the production of viral particles.
  • a polynucleotide is RNA.
  • a nucleic acid molecule comprises a non- naturally occurring polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, preferably 98%, 99% or 100% identical to SEQ ID NO: 7, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86.
  • a non-naturally occurring nucleic acid molecule encodes a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86.
  • the truncated HBV core antigen consists of SEQ ID NO: 86.
  • HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 8, SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89, respectively, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 8, SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89, preferably 98%, 99% or 100% identical to SEQ ID NO: 8, SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO
  • Exemplary non-naturally occurring nucleic acid molecules encoding a truncated HBV core antigen have the polynucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89.
  • the molecule encoding a truncated HBV core antigen has the polynucleotide sequence of SEQ ID NO: 89.
  • a nucleic acid molecule comprises a non- naturally occurring polynucleotide sequence encoding a HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 9.
  • a non-naturally occurring nucleic acid molecule encodes a HBV polymerase antigen consisting of the amino acid sequence of SEQ ID NO: 9.
  • polynucleotide sequences of the application encoding a HBV Pol antigen comprising the amino acid sequence of SEQ ID NO: 9 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 10, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 10, preferably 98%, 99% or 100% identical to SEQ ID NO: 10.
  • a nucleic acid molecule comprises a non-natural polynucleotide sequence encoding aHBV Pre-S 1 antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3, preferably 98%, 99% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3.
  • a non-natural polynucleotide sequence encoding aHBV Pre-S 1 antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3, such as at least 90%, 91%,
  • Examples of polynucleotide sequences of the application encoding a HBV Pre-S 1 antigen consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, respectively, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 98%, 99% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4.
  • Exemplary non-naturally occurring nucleic acid molecules encoding aHBV Pre-S 1 antigen have the polynucleotide sequence of SEQ
  • a nucleic acid molecule comprises a non- naturally occurring polynucleotide sequence encoding aHBV Pre-S2.S antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 5, preferably 98%, 99% or 100% identical to SEQ ID NO: 5.
  • a non-naturally occurring nucleic acid molecule encodes a HBV Pre-S2.S antigen consisting the amino acid sequence of SEQ ID NO: 5.
  • Examples of polynucleotide sequences of the application encoding a HBV Pre- S2.S antigen consisting of the amino acid sequence of SEQ ID NO: 5 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 6, respectively, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 6, preferably 98%, 99% or 100% identical to SEQ ID NO: 6.
  • a nucleic acid molecule comprises a non- naturally occurring polynucleotide sequence encoding comprising, from 5’ end to 3’ end: a polynucleotide sequence encoding a first HBV antigen, a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and a polynucleotide sequence encoding a second HBV antigen, wherein at least one of the first and second HBV antigens is an HBV surface antigen.
  • a non-naturally occurring polynucleotide sequence encoding comprising, from 5’ end to 3’ end: a polynucleotide sequence encoding a first HBV antigen, a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and a polynucleotide sequence encoding a second
  • the non- naturally occurring polynucleotide sequence further comprises, ordered from the 5’- to 3’- end: a second IRES element or a polynucleotide sequence encoding a second autoprotease peptide operably linked to the 3’ end of the polynucleotide sequence encoding the second HBV antigen, and a polynucleotide sequence encoding a third HBV antigen.
  • the non-naturally occurring polynucleotide sequence further comprises, ordered from the 5’- to 3’-end: a third IRES element or a polynucleotide sequence encoding a third autoprotease peptide operably linked to the 3’ end of the polynucleotide sequence encoding the third HBV antigen, and a polynucleotide sequence encoding a fourth HBV antigen.
  • each of the first, second, third and fourth HBV antigens are independently selected from the group consisting of: (i) a first HBV surface antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 1; (ii) a second HBV surface antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 3; (i) a first HBV surface anti
  • the HBV core antigen comprises, preferably consists of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86.
  • each of the polynucleotide sequences encoding the first, second, third and fourth HBV antigens are independently selected from the group consisting of: (i) a polynucleotide sequence encoding the first HBV PreSl antigen having a sequence that is at least 90% identical to SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2; (ii) a polynucleotide sequence encoding the second HBV PreSl antigen having a sequence that is at least 90% identical to SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%,
  • the polynucleotide sequence encoding the HBV core antigen comprises, preferably consists of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89.
  • each of the first, second and third autoprotease peptides independently comprise a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2 A (E2A), a Thosea asigna virus 2 A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof.
  • P2A porcine teschovirus-1 2A
  • FMDV foot-and-mouth disease virus
  • E2A Equine Rhinitis A Virus
  • T2A a Thosea asigna virus 2 A
  • BmCPV2A cytoplasmic polyhedrosis virus 2A
  • BmIFV2A Flacherie Virus 2 A
  • each of the first, second and third autoprotease peptides comprise the peptide sequence of P2A, such as a P2A sequence of SEQ ID NO: 11.
  • the polynucleotide sequence encoding the P2A peptide sequence is SEQ ID NO: 12.
  • each of the first, second and third IRES are derived from encephalomyocarditis virus (EMCV) or Enterovirus 71 (EV71), preferably each of the first, second and third IRES comprise the polynucleotide sequence of SEQ ID NO: 13 or 14.
  • a nucleic acid molecule comprises a non- naturally occurring polynucleotide sequence encoding comprising, from 5’ end to 3’ end: (1) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9; (2) a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P
  • each HBV antigen is independently operably linked to or contains a signal peptide sequence for secretion. Any suitable signal peptide sequence can be used.
  • a signal peptide is operably linked to the N-terminus of the antigen sequence.
  • the signal peptide contains the amino acid sequence of SEQ ID NO: 77, preferably, the signal peptide is encoded by a nucleotide sequence of SEQ ID NO: 90.
  • the nucleic acid molecule comprises a non-naturally occurring polynucleotide sequence of any one of SEQ ID NOs: 15 to 54. In some embodiments, the nucleic acid molecule comprises at least two non-naturally occurring polynucleotide sequences of SEQ ID NOs: 15 to 54.
  • the application also relates to a vector comprising a non-naturally occurring polynucleotide encoding an HBV antigen.
  • a “vector” is a nucleic acid molecule used to carry genetic material into another cell, where it can be replicated and/or expressed. Any vector known to those skilled in the art in view of the present disclosure can be used. Examples of vectors include, but are not limited to, plasmids, viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs).
  • a vector is a DNA plasmid.
  • a vector can be a DNA vector or an RNA vector.
  • One of ordinary skill in the art can construct a vector of the application through standard recombinant techniques in view of the present disclosure.
  • a vector of the application can be an expression vector.
  • expression vector refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed.
  • Expression vectors include, but are not limited to, vectors for recombinant protein expression, such as a DNA plasmid or a viral vector, and vectors for delivery of nucleic acid into a subject for expression in a tissue of the subject, such as a DNA plasmid or a viral vector. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • Vectors of the application can contain a variety of regulatory sequences.
  • regulatory sequence refers to any sequence that allows, contributes or modulates the functional regulation of the nucleic acid molecule, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (i. e. , mRNA) into the host cell or organism.
  • this term encompasses promoters, enhancers and other expression control elements (e.g., polyadenylation signals and elements that affect mRNA stability).
  • a vector is a non- viral vector.
  • non-viral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc.
  • a non-viral vector is a DNA plasmid.
  • DNA plasmids used for expression of an encoded polynucleotide typically comprise an origin of replication, a multiple cloning site, and a selectable marker, which for example, can be an antibiotic resistance gene.
  • DNA plasmids suitable that can be used include, but are not limited to, commercially available expression vectors for use in well- known expression systems (including both prokaryotic and eukaryotic systems), such as pSE420 (Invitrogen, San Diego, Calif), which can be used for production and/or expression of protein in Escherichia coli; pYES2 (Invitrogen, Thermo Fisher Scientific), which can be used for production and/or expression in Saccharomyces cerevisiae strains of yeast;
  • MAXBAC® complete baculovirus expression system (Thermo Fisher Scientific), which can be used for production and/or expression in insect cells; pcDNATM or pcDNA3TM (Life Technologies, Thermo Fisher Scientific), which can be used for high level constitutive protein expression in mammalian cells; and pVAX or pVAX-1 (Life Technologies, Thermo Fisher Scientific), which can be used for high-level transient expression of a protein of interest in most mammalian cells.
  • the backbone of any commercially available DNA plasmid can be modified to optimize protein expression in the host cell, such as to reverse the orientation of certain elements (e.g., origin of replication and/or antibiotic resistance cassette), replace a promoter endogenous to the plasmid (e.g., the promoter in the antibiotic resistance cassette), and/or replace the polynucleotide sequence encoding transcribed proteins (e.g., the coding sequence of the antibiotic resistance gene), by using routine techniques and readily available starting materials. (See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)).
  • a DNA plasmid is an expression vector suitable for protein expression in mammalian host cells.
  • Expression vectors suitable for protein expression in mammalian host cells include, but are not limited to, pcDNATM, pcDNA3TM, pVAX, pVAX-1, ADV AX, NTC8454, etc.
  • an expression vector is based on pVAX-1, which can be further modified to optimize protein expression in mammalian cells.
  • pVAX-1 is commonly used plasmid in DNA vaccines and contains a strong human intermediate early cytomegalovirus (CMV-IE) promoter followed by the bovine growth hormone (bGH)-derived polyadenylation sequence (pA).
  • pVAX-1 further contains a pUC origin of replication and kanamycin resistance gene driven by a small prokaryotic promoter that allows for bacterial plasmid propagation.
  • CMV-IE human intermediate early cytomegalovirus
  • bGH bovine growth hormone
  • pA bo
  • a vector of the application can also be a viral vector.
  • viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that has been rendered non-infectious, but still contains viral promoters and transgenes, thus allowing for translation of the transgene through a viral promoter. Because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection.
  • a vector as described herein is, for instance, a recombinant adenovirus, a recombinant retrovirus, a recombinant pox virus such as a vaccinia virus (e.g., Modified Vaccinia Ankara (MV A)), a recombinant alphavirus such as Semliki forest virus, a recombinant paramyxovirus, such as a recombinant measles virus, or another recombinant virus.
  • a recombinant adenovirus e.g., a Modified Vaccinia Ankara (MV A)
  • MV A Modified Vaccinia Ankara
  • viral vectors examples include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors, etc.
  • a vector as described herein is an MVA vector.
  • the vector can also be anon-viral vector.
  • a viral vector is an adenovirus vector, e.g., a recombinant adenovirus vector.
  • a recombinant adenovirus vector can for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV) or rhesus adenovirus (rhAd).
  • an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc.
  • an adenovirus vector is a rhAd vector, e.g. rhAd51, rhAd52 or rhAd53.
  • a recombinant viral vector useful for the application can be prepared using methods known in the art in view of the present disclosure. For example, in view of the degeneracy of the genetic code, several nucleic acid sequences can be designed that encode the same polypeptide.
  • a polynucleotide encoding an HBV antigen of the application can optionally be codon-optimized to ensure proper expression in the host cell (e.g., bacterial or mammalian cells). Codon-optimization is a technology widely applied in the art, and methods for obtaining codon-optimized polynucleotides will be well known to those skilled in the art in view of the present disclosure.
  • a vector of the application e.g., a DNA plasmid or a viral vector (particularly an adenoviral vector), can comprise any regulatory elements to establish conventional function(s) of the vector, including but not limited to replication and expression of the HBV antigen(s) encoded by the polynucleotide sequence of the vector.
  • a vector can comprise one or more expression cassettes.
  • An “expression cassette” is part of a vector that directs the cellular machinery to make RNA and protein.
  • An expression cassette typically comprises three components: a promoter sequence, an open reading frame, and a 3 ’-untranslated region (UTR) optionally comprising a poly adenylation signal.
  • An open reading frame (ORF) is a reading frame that contains a coding sequence of a protein of interest (e.g., HBV antigen) from a start codon to a stop codon.
  • Regulatory elements of the expression cassette can be operably linked to a polynucleotide sequence encoding an HBV antigen of interest.
  • the term “operably linked” is to be taken in its broadest reasonable context and refers to a linkage of polynucleotide elements in a functional relationship.
  • a polynucleotide is “operably linked” when it is placed into a functional relationship with another polynucleotide.
  • a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Any components suitable for use in an expression cassette described herein can be used in any combination and in any order to prepare vectors of the application.
  • a vector can comprise a promoter sequence, preferably within an expression cassette, to control expression of an HBV antigen of interest.
  • the term “promoter” is used in its conventional sense and refers to a nucleotide sequence that initiates the transcription of an operably linked nucleotide sequence.
  • a promoter is located on the same strand near the nucleotide sequence it transcribes. Promoters can be a constitutive, inducible, or repressible. Promoters can be naturally occurring or synthetic.
  • a promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
  • a promoter can be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source).
  • the promoter can be endogenous to the plasmid (homologous) or derived from other sources (heterologous).
  • the promoter is located upstream of the polynucleotide encoding an HBV antigen within an expression cassette.
  • promoters examples include, but are not limited to, a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter (CMV-IE), Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • HSV human immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • LTR long terminal repeat
  • AMV avian leukosis virus
  • CMV cytomegalovirus
  • a promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein.
  • a promoter can also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic.
  • a promoter is a 26S subgenomic promoter or T7 promoter.
  • a nucleotide sequence of an exemplary 26S subgenomic promoter is shown in SEQ ID NO: 62.
  • a nucleotide sequence of an exemplary T7 promoter is shown in SEQ ID NO: 73.
  • a vector can comprise additional polynucleotide sequences that stabilize the expressed transcript, enhance nuclear export of the RNA transcript, and/or improve transcriptional-translational coupling.
  • sequences include poly adenylation signals and enhancer sequences.
  • a polyadenylation signal is typically located downstream of the coding sequence for a protein of interest (e.g., an HBV antigen) within an expression cassette of the vector.
  • Enhancer sequences are regulatory DNA sequences that, when bound by transcription factors, enhance the transcription of an associated gene.
  • An enhancer sequence is preferably located upstream of the polynucleotide sequence encoding an HBV antigen, but downstream of a promoter sequence within an expression cassette of the vector.
  • the polyadenylation signal can be a SV40 polyadenylation signal (e.g., SEQ ID NO: 64), LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human [3-globin polyadenylation signal.
  • a polyadenylation signal is a SV40 polyadenylation signal.
  • a nucleotide sequence of an exemplary SV40 polyadenylation signal is shown in SEQ ID NO: 64.
  • an enhancer sequence can be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as one from CMV, HA, RSV, or EBV.
  • a viral enhancer such as one from CMV, HA, RSV, or EBV.
  • WPRE Woodchuck HBV Post-transcriptional regulatory element
  • ApoAI intron/exon sequence derived from human apolipoprotein Al precursor
  • HTLV-1) long terminal repeat (LTR) untranslated R-U5 domain of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR), a splicing enhancer, a synthetic rabbit [3-globin intron, or any combination thereof.
  • a vector can comprise a polynucleotide sequence encoding a signal peptide sequence.
  • the polynucleotide sequence encoding the signal peptide sequence is located upstream of the polynucleotide sequence encoding an HBV antigen.
  • Signal peptides typically direct localization of a protein, facilitate secretion of the protein from the cell in which it is produced, and/or improve antigen expression and cross-presentation to antigen- presenting cells.
  • a signal peptide can be present at the N-terminus of an HBV antigen when expressed from the vector, but is cleaved off by signal peptidase, e.g., upon secretion from the cell.
  • a signal peptide can be a cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as a Cystatin S signal peptide, an Ig heavy chain gamma signal peptide SPIgG, or an Ig heavy chain epsilon signal peptide SPIgE.
  • Ig immunoglobulin
  • a vector such as a DNA plasmid
  • Bacterial origins of replication and antibiotic resistance cassettes can be located in a vector in the same orientation as the expression cassette encoding an HBV antigen, or in the opposite (reverse) orientation.
  • An origin of replication (ORI) is a sequence at which replication is initiated, enabling a plasmid to reproduce and survive within cells. Examples of ORIs suitable for use in the application include, but are not limited to ColEl, pMBl, pUC, pSClOl, R6K, and 15A, preferably pUC.
  • Expression cassettes for selection and maintenance in bacterial cells typically include a promoter sequence operably linked to an antibiotic resistance gene.
  • the promoter sequence operably linked to an antibiotic resistance gene differs from the promoter sequence operably linked to a polynucleotide sequence encoding a protein of interest, e.g., HBV antigen.
  • the antibiotic resistance gene can be codon optimized, and the sequence composition of the antibiotic resistance gene is normally adjusted to bacterial, e.g., E. coli, codon usage.
  • Any antibiotic resistance gene known to those skilled in the art in view of the present disclosure can be used, including, but not limited to, kanamycin resistance gene (Kan 1 ), ampicillin resistance gene (Amp r ), and tetracycline resistance gene (Tet 1 ), as well as genes conferring resistance to chloramphenicol, bleomycin, spectinomycin, carbenicillin, etc.
  • Kan 1 kanamycin resistance gene
  • Amicillin resistance gene Amicillin resistance gene
  • Tet 1 tetracycline resistance gene
  • the polynucleotides and expression vectors encoding the HBV antigens of the application can be made by any method known in the art in view of the present disclosure.
  • a polynucleotide encoding an HBV antigen can be introduced or “cloned” into an expression vector using standard molecular biology techniques, e.g., polymerase chain reaction (PCR), etc., which are well known to those skilled in the art.
  • PCR polymerase chain reaction
  • the vector is a self-replicating RNA replicon.
  • selfreplicating RNA molecule which is used interchangeably with “self-amplifying RNA molecule” or “RNA replicon” or “replicon RNA” or “saRNA,” refers to RNA which contains all of the genetic information required for directing its own amplification or self-replication within a permissive cell, which can be a human, mammalian, or animal cell.
  • a selfreplicating RNA molecule resembles mRNA. It is single-stranded, 5 '-capped, and 3 '-polyadenylated and is of positive orientation.
  • the RNA molecule 1) encodes polymerase, replicase, or other proteins which can interact with viral or host cell- derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process; and 2) contain cis-acting RNA sequences required for replication and transcription of the subgenomic replicon-encoded RNA.
  • the delivered RNA leads to the production of multiple daughter RNAs.
  • These daughter RNAs, as well as collinear subgenomic transcripts can be translated themselves to provide in situ expression of a gene of interest, or can be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the gene of interest.
  • the overall result of this sequence of transcriptions is a huge amplification in the number of the introduced replicon RNAs and so the encoded gene of interest becomes a major polypeptide product of the cells.
  • the RNA replicon 1) encodes an RNA-dependent RNA polymerase, which may interact with viral or host cell-derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process, and the non-structural proteins nsPl, nsP2, nsP3, nsP4; and 2) contains cis-acting RNA sequences required for replication and transcription of the genomic and subgenomic RNAs, such as 3’ and 5’ untranslated regions (UTRs; alphavirus nucleotide sequences for non-structural protein-mediated amplification), and/or a subgenomic promoter.
  • UTRs alphavirus nucleotide sequences for non-structural protein-mediated amplification
  • RNA replicon molecule typically contains the following ordered elements: 5' viral RNA sequence(s) required in cis for replication (e.g. a 5’ UTR and a 5’ CSE), sequences coding for biologically active nonstructural proteins (e.g. nsP1234), a promoter for transcribing the subgenomic RNA, 3' viral sequences required in cis for replication (e.g.
  • RNA replicon can refer to a positive sense (or message sense) molecule and the RNA replicon can be of a length different from that of any known, naturally-occurring RNA viruses.
  • the RNA replicon can lack (or not contain) the sequence(s) of at least one (or all) of the structural viral proteins (e.g. nucleocapsid protein C, and envelope proteins P62, 6K, and El).
  • the sequences encoding one or more structural genes can be substituted with one or more heterologous sequences such as, for example, a coding sequence for at least one heterologous protein or peptide (or other gene of interest (GOI)).
  • an RNA replicon of the application comprises, ordered from the 5’- to 3’-end: (1) a 5’ untranslated region (5’-UTR) required for nonstructural protein-mediated amplification of an RNA virus; (2) a polynucleotide sequence encoding at least one, preferably all, of non-structural proteins of the RNA virus; (3) a subgenomic promoter of the RNA virus; (4) a polynucleotide sequence encoding an HBV antigen; and (5) a 3’ untranslated region (3 ’-UTR) required for nonstructural protein-mediated amplification of the RNA virus.
  • a self-replicating RNA molecule encodes an enzyme complex for self-amplification (replicase polyprotein) comprising an RNA-dependent RNA- polymerase function, helicase, capping, and poly-adenylating activity.
  • the viral structural genes downstream of the replicase which are under control of a subgenomic promoter, can be replaced by an HBV antigen.
  • the replicase Upon transfection, the replicase is translated immediately, interacts with the 5' and 3' termini of the genomic RNA, and synthesizes complementary genomic RNA copies. Those act as templates for the synthesis of novel positive-stranded, capped, and poly-adenylated genomic copies, and subgenomic transcripts.
  • Subgenomic RNA is an RNA molecule of a length or size which is smaller than the genomic RNA from which it was derived.
  • the viral subgenomic RNA can be transcribed from an internal promoter, whose sequences reside within the genomic RNA or its complement. Transcription of a subgenomic RNA can be mediated by viral-encoded polymerase(s) associated with host cell-encoded proteins, ribonucleoprotein(s), or a combination thereof.
  • Numerous RNA viruses generate subgenomic mRNAs (sgRNAs) for expression of their 3'-proximal genes.
  • an HBV antigen is expressed under the control of a subgenomic promoter.
  • the subgenomic RNA can be placed under control of internal ribosome entry site (IRES) derived from encephalomyocarditis viruses (EMCV), Bovine Viral Diarrhea Viruses (BVDV), polioviruses, Foot-and-mouth disease viruses (FMD), enterovirus 71 (EV71), or hepatitis C viruses.
  • IRS internal ribosome entry site
  • EMCV encephalomyocarditis viruses
  • BVDV Bovine Viral Diarrhea Viruses
  • FMD Foot-and-mouth disease viruses
  • EV71 enterovirus 71
  • hepatitis C viruses enterovirus 71
  • Subgenomic promoters range from 24 nucleotide (Sindbis virus) to over 100 nucleotides (Beet necrotic yellow vein virus) and are usually found upstream of the transcription start.
  • the RNA replicon includes the coding sequence for at least one, at least two, at least three, or at least four nonstructural viral proteins (e.g. nsPl, nsP2, nsP3, nsP4).
  • Alphavirus genomes encode non-structural proteins nsPl, nsP2, nsP3, and nsP4, which are produced as a single polyprotein precursor, sometimes designated P1234 (or nsPl-4 or nsP1234), and which is cleaved into the mature proteins through proteolytic processing.
  • nsPl can be about 60 kDa in size and may have methyltransferase activity and be involved in the viral capping reaction.
  • nsP2 has a size of about 90 kDa and may have helicase and protease activity while nsP3 is about 60 kDa and contains three domains: a macrodomain, a central (or alphavirus unique) domain, and a hypervariable domain (HVD).
  • nsP4 is about 70 kDa in size and contains the core RNA-dependent RNA polymerase (RdRp) catalytic domain. After infection the alphavirus genomic RNA is translated to yield a Pl 234 polyprotein, which is cleaved into the individual proteins.
  • RdRp RNA-dependent RNA polymerase
  • RNA replicon includes the coding sequence for a portion of the at least one nonstructural viral protein.
  • the RNA replicon can include about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or a range between any two of these values, of the encoding sequence for the at least one nonstructural viral protein.
  • the RNA replicon can include the coding sequence for a substantial portion of the at least one nonstructural viral protein.
  • a “substantial portion” of a nucleic acid sequence encoding a nonstructural viral protein comprises enough of the nucleic acid sequence encoding the nonstructural viral protein to afford putative identification of that protein, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (see, for example, in “Basic Local Alignment Search Tool”; Altschul S F et al., J. Mol. Biol. 215:403-410, 1993).
  • the RNA replicon can include the entire coding sequence for the at least one nonstructural protein.
  • the RNA replicon comprises substantially all the coding sequence for the native viral nonstructural proteins.
  • the one or more nonstructural viral proteins are derived from the same virus. In other embodiments, the one or more nonstructural proteins are derived from different viruses.
  • the RNA replicon can be derived from any suitable plus-strand RNA viruses, such as alphaviruses or flaviviruses.
  • the RNA replicon is derived from alphaviruses.
  • alphavirus describes enveloped single-stranded positive sense RNA viruses of the family Togaviridae.
  • the genus alphavirus contains approximately 30 members, which can infect humans as well as other animals.
  • Alphavirus particles typically have a 70 nm diameter, tend to be spherical or slightly pleomorphic, and have a 40 nm isometric nucleocapsid.
  • the total genome length of alphaviruses ranges between 11,000 and 12,000 nucleotides and has a 5'cap and 3' poly-A tail.
  • ORF's open reading frames
  • the ns ORF encodes proteins (nsPl-nsP4) necessary for transcription and replication of viral RNA.
  • the structural ORF encodes three structural proteins: the core nucleocapsid protein C, and the envelope proteins P62 and El that associate as a heterodimer.
  • the viral membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells through membrane fusion.
  • the four ns protein genes are encoded by genes in the 5' two-thirds of the genome, while the three structural proteins are translated from a subgenomic mRNA colinear with the 3' one-third of the genome.
  • the self-replicating RNA useful for the invention is an RNA replicon derived from an alphavirus virus species.
  • the alphavirus RNA replicon is of an alphavirus belonging to the VEEV/EEEV group, or the SF group, or the SIN group.
  • SF group alphaviruses include Semliki Forest virus, O'Nyong-Nyong virus, Ross River virus, Middelburg virus, Chikungunya virus, Barmah Forest virus, Getah virus, Mayaro virus, Sagiyama virus, Bebaru virus, and Una virus.
  • SIN group alphaviruses include Sindbis virus, Girdwood S. A.
  • VEEV/EEEV group alphaviruses include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), and Una virus (UNAV).
  • Non-limiting examples of alphavirus species include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), and Buggy Creek virus.
  • EEEV
  • the alphavirus RNA replicon is of a Sindbis virus (SIN), a Semliki Forest virus (SFV), a Ross River virus (RRV), a Venezuelan equine encephalitis virus (VEEV), or an Eastern equine encephalitis virus (EEEV).
  • the alphavirus RNA replicon is of a Venezuelan equine encephalitis virus (VEEV).
  • a self-replicating RNA molecule comprises a polynucleotide encoding one or more nonstructural proteins nsPl-4, a subgenomic promoter, such as 26S subgenomic promoter, and a gene of interest encoding an HBV antigen or a fragment thereof described herein.
  • a self-replicating RNA molecule can have a 5' cap (e.g. a 7-methylguanosine). This cap can enhance in vivo translation of the RNA.
  • the 5' nucleotide of a self-replicating RNA molecule useful with the invention can have a 5' triphosphate group. In a capped RNA this can be linked to a 7-methylguanosine via a 5'-to-5' bridge. A 5' triphosphate can enhance RIG-I binding.
  • a self-replicating RNA molecule can have a 3' poly-A tail. It can also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3' end.
  • a poly-A polymerase recognition sequence e.g. AAUAAA
  • the RNA replicon can lack (or not contain) the coding sequence(s) of at least one (or all) of the structural viral proteins (e.g. nucleocapsid protein C, and envelope proteins P62, 6K, and El).
  • the sequences encoding one or more structural genes can be substituted with one or more heterologous sequences such as, for example, a coding sequence for an HBV antigen or a fragment thereof described herein.
  • a self-replicating RNA vector of the application comprises one or more features to confer a resistance to the translation inhibition by the innate immune system or to otherwise increase the expression of the GOI (e.g., an HBV antigen).
  • the GOI e.g., an HBV antigen
  • the RNA sequence can be codon optimized to improve translation efficiency.
  • the RNA molecule can be modified by any method known in the art in view of the present disclosure to enhance stability and/or translation, such by adding a polyA tail, e.g., of at least 30 adenosine residues; and/or capping the 5-end with a modified ribonucleotide, e.g., 7-methylguanosine cap, which can be incorporated during RNA synthesis or enzymatically engineered after RNA transcription.
  • a polyA tail e.g., of at least 30 adenosine residues
  • a modified ribonucleotide e.g., 7-methylguanosine cap
  • an RNA replicon of the application comprises, ordered from the 5’ - to 3 ’-end, (1) an alphavirus 5’ untranslated region (5’-UTR), (2) a 5’ replication sequence of an alphavirus non-structural gene nspl, (3) a downstream loop (DLP) motif of a virus species, (4) a polynucleotide sequence encoding a fourth autoprotease peptide, (5) a polynucleotide sequence encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4, (6) an alphavirus subgenomic promoter, (7) the non-naturally occurring polynucleotide sequence encoding one or more HBV antigens of the application, (8) an alphavirus 3' untranslated region (3' UTR), and (9) optionally, a poly adenosine sequence.
  • an alphavirus 5’ untranslated region 5’-UTR
  • DLP downstream loop
  • a self-replicating RNA vector of the application comprises a downstream loop (DLP) motif of a virus species.
  • DLP downstream loop
  • a “downstream loop” or “DLP motif’ refers to a polynucleotide sequence comprising at least one RNA stemloop, which when placed downstream of a start codon of an open reading frame (ORF) provides increased translation the ORF compared to an otherwise identical construct without the DLP motif.
  • ORF open reading frame
  • members of the Alphavirus genus can resist the activation of antiviral RNA-activated protein kinase (PKR) by means of a prominent RNA structure present within in viral 26S transcripts, which allows an eIF2-independent translation initiation of these mRNAs.
  • PTR antiviral RNA-activated protein kinase
  • This structure is located downstream from the AUG in SINV 26S mRNA.
  • the DLP is also detected in Semliki Forest virus (SFV).
  • SFV Semliki Forest virus
  • Similar DLP structures have been reported to be present in at least 14 other members of the Alphavirus genus including New World (for example, MAYV, UNAV, EEEV (NA), EEEV (SA), AURAV) and Old World (SV, SFV, BEBV, RRV, SAG, GETV, MIDV, CHIKV, and ONNV) members.
  • New World for example, MAYV, UNAV, EEEV (NA), EEEV (SA), AURAV
  • Old World SV, SFV, BEBV, RRV, SAG, GETV, MIDV, CHIKV, and ONNV
  • the predicted structures of these Alphavirus 26S mRNAs were constructed based on SHAPE (selective 2'-hydroxyl acylation and primer extension) data (Toribio et al.
  • a replicon RNA of the application comprises a DLP motif exhibiting at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequences set forth in SEQ ID NO: 57.
  • the self-replicating RNA molecule also contains a coding sequence for an autoprotease peptide operably linked downstream of the DLP motif and upstream of the coding sequences of the nonstructural proteins (e.g., one or more of nspl-4) or gene of interest (e.g., an HBV antigen described herein).
  • a coding sequence for an autoprotease peptide operably linked downstream of the DLP motif and upstream of the coding sequences of the nonstructural proteins (e.g., one or more of nspl-4) or gene of interest (e.g., an HBV antigen described herein).
  • a replicon RNA of the application comprises a coding sequence for P2A having the amino acid sequence of SEQ ID NO: 11.
  • the coding sequence exhibits at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequences set forth in SEQ ID NO: 12.
  • any of the replicons of the invention can also comprise a 5’ and a 3’ untranslated region (UTR).
  • the UTRs can be wild type New World or Old World alphavirus UTR sequences, or a sequence derived from any of them.
  • the 5’ UTR can be of any suitable length, such as about 60 nt or 50-70 nt or 40-80 nt.
  • the 5’ UTR can also have conserved primary or secondary structures (e.g. one or more stem- loop(s)) and can participate in the replication of alphavirus or of replicon RNA.
  • the 3’ UTR can be up to several hundred nucleotides, for example it can be 50- 900 or 100-900 or 50-800 or 100-700 or 200 nt - 700 nt.
  • the ‘3 UTR also can have secondary structures, e.g. a step loop, and can be followed by a polyadenylate tract or poly-A tail.
  • the 5’ and 3’ untranslated regions can be operably linked to any of the other sequences encoded by the replicon.
  • the UTRs can be operably linked to a promoter and/or sequence encoding a heterologous protein or peptide by providing sequences and spacing necessary for recognition and transcription of the other encoded sequences.
  • the poly adenylation signal can be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human [3- globin polyadenylation signal.
  • bGH bovine growth hormone
  • hGH human growth hormone
  • a self-repli eating RNA replicon of the application comprises a modified 5’ untranslated region (5'-UTR), preferably the RNA replicon is devoid of at least a portion of a nucleic acid sequence encoding viral structural proteins.
  • the modified 5'-UTR can comprise one or more nucleotide substitutions at position 1, 2, 4, or a combination thereof.
  • the modified 5'-UTR comprises a nucleotide substitution at position 2, more preferably, the modified 5'-UTR has a U->G or U->A substitution at position 2.
  • a replicon RNA of the application comprises a 5'-UTR exhibiting at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequences set forth in SEQ ID NO: 55.
  • an RNA replicon of the application comprises, ordered from the 5’- to 3’-end, (1) a 5’-UTR having the polynucleotide sequence of SEQ ID NO: 55, (2) a 5’ replication sequence having the polynucleotide sequence of SEQ ID NO: 56, (3) a DLP motif comprising the polynucleotide sequence of SEQ ID NO: 57, (4) a polynucleotide sequence encoding a P2A sequence of SEQ ID NO: 11, (5) polynucleotide sequences encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4, having the nucleic acid sequences of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively, (6) a subgenomic promoter having polynucleotide sequence of SEQ ID NO: 62, (7)
  • the polynucleotide sequence encoding the P2A sequence comprises SEQ ID NO: 12
  • the non- naturally occurring polynucleotide sequence comprises the polynucleotide sequence of any one of SEQ ID NOs: 15 to 54
  • the RNA replicon further comprises a poly adenosine sequence.
  • the poly adenosine sequence has the sequence of SEQ ID NO: 64, at the 3 ’-end of the replicon.
  • an RNA replicon of the application comprises the polynucleotide sequence of any one of SEQ ID NOs: 65 to 72.
  • an RNA replicon of the application comprises a polynucleotide sequence encoding a signal peptide sequence.
  • the polynucleotide sequence encoding the signal peptide sequence is located upstream of or at the 5 ’-end of the polynucleotide sequence encoding an HBV antigen, such as an HBV PreSl antigen, an HBV core antigen and an HBV pol antigen.
  • HBV antigen such as an HBV PreSl antigen, an HBV core antigen and an HBV pol antigen.
  • Signal peptides typically direct localization of a protein, facilitate secretion of the protein from the cell in which it is produced, and/or improve antigen expression and cross-presentation to antigen-presenting cells.
  • a signal peptide can be present at the N-terminus of an HBV antigen when expressed from the replicon, but is cleaved off by signal peptidase, e.g., upon secretion from the cell.
  • An expressed protein in which a signal peptide has been cleaved is often referred to as the “mature protein.” Any signal peptide known in the art in view of the present disclosure can be used.
  • a signal peptide can be a cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as a Cystatin S signal peptide, an Ig heavy chain gamma signal peptide SPIgG, an Ig heavy chain epsilon signal peptide SPIgE, or a short leader peptide sequence.
  • Ig immunoglobulin
  • An exemplary amino acid sequence of a signal peptide is shown in SEQ ID NO: 77.
  • RNA replicons disclosed herein can be engineered, synthetic, or recombinant RNA replicons.
  • the term recombinant means any molecule (e.g. DNA, RNA, etc.), that is or results, however indirectly, from human manipulation of a polynucleotide.
  • a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector.
  • a recombinant RNA replicon can be one or more of the following: 1) synthesized or modified in vitro, for example, using chemical or enzymatic techniques (for example, by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse tianscription, transcription, base modification (including, e.g., methylation), or recombination (including homologous and sitespecific recombination) of nucleic acid molecules; 2) conjoined nucleotide sequences that are not conjoined in nature; 3) engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleotide sequence; and 4) manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleotide sequence.
  • any of the components or sequences of the RNA replicon can be operably linked to any other of the components or sequences.
  • the components or sequences of the RNA replicon can be operably linked for the expression of at least one heterologous protein or peptide (or biotherapeutic) in a host cell or treated organism and/or for the ability of the replicon to self-replicate.
  • the term “operably linked” denotes a functional linkage between two or more sequences that are configured so as to perform their usual function.
  • a promoter or UTR operably linked to a coding sequence is capable of effecting the transcription and expression of the coding sequence when the proper enzymes are present.
  • the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof.
  • an operable linkage between an RNA sequence encoding a heterologous protein or peptide and a regulatory sequence is a functional link that allows for expression of the polynucleotide of interest.
  • Operably linked can also refer to sequences such as the sequences encoding nsPl-4, the UTRs, promoters, and other sequences encoding in the RNA replicon, are linked so that they enable transcription and translation of the biotherapeutic molecule and/or replication of the replicon.
  • the UTRs can be operably linked by providing sequences and spacing necessary for recognition and translation by a ribosome of other encoded sequences.
  • the RNA replicons of the invention can be derived from alphavirus genomes, meaning that they have some of the structural characteristics of alphavirus genomes, or be similar to them.
  • the RNA replicons of the invention can be modified alphavirus genomes.
  • one or more sequences of the replicon can be provided “in trans,” i.e., the sequences of the replicon are provided on more than one RNA molecule.
  • all of the sequences of the replicon are present on a single RNA molecule, which can also be administered to a mammal to be treated as described herein.
  • percent identity or “homology” or “shared sequence identity” or “percent (%) sequence identity” with respect to nucleic acid or polypeptide sequences are defined as the percentage of nucleotide or amino acid residues in the candidate sequence that are identical with the known polynucleotides or polypeptides, after aligning the sequences for maximum percent identity and introducing gaps, if necessary, to achieve the maximum percent homology.
  • N-terminal or C-terminal insertions or deletions shall not be construed as affecting homology, and internal deletions and/or insertions into the nucleotide or polypeptide sequence of less than about 30, less than about 20, or less than about 10 or less than 5 amino acid residues shall not be construed as affecting homology.
  • Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx (Altschul (1997), Nucleic Acids Res. 25, 3389-3402, and Karlin (1990), Proc. Natl. Acad. Sci.
  • the default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff (1992), Proc. Natl. Acad. Sci. USA 89, 10915-10919), recommended for query sequences over 85 in length (nucleotide bases or amino acids).
  • the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N can be +5 and -4, respectively.
  • M i.e., the reward score for a pair of matching residues
  • N i.e., the penalty score for mismatching residues
  • the default values for M and N can be +5 and -4, respectively.
  • Sequences disclosed therefore include polynucleotide or polypeptide sequences having sequence identities of at least 40%, at least 45%, at least 50%, at least 55%, of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%, for example at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% or 85-99% or 85-95% or 90-99% or 95-99% or 97-99% or 98-99% sequence identity with the full-length polynucleotide or polypeptide sequence of any polynucleotide or polypeptide sequence described herein, respectively, such as SEQ ID NOs: 1-90, and fragments thereof.
  • fragments or portions of any of the sequences disclosed herein can include sequences having at least 5 or at least 7 or at least 10, or at least 20, or at least 30, at least 50, at least 75, at least 100, at least 125, 150 or more or 5-10 or 10-12 or 10-15 or 15-20 or 20-40 or 20-50 or 30-50 or 30-75 or 30-100 amino acid or nucleic acid residues of the entire sequence, or at least 100 or at least 200 or at least 300 or at least 400 or at least 500 or at least 600 or at least 700 or at least 800 or at least 900 or at least 1000 or 100-200 or 100-500 or 100-1000 or 500- 1000 amino acid or nucleic acid residues, or any of these amounts but less than 500 or less than 700 or less than 1000 or less than 2000 consecutive amino acids or nucleic acids of any of SEQ ID NOs: 1-90 or of any fragment disclosed herein.
  • variants of such sequences e.g., where at least one or two or three or four or five amino acid residues have been inserted N- and/or C-terminal to, and/or within, the disclosed sequence(s) which contain(s) the insertion and substitution, and nucleic acid sequences encoding such variants.
  • Contemplated variants can additionally or alternately include those containing predetermined mutations by, e.g., homologous recombination or site-directed or PCR mutagenesis, and the corresponding polypeptides or nucleic acids of other species, including, but not limited to, those described herein, the alleles or other naturally occurring variants of the family of polypeptides or nucleic acids which contain an insertion and substitution; and/or derivatives wherein the polypeptide has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid which contains the insertion and substitution (for example, a detectable moiety such as an enzyme).
  • the nucleic acid sequences described herein can be RNA sequences.
  • the RNA replicons of the invention can include an RNA sequence encoding at least one protein or peptide that is heterologous to an alphavirus and can also be (but is not necessarily) heterologous to the human, mammal, or animal that expresses the RNA sequence in the body.
  • the replicons can have RNA sequence(s) encoding two or three or four or more heterologous proteins or peptides.
  • the heterologous protein or peptide is an HBV antigen as described herein.
  • the sequence encoding the heterologous protein or peptide can be operably linked to one or more other sequences of the replicon (e.g. a promoter or 5’ or 3’ UTR sequences), and can be under the control of a sub-genomic promoter so that the heterologous protein or peptide is expressed in the human, mammal, or animal.
  • the RNA replicon of the invention can have an RNA sequence encoding a heterologous protein or peptide (e.g. a monoclonal antibody or a biotherapeutic protein or peptide), RNA sequences encoding amino acid sequences derived from wild type alphavirus nsPl, nsP2, nsP3, and nsP4 protein sequences, and 5’ and 3’ UTR sequences (for non-structural protein-mediated amplification).
  • the RNA replicons can also have a 5’ cap and a poly adenylate (or poly -A) tail.
  • the immunogenicity of a heterologous protein or peptide can be determined by a number of assays known to persons of ordinary skill, for example immunostaining of intracellular cytokines or secreted cytokines by epitope-specific T-cell populations, or by quantifying frequencies and total numbers of epitope-specific T-cells and characterizing their differentiation and activation state, e.g., short-lived effector and memory precursor effector CD8+ T-cells. Immunogenicity can also be determined by measuring an antibody-mediated immune response, e.g. the production of antibodies by measuring serum IgA or IgG titers.
  • the RNA replicons of the application can optionally further encode one or more heterologous proteins or peptides that can be any protein or peptide, including but not limited to, cytokines, growth factors, immunoglobulins, monoclonal antibodies (including Fab antigen-binding fragments, Fc fusion proteins), hormones, interferons, interleukins, regulatory peptides and proteins.
  • heterologous proteins or peptides can be any protein or peptide, including but not limited to, cytokines, growth factors, immunoglobulins, monoclonal antibodies (including Fab antigen-binding fragments, Fc fusion proteins), hormones, interferons, interleukins, regulatory peptides and proteins.
  • the heterologous protein or peptide can be encoded by an RNA sequence of up to 5 kb or up to 6 kb or up to 7 kb or up to 8 kb, or up to 9 kb or up to 10 kb or up to 11 kb or up to 12 kb.
  • the heterologous protein can also be a single-chain antibody molecule.
  • the alphavirus replicons of the invention can also have a sub-genomic promoter for expression of the heterologous protein or peptide.
  • the term “subgenomic promoter,” as used herein, refers to a promoter of a subgenomic mRNA of a viral nucleic acid.
  • an “alphavirus subgenomic promoter” is a promoter as originally defined in an alphavirus genome that directs transcription of a subgenomic messenger RNA as part of the alphavirus replication process.
  • heterologous when used in reference to a polynucleotide, a gene, a nucleic acid, a polypeptide, a protein, or an enzyme, refers to a polynucleotide, gene, a nucleic acid, polypeptide, protein, or an enzyme that is not derived from the host species.
  • heterologous gene or “heterologous nucleic acid sequence” as used herein, refers to a gene or nucleic acid sequence from a different species than the species of the host organism it is introduced into. Heterologous sequences can also be synthetic and not derived from an organism or not found in Nature.
  • a gene regulatory sequence or to an auxiliary nucleic acid sequence used for manipulating expression of a gene sequence e.g. a 5' untranslated region, 3' untranslated region, poly A addition sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genome homology region, recombination site, etc.
  • heterologous means that the regulatory or auxiliary sequence or sequence encoding a protein domain or localization sequence is from a different source than the gene with which the regulatory or auxiliary nucleic acid sequence or nucleic acid sequence encoding a protein domain or localization sequence is juxtaposed in a genome, chromosome or episome.
  • a promoter operably linked to a gene to which it is not operably linked to in its natural state is referred to herein as a “heterologous promoter,” even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked.
  • heterologous when referring to a protein localization sequence or protein domain of an engineered protein, “heterologous” means that the localization sequence or protein domain is derived from a protein different from that into which it is incorporated by genetic engineering.
  • non-naturally occurring refers to a nucleic acid molecule or non-naturally occurring polynucleotide sequence that has been altered through human intervention.
  • a recombinant nucleic acid molecule 1) has been synthesized or modified in vitro, for example, using chemical or enzymatic techniques (for example, by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, e.g., methylation), or recombination (including homologous and site-specific recombination) of nucleic acid molecules; 2) includes conjoined nucleotide sequences that are not conjoined in nature, 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleic acid molecule sequence, and/or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleic acid sequence.
  • chemical or enzymatic techniques for example, by
  • a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector or that has been integrated into an RNA replicon.
  • an RNA replicon of the invention comprises, ordered from the 5’ - to 3 ’-end: a 5’ untranslated region (5’-UTR) required for nonstructural protein- mediated amplification of an RNA virus; a polynucleotide sequence encoding at least one, preferably all, of non-structural proteins of the RNA virus; a subgenomic promoter of the RNA virus; a non-naturally occurring polynucleotide sequence described herein; and a 3’ untranslated region (3’-UTR) required for nonstructural protein-mediated amplification of the RNA virus.
  • 5’-UTR 5’ untranslated region required for nonstructural protein- mediated amplification of an RNA virus
  • a polynucleotide sequence encoding at least one, preferably all, of non-structural proteins of the RNA virus
  • a subgenomic promoter of the RNA virus a non-naturally occurring polynucleotide sequence described herein
  • an RNA replicon of the invention comprises, ordered from the 5’ - to 3 ’-end: an alphavirus 5’ untranslated region (5’-UTR); a 5’ replication sequence of an alphavirus non-structural gene nspl; a downstream loop (DLP) motif of a virus species; a polynucleotide sequence encoding a fourth autoprotease peptide; a polynucleotide sequence encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4; an alphavirus subgenomic promoter; a non-naturally occurring polynucleotide sequence described herein; an alphavirus 3' untranslated region (3' UTR); and, optionally, a poly adenosine sequence.
  • an alphavirus 5’ untranslated region 5’-UTR
  • a 5’ replication sequence of an alphavirus non-structural gene nspl a downstream loop (D
  • the DLP motif is from a virus species selected from the group consisting of Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MTDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (U AV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndum
  • EEEV Eastern e
  • the fourth autoprotease peptide is selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2 A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof.
  • the fourth autoprotease peptide comprises the peptide sequence of P2A.
  • the fourth autoprotease peptide comprises SEQ ID NO: 11.
  • a polynucleotide sequence encoding a fourth autoprotease peptide comprises SEQ ID NO: 12. In some embodiments, a polynucleotide sequence encoding a fourth autoprotease peptide consists of SEQ ID NO: 12.
  • an RNA replicon of the invention comprises, ordered from the 5’- to 3’-end: a 5’-UTR having the polynucleotide sequence of SEQ ID NO: 55; a 5’ replication sequence having the polynucleotide sequence of SEQ ID NO: 56; a DLP motif comprising the polynucleotide sequence of SEQ ID NO: 57; a polynucleotide sequence encoding a P2A sequence of SEQ ID NO: 11; polynucleotide sequences encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4, as those encoded by the nucleic acid sequences of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively; a subgenomic promoter having polynucleotide sequence of SEQ ID NO: 62; a non-naturally occurring
  • the polynucleotide sequence encoding the P2A sequence comprises SEQ ID NO: 12
  • the non-naturally occurring polynucleotide sequence comprises the polynucleotide sequence of any one of SEQ ID NOs: 15 to 54
  • the RNA replicon further comprises a poly adenosine sequence.
  • the poly adenosine sequence has the sequence of SEQ ID NO: 64 at the 3’-end of the replicon.
  • an RNA replicon of the invention comprises the polynucleotide sequence of any one of SEQ ID NOs: 65 to 72.
  • a nucleic acid molecule comprising a DNA sequence encoding an RNA replicon disclosed herein further comprises a T7 promoter operably linked to the 5 ’-end of the DNA sequence. More preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO: 73.
  • RNA replicon of the application comprising transcribing a nucleic acid molecule comprising a DNA sequence encoding a RNA replicon disclosed herein.
  • the nucleic acid molecule is transcribed in vivo. In some embodiments, the nucleic acid molecule is transcribed in vitro.
  • the application also provides cells, preferably isolated cells, comprising any of the polynucleotides and vectors described herein.
  • the cells can, for instance, be used for recombinant protein production, or for the production of viral particles.
  • the cells can be used for production of an RNA replicon.
  • Host cells comprising a RNA replicon or a nucleic acid encoding the RNA replicon of the application also form part of the invention.
  • the HBV antigens may be produced through recombinant DNA technology involving expression of the molecules in host cells, e.g., Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6 cells, or yeast, fungi, insect cells, and the like, or transgenic animals or plants.
  • the cells are from a multicellular organism, in certain embodiments they are of vertebrate or invertebrate origin.
  • the cells are mammalian cells, such as human cells, or insect cells.
  • the production of a recombinant protein, such the HBV antigens of the invention, in a host cell comprises the introduction of a heterologous nucleic acid molecule encoding the protein in expressible format into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid molecule and allowing expression of the protein in said cell.
  • the nucleic acid molecule encoding a protein in expressible format may be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like.
  • promoters can be used to obtain expression of a gene in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed. Further regulatory sequences may be added. Many promoters can be used for expression of a transgene(s), and are known to the skilled person, e.g. these may comprise viral, mammalian, synthetic promoters, and the like.
  • a non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is a CMV -promoter (US 5,385,839), e.g. the CMV immediate early promoter, for instance comprising nt.
  • a polyadenylation signal for example the bovine growth hormone polyA signal (US 5,122,458), may be present behind the transgene(s).
  • a polyadenylation signal for example the bovine growth hormone polyA signal (US 5,122,458)
  • a polyadenylation signal for example the bovine growth hormone polyA signal (US 5,122,458)
  • pcDNA and pEF vector series of Invitrogen pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc, which can be used to recombinantly express the protein of interest, or to obtain suitable promoters and/or transcription terminator sequences, polyA sequences, and the like.
  • the cell culture can be any type of cell culture, including adherent cell culture, e.g., cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. More recently, continuous processes based on perfusion principles are becoming more common and are also suitable. Suitable culture media are also well known to the skilled person and can generally be obtained from commercial sources in large quantities, or custom-made according to standard protocols. Culturing can be done for instance in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems and the like. Suitable conditions for culturing cells are known (see e.g.
  • Cell culture media are available from various vendors, and a suitable medium can be routinely chosen for a host cell to express the protein of interest, here the HBV antigens.
  • the suitable medium may or may not contain serum.
  • Embodiments of the application thus also relate to a method of making an HBV antigen of the application.
  • the method comprises transfecting a host cell with an expression vector comprising a polynucleotide encoding an HBV antigen of the application operably linked to a promoter, growing the transfected cell under conditions suitable for expression of the HBV antigen, and optionally purifying or isolating the HBV antigen expressed in the cell.
  • the HBV antigen can be isolated or collected from the cell by any method known in the art including affinity chromatography, size exclusion chromatography, etc. Techniques used for recombinant protein expression will be well known to one of ordinary skill in the art in view of the present disclosure.
  • the expressed HBV antigens can also be studied without purifying or isolating the expressed protein, e.g., by analyzing the supernatant of cells transfected with an expression vector encoding the HBV antigen and grown under conditions suitable for expression of the HBV antigen.
  • non-naturally occurring or recombinant polypeptides comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, 84, 85 or 86, or SEQ ID NO: 9.
  • isolated nucleic acid molecules encoding these sequences, vectors comprising these sequences operably linked to a promoter, and compositions comprising the polypeptide, polynucleotide, or vector are also contemplated by the application.
  • a recombinant polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, 84, 85 or 86, or SEQ ID NO: 9, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, 84, 85 or 86, or SEQ ID NO: 9.
  • a non-naturally occurring or recombinant polypeptide consists of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 84, 85, 85 or 86, or SEQ ID NO:
  • the RNAi component comprises one or more RNAi agents.
  • Each RNAi agent disclosed herein includes at least a sense strand and an antisense strand.
  • the sense strand and the antisense strand can be partially, substantially, or fully complementary to each other.
  • the length of the RNAi agent sense and antisense strands described herein each can be 16 to 30 nucleotides in length.
  • the sense and antisense strands are independently 17 to 26 nucleotides in length.
  • the sense and antisense strands are independently 19 to 26 nucleotides in length.
  • the sense and antisense strands are independently 21 to 26 nucleotides in length.
  • the sense and antisense strands are independently 21 to 24 nucleotides in length.
  • the sense and antisense strands can be either the same length or different lengths.
  • the HBV RNAi agents disclosed herein have been designed to include antisense strand sequences that are at least partially complementary to a sequence in the HBV genome that is conserved across the majority of known serotypes of HBV.
  • the RNAi agents described herein upon delivery to a cell expressing HBV, inhibit the expression of one or more HBV genes in vivo or in vitro.
  • An RNAi agent includes a sense strand (also referred to as a passenger strand) that includes a first sequence, and an antisense strand (also referred to as a guide strand) that includes a second sequence.
  • a sense strand of the HBV RNAi agents described herein includes a core stretch having at least about 85% identity to a nucleotide sequence of at least 16 consecutive nucleotides in an HBV mRNA.
  • the sense strand core nucleotide stretch having at least about 85% identity to a sequence in an HBV mRNA is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.
  • An antisense strand of an HBV RNAi agent comprises a nucleotide sequence having at least about 85% complementary over a core stretch of at least 16 consecutive nucleotides to a sequence in an HBV mRNA and the corresponding sense strand.
  • the antisense strand core nucleotide sequence having at least about 85% complementarity to a sequence in an HBV mRNA or the corresponding sense strand is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.
  • the RNAi component comprises a first RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107, or a second RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109,
  • the RNAi component comprises a first RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO: 99, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107, and a second RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO
  • the first and the second RNAi agents disclosed herein comprise any of the sequences in Table 1.
  • RNAi agents are delivered to target cells or tissues using any oligonucleotide delivery technology known in the art.
  • Nucleic acid delivery methods include, but are not limited to, by encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, proteinaceous vectors or Dynamic Polyconjugates (DPCs) (see, for example WO 2000/053722, WO 2008/0022309, WO 2011/104169, and WO 2012/083185, each of which is incorporated herein by reference).
  • DPCs Dynamic Polyconjugates
  • an HBV RNAi agent is delivered to target cells or tissues by covalently linking the RNAi agent to a targeting group.
  • the targeting group can include a cell receptor ligand, such as an asialoglycoprotein receptor (ASGPr) ligand.
  • ASGPr asialoglycoprotein receptor
  • an ASGPr ligand includes or consists of a galactose derivative cluster.
  • a galactose derivative cluster includes an N-acetyl-galactosamine trimer or an N-acetyl-galactosamine tetramer.
  • a galactose derivative cluster is an N-acetyl-galactosamine trimer or an N-acetyl-galactosamine tetramer.
  • a targeting group can be linked to the 3' or 5' end of a sense strand or an antisense strand of an HBV RNAi agent. In some embodiments, a targeting group is linked to the 3' or 5' end of the sense strand. In some embodiments, a targeting group is linked to the 5’ end of the sense strand. In some embodiments, a targeting group is linked to the RNAi agent via a linker.
  • the RNAi component comprises a combination or cocktail of a first and a second RN Ai agent having different nucleotide sequences.
  • the first and the second RNAi agents are each separately and independently linked to targeting groups.
  • the first and the second RNAi agents are each linked to targeting groups comprised of N-acetyl-galactosamines.
  • each of the RNAi agents is linked to the same targeting group.
  • each of the RNAi agents is linked to different targeting groups, such as targeting groups having different chemical structures.
  • targeting groups are linked to the first and the second RNAi agents without the use of an additional linker.
  • the targeting group is designed having a linker readily present to facilitate the linkage to the first or the second RN Ai agent.
  • the first and the second RNAi agents may be linked to the targeting groups using the same linkers.
  • the first and the second RNAi agents are linked to the targeting groups using different linkers.
  • the non-nucleotide group can be covalently linked to the 3' and/or 5' end of either the sense strand and/or the antisense strand.
  • the first or second RNAi agent contains a non-nucleotide group linked to the 3' and/or 5' end of the sense strand.
  • a non-nucleotide group is linked to the 5' end of the first or second RNAi agent sense strand.
  • a non-nucleotide group may be linked directly or indirectly to the first or second RNAi agent via a linker/linking group.
  • a non-nucleotide group is linked to the first or second RNAi agent via a labile, cleavable, or reversible bond or linker.
  • Targeting groups and linking groups include the following, for which their chemical structures are provided below in Table 2: (PAZ), (NAGI 3), (NAG13)s, (NAGI 8), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NA
  • the first or the second RNAi agent contains one or more modified nucleotides.
  • a “modified nucleotide” is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide).
  • at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides.
  • modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides (represented herein as Ab), 2'-modified nucleotides, 3' to 3' linkages (inverted) nucleotides (represented herein as invdN, invN, invn, invAb), non-natural base-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2',3'-seco nucleotide mimics (unlocked nucleobase analogues, represented herein as NUNA or NUNA), locked nucleotides (represented herein as NLNA or NLNA), 3'-O-methoxy (2' intemucleoside linked) nucleotides (represented herein as 3'-OMen), 2'-F-Arabino nucleotides (represented herein as NfANA or NfANA), 5
  • 2'-modified nucleotides include, but are not limited to, 2'-O-methyl nucleotides (represented herein as a lower case letter 'n' in a nucleotide sequence), 2'-deoxy-2'-fluoro nucleotides (represented herein as Nf, also represented herein as 2'-fluoro nucleotide), 2'-deoxy nucleotides (represented herein as dN), 2'-methoxy ethyl (2'-O-2-methoxylethyl) nucleotides (represented herein as NM or 2'-MOE), 2'-amino nucleotides, and 2'-alkyl nucleotides.
  • 2'-O-methyl nucleotides represented herein as a lower case letter 'n' in a nucleotide sequence
  • 2'-deoxy-2'-fluoro nucleotides represented herein as Nf, also represented herein as 2'-fluoro nu
  • RNAi agent sense strands and antisense strands may be synthesized and/or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide.
  • Modified nucleobases include synthetic and natural nucleobases, such as 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, (e.g, 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me- C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6- methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cyto
  • all or at least 90% of the nucleotides of the first or the second RNAi agent are modified nucleotides.
  • an RNAi agent wherein at least 90% of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides.
  • a sense strand wherein at least 90% of the nucleotides present are modified nucleotides, is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides.
  • an antisense sense strand wherein at least 90% of the nucleotides present are modified nucleotides, is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides.
  • one or more nucleotides of an RNAi agent is a ribonucleotide.
  • one or more nucleotides of the first or the second RNAi agent are linked by non-standard linkages or backbones (i.e., modified intemucleoside linkages or modified backbones).
  • a modified intemucleoside linkage is a non-phosphate-containing covalent intemucleoside linkage.
  • Modified intemucleoside linkages or backbones include, but are not limited to, 5 ’-phosphorothioate groups (represented herein as a lower case “s”), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g, methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphorami dates), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nu
  • a modified intemucleoside linkage or backbone lacks a phosphorus atom.
  • Modified intemucleoside linkages lacking a phosphoms atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages.
  • modified intemucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene- containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.
  • a sense strand of the first or the second RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages
  • an antisense strand of the first or the second RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages
  • both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages.
  • a sense strand of the first or the second RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages
  • an antisense strand of the first or the second RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages
  • both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.
  • the first or the second RNAi agent sense strand contains at least two phosphorothioate intemucleoside linkages. In some embodiments, the at least two phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, the at least two phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6- 8 from the 5' end of the sense strand. In some embodiments, the first or the second RNAi agent antisense strand contains four phosphorothioate intemucleoside linkages.
  • the four phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 5' end of the sense strand and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end.
  • the first or the second RNAi agent contains at least two phosphorothioate intemucleoside linkages in the sense strand and three or four phosphorothioate intemucleoside linkages in the antisense strand.
  • the first or the second RNAi agent contains one or more modified nucleotides and one or more modified intemucleoside linkages. In some embodiments, a 2'-modified nucleoside is combined with modified intemucleoside linkage. In some embodiments, the first and the second RNAi agents disclosed herein comprise any of the modified sequences in Table 3.
  • A adenosine-3 '-phosphate
  • C cytidine-3 '-phosphate
  • G guanosine-3 '-phosphate
  • Nf any 2'-fluoro modified nucleotide
  • Afs 2'-fluoroadenosine-3'-phosporothioate
  • Cf 2'-fluorocytidine-3 '-phosphate
  • Cfs 2'-fluorocytidine-3 '-phosphorothioate
  • NUNA 2',3'-seco nucleotide mimics (unlocked nucleobase analogs)
  • NLNA locked nucleotide
  • NfANA 2'-F-Arabino nucleotide
  • NM 2'-methoxyethyl nucleotide
  • AM 2'-methoxyethyladenosine-3'-phosphate
  • AMs 2'-methoxyethyladenosine-3'-phosphorothioate
  • TM 2'-methoxyethylthymidine-3'-phosphate
  • TMs 2'-methoxyethylthymidine-3'-phosphorothioate
  • the first RNAi agent comprises SEQ ID NO:97 and SEQ ID NO: 106. In some embodiments, the first RNAi agent comprises SEQ ID NO: 108 and SEQ ID NO: 106. In some embodiments, the first RNAi agent comprises SEQ ID NO:99 and SEQ ID NO: 107. In some embodiments, the first RNAi agent comprises SEQ ID NO:93 and SEQ ID NO: 102, 103, or 105. In some embodiments, the first RNAi agent comprises SEQ ID NO:94 and SEQ ID NO: 102, 103, or 105. In some embodiments, the first RNAi agent comprises SEQ ID NO:95 and SEQ ID NO: 102, 103, or 105.
  • the first RNAi agent comprises SEQ ID NO:96 and SEQ ID NO: 106.
  • the second RNAi agent comprises SEQ ID NO: 101 and SEQ ID NO: 111.
  • the second RNAi agent comprises SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:97 and SEQ ID NO: 106 and a second RNAi agent comprising SEQ ID NO: 101 and SEQ ID NO: 111.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 108 and SEQ ID NO: 106 and a second RNAi agent comprising SEQ ID NO: 101 and SEQ ID NO: 111.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:99 and SEQ ID NO: 107 and a second RNAi agent comprising SEQ ID NO: 101 and SEQ ID NO: 111.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:93 and SEQ ID NO: 102, 103, or 105 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:94 and SEQ ID NO: 102, 103, or 105 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108,
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:95 and SEQ ID NO: 102, 103, or 105 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:96 and SEQ ID NO: 106 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or
  • the RNAi component comprises a first and a second RNAi agent in a ratio of about 1 : 1, 2: 1, 3: 1, 4: 1 or 5: 1.
  • the two HBV RNAi agents are administered in a ratio of about 2:1.
  • ratio is intended in accordance with its ordinary meaning in the field, e.g., as a weight ratio or as a molar ratio.
  • the first and the second RNAi agents are each independently conjugated to (NAG37)s, (NAG31)s, or (NAG25)s, the first RNAi agent comprises an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 103 or SEQ ID NO: 105, the second RNAi agent comprises an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 108 or SEQ ID NO: 110.
  • the first HBV RNAi agent comprises or consists of the duplex of SEQ ID NO: 94 and 103 linked to (NAG37)s shown as a sodium salt having the structure represented by the following:
  • the first HBV RNAi agent comprises or consists of the duplex of SEQ ID NO: 94 and 105 linked to (NAG25)s shown as a sodium salt having the structure represented by the following: [0246] In some embodiments, the first HBV RNAi agent comprises or consists of the duplex of SEQ ID NO: 94 and 103 linked to (NAG37)s shown as a free acid having the structure represented by the following: [0247] In some embodiments, the first HBV RNAi agent comprises or consists of the duplex of SEQ ID NO: 100 and 108 linked to (NAG37)s shown as a sodium salt having the structure represented by the following: [0248] In some embodiments, the first HBV RNAi agent comprises or consists of the duplex of SEQ ID NO: 100 and 110 linked to (NAG25)s shown as a sodium salt having the structure represented by the following: [0249] In some embodiments, the first HBV RNAi agent comprises or consists of the duplex of SEQ ID NO: 94 and
  • the RNAi component is administered to the subject subcutaneously or orally, more particularly subcutaneously.
  • the RNAi component is in a salt form (e.g., in a sodium salt form) or is in a free acid form.
  • composition comprising an RNAi component and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, wherein
  • RNAi component comprises
  • a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
  • a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; and
  • nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3’-end:
  • IVS internal ribosome entry sequence
  • first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
  • the application also relates to compositions, pharmaceutical compositions, immunogenic combinations, and more particularly vaccines, comprising one or more HBV antigens, polynucleotides, and/or vectors encoding one more HBV antigens according to the application.
  • Any of the HBV antigens, polynucleotides (including RNA and DNA), and/or vectors of the application described herein can be used in the compositions, pharmaceutical compositions, immunogenic combinations, and vaccines of the application.
  • compositions comprising any nucleic acid molecule, vector, or RNA replicon described herein, together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is non-toxic and should not interfere with the efficacy of the active ingredient.
  • Pharmaceutically acceptable carriers can include one or more excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings.
  • suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like.
  • suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
  • the aqueous solution/suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives.
  • compositions of the application can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections.
  • the parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection.
  • Pharmaceutical compositions of the application can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal.
  • compositions of the application are formulated for parental injection, preferably subcutaneous, intradermal injection, or intramuscular injection, more preferably intramuscular injection.
  • administration can be by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection.
  • Intramuscular injection can be combined with electroporation, i.e., application of an electric field to facilitate delivery of the DNA plasmids to cells.
  • electroporation refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane.
  • in vivo electroporation electrical fields of appropriate magnitude and duration are applied to cells, inducing a transient state of enhanced cell membrane permeability, thus enabling the cellular uptake of molecules unable to cross cell membranes on their own. Creation of such pores by electroporation facilitates passage of biomolecules, such as plasmids, oligonucleotides, siRNAs, drugs, etc., from one side of a cellular membrane to the other.
  • In vivo electroporation for the delivery of DNA vaccines has been shown to significantly increase plasmid uptake by host cells, while also leading to mild-to-moderate inflammation at the injection site.
  • electroporation is combined with intramuscular injection.
  • electroporation is also possible to combine electroporation with other forms of parenteral administration, e.g., intradermal injection, subcutaneous injection, etc.
  • Electroporation can be accomplished using an in vivo electroporation device.
  • electroporation devices and electroporation methods that can facilitate delivery of compositions and immunogenic combinations of the application, include CELLECTRA® (Inovio Pharmaceuticals, Blue Bell, PA), Eigen electroporator (Inovio Pharmaceuticals, Inc.) Tri-GridTM delivery system (Ichor Medical Systems, Inc., San Diego, CA 92121) and those described in U.S. Patent No. 7,664,545, U.S. Patent No. 8,209,006, U.S. Patent No. 9,452,285, U.S. Patent No. 5,273,525, U.S. Patent No. 6,110,161, U.S. Patent No. 6,261,281, U.S. Patent No. 6,958,060, and U.S. Patent No.
  • in vivo electroporation devices are described in International Patent Application entitled “Method and Apparatus for the Delivery of Hepatitis B Virus (HBV) Vaccines,” filed on the same day as this application with the Attorney Docket Number 688097-405WO, the contents of which are hereby incorporated by reference in their entireties.
  • HBV Hepatitis B Virus
  • a pulsed electric field for instance as described in, e.g., U.S. Patent No. 6,697,669, which is herein incorporated by reference in its entirety.
  • a pharmaceutical composition or immunogenic combination comprises one or more DNA plasmids
  • the method of administration is transdermal.
  • Transdermal administration can be combined with epidermal skin abrasion to facilitate delivery of the DNA plasmids to cells.
  • a dermatological patch can be used for epidermal skin abrasion. Upon removal of the dermatological patch, the composition or immunogenic combination can be deposited on the abraised skin.
  • Methods of delivery are not limited to the above described embodiments, and any means for intracellular delivery can be used.
  • Other methods of intracellular delivery contemplated by the methods of the application include, but are not limited to, liposome encapsulation, lipoplexes, nanoparticles, etc.
  • an RNA replicon of the application can be formulated in an immunogenic composition that comprises one or more lipid molecules, preferably positively charged lipid molecules.
  • an RNA replicon of the disclosure can be formulated using one or more liposomes, lipoplexes, and/or lipid nanoparticles.
  • liposome or lipid nanoparticle formulations described herein can comprise a poly cationic composition.
  • the formulations comprising a poly cationic composition can be used for the delivery of the RNA replicon described herein in vivo and/or ex vitro.
  • compositions for administration will typically comprise a buffered solution in a pharmaceutically acceptable carrier, e.g., an aqueous carrier such as buffered saline and the like, e.g., phosphate buffered saline (PBS).
  • a pharmaceutically acceptable carrier e.g., an aqueous carrier such as buffered saline and the like, e.g., phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the plasmid DNA can be present in a concentration of, e.g., 0.5 mg/mL to 5 mg/mL, such as 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, or 5 mg/mL, preferably at 1 mg/mL.
  • a pharmaceutical composition of the application comprising an RNA replicon can be administered in a concentration of, e.g., about 20 pg/mL to about 200 pg/mL, such as 20 pg/mL.
  • a pharmaceutical composition of the application comprising an RNA replicon can be administered in a concentration below 20 pg/mL. In some embodiments, a pharmaceutical composition of the application comprising an RNA replicon can be administered in a concentration above 200 pg/mL.
  • compositions of the application can be formulated as a vaccine (also referred to as an “immunogenic composition”) according to methods well known in the art. Such compositions can include adjuvants to enhance immune responses.
  • the optimal ratios of each component in the formulation can be determined by techniques well known to those skilled in the art in view of the present disclosure.
  • a pharmaceutical composition, composition, or immunogenic combination is a DNA vaccine.
  • DNA vaccines typically comprise bacterial plasmids containing a polynucleotide encoding an antigen of interest under control of a strong eukaryotic promoter. Once the plasmids are delivered to the cell cytoplasm of the host, the encoded antigen is produced and processed endogenously. The resulting antigen typically induces both humoral and cell-medicated immune responses.
  • DNA vaccines are advantageous at least because they offer improved safety, are temperature stable, can be easily adapted to express antigenic variants, and are simple to produce. Any of the DNA plasmids of the application can be used to prepare such a DNA vaccine.
  • RNA vaccines typically comprise at least one single-stranded RNA molecule encoding an antigen of interest, e.g., HBV antigen. Once the RNA is delivered to the cell cytoplasm of the host, the encoded antigen is produced and processed endogenously, inducing both humoral and cell-mediated immune responses, similar to a DNA vaccine.
  • the RNA sequence can be codon optimized to improve translation efficiency.
  • RNA molecule can be modified by any method known in the art in view of the present disclosure to enhance stability and/or translation, such by adding a polyA tail, e.g., of at least 30 adenosine residues; and/or capping the 5-end with a modified ribonucleotide, e.g., 7-methylguanosine cap, which can be incorporated during RNA synthesis or enzymatically engineered after RNA transcription.
  • An RNA vaccine can also be self-replicating RNA vaccine developed from an alphavirus expression vector.
  • Selfreplicating RNA vaccines comprise a replicase RNA molecule derived from a virus belonging to the alphavirus family with a subgenomic promoter that controls replication of the HBV antigen RNA followed by an artificial poly A tail located downstream of the replicase.
  • an adjuvant is included in a pharmaceutical composition of the application or co-administered with a pharmaceutical composition of the application.
  • Use of an adjuvant is optional and can further enhance immune responses when the composition is used for vaccination purposes.
  • Adjuvants suitable for co-administration or inclusion in compositions in accordance with the application should preferably be ones that are potentially safe, well tolerated, and effective in humans.
  • An adjuvant can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti- PD1, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/or TLR8 agonists), RIG-1 agonists, IL- 15 superagonists (Aitor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant, and IL-7-hyFc.
  • immune checkpoint inhibitors e.g., anti- PD1, anti-TIM-3, etc.
  • toll-like receptor agonists e.g., TLR7 agonists and/or TLR8 agonists
  • RIG-1 agonists e.g., IL- 15 superagonists (Aitor Bioscience)
  • mutant IRF3 and IRF7 genetic adjuvants e.g., STING
  • the application also provides methods of making pharmaceutical compositions and immunogenic combinations of the application.
  • a method of producing a pharmaceutical composition or immunogenic combination comprises mixing an isolated polynucleotide encoding an HBV antigen, vector, and/or polypeptide of the application with one or more pharmaceutically acceptable carriers.
  • One of ordinary skill in the art will be familiar with conventional techniques used to prepare such compositions.
  • the method of administration is a lipid composition, such as a lipid nanoparticle (LNP) or a liposome.
  • Lipid compositions preferably lipid nanoparticles or liposomes, that can be used to deliver a therapeutic product (such as one or more nucleic acid molecules of the invention), include, but are not limited to, liposomes or lipid vesicles, wherein an aqueous volume is encapsulated by amphipathic lipid bilayers, or wherein the lipids coat an interior that comprises a therapeutic product; or lipid aggregates or micelles, wherein the lipid-encapsulated therapeutic product is contained within a relatively disordered lipid mixture.
  • the lipid composition can provide the therapeutic product (such as one or more nucleic acid molecules of the invention) with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the therapeutic product is fully encapsulated in the lipid particle (e.g., to form an LNP).
  • Lipid compositions of this invention can comprise one or more lipids selected from cationic lipids, anionic lipids, zwitterionic lipids, neutral lipids, steroids, polymer conjugated lipids, phospholipids, glycolipids, and any combination of the foregoing.
  • the lipids can be saturated or unsaturated.
  • a mixture can comprise both saturated and unsaturated lipids.
  • the lipid compositions can be substantially free of liposomes or can contain liposomes.
  • the use of at least one unsaturated lipid for preparing liposomes is preferred. If an unsaturated lipid has two tails, both tails can be unsaturated, or it can have one saturated tail and one unsaturated tail.
  • the lipids and nucleic acid molecules can be mixed and configured in any suitable structures.
  • the lipid compositions comprise a cationic lipid to encapsulate and/or enhance the delivery of a nucleic acid molecule, such as a DNA or RNA molecule of the invention, into the target cell.
  • the cationic lipid can be any lipid species that carries a net positive charge at a selected pH, such as physiological pH.
  • the cationic lipids such as ionizable amino lipids, promote selfassembly of the components into macromolecular nanoparticles that encapsulate the nucleic acid (DNA and/or RNA).
  • the nucleic acid-containing nanoparticles are efficiently taken up into target cells by endocytosis. Once inside the endosome, the positively-charged lipid nanoparticles interact with the negatively-charged endosome membrane, causing disruption of the compartment and release of the nucleic acid molecules into the cytoplasm, where the nucleic acid molecules can be expressed.
  • cationic lipids have been described in the literature, many of which are commercially available.
  • suitable cationic lipids for use in the compositions and methods of the invention include l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2- DiLinoleyloxy-,N,N-dimethylaminopropane (DLinDMA), and 1,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA).
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DLinDMA 1,2- DiLinoleyloxy-,N,N-dimethylaminopropane
  • DLenDMA 1,2-Dilinolenyloxy-N,N- dimethylaminopropane
  • the pKa of formulated cationic lipids is correlated with the effectiveness of lipid particles for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)).
  • the preferred range of pKa is ⁇ 5 to ⁇ 7.
  • the cationic lipid is a compound of Formula (I):
  • R 1 is a substituted alkyl consisting of 10 to 31 carbons
  • R 2 is a linear alkyl, alkenyl or alkynyl consisting of 2 to 20 carbons
  • R 3 is a linear or branched alkane consisting of 1 to 6 carbons
  • R 4 and R 5 are the same or different, each a hydrogen or a linear or branched alkyl consisting of 1 to 6 carbons
  • L 1 and L 2 are the same or different, each a linear alkane of 1 to 20 carbons or a linear alkene of 2 to 20 carbons
  • X 1 is S or O; or a salt or solvate thereof.
  • Exemplary compounds of formula (I), their synthesis and uses thereof are described in US2018/0169268, all of which are herein incorporated by reference.
  • the cationic lipid is a compound of formula (II): wherein R 1 is a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons; L 1 is linear alkane of 1 to 15 carbons; R 2 is a linear alkyl or alkenyl of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons or a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons; L 2 is a linear alkane of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons; X is O or S; R 3 is a linear alkane of 1, 2, 3, 4, 5, or 6 carbons; and R 4 and R 5 are the same or different, each a linear or branched, noncyclic alkyl of 1, 2, 3, 4, 5, or 6 carbons; or a pharmaceutically acceptable salt or solvate thereof.
  • R 1 is a branched, non
  • the cationic lipid is a compound of formula (III), (IV) or (V):
  • R comprises a biologically active molecule
  • L 1 , L 2 , and L 3 independently for each occurrence comprise a ligand selected from the group consisting of a carbohydrate, a polypeptide, or a lipophile; a pharmaceutically acceptable salt thereof; or a pharmaceutical composition thereof.
  • ligand selected from the group consisting of a carbohydrate, a polypeptide, or a lipophile
  • a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.
  • R' is, at each occurrence, independently H or C 1 -C 12 alkyl; a 1 and a 2 are, at each occurrence, independently an integer from 3 to 12; b 1 and b 2 are, at each occurrence, independently 0 or 1; c 1 and c 2 are, at each occurrence, independently an integer from 2 to 12; d 1 and d 2 are, at each occurrence, independently an integer from 2 to 12; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein a 1 , a 2 , c 1 , c 2 , d 1 and d 2 are selected such that the sum of a 1 +c 1 +d 1 is an integer from 18 to 30, and the sum of a 2 +c 2 +d 2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxy carbony
  • compositions of the application include, but are not limited to, those described in W02019/036030, W02019/036028, WO2019/036008, WO2019/036000, US2016/0376224, US2017/0119904, W02018/200943 and WO2018/191657, the relevant contents on the lipids, their synthesis and uses are herein incorporated by reference in their entireties.
  • the lipid nanoparticles can be prepared by including multi-component lipid mixtures of varying ratios employing one or more cationic lipids, non-cationic lipids and polyethylene glycol (PEG) - modified, or pegylated, lipids, i.e. the lipid is modified by covalent attachment of a polyethylene glycol.
  • PEG provides the liposomes with a coat which can confer favorable pharmacokinetic characteristics e.g. it can increase stability and prevent non-specific adsorption of the liposomes.
  • the PEG has an average molecular mass of 1 kDa to 12 kDa, such as 1 , 2, 3. 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 kDa.
  • the length of the PEG can affect in vivo expression of encapsulated RNA, and that PEG with a molecular weight below 1 kDa (e.g. 500 or 750 Da) does not form stable liposomes. See, e.g., US2014/0255472, the relevant content of which is incorporated herein by reference.
  • the lipid formulations can include anionic lipids.
  • the anionic lipids can be any lipid species that carries a net negative charge at a selected pH, such as physiological pH.
  • the anionic lipids when combined with cationic lipids, are used to reduce the overall surface charge of LNPs and liposomes and to introduce pH-dependent disruption of the LNP or liposome bilayer structure, facilitating nucleotide release.
  • Several anionic lipids have been described in the literature, many of which are commercially available.
  • suitable anionic lipids for use in the compositions and methods of the invention include 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, and palmitoyloleyolphosphatidylglycerol (POPG).
  • DOPE 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine
  • DOPE 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine
  • phosphatidylglycerol 1,2-dioleoyl- sn-glycero-3
  • the lipid formulations can also include a lipid bilayer stabilizing component.
  • Bilayer stabilizing components can be used to inhibit aggregation of LNPs, but bilayer stabilizing components are not limited to this function.
  • conjugated lipids such as PEG-lipid conjugates and cationic-polymer-lipid conjugates can be used to inhibit the aggregation of LNPs or liposomes.
  • concentration of the bilayer stabilizing component By controlling the composition and concentration of the bilayer stabilizing component, one can control the rate at which the bilayer stabilizing component exchanges out of the liposome and, in turn, the rate at which the liposome becomes fusogenic.
  • fusogenic refers to the ability of a liposome or other drug delivery system to fuse with membranes of a cell.
  • the rate at which the liposome becomes fusogenic can be varied, for example, by varying the concentration of the bilayer stabilizing component, by varying the molecular weight of the polyethyleneglycol, or by varying the chain length and degree of saturation of the acyl chain groups on the phosphatidylethanolamine or the ceramide.
  • other variables including, for example, pH, temperature, ionic strength, etc. can be used to vary and/or control the rate at which the liposome becomes fusogenic. Other methods which can be used to control the rate at which the liposome becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure.
  • LNPs and liposomes can be prepared using methods known in the art in view of the present disclosure.
  • the LNPs can be prepared using ethanol injection or dilution, thin film hydration, freeze-thaw, French press or membrane extrusion, diafiltration, sonication, detergent dialysis, ether infusion, and reverse phase evaporation.
  • One useful method of preparing liposomes involves mixing (i) an ethanolic solution of the lipids (ii) an aqueous solution of the nucleic acid and (iii) buffer, followed by mixing, equilibration, dilution and purification.
  • Preferred liposomes of the invention e.g.
  • liposomes with a preferred diameter are obtainable by this mixing process.
  • mixing can be performed using a process in which two feed streams of aqueous nucleic acid solution are combined in a single mixing zone with one stream of an ethanolic lipid solution, all at the same flow rate e.g. in a microfluidic channel as described below.
  • compositions, and methods to create liposomes are described in US 2014/0255472, which is hereby incorporated by reference in its entirety.
  • lipids, lipid compositions, and methods to create lipid carriers for delivering active nucleic acid molecules are described in: US2017/0190661, US2006/0008910, US2015/0064242, US2005/0064595, WO/2019/036030, US2019/0022247, WO/2019/036028, WO/2019/036008, WO/2019/036000, US2016/0376224, US2017/0119904, WO/2018/200943,
  • Liposomes are microscopic vesicles including at least one concentric lipid bilayer. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex. In particular embodiments, liposomes provide a lipid composition that is an outer layer surrounding a porous nanoparticle.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PI phosphatidylinositol
  • SM sphingomyelin
  • DOPE dioleoylphosphatidylethanolamine
  • lipids capable of producing a stable liposome are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebro sides, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4- (N-maleimido-methyl)cyclohexane-
  • HSPC hydrogenated soy phosphati
  • Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, DDAB, dioctadecyl dimethyl ammonium chloride (DODAC), 1 ,2-dimyristoyl-3-trimethylammonium propane (DMTAP), DOTAP, DOTMA, DC-Choi, phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol, DOPG, and dicetylphosphate.
  • DODAC
  • lipids used to create liposomes disclosed herein include cholesterol, hydrogenated soy phosphatidylcholine (HSPC) and, the derivatized vesicle-forming lipid PEG-DSPE.
  • HSPC hydrogenated soy phosphatidylcholine
  • PEG-DSPE the derivatized vesicle-forming lipid PEG-DSPE.
  • Embodiments of the application also contemplate administering an effective amount of a pharmaceutical composition or immunogenic combination to a subject, and subsequently administering another dose of an effective amount of a pharmaceutical composition or immunogenic combination to the same subject, in a so-called prime-boost regimen.
  • a pharmaceutical composition or immunogenic combination of the application is a primer vaccine used for priming an immune response.
  • a pharmaceutical composition or immunogenic combination of the application is a booster vaccine used for boosting an immune response.
  • the priming and boosting vaccines of the application can be used in the methods of the application described herein. This general concept of a prime-boost regimen is well known to the skilled person in the vaccine field.
  • compositions and immunogenic combinations of the application described herein can be used as priming and/or boosting vaccines for priming and/or boosting an immune response against HBV.
  • methods for vaccinating a subject comprise administering to the subject a pharmaceutical composition comprising a nucleic acid molecule, vector, or RNA replicon of the application, and administering to the subject a second composition comprising a nucleic acid molecule encoding at least one identical HBV antigen as a prime-boost regimen.
  • a pharmaceutical composition or immunogenic combination of the application can be administered for priming immunization.
  • the pharmaceutical composition or immunogenic combination can be re-administered for boosting immunization. Further booster administrations of the pharmaceutical composition or vaccine combination can optionally be added to the regimen, as needed.
  • An adjuvant can be present in a pharmaceutical composition of the application used for boosting immunization, present in a separate composition to be administered together with the pharmaceutical composition or immunogenic combination of the application for the boosting immunization, or administered on its own as the boosting immunization.
  • the adjuvant is preferably used for boosting immunization.
  • An illustrative and non-limiting example of a prime-boost regimen includes administering a single dose of an effective amount of a pharmaceutical composition or immunogenic combination of the application to a subject to prime the immune response; and subsequently administering another dose of an effective amount of a pharmaceutical composition or immunogenic combination of the application to boost the immune response, wherein the boosting immunization is first administered about two, four, six, or eight weeks, preferably four weeks after the priming immunization is initially administered.
  • the antigens in the priming and boosting compositions need not to be identical but should share antigens or be substantially similar to each other.
  • the vector of priming composition is a replicon, and the vector of the boosting composition is different from the priming composition, e.g., an adenovirus vector, Modified Vaccinia Ankara (MV A) vector, DNA, or protein.
  • the vector of the boosting composition is a replicon, and the vector of the priming composition is different from the boosting composition, e.g., an adenovirus vector, Modified Vaccinia Ankara (MV A) vector, DNA, or protein.
  • the priming and boosting compositions of the invention can each comprise one, two, three, four, or up to five doses.
  • HBV Hepatitis B Virus
  • methods for enhancing an immune response in a subject with a HBV infection methods for decreasing viral replication in a subject with a HBV infection; methods for decreasing expression of one or more HBV polypeptide(s) in a subject in need thereof; methods for modulating Hepatitis B viral (HBV) capsid assembly or disassembly in a subject with a HBV infection; and/or methods for targeted killing of hepatocytes containing integrated viral DNA or extrachromosomal viral DNA in a subject with a HBV infection comprising administering a pharmaceutical composition comprising one or more HBV RNAi agents that can be administered in a number of ways depending upon whether local or systemic treatment is desired.
  • HBV Hepatitis B Virus
  • Administration can be, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, subdermal (e.g., via an implanted device), and intraparenchymal administration.
  • the pharmaceutical compositions described herein are administered by subcutaneous injection.
  • methods described herein comprise one or more HBV RNAi agents, wherein the one or more HBV agents are prepared as pharmaceutical compositions or formulations.
  • pharmaceutical compositions include at least one HBV RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of the target mRNA in a target cell, a group of cells, a tissue, or an organism.
  • the pharmaceutical compositions can be used to treat a subject having a disease or disorder that would benefit from reduction in the level of the target mRNA, or inhibition in expression of the target gene.
  • the pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene.
  • the method includes administering an HBV RNAi agent linked to a targeting ligand as described herein, to a subject to be treated.
  • one or more pharmaceutically acceptable excipients are added to the pharmaceutical compositions including an HBV RNAi agent, thereby forming a pharmaceutical formulation suitable for in vivo delivery to a human.
  • compositions that include an HBV RNAi agent and methods disclosed herein may decrease the level of the target mRNA in a cell, group of cells, group of cells, tissue, or subject, including: administering to the subject a therapeutically effective amount of a herein described HBV RNAi agent, thereby inhibiting the expression of a target mRNA in the subject.
  • the described pharmaceutical compositions including an HBV RNAi agent are used for treating or managing clinical presentations associated with HBV infection.
  • a therapeutically or prophylactically effective amount of one or more of pharmaceutical compositions is administered to a subject in need of such treatment, prevention or management.
  • administration of any of the disclosed HBV RNAi agents can be used to decrease the number, severity, and/or frequency of symptoms of a disease in a subject.
  • the described pharmaceutical compositions including an HBV RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder that would benefit from reduction or inhibition in expression of HBV mRNA.
  • the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions including an HBV RNAi agent thereby treating the symptom.
  • the subject is administered a prophylactically effective amount of one or more HBV RNAi agents, thereby preventing the at least one symptom.
  • the route of administration is the path by which an HBV RNAi agent is brought into contact with the body.
  • methods of administering drugs and nucleic acids for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein.
  • the HBV RNAi agents disclosed herein can be administered via any suitable route in a preparation appropriately tailored to the particular route.
  • herein described pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, intraarticularly, or intraperitoneally. In some embodiments, there herein described pharmaceutical compositions via subcutaneous injection.
  • compositions including an HBV RNAi agent described herein can be delivered to a cell, group of cells, tumor, tissue, or subject using oligonucleotide delivery technologies known in the art.
  • any suitable method recognized in the art for delivering a nucleic acid molecule in vitro or in vivo can be adapted for use with a composition described herein.
  • delivery can be by local administration, (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.
  • the compositions are administered by subcutaneous or intravenous infusion or injection.
  • the herein described pharmaceutical compositions may comprise one or more pharmaceutically acceptable excipients.
  • the pharmaceutical compositions described herein can be formulated for administration to a subject.
  • a pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients.
  • Pharmaceutically acceptable excipients are substances other than the Active Pharmaceutical ingredient (API, therapeutic product, e.g., HBV RNAi agent) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage.
  • Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use.
  • a pharmaceutically acceptable excipient may or may not be an inert substance.
  • Excipients include, but are not limited to: absorption enhancers, anti -adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension.
  • Liposomal formulations or biodegradable polymer systems can also be used to present the drug for both intra-articular and ophthalmic administration.
  • the active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polygly colic acid, collagen, poly orthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • the HBV RNAi agents can be formulated in compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • a pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions.
  • additional components include, but are not limited to, anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.).
  • additional components include, but are not limited to, anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.).
  • RNAi agents may be used as “pharmaceutical compositions.”
  • “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an RNAi agent to produce a pharmacological, therapeutic or preventive result.
  • an effective amount of an active compound will be in the range of from about 0.1 to about 100 mg/kg of body weight/day, e.g., from about 1.0 to about 50 mg/kg of body weight/day. In some embodiments, an effective amount of an active compound will be in the range of from about 0.25 to about 5 mg/kg of body weight per dose. In some embodiments, an effective amount of an active compound will be in the range of about 40- 1000 mg, more particularly about 25-400 mg per 1-18 weeks or 1-6 months. In some embodiments, an effective amount of an active compound will be in the range of 50-250 mg per 4 weeks (Q4W) or per one month (Q1M).
  • an effective amount of an active compound will be in the range of 50-250 mg per 8 weeks (Q8W) or per two months (Q2M). In some embodiments, an effective amount of an active compound will be in the range of 50-250 mg per 12 weeks (Q12W) or per three months (Q3M). In some embodiments, an effective amount of an active ingredient will be in the range of from about 0.5 to about 3 mg/kg of body weight per dose. In some embodiments, an effective amount of an active ingredient will be in the range of from about 25-400 mg per dose.
  • an effective amount of an active ingredient will be in the range of from about 50-125 mg per dose. In some embodiments, an effective amount of an active ingredient will be about 100 mg or 200 mg per dose.
  • the amount administered will also likely depend on such variables as the overall health status of the patient, the relative biological efficacy of the compound delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. Also, it is to be understood that the initial dosage administered can be increased beyond the above upper level in order to rapidly achieve the desired blood-level or tissue level, or the initial dosage can be smaller than the optimum.
  • an effective amount of the RNAi component is in the range of about 25-600 mg per dose. In some embodiments, an effective amount of the RNAi component is in the range of about 25-50 mg, about 50-75 mg, about 75-100 mg, about 100- 150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, about 300-400 mg, about 400-500 mg or about 500-600 mg per dose.
  • an effective amount of the RNAi component is about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg or about 600 mg per dose. In some embodiments, an effective amount of the RNAi component is about 25 mg, about 35mg, about 40 mg, about 50 mg, about 100 mg or about 200 mg per dose.
  • the one or more (e.g. , at least two) HBV RNAi agents described herein can be formulated into one single composition or separate individual compositions.
  • the HBV RNAi agents in separate individual compositions can be formulated with the same or different excipients and carriers.
  • the HBV RNAi agents in separate individual compositions agents can be administered through same or different administration routes.
  • the HBV RNAi agents are administered subcutaneously.
  • compositions described herein including an HBV RNAi agent can be combined with an excipient or with a second therapeutic agent or treatment including, but not limited to: a second or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, and/or a vaccine.
  • HBV RNAi agents when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensers.
  • the pharmaceutical compositions described herein may be packaged in pre-filled syringes or vials.
  • the composition comprises an effective amount of an RNAi component in the range of about 40-1000 mg and an effective amount of the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence in the range of about 10-300 pg per dose.
  • kits comprising an effective amount of an RNAi component and an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens or a pharmaceutically acceptable salt thereof.
  • the kit further comprises a package insert including, without limitation, appropriate instructions for preparation and administration of the formulation, side effects of the formulation, and any other relevant information.
  • the instructions can be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, optical disc or directions to internet-based instructions.
  • kits for treating an individual who suffers from or is susceptible to the conditions described herein comprising a first container comprising a dosage amount of a composition or formulation as disclosed herein, and a package insert for use.
  • the container can be any of those known in the art and appropriate for storage and delivery of intravenous formulation.
  • the kit further comprises a second container comprising a pharmaceutically acceptable carrier, diluent, adjuvant, etc. for preparation of the formulation to be administered to the individual.
  • the kit comprises one or more doses of the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens in the range of about 10
  • an effective amount of the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens can be administered to the subject in a dose below 10
  • the kit comprises one or more doses of the RNAi component in the range of about 25-600 mg per dose. In some embodiments, the kit comprises one or more doses of the RNAi component in the range of about 25-50 mg, about 50-75 mg, about 75-100 mg, about 100-150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, about 300-400 mg, about 400-500 mg or about 500-600 mg per dose.
  • the kit comprises one or more doses of the RNAi component of about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg or about 600 mg per dose.
  • the kit comprises one or more doses of the RNAi component of about 25 mg, about 35mg, about 40 mg, about 50 mg, about 100 mg or about 200 mg per dose.
  • the kit contains an RNAi component useful for the invention, such as those described herein, for once monthly (or every four weeks) administration to a subject in a dose of about 40-1000 mg, more particularly about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg; more particularly 200 mg; and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, such as those described herein, for administration to a subject in a dose of about 10-300 pg, more particularly 20-200 pg, wherein said dose is administered up to five (5) times.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered in up to five (5) doses, up to three (3) doses, or in two (2) doses.
  • the first dose is a priming dose
  • the second dose is a boosting dose.
  • the kit further comprises instructions for using the RNAi component and the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens contained therein for administration to treat a subject with an HBV infection, in particular, a subject having a chronic HBV infection.
  • the kit further comprises instructions for using the RNAi component and the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens contained therein for administration to a subject with an HBV infection to enhance the immune response, to decrease viral replication, to decrease expression of one or more HBV polypeptide(s), and/or to increase the targeted killing of hepatocytes comprising integrated viral DNA and/or extrachromosomal viral DNA.
  • kits may also be provided that contain sufficient dosages of the compositions described herein (including pharmaceutical compositions thereof) to provide effective treatment for an individual for an extended period, such as 1-3 days, 1-5 days, a week, 2 weeks, 3, weeks, 4 weeks, 6 weeks, 8 weeks, 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles or more.
  • one cycle of treatment is about 1-24 months, about 1-3 months, about 3-6 months, about 6-9 months, about 9-12 months, about 12-18 months, about 18-21 months or about 21-24 months.
  • one cycle of treatment is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 21 months or about 24 months.
  • kits can also include multiple doses and may be packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
  • the kits may include a dosage amount of at least one composition as disclosed herein.
  • a method for treating a Hepatitis B viral (HBV) infection in a subject in need thereof comprises administering to the subject an effective amount of an RNAi component and a nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens, such as those described herein.
  • a method for enhancing an immune response in a subject with an HBV infection comprising administering to the subject an effective amount of an RNAi component and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, such as those described herein.
  • Also provided herein is a method for decreasing viral replication in a subject with a HBV infection, wherein the method comprises administering to the subject an effective amount of an RNAi component and a nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens, such as those described herein. Also provided is a method for decreasing the expression of one or more HBV polypeptide(s), more particularly of one or more polypeptide(s) selected from HBsAg and HBeAg, in a subject in need thereof, such as those described herein.
  • Also provided herein is a method for increasing the targeted killing of hepatocytes comprising integrated viral DNA and/or extrachromosomal viral DNA in a subject with an HBV infection, the method comprises administering to the subject an effective amount of an RNAi component and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, such as those described herein.
  • the HBV infection is a chronic HBV infection.
  • administering the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens decreases the immune tolerogenicity of the subject to HBV, more particularly the immune tolerogenicity of the liver of the subject to HBV. Decreasing the immune tolerogenicity of the subject to HBV or the immune tolerogenicity of the liver of the subject to HBV infection is characterized by the reactivation of the immune system in the subject with the chronic HBV infection.
  • Reactivation of the immune system can occur after administration of the effective amount the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, which serves to decrease or reduce the level of HBsAg in the subject, which allows for the reactivation of the immune system.
  • LLoQ lower limit of
  • the at least one of, at least two of, at least three of, at least four of, or five of features (i), (ii), (iii), (iv), and (v) are still met six (6) months after the end of treatment.
  • administering the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens at least until the subject meets at least one of, at least two of, at least three of, or four of the following features: (i) a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 lU/mL, more particularly is lower than 15 lU/mL, more particularly is lower than 10 lU/mL; (ii) a serum ALT concentration lower than 3 times the upper normal limit, or lower than 129 U/L if the subject is a
  • LLoQ lower limit of quantification
  • the method comprises administering the effective amount of the pharmaceutical composition comprising the RNAi component at least until the serum HBsAg level of the subject decreases down to 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly down to 10 lU/mL or lower.
  • the method comprises administering, more particularly start administering, the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens after the serum HBsAg level of the patient has decreased down to 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly to 10 lU/mL or lower.
  • the serum HBsAg level in the subject is reduced at a faster rate, is reduced at a greater level, and/or is reduced for a greater time period than in a subject in which only an effective amount of the pharmaceutical composition comprising the RNAi component is administered or an effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered.
  • enhancing an immune response in the subject results in an increase in an innate immune system and/or an adaptive immune system.
  • the increase in the innate immune system results in an increase in the expression of one or several from among an interferon stimulated gene (ISG), an interferon gamma-induced protein 10 (IP 10), an interferon alpha (IFNa), an interleukin 12 (IL 12), or an interleukin-6 (IL-6).
  • ISG interferon stimulated gene
  • IP 10 interferon gamma-induced protein 10
  • IFNa interferon alpha
  • IL 12 interleukin 12
  • IL-6 interleukin-6
  • the increase in the adaptive immune system results in an increase in the level of HBV-specific T cell responses and/or an increased activation of the HBV-specific T cells.
  • Activation of HBV-specific T cells can be measured by any means that the person of average skill in the art may find appropriate, for example, by interferon gamma production (e.g., an interferon gamma ELISPOT).
  • the increase in expression of the at least one of ISG, IP10, IFNa, IL12, or IL-6; the increase in the level of HBV-specific T cell responses; and/or the increased activation of HBV-specific T cells can be compared to a control.
  • the control can be an expression level of ISG, IP10, IFNa, IL12, or IL-6; the level of HBV-specific T cell responses; the level of activation of HBV-specific T cells from a sample from the subject prior to administration of the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens.
  • control can be the expression level of ISG, IP 10, IFNa, IL12, or IL-6; the level of HBV-specific T cell responses; and/or the level of activation of HBV-specific T cells from a subject administered the effective amount of the pharmaceutical composition comprising the RNAi component, but not the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens; or from a subject administered the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, but not the effective amount of the pharmaceutical composition comprising the RNAi component.
  • a person skilled in the art will understand the proper control for determining an increase in the expression level of ISG, IP 10, IFNa, IL 12, or IL-6; an increase in the level of HBV-specific T cell responses; and/or an increased activation level of HBV-specific T cells in the subject.
  • enhancing an immune response in the subject results in an increase in the immunocompetence of the subject.
  • An increase in the immunocompetence of the subject can, for example, result in an increase in the level of HBV-specific T cells, B cells, or NK cells in the liver and/or an increased activation of HBV-specific T cells, B cells, or NK cells in the liver.
  • An increase in the immunocompetence of the subject can, for example, result in an increase in NK cells, T cells, or B cells in a peripheral immune cell compartment and/or an increased activation of NK cells, T cells, or B cells in the peripheral immune cell compartment.
  • An increase in the immunocompetence of the subject can, for example, result in a decrease of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or in a peripheral immune cell compartment; and/or a decreased immunosuppressive activity of MDSCs in the lever and/or in a peripheral immune cell compartment; and/or a decrease of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or in a peripheral immune cell compartment and/or a decreased immunosuppressive activity of Treg in the liver and/or in a peripheral immune cell compartment.
  • MDSCs Myeloid Derived Suppressive Cells
  • Treg regulatory T cells
  • Treg regulatory T cells
  • the increase in the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the increase in the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; the decrease in the level of or decreased immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment; and/or the decrease in the level of or decreased immunosuppressive activity of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or in a peripheral immune cell compartment can, for example, be determined by comparing to a control.
  • the control can be the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; the level of or immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment; and/or the level of or activity of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or in a peripheral immune cell compartment from a sample from the subject prior to administration of the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens.
  • MDSCs Myeloid Derived Suppressive Cells
  • control can be the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; the level of or immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment; and/or the level of or activity of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or in a peripheral immune cell compartment from a subject administered the effective amount of the pharmaceutical composition comprising the RNAi component, but not the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens; or from a subject administered the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, but not the effective amount of
  • a person skilled in the art will understand the proper control for determining an increase in the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; a decrease in the level of or immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment; and/or a decrease in the level of or activity of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or in a peripheral immune cell compartment in the subject.
  • MDSCs Myeloid Derived Suppressive Cells
  • Activation of HBV-specific NK cells can be measured by any means that the person of average skill in the art may find appropriate, for example, by interferon gamma production (e.g., interferon gamma ELISPOT).
  • Activation of HBV-specific B cells can be measured by any means that the person of average skill in the art may find appropriate, for example, by anti-HBs antibody production (e.g., anti-HBs ELISPOT).
  • the immunosuppressive activity of MDSCs can be measured by any means that the person of average skill in the art may find appropriate, for example, by arginase expression.
  • decreasing the viral replication in the subject results in a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 lU/mL, more particularly is lower than 15 lU/mL, more particularly is lower than 10 lU/mL.
  • the targeted hepatocytes comprise cccDNA. In some embodiments, the targeted hepatocytes comprise integrated HBV DNA.
  • the RNAi component comprises: (i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and (ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:
  • the first RNAi agent comprises SEQ ID NO:97 and SEQ ID NO: 106. In some embodiments, the first RNAi agent comprises SEQ ID NO:98 and SEQ ID NO: 106. In some embodiments, the first RNAi agent comprises SEQ ID NO:99 and SEQ ID NO: 107. In some embodiments, the first RNAi agent comprises SEQ ID NO:93 and SEQ ID NO: 102, 103, or 105. In some embodiments, the first RNAi agent comprises SEQ ID NO:94 and SEQ ID NO: 102, 103, or 105. In some embodiments, the first RNAi agent comprises SEQ ID NO:95 and SEQ ID NO: 102, 103, or 105.
  • the first RNAi agent comprises SEQ ID NO:96 and SEQ ID NO: 104.
  • the second RNAi agent comprises SEQ ID NO: 101 and SEQ ID NO: 111.
  • the second RNAi agent comprises SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:97 and SEQ ID NO: 106 and a second RNAi agent comprising SEQ ID NO: 101 and SEQ ID NO: 111.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:98 and SEQ ID NO: 106 and a second RNAi agent comprising SEQ ID NO: 101 and SEQ ID NO: 111.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:99 and SEQ ID NO: 107 and a second RNAi agent comprising SEQ ID NO: 101 and SEQ ID NO: 111.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:93 and SEQ ID NO: 102, 103, or 105 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:94 and SEQ ID NO: 102, 103, or 105 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:95 and SEQ ID NO: 102, 103, or 105 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:96 and SEQ ID NO: 104 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110.
  • the RNAi component comprises a first RNAi agent comprising SEQ ID NO:94 and SEQ ID NO: 103 or SEQ ID NO: 105 and the second RNAi agent comprising SEQ ID NO: 108 or SEQ ID NO: 110 and SEQ ID NO: 100.
  • the two HBV RNAi agents are administered in a ratio of about 1:1, 2:1, 3:1, 4:1 or 5:1. In some embodiments, the two HBV RNAi agents are administered in a ratio of about 2: 1.
  • the two HBV RNAi agents are administered in a combined amount of about 25-75 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1: 1, about 4:1, about 5: 1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 35-40 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1:1, about 4:1, about 5:1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50-125 mg per dose administration and in the ratio of about 2: 1, about 3:1, about 1:1, about 4:1, about 5:1 or about 1:2.
  • the two HBV RNAi agents are administered in a combined amount of about 75-150 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1:1, about 4:1, about 5 : 1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 100-200 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1:1, about 4:1, about 5:1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 150-250 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1:1, about 4:1, about 5 : 1 or about 1:2.
  • the two HBV RNAi agents are administered in a combined amount of about 200-300 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1:1, about 4:1, about 5:1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 300-400 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1:1, about 4:1, about 5 : 1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50-100 mg per dose administration and in the ratio of about 2: 1, about 3:1, about 1:1, about 4:1, about 5:1 or about 1:2.
  • the two HBV RNAi agents are administered in a combined amount of about 25-400 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 25-75 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 35-40 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50-125 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 75-150 mg per dose administration and in the ratio of about 2:1.
  • the two HBV RNAi agents are administered in a combined amount of about 100-200 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 125-225 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 150-250 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 200-300 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 300-400 mg per dose administration and in the ratio of about 2:1.
  • the two HBV RNAi agents are administered in a combined amount of about 100 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 25 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 35 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 40 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50 mg per dose administration and in the ratio of about 2:1.
  • the two HBV RNAi agents are administered in a combined amount of about 75 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 200 mg per dose administration and in the ratio of about 2: 1.
  • the first RNAi agent is administered in an amount of about 3-650 mg per dose administration, and the second RNAi agent is administered in an amount of about 2-325 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 15-150 mg per dose administration, and the second RNAi agent is administered in an amount of about 5-75 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 35-265 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 50-75 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 15-75 mg per dose administration.
  • the second RNAi agent is administered in an amount of about 20-125 mg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 25-50 mg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 5-40 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 17 mg per dose administration, and the second RNAi agent is administered in an amount of about 8 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 23 mg per dose administration, and the second RNAi agent is administered in an amount of about 12 mg per dose administration.
  • the first RNAi agent is administered in an amount of about 27 mg per dose administration, and the second RNAi agent is administered in an amount of about 13 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 33 mg per dose administration, and the second RNAi agent is administered in an amount of about 17 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 67 mg per dose administration, and the second RNAi agent is administered in an amount of about 33 mg per dose administration.
  • two RNAi agents are administered at a combined dose of 25-400 mg per dose administration.
  • two RNAi agents are administered at a combined dose of 25-400 mg
  • the first RNAi agent is administered with the second RNAi agent at a ratio of 1 : 1.
  • the dose of each of the first and second RNAi agents is in an amount of about 12 mg for a combined dose of about 25 mg.
  • the dose of each of the first and second RNAi agents is in an amount of about 17 mg for a combined dose of about 35 mg.
  • the dose of each of the first and second RNAi agents is in an amount of about 20 mg for a combined dose of about 40 mg.
  • the dose of each of the first and second RNAi agents is in an amount of about 25 mg for a combined dose of about 50 mg. In an embodiment, the dose of each of the first and second RNAi agents is in an amount of about 50 mg for a combined dose of about 100 mg. In an embodiment, the dose of each of the first and second RNAi agents is in an amount of about 100 mg for a combined dose of about 200 mg. In an embodiment, the dose of each of the first and second RNAi agents is in an amount of about 150 mg for a combined dose of about 300 mg. In an embodiment, the dose of each of the first and second RNAi agents is in an amount of about 200 mg for a combined dose of about 400 mg.
  • two RNAi agents are administered at a combined dose of 25- 400 mg per dose, and the first RNAi agent is administered with the second RNAi agent at a ratio of 2: 1.
  • the dose of the first RNAi agent is in an amount of about 16 mg, and the dose of the second RNAi agent is in an amount of about 8 mg for a combined dose of about 25 mg.
  • the dose of the first RNAi agent is in an amount of about 24 mg, and the dose of the second RNAi agent is in an amount of about 12 mg for a combined dose of about 35 mg.
  • the dose of the first RNAi agent is in an amount of about 27 mg, and the dose of the second RNAi agent is in an amount of about 13 mg for a combined dose of about 40 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 33 mg, and the dose of the second RNAi agent is in an amount of about 17 mg for a combined dose of about 50 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 65 mg, and the dose of the second RNAi agent is in an amount of about 35 mg for a combined dose of about 100 mg.
  • the dose of the first RNAi agent is in an amount of about 133 mg, and the dose of the second RNAi agent is in an amount of about 67 mg for a combined dose of about 200 mg.
  • the dose of the first RNAi agent is in an amount of about 200 mg, and the dose of the second RNAi agent is in an amount of about 100 mg for a combined dose of about 300 mg.
  • the dose of the first RNAi agent is in an amount of about 270 mg, and the dose of the second RNAi agent is in an amount of about 135 mg for a combined dose of about 400 mg.
  • two RNAi agents are administered at a combined dose of 25- 400 mg per dose, the first RNAi agent is administered with the second RNAi agent at a ratio of 3 : 1.
  • the dose of the first RNAi agent is in an amount of about 18 mg, and the dose of the second RNAi agent is in an amount of about 6 mg for a combined dose of about 25 mg.
  • the dose of the first RNAi agent is in an amount of about 27 mg, and the dose of the second RNAi agent is in an amount of about 9 mg for a combined dose of about 35 mg.
  • the dose of the first RNAi agent is in an amount of about 30 mg, and the dose of the second RNAi agent is in an amount of about 10 mg for a combined dose of about 40 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 36 mg, and the dose of the second RNAi agent is in an amount of about 12 mg for a combined dose of about 50 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 75 mg, and the dose of the second RNAi agent is in an amount of about 25 mg for a combined dose of about 100 mg.
  • the dose of the first RNAi agent is in an amount of about 150 mg, and the dose of the second RNAi agent is in an amount of about 50 mg for a combined dose of about 200 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 225 mg, and the dose of the second RNAi agent is in an amount of about 75 mg for a combined dose of about 300 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 300 mg, and the dose of the second RNAi agent is in an amount of about 100 mg for a combined dose of about 400 mg.
  • the first RNAi agent and the second RNAi agent are administered in a combined amount of about 25-400 mg per dose administration. In some embodiments, the first RNAi agent and the second RNAi agent are administered in a combined amount of about 25-50 mg, 50-75 mg, 75-100 mg, 100-125 mg, 125-150 mg, 150- 175 mg, 175-200 mg, 200-225 mg, 225-250 mg, 250-275 mg, 275-300 mg, 300-325 mg, 325- 350 mg, 350-375 mg, 375-400 mg, 25-75 mg, 50-100 mg, 100-150 mg, 150-200 mg, 200-250 mg, 250-300 mg, 300-350 mg, 350-400 mg, 25-100 mg, 50-150 mg, 100-200 mg, 150-250 mg, 200-300 mg, 300-400 mg, 25-200 mg, or 200-400 mg per dose administration.
  • the first RNAi agent to the second RNAi agent are administered in a combined amount of about 25 mg, about 50 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, or about 400 mg per dose administration.
  • the first RNAi agent and the second RNAi agent are administered in a combined amount of about 50 mg, about 75 mg, about 100 mg, or about 125 mg per dose administration.
  • the first RNAi agent and the second RNAi agent are administered in a combined amount of about 25 mg, about 35 mg, about 40 mg, or about 200 mg per dose administration.
  • the two HBV RNAi agents are administered in a combined amount of about 1-10 mg/kg per dose administration. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 1-5 mg/kg per dose administration.
  • the two HBV RNAi agents are administered in a combined amount of about 1-1.5 mg/kg, about 1.5-2.0 mg/kg, about 2.0-2.5 mg/kg, about 2.5-3.0 mg/kg, about 3.0-3.5 mg/kg, about 3.5-4.0 mg/kg, about 4.0-4.5 mg/kg, about 4.5-5.0 mg/kg, about 5.0-5.5 mg/kg, about 5.5-6.0 mg/kg, about 6.0-6.5 mg/kg, about 6.5-7.0 mg/kg, about 7.0-7.5 mg/kg, about 7.5-8.0 mg/kg, about 8.0-8.5 mg/kg, about 8.5-9.0 mg/kg, about 9.0-9.5 mg/kg, about 9.5-10 mg/kg, about 1-2.5 mg/kg, about 2.5-5.0 mg/kg, about 5.0-7.5 mg/kg, about 7.5-10 mg/kg, about 1-5.0 mg/kg, or about 5.0-10 mg/kg per dose administration.
  • the first RNAi agent is administered in an amount of about 0.6-7 mg/kg per dose administration, and the second RNAi agent is administered in an amount of about 0.3-5 mg/kg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 0.5-2.5 mg/kg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 0.3- 1.5 mg/kg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 0.6-5 mg/kg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 1-2.5 mg/kg per dose administration. [0355] In some embodiments, the two RNAi agents are administered in about 1-18 week intervals.
  • the two RNAi agents are administered in about 1-week intervals, about 2-week intervals, about 3-week intervals, about 4-week intervals, about 5- week intervals, about 6-week intervals, about 7-week intervals, about 8-week intervals, about 9-week intervals, about 10-week intervals, about 11-week intervals, about 12-week intervals, about 13 -week intervals, about 14-week intervals, about 15 -week intervals, about 16-week intervals, about 17-week intervals, or about 18-week intervals. In some embodiments, the two RNAi agents are administered in about 1-6 month intervals.
  • the two RNAi agents are administered in about 1 -month intervals, about 2-month intervals, about 3-month intervals, about 4-month intervals, about 5-month intervals, or about 6-month intervals. In some embodiments, the two RNAi agents are administered in about 4-week intervals or 1 -month intervals. In some embodiments, the two RNAi agents are administered once per month.
  • the first RNAi agent and the second RNAi agent are administered for a duration of about 1-12 months. In some embodiments, the first RNAi agent and the second RNAi agent are administered for a duration of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months or at least about 12 months. In some embodiments, the first RNAi agent and the second RNAi agent are administered for a duration of about 1-18 weeks.
  • the first RNAi agent and the second RNAi agent are administered for a duration of at least about 1 week, at least about 5 weeks, at least about 10 weeks, at least about 15 weeks, at least about 20 weeks, at least about 25 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at least about 50 weeks, at least about 55 weeks, at least about 60 weeks, at least about 65 weeks, at least about 70 weeks, at least about 75 weeks, at least about 80 weeks, at least about 90 weeks, or at least 96 weeks.
  • the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg per dose administration.
  • the first RNAi agent and the second RNAi agent are administered at a combined dose of 25- 400 mg
  • the first RNAi agent is administered with the second RNAi agent at a ratio of 1: 1.
  • the dose of the first RNAi agent is administered with the second RNAi agent is in an amount of about 12 mg for a combined dose of about 25 mg.
  • the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 17 mg for a combined dose of about 35 mg.
  • the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 20 mg for a combined dose of about 40 mg. In an embodiment, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 25 mg for a combined dose of about 50 mg. In an embodiment, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 50 mg for a combined dose of about 100 mg. In an embodiment, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 100 mg for a combined dose of about 200 mg.
  • the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 150 mg for a combined dose of about 300 mg. In an embodiment, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 200 mg for a combined dose of about 400 mg. [0358] In an embodiment, the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg per dose, and the second RNAi agent is administered with the first RNAi agent at a ratio of 1 :2. In an embodiment, the dose of the first RNAi agent is in an amount of about 16 mg, and the dose of the second RNAi agent is in an amount of about 8 mg for a combined dose of about 25 mg.
  • the dose of the second RNAi agent is in an amount of about 12 mg, and the dose of the first RNAi agent is in an amount of about 24 mg for a combined dose of about 35 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 27 mg, and the dose of the second RNAi agent is in an amount of about 13 mg for a combined dose of about 40 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 33 mg, and the dose of the second RNAi agent is in an amount of about 17 mg for a combined dose of about 50 mg.
  • the dose of the second RNAi agent is in an amount of about 35 mg, and the dose of the first RNAi agent is in an amount of about 65 mg for a combined dose of about 100 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 67 mg, and the dose of the first RNAi agent is in an amount of about 133 mg for a combined dose of about 200 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 100 mg, and the dose of the first RNAi agent is in an amount of about 200 mg for a combined dose of about 300 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 135 mg, and the dose of the first RNAi agent is in an amount of about 270 mg for a combined dose of about 400 mg.
  • the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg per dose, the second RNAi agent is administered with the first RNAi agent at a ratio of 1 :3.
  • the dose of the first RNAi agent is in an amount of about 18 mg, and the dose of the second RNAi agent is in an amount of about 6 mg for a combined dose of about 25 mg.
  • the dose of the second RNAi agent is in an amount of about 9 mg, and the dose of the first RNAi agent is in an amount of about 27 mg for a combined dose of about 35 mg.
  • the dose of the first RNAi agent is in an amount of about 30 mg, and the dose of the second RNAi agent is in an amount of about 10 mg for a combined dose of about 40 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 36 mg, and the dose of the second RNAi agent is in an amount of about 12 mg for a combined dose of about 50 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 25 mg, and the dose of the first RNAi agent is in an amount of about 75 mg for a combined dose of about 100 mg.
  • the dose of the second RNAi agent is in an amount of about 50 mg, and the dose of the first RNAi agent is in an amount of about 150 mg for a combined dose of about 200 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 75 mg, and the dose of the first RNAi agent is in an amount of about 225 mg for a combined dose of about 300 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 100 mg, and the dose of the first RNAi agent is in an amount of about 300 mg for a combined dose of about 400 mg.
  • about 1 mg/kg (mpk) of the first RNAi agent and about 1 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 1.5 mg/kg of the first RNAi agent and about 1.5 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 2.0 mg/kg of the first RNAi agent and about 1.0 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 3.0 mg/kg of the first RNAi agent and about 1.0 mg/kg of the second RNAi agent are administered to a subject in need thereof.
  • about 3.2 mg/kg of the first RNAi agent and about 0.8 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 2.7 mg/kg of the first RNAi agent and about 1.3 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 4.0 mg/kg of the first RNAi agent and about 1.0 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 3.3 mg/kg of the first RNAi agent and about 1.7 mg/kg of the second RNAi agent are administered to a subject in need thereof.
  • between about 0.05 and about 5 mg/kg of the first RNAi agent and between about 0.05 and about 5 mg/kg of the second RNAi agent are administered to a subject in need thereof.
  • about the first RNAi agent and about the second RNAi agent are administered separately (e.g., in separate injections).
  • the respective dose of the first RNAi agent and the respective dose of the second RNAi agent are administered together (e.g., in the same injection).
  • the respective dose of the first RNAi agent and the respective dose of the second RNAi agent are prepared in a single pharmaceutical composition.
  • the RNAi component is administered to the subject once monthly in a dose of about 40-350 mg, such as about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg, more particularly 200 mg.
  • nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end, is as follows:
  • IVS internal ribosome entry sequence
  • first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
  • an effective amount when used with reference to a nucleic acid molecule, vector, or RNA replicon can range from about 10 pg of nucleic acid molecule, vector, or RNA replicon to about 300 pg of nucleic acid molecule, vector, or RNA replicon.
  • an effective amount of a nucleic acid molecule, vector, or RNA replicon is about 10 pg to about 300 pg, more particularly about 20 pg to about 200 pg.
  • An effective amount can be from one nucleic acid molecule, vector, or RNA replicon, or from multiple nucleic acid molecules, vectors, or RNA replicons.
  • An effective amount can be administered in a single composition, or in multiple compositions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compositions (e.g., tablets, capsules or injectables, or any composition adapted to intradermal delivery, e.g., to intradermal delivery using an intradermal delivery patch), wherein the administration of the multiple capsules or injections collectively provides a subject with an effective amount. It is also possible to administer an effective amount to a subject, and subsequently administer another dose of an effective amount to the same subject, in a so- called prime-boost regimen. This general concept of a prime-boost regimen is well known to the skilled person in the vaccine field. Further booster administrations can optionally be added to the regimen, as needed.
  • An immunogenic combination comprising two vectors, e.g., a first vector encoding a first HBV antigen and second vector encoding a second HBV antigen can be administered to a subject by mixing both vectors and delivering the mixture to a single anatomic site. Alternatively, two separate immunizations each delivering a single expression vector can be performed.
  • the first vector and the second vector can be administered in a ratio of 10:1 to 1:10, by weight, such as 10:1, 9:1, 8:1, 7:1, 6:1, 5: 1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, by weight.
  • the first and second vectors are administered in a ratio of 1:1, by weight.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose in the range of about 10 pg to about 300 pg, more particularly abouut 20 pg to about 200 pg.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered for a duration of at least about 1 week, at least about 5 weeks, at least about 10 weeks, at least about 15 weeks, at least about 20 weeks, at least about 25 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at least about 50 weeks, at least about 55 weeks.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered for a duration of about 12 weeks or of about 24 weeks.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered once every 3 weeks, once every month (Q4W), once every six weeks (Q6W), or once every eight weeks (Q8W).
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is formulated for subcutaneous, intradermal injection, or intramuscular injection, more preferably intramuscular injection.
  • the nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens is formulated in in a liquid form, such as suspensions, solutions, emulsions, or syrups, or may be lyophilized.
  • the RNAi component is formulated for subcutaneous injection.
  • the RNAi component is formulated in in a liquid form, such as suspensions, solutions, emulsions, or may be lyophilized.
  • the RNAi component and the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens are administered simultaneously or intermittently. In some embodiments, the RNAi component and the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens are administered and formulated separately and administered with different dosing frequencies. In some embodiments, the RNAi component and the nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens are formulated as one or separate compositions.
  • the RNAi component is formulated as a solution and administered once per month or once every four weeks via subcutaneous injection.
  • the nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens is formulated as an intramuscular injection and administered in up to five (5) doses, preferably at least three doses.
  • the RNAi component is administered in the amount of about 50-250 mg once a month via subcutaneous injection while the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20-200 pg in the form of a liquid solution at least six (6) months after the administration of the RNAi component has started, wherein said dose is administered up five (5) timese.
  • the RNAi component is administered in the amount of about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200 mg.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg, wherein said dose is administered up to five (5) times.
  • the RNAi component is administered in the amount of about 100 mg once a month or once every four weeks via subcutaneous injection while the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg in the form of a liquid solution at least six (6) months after the administration of the RNAi component has started, wherein said dose is administered up to five (5) times.
  • the RNAi component is administered in the amount of about 25-200 mg once a month or once every four weeks via subcutaneous injection while the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg in the form of a liquid solution at least six (6) months after the administration of the RNAi component has started, wherein said dose is administered up to five (5) times.
  • the RNAi component is administered in the amount of about 35 mg, about 40 mg, about 50 mg, about 100 mg, or about 200 mg.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg in the form of a liquid solution, wherein said dose is administered up to five (5) times.
  • the RNAi component is administered in the amount of about 40 mg once a month or once every four weeks via subcutaneous injection while the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg in the form of a liquid solution at least six (6) months after the administration of the RNAi component has started, wherein said dose is administered up to five (5) times.
  • the RNAi component is administered in the amount of about 100 mg once a month or once every four weeks via subcutaneous injection while the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg in the form of a liquid solution at least six (6) months after the administration of the RNAi component has started, wherein said dose is administered up to five (5) times.
  • the RNAi component is administered in the amount of about 200 mg once a month or once every four weeks via subcutaneous injection while the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg in the form of a liquid solution at least six (6) months after the administration of the RNAi component has started, wherein said dose is administered up to five (5) times.
  • a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, such as any of those described herein, is administered to the subject once or twice at a dose of about 20 pg to about 200 pg.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject about six (6) months after the administration of the RNAi component has started.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject about six (6) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject up to five (5) times over the course of six (6) months in total.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject about three (3) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject up to three (3) times over the course of three (3) months in total.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject about six (6) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject up to three (3) times over the course of about three (3) months in total.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject about eight (8) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject up to three (3) times over the course of about three (3) months in total.
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject about three (3) months after the administration of the RNAi component has started, wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject for about three (3) months in total, wherein the administration of the RNAi component continues, e.g., for about six (6) months, after the administration of the up to three (3) doses of the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens has been stopped.
  • Methods according to embodiments of the application further comprises administering to the subject in need thereof another immunogenic agent (such as another HBV antigen or other antigen) or another anti-HBV agent (such as a nucleoside analog or other anti-HBV agent) in combination with a pharmaceutical composition of the application.
  • another immunogenic agent such as another HBV antigen or other antigen
  • another anti-HBV agent such as a nucleoside analog or other anti-HBV agent
  • another anti-HBV agent or immunogenic agent can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PDl, anti- PDL1, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/oror TLR8 agonists), RIG-1 agonists, IL- 15 superagonists (Aitor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant, IL-7-hyFc; CAR-T which bind HBV env (S-CAR cells); capsid assembly modulators; cccDNA inhibitors, HBV polymerase inhibitors (e.g., entecavir and tenofovir).
  • the one or other anti-HBV active agents can be, for example, a small molecule, an antibody or antigen binding fragment thereof, a polypeptide,
  • the method further comprises administering a nucleoside analog.
  • the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof.
  • the nucleoside analog is entecavir and it is administered in a daily dose in the amount of about 0.01-5 mg, about 0.01-0.05 mg, about 0.05-0.1 mg, about 0.1-0.5 mg, about 0.5-1 mg, about 1-2 mg, about 2-3 mg, about 3-4 mg or about 4-5 mg.
  • the nucleoside analog is entecavir and it is administered in a daily dose in the amount of about 0.5 mg. In some embodiments, the nucleoside analog is tenofovir disoproxil fumarate and it is administered in a daily dose in the amount of about 100-500 mg, about 100-150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, 300-400 mg, about 400-500 mg.
  • the nucleoside analog is tenofovir disoproxil fumarate and it is administered in a daily dose in the amount of about 300 mg In some embodiments, the nucleoside analog is tenofovir alafenamide and it is administered in a daily dose in the amount of about 5-100 mg, about 5-25 mg, about 25-50 mg, about 50-75 or about 75-100 mg. In some embodiments, the nucleoside analog is tenofovir alafenamide and it is administered in a daily dose in the amount of about 25 mg.
  • the nucleoside analog is lamivudine and it is administered in a daily dose in the amount of about 50-600 mg, about SO- SOO mg, about 100-300 mg, about 100-500 mg, about 150-400 mg, about 200-350, or about 250-300 mg. In some embodiments, the nucleoside analog is lamivudine and it is administered in a daily dose in the amount of 100 mg, 150 mg, or 300 mg. In some embodiments, the nucleoside analog is telbivudine and it is administered in a daily dose in the amount of about 300-800 mg, about 400-700 mg, about 300-600 mg, about 300-400 mg, about 400-500 mg, or about 500-600 mg.
  • the nucleoside analog is telbivudine and it is administered in a daily dose in the amount of 600 mg.
  • the patients have been exposed to the nucleoside analog prior to the combination therapy.
  • the patients have been administered the nucleoside analog for at least 1 month, at least 3 months, at least 6 months, or at least 1 year prior to receiving the combination therapy.
  • the other agent is a nucleic acid polymer (NAP).
  • NAP nucleic acid polymer
  • the NAP can, for example, be selected from REP2139 or REP2165.
  • REP2139 has a sequence of (A,5'MeC) 20 with each linkage being phosphorothioated and every ribose being 2’0 methylated (which is disclosed as SEQ ID NOTO in WO2016/04525, the content of which is incorporated herein by reference in its entirety).
  • REP2165 has a sequence of (A,5'MeC) 20 with each linkage being phosphorothioated, every rbose being 2’0 methylated except adenosines at positions 11, 21, and 31, where riboses are 2’OH (which is disclosed as SEQ ID NO: 13 in WO2016/04525).
  • the NAP can also be other exemplary nucleic acid polymers, which include, but are not limited to, REP2006, REP2031, REP2055, STOPSTM (S-antigen transport-inhibiting oligonucleotide polymers), and those disclosed in Patent Application Publication Nos. WO200424919; WO201221985; and WO202097342 and U.S. Patent Nos. 7,358,068; 8,008,269; 8,008,270; and 8,067,385, the content of each is incorporated herein by reference in its entirety.
  • the patients are screened for HBeAg status prior to administration of the combination therapy.
  • the patients are HBeAg positive.
  • the patients are HBeAg negative.
  • the patients are screened for immune tolerance prior to administration of the combination therapy.
  • the HBsAg level in the patient is reduced by at least about logio 0.5, about logio 0.75, about logio 1, about logio 1.25, about logio 1.5, about logio 1.75, about logio 2 or about logio 2.5 from base line on Day 1.
  • the HBeAg level in the patient is reduced by at least about logio 0.5, about logio 0.75, about logio 1, about logio 1.25, about logio 1.5, about logio 1.75, about logio 2 or about logio 2.5 from base line on Day 1.
  • the HBcrAg level in the patient is reduced by at least about logio 0.5, about logio 0.75, about logio 1, about logio 1.25, about logio 1.5, about logio 1.75, about logio 2 or about logio 2.5 from base line on Day 1.
  • the HBV DNA level in the patient is reduced by at least about logio 0.5, about logio 1, about logio 1.5, about logio 2, about logio 3, about logio 4, about logio 5 or about logio 7.5 from base line on Day 1.
  • the HBV RNA level in the patient is reduced by at least about logio 0.5, about logio 0.75, about logio 1, about logio 1.25, about logio 1.5, about logio 1.75, about logio 2 or about logio 2.5 from base line on Day 1.
  • the application also relates to an effective amount of an RNAi component and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, optionally a nucleoside analog, each of which as that described herein, in the manufacture of a medicament for treating a Hepatitis B viral (HBV) infection in a subject; enhancing an immune response in a subject with an HBV infection; decreasing viral replication in a subject with HBV; decreasing the expression of one or more HBV polypeptide(s), more particularly of one or more polypeptide(s) selected from HBsAg and HBeAg, in a subject in need thereof; and/or increasing the targeted killing of hepatocytes comprising integrated viral DNA and/or extrachromosomal viral DNA in a subject with a HBV infection.
  • HBV Hepatitis B viral
  • the RNAi component is for once monthly or once every four weeks administration to a subject in a dose of about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg, more particularly 200 mg;
  • the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is for administration to a subject at a dose of about 20-200 p.g, wherein said dose is administered up to five (5) times.
  • the medicament is for administering to a subject infected by HBV, in particular, a subject having a chronic HBV infection.
  • Embodiment 1 is a combination for use in treating a Hepatitis B Virus (HBV) infection, more particularly chronic HBV infection (CHB) with or without viral co-infection, and/or for treating chronic HDV infection, in a subject in need thereof, comprising:
  • RNAi component having:
  • a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
  • a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; and
  • nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
  • IVS internal ribosome entry sequence
  • first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
  • Embodiment 2 is a combination for use in enhancing an immune response in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection, comprising: (a) an effective amount of a pharmaceutical composition comprising an RNAi component having:
  • a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
  • a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; and
  • nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
  • IVS internal ribosome entry sequence
  • first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
  • Embodiment 3 is a combination for use in decreasing viral replication in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection, comprising:
  • RNAi component having:
  • RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and (ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,
  • nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
  • IVS internal ribosome entry sequence
  • first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
  • Embodiment 4 is a combination for use in decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide(s), more particularly of one or more polypeptide(s) selected from HBsAg and HBeAg, in a subject in need thereof, comprising:
  • RNAi component having:
  • a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
  • a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; and
  • nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
  • HBV hepatitis B virus
  • IVS internal ribosome entry sequence
  • first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
  • Embodiment 5 is a combination for use in increasing the targeted killing of hepatocytes containing integrated viral DNA or extrachromosomal viral DNA in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection, comprising:
  • RNAi component having:
  • a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
  • a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; and
  • nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
  • IVS internal ribosome entry sequence
  • Embodiment 6 is the combination for use of any one of embodiments 1-5, wherein the HBV infection is a chronic HBV infection.
  • Embodiment 7 is the combination for use of any one of embodiments 1 -6, wherein the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens decrease the immune tolerogenicity of the subject to HBV, more particularly the immune tolerogenicity of the liver of the subject to HBV.
  • Embodiment 8 is the combination for use of any one of embodiments 1-7, wherein the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens are administered to the subject at least until the subject meets at least one of, at least two of, at least three of, at least four of, or the five following features: i. a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 lU/mL, more particularly is lower than 15 lU/mL, more particularly is lower than 10 lU/ml; ii.
  • LLoQ lower limit of quantification
  • Embodiment 9 is the combination for use of embodiment 8, wherein at least one of, at least two of, at least three of, at least four of, or the five features i., ii., iii., iv., and v. are still met 6 months after the end of the treatment.
  • Embodiment 10 is the combination for use of embodiment 8, wherein the serum HBsAg level is 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly of 10 lU/mL or lower.
  • Embodiment 11 is the combination for use of any one of embodiments 1-10, wherein the effective amount of the pharmaceutical composition comprising the RNAi component is administered to the subject at least until the serum HBsAg level of the subject decreases down to 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly down to 10 lU/mL or lower.
  • Embodiment 12 is the combination for use of any one of embodiments 1-11, wherein the effective amount of the nucleic acid molecule comprising a non-naturally occurring polynucleotide is administered to the subject after the serum HBsAg level of the patient has decreased down to 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly to 10 lU/mL or lower.
  • Embodiment 13 is the combination for use of any one of embodiments 8-12, wherein the serum HBsAg level in the subject is reduced at a faster rate, is reduced at a greater level, and/or is reduced for a greater time period than in a subject in which only an effective amount of the pharmaceutical composition comprising the RNAi component is administered or an effective amount of the nucleic acid molecule comprising a non-naturally occurring polynucleotide is administered.
  • Embodiment 14 is the combination for use of embodiment 2, wherein enhancing an immune response in the subject results in an increase in an innate immune system response and/or an adaptive immune system response.
  • Embodiment 15 is the combination for use of embodiment 14, wherein the increase in the innate immune system response results in an increase in the expression of one or several from among an interferon stimulated gene (ISG), an interferon gamma-induced protein 10 (IP10), an interferon alpha (IFNoc), an interleukin 12, or an interleukin-6 (IL-6).
  • ISG interferon stimulated gene
  • IP10 interferon gamma-induced protein 10
  • IFNoc interferon alpha
  • IL-6 interleukin-6
  • Embodiment 16 is the combination for use of embodiment 14, wherein the increase in the adaptive immune system response results in an increase in HBV-specific T cell responses and/or an increased activation of the HBV-specific T cells.
  • Embodiment 17 is the combination for use of embodiment 2, wherein enhancing an immune response in the subject results in an increase in the immunocompetence of the subject.
  • Embodiment 18 is the combination for use of embodiment 17, wherein the increase in the immunocompetence of the subject results in an increase of HBV-specific T cells, B cells, or NK cells in the liver and/or an increased activation of HBV-specific T cells, B cells, or NK cells in the liver.
  • Embodiment 19 is the combination for use of embodiment 17 or 18, wherein the increase in the immunocompetence of the subject results in an increase in NK cells, T cells, or B cells in a peripheral immune cell compartment and/or an increased activation of NK cells, T cells, or B cells in a peripheral immune cell compartment.
  • Embodiment 20 is the combination for use of any one of embodiments 17-19, wherein the increase in the immunocompetence of the subject results in a decrease of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or in a peripheral immune cell compartment and/or a decreased immunosuppressive activity of MDSCs in the liver and/or in a peripheral immune cell compartment, and/or a decrease of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or peripheral immune cell compartment and/or a decreased immunosuppressive activity of Treg in the liver and/or a peripheral immune cell compartment.
  • MDSCs Myeloid Derived Suppressive Cells
  • Treg regulatory T cells
  • Treg regulatory T cells
  • Embodiment 21 is the combination for use of embodiment 3, wherein decreasing viral replication in the subject results in a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 lU/mL, more particularly is lower than 15 lU/mL, more particularly is lower than 10 lU/mL.
  • LLoQ lower limit of quantification
  • Embodiment 22 is the combination for use of embodiment 5, wherein the targeted hepatocytes comprise cccDNA.
  • Embodiment 23 is the combination for use of embodiment 5 or 22, wherein the targeted hepatocytes comprise integrated HBV DNA.
  • Embodiment 24 is the combination for use of any one of embodiments 1-23, wherein the first or the second RNAi agent comprises at least one modified nucleotide and/or at least one modified intemucleoside linkage.
  • Embodiment 25 is the combination for use of any one of embodiments 1-24, wherein at least 90% of the nucleotides in the first and the second RNAi agents are modified nucleotides.
  • Embodiment 26 is the combination for use of any one of embodiments 1-25, wherein the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent.
  • Embodiment 27 is the combination for use of embodiment 26, wherein the targeting ligand comprises N-acetyl-galactosamine.
  • Embodiment 28 is the combination for use of embodiment 27, wherein the targeting ligand is selected from the group consisting of (NAGI 3), (NAG13)s, (NAGI 8), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), and (NAG39)s.
  • the targeting ligand is selected
  • Embodiment 29 is the combination for use of embodiment 28, wherein the targeting ligand is (NAG25), (NAG25)s, (NAG31), (NAG31)s, (NAG37), or (NAG37)s.
  • Embodiment 30 is the combination for use of any one of embodiments 26-29, wherein the targeting ligand is conjugated to the sense strand of the first or the second RNAi agent.
  • Embodiment 31 is the combination for use of embodiment 30, wherein the targeting ligand is conjugated to the 5’ terminus of the sense stand of the first or the second RNAi agent.
  • Embodiment 32 is the combination for use of any one of embodiments 1-31, wherein the first and the second RNAi agents independently comprise a duplex selected from the group consisting of:
  • Embodiment 33 is the combination for use of any one of embodiments 1-32, wherein the first and the second RNAi agents are each independently conjugated to a targeting ligand comprising N-acetyl-galactosamine, and the first and the second RNAi agents independently comprise a duplex selected from the group consisting of:
  • Embodiment 34 is the combination for use of any one of embodiments 1-33, wherein the ratio of the first RNAi agent to the second RNAi agent by weight is in the range of about 1:2 to about 5:1.
  • Embodiment 35 is the combination for use of any one of embodiments 1-34, wherein one of the first or second HBV antigens is an HBV core or an HBV pol antigen.
  • Embodiment 36 is the combination for use of any one of embodiments 1-35, wherein the non-naturally occurring polynucleotide sequence further comprises, ordered from the 5’ - to 3 ’-end:
  • Embodiment 37 is the combination for use of embodiment 36, wherein the non- naturally occurring polynucleotide sequence further comprises, ordered from the 5’- to 3’- end:
  • a third IRES element or a polynucleotide sequence encoding a third autoprotease peptide operably linked to the 3’ end of the polynucleotide sequence encoding the third HBV antigen and (7) a polynucleotide sequence encoding a fourth HBV antigen independently selected from the group consisting of an HBV core antigen, an HBV pol antigen, and an HBV surface antigen.
  • Embodiment 38 is the combination for use of embodiment 37, wherein each of the first, second, third and fourth HBV antigens is different from each other.
  • Embodiment 39 is the combination for use of embodiment 37 or 38, wherein each of the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
  • a first HBV Pre-S 1 antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: i;
  • a second HBV Pre-S 1 antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 3;
  • an HBV PreS2.S antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 5;
  • an HBV core antigen comprising, preferably consisting of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7; and
  • an HBV pol antigen comprising, preferably consisting of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 9, preferably, each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a signal peptide, and the HBV PreS2.S antigen comprises an internal signal peptide.
  • Embodiment 40 is the combination for use of embodiment 39, wherein the HBV core antigen comprises, preferably consists of, an amino acid sequence that is at least 98% identical to at least one of SEQ ID NOs: 84, 85, or 86, such as at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 84, 85, or 86.
  • Embodiment 41 is the combination for use of embodiment 39 or 40, wherein the last five C-terminal amino acids of the HBV core antigen comprise a VVR amino acid sequence, more particularly a VVRR (SEQ ID NO: 91) amino acid sequence, more particularly a VVRRR (SEQ ID NO: 92) amino acid sequence.
  • VVRR SEQ ID NO: 91
  • VVRRR SEQ ID NO: 92
  • Embodiment 42 is the combination for use of embodiment 39, wherein each of the
  • HBV surface antigen, the HBV core antigen and the HBV pol antigen comprises:
  • Embodiment 43 is the combination for use of embodiment 42, wherein each of the
  • HBV surface antigens, the HBV core antigen and the HBV pol antigen comprises one or more epitopes for HL A- A* 11:01.
  • Embodiment 44 is the combination for use of embodiment 39, wherein each of the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
  • HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86;
  • Embodiment 45 is the combination for use of any one of embodiments 37-44, wherein each of the polynucleotide sequences encoding the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
  • a polynucleotide sequence encoding the first HBV Pre-S 1 antigen having a sequence that is at least 90% identical to SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2;
  • a polynucleotide sequence encoding the second HBV Pre-S 1 antigen having a sequence that is at least 90% identical to SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4;
  • a polynucleotide sequence encoding the HBV PreS2.S antigen having a sequence that is at least 90% identical to SEQ ID NO: 6, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 6;
  • a polynucleotide sequence encoding the HBV core antigen having a sequence that is at least 90% identical to SEQ ID NO: 8, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 8; and
  • the polynucleotide sequence encoding the HBV pol antigen having a sequence that is at least 90% identical to SEQ ID NO: 10, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 10; preferably, the polynucleotide sequence encoding each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a polynucleotide sequence encoding a signal peptide, and the HBV PreS2.S antigen comprises an internal signal peptide.
  • Embodiment 46 is the combination for use of embodiment 45, wherein each of the polynucleotide sequences encoding the first, second, third and fourth HBV antigens is independently selected from the group consisting of: (i) a polynucleotide sequence encoding the first HBV Pre-S 1 antigen consisting of the sequence of SEQ ID NO: 2;
  • Embodiment 47 is the combination for use of any one of embodiments 37-46, wherein each of the first, second and third autoprotease peptides independently comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2 A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof, preferably, each of the first, second and third autoprotease peptides comprise the peptide sequence of P2A, such as a P2A sequence of SEQ ID NO: 11.
  • Embodiment 48 is the combination for use of any one of embodiments 37-47, wherein each of the first, second and third IRES is derived from encephalomyocarditis virus (EMCV) or Enterovirus 71 (EV71), preferably each of the first, second and third IRES comprises the polynucleotide sequence of SEQ ID NO: 13 or 14.
  • EMCV encephalomyocarditis virus
  • EV71 Enterovirus 71
  • Embodiment 49 is the combination for use of embodiment 1, wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprises, ordered from the 5’ - to 3 ’-end:
  • (6) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- S1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
  • S antigen having the amino acid sequence of SEQ ID NO: 5 preferably, the polynucleotide sequence encoding each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a polynucleotide sequence encoding a signal peptide, such as the signal peptide comprising the amino acid sequence of S
  • Embodiment 50 is the combination for use of embodiment 49, wherein the non- naturally occurring polynucleotide sequence comprises a sequence of any one of SEQ ID NOs: 15 to 54.
  • Embodiment 51 is the combination for use of any one of embodiments 1-50, wherein a vector comprises the non-naturally occurring polynucleotide sequence.
  • Embodiment 52 is the combination for use of embodiment 51, wherein the vector is a DNA plasmid, a DNA viral vector, or an RNA viral vector.
  • Embodiment 53 is the combination for use of any one of embodiment 1-50, wherein an RNA replicon comprises the non-naturally polynucleotide sequence, and wherein the RNA replicon comprises, ordered from the 5’ - to 3 ’-end:
  • RNA virus (1) a 5’ untranslated region (5’-UTR) required for nonstructural protein-mediated amplification of an RNA virus;
  • RNA virus a 3’ untranslated region (3’-UTR) required for nonstructural protein-mediated amplification of the RNA virus.
  • Embodiment 54 is the combination for use of any one of embodiments 1-50, wherein an RNA replicon comprises the non-naturally polynucleotide sequence, and wherein the RNA replicon comprises, ordered from the 5’ - to 3 ’-end:
  • an alphavirus 3' untranslated region (3' UTR); and (9) optionally, a poly adenosine sequence.
  • Embodiment 55 is the combination for use of embodiment 54, wherein the DLP motif is from a virus species selected from the group consisting of Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MTDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (U AV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (B ABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJ
  • Embodiment 56 is the combination for use of embodiment 55, wherein the fourth autoprotease peptide is selected from the group consisting of porcine tesehovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof, preferably, the fourth autoprotease peptide comprises the peptide sequence of P2A.
  • P2A porcine tesehovirus-1 2A
  • FMDV foot-and-mouth disease virus
  • E2A Equine Rhinitis A Virus
  • T2A a cytoplasmic polyhedrosis virus 2A
  • BmCPV2A cytoplasmic polyhedrosis virus 2
  • Embodiment 57 is the combination for use of any one of embodiments 1-50, wherein an RNA replicon comprises the non-naturally polynucleotide sequence, and wherein the RNA replicon comprises, ordered from the 5’ - to 3 ’-end:
  • Embodiment 58 is the combination for use of embodiment 57, wherein:
  • the polynucleotide sequence encoding the P2A sequence comprises SEQ ID NO: 12
  • the non-naturally occurring polynucleotide sequence comprises the polynucleotide sequence of any one of SEQ ID NOs: 15 to 54
  • the RNA replicon further comprises a poly adenosine sequence, preferably the poly adenosine sequence has the sequence of SEQ ID NO: 64, at the 3’-end of the replicon.
  • Embodiment 59 is the combination for use of any one of embodiments 53-58, wherein the RNA replicon comprises the polynucleotide sequence of any one of SEQ ID NOs: 65 to 72.
  • Embodiment 60 is the combination for use of any one of embodiments 53-58, wherein a nucleic acid molecule comprises a DNA sequence encoding the RNA replicon, preferably wherein the nucleic acid molecule further comprises a T7 promoter operably linked to the 5 ’-end of the DNA sequence, more preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO: 73.
  • Embodiment 61 is the combination for use of any one of embodiments 1-60, wherein a pharmaceutical composition comprises the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence, the vector, or the RNA replicon, and a pharmaceutically acceptable carrier.
  • Embodiment 62 is the combination for use of embodiment 61, wherein the pharmaceutical composition further comprises:
  • polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85 or 86.
  • Embodiment 63 is the combination for use of any one of embodiments 1-62, wherein the RNAi component is administered to the subject once monthly (Q4W) in a dose of about 40-1000 mg, more particularly of about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg, more particularly 200 mg.
  • Embodiment 64 is the combination for use of any one of embodiments 1-63, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject at a concentration of about 10-300 mg/mL, more particularly of about 20-200 ⁇ g/mL in a dose, wherein said dose is administered up to five (5) times.
  • Embodiment 65 is the combination for use of any one of embodiments 1-64, wherein the RNAi component is administered to the subject via intravenous or subcutaneous injection.
  • Embodiment 66 is the combination for use of any one of embodiments 1-65, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject via intramuscular injection.
  • Embodiment 67 is the combination for use of any one of embodiments 1-66, wherein the RNAi component is administered simultaneously or sequentially with the nucleic acid molecule comprising the non-naturally occurring polynucleotide.
  • Embodiment 68 is the combination for use of any one of embodiments 1-66, wherein the RNAi component is administered separately from the nucleic acid molecule comprising the non-naturally occurring polynucleotide.
  • Embodiment 69 is the combination for use of embodiment 68, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about six (6) months after the administration of the RNAi component has started.
  • Embodiment 70 is the combination for use of embodiment 68 or 69, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about six (6) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to five (5) times over the course of about six (6) months in total.
  • Embodiment 71 is the combination for use of embodiment 68 or 69, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about three (3) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to three (3) times over the course of about three (3) months in total.
  • Embodiment 72 is the combination for use of embodiment 68 or 69, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about six (6) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to three (3) times over the course of about three (3) months in total.
  • Embodiment 73 is the combination for use of embodiment 68 or 69, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about eight (8) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to three (3) times over the course of about three (3) months in total.
  • Embodiment 74 is the combination for use of embodiment 68 or 69, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about three (3) months after the administration of the RNAi component has started, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to three (3) times over the course of about three (3) months in total, and wherein the administration of the RNAi component continues, e.g., for about six (6) months, after the administration of the nucleic acid molecule comprising the non-naturally occurring polynucleotide has been stopped.
  • Embodiment 75 is the combination for use of any one of embodiments 1-74 further comprising administering to the subject a nucleoside analog or a nucleotide analog.
  • Embodiment 76 is the combination for use of embodiment 75, wherein the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof.
  • Embodiment 77 is the combination for use of embodiment 76, wherein entecavir is administered to the subject in a daily dose of about 0.1-5 mg.
  • Embodiment 78 is the combination for use of embodiment 76, wherein tenofovir is administered to the subject in a daily dose of about 5-50 mg of tenofovir alafenamide or about 200-500 mg of tenofovir disoproxil fumarate.
  • Embodiment 79 is the combination for use of embodiment 76, wherein lamivudine is administered to the subject in a daily dose of about 100 mg, about 150 mg or about 300 mg.
  • Embodiment 80 is the combination for use of embodiment 76, wherein telbivudine is administered to the subject in a daily dose of about 600 mg.
  • Embodiment 81 is the combination for use of any one of embodiments 1-80, wherein the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the compound of formula (I) is administered to the subject for 10-96 weeks, more particularly 12-72 weeks, more particularly 12-60 weeks, more particularly 12-52 weeks, more particularly 48 weeks.
  • Embodiment 82 is the combination for use of embodiment 81, further comprising administration of a nucleoside analog or a nucleotide analog, such as entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof to the subject, and wherein the administration of the nucleoside or nucleotide analog is optionally being continued once the administration of the effective amount the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising the non- naturally occurring polynucleotide stops.
  • a nucleoside analog or a nucleotide analog such as entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof to the subject, and wherein the administration of the nucleoside
  • Embodiment 83 is an RNAi component for use in combination with a nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence in the treatment of a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without a viral co-infection, and/or in the treatment of a chronic Hepatitis D Virus (HDV) infection, wherein the RNAi component and the nucleic acid molecule comprising the non- naturally occurring polynucleotide sequence are as defined in any one of embodiments 1-82, and wherein the RNAi component optionally is indicated for simultaneous, sequential, or separate administration with the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence.
  • HBV Hepatitis B Virus
  • CHB chronic HBV infection
  • HDV chronic Hepatitis D Virus
  • Embodiment 84 is a nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence for use in combination with an RNAi component in the treatment of a Hepatits B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without a viral co-infection, and/or in the treatment of a chronic Hepatitis D Virus (HDV) infection, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence and the RNAi component are as defined in any one of embodiments 1-82, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is indicated for simultaneous, sequential, or separate administration with the RNAi component.
  • HBV Hepatits B Virus
  • CHB chronic HBV infection
  • HDV chronic Hepatitis D Virus
  • Embodiment 85 is an effective amount of an RNAi component and a nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence in the manufacture of a medicament for inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof, wherein the RNAi component and the nucleic acid molecule comprising the non- naturally occurring polynucleotide sequence are as defined in any one of embodiments 1-82.
  • Example 1 Efficacy and Immunogenicity of the self-replicating RNA HBV therapeutic vaccine together with a liver-specific HBV targeted siRNA
  • mice are inoculated with AAV -HBV (10 11 viral genomes/mouse) to establish chronic infection.
  • AAV -HBV 11 viral genomes/mouse
  • HBsAg levels are measured and animals with serum HBsAg levels reflective of those seen in CHB patients (10 3 - 10 4 lU/mL) are distributed into a 1 st cohort to be monitored for efficacy and a 2 nd cohort to examine immunogenicity .
  • siRNA is a mixture of a first RNAi agent and a second RNAi agent as described in Table 3 (e.g., the SEQ ID NO: 94 and SEQ ID NO: 103 or SEQ ID NO: 105 duplex in a 2: 1 molar miture with the SEQ ID NO: 100 and SEQ ID NO: 108 or SEQ ID NO: 110 duplex), which are each independently conjugated to (NAG37)s or (NAG25)s, e.g., on the 5’ end of their respective sense strands, which may further carry an invAb at the 5’ or 3’ end of the sense strand.
  • Table 3 e.g., the SEQ ID NO: 94 and SEQ ID NO: 103 or SEQ ID NO: 105 duplex in a 2: 1 molar miture with the SEQ ID NO: 100 and SEQ ID NO: 108 or SEQ ID NO: 110 duplex
  • the HBV replicon therapeutic vaccine is provided:
  • one of the two bigenic constructs provides the Core coding sequence (e.g. SEQ ID NO: 8) and the Pol coding sequence (e.g. SEQ ID NO: 10) linked by a P2A or IRES coding sequence (e.g.
  • SEQ ID NO: 12, 13 or 14 wherein the other of the two bigenic constructs provides the PreS2.S coding sequence (e.g., SEQ ID NO: 6) and the preSl coding sequence (e.g., SEQ ID NO: 4) linked by a P2A or IRES coding sequence (e.g., SEQ ID NO: 12, 13 or 14), e.g., the mixture of the bigenic construct of SEQ ID NO: 68 and of the bigenic construct of SEQ ID NO: 65 or 66; or the mixture of the bigenic construct of SEQ ID NO: 67 and of the bigenic construct of SEQ ID NO: 66 or 65; or
  • tetracistronic construct providing the Core, Pol, preS2.S and PreSl coding sequences (SEQ ID NO: 8, 10, 6 and 4) linked together by P2A or IRES coding sequences (e.g. SEQ ID NO: 12, 13 or 14) (e.g., the tetracistronic organization shown in FIG. 2B), e.g., the tetracistronic construct of SEQ ID NO: 70, 71, 69 or 72.
  • the construct(s) is(are) optionally formulated in LNPs e.g., as described in US2014255472A1).
  • Two 10 microgram doses of the therapeutic vaccine are given via intramuscular injection 6 weeks apart with an optional 3rd dose administered if HBsAg levels are observed to rebound.
  • the first dose of replicon vaccine is administered the same day as the last dose of siRNA treatment.
  • Serum is collected at the time points indicated in the study design in FIGs. 1 A-1C in order to monitor serum cytokines as well as HBV DNA, HBsAg andHBeAg levels.
  • EXAMPLE 2 Therapeutic efficacy and immunogenicity of combination siRNA and replicon therapeutic vaccine in AAV-HBV mouse model.
  • example 1 has been implemented with the tetra-3 construct as follows.
  • the therapeutic efficacy of siRNA targeting HBsAg in combination with HBV tetra-3 replicon TxVx was evaluated in the AAV-HBV mouse model.
  • Two cohorts (immunogenicity cohort and efficacy cohort) of C57/BL6 mice were injected with 1.0x10 11 genomic equivalents of AAV vector expressing 1.2 genomes of HBV genotype D by IV injection.
  • the immunogenicity cohort was sacrificed two weeks after the final replicon dose and spleen and liver were harvested for assessment of HBV-specific, IFN-y + T-cell responses (ELISpot; Mabtech) while the efficacy cohort was monitored for serum HBsAg levels for 14 weeks following the last replicon dose.
  • ELISpot IFN-y + T-cell responses
  • HBV tetra-3 is the following construct: a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: r 86, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3; and where HBV siRNA is the 2: 1 (molar) mixture of a
  • HBsAg serum concentrations were lower in mice that received HBV siRNA + HBV tetra-3 replicon (group 1) compared to mice that received HBV siRNA + control replicon.
  • each mouse’s serum HBsAg concentration was normalized to their baseline HBsAg concentration.
  • Delta HBsAg concentrations were not significantly different in HBV siRNA + HBV tetra-3 replicon treated mice and HBV siRNA + control replicon treated mice at any timepoint after initiation of treatment (FIG. 4B).
  • HBV siRNA + HBV tetra-3 replicon combination therapy enhanced HBV-specific T cell responses compared to monotherapy
  • the immunogenicity cohort was sacrificed, and spleens were harvested two weeks after the second replicon dose.
  • Splenocytes were isolated by mechanical digestion and HBV-specific IFN ⁇ T cell responses were determined by ELISpot. Briefly, 200k cells/well were cultured in the presence of 2 ug/mL of overlapping peptides (15mers overlapping by 9 amino acids) covering pol, core, preSl and preS2.S for 18-24 hrs and ELISpot plates were processed according to the manufacturer’s instructions (Mabtech). As shown in FIG.
  • HBV-specific IFNy T cells were only detected from mice that received HBV tetra-3 replicon.

Abstract

Described are RNA interference (RNAi) agents for inhibiting the expression of Hepatitis B Virus (HBV) used in combination with nucleic acid molecules encoding HBV surface antigens, HBV core antigens, and HBV polymerase antigens, and methods of administering same. The HBV RNAi agents and nucleic acid molecules are administered to effectively inhibit HBV gene expression and to treat HBV infections.

Description

COMBINATION THERAPY FOR TREATING HEPATITIS B VIRUS INFECTION
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Application No. 63/127,430, filed December 18, 2020, the disclosure of which is incorporated herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY [0002] This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “065814.68WO1 Sequence Listing” and a creation date of November 19, 2021 and having a size of 424 kb. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.
FIELD OF INVENTION
[0003] The present disclosure relates generally to compositions and kits comprising an RNA interference (RNAi) component and an RNA replicon vaccine and their uses for treating hepatitis B virus infection or inhibiting the expression of at least one hepatitis B virus gene.
BACKGROUND
[0004] The hepatitis B virus (HBV) is a hepatotrophic, double-stranded DNA containing virus. Although DNA is the genetic material, the replication cycle involves a reverse transcription step to copy a pregenomic RNA into DNA. Hepatitis B virus is classified as one member of the Hepadnaviruses and belongs to the family of Hepadnaviridae. The primary infection of adult humans with hepatitis B virus causes an acute hepatitis with symptoms of organ inflammation, fever, jaundice and increased liver transaminases in blood. Those patients that are not able to overcome the virus infection suffer a chronic disease progression over many years with increased risk of developing cirrhotic liver or liver cancer. Perinatal transmission from hepatitis B virus-infected mothers to newborns also leads to chronic hepatitis.
[0005] Upon uptake by hepatocytes, the nucleocapsid is transferred to the nucleus and DNA is released. There, the DNA strand synthesis is completed and gaps repaired to give the covalently closed circular (ccc) supercoiled DNA of 3.2kb. The cccDNA serves as a template for transcription of five major viral mRNAs, which are 3.5, 3.5, 2.4, 2.1 and 0.7 kb long. All mRNAs are 5'-capped and polyadenylated at the 3'-end. There is sequence overlap at the 3'- end between all five mRNAs.
[0006] One 3.5 kb mRNA serves as template for core protein and polymerase production. In addition, the same transcript serves as a pre-genomic replication intermediate and allows the viral polymerase to initiate the reverse transcription into DNA. Core protein is needed for nucleocapsid formation. The other 3.5 kb mRNA encodes pre-core, the secretable e-antigen (HBeAg). In the absence of replication inhibitors, the abundance of e-antigen in blood correlates with Hepatitis B Virus replication in liver and serves as an important diagnostic marker for monitoring the disease progression.
[0007] The 2.4 and 2.1 kb mRNAs carry the open reading frames (“ORF”) pre-Sl, pre-S2 and S for expression of viral large, medium and small surface antigen. The s-antigen is associated with infectious, complete particles. In addition, blood of infected patients also contains non-infectious particles derived from s-antigen alone, free of genomic DNA or polymerase. The function of these particles is not fully understood. The complete and lasting depletion of detectable s-antigen in blood is considered as a reli able indicator for hepatitis B virus clearance.
[0008] The 0.7 kb mRNA encodes the X protein. This gene product is important for efficient transcription of viral genes and also acts as a transactivator on host gene expression. The latter activity seems to be important for hepatocyte transformation during development of liver cancer.
[0009] Patients with detectable s-antigen (HBsAg), e-antigen (HBeAg), and/or viral DNA in the blood for more than 6 months are considered chronically infected. Chronic HBV infection can be classified into five phases: (I) HBeAg-positive chronic infection, (II) HBeAg-positive chronic hepatitis, (III) HBeAg-negative chronic infection, (IV) HBeAg- negative chronic hepatitis and (V) HBsAg-negative phase. All patients with chronic HBV infection are at increased risk of progression to cirrhosis and hepatocellular carcinoma (HCC), depending on host and viral factors (Lampertico etal., J Hepatol., 2017, 67(2):370- 398). At the present, a complete sterilizing cure, i.e., viral eradication from the host, is unlikely to be feasible (Lok et al., J Hepatol., 2017, 67(4): 847-861 ). The main goal of therapy is to improve survival and quality of life by preventing disease progression, and consequently HCC development. The induction of long-term suppression of HBV replication represents the main endpoint of current treatment strategies, while HBsAg loss is an optimal endpoint. [0010] Nucleoside analogs as inhibitors of reverse transcriptase activity are typically the first treatment option for many patients. Long term administration of lamivudine, tenofovir, and/or entecavir has been shown to suppress hepatitis B virus replication, sometimes to undetectable levels, with improvement of liver function and reduction of liver inflammation typically seen as the most important benefits. However, only few patients achieve complete and lasting remission after the end of treatment. Furthermore, the hepatitis B virus develops drug resistance with increasing duration of treatment. This is especially difficult for patients co-infected with hepatitis B and human immunodeficiency virus (HIV). Both viruses are susceptible to nucleoside analogue drugs and may co-develop resistance.
[0011] Pegylated interferon-alpha (IFN) has been used to treat mild to moderate chronic hepatitis B patients. However, current treatment of chronic hepatitis B has limited efficacy (Erha et al., Gut. 2005 Jul; 54(7): 1009-1013). For example, the Asian genotype B gives very poor response rates. Co-infection with hepatitis D virus (HDV) or human immunodeficiency virus has been shown to render interferon-alpha therapy completely ineffective. Patients with strong liver damage and heavy fibrotic conditions are not qualified for interferon-alpha therapy.
[0012] Certain hepatitis B virus-specific RNA interference (RN Ai) agents have been previously shown to inhibit expression of HBV gene expression. For example, U.S. Patent Application Publication No. 2013/0005793, to Chin et al., which is incorporated herein by reference in its entirety, discloses certain double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of hepatitis B virus gene.
[0013] Several combinations have been tried in the treatment of HBV in various settings (Paul, Curr Hepat Rep. 2011 Jun; 10(2): 98-105). However, various combination therapies for HBeAg-positive chronic HBV or HBeAg-negative hepatitis B have not established a benefit over monotherapy. Id.
[0014] There is a need for improved HBV therapy that can overcome at least one of the disadvantages of existing treatment options, such as are toxicity, mutagenicity, lack of selectivity, poor efficacy, poor bioavailability, and difficulty of synthesis, while providing additional benefits such as increased potency or an increased safety window.
[0015] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety. BRIEF SUMMARY
[0016] Provided herein is a method of treating a Hepatitis B viral (HBV) infection in a subject, enhancing an immune response in a subject with a Hepatitis B viral (HBV) infection, decreasing viral replication in a subject with a Hepatitis B viral (HBV) infection, decreasing expression of one or more Hepatitis B Virus (HBV) polypeptide(s), more particularly of one or more polypeptide(s) from HBsAg and HBeAg, in a subject in need thereof, and/or increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with Hepatitis B viral (HBV) infection, wherein the method comprises administering to the subject:
(a) an effective amount of a pharmaceutical composition comprising an RNAi component having:
(i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
(ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111;
(b) an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
(1) a polynucleotide sequence encoding a first hepatitis B virus (HBV) antigen,
(2) a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and
(3) a polynucleotide sequence encoding a second HBV antigen, wherein the first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
[0017] In some embodiments, the method further comprises administering to the subject another agent for treating infection caused by hepatitis B virus HBV. The other agent can be a nucleoside analog. In some embodiments, the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof.
[0018] In some embodiments, the other agent is a nucleic acid polymer (NAP). The NAP can, for example, be selected from REP2139 or REP2165. REP2139 has a sequence of (A,5'MeC)20 with each linkage being phosphorothioated and every ribose being 2’0 methylated (which is disclosed as SEQ ID NOTO in WO2016/04525, the content of which is incorporated herein by reference in its entirety). REP2165 has a sequence of (A,5'MeC)20 with each linkage being phosphorothioated, every rbose being 2’0 methylated except adenosines at positions 11, 21, and 31, where riboses are 2’OH (which is disclosed as SEQ ID NO: 13 in WO2016/04525).
[0019] The NAP can also be other exemplary nucleic acid polymers, which include, but are not limited to, REP2006, REP2031, REP2055, STOPS™ (S-antigen transport-inhibiting oligonucleotide polymers), and those disclosed in Patent Application Publication Nos. WO200424919; WO201221985; and WO202097342 and U.S. Patent Nos. 7,358,068;
8,008,269; 8,008,270; and 8,067,385, the content of each is incorporated herein by reference in its entirety.
[0020] In some embodiments, the subject has chronic HBV infection.
[0021] Another general aspect of the application relates to a combination or a kit for use in treating a HBV infection, such as a chronic HBV infection (CHB), with or without viral co-infection, e.g., with or without co-infection with HDV and/or HCV and/or HIV, more particularly with or without co-infection with at least HDV, and/or for treating chronic HDV infection (CHD) in a subject in need thereof, comprising:
(a) a pharmaceutical composition comprising an RNAi component having:
(i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
(ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; (b) a pharmaceutical composition comprising an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3’-end:
(1) a polynucleotide sequence encoding a first hepatitis B virus (HBV) antigen,
(2) a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and
(3) a polynucleotide sequence encoding a second HBV antigen, wherein the first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen. [0022] In some embodiments, the combination or kit is for use in enhancing an immune response, decreasing viral replication, and/or decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptides, more particularly of one or more polypeptide(s) selected from HBsAg and HBeAg, in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection.
[0023] In some embodiments, the combination or kit further comprises another agent for treating infection caused by hepatitis B virus HBV. The other agent can be a nucleoside analog. In some embodiments, the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof.
[0024] In some embodiments, the other agent is a nucleic acid polymer (NAP). The NAP can, for example, be selected from REP2139 or REP2165. REP2139 has a sequence of (A,5'MeC)20 with each linkage being phosphorothioated and every ribose being 2’0 methylated (which is disclosed as SEQ ID NOTO in WO2016/04525, the content of which is incorporated herein by reference in its entirety). REP2165 has a sequence of (A,5'MeC)20 with each linkage being phosphorothioated, every rbose being 2’0 methylated except adenosines at positions 11, 21, and 31, where riboses are 2’OH (which is disclosed as SEQ ID NO: 13 in WO2016/04525).
[0025] The NAP can also be other exemplary nucleic acid polymers, which include, but are not limited to, REP2006, REP2031, REP2055, STOPS™ (S-antigen transport-inhibiting oligonucleotide polymers), and those disclosed in Patent Application Publication Nos. WO200424919; WO201221985; and WO202097342 and U.S. Patent Nos. 7,358,068;
8,008,269; 8,008,270; and 8,067,385, the content of each is incorporated herein by reference in its entirety. [0026] Also provided herein is an effective amount of an RNAi component and a nucleic acid molecule comprising a non-naturally occurring polynucleotide, optionally a nucleoside analog, in the manufacture of a medicament for treating a Hepatitis B viral (HBV) infection in a subject, enhancing an immune response in a subject with a HBV infection, decreasing viral replication in a subject with a HBV infection, decreasing the expression of one or more HBV polypeptide(s), more particularly of one or more polypeptide(s) from HBsAg and HBeAg, in a subject in need thereof, modulating Hepatitis B viral (HBV) capsid assembly or disassembly in a subject with a HBV infection, and/or increasing the targeted killing of hepatocytes comprising integrated viral DNA or extrachromosomal DNA in a subject with a HBV infection, wherein:
(a) the RNAi component comprises:
(i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
(ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111 ; and
(b) the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3’-end:
(1) a polynucleotide sequence encoding a first hepatitis B virus (HBV) antigen,
(2) a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and
(3) a polynucleotide sequence encoding a second HBV antigen, wherein the first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen; and
(c) the nucleoside analog is preferably entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof. [0027] In other embodiments, the treatment comprises a first phase conducted before a second phase, and a) the first phase comprises administering the RNAi component to the subject to thereby decrease the HBsAg to a level low enough to allow recovery of T cell function, preferably to a serum HBsAg level of less than 1000, 100, 10, or 1 lU/mL; and b) the second phase comprises administering the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence to the subject.
[0028] In certain embodiments, the second phase does not comprise administering the RNAi component to the subject. In other embodiments, the second phase further comprises administering the RNAi component to the subject. The first phase of the treatment can last about 1-24 months, such as 1-12 months, 1-3 months, 4-6 months, 7-9 months, 10-12 months, or any period of time in between. The second phase of the treatment can last about 1-24 months, such as 1-12 months, 4-6 months, 7-9 months, 10-12 months, 13-18 months, 19-24 months, or any period of time in between. In some embodiments, the second phase comprises administering the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence and administering the RNAi component to the subject over the same treatment period. In other embodiments, the second phase comprises administering the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence and administering the RNAi component to the subject over different treatment periods, wherein the administration of the RNAi component stops first, and the administration of the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence continues, for example, for an additional 1 month, 2-3 months, 4-5 months, 6 or more months, or any period of time in between, until the end of the second phase.
[0029] In some embodiments, the RNAi component is administered subcutaneously or intravenously, preferably subcutaneously, at an amount of about 40-1000 mg per dose, more particularly about 40-250 mg per dose, such as about 100-200 mg per dose, more particularly about 200 mg per dose, and it is administered weekly, every two weeks, every 4 weeks, monthly, every 2 months, or every 3 months, preferably every 4 weeks or monthly.
[0030] In some embodiments, a subject has achieved at least one of the following features a)-e), more particularly more than one of the following features a)-e), more particularly at least features a), b) and c), more particularly all of features a)-d), during or after the treatment with a combination according to an embodiment of the application: a) decreased HBV replication as measured by serum HBV DNA level, preferably undetectable serum HBV DNA level; b) decreased expression of one or more HBV polypeptide(s), preferably decreased expression of HBsAg as measured by serum HBsAg level, preferably undetectable serum HBsAg level; c) enhanced HBV-specific T cell responses; d) loss of HBeAg or serocoversion for HBeAg, if the subject is HBeAg positive before the treatment; and e) seroconversion for HBsAg, preferably, the subject has achieved functional cure after the treatment; more preferably, when administered to a cohort of CHB patients, a greater proportion of the CHB patients have achieved at least one of a) to e) after the treatment compared to an otherwise identical treatment in a clinically comparable cohort of CHB patients with only one of the RNAi component and the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence, for example, about 30-50% of the CHB patients have achieved functional cure after the treatment.
[0031] In certain embodiments, a combination according to an embodiment of the application is for use in decreasing viral replication, as measured by serum HBV DNA, in the subject with CHB, wherein the subject is not already suppressed by nucleoside/nucleotide analogue therapy. The combination can also be used for decreasing the expression of one or more HBV polypeptides in the subject with CHB, such as HBsAg in serum of the subject. The combination can further be used for bringing about an enhanced HBV-specific T cell response, which can be enhanced in a quantitative and/or qualitative manner, in the subject with CHB. The enhanced T cell response in the subject with CHB can result in one or more of the following: a) greater number of HBV-specific T cells; b) greater breadth of HBV-specific T cells in that more HBV antigens are recognized by the T cells; c) greater function of HBV-specific T cells in that when re-stimulated with HBV antigens (such as HBV peptide pools), the T cells secrete greater number of cytokines, e.g., polyfunctional T cells; d) enhanced clearance of HBV-infected cells, such as cells containing cccDNA, or of non-infected cells that have integrated HBV DNA; and e) functional cure in the subject with CHB through enhancing immune responses, especially HBV-specific T cell responses.
[0032] In some embodiments, a combination according to an embodiment of the application is for use in treating a subject co-infected with CHB and another chronic infection with at least one of: hepatitis D virus (HDV); hepatitis C virus (HCV); or human immunodeficiency virus (HIV). The combination can be used in a method of decreasing the serum levels of HDV RNA in a subject chronically co-infected with both HBV and HDV; a method of normalizing alanine aminotransferase (ALT) level in a subject chronically coinfected with HBV and HDV; or a method of eradicating HDV infection in a subject chronically co-infected with HBV and HDV.
[0033] In any of the methods or other disclosures herein, in one variation the first or the second RNAi agent comprises at least one modified nucleotide or at least one modified intemucleoside linkage. In another variation, substantially all of the nucleotides in the first and the second RNAi agents are modified nucleotides. In a further variation, the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent. In one aspect, the targeting ligand comprises N-acetyl-galactosamine. In a particular aspect, the targeting ligand is selected from the group consisting of (NAGI 3), (NAG13)s, (NAGI 8), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), and (NAG39)s. In one variation, the targeting ligand is (NAG25), (NAG25)s, (NAG31), (NAG31)s, (NAG37), or (NAG37)s. In another variation, the targeting ligand is conjugated to the sense strand of the first or the second RNAi agent. In another variation, the targeting ligand is conjugated to the 5’ terminus of the sense stand of the first or the second RNAi agent. In a further variation, the first and the second RNAi agents further comprise a targeting ligand that is conjugated to each of the first and the second RNAi agents. In one aspect, the targeting ligand comprises N-acetyl-galactosamine. In a particular aspect, the targeting ligand is selected from the group consisting of (NAGI 3), (NAG13)s, (NAGI 8), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), and (NAG39)s. In one variation, the targeting ligand is (NAG25), (NAG25)s, (NAG31), (NAG31)s, (NAG37), or (NAG37)s. In another variation, the targeting ligand is conjugated to the sense strand of each of the first and second RNAi agents. In another variation, the targeting ligand is conjugated to the 5’ terminus of the sense stand of each of the first and the second RNAi agents. In still another variation, the first and the second RNAi agents independently comprise a duplex selected from the group consisting of: an antisense strand comprising SEQ ID NO:93 and a sense strand comprising SEQ ID NO: 102; an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 103; an antisense strand comprising SEQ ID NO: 95 and a sense strand comprising SEQ ID NO: 103; an antisense strand comprising SEQ ID NO:96 and a sense strand comprising SEQ ID NO: 104; an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 108; an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 109; an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 105; and an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 110. In a particular variation, the first and the second RNAi agents are each independently conjugated to a targeting ligand comprising N-acetyl- galactosamine, and the first and the second RNAi agents independently comprise a duplex selected from the group consisting of: an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 103; an antisense strand comprising SEQ ID NO:96 and a sense strand comprising SEQ ID NO: 104; an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 108; an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 105; and an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 110. In still another variation, the ratio of the first RNAi agent to the second RNAi agent by weight is in the range of about 1 :2 to about 5: 1. In another variation, the ratio of the first RNAi agent to the second RNAi agent by weight is about 2: 1. In still another variation, the molar ratio of the first RNAi agent to the second RNAi agent is in the range of about 1 :2 to about 5: 1. In another variation, the molar ratio of the first RNAi agent to the second RNAi agent is about 2: 1. In certain aspects, the first and the second RNAi agents are each independently conjugated to (NAG37)s, the first RNAi agent comprises an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 103, and the second RNAi agent comprises an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 108.
[0034] In any of the methods or other disclosures herein, one of the first or second HBV antigens is an HBV core or an HBV pol antigen. [0035] In any of the methods or other disclosures herein, the non-naturally occurring polynucleotide sequence further comprises, ordered from the 5’ - to 3 ’-end:
(4) a second IRES element or a polynucleotide sequence encoding a second autoprotease peptide operably linked to the 3’ end of the polynucleotide sequence encoding the second HBV antigen, and
(5) a polynucleotide sequence encoding a third HBV antigen independently selected from the group consisting of an HBV core antigen, an HBV pol antigen, and an HBV surface antigen.
[0036] In any of the methods or other disclosures herein, the non-naturally occurring polynucleotide sequence further comprises, ordered from the 5’ - to 3 ’-end:
(6) a third IRES element or a polynucleotide sequence encoding a third autoprotease peptide operably linked to the 3’ end of the polynucleotide sequence encoding the third HBV antigen, and
(7) a polynucleotide sequence encoding a fourth HBV antigen independently selected from the group consisting of an HBV core antigen, an HBV pol antigen, and an HBV surface antigen.
[0030] In any of the methods or other disclosures herein, each of the first, second, third and fourth HBV antigens is different from each other.
[0031] In any of the methods or other disclosures herein, each of the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
(i) a first HBV Pre-S 1 antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: i;
(ii) a second HBV Pre-S 1 antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 3;
(iii) an HBV PreS2.S antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 5;
(iv) an HBV core antigen comprising, preferably consisting of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7; and
(v) an HBV polymerase antigen comprising, preferably consisting of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 9, preferably, each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a signal peptide, and the HBV PreS2.S antigen comprises an internal signal peptide.
[0032] In any of the methods or other disclosures herein, the HBV core antigen comprises, preferably consists of, an amino acid sequence that is at least 98% identical to at least one of SEQ ID NOs: 84, 85, or 86, such as at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 84, 85, or 86. In some embodiments, the last five C-terminal amino acids of the HBV core antigen comprise a VVR amino acid sequence, more particularly a VVRR (SEQ ID NO: 91) amino acid sequence, more particularly a VVRRR (SEQ ID NO: 92) amino acid sequence.
[0033] In any of the methods or other disclosures herein, each of the HBV surface antigen, the HBV core antigen and the HBV pol antigen comprises:
(i) a consensus sequence for HBV genotypes A, B, C and D; and/or
(ii) one or more epitopes for HL A-A* 11:01, HLA-A*24:02, HLA-A*02:01, HLA- A*A2402, HLA-A*A0101, or HLA-B*40:01.
[0034] In any of the methods or other disclosures herein, each of the HBV surface antigens, the HBV core antigen and the HBV pol antigen comprises one or more epitopes for HLA- A*ll:01.
[0035] In any of the methods or other disclosures herein, each of the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
(i) the first HBV Pre-S 1 antigen consisting of the amino acid sequence of SEQ ID NO: i; (ii) the second HBV Pre-S 1 antigen consisting of the amino acid sequence of SEQ ID NO: 3;
(iii) the HBV PreS2.S antigen consisting of the amino acid sequence of SEQ ID NO: 5;
(iv) the HBV core antigen consists of the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86; and
(v) the HBV pol antigen consisting of the amino acid sequence of SEQ ID NO: 9, preferably, each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a signal peptide, such as the signal peptide comprising the amino acid sequence of SEQ ID NO: 77.
[0036] In any of the methods or other disclosures herein, each of the polynucleotide sequences encoding the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
(i) a polynucleotide sequence encoding the first HBV Pre-S 1 antigen having a sequence that is at least 90% identical to SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2;
(ii) a polynucleotide sequence encoding the second HBV Pre-S 1 antigen having a sequence that is at least 90% identical to SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4;
(iii) a polynucleotide sequence encoding the HBV PreS2.S antigen having a sequence that is at least 90% identical to SEQ ID NO: 6, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 6;
(iv) a polynucleotide sequence encoding the HBV core antigen having a sequence that is at least 90% identical to SEQ ID NO: 8, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 8; and
(v) the polynucleotide sequence encoding the HBV pol antigen having a sequence that is at least 90% identical to SEQ ID NO: 10, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 10, preferably, the polynucleotide sequence encoding each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a polynucleotide sequence encoding a signal peptide, and the HBV PreS2.S antigen comprises an internal signal peptide.
[0037] In any of the methods or other disclosures herein, each of the polynucleotide sequences encoding the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
(i) a polynucleotide sequence encoding the first HBV Pre-S 1 antigen consisting of the sequence of SEQ ID NO: 2;
(ii) a polynucleotide sequence encoding the second HBV Pre-S 1 antigen consisting of the sequence of SEQ ID NO: 4;
(iii) a polynucleotide sequence encoding the HBV PreS2.S antigen consisting of the sequence of SEQ ID NO: 6;
(iv) a polynucleotide sequence encoding the HBV core antigen consisting of the sequence of any one of SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89; and
(v) a polynucleotide sequence encoding the HBV pol antigen consisting of the sequence of SEQ ID NO: 10; preferably, the polynucleotide sequence encoding each of the first and second HBV Pre- S1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a polynucleotide encoding a signal peptide, such as the polynucleotide comprising the sequence of SEQ ID NO: 90.
[0038] In any of the methods or other disclosures herein, each of the first, second and third autoprotease peptides independently comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2 A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof. Preferably, each of the first, second and third autoprotease peptides comprises the peptide sequence of P2A, such as a P2A sequence of SEQ ID NO: 11.
[0039] In any of the methods or other disclosures herein, each of the first, second and third IRES is derived from encephalomyocarditis virus (EMCV) or Enterovirus 71 (EV71).
Preferably, each of the first, second and third IRES comprises the polynucleotide sequence of SEQ ID NO: 13 or 14. [0040] In any of the methods or other disclosures herein, the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence comprises, ordered from the 5’- to 3’-end:
(1) a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5;
(2) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV Pre-Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(3) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(4) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(5) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(6) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(7) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(8) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3; (9) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(10) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(11) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(12) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(13) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(14) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(15) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(16) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(17) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5;
(18) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5;
(19) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(20) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(21) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5; or (22) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, preferably, the polynucleotide sequence encoding each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a polynucleotide sequence encoding a signal peptide, such as the signal peptide comprising the amino acid sequence of SEQ ID NO: 77.
[0041] In any of the methods or other disclosures herein, the nucleic acid molecule comprises the non-naturally occurring polynucleotide sequence of any one of SEQ ID NOs: 15 to 54. [0042] In any of the methods or other disclosures herein, the application relates to a vector comprising a non-naturally occurring polynucleotide sequence of the application.
[0043] In any of the methods or other disclosures herein, the vector is a DNA plasmid. In another embodiment, the vector is a DNA viral vector or an RNA viral vector.
[0044] In any of the methods or other disclosures herein, the application relates to a RNA replicon, comprising, ordered from the 5’- to 3’-end:
(1) a 5’ untranslated region (5’-UTR) required for nonstructural protein-mediated amplification of an RNA virus;
(2) a polynucleotide sequence encoding at least one, preferably all, of non-structural proteins of the RNA virus;
(3) a subgenomic promoter of the RNA virus;
(4) a non-naturally occurring polynucleotide sequence of the application; and
(5) a 3’ untranslated region (3’-UTR) required for nonstructural protein-mediated amplification of the RNA virus.
[0045] In any of the methods or other disclosures herein, the application relates to an RNA replicon, comprising, ordered from the 5’- to 3’-end,
(1) an alphavirus 5’ untranslated region (5’-UTR),
(2) a 5’ replication sequence of an alphavirus non-structural gene nspl,
(3) a downstream loop (DLP) motif of a virus species,
(4) a polynucleotide sequence encoding a fourth autoprotease peptide, (5) a polynucleotide sequence encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4,
(6) an alphavirus subgenomic promoter,
(7) a non-naturally occurring polynucleotide sequence of the application,
(8) an alphavirus 3' untranslated region (3' UTR), and
(9) optionally, a poly adenosine sequence.
[0046] In any of the methods or other disclosures herein, the DLP motif is from a virus species selected from the group consisting of Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MTDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (U AV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), and Buggy Creek virus.
[0047] In any of the methods or other disclosures herein, the fourth autoprotease peptide is selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof. Preferably, the fourth autoprotease peptide comprises the peptide sequence of P2A.
[0048] In any of the methods or other disclosures herein, the application relates to an RNA replicon, comprising, ordered from the 5’- to 3’-end,
(1) a 5’-UTR having the polynucleotide sequence of SEQ ID NO: 55,
(2) a 5’ replication sequence having the polynucleotide sequence of SEQ ID NO: 56,
(3) a DLP motif comprising the polynucleotide sequence of SEQ ID NO: 57,
(4) a polynucleotide sequence encoding a P2A sequence of SEQ ID NO: 11,
(5) polynucleotide sequences encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4 having the nucleic acid sequences of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively,
(6) a subgenomic promoter having polynucleotide sequence of SEQ ID NO: 62,
(7) a non-naturally occurring polynucleotide sequence of the application, and
(8) a 3' UTR having the polynucleotide sequence of SEQ ID NO: 63. [0049] In any of the methods or other disclosures herein, the polynucleotide sequence encoding the P2A sequence comprises SEQ ID NO: 12, the non-naturally occurring polynucleotide sequence comprises the polynucleotide sequence of any one of SEQ ID NOs: 15 to 54, and the RNA replicon further comprises a poly adenosine sequence at the 3 ’-end of the replicon. Preferably, the poly adenosine sequence has the sequence of SEQ ID NO: 64. [0050] In any of the methods or other disclosures herein, the application relates to an RNA replicon comprising the polynucleotide sequence of any one of SEQ ID NOs: 65 to 72.
[0051] In any of the methods or other disclosures herein, the application relates to a nucleic acid molecule comprising a DNA sequence encoding an RNA replicon of the application. Preferably, the nucleic acid further comprises a T7 promoter operably linked to the 5 ’-end of the DNA sequence. More preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO: 73.
[0052] In any of the methods or other disclosures herein, the application relates to a pharmaceutical composition comprising a nucleic acid molecule, a vector, or an RNA replicon of the application, and a pharmaceutically acceptable carrier. In an embodiment, the pharmaceutical composition further comprises: (1) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9; or (2) a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85 or 86.
[0053] In any of the methods or other disclosures herein, the application relates to a method for vaccinating a subject against HBV, the method comprising administering to the subject a combination of pharmaceutical compositions of the application. In an embodiment, the method comprises administering to the subject a first composition comprising an RNAi component, a second composition comprising a nucleic acid molecule comprising a non- naturrally occurring polynucleotide sequence encoding one or more HBV antigens, and a third composition comprising a nucleic acid molecule encoding at least one identical HBV antigen, wherein the second and third composition comprise a prime-boost regimen. In some embodiments, the prime-boost regimen comprises a priming composition comprising an RNA replicon of the application and a boosting composition comprising a vector which is not an RNA replicon and which encodes at least one identical HBV epitope, preferably, at least one identical HBV antigen, as the priming composition. In an embodiment, the boosting composition comprises a Modified Vaccinia Ankara (MV A) vector, an adenovirus vector, or a plasmid vector. In some embodiments, the prime-boost regimen comprises a boosting composition comprising an RNA replicon of the application and a priming composition comprising a vector which is not an RNA replicon and which encodes at least one identical HBV epitope, such as at least one identical HLA epitope, preferably, at least one identical HBV antigen, as the boosting composition. In an embodiment, the priming composition comprises a Modified Vaccinia Ankara (MV A) vector, an adenovirus vector, or a plasmid vector.
[0054] In another general aspect, the application relates to an isolated host cell comprising a nucleic acid molecule, a vector, or an RNA replicon of the application.
[0055] In another general aspect, the application relates to a method of producing an RNA replicon, comprising transcribing a nucleic acid of the application, in vivo or in vitro.
[0056] Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.
[0058] FIGs. 1A-1C show the experimental design for the RNAi and RNA replicon combination therapy. FIG. 1 A shows the groups to be tested with the combination therapy. FIG. IB shows provides subject and readout information for the combination therapy experiment. FIG. 1C shows a schematic of the experimental design.
[0059] FIGs. 2A-2B show schematics of antigen designs (FIG. 2 A) and bicistronic and tetracistronic vaccine designs (FIG. 2B). The antigen designs (FIG. 2A) include Pol with inactivating point mutations, truncated Core with C terminal deletion (SEQ ID NO:76), PreS2.S, and PreSl showing the cystatin S Signal peptide (SEQ ID NO:77). [0060] FIGs. 3A-3F show graphs of HBsAg serum concentrations (geometric mean ± 95% confidence intervals) in mice that were administered HBV siRNA + HBV tetra-3 replicon (Group 1) compared to mice that received HBV siRNA + control replicon (Group 4) at wk 0 (immediately prior to first siRNA dose; FIG. 3A & 3D); wk 6 (immediately prior to the first replicon dose; FIG. 3B & 3E) and wk 14 (2 wks after the last replicon dose; FIG. 3C & 3F). FIGs. 3A-3C show serum HBsAg concentrations for mice from the efficacy and immunogenicity cohorts combined. FIGs. 3D-3F show HBsAg serum concentrations in mice in the efficacy (E) and immunogenicity (I) cohorts separately.
[0061] FIGs. 4A-4B show graphs demonstrating HBsAg serum concentration in mice that were administered HBV siRNA + HBV tetra-3 replicon compared to HBV siRNA + control replicon over time. FIG. 4A shows absolute HBsAg concentrations. FIG. 4B shows delta HBsAg concentrations normalized to wk 0 HBsAg concentrations. Closed circle, HBV siRNA + HBV replicon (n=8); closed square, AAT siRNA + HBV replicon (n=10); closed triangle, vehicle + HBV replicon (n=10); closed inverted triangle, HBV siRNA + rFF replicon (n=10); closed diamond, AAT siRNA + rFF replicon (n=10); open circle, vehicle + vehicle (n=9). Asterisks indicate statistically significant difference (P<0.05) calculated by Mann- Whitney test comparing HBV siRNA + HBV replicon and HBV siRNA + rFF replicon.
[0062] FIG. 5 shows a graph of HBV-specific IFNg T cell responses in mice treated with HBV siRNA + HBV tetra-3 replicon or AAT siRNA + HBV tetra-3 -replicon as compared to mice that received the siRNA vehicle + replicon. Statistical significance was determined by Mann- Whitney test).
DETAILED DESCRIPTION
[0059] The following description is presented to enable a person of ordinary skill in the art to make and use the various embodiments. Descriptions of specific compositions, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown but are to be accorded the scope consistent with the claims. [0060] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.
[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth fully herein.
[0062] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the,” include plural reference unless the context clearly dictates otherwise.
[0063] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.
[0064] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having.”
[0065] When used herein “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned terms of “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment of the application can be replaced with the term “consisting of’ or “consisting essentially of’ to vary scopes of the disclosure.
[0066] As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”
[0067] Unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1 mg/mL to 10 mg/mL includes 0.9 mg/mL to 11 mg/mL. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
[0068] By “optional” or “optionally” is meant that the event described subsequent thereto may or may not happen. This term encompasses the cases that the event may or may not happen.
[0069] An “epitope” as used herein is a set of amino acid residues that form a site recognized by an immunoglobulin, T cell receptor or human leukocyte antigen (HLA) molecule.
[0070] The HLA proteins are encoded by clusters of genes that form a region located on chromosome 6 known as the Major Histocompatibility Complex (MHC), in recognition of the important role of the proteins encoded by the MHC loci in graft rejection. Accordingly, the HLA proteins are also referred to as MHC proteins. HLA or MHC proteins are cell surface glycoproteins that bind peptides at intracellular locations and deliver them to the cell surface, where the combined ligand is recognized by a T cell. Class I MHC proteins are found on virtually all of the nucleated cells of the body. The class I MHC proteins bind peptides present in the cytosol and form peptide-MHC protein complexes that are presented at the cell surface, where they are recognized by cytotoxic CD8+ T cells. Class II MHC proteins are usually found only on antigen-presenting cells such as B lymphocytes, macrophages, and dendritic cells. Each MHC Class I receptor consists of a variable a chain and a relatively conserved P2-microglobulin chain. Three different, highly polymorphic class I a chain genes have been identified. These are called HLA- A, HLA-B, and HLA-C. Variations in the a chain accounts for all of the different class I MHC genes in the population. [0071] As used herein, the half maximal effective concentration (EC50) is intended in accordance with its general meaning in the field. It may more particularly refer to the concentration of a compound which induces a response halfway between the baseline and maximum, typically after a specified exposure time. The EC50 value is commonly used as a measure of a compound’s potency, with a lower value generally indicating a higher potency. [0072] As used herein, a “non-naturally occurring” nucleic acid or polypeptide refers to a nucleic acid or polypeptide that does not occur in nature. A “non-naturally occurring” nucleic acid or polypeptide can be synthesized, treated, fabricated, and/or otherwise manipulated in a laboratory and/or manufacturing setting. In some cases, a non-naturally occurring nucleic acid or polypeptide can comprise a naturally-occurring nucleic acid or polypeptide that is treated, processed, or manipulated to exhibit properties that were not present in the naturally-occurring nucleic acid or polypeptide, prior to treatment. As used herein, a “non-naturally occurring” nucleic acid or polypeptide can be a nucleic acid or polypeptide isolated or separated from the natural source in which it was discovered, and it lacks covalent bonds to sequences with which it was associated in the natural source. A “non-naturally occurring” nucleic acid or polypeptide can be made recombinantly or via other methods, such as chemical synthesis.
[0073] As used herein, the term “priming composition,” “priming immunization,” or “prime immunization” refers to primary antigen stimulation by using a first composition of the invention. Specifically, the term “priming” or “potentiating” an immune response, as used herein, refers to a first immunization using an antigen which induces an immune response to the desired antigen and recalls a higher level of immune response to the desired antigen upon subsequent re-immunization with the same antigen. As used herein, the term “boosting composition,” “boosting immunization,” or “boost immunization” refers to an additional immunization administered to, or effective in, a mammal after the primary immunization. Specifically, the term “boosting” an immune response, as used herein, refers to the administration of a composition delivering the same antigen as encoded in the priming immunization.
[0074] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions.
[0075] The term “pharmaceutically acceptable salt” refers to a salt of any of the compounds or compositions herein which are known to be non-toxic and are commonly used in the pharmaceutical literature. In some embodiments, the pharmaceutically acceptable salt of a compound retains the biological effectiveness of the compounds described herein and are not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts can be found in Berge et al., Pharmaceutical Salts, J. Pharmaceutical Sciences, January 1977, 66(1), 1-19. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 2 -hydroxy ethylsulfonic acid, p- toluenesulfonic acid, stearic acid and salicylic acid. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines; substituted amines including naturally occurring substituted amines; cyclic amines; and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, tri ethyl amine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts.
[0076] The pharmaceutically acceptable salts according to the invention may be prepared from the parent compound containing acidic or basic group through conventional chemical procedures. Generally, such salts can be prepared through the reaction of the compounds in the form of free acid or base with stoichiometric appropriate base or acid in water, organic solvent or the mixture thereof. Typically, nonaqueous medium like ether, ethyl acetate, ethanol, isopropanol, acetonitrile etc. are preferable.
[0077] The terms “patient” and “subject” refer to a human or a great ape (e.g., chimpanzee, orangutan, bonobo, gorilla). In some embodiments, the patient or subject is a human that has been or will be the object of treatment, observation or experiment. The combinations, compositions, and methods described herein can be useful in both human therapy and veterinary applications for species that can be chronically infected by HBV. In some embodiments, the subject has an HBV infection, more particularly a chronic HBV infection (CHB). The subject can have a CHB, with or without viral co-infection. As used herein, a “viral co-infection” refers to an infection with at least two types of virus. A “viral co-infection” can be an infection with at least two types of virus simultaneously. A “viral coinfection” can also be a superinfection, wherein an infection with one or more types of virus is in addition to a pre-existing infection with one or more other types of virus. In some embodiments, the subject has an HBV-HDV co-infection, an HBV -HIV co-infection, or an HBV-HCV co-infection. Preferably, the subject has a CHB-HDV co-infection, a CHB-HIV co-infection, or a CHB-HCV co-infection. More preferably, the subject has a viral coinfection with CHB and chronic HDV.
[0078] As used herein “hepatitis B virus” or “HBV” refers to a specific virus of the hepadnaviridae family. HBV is a small (e.g., 3.2 kb) hepatotropic DNA virus that encodes four open reading frames and seven proteins. The seven proteins encoded by HBV include small (S), middle (M), and large (L) surface antigens (HBsAg) or envelope (Env) proteins, pre-core protein, core protein, viral polymerase (Pol), and HBx protein. HBV expresses three surface antigens, or envelope proteins, L, M, and S, with S being the smallest and L being the largest, and M in the middle. The L protein is encoded by the Pre-S 1-Pre-S2-S domains, the M protein the Pre-S2-S domains and the S protein only the S domain. Core protein is the subunit of the viral nucleocapsid. Pol is needed for synthesis of viral DNA (reverse transcriptase, RNaseH, and primer), which takes place in nucleocapsids localized to the cytoplasm of infected hepatocytes. PreCore is the core protein with an N-terminal signal peptide and is proteolytically processed at its N and C termini before secretion from infected cells, as the so-called hepatitis B e-antigen (HBeAg). HBx protein is required for efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a viral structural protein. All viral proteins of HBV have their own mRNA except for core and polymerase, which share an mRNA. With the exception of the protein pre-Core, none of the HBV viral proteins are subject to post-translational proteolytic processing.
[0079] The HBV virion contains a viral envelope, nucleocapsid, and single copy of the partially double-stranded DNA genome. The nucleocapsid comprises 120 dimers of core protein and is covered by a lipid membrane embedded with the S, M, and L viral envelope or surface antigen proteins. After entry into the cell, the virus is uncoated and the capsidcontaining relaxed circular DNA (rcDNA) with covalently bound viral polymerase migrates to the nucleus. During that process, phosphorylation of the core protein induces structural changes, exposing a nuclear localization signal enabling interaction of the capsid with so- called importins. These importins mediate binding of the core protein to nuclear pore complexes upon which the capsid disassembles and polymerase/rcDNA complex is released into the nucleus. Within the nucleus the rcDNA becomes deproteinized (removal of polymerase) and is converted by host DNA repair machinery to a covalently closed circular DNA (cccDNA) genome (further converted into a minichromosome with the addition of histones and other host factors) from which overlapping transcripts encode for HBeAg, HBsAg, Core protein, viral polymerase and HBx protein. Core protein, viral polymerase, and pre-genomic RNA (pgRNA) associate in the cytoplasm and self-assemble into immature pgRNA-containing capsid particles, which further convert into mature rcDNA-capsids and function as a common intermediate that is either enveloped and secreted as infectious virus particles or transported back to the nucleus to replenish and maintain a stable cccDNA pool. [0080] To date, HBV is divided into four main serotypes (adr, adw, ayr, ayw) based on antigenic epitopes present on the envelope proteins, and into eight genotypes (A, B, C, D, E, F, G, and H) based on the sequence of the viral genome. The HBV genotypes are distributed over different geographic regions. For example, the most prevalent genotypes in Asia are genotypes B and C. Genotype D is dominant in Africa, the Middle East, and India, whereas genotype A is widespread in Northern Europe, sub-Saharan Africa, and West Africa.
[0081] As used herein, a patient or subject having a “chronic hepatitis B virus (CHB) infection,” “CHB,” “CHB infection,” or “CHB virus infection” refers to the ordinary meaning in the art, more particularly to a patient or subject chronically infected with HBV and having detectable HBsAg (with or without HBeAg) in the blood for six or more months after HBV detection. CHB infection can be classified into four phases which typically, but not always, progress from one to the next: (I) HBeAg-positive chronic infection (previously known as immune tolerant), (II) HBeAg-positive chronic hepatitis (previously known as immune active), (III) HBeAg-negative chronic infection (previously known as inactive carrier), and (IV) HBeAg-negative chronic hepatitis (previously known as reactivation). The different phases of chronic HBV infection can also be characterized by differences in viral load, liver enzyme levels (necroinflammatory activity), HBeAg, or HBsAg load or presence of antibodies to these antigens. cccDNA levels in untreated subjects stay relatively constant at approximately 10 to 50 copies per cell but may be as low as 1 to 2 copies per cell when suppressed by nucleos(t)ide analogue therapy, even though viremia can vary considerably. The persistence of the cccDNA species leads to chronicity. In some embodiment, a chronic HBV infection can be characterized the laboratory criteria published by the Centers for Disease Control and Prevention (CDC), such as: (i) negative for IgM antibodies to hepatitis B core antigen (IgM anti-HBc) and positive for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), or nucleic acid test for hepatitis B virus DNA, or (ii) positive for HBsAg or nucleic acid test for HBV DNA, or positive for HBeAg two times at least 6 months apart. [0082] The term “therapeutically effective amount” or “effective amount” refers to that amount of each component of the combination disclosed and/or described herein that is sufficient to affect treatment, as defined herein, when administered to a subject in need of such treatment. The therapeutically effective amount will vary depending upon, for example, the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the particular combination and/or composition, the dosing regimen to be followed, timing of administration, the manner of administration, all of which can readily be determined by one of ordinary skill in the art. The therapeutically effective amount can be ascertained experimentally, for example by assaying blood concentration of each component of the combination, or theoretically, by calculating bioavailability by one of ordinary skill in the art in view of the present disclosure.
[0083] In particular embodiments of the application, a therapeutically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an HBV infection or a symptom associated therewith; (ii) reduce the duration of an HBV infection or symptom associated therewith; (iii) prevent the progression of an HBV infection or symptom associated therewith; (iv) cause regression of an HBV infection or symptom associated therewith; (v) prevent the development or onset of an HBV infection, or symptom associated therewith; (vi) treat or retreat a chronic HBV infection that recurs due to relapse after functional cure is achieved or symptom associated therewith; (vii) prevent the recurrence of an HBV infection or symptom associated therewith; (viii) reduce hospitalization of a subject having an HBV infection; (ix) reduce hospitalization length of a subject having an HBV infection; (x) increase the survival of a subject with an HBV infection; (xi) eliminate an HBV infection in a subject; (xii) inhibit or reduce HBV replication in a subject; and/or (xiii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.
[0084] A therapeutically effective amount can also be an amount of the combination and/or composition sufficient to reduce HBsAg levels consistent with evolution to clinical seroconversion; achieve sustained HBsAg clearance associated with reduction of infected hepatocytes by a subject’s immune system; induce HBV-antigen specific activated T-cell populations; and/or achieve persistent loss of HBsAg during or after treatment that then preferably persists at 6 months or more after the end of treatment, most preferably for life. Examples of a target index include a serum HBsAg level that is below a threshold of, e.g., 100 lU/mL of HBsAg and/or HBV-specific CD8 T cells responses of greater numbers or of greater polyfunctionality than, e.g., at the beginning of the treatment. Additional examples of target indexes include, but are not limited to, serum HBV DNA levels lower than the lower limit of quantification (LLoQ) or lower than 20 lU/mL, more particularly lower than 15 lU/mL, more particularly lower than 10 lU/mL; serum ALT concentration lower than 3 times the upper normal limit, or lower than 129 U/L if the subject is a male subject, or lower than 108 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 120 U/L if the subject is a male subject or lower than 105 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 90 U/L if the subject is a male subject or lower than 57 U/L if the subject is a female subject; HBeAg-negative serum; serum HBsAg level of 1000 lU/ml or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly of 10 lU/mL or lower, which would be considered normalized; HBs seroconversion; and/or core-related antigen (crag) below LLoQ.
[0085] The phrase “inducing an immune response” when used with reference to the methods described herein encompasses providing a therapeutic immunity for treating against a pathogenic agent, e.g., HBV. In an embodiment, “inducing an immune response” means producing an immunity in a subject in need thereof, e.g., to provide a therapeutic effect against a disease, such as HBV infection. In certain embodiments, “inducing an immune response” refers to causing or improving cellular immunity, e.g., HBV-specific CD4+ and CD8+ T cell responses. In certain embodiments, this T cell response can bring about functional cure for the treated patient who has CHB. In certain embodiments, “inducing an immune response” refers to causing or improving a humoral immune response against HBV infection. In certain embodiments, “inducing an immune response” refers to causing or improving a cellular and a humoral immune response against HBV infection.
[0086] As used herein, the term “protective immunity” or “protective immune response” means that the vaccinated subject can control an infection with the pathogenic agent against which the vaccination was done. Usually, the subject having developed a “protective immune response” develops only mild to moderate clinical symptoms or no symptoms at all. Usually, a subject having a “protective immune response” or “protective immunity” against a certain agent will not die as a result of the infection with said agent.
[0087] As used herein, the term “functional cure” or “FC” refers to a state of a subject who had CHB where the serum of the subject remains free of detectable HBV DNA and HBsAg after the subject is off all HBV treatment(s) for at least 6 months. For example, the serum of the subject with FC has undetectable HBV DNA and is HBsAg-negative 6 months after the end of HBV treatment. The HBsAg loss in a subject with FC can be with or without HBsAg seroconversion. In some embodiment, the FC lasts at least 1 year, preferably at least 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years. Most preferably, the FC lasts for life.
[0088] As used herein, the term “recovery of T cell function” refers to the re-activation of exhausted and otherwise non-functional HBV-specific T cells such that they are able to perform their normal effector functions. Examples of recovered HBV-specific T cell response include, but are not limited to, killing or non-cytolytic control of HBV-infected hepatocytes, killing of hepatocytes with integrated HBV DNA expressing HBsAg and, once FC is achieved, performing surveillance and killing infected cells that reactivate over time. The net effect is to keep the serum free of virus products, such as HBV DNA, HBsAg and other HBV proteins.
[0089] As used herein, the terms and phrases “in combination,” “in combination with,” “co-delivery,” and “administered together with” in the context of the administration of two or more therapies or components to a subject refers to simultaneous administration or subsequent administration of two or more therapies or components, such as two vectors, e.g., DNA plasmids, peptides, or a therapeutic combination and an adjuvant. “Simultaneous administration” can be administration of the two or more therapies or components at least within the same day. When two components are “administered together with” or “administered in combination with,” they can be administered in separate compositions sequentially within a short time period, such as 24, 20, 16, 12, 8 or 4 hours, or within 1 hour, or they can be administered in a single composition at the same time. “Subsequent administration” can be administration of the two or more therapies or components in the same day or on separate days. The use of the term “in combination with” does not restrict the order in which therapies or components are administered to a subject. For example, a first therapy or component (e.g., an RNAi component) can be administered prior to (e.g., 5 minutes to one hour before), concomitantly with or simultaneously with, or subsequent to (e.g., 5 minutes to one hour after) the administration of a second therapy or component (e.g., a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence). In some embodiments, a first therapy or component (e.g., an RNAi component) and a second therapy or component (e.g., a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence) are administered in the same composition. In other embodiments, a first therapy or component (e.g., an RNAi component) and a second therapy or component (e.g., a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence) are administered in separate compositions, such as two separate compositions.
[0090] Thus, in a further general aspect, the application relates to a method for treating an HBV infection in an individual in need thereof, comprising administering to the individual an RNAi component, as described herein under 1), and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence as described herein under 2).
[0091] In another general aspect, the application relates to an RNAi component, as described herein under 1), for use in treating an HBV infection in a subject in need thereof, in combination with a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence as described herein under 2).
[0092] In an additional general aspect, the application relates to the use of an RNAi component, as described herein under 1), in the manufacture of a medicament for the treatment of hepatitis B Virus (HBV) infection, wherein the medicament is for administration, or to be administered, in combination with a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence, as described herein under 2). [0093] In an attempt to help the reader of the application, the description has been separated in various paragraphs or sections or is directed to various embodiments of the application. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiments. To the contrary, one skilled in the art will understand that the description has broad application and encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples. For example, while embodiments of RNAi component described herein may contain particular components arranged in a particular order, those having ordinary skill in the art will appreciate that the concepts disclosed herein may equally apply to other components arranged in other orders that can be used in RNAi of the application. The application contemplates use of any of the applicable components in any combination that can be used the application, whether or not a particular combination is expressly described. The invention generally relates to a therapeutic combination comprising one or more HBV RNAi and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence.
COMBINATION
[0094] Provided herein is a combination of an effective amount of an RNAi component and an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens or a pharmaceutically acceptable salt thereof.
[0095] The combination of an effective amount of an RNAi component and an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens can be for use in treating a HBV infection, such as a chronic HBV infection (CHB), with or without viral co-infection, e.g., with or without co-infection with HDV and/or HCV and/or HIV, more particularly with or without co-infection with at least HDV, and/or for treating chronic HDV infection (CHD) in a subject in need thereof.
[0096] In some embodiments, the combination is for use in enhancing an immune response, decreasing viral replication, and/or decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptides, more particularly of one or more polypeptide(s) selected from HBsAg and HBeAg, in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection.
[0097] In some embodiments, provided is an RNAi component for use in combination with a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens in the treatment of a Hepatitis B Virus infection, more particularly a chomic HBV infection (CHB) with or without a viral co-infection, and/or in the treatment of a chronic Hepatitis D Virus (HDV) infection, wherein the RNAi component and the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens are as defined herein, and wherein the RNAi component optionally is indicated for simultaneous, sequential, or separate administration with the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens.
[0098] In some embodiments, provided is a nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence comprising one or more HBV antigens for use in combination with an RNAi component in the treatment of a Hepatitis B Virus infection, more particularly a chomic HBV infection (CHB) with or without a viral co-infection, and/or in the treatment of a chronic Hepatitis D Virus (HDV) infection, wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens and the RNAi component are as defined herein, and wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising one or more HBV antigens optionally is indicated for simultaneous, sequential, or separate administration with the RNAi component.
Hepatitis B Virus (HBV)
[0099] As used herein “hepatitis B virus” or “HBV” refers to a virus of the hepadnaviridae family. HBV is a small (e.g., 3.2 kb) hepatotropic DNA virus that encodes four open reading frames and seven proteins. The seven proteins encoded by HBV include small (S), medium (M), and large (L) surface antigen (HBsAg) or envelope (Env) proteins, pre-Core protein, core protein, viral polymerase (Pol), and HBx protein. HBV expresses three surface antigens, or envelope proteins, L, M, and S, with S being the smallest and L being the largest. The extra domains in the M and L proteins are named Pre-S2 and Pre-S 1, respectively. Core protein is the subunit of the viral nucleocapsid. Pol is needed for synthesis of viral DNA (reverse transcriptase, RNaseH, and primer), which takes place in nucleocapsids localized to the cytoplasm of infected hepatocytes. PreCore is the core protein with an N-terminal signal peptide and is proteolytically processed at its N and C termini before secretion form infected cells, as the so-called hepatitis B e-antigen (HBeAg). HBx protein is required for efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a viral structural protein. All viral proteins of HBV have their own mRNA except for core and polymerase, which share an mRNA. With the exception of the protein pre-Core, none of the HBV viral proteins are subject to post-translational proteolytic processing.
[0100] The HBV virion contains a viral envelope, nucleocapsid, and single copy of the partially double-stranded DNA genome. The nucleocapsid comprises 120 dimers of core protein and is covered by a capsid membrane embedded with the S, M, and L viral envelope or surface antigen proteins. After entry into the cell, the virus is uncoated and the capsidcontaining relaxed circular DNA (rcDNA) with covalently bound viral polymerase migrates to the nucleus. During that process, phosphorylation of the Core protein induces structural changes, exposing a nuclear localization signal enabling interaction of the capsid with so- called importins. These importins mediate binding of the core protein to nuclear pore complexes upon which the capsid disassembles and polymerase/rcDNA complex is released into the nucleus. Within the nucleus the rcDNA becomes deproteinized (removal of polymerase) and is converted by host DNA repair machinery to a covalently closed circular DNA (cccDNA) genome from which overlapping transcripts encode for HBeAg, HBsAg, Core protein, viral polymerase and HBx protein. Core protein, viral polymerase, and pre- genomic RNA (pgRNA) associate in the cytoplasm and self-assemble into immature pgRNA- containing capsid particles, which further convert into mature rcDNA-capsids and function as a common intermediate that is either enveloped and secreted as infections virus particles or transported back to the nucleus to replenish and maintain a stable cccDNA pool.
[0101] To date, HBV is divided into four serotypes (adr, adw, ayr, ayw) based on antigenic epitopes present on the envelope proteins, and into eight genotypes (A, B, C, D, E, F, G, and H) based on the sequence of the viral genome. The HBV genotypes are distributed over different geographic regions. For example, the most prevalent genotypes in Asia are genotypes B and C. Genotype D is dominant in Africa, the Middle East, and India, whereas genotype A is widespread in Northern Europe, sub-Saharan Africa, and West Africa. HBV Antigens
[0102] As used herein, the terms “HBV antigen,” “antigenic polypeptide of HBV,” “HBV antigenic polypeptide,” “HBV antigenic protein,” “HBV immunogenic polypeptide,” and “HBV immunogen” all refer to a polypeptide capable of inducing an immune response against an HBV in a subject. The induced response can be a humoral and/or cellular mediated response. The HBV antigen can be a polypeptide of HBV, a fragment or epitope thereof, or a combination of multiple HBV polypeptides, portions or derivatives thereof. An HBV antigen is capable of raising in a host a protective immune response, e.g., inducing an immune response against a viral disease or infection, and/or producing an immunity (i.e., vaccinates) a subject against a viral disease or infection, that protects the subject against the viral disease or infection. For example, an HBV antigen can comprise a polypeptide or immunogenic fragment(s) thereof from any HBV protein, such as HBeAg, pre-core protein, HBsAg (S, M, or L proteins), core protein, viral polymerase, or HBx protein derived from any HBV genotype, e.g., genotype A, B, C, D, E, F, G, and/or H, or combination thereof.
(1) HBV Core Antigen
[0103] As used herein, each of the terms “HBV core antigen,” “HBeAg” and “core antigen” refers to an HBV antigen capable of inducing an immune response against an HBV core protein in a subject. The induced immune response can be a humoral and/or cellular mediated response. Each of the terms “core,” “core polypeptide,” and “core protein” refers to the HBV viral core protein. Full-length core antigen is typically 183 amino acids in length and includes an assembly domain (amino acids 1 to 149) and a nucleic acid binding domain (amino acids 150 to 183). The 34-residue nucleic acid binding domain is required for pre- genomic RNA encapsidation. This domain also functions as a nuclear import signal. It comprises 17 arginine residues and is highly basic, consistent with its function. HBV core protein is dimeric in solution, with the dimers self-assembling into icosahedral capsids. Each dimer of core protein has four a-helix bundles flanked by an a-helix domain on either side. Truncated HBV core proteins lacking the nucleic acid binding domain are also capable of forming capsids.
[0104] In an embodiment of the application, an HBV antigen is a truncated HBV core antigen. As used herein, a “truncated HBV core antigen,” refers to an HBV antigen that does not contain the entire length of an HBV core protein but is capable of inducing an immune response against the HBV core protein in a subject. For example, an HBV core antigen can be modified to delete one or more amino acids of the highly positively charged (arginine rich) C-terminal nucleic acid binding domain of the core antigen, which typically contains seventeen arginine (R) residues. A truncated HBV core antigen of the application is preferably a C-terminally truncated HBV core protein which does not comprise the HBV core nuclear import signal and/or a truncated HBV core protein from which the C-terminal HBV core nuclear import signal has been deleted. In an embodiment, a truncated HBV core antigen comprises a deletion in the C-terminal nucleic acid binding domain, such as a deletion of 1 to 34 amino acid residues of the C-terminal nucleic acid binding domain, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acid residues, preferably a deletion of 31-34 C-terminal amino acid residues of the C-terminal nucleic acid binding domain. In a preferred embodiment, a truncated HBV core antigen comprises a deletion in the C-terminal nucleic acid binding domain, preferably a deletion of 31 C-terminal amino acid residues of the C-terminal nucleic acid binding domain.
[0105] In some embodiments, an HBV core antigen amino acid sequence is operably linked to a signal peptide for secretion. Any suitable signal peptide can be used. In one embodiment, an HBV core antigen is operably linked at its N-terminus to a Cystatin S precursor signal peptide, to enhance secretion. In a particular embodiment, the Cystatin S precursor signal peptide has the amino acid sequence of SEQ ID NO: 77. In another particular embodiment, a coding sequence of an HBV core antigen is operably linked to a coding sequence of Cystatin S precursor signal peptide having the polynucleotide sequence of SEQ ID NO: 90.
[0106] An HBV core antigen of the application can be a consensus sequence derived from multiple HBV genotypes (e.g., genotypes A, B, C, D, E, F, G, and H). As used herein, “consensus sequence” means an artificial sequence of amino acids based on an alignment of amino acid sequences of homologous proteins as determined by an alignment of amino acid sequences of homologous proteins. The alignment can be conducted using methods or algorithm known in the art, such as using Clustal Omega. It can be the calculated order of most frequent amino acid residues, found at each position in a sequence alignment, based upon sequences of HBV antigens (e.g., core, pol, etc.) from at least 100 natural HBV isolates. A consensus sequence can be non-naturally occurring and different from the native viral sequences. Consensus sequences can be designed by aligning multiple HBV antigen sequences from different sources using a multiple sequence alignment tool, and at variable alignment positions, selecting the most frequent amino acid. Preferably, a consensus sequence of an HBV antigen is derived from HBV genotypes A, B, C, and D. The term “consensus antigen” is used to refer to an antigen having consensus sequence.
[0107] An exemplary truncated HBV core antigen according to the application lacks the nucleic acid binding function and is capable of inducing an immune response in a mammal against at least two HBV genotypes. Preferably a truncated HBV core antigen is capable of inducing a T cell response in a mammal against at least HBV genotypes A, B, C and D. More preferably, a truncated HBV core antigen is capable of inducing a CD8 T cell response in a human subject against at least HBV genotypes A, B, C and D.
[0108] In some embodiments, an HBV core antigen of the application comprises one or more T cell epitopes for MHC class I HLA alleles. In some embodiments, an HBV core antigen comprises one or more epitopes selected from the group consisting of HLA- A* 11:01 epitopes, HLA-A*02:01 epitopes, HLA-A*A0101 epitopes, and HLA-B*40:01 epitopes. Preferably, an HBV core antigen comprises two or more, such as 2, 3, or 4, of T cell epitopes selected from the group consisting of HLA-A*11:01 epitopes, HLA-A*02:01 epitopes, HLA- A*A0101 epitopes, and HL A-B* 40: 01 epitopes. More preferably, an HBV core antigen comprises HLA-A*ll:01, HLA-A*02:01, HLA-A*A0101, and HLA-B*40:01 T cell epitopes. More preferably, an HBV core antigen comprises one or more HLA-A* 11:01 epitope(s).
[0109] In some embodiments, an HBV core antigen of the application is a consensus antigen, preferably a consensus antigen derived from at least two, preferably all, of HBV genotypes A, B, C, and D, more preferably a truncated consensus antigen derived from HBV genotypes A, B, C, and D. An exemplary truncated HBV core consensus antigen according to the application consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 86, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 86. In some embodiments, the HBV core antigen comprises, preferably consists of, an amino acid sequence that is at least 98% identical to SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86. SEQ ID NO: 86 is a core consensus antigen derived from HBV genotypes A, B, C, and D. SEQ ID NO: 7 contains a 34-amino acid C-terminal deletion of the highly positively charged (arginine rich) nucleic acid binding domain of the native core antigen. In some preferred embodiments, an HBV core antigen of the application retains one or more of the C-terminal arginine residues and has the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, to thereby restore the HLA-A* 11:01 epitope in the HBV core antigen. In some embodiments, the last five C-terminal amino acids of an HBV core antigen comprise a VVR amino acid sequence, more particularly a VVRR (SEQ ID NO: 91) amino acid sequence, more particularly a VVRRR (SEQ ID NO: 92) amino acid sequence.
[0110] In a particular embodiment of the application, an HBV core antigen is a truncated HBV antigen consisting of the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86. In another particular embodiment, an HBV core antigen is encoded by a polynucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89, respectively. Preferably, the HBV core antigen consists of the amino acid sequence of SEQ ID NO: 86 and is encoded by the polynucleotide sequence of SEQ ID NO: 89. In some embodiments, an HBV core antigen consists of an amino acid sequence, or is encoded by a polynucleotide sequence, as described in U.S. Patent Application Publication No. US2019/0185828, the content of which is herein incorporated by reference in its entirety. (2) HBV Polymerase Antigen
[oni] As used herein, the term “HBV polymerase antigen,” “HBV Pol antigen,” or “HBV pol antigen” refers to an HBV antigen capable of inducing an immune response against an HBV polymerase in a subject. The immune response can be a humoral and/or cellular mediated response. Each of the terms “polymerase,” “polymerase polypeptide,” “Pol,” and “pol” refers to the HBV viral DNA polymerase. The HBV viral DNA polymerase has four domains, including, from the N terminus to the C terminus, a terminal protein (TP) domain, which acts as a primer for minus-strand DNA synthesis; a spacer that is nonessential for the polymerase functions; a reverse transcriptase (RT) domain for transcription; and an RNase H domain. [0112] In an embodiment of the application, an HBV antigen comprises an HBV Pol antigen, or any immunogenic fragment or combination thereof. An HBV Pol antigen can contain further modifications to improve immunogenicity of the antigen, such as by introducing mutations into the active sites of the polymerase and/or RNase domains to decrease or substantially eliminate certain enzymatic activities.
[0113] Preferably, an HBV Pol antigen of the invention does not have reverse transcriptase activity and RNase H activity and is capable of inducing an immune response in a mammal against at least two HBV genotypes. Preferably, an HBV Pol antigen is capable of inducing a T cell response in a mammal against at least two, preferably all, of HBV genotypes A, B, C and D. More preferably, a HBV Pol antigen is capable of inducing a CD8 T cell response in a human subject against at least HBV genotypes A, B, C and D.
[0114] Thus, in some embodiments, an HBV Pol antigen is an inactivated Pol antigen. In an embodiment, an inactivated HBV Pol antigen comprises one or more amino acid mutations in the active site of the polymerase domain. In another embodiment, an inactivated HBV Pol antigen comprises one or more amino acid mutations in the active site of the RNaseH domain. In a preferred embodiment, an inactivated HBV pol antigen comprises one or more amino acid mutations in the active site of both the polymerase domain and the RNaseH domain. For example, the “YXDD” motif (SEQ ID NO: 74) in the polymerase domain of an HBV pol antigen that can be required for nucleotide/metal ion binding can be mutated, e.g., by replacing one or more of the aspartate residues (D) with asparagine residues (N), eliminating or reducing metal coordination function, thereby decreasing or substantially eliminating reverse transcriptase function. Alternatively, or in addition to mutation of the “YXDD” motif (SEQ ID NO: 74), the “DEDD” motif (SEQ ID NO: 75) in the RNaseH domain of an HBV pol antigen required for Mg2+ coordination can be mutated, e.g., by replacing one or more aspartate residues (D) with asparagine residues (N) and/or replacing the glutamate residue (E) with glutamine (Q), thereby decreasing or substantially eliminating RNaseH function. In a particular embodiment, an HBV pol antigen is modified by (1) mutating the aspartate residues (D) to asparagine residues (N) in the “YXDD” motif (SEQ ID NO: 74) of the polymerase domain; and (2) mutating the first aspartate residue (D) to an asparagine residue (N) and the first glutamate residue (E) to a glutamine residue (N) in the “DEDD” motif (SEQ ID NO: 75) of the RNaseH domain, thereby decreasing or substantially eliminating both the reverse transcriptase and RNaseH functions of the pol antigen.
[0115] In some embodiments, an HBV pol antigen amino acid sequence is operably linked to a signal peptide for secretion. Any suitable signal peptides can be used. In one embodiment, an HBV pol antigen is operably linked at its N-terminus to a Cystatin S precursor signal peptide, to enhance secretion. In a particular embodiment, the Cystatin S precursor signal peptide has the amino acid sequence of SEQ ID NO: 77. In another particular embodiment, a coding sequence of an HBV pol antigen is operably linked to a coding sequence of Cystatin S precursor signal peptide having the polynucleotide sequence of SEQ ID NO: 90.
[0116] In some embodiments, an HBV pol antigen of the application comprises one or more T cell epitopes for MHC class I HLA alleles. In some embodiments, an HBV pol antigen comprises one or more epitopes selected from the group consisting of HLA- A* 11:01 epitopes, HLA-A*24:02 epitopes, HLA-A*02:01 epitopes, and HLA-A*A0101 epitopes. Preferably, an HBV pol antigen comprises two or more, such as 2, 3, or 4, T cell epitopes selected from the group consisting of HLA-A*11:01 epitopes, HLA-A*24:02 epitopes, HLA- A*02:01 epitopes, and HLA-A*A0101. More preferably, an HBV pol antigen comprises HLA-A*ll:01, HLA-A*24:02, HLA-A*02:01, and HLA- A* A0101 T cell epitopes. More preferably, an HBV pol antigen comprises one or more HLA-A*11:01 epitope(s).
[0117] In a preferred embodiment of the application, an HBV pol antigen is a consensus antigen, preferably a consensus antigen derived from at least two, preferably all, of HBV genotypes A, B, C, and D, more preferably an inactivated consensus antigen derived from HBV genotypes A, B, C, and D. An exemplary HBV pol consensus antigen according to the application comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 9, preferably at least 98% identical to SEQ ID NO: 9, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 9. SEQ ID NO: 9 is a pol consensus antigen derived from HBV genotypes A, B, C, and D comprising four mutations located in the active sites of the polymerase and RNaseH domains. In particular, the four mutations include mutation of the aspartic acid residues (D) to asparagine residues (N) in the “YXDD” motif (SEQ ID NO: 74) of the polymerase domain; and mutation of the first aspartate residue (D) to an asparagine residue (N) and mutation of the glutamate residue (E) to a glutamine residue (Q) in the “DEDD” (SEQ ID NO: 75) motif of the RNaseH domain.
[0118] In a particular embodiment of the application, an HBV pol antigen comprises the amino acid sequence of SEQ ID NO: 9. Preferably, an HBV pol antigen consists of the amino acid sequence of SEQ ID NO: 9. In another particular embodiment, an HBV pol antigen is encoded by a polynucleotide sequence of SEQ ID NO: 10. In some embodiments, an HBV pol antigen contains or consists of an amino acid sequence, or is encoded by a polynucleotide sequence as described in U.S. Patent Application Publication No.
US2019/0185828, the content of which is herein incorporated by reference in its entirety. (3) HBV Surface Antigens
[0119] As used herein, each of the terms “HBV surface antigen,” “surface antigen,” “HBV envelope antigen,” “envelope antigen,” and “env antigen” refers to an HBV antigen capable of inducing or eliciting an immune response against one or more HBV surface antigens or envelope proteins in a subject. The immune response can be a humoral and/or cellular mediated response. Each of the terms “HBV surface protein,” “surface protein,” “HBV envelope protein,” and “envelope protein” refers to HBV viral surface or envelope proteins. HBV expresses three surface antigens, or envelope proteins. Gene S is the gene of the HBV genome that encodes the surface antigens. The surface antigen gene is one long open reading frame but contains three in frame “start” (ATG) codons that divide the gene into three sections, pre-Sl, pre-S2, and S. Because of the multiple start codons, polypeptides of three different sizes called large (L) or L-surface antigen, middle (M) or M-surface antigen, and small (S) or S-surface antigen are produced, also named the HBV L, M and S envelope proteins. Two different promoters (PreSl and PreS2) drive transcription of the L, M, and S- surface antigen coding sequences resulting in three different translated proteins, the L, M and S envelope proteins. The PreS2 promoter is sometimes referred to as the PreS2/S promoter since it is driving M-surface antigen and S-surface antigen transcription separately. The amino acid sequence of the L-surface antigen is in-frame with the M and S-surface antigen sequences. Thus, the L-surface antigen contains the M- and S-surface antigen domains and the M-surface antigen includes the S-surface antigen domain. The L-, M- and S-surface antigen are co-C-terminal and share the entire S domain. Relative to S, M has an additional domain, pre-S2, at its N terminus, and relative to M, L has a pre-Sl domain.
[0120] In some embodiments, an HBV antigen is an HBV PreSl antigen, which is encoded by a pre-Sl gene section and contains only the Pre-Sl domain of the L antigen. The PreSl antigen can have various lengths, such as having 99 to 109 amino acids. An HBV PreSl antigen of the application can contain the sequence of any naturally occurring PreSl domain, and variants or derivatives thereof.
[0121] In other embodiments, an HBV antigen is an HBV PreS2.S antigen, which is encoded by the pre-S2 and S gene sections and contains the PreS2 domain and the S domain. The PreS2 domain can be about 55 amino acids long and the S-domain can contain about 226 amino acids. An HBV PreS2.S antigen of the application can contain the sequences of any of the naturally occurring PreS2 and S domains, and variants or derivatives thereof. In some embodiments, an internal signal peptide of PreS2.S is left intact to facilitate secretion PreS2.S protein products of the HBV M and HBV S antigens. In one embodiment, an HBV PreS2.S antigen is an HBV M surface antigen. In another embodiment, an HBV PreS2.S antigen is an HBV S surface antigen. In yet another embodiment, an HBV PreS2.S antigen encompasses an HBV M surface antigen and an HBV S surface antigen.
[0122] In some embodiments, an HBV surface antigen amino acid sequence is operably linked to or contains a signal peptide for secretion. Any suitable signal peptides can be used. In one embodiment, an HBV Pre-S 1 antigen amino acid sequence is operably linked at its N- terminus to a Cystatin S precursor signal peptide to enhance secretion. In a particular embodiment, the Cystatin S precursor signal peptide has the amino acid sequence of SEQ ID NO: 77. In another particular embodiment, a coding sequence of an HBV Pre-S 1 antigen is operably linked to a coding sequence of Cystatin S precursor signal peptide having the polynucleotide sequence of SEQ ID NO: 90.
[0123] In an embodiment of the application, an HBV antigen comprises an HBV surface antigen, or any immunogenic fragment or combination thereof. An HBV surface antigen is capable of inducing an immune response in a subject against at least one of L-surface antigen, M-surface antigen, and S-surface antigen proteins. Preferably, an HBV surface antigen, such as a Pre-S 1 or PreS2.S antigen, is a consensus antigen, preferably a consensus antigen derived from at least two HBV genotypes A, B, C, and D, and more preferably a consensus antigen derived from HBV genotypes A, B, C, and D.
[0124] In some embodiments, an HBV surface antigen of the application comprises one or more T cell epitopes for MHC class I HLA alleles. In some embodiments, an HBV surface antigen comprises one or more T cell epitopes selected from the group consisting of HLA- A* 11:01 epitopes, HLA-A*24:02 epitopes, and HLA-A*A2402 epitopes. Preferably, an HBV Pre-S 1 antigen comprises one or more T cell epitopes selected from the group consisting of HLA-A*11 :01 epitopes and HLA-A*24:02 epitopes. More preferably, an HBV Pre-Sl antigen comprises HLA-A*ll:01 and HLA-A*24:02 T cell epitopes. More preferably, an HBV Pre-Sl antigen comprises one or more HLA- A*11:01 epitope(s). Preferably, an HBV PreS2.S antigen comprises one or more T cell epitopes selected from the group consisting of HLA-A*l l:01 epitopes, HLA-A*24:02 epitopes, and HLA-A*A2402 epitopes. More preferably, an HBV PreS2.S antigen comprises HLA-A*ll:01, HLA-A*24:02, and HLA-A*A2402 T cell epitopes. More preferably, an HBV PreS2.S antigen comprises one or more HLA-A* 11:01 epitope(s).
[0125] In some embodiments of the application, an HBV surface antigen is a Pre-S 1 antigen. An exemplary Pre-S 1 antigen according to the application comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments of the application, an HBV surface antigen is a Pre-S2.S antigen. An exemplary Pre-S2.S antigen according to the application comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 5.
[0126] In a particular embodiment of the application, an HBV surface antigen is a Pre-S 1 antigen consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In another particular embodiment, an HBV surface antigen is encoded by a polynucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In another particular embodiment, an HBV surface antigen is a Pre-S2.S antigen consisting of the amino acid sequence of SEQ ID NO: 5. In another particular embodiment, an HBV surface antigen is encoded by a polynucleotide sequence of SEQ ID NO: 6.
[0127] In some embodiments of the application, an HBV surface antigen is an S-surface antigen. An exemplary S-surface antigen according to the application consists of an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 79, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 79. SEQ ID NO: 81 is an HBV consensus S-surface antigen derived from HBV genotypes A, B, C, and D. In a particular embodiment of the application, an S-surface antigen consists of the amino acid sequence of SEQ ID NO: 79. In another particular embodiment, an HBV surface antigen is encoded by a polynucleotide sequence of SEQ ID NO: 78.
[0128] In some embodiments, an HBV surface antigen is M-surface antigen, or any immunogenic fragment or combination thereof. Preferably, the M-surface antigen is a consensus antigen, preferably a consensus antigen derived from at least two, preferably all, of HBV genotypes A, B, C, and D, and more preferably a consensus antigen derived from HBV genotypes A, B, C, and D. Preferably, the M- surface antigen is capable of inducing or eliciting an immune response against M-surface antigen in a subject.
[0129] An exemplary M-surface antigen according to the application comprises or consists of an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 82, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO:
82. SEQ ID NO: 82 is an HBV consensus M-surface antigen derived from HBV genotypes A, B, C, and D. In a particular embodiment of the application, an M-surface antigen consists of the amino acid sequence of SEQ ID NO: 82.
[0130] In some embodiments, an HBV surface antigen is an L-surface antigen, or any immunogenic fragment or combination thereof. Preferably, the L-surface antigen is a consensus antigen, preferably a consensus antigen derived from at least two, preferably all, of HBV genotypes A, B, C, and D, and more preferably a consensus antigen derived from HBV genotypes A, B, C, and D. Preferably, the L-surface antigen is capable of inducing or eliciting an immune response against L-surface antigen in a subject.
[0131] An exemplary L-surface antigen according to the application comprises or consists of an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 83, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO:
83. SEQ ID NO: 83 is an HBV consensus L-surface antigen derived from HBV genotypes A, B, C, and D. In a particular embodiment of the application, an M-surface antigen consists of the amino acid sequence of SEQ ID NO: 83.
[0132] In some embodiments, an HBV surface antigen comprises a portion of any one of the L-, M-, and S-surface antigens, or any combination thereof. For example, an HBV surface antigen can comprise or consist of the N-terminal L-surface antigen domain. An HBV surface antigen can also comprise or consist of the M-surface antigen domain. An HBV surface antigen can also comprise or consist of the N-terminal L-surface antigen domain and the M- surface antigen domain. An HBV surface antigen can also comprise or consist of the N- terminal L-surface antigen domain, the M-surface antigen domain, and a portion of the S- surface antigen domain.
[0133] An exemplary example of such a surface antigen according to the application consists of an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 81, such as at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 81, preferably at least 98% identical to SEQ ID NO: 81, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 81. SEQ ID NO: 81 is a consensus antigen derived from HBV genotypes A, B, C, and D, containing the N-terminal L-surface antigen domain, the entire M- surface antigen domain, and a 15 -amino acid C-terminal tail from the S-surface antigen domain. In a particular embodiment of the application, an HBV surface antigen consists of the amino acid sequence of SEQ ID NO: 81. In another particular embodiment, an HBV surface antigen is encoded by a polynucleotide sequence of SEQ ID NO: 80 that encodes an HBV surface antigen. In some embodiments, an HBV surface antigen consists of an amino acid sequence, or is encoded by a polynucleotide sequence, described in European Patent Application No. 19180926, the content of which is herein incorporated by reference in its entirety.
Polynucleotides and Vectors
[0134] In another general aspect, the application provides a nucleic acid molecule comprising a non-naturally polynucleotide sequence encoding an HBV antigen according to the application, and a vector comprising the non-naturally occurring nucleic acid. A nucleic acid molecule can comprise any non-naturally occurring polynucleotide sequence encoding an HBV antigen of the application, which can be made using methods known in the art in view of the present disclosure. Preferably, a non-naturally occurring polynucleotide encodes at least one of a truncated HBV core antigen, an HBV polymerase antigen, an HBV Pre-S 1 antigen, and an HBV Pre-S2.S antigen of the application. A polynucleotide can be in the form of RNA or in the form of DNA obtained by recombinant techniques (e.g., cloning) or produced synthetically (e.g., chemical synthesis). The DNA can be single-stranded or double-stranded or can contain portions of both double-stranded and single-stranded sequence. The DNA can, for example, comprise genomic DNA, cDNA, or combinations thereof. The polynucleotide can also be a DNA/RNA hybrid. The polynucleotides and vectors of the application can be used for recombinant protein production, expression of the protein in host cell, or the production of viral particles. Preferably, a polynucleotide is RNA. [0135] In an embodiment of the application, a nucleic acid molecule comprises a non- naturally occurring polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, preferably 98%, 99% or 100% identical to SEQ ID NO: 7, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86. In a particular embodiment of the application, a non-naturally occurring nucleic acid molecule encodes a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86. Preferably, the truncated HBV core antigen consists of SEQ ID NO: 86.
[0136] Examples of polynucleotide sequences of the application encoding a truncated
HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 8, SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89, respectively, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 8, SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89, preferably 98%, 99% or 100% identical to SEQ ID NO: 8, SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89. Exemplary non-naturally occurring nucleic acid molecules encoding a truncated HBV core antigen have the polynucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89. Preferably, the molecule encoding a truncated HBV core antigen has the polynucleotide sequence of SEQ ID NO: 89.
[0137] In an embodiment of the application, a nucleic acid molecule comprises a non- naturally occurring polynucleotide sequence encoding a HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 9. In a particular embodiment of the application, a non-naturally occurring nucleic acid molecule encodes a HBV polymerase antigen consisting of the amino acid sequence of SEQ ID NO: 9.
[0138] Examples of polynucleotide sequences of the application encoding a HBV Pol antigen comprising the amino acid sequence of SEQ ID NO: 9 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 10, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 10, preferably 98%, 99% or 100% identical to SEQ ID NO: 10. Exemplary non- naturally occurring nucleic acid molecules encoding a HBV pol antigen have the polynucleotide sequence of SEQ ID NO: 10. [0139] In an embodiment of the application, a nucleic acid molecule comprises a non- naturally occurring polynucleotide sequence encoding aHBV Pre-S 1 antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3, preferably 98%, 99% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3. In a particular embodiment of the application, a non-naturally occurring nucleic acid molecule encodes aHBV Pre-S 1 antigen consisting the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
[0140] Examples of polynucleotide sequences of the application encoding a HBV Pre-S 1 antigen consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4, respectively, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4, preferably 98%, 99% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4. Exemplary non-naturally occurring nucleic acid molecules encoding aHBV Pre-S 1 antigen have the polynucleotide sequence of SEQ ID NOs: 2 or 4.
[0141] In an embodiment of the application, a nucleic acid molecule comprises a non- naturally occurring polynucleotide sequence encoding aHBV Pre-S2.S antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 5, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 5, preferably 98%, 99% or 100% identical to SEQ ID NO: 5. In a particular embodiment of the application, a non-naturally occurring nucleic acid molecule encodes a HBV Pre-S2.S antigen consisting the amino acid sequence of SEQ ID NO: 5.
[0142] Examples of polynucleotide sequences of the application encoding a HBV Pre- S2.S antigen consisting of the amino acid sequence of SEQ ID NO: 5 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 6, respectively, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 6, preferably 98%, 99% or 100% identical to SEQ ID NO: 6.
Exemplary non-naturally occurring nucleic acid molecules encoding a HBV Pre-S2.S antigen have the polynucleotide sequence of SEQ ID NO: 6. [0143] In an embodiment of the application, a nucleic acid molecule comprises a non- naturally occurring polynucleotide sequence encoding comprising, from 5’ end to 3’ end: a polynucleotide sequence encoding a first HBV antigen, a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and a polynucleotide sequence encoding a second HBV antigen, wherein at least one of the first and second HBV antigens is an HBV surface antigen. In some embodiments, the non- naturally occurring polynucleotide sequence further comprises, ordered from the 5’- to 3’- end: a second IRES element or a polynucleotide sequence encoding a second autoprotease peptide operably linked to the 3’ end of the polynucleotide sequence encoding the second HBV antigen, and a polynucleotide sequence encoding a third HBV antigen. In some embodiments, the non-naturally occurring polynucleotide sequence further comprises, ordered from the 5’- to 3’-end: a third IRES element or a polynucleotide sequence encoding a third autoprotease peptide operably linked to the 3’ end of the polynucleotide sequence encoding the third HBV antigen, and a polynucleotide sequence encoding a fourth HBV antigen.
[0144] In some embodiments, each of the first, second, third and fourth HBV antigens are independently selected from the group consisting of: (i) a first HBV surface antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 1; (ii) a second HBV surface antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 3; (iii) a third HBV surface antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 5; (iv) an HBV core antigen comprising, preferably consisting of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7; and (v) an HBV polymerase antigen comprising, preferably consisting of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 9. In some embodiments, the HBV core antigen comprises, preferably consists of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86.
[0145] In some embodiments, each of the polynucleotide sequences encoding the first, second, third and fourth HBV antigens are independently selected from the group consisting of: (i) a polynucleotide sequence encoding the first HBV PreSl antigen having a sequence that is at least 90% identical to SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2; (ii) a polynucleotide sequence encoding the second HBV PreSl antigen having a sequence that is at least 90% identical to SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4; (iii) a polynucleotide sequence encoding an HBV PreS2.S antigen having a sequence that is at least 90% identical to SEQ ID NO: 6, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%; 99.9% or 100% identical to SEQ ID NO: 6; (iv) a polynucleotide sequence encoding the HBV polymerase antigen having a sequence that is at least 90% identical to SEQ ID NO: 8, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 8; and (v) the polynucleotide sequence encoding the HBV core antigen having a sequence that is at least 90% identical to SEQ ID NO: 10, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide sequence encoding the HBV core antigen comprises, preferably consists of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89. [0146] In some embodiments, each of the first, second and third autoprotease peptides independently comprise a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2 A (E2A), a Thosea asigna virus 2 A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof. Preferably, each of the first, second and third autoprotease peptides comprise the peptide sequence of P2A, such as a P2A sequence of SEQ ID NO: 11. Preferably, the polynucleotide sequence encoding the P2A peptide sequence is SEQ ID NO: 12.
[0147] In some embodiments, each of the first, second and third IRES are derived from encephalomyocarditis virus (EMCV) or Enterovirus 71 (EV71), preferably each of the first, second and third IRES comprise the polynucleotide sequence of SEQ ID NO: 13 or 14.
[0148] In an embodiment of the application, a nucleic acid molecule comprises a non- naturally occurring polynucleotide sequence encoding comprising, from 5’ end to 3’ end: (1) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9; (2) a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86; (3) a polynucleotide sequence encoding an HBV Pre-S 1 antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5; (4) a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV Pre-Sl antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3; (5) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3; (6) a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3; (7) a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-S 1 antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9; (8) a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-S 1 antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86; (9) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3; (10) a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-Sl antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3; (11) a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-Sl antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9; (12) a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-S 1 antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86; (13) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3; (14) a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-Sl antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3; (15) a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-Sl antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9; (16) a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre-S 1 antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86; (17) a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 86; (18) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9; (19) a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5; (20) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5; (21) a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86; (22) a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9; (23) a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5; and (24) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of SEQ ID NO: 7, preferably, consisting of the amino acid sequence of SEQ ID NO: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV PreS2.S antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 5. [0149] In some embodiments, each HBV antigen is independently operably linked to or contains a signal peptide sequence for secretion. Any suitable signal peptide sequence can be used. Preferably, for each of an HBV PreSl antigen, an HBV core antigen and an HBV pol antigen, a signal peptide is operably linked to the N-terminus of the antigen sequence. In some embodiments, the signal peptide contains the amino acid sequence of SEQ ID NO: 77, preferably, the signal peptide is encoded by a nucleotide sequence of SEQ ID NO: 90.
[0150] In some embodiments, the nucleic acid molecule comprises a non-naturally occurring polynucleotide sequence of any one of SEQ ID NOs: 15 to 54. In some embodiments, the nucleic acid molecule comprises at least two non-naturally occurring polynucleotide sequences of SEQ ID NOs: 15 to 54.
[0151] The application also relates to a vector comprising a non-naturally occurring polynucleotide encoding an HBV antigen. As used herein, a “vector” is a nucleic acid molecule used to carry genetic material into another cell, where it can be replicated and/or expressed. Any vector known to those skilled in the art in view of the present disclosure can be used. Examples of vectors include, but are not limited to, plasmids, viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs). Preferably, a vector is a DNA plasmid. A vector can be a DNA vector or an RNA vector. One of ordinary skill in the art can construct a vector of the application through standard recombinant techniques in view of the present disclosure.
[0152] A vector of the application can be an expression vector. As used herein, the term “expression vector” refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed. Expression vectors include, but are not limited to, vectors for recombinant protein expression, such as a DNA plasmid or a viral vector, and vectors for delivery of nucleic acid into a subject for expression in a tissue of the subject, such as a DNA plasmid or a viral vector. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
[0153] Vectors of the application can contain a variety of regulatory sequences. As used herein, the term “regulatory sequence” refers to any sequence that allows, contributes or modulates the functional regulation of the nucleic acid molecule, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (i. e. , mRNA) into the host cell or organism. In the context of the disclosure, this term encompasses promoters, enhancers and other expression control elements (e.g., polyadenylation signals and elements that affect mRNA stability).
[0154] In some embodiments of the application, a vector is a non- viral vector. Examples of non-viral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. Preferably, a non-viral vector is a DNA plasmid. A “DNA plasmid,” which is used interchangeably with “DNA plasmid vector,” “plasmid DNA,” or “plasmid DNA vector,” refers to a double-stranded and generally circular DNA sequence that is capable of autonomous replication in a suitable host cell. DNA plasmids used for expression of an encoded polynucleotide typically comprise an origin of replication, a multiple cloning site, and a selectable marker, which for example, can be an antibiotic resistance gene. Examples of DNA plasmids suitable that can be used include, but are not limited to, commercially available expression vectors for use in well- known expression systems (including both prokaryotic and eukaryotic systems), such as pSE420 (Invitrogen, San Diego, Calif), which can be used for production and/or expression of protein in Escherichia coli; pYES2 (Invitrogen, Thermo Fisher Scientific), which can be used for production and/or expression in Saccharomyces cerevisiae strains of yeast;
MAXBAC® complete baculovirus expression system (Thermo Fisher Scientific), which can be used for production and/or expression in insect cells; pcDNA™ or pcDNA3™ (Life Technologies, Thermo Fisher Scientific), which can be used for high level constitutive protein expression in mammalian cells; and pVAX or pVAX-1 (Life Technologies, Thermo Fisher Scientific), which can be used for high-level transient expression of a protein of interest in most mammalian cells. The backbone of any commercially available DNA plasmid can be modified to optimize protein expression in the host cell, such as to reverse the orientation of certain elements (e.g., origin of replication and/or antibiotic resistance cassette), replace a promoter endogenous to the plasmid (e.g., the promoter in the antibiotic resistance cassette), and/or replace the polynucleotide sequence encoding transcribed proteins (e.g., the coding sequence of the antibiotic resistance gene), by using routine techniques and readily available starting materials. (See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)).
[0155] Preferably, a DNA plasmid is an expression vector suitable for protein expression in mammalian host cells. Expression vectors suitable for protein expression in mammalian host cells include, but are not limited to, pcDNA™, pcDNA3™, pVAX, pVAX-1, ADV AX, NTC8454, etc. Preferably, an expression vector is based on pVAX-1, which can be further modified to optimize protein expression in mammalian cells. pVAX-1 is commonly used plasmid in DNA vaccines and contains a strong human intermediate early cytomegalovirus (CMV-IE) promoter followed by the bovine growth hormone (bGH)-derived polyadenylation sequence (pA). pVAX-1 further contains a pUC origin of replication and kanamycin resistance gene driven by a small prokaryotic promoter that allows for bacterial plasmid propagation.
[0156] A vector of the application can also be a viral vector. In general, viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that has been rendered non-infectious, but still contains viral promoters and transgenes, thus allowing for translation of the transgene through a viral promoter. Because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. In certain embodiments, a vector as described herein is, for instance, a recombinant adenovirus, a recombinant retrovirus, a recombinant pox virus such as a vaccinia virus (e.g., Modified Vaccinia Ankara (MV A)), a recombinant alphavirus such as Semliki forest virus, a recombinant paramyxovirus, such as a recombinant measles virus, or another recombinant virus. Examples of viral vectors that can be used include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors, etc. In certain embodiments, a vector as described herein is an MVA vector. The vector can also be anon-viral vector.
[0157] In some embodiments, a viral vector is an adenovirus vector, e.g., a recombinant adenovirus vector. A recombinant adenovirus vector can for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV) or rhesus adenovirus (rhAd). Preferably, an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc. In other embodiments, an adenovirus vector is a rhAd vector, e.g. rhAd51, rhAd52 or rhAd53. A recombinant viral vector useful for the application can be prepared using methods known in the art in view of the present disclosure. For example, in view of the degeneracy of the genetic code, several nucleic acid sequences can be designed that encode the same polypeptide. A polynucleotide encoding an HBV antigen of the application can optionally be codon-optimized to ensure proper expression in the host cell (e.g., bacterial or mammalian cells). Codon-optimization is a technology widely applied in the art, and methods for obtaining codon-optimized polynucleotides will be well known to those skilled in the art in view of the present disclosure. [0158] A vector of the application, e.g., a DNA plasmid or a viral vector (particularly an adenoviral vector), can comprise any regulatory elements to establish conventional function(s) of the vector, including but not limited to replication and expression of the HBV antigen(s) encoded by the polynucleotide sequence of the vector. Regulatory elements include, but are not limited to, a promoter, an enhancer, a polyadenylation signal, translation stop codon, a ribosome binding element, a transcription terminator, selection markers, origin of replication, etc. A vector can comprise one or more expression cassettes. An “expression cassette” is part of a vector that directs the cellular machinery to make RNA and protein. An expression cassette typically comprises three components: a promoter sequence, an open reading frame, and a 3 ’-untranslated region (UTR) optionally comprising a poly adenylation signal. An open reading frame (ORF) is a reading frame that contains a coding sequence of a protein of interest (e.g., HBV antigen) from a start codon to a stop codon. Regulatory elements of the expression cassette can be operably linked to a polynucleotide sequence encoding an HBV antigen of interest. As used herein, the term “operably linked” is to be taken in its broadest reasonable context and refers to a linkage of polynucleotide elements in a functional relationship. A polynucleotide is “operably linked” when it is placed into a functional relationship with another polynucleotide. For instance, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. Any components suitable for use in an expression cassette described herein can be used in any combination and in any order to prepare vectors of the application.
[0159] A vector can comprise a promoter sequence, preferably within an expression cassette, to control expression of an HBV antigen of interest. The term “promoter” is used in its conventional sense and refers to a nucleotide sequence that initiates the transcription of an operably linked nucleotide sequence. A promoter is located on the same strand near the nucleotide sequence it transcribes. Promoters can be a constitutive, inducible, or repressible. Promoters can be naturally occurring or synthetic. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source). For example, if the vector to be employed is a DNA plasmid, the promoter can be endogenous to the plasmid (homologous) or derived from other sources (heterologous). Preferably, the promoter is located upstream of the polynucleotide encoding an HBV antigen within an expression cassette. [0160] Examples of promoters that can be used include, but are not limited to, a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter (CMV-IE), Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. A promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. A promoter can also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Preferably, a promoter is a 26S subgenomic promoter or T7 promoter. A nucleotide sequence of an exemplary 26S subgenomic promoter is shown in SEQ ID NO: 62. A nucleotide sequence of an exemplary T7 promoter is shown in SEQ ID NO: 73.
[0161] A vector can comprise additional polynucleotide sequences that stabilize the expressed transcript, enhance nuclear export of the RNA transcript, and/or improve transcriptional-translational coupling. Examples of such sequences include poly adenylation signals and enhancer sequences. A polyadenylation signal is typically located downstream of the coding sequence for a protein of interest (e.g., an HBV antigen) within an expression cassette of the vector. Enhancer sequences are regulatory DNA sequences that, when bound by transcription factors, enhance the transcription of an associated gene. An enhancer sequence is preferably located upstream of the polynucleotide sequence encoding an HBV antigen, but downstream of a promoter sequence within an expression cassette of the vector. [0162] Any poly adenylation signal known to those skilled in the art in view of the present disclosure can be used. For example, the polyadenylation signal can be a SV40 polyadenylation signal (e.g., SEQ ID NO: 64), LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human [3-globin polyadenylation signal. Preferably, a polyadenylation signal is a SV40 polyadenylation signal. A nucleotide sequence of an exemplary SV40 polyadenylation signal is shown in SEQ ID NO: 64.
[0163] Any enhancer sequence known to those skilled in the art in view of the present disclosure can be used. For example, an enhancer sequence can be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as one from CMV, HA, RSV, or EBV. Examples of particular enhancers include, but are not limited to, Woodchuck HBV Post-transcriptional regulatory element (WPRE), intron/exon sequence derived from human apolipoprotein Al precursor (ApoAI), untranslated R-U5 domain of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR), a splicing enhancer, a synthetic rabbit [3-globin intron, or any combination thereof.
[0164] A vector can comprise a polynucleotide sequence encoding a signal peptide sequence. Preferably, the polynucleotide sequence encoding the signal peptide sequence is located upstream of the polynucleotide sequence encoding an HBV antigen. Signal peptides typically direct localization of a protein, facilitate secretion of the protein from the cell in which it is produced, and/or improve antigen expression and cross-presentation to antigen- presenting cells. A signal peptide can be present at the N-terminus of an HBV antigen when expressed from the vector, but is cleaved off by signal peptidase, e.g., upon secretion from the cell. An expressed protein in which a signal peptide has been cleaved is often referred to as the “mature protein.” Any signal peptide known in the art in view of the present disclosure can be used. For example, a signal peptide can be a cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as a Cystatin S signal peptide, an Ig heavy chain gamma signal peptide SPIgG, or an Ig heavy chain epsilon signal peptide SPIgE.
[0165] A vector, such as a DNA plasmid, can also include a bacterial origin of replication and an antibiotic resistance expression cassette for selection and maintenance of the plasmid in bacterial cells, e.g., E. coli. Bacterial origins of replication and antibiotic resistance cassettes can be located in a vector in the same orientation as the expression cassette encoding an HBV antigen, or in the opposite (reverse) orientation. An origin of replication (ORI) is a sequence at which replication is initiated, enabling a plasmid to reproduce and survive within cells. Examples of ORIs suitable for use in the application include, but are not limited to ColEl, pMBl, pUC, pSClOl, R6K, and 15A, preferably pUC.
[0166] Expression cassettes for selection and maintenance in bacterial cells typically include a promoter sequence operably linked to an antibiotic resistance gene. Preferably, the promoter sequence operably linked to an antibiotic resistance gene differs from the promoter sequence operably linked to a polynucleotide sequence encoding a protein of interest, e.g., HBV antigen. The antibiotic resistance gene can be codon optimized, and the sequence composition of the antibiotic resistance gene is normally adjusted to bacterial, e.g., E. coli, codon usage. Any antibiotic resistance gene known to those skilled in the art in view of the present disclosure can be used, including, but not limited to, kanamycin resistance gene (Kan1), ampicillin resistance gene (Ampr), and tetracycline resistance gene (Tet1), as well as genes conferring resistance to chloramphenicol, bleomycin, spectinomycin, carbenicillin, etc. [0167] The polynucleotides and expression vectors encoding the HBV antigens of the application can be made by any method known in the art in view of the present disclosure. For example, a polynucleotide encoding an HBV antigen can be introduced or “cloned” into an expression vector using standard molecular biology techniques, e.g., polymerase chain reaction (PCR), etc., which are well known to those skilled in the art.
RNA Replicons
[0168] Preferably, the vector is a self-replicating RNA replicon. As used herein, “selfreplicating RNA molecule,” which is used interchangeably with “self-amplifying RNA molecule” or “RNA replicon” or “replicon RNA” or “saRNA,” refers to RNA which contains all of the genetic information required for directing its own amplification or self-replication within a permissive cell, which can be a human, mammalian, or animal cell. A selfreplicating RNA molecule resembles mRNA. It is single-stranded, 5 '-capped, and 3 '-polyadenylated and is of positive orientation. To direct its own replication, the RNA molecule 1) encodes polymerase, replicase, or other proteins which can interact with viral or host cell- derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process; and 2) contain cis-acting RNA sequences required for replication and transcription of the subgenomic replicon-encoded RNA. Thus, the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, can be translated themselves to provide in situ expression of a gene of interest, or can be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the gene of interest. The overall result of this sequence of transcriptions is a huge amplification in the number of the introduced replicon RNAs and so the encoded gene of interest becomes a major polypeptide product of the cells.
[0169] The RNA replicon 1) encodes an RNA-dependent RNA polymerase, which may interact with viral or host cell-derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process, and the non-structural proteins nsPl, nsP2, nsP3, nsP4; and 2) contains cis-acting RNA sequences required for replication and transcription of the genomic and subgenomic RNAs, such as 3’ and 5’ untranslated regions (UTRs; alphavirus nucleotide sequences for non-structural protein-mediated amplification), and/or a subgenomic promoter. These sequences can be bound during the process of replication to self-encoded proteins, or non-self-encoded cell-derived proteins, nucleic acids or ribonucleoproteins, or complexes between any of these components. In some embodiments, a modified RNA replicon molecule typically contains the following ordered elements: 5' viral RNA sequence(s) required in cis for replication (e.g. a 5’ UTR and a 5’ CSE), sequences coding for biologically active nonstructural proteins (e.g. nsP1234), a promoter for transcribing the subgenomic RNA, 3' viral sequences required in cis for replication (e.g. 3’ UTR), and a polyadenylate tract, and optionally, a sequence (or two or more sequences) encoding a heterologous protein or peptide after or under the control of a sub-genomic promoter. Further, the term RNA replicon can refer to a positive sense (or message sense) molecule and the RNA replicon can be of a length different from that of any known, naturally-occurring RNA viruses. In any of the embodiments of the present disclosure, the RNA replicon can lack (or not contain) the sequence(s) of at least one (or all) of the structural viral proteins (e.g. nucleocapsid protein C, and envelope proteins P62, 6K, and El). In these embodiments, the sequences encoding one or more structural genes can be substituted with one or more heterologous sequences such as, for example, a coding sequence for at least one heterologous protein or peptide (or other gene of interest (GOI)).
[0170] In certain embodiments, an RNA replicon of the application comprises, ordered from the 5’- to 3’-end: (1) a 5’ untranslated region (5’-UTR) required for nonstructural protein-mediated amplification of an RNA virus; (2) a polynucleotide sequence encoding at least one, preferably all, of non-structural proteins of the RNA virus; (3) a subgenomic promoter of the RNA virus; (4) a polynucleotide sequence encoding an HBV antigen; and (5) a 3’ untranslated region (3 ’-UTR) required for nonstructural protein-mediated amplification of the RNA virus.
[0171] In certain embodiments, a self-replicating RNA molecule encodes an enzyme complex for self-amplification (replicase polyprotein) comprising an RNA-dependent RNA- polymerase function, helicase, capping, and poly-adenylating activity. The viral structural genes downstream of the replicase, which are under control of a subgenomic promoter, can be replaced by an HBV antigen. Upon transfection, the replicase is translated immediately, interacts with the 5' and 3' termini of the genomic RNA, and synthesizes complementary genomic RNA copies. Those act as templates for the synthesis of novel positive-stranded, capped, and poly-adenylated genomic copies, and subgenomic transcripts. Amplification eventually leads to very high RNA copy numbers of up to 2 x 105 copies per cell. Thus, much lower amounts of saRNA compared to conventional mRNA suffice to achieve effective gene transfer and protective vaccination (Beissert et al., Hum Gene Ther. 2017, 28(12): 1138-1146).
[0172] Subgenomic RNA is an RNA molecule of a length or size which is smaller than the genomic RNA from which it was derived. The viral subgenomic RNA can be transcribed from an internal promoter, whose sequences reside within the genomic RNA or its complement. Transcription of a subgenomic RNA can be mediated by viral-encoded polymerase(s) associated with host cell-encoded proteins, ribonucleoprotein(s), or a combination thereof. Numerous RNA viruses generate subgenomic mRNAs (sgRNAs) for expression of their 3'-proximal genes.
[0173] In some embodiments of the present disclosure, an HBV antigen is expressed under the control of a subgenomic promoter. In certain embodiments, instead of the native subgenomic promoter, the subgenomic RNA can be placed under control of internal ribosome entry site (IRES) derived from encephalomyocarditis viruses (EMCV), Bovine Viral Diarrhea Viruses (BVDV), polioviruses, Foot-and-mouth disease viruses (FMD), enterovirus 71 (EV71), or hepatitis C viruses. Subgenomic promoters range from 24 nucleotide (Sindbis virus) to over 100 nucleotides (Beet necrotic yellow vein virus) and are usually found upstream of the transcription start.
[0174] In some embodiments, the RNA replicon includes the coding sequence for at least one, at least two, at least three, or at least four nonstructural viral proteins (e.g. nsPl, nsP2, nsP3, nsP4). Alphavirus genomes encode non-structural proteins nsPl, nsP2, nsP3, and nsP4, which are produced as a single polyprotein precursor, sometimes designated P1234 (or nsPl-4 or nsP1234), and which is cleaved into the mature proteins through proteolytic processing. nsPl can be about 60 kDa in size and may have methyltransferase activity and be involved in the viral capping reaction. nsP2 has a size of about 90 kDa and may have helicase and protease activity while nsP3 is about 60 kDa and contains three domains: a macrodomain, a central (or alphavirus unique) domain, and a hypervariable domain (HVD). nsP4 is about 70 kDa in size and contains the core RNA-dependent RNA polymerase (RdRp) catalytic domain. After infection the alphavirus genomic RNA is translated to yield a Pl 234 polyprotein, which is cleaved into the individual proteins. In disclosing the nucleic acid or polypeptide sequences herein, for example sequences of nsPl, nsP2, nsP3, nsP4, also disclosed are sequences considered to be based on or derived from the original sequence.
[0175] In some embodiments, RNA replicon includes the coding sequence for a portion of the at least one nonstructural viral protein. For example, the RNA replicon can include about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or a range between any two of these values, of the encoding sequence for the at least one nonstructural viral protein. In some embodiments, the RNA replicon can include the coding sequence for a substantial portion of the at least one nonstructural viral protein. As used herein, a “substantial portion” of a nucleic acid sequence encoding a nonstructural viral protein comprises enough of the nucleic acid sequence encoding the nonstructural viral protein to afford putative identification of that protein, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (see, for example, in “Basic Local Alignment Search Tool”; Altschul S F et al., J. Mol. Biol. 215:403-410, 1993). In some embodiments, the RNA replicon can include the entire coding sequence for the at least one nonstructural protein. In some embodiments, the RNA replicon comprises substantially all the coding sequence for the native viral nonstructural proteins. In certain embodiments, the one or more nonstructural viral proteins are derived from the same virus. In other embodiments, the one or more nonstructural proteins are derived from different viruses.
[0176] The RNA replicon can be derived from any suitable plus-strand RNA viruses, such as alphaviruses or flaviviruses. Preferably, the RNA replicon is derived from alphaviruses. The term “alphavirus” describes enveloped single-stranded positive sense RNA viruses of the family Togaviridae. The genus alphavirus contains approximately 30 members, which can infect humans as well as other animals. Alphavirus particles typically have a 70 nm diameter, tend to be spherical or slightly pleomorphic, and have a 40 nm isometric nucleocapsid. The total genome length of alphaviruses ranges between 11,000 and 12,000 nucleotides and has a 5'cap and 3' poly-A tail. There are two open reading frames (ORF's) in the genome, non-structural (ns) and structural. The ns ORF encodes proteins (nsPl-nsP4) necessary for transcription and replication of viral RNA. The structural ORF encodes three structural proteins: the core nucleocapsid protein C, and the envelope proteins P62 and El that associate as a heterodimer. The viral membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells through membrane fusion. The four ns protein genes are encoded by genes in the 5' two-thirds of the genome, while the three structural proteins are translated from a subgenomic mRNA colinear with the 3' one-third of the genome.
[0177] In some embodiments, the self-replicating RNA useful for the invention is an RNA replicon derived from an alphavirus virus species. In some embodiments, the alphavirus RNA replicon is of an alphavirus belonging to the VEEV/EEEV group, or the SF group, or the SIN group. Non-limiting examples of SF group alphaviruses include Semliki Forest virus, O'Nyong-Nyong virus, Ross River virus, Middelburg virus, Chikungunya virus, Barmah Forest virus, Getah virus, Mayaro virus, Sagiyama virus, Bebaru virus, and Una virus. Nonlimiting examples of SIN group alphaviruses include Sindbis virus, Girdwood S. A. virus, South African Arbovirus No. 86, Ockelbo virus, Aura virus, Babanki virus, Whataroa virus, and Kyzylagach virus. Non-limiting examples of VEEV/EEEV group alphaviruses include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), and Una virus (UNAV).
[0178] Non-limiting examples of alphavirus species include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), and Buggy Creek virus. Virulent and avirulent alphavirus strains are both suitable. In some embodiments, the alphavirus RNA replicon is of a Sindbis virus (SIN), a Semliki Forest virus (SFV), a Ross River virus (RRV), a Venezuelan equine encephalitis virus (VEEV), or an Eastern equine encephalitis virus (EEEV). In some embodiments, the alphavirus RNA replicon is of a Venezuelan equine encephalitis virus (VEEV).
[0179] In certain embodiments, a self-replicating RNA molecule comprises a polynucleotide encoding one or more nonstructural proteins nsPl-4, a subgenomic promoter, such as 26S subgenomic promoter, and a gene of interest encoding an HBV antigen or a fragment thereof described herein.
[0180] A self-replicating RNA molecule can have a 5' cap (e.g. a 7-methylguanosine). This cap can enhance in vivo translation of the RNA.
[0181] The 5' nucleotide of a self-replicating RNA molecule useful with the invention can have a 5' triphosphate group. In a capped RNA this can be linked to a 7-methylguanosine via a 5'-to-5' bridge. A 5' triphosphate can enhance RIG-I binding.
[0182] A self-replicating RNA molecule can have a 3' poly-A tail. It can also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3' end.
[0183] In any of the embodiments of the present disclosure, the RNA replicon can lack (or not contain) the coding sequence(s) of at least one (or all) of the structural viral proteins (e.g. nucleocapsid protein C, and envelope proteins P62, 6K, and El). In these embodiments, the sequences encoding one or more structural genes can be substituted with one or more heterologous sequences such as, for example, a coding sequence for an HBV antigen or a fragment thereof described herein.
[0184] In certain embodiments, a self-replicating RNA vector of the application comprises one or more features to confer a resistance to the translation inhibition by the innate immune system or to otherwise increase the expression of the GOI (e.g., an HBV antigen).
[0185] In certain embodiments, the RNA sequence can be codon optimized to improve translation efficiency. The RNA molecule can be modified by any method known in the art in view of the present disclosure to enhance stability and/or translation, such by adding a polyA tail, e.g., of at least 30 adenosine residues; and/or capping the 5-end with a modified ribonucleotide, e.g., 7-methylguanosine cap, which can be incorporated during RNA synthesis or enzymatically engineered after RNA transcription.
[0186] In certain embodiments, an RNA replicon of the application comprises, ordered from the 5’ - to 3 ’-end, (1) an alphavirus 5’ untranslated region (5’-UTR), (2) a 5’ replication sequence of an alphavirus non-structural gene nspl, (3) a downstream loop (DLP) motif of a virus species, (4) a polynucleotide sequence encoding a fourth autoprotease peptide, (5) a polynucleotide sequence encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4, (6) an alphavirus subgenomic promoter, (7) the non-naturally occurring polynucleotide sequence encoding one or more HBV antigens of the application, (8) an alphavirus 3' untranslated region (3' UTR), and (9) optionally, a poly adenosine sequence.
[0187] In certain embodiments, a self-replicating RNA vector of the application comprises a downstream loop (DLP) motif of a virus species. As used herein, a “downstream loop” or “DLP motif’ refers to a polynucleotide sequence comprising at least one RNA stemloop, which when placed downstream of a start codon of an open reading frame (ORF) provides increased translation the ORF compared to an otherwise identical construct without the DLP motif. As an example, members of the Alphavirus genus can resist the activation of antiviral RNA-activated protein kinase (PKR) by means of a prominent RNA structure present within in viral 26S transcripts, which allows an eIF2-independent translation initiation of these mRNAs. This structure, called the downstream loop (DLP), is located downstream from the AUG in SINV 26S mRNA. The DLP is also detected in Semliki Forest virus (SFV). Similar DLP structures have been reported to be present in at least 14 other members of the Alphavirus genus including New World (for example, MAYV, UNAV, EEEV (NA), EEEV (SA), AURAV) and Old World (SV, SFV, BEBV, RRV, SAG, GETV, MIDV, CHIKV, and ONNV) members. The predicted structures of these Alphavirus 26S mRNAs were constructed based on SHAPE (selective 2'-hydroxyl acylation and primer extension) data (Toribio et al., Nucleic Acids Res. May 19; 44(9):4368-80, 2016), the content of which is hereby incorporated by reference). Stable stem-loop structures were detected in all cases except for CHIKV and ONNV, whereas MAYV and EEEV showed DLPs of lower stability (Toribio et al., 2016 supra). In the case of Sindbis virus, the DLP motif is found in the first 150 nt of the Sindbis subgenomic RNA. The hairpin is located downstream of the Sindbis capsid AUG initiation codon (AUG is collated at nt 50 of the Sindbis subgenomic RNA). Previous studies of sequence comparisons and structural RNA analysis revealed the evolutionary conservation of DLP in SINV and predicted the existence of equivalent DLP structures in many members of the Alphavirus genus (see e.g., Ventoso, J. Virol. 9484-9494, Vol. 86, September 2012). Examples of a self-replicating RNA vector comprising a DLP motif are described in US Patent Application Publication US2018/0171340 and the International Patent Application Publication W02018106615, the content of which is incorporated herein by reference in its entirety. In some embodiments, a replicon RNA of the application comprises a DLP motif exhibiting at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequences set forth in SEQ ID NO: 57.
[0188] In one embodiment, the self-replicating RNA molecule also contains a coding sequence for an autoprotease peptide operably linked downstream of the DLP motif and upstream of the coding sequences of the nonstructural proteins (e.g., one or more of nspl-4) or gene of interest (e.g., an HBV antigen described herein). Examples of the autoprotease peptide include, but are not limited to, a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), and a combination thereof. In some embodiments, a replicon RNA of the application comprises a coding sequence for P2A having the amino acid sequence of SEQ ID NO: 11. Preferably, the coding sequence exhibits at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequences set forth in SEQ ID NO: 12.
[0189] Any of the replicons of the invention can also comprise a 5’ and a 3’ untranslated region (UTR). The UTRs can be wild type New World or Old World alphavirus UTR sequences, or a sequence derived from any of them. In various embodiments the 5’ UTR can be of any suitable length, such as about 60 nt or 50-70 nt or 40-80 nt. In some embodiments the 5’ UTR can also have conserved primary or secondary structures (e.g. one or more stem- loop(s)) and can participate in the replication of alphavirus or of replicon RNA. In some embodiments the 3’ UTR can be up to several hundred nucleotides, for example it can be 50- 900 or 100-900 or 50-800 or 100-700 or 200 nt - 700 nt. The ‘3 UTR also can have secondary structures, e.g. a step loop, and can be followed by a polyadenylate tract or poly-A tail. In any of the embodiments of the invention the 5’ and 3’ untranslated regions can be operably linked to any of the other sequences encoded by the replicon. The UTRs can be operably linked to a promoter and/or sequence encoding a heterologous protein or peptide by providing sequences and spacing necessary for recognition and transcription of the other encoded sequences. Any polyadenylation signal known to those skilled in the art in view of the present disclosure can be used. For example, the poly adenylation signal can be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human [3- globin polyadenylation signal.
[0190] In another embodiment, a self-repli eating RNA replicon of the application comprises a modified 5’ untranslated region (5'-UTR), preferably the RNA replicon is devoid of at least a portion of a nucleic acid sequence encoding viral structural proteins. For example, the modified 5'-UTR can comprise one or more nucleotide substitutions at position 1, 2, 4, or a combination thereof. Preferably, the modified 5'-UTR comprises a nucleotide substitution at position 2, more preferably, the modified 5'-UTR has a U->G or U->A substitution at position 2. Examples of such self-replicating RNA molecules are described in US Patent Application Publication US2018/0104359 and the International Patent Application Publication WO2018075235, the content of which is incorporated herein by reference in its entirety. In some embodiments, a replicon RNA of the application comprises a 5'-UTR exhibiting at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequences set forth in SEQ ID NO: 55.
[0191] In some embodiments, an RNA replicon of the application comprises, ordered from the 5’- to 3’-end, (1) a 5’-UTR having the polynucleotide sequence of SEQ ID NO: 55, (2) a 5’ replication sequence having the polynucleotide sequence of SEQ ID NO: 56, (3) a DLP motif comprising the polynucleotide sequence of SEQ ID NO: 57, (4) a polynucleotide sequence encoding a P2A sequence of SEQ ID NO: 11, (5) polynucleotide sequences encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4, having the nucleic acid sequences of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively, (6) a subgenomic promoter having polynucleotide sequence of SEQ ID NO: 62, (7) a non-naturally occurring polynucleotide sequence as described herein, and (8) a 3' UTR having the polynucleotide sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide sequence encoding the P2A sequence comprises SEQ ID NO: 12, the non- naturally occurring polynucleotide sequence comprises the polynucleotide sequence of any one of SEQ ID NOs: 15 to 54, and the RNA replicon further comprises a poly adenosine sequence. Preferably the poly adenosine sequence has the sequence of SEQ ID NO: 64, at the 3 ’-end of the replicon.
[0192] In some preferred embodiments, an RNA replicon of the application comprises the polynucleotide sequence of any one of SEQ ID NOs: 65 to 72.
[0193] In some embodiments, an RNA replicon of the application comprises a polynucleotide sequence encoding a signal peptide sequence. Preferably, the polynucleotide sequence encoding the signal peptide sequence is located upstream of or at the 5 ’-end of the polynucleotide sequence encoding an HBV antigen, such as an HBV PreSl antigen, an HBV core antigen and an HBV pol antigen. Signal peptides typically direct localization of a protein, facilitate secretion of the protein from the cell in which it is produced, and/or improve antigen expression and cross-presentation to antigen-presenting cells. A signal peptide can be present at the N-terminus of an HBV antigen when expressed from the replicon, but is cleaved off by signal peptidase, e.g., upon secretion from the cell. An expressed protein in which a signal peptide has been cleaved is often referred to as the “mature protein.” Any signal peptide known in the art in view of the present disclosure can be used. For example, a signal peptide can be a cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as a Cystatin S signal peptide, an Ig heavy chain gamma signal peptide SPIgG, an Ig heavy chain epsilon signal peptide SPIgE, or a short leader peptide sequence. An exemplary amino acid sequence of a signal peptide is shown in SEQ ID NO: 77.
[0194] In various embodiments the RNA replicons disclosed herein can be engineered, synthetic, or recombinant RNA replicons. As used herein, the term recombinant means any molecule (e.g. DNA, RNA, etc.), that is or results, however indirectly, from human manipulation of a polynucleotide. As non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector. As non-limiting examples, a recombinant RNA replicon can be one or more of the following: 1) synthesized or modified in vitro, for example, using chemical or enzymatic techniques (for example, by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse tianscription, transcription, base modification (including, e.g., methylation), or recombination (including homologous and sitespecific recombination) of nucleic acid molecules; 2) conjoined nucleotide sequences that are not conjoined in nature; 3) engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleotide sequence; and 4) manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleotide sequence.
[0195] Any of the components or sequences of the RNA replicon can be operably linked to any other of the components or sequences. The components or sequences of the RNA replicon can be operably linked for the expression of at least one heterologous protein or peptide (or biotherapeutic) in a host cell or treated organism and/or for the ability of the replicon to self-replicate. The term “operably linked” denotes a functional linkage between two or more sequences that are configured so as to perform their usual function. Thus, a promoter or UTR operably linked to a coding sequence is capable of effecting the transcription and expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, an operable linkage between an RNA sequence encoding a heterologous protein or peptide and a regulatory sequence (for example, a promoter or UTR) is a functional link that allows for expression of the polynucleotide of interest. Operably linked can also refer to sequences such as the sequences encoding nsPl-4, the UTRs, promoters, and other sequences encoding in the RNA replicon, are linked so that they enable transcription and translation of the biotherapeutic molecule and/or replication of the replicon. The UTRs can be operably linked by providing sequences and spacing necessary for recognition and translation by a ribosome of other encoded sequences.
[0196] The RNA replicons of the invention can be derived from alphavirus genomes, meaning that they have some of the structural characteristics of alphavirus genomes, or be similar to them. The RNA replicons of the invention can be modified alphavirus genomes. In some embodiments of the replicons disclosed herein one or more sequences of the replicon can be provided “in trans,” i.e., the sequences of the replicon are provided on more than one RNA molecule. In other embodiments all of the sequences of the replicon are present on a single RNA molecule, which can also be administered to a mammal to be treated as described herein.
[0197] As used herein, the terms “percent identity” or “homology” or “shared sequence identity” or “percent (%) sequence identity” with respect to nucleic acid or polypeptide sequences are defined as the percentage of nucleotide or amino acid residues in the candidate sequence that are identical with the known polynucleotides or polypeptides, after aligning the sequences for maximum percent identity and introducing gaps, if necessary, to achieve the maximum percent homology. N-terminal or C-terminal insertions or deletions shall not be construed as affecting homology, and internal deletions and/or insertions into the nucleotide or polypeptide sequence of less than about 30, less than about 20, or less than about 10 or less than 5 amino acid residues shall not be construed as affecting homology. Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx (Altschul (1997), Nucleic Acids Res. 25, 3389-3402, and Karlin (1990), Proc. Natl. Acad. Sci. USA 87, 2264-2268), which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified, and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul (1994), Nature Genetics 6, 119-129. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix, and filter (low complexity) can be at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff (1992), Proc. Natl. Acad. Sci. USA 89, 10915-10919), recommended for query sequences over 85 in length (nucleotide bases or amino acids).
[0198] For blastn, designed for comparing nucleotide sequences, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N can be +5 and -4, respectively. Four blastn parameters can be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=l (generates word hits at every winkth position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings for comparison of amino acid sequences can be: Q=9; R=2; wink=l; and gapw=32. A BESTFIT® comparison between sequences, available in the GCG package version 10.0, can use DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty), and the equivalent settings in protein comparisons can be GAP=8 and LEN=2. [0199] In disclosing the nucleic acid or polypeptide sequences herein, for example sequences of viral capsid enhancers, autoprotease peptides, subgenomic promoters, nonstructural proteins, HBV antigens, also disclosed are sequences considered to be based on or derived from the original sequence. Sequences disclosed therefore include polynucleotide or polypeptide sequences having sequence identities of at least 40%, at least 45%, at least 50%, at least 55%, of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%, for example at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% or 85-99% or 85-95% or 90-99% or 95-99% or 97-99% or 98-99% sequence identity with the full-length polynucleotide or polypeptide sequence of any polynucleotide or polypeptide sequence described herein, respectively, such as SEQ ID NOs: 1-90, and fragments thereof. Also disclosed are fragments or portions of any of the sequences disclosed herein. Fragments or portions of sequences can include sequences having at least 5 or at least 7 or at least 10, or at least 20, or at least 30, at least 50, at least 75, at least 100, at least 125, 150 or more or 5-10 or 10-12 or 10-15 or 15-20 or 20-40 or 20-50 or 30-50 or 30-75 or 30-100 amino acid or nucleic acid residues of the entire sequence, or at least 100 or at least 200 or at least 300 or at least 400 or at least 500 or at least 600 or at least 700 or at least 800 or at least 900 or at least 1000 or 100-200 or 100-500 or 100-1000 or 500- 1000 amino acid or nucleic acid residues, or any of these amounts but less than 500 or less than 700 or less than 1000 or less than 2000 consecutive amino acids or nucleic acids of any of SEQ ID NOs: 1-90 or of any fragment disclosed herein. Also disclosed are variants of such sequences, e.g., where at least one or two or three or four or five amino acid residues have been inserted N- and/or C-terminal to, and/or within, the disclosed sequence(s) which contain(s) the insertion and substitution, and nucleic acid sequences encoding such variants. Contemplated variants can additionally or alternately include those containing predetermined mutations by, e.g., homologous recombination or site-directed or PCR mutagenesis, and the corresponding polypeptides or nucleic acids of other species, including, but not limited to, those described herein, the alleles or other naturally occurring variants of the family of polypeptides or nucleic acids which contain an insertion and substitution; and/or derivatives wherein the polypeptide has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid which contains the insertion and substitution (for example, a detectable moiety such as an enzyme). The nucleic acid sequences described herein can be RNA sequences.
Heterologous proteins and peptides [0200] The RNA replicons of the invention can include an RNA sequence encoding at least one protein or peptide that is heterologous to an alphavirus and can also be (but is not necessarily) heterologous to the human, mammal, or animal that expresses the RNA sequence in the body. In any embodiment the replicons can have RNA sequence(s) encoding two or three or four or more heterologous proteins or peptides. In some embodiments, the heterologous protein or peptide is an HBV antigen as described herein. In any of the embodiments the sequence encoding the heterologous protein or peptide can be operably linked to one or more other sequences of the replicon (e.g. a promoter or 5’ or 3’ UTR sequences), and can be under the control of a sub-genomic promoter so that the heterologous protein or peptide is expressed in the human, mammal, or animal.
[0201] In one embodiment the RNA replicon of the invention can have an RNA sequence encoding a heterologous protein or peptide (e.g. a monoclonal antibody or a biotherapeutic protein or peptide), RNA sequences encoding amino acid sequences derived from wild type alphavirus nsPl, nsP2, nsP3, and nsP4 protein sequences, and 5’ and 3’ UTR sequences (for non-structural protein-mediated amplification). The RNA replicons can also have a 5’ cap and a poly adenylate (or poly -A) tail.
[0202] The immunogenicity of a heterologous protein or peptide can be determined by a number of assays known to persons of ordinary skill, for example immunostaining of intracellular cytokines or secreted cytokines by epitope-specific T-cell populations, or by quantifying frequencies and total numbers of epitope-specific T-cells and characterizing their differentiation and activation state, e.g., short-lived effector and memory precursor effector CD8+ T-cells. Immunogenicity can also be determined by measuring an antibody-mediated immune response, e.g. the production of antibodies by measuring serum IgA or IgG titers. [0203] In addition to the HBV antigens of the application, the RNA replicons of the application can optionally further encode one or more heterologous proteins or peptides that can be any protein or peptide, including but not limited to, cytokines, growth factors, immunoglobulins, monoclonal antibodies (including Fab antigen-binding fragments, Fc fusion proteins), hormones, interferons, interleukins, regulatory peptides and proteins.
[0204] In some embodiments the heterologous protein or peptide can be encoded by an RNA sequence of up to 5 kb or up to 6 kb or up to 7 kb or up to 8 kb, or up to 9 kb or up to 10 kb or up to 11 kb or up to 12 kb. The heterologous protein can also be a single-chain antibody molecule.
[0205] The alphavirus replicons of the invention can also have a sub-genomic promoter for expression of the heterologous protein or peptide. The term “subgenomic promoter,” as used herein, refers to a promoter of a subgenomic mRNA of a viral nucleic acid. As used herein, an “alphavirus subgenomic promoter” is a promoter as originally defined in an alphavirus genome that directs transcription of a subgenomic messenger RNA as part of the alphavirus replication process.
[0206] The term “heterologous” when used in reference to a polynucleotide, a gene, a nucleic acid, a polypeptide, a protein, or an enzyme, refers to a polynucleotide, gene, a nucleic acid, polypeptide, protein, or an enzyme that is not derived from the host species. For example, “heterologous gene” or “heterologous nucleic acid sequence” as used herein, refers to a gene or nucleic acid sequence from a different species than the species of the host organism it is introduced into. Heterologous sequences can also be synthetic and not derived from an organism or not found in Nature. When referring to a gene regulatory sequence or to an auxiliary nucleic acid sequence used for manipulating expression of a gene sequence (e.g. a 5' untranslated region, 3' untranslated region, poly A addition sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genome homology region, recombination site, etc.) or to a nucleic acid sequence encoding a protein domain or protein localization sequence, “heterologous” means that the regulatory or auxiliary sequence or sequence encoding a protein domain or localization sequence is from a different source than the gene with which the regulatory or auxiliary nucleic acid sequence or nucleic acid sequence encoding a protein domain or localization sequence is juxtaposed in a genome, chromosome or episome. Thus, a promoter operably linked to a gene to which it is not operably linked to in its natural state (for example, in the genome of a non- genetically engineered organism) is referred to herein as a “heterologous promoter,” even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked. Similarly, when referring to a protein localization sequence or protein domain of an engineered protein, “heterologous” means that the localization sequence or protein domain is derived from a protein different from that into which it is incorporated by genetic engineering.
[0207] The term “non-naturally occurring,” “recombinant,” or “engineered” nucleic acid molecule or polynucleotide sequence, as used herein, refers to a nucleic acid molecule or non-naturally occurring polynucleotide sequence that has been altered through human intervention. As non-limiting examples, a recombinant nucleic acid molecule: 1) has been synthesized or modified in vitro, for example, using chemical or enzymatic techniques (for example, by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, e.g., methylation), or recombination (including homologous and site-specific recombination) of nucleic acid molecules; 2) includes conjoined nucleotide sequences that are not conjoined in nature, 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleic acid molecule sequence, and/or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleic acid sequence. As non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector or that has been integrated into an RNA replicon.
[0208] In some embodiments, an RNA replicon of the invention comprises, ordered from the 5’ - to 3 ’-end: a 5’ untranslated region (5’-UTR) required for nonstructural protein- mediated amplification of an RNA virus; a polynucleotide sequence encoding at least one, preferably all, of non-structural proteins of the RNA virus; a subgenomic promoter of the RNA virus; a non-naturally occurring polynucleotide sequence described herein; and a 3’ untranslated region (3’-UTR) required for nonstructural protein-mediated amplification of the RNA virus.
[0209] In some embodiments, an RNA replicon of the invention comprises, ordered from the 5’ - to 3 ’-end: an alphavirus 5’ untranslated region (5’-UTR); a 5’ replication sequence of an alphavirus non-structural gene nspl; a downstream loop (DLP) motif of a virus species; a polynucleotide sequence encoding a fourth autoprotease peptide; a polynucleotide sequence encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4; an alphavirus subgenomic promoter; a non-naturally occurring polynucleotide sequence described herein; an alphavirus 3' untranslated region (3' UTR); and, optionally, a poly adenosine sequence. [0210] In some embodiments, the DLP motif is from a virus species selected from the group consisting of Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MTDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (U AV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), and Buggy Creek virus.
[0211] In some embodiments, the fourth autoprotease peptide is selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2 A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof. Preferably, the fourth autoprotease peptide comprises the peptide sequence of P2A. In some embodiments, the fourth autoprotease peptide comprises SEQ ID NO: 11. In some embodiments, a polynucleotide sequence encoding a fourth autoprotease peptide comprises SEQ ID NO: 12. In some embodiments, a polynucleotide sequence encoding a fourth autoprotease peptide consists of SEQ ID NO: 12.
[0212] In some embodiments, an RNA replicon of the invention comprises, ordered from the 5’- to 3’-end: a 5’-UTR having the polynucleotide sequence of SEQ ID NO: 55; a 5’ replication sequence having the polynucleotide sequence of SEQ ID NO: 56; a DLP motif comprising the polynucleotide sequence of SEQ ID NO: 57; a polynucleotide sequence encoding a P2A sequence of SEQ ID NO: 11; polynucleotide sequences encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4, as those encoded by the nucleic acid sequences of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively; a subgenomic promoter having polynucleotide sequence of SEQ ID NO: 62; a non-naturally occurring polynucleotide sequence disclosed herein; and a 3' UTR having the polynucleotide sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide sequence encoding the P2A sequence comprises SEQ ID NO: 12, the non-naturally occurring polynucleotide sequence comprises the polynucleotide sequence of any one of SEQ ID NOs: 15 to 54, and the RNA replicon further comprises a poly adenosine sequence. Preferably, the poly adenosine sequence has the sequence of SEQ ID NO: 64 at the 3’-end of the replicon.
[0213] In some embodiments, an RNA replicon of the invention comprises the polynucleotide sequence of any one of SEQ ID NOs: 65 to 72.
[0214] In some embodiments, a nucleic acid molecule comprising a DNA sequence encoding an RNA replicon disclosed herein further comprises a T7 promoter operably linked to the 5 ’-end of the DNA sequence. More preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO: 73.
[0215] Also provided are methods of producing an RNA replicon of the application, comprising transcribing a nucleic acid molecule comprising a DNA sequence encoding a RNA replicon disclosed herein. In some embodiments, the nucleic acid molecule is transcribed in vivo. In some embodiments, the nucleic acid molecule is transcribed in vitro.
Cells and Polypeptides
[0216] The application also provides cells, preferably isolated cells, comprising any of the polynucleotides and vectors described herein. The cells can, for instance, be used for recombinant protein production, or for the production of viral particles. In some embodiments, the cells can be used for production of an RNA replicon.
[0217] Host cells comprising a RNA replicon or a nucleic acid encoding the RNA replicon of the application also form part of the invention. The HBV antigens may be produced through recombinant DNA technology involving expression of the molecules in host cells, e.g., Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6 cells, or yeast, fungi, insect cells, and the like, or transgenic animals or plants. In certain embodiments, the cells are from a multicellular organism, in certain embodiments they are of vertebrate or invertebrate origin. In certain embodiments, the cells are mammalian cells, such as human cells, or insect cells. In general, the production of a recombinant protein, such the HBV antigens of the invention, in a host cell comprises the introduction of a heterologous nucleic acid molecule encoding the protein in expressible format into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid molecule and allowing expression of the protein in said cell. The nucleic acid molecule encoding a protein in expressible format may be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like. The person skilled in the art is aware that various promoters can be used to obtain expression of a gene in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed. Further regulatory sequences may be added. Many promoters can be used for expression of a transgene(s), and are known to the skilled person, e.g. these may comprise viral, mammalian, synthetic promoters, and the like. A non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is a CMV -promoter (US 5,385,839), e.g. the CMV immediate early promoter, for instance comprising nt. -735 to +95 from the CMV immediate early gene enhancer/promoter. A polyadenylation signal, for example the bovine growth hormone polyA signal (US 5,122,458), may be present behind the transgene(s). Alternatively, several widely used expression vectors are available in the art and from commercial sources, e.g. the pcDNA and pEF vector series of Invitrogen, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc, which can be used to recombinantly express the protein of interest, or to obtain suitable promoters and/or transcription terminator sequences, polyA sequences, and the like.
[0218] The cell culture can be any type of cell culture, including adherent cell culture, e.g., cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. More recently, continuous processes based on perfusion principles are becoming more common and are also suitable. Suitable culture media are also well known to the skilled person and can generally be obtained from commercial sources in large quantities, or custom-made according to standard protocols. Culturing can be done for instance in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems and the like. Suitable conditions for culturing cells are known (see e.g. Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley -Liss Inc., 2000, ISBN 0-471-34889-9)). Cell culture media are available from various vendors, and a suitable medium can be routinely chosen for a host cell to express the protein of interest, here the HBV antigens. The suitable medium may or may not contain serum.
[0219] Embodiments of the application thus also relate to a method of making an HBV antigen of the application. The method comprises transfecting a host cell with an expression vector comprising a polynucleotide encoding an HBV antigen of the application operably linked to a promoter, growing the transfected cell under conditions suitable for expression of the HBV antigen, and optionally purifying or isolating the HBV antigen expressed in the cell. The HBV antigen can be isolated or collected from the cell by any method known in the art including affinity chromatography, size exclusion chromatography, etc. Techniques used for recombinant protein expression will be well known to one of ordinary skill in the art in view of the present disclosure. The expressed HBV antigens can also be studied without purifying or isolating the expressed protein, e.g., by analyzing the supernatant of cells transfected with an expression vector encoding the HBV antigen and grown under conditions suitable for expression of the HBV antigen.
[0220] Thus, also provided are non-naturally occurring or recombinant polypeptides comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, 84, 85 or 86, or SEQ ID NO: 9. As described above and below, isolated nucleic acid molecules encoding these sequences, vectors comprising these sequences operably linked to a promoter, and compositions comprising the polypeptide, polynucleotide, or vector are also contemplated by the application.
[0221] In an embodiment of the application, a recombinant polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, 84, 85 or 86, or SEQ ID NO: 9, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, 84, 85 or 86, or SEQ ID NO: 9. Preferably, a non-naturally occurring or recombinant polypeptide consists of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 84, 85 or 86, or SEQ ID NO: 9.
RNAi Component
[0222] In one aspect, the RNAi component comprises one or more RNAi agents. Each RNAi agent disclosed herein includes at least a sense strand and an antisense strand. The sense strand and the antisense strand can be partially, substantially, or fully complementary to each other. The length of the RNAi agent sense and antisense strands described herein each can be 16 to 30 nucleotides in length. In some embodiments, the sense and antisense strands are independently 17 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 19 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21 to 24 nucleotides in length. The sense and antisense strands can be either the same length or different lengths. The HBV RNAi agents disclosed herein have been designed to include antisense strand sequences that are at least partially complementary to a sequence in the HBV genome that is conserved across the majority of known serotypes of HBV. The RNAi agents described herein, upon delivery to a cell expressing HBV, inhibit the expression of one or more HBV genes in vivo or in vitro.
[0223] An RNAi agent includes a sense strand (also referred to as a passenger strand) that includes a first sequence, and an antisense strand (also referred to as a guide strand) that includes a second sequence. A sense strand of the HBV RNAi agents described herein includes a core stretch having at least about 85% identity to a nucleotide sequence of at least 16 consecutive nucleotides in an HBV mRNA. In some embodiments, the sense strand core nucleotide stretch having at least about 85% identity to a sequence in an HBV mRNA is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. An antisense strand of an HBV RNAi agent comprises a nucleotide sequence having at least about 85% complementary over a core stretch of at least 16 consecutive nucleotides to a sequence in an HBV mRNA and the corresponding sense strand. In some embodiments, the antisense strand core nucleotide sequence having at least about 85% complementarity to a sequence in an HBV mRNA or the corresponding sense strand is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.
[0224] In some embodiments, the RNAi component comprises a first RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107, or a second RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111. In some embodiments, the RNAi component comprises a first RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO: 99, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107, and a second RNAi agent comprising an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111.
[0225] In some embodiments, the first and the second RNAi agents disclosed herein comprise any of the sequences in Table 1.
Table 1. Exemplary sequences for first and second RNAi agents
Figure imgf000085_0001
Targeting Group [0226] In some embodiments, the RNAi agents are delivered to target cells or tissues using any oligonucleotide delivery technology known in the art. Nucleic acid delivery methods include, but are not limited to, by encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, proteinaceous vectors or Dynamic Polyconjugates (DPCs) (see, for example WO 2000/053722, WO 2008/0022309, WO 2011/104169, and WO 2012/083185, each of which is incorporated herein by reference). In some embodiments, an HBV RNAi agent is delivered to target cells or tissues by covalently linking the RNAi agent to a targeting group. In some embodiments, the targeting group can include a cell receptor ligand, such as an asialoglycoprotein receptor (ASGPr) ligand. In some embodiments, an ASGPr ligand includes or consists of a galactose derivative cluster. In some embodiments, a galactose derivative cluster includes an N-acetyl-galactosamine trimer or an N-acetyl-galactosamine tetramer. In some embodiments, a galactose derivative cluster is an N-acetyl-galactosamine trimer or an N-acetyl-galactosamine tetramer.
[0227] A targeting group can be linked to the 3' or 5' end of a sense strand or an antisense strand of an HBV RNAi agent. In some embodiments, a targeting group is linked to the 3' or 5' end of the sense strand. In some embodiments, a targeting group is linked to the 5’ end of the sense strand. In some embodiments, a targeting group is linked to the RNAi agent via a linker.
[0228] In some embodiments, the RNAi component comprises a combination or cocktail of a first and a second RN Ai agent having different nucleotide sequences. In some embodiments, the first and the second RNAi agents are each separately and independently linked to targeting groups. In some embodiments, the first and the second RNAi agents are each linked to targeting groups comprised of N-acetyl-galactosamines. In some embodiments, when first and the second RNAi agents are included in a composition, each of the RNAi agents is linked to the same targeting group. In some embodiments, when first and the second RNAi agents are included in a composition, each of the RNAi agents is linked to different targeting groups, such as targeting groups having different chemical structures.
[0229] In some embodiments, targeting groups are linked to the first and the second RNAi agents without the use of an additional linker. In some embodiments, the targeting group is designed having a linker readily present to facilitate the linkage to the first or the second RN Ai agent. In some embodiments, when the first and the second RNAi agents are included in a composition, the first and the second RNAi agents may be linked to the targeting groups using the same linkers. In some embodiments, when the first and the second RNAi agents are included in a composition, the first and the second RNAi agents are linked to the targeting groups using different linkers.
[0230] Examples of targeting groups and linking groups are provided in Table 2. The non-nucleotide group can be covalently linked to the 3' and/or 5' end of either the sense strand and/or the antisense strand. In some embodiments, the first or second RNAi agent contains a non-nucleotide group linked to the 3' and/or 5' end of the sense strand. In some embodiments, a non-nucleotide group is linked to the 5' end of the first or second RNAi agent sense strand. A non-nucleotide group may be linked directly or indirectly to the first or second RNAi agent via a linker/linking group. In some embodiments, a non-nucleotide group is linked to the first or second RNAi agent via a labile, cleavable, or reversible bond or linker. [0231] Targeting groups and linking groups include the following, for which their chemical structures are provided below in Table 2: (PAZ), (NAGI 3), (NAG13)s, (NAGI 8), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), (NAG39)s. Each sense strand and/or antisense strand can have any targeting groups or linking groups listed above, as well as other targeting or linking groups, conjugated to the 5' and/or 3' end of the sequence.
Table 2. Structures Representing Various Modified Nucleotides, Targeting Groups,
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Modified Nucleotides
[0232] In some embodiments, the first or the second RNAi agent contains one or more modified nucleotides. As used herein, a “modified nucleotide” is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides (represented herein as Ab), 2'-modified nucleotides, 3' to 3' linkages (inverted) nucleotides (represented herein as invdN, invN, invn, invAb), non-natural base-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2',3'-seco nucleotide mimics (unlocked nucleobase analogues, represented herein as NUNA or NUNA), locked nucleotides (represented herein as NLNA or NLNA), 3'-O-methoxy (2' intemucleoside linked) nucleotides (represented herein as 3'-OMen), 2'-F-Arabino nucleotides (represented herein as NfANA or NfANA), 5'-Me, 2'-fluoro nucleotide (represented herein as 5Me-Nf), morpholino nucleotides, vinyl phosphonate deoxyribonucleotides (represented herein as vpdN), vinyl phosphonate containing nucleotides, and cyclopropyl phosphonate containing nucleotides (cPrpN). 2'-modified nucleotides (i.e. a nucleotide with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides (represented herein as a lower case letter 'n' in a nucleotide sequence), 2'-deoxy-2'-fluoro nucleotides (represented herein as Nf, also represented herein as 2'-fluoro nucleotide), 2'-deoxy nucleotides (represented herein as dN), 2'-methoxy ethyl (2'-O-2-methoxylethyl) nucleotides (represented herein as NM or 2'-MOE), 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification can be incorporated in the first or second RNAi agent or even in a single nucleotide thereof. The RNAi agent sense strands and antisense strands may be synthesized and/or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide. [0233] Modified nucleobases include synthetic and natural nucleobases, such as 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, (e.g, 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me- C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6- methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3 -deazaadenine.
[0234] In some embodiments, all or at least 90% of the nucleotides of the first or the second RNAi agent are modified nucleotides. As used herein, an RNAi agent wherein at least 90% of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides. As used herein, a sense strand, wherein at least 90% of the nucleotides present are modified nucleotides, is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides. As used herein, an antisense sense strand, wherein at least 90% of the nucleotides present are modified nucleotides, is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides. In some embodiments, one or more nucleotides of an RNAi agent is a ribonucleotide.
Modified Internucleoside Linkages
[0235] In some embodiments, one or more nucleotides of the first or the second RNAi agent are linked by non-standard linkages or backbones (i.e., modified intemucleoside linkages or modified backbones). In some embodiments, a modified intemucleoside linkage is a non-phosphate-containing covalent intemucleoside linkage. Modified intemucleoside linkages or backbones include, but are not limited to, 5 ’-phosphorothioate groups (represented herein as a lower case “s”), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g, methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphorami dates), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, a modified intemucleoside linkage or backbone lacks a phosphorus atom. Modified intemucleoside linkages lacking a phosphoms atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. In some embodiments, modified intemucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene- containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.
[0236] In some embodiments, a sense strand of the first or the second RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of the first or the second RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, a sense strand of the first or the second RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, an antisense strand of the first or the second RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.
[0237] In some embodiments, the first or the second RNAi agent sense strand contains at least two phosphorothioate intemucleoside linkages. In some embodiments, the at least two phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, the at least two phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6- 8 from the 5' end of the sense strand. In some embodiments, the first or the second RNAi agent antisense strand contains four phosphorothioate intemucleoside linkages. In some embodiments, the four phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 5' end of the sense strand and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end. In some embodiments, the first or the second RNAi agent contains at least two phosphorothioate intemucleoside linkages in the sense strand and three or four phosphorothioate intemucleoside linkages in the antisense strand.
[0238] In some embodiments, the first or the second RNAi agent contains one or more modified nucleotides and one or more modified intemucleoside linkages. In some embodiments, a 2'-modified nucleoside is combined with modified intemucleoside linkage. In some embodiments, the first and the second RNAi agents disclosed herein comprise any of the modified sequences in Table 3.
Table 3. Exemplary modified sequences for first and second RNAi agents
Figure imgf000104_0001
Figure imgf000105_0001
A = adenosine-3 '-phosphate; C = cytidine-3 '-phosphate; G = guanosine-3 '-phosphate;
U = uridine-3 '-phosphate n = any 2'-0Me modified nucleotide a = 2'-O-methyladenosine-3 '-phosphate as = 2'-O-methyladenosine-3'-phosphorothioate c = 2'-O-methylcytidine-3 '-phosphate cs = 2'-O-methylcytidine-3'-phosphorothioate g = 2'-O-methylguanosine-3'-phosphate gs = 2'-O-methylguanosine-3'-phosphorothioate t = 2'-O-methyl-5-methyluridine-3'-phosphate ts = 2'-O-methyl-5-methyluridine-3'-phosphorothioate u = 2'-O-methyluridine-3 '-phosphate us = 2'-O-methyluridine-3 '-phosphorothioate
Nf = any 2'-fluoro modified nucleotide
Af = 2'-fluoroadenosine-3'-phosphate
Afs = 2'-fluoroadenosine-3'-phosporothioate Cf = 2'-fluorocytidine-3 '-phosphate Cfs = 2'-fluorocytidine-3 '-phosphorothioate
Gf = 2'-fluoroguanosine-3 '-phosphate
Gfs = 2'-fluoroguanosine-3 '-phosphorothioate Tf = 2'-fluoro-5'-methyluridine-3'-phosphate Tfs = 2'-fluoro-5'-methyluridine-3'-phosphorothioate
Uf = 2'-fluorouridine-3 '-phosphate
Ufs = 2'-fluorouridine-3 '-phosphorothioate dN = any 2'-deoxyribonucleotide dT = 2'-deoxythymidine-3 '-phosphate
NUNA = 2',3'-seco nucleotide mimics (unlocked nucleobase analogs) NLNA = locked nucleotide NfANA = 2'-F-Arabino nucleotide
NM = 2'-methoxyethyl nucleotide AM = 2'-methoxyethyladenosine-3'-phosphate AMs = 2'-methoxyethyladenosine-3'-phosphorothioate
TM = 2'-methoxyethylthymidine-3'-phosphate TMs = 2'-methoxyethylthymidine-3'-phosphorothioate
R = ribitol (invdN) = any inverted deoxy ribonucleotide (3 '-3' linked nucleotide) (invAb) = inverted (3'-3' linked) abasic deoxyribonucleotide, see Table 6
(invAb)s = inverted (3'-3' linked) abasic deoxyribonucleotide-5'- phosphorothioate, see Table 6
(invn) = any inverted 2'-OMe nucleotide (3 '-3' linked nucleotide) s = phosphorothioate linkage
[0239] In some embodiments, the first RNAi agent comprises SEQ ID NO:97 and SEQ ID NO: 106. In some embodiments, the first RNAi agent comprises SEQ ID NO: 108 and SEQ ID NO: 106. In some embodiments, the first RNAi agent comprises SEQ ID NO:99 and SEQ ID NO: 107. In some embodiments, the first RNAi agent comprises SEQ ID NO:93 and SEQ ID NO: 102, 103, or 105. In some embodiments, the first RNAi agent comprises SEQ ID NO:94 and SEQ ID NO: 102, 103, or 105. In some embodiments, the first RNAi agent comprises SEQ ID NO:95 and SEQ ID NO: 102, 103, or 105. In some embodiments, the first RNAi agent comprises SEQ ID NO:96 and SEQ ID NO: 106. In some embodiments, the second RNAi agent comprises SEQ ID NO: 101 and SEQ ID NO: 111. In some embodiments, the second RNAi agent comprises SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110.
[0240] In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:97 and SEQ ID NO: 106 and a second RNAi agent comprising SEQ ID NO: 101 and SEQ ID NO: 111. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO: 108 and SEQ ID NO: 106 and a second RNAi agent comprising SEQ ID NO: 101 and SEQ ID NO: 111. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:99 and SEQ ID NO: 107 and a second RNAi agent comprising SEQ ID NO: 101 and SEQ ID NO: 111.
[0241] In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:93 and SEQ ID NO: 102, 103, or 105 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:94 and SEQ ID NO: 102, 103, or 105 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108,
109, or 110. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:95 and SEQ ID NO: 102, 103, or 105 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:96 and SEQ ID NO: 106 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or
110. [0242] In some embodiments, the RNAi component comprises a first and a second RNAi agent in a ratio of about 1 : 1, 2: 1, 3: 1, 4: 1 or 5: 1. In some embodiments, the two HBV RNAi agents are administered in a ratio of about 2:1. Throughout the application, ratio is intended in accordance with its ordinary meaning in the field, e.g., as a weight ratio or as a molar ratio. [0243] In some embodiments, the first and the second RNAi agents are each independently conjugated to (NAG37)s, (NAG31)s, or (NAG25)s, the first RNAi agent comprises an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 103 or SEQ ID NO: 105, the second RNAi agent comprises an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 108 or SEQ ID NO: 110.
[0244] In some embodiments, the first HBV RNAi agent comprises or consists of the duplex of SEQ ID NO: 94 and 103 linked to (NAG37)s shown as a sodium salt having the structure represented by the following:
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
[0245] In some embodiments, the first HBV RNAi agent comprises or consists of the duplex of SEQ ID NO: 94 and 105 linked to (NAG25)s shown as a sodium salt having the structure represented by the following:
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
[0246] In some embodiments, the first HBV RNAi agent comprises or consists of the duplex of SEQ ID NO: 94 and 103 linked to (NAG37)s shown as a free acid having the structure represented by the following:
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
[0247] In some embodiments, the first HBV RNAi agent comprises or consists of the duplex of SEQ ID NO: 100 and 108 linked to (NAG37)s shown as a sodium salt having the structure represented by the following:
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
[0248] In some embodiments, the first HBV RNAi agent comprises or consists of the duplex of SEQ ID NO: 100 and 110 linked to (NAG25)s shown as a sodium salt having the structure represented by the following:
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
[0249] In some embodiments, the first HBV RNAi agent comprises or consists of the duplex of SEQ ID NO: 100 and 108 linked to (NAG37)s shown as a free acid having the structure represented by the following:
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
[0250] In some embodiments, an HBV RNAi agent disclosed herein consists of or comprises the duplex of SEQ ID NO: 112 and 113 linked to (NAG25)s shown as a sodium salt having the structure represented by the following:
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
[0251] In some embodiments, an HBV RNAi agent disclosed herein consists of or comprises the duplex of SEQ ID NO: 114 and 115 linked to (NAG3 l)s shown as a sodium salt having the structure represented by the following:
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
[0252] In certain embodiments of the application, the RNAi component is administered to the subject subcutaneously or orally, more particularly subcutaneously. In certain embodiments of the application, the RNAi component is in a salt form (e.g., in a sodium salt form) or is in a free acid form.
[0253] The combinations described herein can be used in any methods or kits described below.
COMPOSITION [0254] Also provided herein is a composition comprising an RNAi component and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, wherein
(a) the RNAi component comprises
(i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
(ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; and
(b) the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3’-end:
(1) a polynucleotide sequence encoding a first hepatitis B virus (HBV) antigen,
(2) a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and
(3) a polynucleotide sequence encoding a second HBV antigen, wherein the first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
Nucleic Acid Molecule Pharmaceutical Compositions
[0255] The application also relates to compositions, pharmaceutical compositions, immunogenic combinations, and more particularly vaccines, comprising one or more HBV antigens, polynucleotides, and/or vectors encoding one more HBV antigens according to the application. Any of the HBV antigens, polynucleotides (including RNA and DNA), and/or vectors of the application described herein can be used in the compositions, pharmaceutical compositions, immunogenic combinations, and vaccines of the application.
[0256] The application provides, for example, a pharmaceutical composition comprising any nucleic acid molecule, vector, or RNA replicon described herein, together with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is non-toxic and should not interfere with the efficacy of the active ingredient. Pharmaceutically acceptable carriers can include one or more excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings. The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes. For liquid injectable preparations, for example, suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like. For solid oral preparations, for example, powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. For nasal sprays/inhalant mixtures, the aqueous solution/suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives.
[0257] Pharmaceutical compositions of the application can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections. The parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection. Pharmaceutical compositions of the application can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal.
[0258] In a preferred embodiment of the application, pharmaceutical compositions of the application are formulated for parental injection, preferably subcutaneous, intradermal injection, or intramuscular injection, more preferably intramuscular injection.
[0259] In some embodiments of the application in which a pharmaceutical composition or immunogenic combination comprises one or more replicons, administration can be by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection. Intramuscular injection can be combined with electroporation, i.e., application of an electric field to facilitate delivery of the DNA plasmids to cells. As used herein, the term “electroporation” refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane. During in vivo electroporation, electrical fields of appropriate magnitude and duration are applied to cells, inducing a transient state of enhanced cell membrane permeability, thus enabling the cellular uptake of molecules unable to cross cell membranes on their own. Creation of such pores by electroporation facilitates passage of biomolecules, such as plasmids, oligonucleotides, siRNAs, drugs, etc., from one side of a cellular membrane to the other. In vivo electroporation for the delivery of DNA vaccines has been shown to significantly increase plasmid uptake by host cells, while also leading to mild-to-moderate inflammation at the injection site.
[0260] In a typical embodiment, electroporation is combined with intramuscular injection. However, it is also possible to combine electroporation with other forms of parenteral administration, e.g., intradermal injection, subcutaneous injection, etc.
[0261] Administration of a pharmaceutical composition, immunogenic combination or vaccine of the application via electroporation can be accomplished using electroporation devices that can be configured to deliver to a desired tissue of a mammal a pulse of energy effective to cause reversible pores to form in cell membranes. The electroporation device can include an electroporation component and an electrode assembly or handle assembly. The electroporation component can include one or more of the following components of electroporation devices: controller, current waveform generator, impedance tester, waveform logger, input element, status reporting element, communication port, memory component, power source, and power switch. Electroporation can be accomplished using an in vivo electroporation device. Examples of electroporation devices and electroporation methods that can facilitate delivery of compositions and immunogenic combinations of the application, include CELLECTRA® (Inovio Pharmaceuticals, Blue Bell, PA), Eigen electroporator (Inovio Pharmaceuticals, Inc.) Tri-Grid™ delivery system (Ichor Medical Systems, Inc., San Diego, CA 92121) and those described in U.S. Patent No. 7,664,545, U.S. Patent No. 8,209,006, U.S. Patent No. 9,452,285, U.S. Patent No. 5,273,525, U.S. Patent No. 6,110,161, U.S. Patent No. 6,261,281, U.S. Patent No. 6,958,060, and U.S. Patent No. 6,939,862, U.S. Patent No. 7,328,064, U.S. Patent No. 6,041,252, U.S. Patent No. 5,873,849, U.S. Patent No. 6,278,895, U.S. Patent No. 6,319,901, U.S. Patent No. 6,912,417, U.S. Patent No. 8,187,249, U.S. Patent No. 9,364,664, U.S. Patent No. 9,802,035, U.S. Patent No. 6,117,660, and International Patent Application Publication WO2017172838, all of which are herein incorporated by reference in their entireties. Other examples of in vivo electroporation devices are described in International Patent Application entitled “Method and Apparatus for the Delivery of Hepatitis B Virus (HBV) Vaccines,” filed on the same day as this application with the Attorney Docket Number 688097-405WO, the contents of which are hereby incorporated by reference in their entireties. Also contemplated by the application for delivery of the compositions and immunogenic combinations of the application are use of a pulsed electric field, for instance as described in, e.g., U.S. Patent No. 6,697,669, which is herein incorporated by reference in its entirety.
[0262] In other embodiments of the application in which a pharmaceutical composition or immunogenic combination comprises one or more DNA plasmids, the method of administration is transdermal. Transdermal administration can be combined with epidermal skin abrasion to facilitate delivery of the DNA plasmids to cells. For example, a dermatological patch can be used for epidermal skin abrasion. Upon removal of the dermatological patch, the composition or immunogenic combination can be deposited on the abraised skin.
[0263] Methods of delivery are not limited to the above described embodiments, and any means for intracellular delivery can be used. Other methods of intracellular delivery contemplated by the methods of the application include, but are not limited to, liposome encapsulation, lipoplexes, nanoparticles, etc. For example, an RNA replicon of the application can be formulated in an immunogenic composition that comprises one or more lipid molecules, preferably positively charged lipid molecules. In some embodiments, an RNA replicon of the disclosure can be formulated using one or more liposomes, lipoplexes, and/or lipid nanoparticles. In some embodiments, liposome or lipid nanoparticle formulations described herein can comprise a poly cationic composition. In some embodiments, the formulations comprising a poly cationic composition can be used for the delivery of the RNA replicon described herein in vivo and/or ex vitro.
[0264] According to embodiments of the application, pharmaceutical compositions for administration will typically comprise a buffered solution in a pharmaceutically acceptable carrier, e.g., an aqueous carrier such as buffered saline and the like, e.g., phosphate buffered saline (PBS). The compositions and immunogenic combinations can also contain pharmaceutically acceptable substances as required to approximate physiological conditions such as pH adjusting and buffering agents. For example, a pharmaceutical composition of the application comprising plasmid DNA can contain phosphate buffered saline (PBS) as the pharmaceutically acceptable carrier. The plasmid DNA can be present in a concentration of, e.g., 0.5 mg/mL to 5 mg/mL, such as 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, or 5 mg/mL, preferably at 1 mg/mL.
[0265] In some embodiments, a pharmaceutical composition of the application comprising an RNA replicon can be administered in a concentration of, e.g., about 20 pg/mL to about 200 pg/mL, such as 20 pg/mL. 30 pg/mL, 40 pg/mL, 50 pg/mL, 60 pg/mL, 70 pg/mL, 80 pg/mL, 90 pg/mL, 100 pg/mL, 110 pg/mL, 120 pg/mL, 130 pg/mL, 140 pg/mL, 150 pg/mL, 160 pg/mL, 170 pg/mL, 180 pg/mL, 190 pg/mL, or 200 pg/mL. In some embodiments, a pharmaceutical composition of the application comprising an RNA replicon can be administered in a concentration below 20 pg/mL. In some embodiments, a pharmaceutical composition of the application comprising an RNA replicon can be administered in a concentration above 200 pg/mL.
[0266] Pharmaceutical compositions of the application can be formulated as a vaccine (also referred to as an “immunogenic composition”) according to methods well known in the art. Such compositions can include adjuvants to enhance immune responses. The optimal ratios of each component in the formulation can be determined by techniques well known to those skilled in the art in view of the present disclosure.
[0267] In a particular embodiment of the application, a pharmaceutical composition, composition, or immunogenic combination is a DNA vaccine. DNA vaccines typically comprise bacterial plasmids containing a polynucleotide encoding an antigen of interest under control of a strong eukaryotic promoter. Once the plasmids are delivered to the cell cytoplasm of the host, the encoded antigen is produced and processed endogenously. The resulting antigen typically induces both humoral and cell-medicated immune responses. DNA vaccines are advantageous at least because they offer improved safety, are temperature stable, can be easily adapted to express antigenic variants, and are simple to produce. Any of the DNA plasmids of the application can be used to prepare such a DNA vaccine.
[0268] In other particular embodiments of the application, a pharmaceutical composition, composition, or immunogenic combination is an RNA vaccine. RNA vaccines typically comprise at least one single-stranded RNA molecule encoding an antigen of interest, e.g., HBV antigen. Once the RNA is delivered to the cell cytoplasm of the host, the encoded antigen is produced and processed endogenously, inducing both humoral and cell-mediated immune responses, similar to a DNA vaccine. The RNA sequence can be codon optimized to improve translation efficiency. The RNA molecule can be modified by any method known in the art in view of the present disclosure to enhance stability and/or translation, such by adding a polyA tail, e.g., of at least 30 adenosine residues; and/or capping the 5-end with a modified ribonucleotide, e.g., 7-methylguanosine cap, which can be incorporated during RNA synthesis or enzymatically engineered after RNA transcription. An RNA vaccine can also be self-replicating RNA vaccine developed from an alphavirus expression vector. Selfreplicating RNA vaccines comprise a replicase RNA molecule derived from a virus belonging to the alphavirus family with a subgenomic promoter that controls replication of the HBV antigen RNA followed by an artificial poly A tail located downstream of the replicase.
[0269] In certain embodiments, an adjuvant is included in a pharmaceutical composition of the application or co-administered with a pharmaceutical composition of the application. Use of an adjuvant is optional and can further enhance immune responses when the composition is used for vaccination purposes. Adjuvants suitable for co-administration or inclusion in compositions in accordance with the application should preferably be ones that are potentially safe, well tolerated, and effective in humans. An adjuvant can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti- PD1, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/or TLR8 agonists), RIG-1 agonists, IL- 15 superagonists (Aitor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant, and IL-7-hyFc.
[0270] The application also provides methods of making pharmaceutical compositions and immunogenic combinations of the application. A method of producing a pharmaceutical composition or immunogenic combination comprises mixing an isolated polynucleotide encoding an HBV antigen, vector, and/or polypeptide of the application with one or more pharmaceutically acceptable carriers. One of ordinary skill in the art will be familiar with conventional techniques used to prepare such compositions.
Liposomes and Lipid Nanoparticles
[0271] In certain embodiments of the application, the method of administration is a lipid composition, such as a lipid nanoparticle (LNP) or a liposome. Lipid compositions, preferably lipid nanoparticles or liposomes, that can be used to deliver a therapeutic product (such as one or more nucleic acid molecules of the invention), include, but are not limited to, liposomes or lipid vesicles, wherein an aqueous volume is encapsulated by amphipathic lipid bilayers, or wherein the lipids coat an interior that comprises a therapeutic product; or lipid aggregates or micelles, wherein the lipid-encapsulated therapeutic product is contained within a relatively disordered lipid mixture.
[0272] The lipid composition can provide the therapeutic product (such as one or more nucleic acid molecules of the invention) with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the therapeutic product is fully encapsulated in the lipid particle (e.g., to form an LNP). [0273] Lipid compositions of this invention can comprise one or more lipids selected from cationic lipids, anionic lipids, zwitterionic lipids, neutral lipids, steroids, polymer conjugated lipids, phospholipids, glycolipids, and any combination of the foregoing. The lipids can be saturated or unsaturated. A mixture can comprise both saturated and unsaturated lipids. The lipid compositions can be substantially free of liposomes or can contain liposomes. The use of at least one unsaturated lipid for preparing liposomes is preferred. If an unsaturated lipid has two tails, both tails can be unsaturated, or it can have one saturated tail and one unsaturated tail. The lipids and nucleic acid molecules can be mixed and configured in any suitable structures.
[0274] In particular embodiments, the lipid compositions comprise a cationic lipid to encapsulate and/or enhance the delivery of a nucleic acid molecule, such as a DNA or RNA molecule of the invention, into the target cell. The cationic lipid can be any lipid species that carries a net positive charge at a selected pH, such as physiological pH. Without wishing to be bound by the theory, the cationic lipids, such as ionizable amino lipids, promote selfassembly of the components into macromolecular nanoparticles that encapsulate the nucleic acid (DNA and/or RNA). The nucleic acid-containing nanoparticles are efficiently taken up into target cells by endocytosis. Once inside the endosome, the positively-charged lipid nanoparticles interact with the negatively-charged endosome membrane, causing disruption of the compartment and release of the nucleic acid molecules into the cytoplasm, where the nucleic acid molecules can be expressed.
[0275] Several cationic lipids have been described in the literature, many of which are commercially available. For example, suitable cationic lipids for use in the compositions and methods of the invention include l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2- DiLinoleyloxy-,N,N-dimethylaminopropane (DLinDMA), and 1,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA). The pKa of formulated cationic lipids is correlated with the effectiveness of lipid particles for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). The preferred range of pKa is ~5 to ~7.
[0276] In one embodiment, the cationic lipid is a compound of Formula (I):
Figure imgf000147_0001
wherein R1 is a substituted alkyl consisting of 10 to 31 carbons, R2 is a linear alkyl, alkenyl or alkynyl consisting of 2 to 20 carbons, R3 is a linear or branched alkane consisting of 1 to 6 carbons, R4 and R5 are the same or different, each a hydrogen or a linear or branched alkyl consisting of 1 to 6 carbons; L1 and L2 are the same or different, each a linear alkane of 1 to 20 carbons or a linear alkene of 2 to 20 carbons, and X1 is S or O; or a salt or solvate thereof. Exemplary compounds of formula (I), their synthesis and uses thereof are described in US2018/0169268, all of which are herein incorporated by reference.
[0277] In another embodiment, the cationic lipid is a compound of formula (II):
Figure imgf000147_0002
wherein R1 is a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons; L1 is linear alkane of 1 to 15 carbons; R2 is a linear alkyl or alkenyl of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons or a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons; L2is a linear alkane of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons; X is O or S; R3 is a linear alkane of 1, 2, 3, 4, 5, or 6 carbons; and R4 and R5 are the same or different, each a linear or branched, noncyclic alkyl of 1, 2, 3, 4, 5, or 6 carbons; or a pharmaceutically acceptable salt or solvate thereof. Exemplary compounds of formula (II), their synthesis and uses thereof are described in US2018/0170866, all of which are herein incorporated by reference.
[0278] In another embodiment, the cationic lipid is a compound of formula (III), (IV) or (V):
Figure imgf000148_0001
wherein R comprises a biologically active molecule, and L1, L2, and L3 independently for each occurrence comprise a ligand selected from the group consisting of a carbohydrate, a polypeptide, or a lipophile; a pharmaceutically acceptable salt thereof; or a pharmaceutical composition thereof. Exemplary compounds of formula (III), (IV) and (V), their synthesis and uses thereof are described in US2017/0028074, all of which are herein incorporated by reference.
[0279] In another embodiment, the cationic lipid is a compound of formula (VI):
Figure imgf000148_0002
wherein X is a linear or branched alkylene or alkenylene, monocyclic, bicyclic, or tricyclic arene or heteroarene; Y is a bond, an ethene, or an unsubstituted or substituted aromatic or heteroaromatic ring; Z is S or O; L is a linear or branched alkylene of 1 to 6 carbons; R3 and R4 are independently a linear or branched alkyl of 1 to 6 carbons; R1 and R2 are independently a linear or branched alkyl or alkenyl of 1 to 20 carbons; r is 0 to 6; and m, n, p, and q are independently 1 to 18; wherein when n=q, m=p, and R1=R2, then X and Y differ; wherein when X=Y, n=q, m=p, then R1 and R2 differ; wherein when X=Y, n=q, and R1=R2, then m and p differ; and wherein when X=Y, m=p, and R1=R2, then n and q differ; or a pharmaceutically acceptable salt thereof. Exemplary compounds of formula (VI), their synthesis and uses thereof are described in US2017/0190661, all of which are herein incorporated by reference.
[0280] In another embodiment, the cationic lipid is a compound of formula (VII):
Figure imgf000149_0001
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G1 or G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , — C(=O) — , — O — , — S(O)y , — S— S— , — C(=O)S— , SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— , — N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra)— or — N(Ra)C(=O)O— , and the other of G1 or G2 is, at each occurrence, — O(C=O)— , — (C=O)O— , — C(=O)— , — O— , — S(O)y , — S— S‘, — C(=O)S— , — SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— , — N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra) — or — N(Ra)C(=O)O — or a direct bond; L is, at each occurrence, ~O(C=O) — , wherein ~ represents a covalent bond to X; X is CRa; Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; Ra is, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxy carbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl; R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; R1 and R2 have, at each occurrence, the following structure, respectively:
Figure imgf000149_0002
a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2 are, at each occurrence, independently 0 or 1; c1 and c2 are, at each occurrence, independently an integer from 5 to 10; d1 and d2 are, at each occurrence, independently an integer from 5 to 10; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxy carbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
[0281] In another embodiment, the cationic lipid is a compound of formula (VIII):
Figure imgf000150_0001
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G1 or G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , — C(=O) — , — O — , — S(O)y , — S— S— , — C(=O)S— , SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— , — N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra)— or — N(Ra)C(=O)O— , and the other of G1 or G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , — C(=O) — , — O — , — S(O)y , — S— S— , — C(=O)S— , — SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— , — N(Ra)C(=O)N(Ra) — , — OC(=O)N(Ra) — or — N(Ra)C(=O)O — or a direct bond; L is, at each occurrence, ~O(C=O) — , wherein ~ represents a covalent bond to X; X is CRa; Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; Rais, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxy carbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl; R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; R1 and R2 have, at each occurrence, the following structure, respectively:
Figure imgf000150_0002
R' is, at each occurrence, independently H or C1-C12 alkyl; a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2 are, at each occurrence, independently 0 or 1; c1 and c2 are, at each occurrence, independently an integer from 2 to 12; d1 and d2 are, at each occurrence, independently an integer from 2 to 12; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein a1, a2, c1, c2, d1 and d2 are selected such that the sum of a1+c1+d1 is an integer from 18 to 30, and the sum of a2+c2+d2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxy carbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
[0282] Exemplary compounds of formula (VII) and (VIII), their synthesis and uses thereof are described in US20190022247, all of which are herein incorporated by reference. [0283] Additional cationic lipids that can be used in compositions of the application include, but are not limited to, those described in W02019/036030, W02019/036028, WO2019/036008, WO2019/036000, US2016/0376224, US2017/0119904, W02018/200943 and WO2018/191657, the relevant contents on the lipids, their synthesis and uses are herein incorporated by reference in their entireties.
[0284] The lipid nanoparticles can be prepared by including multi-component lipid mixtures of varying ratios employing one or more cationic lipids, non-cationic lipids and polyethylene glycol (PEG) - modified, or pegylated, lipids, i.e. the lipid is modified by covalent attachment of a polyethylene glycol. PEG provides the liposomes with a coat which can confer favorable pharmacokinetic characteristics e.g. it can increase stability and prevent non-specific adsorption of the liposomes. In certain embodiments, the PEG has an average molecular mass of 1 kDa to 12 kDa, such as 1 , 2, 3. 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 kDa. For example, it was reported that the length of the PEG can affect in vivo expression of encapsulated RNA, and that PEG with a molecular weight below 1 kDa (e.g. 500 or 750 Da) does not form stable liposomes. See, e.g., US2014/0255472, the relevant content of which is incorporated herein by reference.
[0285] The lipid formulations can include anionic lipids. The anionic lipids can be any lipid species that carries a net negative charge at a selected pH, such as physiological pH. The anionic lipids, when combined with cationic lipids, are used to reduce the overall surface charge of LNPs and liposomes and to introduce pH-dependent disruption of the LNP or liposome bilayer structure, facilitating nucleotide release. Several anionic lipids have been described in the literature, many of which are commercially available. For example, suitable anionic lipids for use in the compositions and methods of the invention include 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, and palmitoyloleyolphosphatidylglycerol (POPG).
[0286] The lipid formulations can also include a lipid bilayer stabilizing component. Bilayer stabilizing components can be used to inhibit aggregation of LNPs, but bilayer stabilizing components are not limited to this function. For example, conjugated lipids such as PEG-lipid conjugates and cationic-polymer-lipid conjugates can be used to inhibit the aggregation of LNPs or liposomes. By controlling the composition and concentration of the bilayer stabilizing component, one can control the rate at which the bilayer stabilizing component exchanges out of the liposome and, in turn, the rate at which the liposome becomes fusogenic. The term “fusogenic” refers to the ability of a liposome or other drug delivery system to fuse with membranes of a cell. For instance, when a polyethyleneglycolphosphatidylethanolamine conjugate or a polyethyleneglycol-ceramide conjugate is used as the bilayer stabilizing component, the rate at which the liposome becomes fusogenic can be varied, for example, by varying the concentration of the bilayer stabilizing component, by varying the molecular weight of the polyethyleneglycol, or by varying the chain length and degree of saturation of the acyl chain groups on the phosphatidylethanolamine or the ceramide. In addition, other variables including, for example, pH, temperature, ionic strength, etc. can be used to vary and/or control the rate at which the liposome becomes fusogenic. Other methods which can be used to control the rate at which the liposome becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure.
[0287] LNPs and liposomes can be prepared using methods known in the art in view of the present disclosure. For example, the LNPs can be prepared using ethanol injection or dilution, thin film hydration, freeze-thaw, French press or membrane extrusion, diafiltration, sonication, detergent dialysis, ether infusion, and reverse phase evaporation. One useful method of preparing liposomes involves mixing (i) an ethanolic solution of the lipids (ii) an aqueous solution of the nucleic acid and (iii) buffer, followed by mixing, equilibration, dilution and purification. Preferred liposomes of the invention, e.g. liposomes with a preferred diameter, are obtainable by this mixing process. To obtain liposomes with the desired diameter(s), mixing can be performed using a process in which two feed streams of aqueous nucleic acid solution are combined in a single mixing zone with one stream of an ethanolic lipid solution, all at the same flow rate e.g. in a microfluidic channel as described below. Further examples, compositions, and methods to create liposomes are described in US 2014/0255472, which is hereby incorporated by reference in its entirety.
[0288] Some examples of lipids, lipid compositions, and methods to create lipid carriers for delivering active nucleic acid molecules, such as those of this invention, are described in: US2017/0190661, US2006/0008910, US2015/0064242, US2005/0064595, WO/2019/036030, US2019/0022247, WO/2019/036028, WO/2019/036008, WO/2019/036000, US2016/0376224, US2017/0119904, WO/2018/200943,
WO/2018/191657, US2014/0255472, and US2013/0195968, the relevant content of each of which is hereby incorporated by reference in its entirety.
[0289] Liposomes are microscopic vesicles including at least one concentric lipid bilayer. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex. In particular embodiments, liposomes provide a lipid composition that is an outer layer surrounding a porous nanoparticle.
[0290] Liposomes can be neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM) and other type of bipolar lipids including dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or more double C=C bonds. Examples of lipids capable of producing a stable liposome, alone, or in combination with other lipid components are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebro sides, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4- (N-maleimido-methyl)cyclohexane-l -carboxylate (DOPE-mal). Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, DDAB, dioctadecyl dimethyl ammonium chloride (DODAC), 1 ,2-dimyristoyl-3-trimethylammonium propane (DMTAP), DOTAP, DOTMA, DC-Choi, phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol, DOPG, and dicetylphosphate. In particular embodiments, lipids used to create liposomes disclosed herein include cholesterol, hydrogenated soy phosphatidylcholine (HSPC) and, the derivatized vesicle-forming lipid PEG-DSPE. [0291] Methods of forming liposomes are described in, for example, US Patent Nos. 4,229,360; 4,224,179; 4,241,046; 4,737,323; 4,078,052; 4,235,871; 4,501,728; and 4,837,028, as well as in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and Hope et al., Chem. Phys. Lip. 40:89 (1986).
Prime/Boost Immunization
[0292] Embodiments of the application also contemplate administering an effective amount of a pharmaceutical composition or immunogenic combination to a subject, and subsequently administering another dose of an effective amount of a pharmaceutical composition or immunogenic combination to the same subject, in a so-called prime-boost regimen. Thus, in an embodiment, a pharmaceutical composition or immunogenic combination of the application is a primer vaccine used for priming an immune response. In another embodiment, a pharmaceutical composition or immunogenic combination of the application is a booster vaccine used for boosting an immune response. The priming and boosting vaccines of the application can be used in the methods of the application described herein. This general concept of a prime-boost regimen is well known to the skilled person in the vaccine field. Any of the pharmaceutical compositions and immunogenic combinations of the application described herein can be used as priming and/or boosting vaccines for priming and/or boosting an immune response against HBV. Preferably, methods for vaccinating a subject comprise administering to the subject a pharmaceutical composition comprising a nucleic acid molecule, vector, or RNA replicon of the application, and administering to the subject a second composition comprising a nucleic acid molecule encoding at least one identical HBV antigen as a prime-boost regimen.
[0293] In some embodiments of the application, a pharmaceutical composition or immunogenic combination of the application can be administered for priming immunization. The pharmaceutical composition or immunogenic combination can be re-administered for boosting immunization. Further booster administrations of the pharmaceutical composition or vaccine combination can optionally be added to the regimen, as needed. An adjuvant can be present in a pharmaceutical composition of the application used for boosting immunization, present in a separate composition to be administered together with the pharmaceutical composition or immunogenic combination of the application for the boosting immunization, or administered on its own as the boosting immunization. In those embodiments in which an adjuvant is included in the regimen, the adjuvant is preferably used for boosting immunization.
[0294] An illustrative and non-limiting example of a prime-boost regimen includes administering a single dose of an effective amount of a pharmaceutical composition or immunogenic combination of the application to a subject to prime the immune response; and subsequently administering another dose of an effective amount of a pharmaceutical composition or immunogenic combination of the application to boost the immune response, wherein the boosting immunization is first administered about two, four, six, or eight weeks, preferably four weeks after the priming immunization is initially administered. Optionally, about 10, 12, or 14 weeks, preferably 12 weeks, after the priming immunization is initially administered, a further boosting immunization of the pharmaceutical composition or immunogenic combination, or other adjuvant, is administered.
[0295] The antigens in the priming and boosting compositions need not to be identical but should share antigens or be substantially similar to each other. In certain embodiments, the vector of priming composition is a replicon, and the vector of the boosting composition is different from the priming composition, e.g., an adenovirus vector, Modified Vaccinia Ankara (MV A) vector, DNA, or protein. In certain embodiments, the vector of the boosting composition is a replicon, and the vector of the priming composition is different from the boosting composition, e.g., an adenovirus vector, Modified Vaccinia Ankara (MV A) vector, DNA, or protein. The priming and boosting compositions of the invention can each comprise one, two, three, four, or up to five doses.
RNAi Agent Pharmaceutical Compositions
[0296] In another aspect, described herein are methods for therapeutic and/or prophylactic treatment of a Hepatitis B Virus (HBV) infection in a subject in need thereof, methods for enhancing an immune response in a subject with a HBV infection; methods for decreasing viral replication in a subject with a HBV infection; methods for decreasing expression of one or more HBV polypeptide(s) in a subject in need thereof; methods for modulating Hepatitis B viral (HBV) capsid assembly or disassembly in a subject with a HBV infection; and/or methods for targeted killing of hepatocytes containing integrated viral DNA or extrachromosomal viral DNA in a subject with a HBV infection comprising administering a pharmaceutical composition comprising one or more HBV RNAi agents that can be administered in a number of ways depending upon whether local or systemic treatment is desired. Administration can be, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, subdermal (e.g., via an implanted device), and intraparenchymal administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.
[0297] In another aspect, methods described herein comprise one or more HBV RNAi agents, wherein the one or more HBV agents are prepared as pharmaceutical compositions or formulations. In some embodiments, pharmaceutical compositions include at least one HBV RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of the target mRNA in a target cell, a group of cells, a tissue, or an organism. The pharmaceutical compositions can be used to treat a subject having a disease or disorder that would benefit from reduction in the level of the target mRNA, or inhibition in expression of the target gene. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene. In one embodiment, the method includes administering an HBV RNAi agent linked to a targeting ligand as described herein, to a subject to be treated. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and/or delivery polymers) are added to the pharmaceutical compositions including an HBV RNAi agent, thereby forming a pharmaceutical formulation suitable for in vivo delivery to a human.
[0298] The pharmaceutical compositions that include an HBV RNAi agent and methods disclosed herein may decrease the level of the target mRNA in a cell, group of cells, group of cells, tissue, or subject, including: administering to the subject a therapeutically effective amount of a herein described HBV RNAi agent, thereby inhibiting the expression of a target mRNA in the subject.
[0299] In some embodiments, the described pharmaceutical compositions including an HBV RNAi agent are used for treating or managing clinical presentations associated with HBV infection. In some embodiments, a therapeutically or prophylactically effective amount of one or more of pharmaceutical compositions is administered to a subject in need of such treatment, prevention or management. In some embodiments, administration of any of the disclosed HBV RNAi agents can be used to decrease the number, severity, and/or frequency of symptoms of a disease in a subject.
[0300] The described pharmaceutical compositions including an HBV RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder that would benefit from reduction or inhibition in expression of HBV mRNA. In some embodiments, the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions including an HBV RNAi agent thereby treating the symptom. In other embodiments, the subject is administered a prophylactically effective amount of one or more HBV RNAi agents, thereby preventing the at least one symptom.
[0301] The route of administration is the path by which an HBV RNAi agent is brought into contact with the body. In general, methods of administering drugs and nucleic acids for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein. The HBV RNAi agents disclosed herein can be administered via any suitable route in a preparation appropriately tailored to the particular route. Thus, herein described pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, intraarticularly, or intraperitoneally. In some embodiments, there herein described pharmaceutical compositions via subcutaneous injection.
[0302] The pharmaceutical compositions including an HBV RNAi agent described herein can be delivered to a cell, group of cells, tumor, tissue, or subject using oligonucleotide delivery technologies known in the art. In general, any suitable method recognized in the art for delivering a nucleic acid molecule in vitro or in vivo) can be adapted for use with a composition described herein. For example, delivery can be by local administration, (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection.
[0303] Accordingly, in some embodiments, the herein described pharmaceutical compositions may comprise one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions described herein can be formulated for administration to a subject.
[0304] As used herein, a pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical ingredient (API, therapeutic product, e.g., HBV RNAi agent) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.
[0305] Excipients include, but are not limited to: absorption enhancers, anti -adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents. [0306] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[0307] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. [0308] Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems can also be used to present the drug for both intra-articular and ophthalmic administration.
[0309] The active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polygly colic acid, collagen, poly orthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
[0310] The HBV RNAi agents can be formulated in compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
[0311] A pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to, anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.). It is also envisioned that cells, tissues or isolated organs that express or comprise the herein defined RNAi agents may be used as “pharmaceutical compositions.” As used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an RNAi agent to produce a pharmacological, therapeutic or preventive result.
[0312] Generally, an effective amount of an active compound will be in the range of from about 0.1 to about 100 mg/kg of body weight/day, e.g., from about 1.0 to about 50 mg/kg of body weight/day. In some embodiments, an effective amount of an active compound will be in the range of from about 0.25 to about 5 mg/kg of body weight per dose. In some embodiments, an effective amount of an active compound will be in the range of about 40- 1000 mg, more particularly about 25-400 mg per 1-18 weeks or 1-6 months. In some embodiments, an effective amount of an active compound will be in the range of 50-250 mg per 4 weeks (Q4W) or per one month (Q1M). The terms “Q4W” and “Q1M”, “per 4 weeks” and “per one month”, and variations thereof, can be used interchangeably throughout this application. In some embodiments, an effective amount of an active compound will be in the range of 50-250 mg per 8 weeks (Q8W) or per two months (Q2M). In some embodiments, an effective amount of an active compound will be in the range of 50-250 mg per 12 weeks (Q12W) or per three months (Q3M). In some embodiments, an effective amount of an active ingredient will be in the range of from about 0.5 to about 3 mg/kg of body weight per dose. In some embodiments, an effective amount of an active ingredient will be in the range of from about 25-400 mg per dose. In some embodiments, an effective amount of an active ingredient will be in the range of from about 50-125 mg per dose. In some embodiments, an effective amount of an active ingredient will be about 100 mg or 200 mg per dose. The amount administered will also likely depend on such variables as the overall health status of the patient, the relative biological efficacy of the compound delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. Also, it is to be understood that the initial dosage administered can be increased beyond the above upper level in order to rapidly achieve the desired blood-level or tissue level, or the initial dosage can be smaller than the optimum.
[0313] In some embodiments, an effective amount of the RNAi component is in the range of about 25-600 mg per dose. In some embodiments, an effective amount of the RNAi component is in the range of about 25-50 mg, about 50-75 mg, about 75-100 mg, about 100- 150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, about 300-400 mg, about 400-500 mg or about 500-600 mg per dose. In some embodiments, an effective amount of the RNAi component is about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg or about 600 mg per dose. In some embodiments, an effective amount of the RNAi component is about 25 mg, about 35mg, about 40 mg, about 50 mg, about 100 mg or about 200 mg per dose.
[0314] The one or more (e.g. , at least two) HBV RNAi agents described herein can be formulated into one single composition or separate individual compositions. In some embodiments, the HBV RNAi agents in separate individual compositions can be formulated with the same or different excipients and carriers. In some embodiments, the HBV RNAi agents in separate individual compositions agents can be administered through same or different administration routes. In some embodiments, the HBV RNAi agents are administered subcutaneously.
[0315] For treatment of disease or for formation of a medicament or composition for treatment of a disease, the pharmaceutical compositions described herein including an HBV RNAi agent can be combined with an excipient or with a second therapeutic agent or treatment including, but not limited to: a second or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, and/or a vaccine.
[0316] The described HBV RNAi agents, when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensers. The pharmaceutical compositions described herein may be packaged in pre-filled syringes or vials.
[0317] In some embodiments, the composition comprises an effective amount of an RNAi component in the range of about 40-1000 mg and an effective amount of the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence in the range of about 10-300 pg per dose.
KIT
[0318] Provided herein is a kit comprising an effective amount of an RNAi component and an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens or a pharmaceutically acceptable salt thereof.
[0319] In another aspect, the kit further comprises a package insert including, without limitation, appropriate instructions for preparation and administration of the formulation, side effects of the formulation, and any other relevant information. The instructions can be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, optical disc or directions to internet-based instructions.
[0320] In another aspect, kits for treating an individual who suffers from or is susceptible to the conditions described herein are provided, comprising a first container comprising a dosage amount of a composition or formulation as disclosed herein, and a package insert for use. The container can be any of those known in the art and appropriate for storage and delivery of intravenous formulation. In certain embodiments, the kit further comprises a second container comprising a pharmaceutically acceptable carrier, diluent, adjuvant, etc. for preparation of the formulation to be administered to the individual.
[0321] In some embodiments, the kit comprises one or more doses of the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens in the range of about 10 |ixg to about 300 |ixg per dose, more particularly about 20 |LLg to about 200 |ixg per dose. In some embodiments, an effective amount of the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens can be administered to the subject in a dose below 10 |LLg- In some embodiments, an effective amount of the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antignes can be administered to the subject in a dose above 300 pg.
[0322] In some embodiments, the kit comprises one or more doses of the RNAi component in the range of about 25-600 mg per dose. In some embodiments, the kit comprises one or more doses of the RNAi component in the range of about 25-50 mg, about 50-75 mg, about 75-100 mg, about 100-150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, about 300-400 mg, about 400-500 mg or about 500-600 mg per dose. In some embodiments, the kit comprises one or more doses of the RNAi component of about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg or about 600 mg per dose. In some embodiments, the kit comprises one or more doses of the RNAi component of about 25 mg, about 35mg, about 40 mg, about 50 mg, about 100 mg or about 200 mg per dose.
[0323] In some embodiments, the kit contains an RNAi component useful for the invention, such as those described herein, for once monthly (or every four weeks) administration to a subject in a dose of about 40-1000 mg, more particularly about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg; more particularly 200 mg; and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, such as those described herein, for administration to a subject in a dose of about 10-300 pg, more particularly 20-200 pg, wherein said dose is administered up to five (5) times. In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered in up to five (5) doses, up to three (3) doses, or in two (2) doses. In certain embodiments, the first dose is a priming dose, and the second dose is a boosting dose.
[0324] In some embodiments, the kit further comprises instructions for using the RNAi component and the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens contained therein for administration to treat a subject with an HBV infection, in particular, a subject having a chronic HBV infection. In some embodiments, the kit further comprises instructions for using the RNAi component and the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens contained therein for administration to a subject with an HBV infection to enhance the immune response, to decrease viral replication, to decrease expression of one or more HBV polypeptide(s), and/or to increase the targeted killing of hepatocytes comprising integrated viral DNA and/or extrachromosomal viral DNA.
[0325] In another aspect, kits may also be provided that contain sufficient dosages of the compositions described herein (including pharmaceutical compositions thereof) to provide effective treatment for an individual for an extended period, such as 1-3 days, 1-5 days, a week, 2 weeks, 3, weeks, 4 weeks, 6 weeks, 8 weeks, 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles or more. In some embodiments, one cycle of treatment is about 1-24 months, about 1-3 months, about 3-6 months, about 6-9 months, about 9-12 months, about 12-18 months, about 18-21 months or about 21-24 months. In some embodiments, one cycle of treatment is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 21 months or about 24 months.
[0326] In some embodiments, the kits can also include multiple doses and may be packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies. In certain embodiments the kits may include a dosage amount of at least one composition as disclosed herein.
METHOD
[0327] Also provided herein is a method for treating a Hepatitis B viral (HBV) infection in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of an RNAi component and a nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens, such as those described herein. Also provided herein is a method for enhancing an immune response in a subject with an HBV infection, wherein the method comprises administering to the subject an effective amount of an RNAi component and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, such as those described herein. Also provided herein is a method for decreasing viral replication in a subject with a HBV infection, wherein the method comprises administering to the subject an effective amount of an RNAi component and a nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens, such as those described herein. Also provided is a method for decreasing the expression of one or more HBV polypeptide(s), more particularly of one or more polypeptide(s) selected from HBsAg and HBeAg, in a subject in need thereof, such as those described herein. Also provided herein is a method for increasing the targeted killing of hepatocytes comprising integrated viral DNA and/or extrachromosomal viral DNA in a subject with an HBV infection, the method comprises administering to the subject an effective amount of an RNAi component and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, such as those described herein.
[0328] In some embodiments, the HBV infection is a chronic HBV infection. In some embodiments, administering the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens decreases the immune tolerogenicity of the subject to HBV, more particularly the immune tolerogenicity of the liver of the subject to HBV. Decreasing the immune tolerogenicity of the subject to HBV or the immune tolerogenicity of the liver of the subject to HBV infection is characterized by the reactivation of the immune system in the subject with the chronic HBV infection. Reactivation of the immune system can occur after administration of the effective amount the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, which serves to decrease or reduce the level of HBsAg in the subject, which allows for the reactivation of the immune system.
[0329] In some embodiments, administering the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens at least until the subject meets at least one of, at least two of, at least three of, at least four of, or the five following features: (i) a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 lU/mL, more particularly is lower than 15 lU/mL, more particularly is lower than 10 lU/mL; (ii) a serum ALT concentration lower than 3 times the upper normal limit, or lower than 129 U/L if the subject is a male subject, or lower than 108 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 120 U/L if the subject is a male subject or lower than 105 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 90 U/L if the subject is a male subject or lower than 57 U/L if the subject is a female subject; (iii) a HBeAg-negative serum, (iv) a serum HBsAg level of 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly of 10 lU/mL or lower; and (v) HBs seroconversion. In some embodiments, the at least one of, at least two of, at least three of, at least four of, or five of features (i), (ii), (iii), (iv), and (v) are still met six (6) months after the end of treatment. [0330] In some embodiments, administering the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens at least until the subject meets at least one of, at least two of, at least three of, or four of the following features: (i) a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 lU/mL, more particularly is lower than 15 lU/mL, more particularly is lower than 10 lU/mL; (ii) a serum ALT concentration lower than 3 times the upper normal limit, or lower than 129 U/L if the subject is a male subject, or lower than 108 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 120 U/L if the subject is a male subject or lower than 105 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 90 U/L if the subject is a male subject or lower than 57 U/L if the subject is a female subject; (iii) a HBeAg-negative serum; and (iv) a serum HBsAg level of 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly of 10 lU/mL or lower. In some embodiments, the at least one of, at least two of, at least three of, or four of features (i), (ii), (iii), and (iv) are still met six (6) months after the end of treatment.
[0331] In some embodiments, the method comprises administering the effective amount of the pharmaceutical composition comprising the RNAi component at least until the serum HBsAg level of the subject decreases down to 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly down to 10 lU/mL or lower. In some embodiments, the method comprises administering, more particularly start administering, the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens after the serum HBsAg level of the patient has decreased down to 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly to 10 lU/mL or lower.
[0332] In some embodiments, the serum HBsAg level in the subject is reduced at a faster rate, is reduced at a greater level, and/or is reduced for a greater time period than in a subject in which only an effective amount of the pharmaceutical composition comprising the RNAi component is administered or an effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered.
[0333] In some embodiments, enhancing an immune response in the subject results in an increase in an innate immune system and/or an adaptive immune system. The increase in the innate immune system results in an increase in the expression of one or several from among an interferon stimulated gene (ISG), an interferon gamma-induced protein 10 (IP 10), an interferon alpha (IFNa), an interleukin 12 (IL 12), or an interleukin-6 (IL-6).
[0334] In some embodiments, the increase in the adaptive immune system results in an increase in the level of HBV-specific T cell responses and/or an increased activation of the HBV-specific T cells. Activation of HBV-specific T cells can be measured by any means that the person of average skill in the art may find appropriate, for example, by interferon gamma production (e.g., an interferon gamma ELISPOT).
[0335] The increase in expression of the at least one of ISG, IP10, IFNa, IL12, or IL-6; the increase in the level of HBV-specific T cell responses; and/or the increased activation of HBV-specific T cells can be compared to a control. The control can be an expression level of ISG, IP10, IFNa, IL12, or IL-6; the level of HBV-specific T cell responses; the level of activation of HBV-specific T cells from a sample from the subject prior to administration of the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens. By way of another example, the control can be the expression level of ISG, IP 10, IFNa, IL12, or IL-6; the level of HBV-specific T cell responses; and/or the level of activation of HBV-specific T cells from a subject administered the effective amount of the pharmaceutical composition comprising the RNAi component, but not the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens; or from a subject administered the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, but not the effective amount of the pharmaceutical composition comprising the RNAi component. A person skilled in the art will understand the proper control for determining an increase in the expression level of ISG, IP 10, IFNa, IL 12, or IL-6; an increase in the level of HBV-specific T cell responses; and/or an increased activation level of HBV-specific T cells in the subject.
[0336] In some embodiments, enhancing an immune response in the subject results in an increase in the immunocompetence of the subject. An increase in the immunocompetence of the subject can, for example, result in an increase in the level of HBV-specific T cells, B cells, or NK cells in the liver and/or an increased activation of HBV-specific T cells, B cells, or NK cells in the liver. An increase in the immunocompetence of the subject can, for example, result in an increase in NK cells, T cells, or B cells in a peripheral immune cell compartment and/or an increased activation of NK cells, T cells, or B cells in the peripheral immune cell compartment. An increase in the immunocompetence of the subject can, for example, result in a decrease of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or in a peripheral immune cell compartment; and/or a decreased immunosuppressive activity of MDSCs in the lever and/or in a peripheral immune cell compartment; and/or a decrease of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or in a peripheral immune cell compartment and/or a decreased immunosuppressive activity of Treg in the liver and/or in a peripheral immune cell compartment.
[0337] The increase in the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the increase in the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; the decrease in the level of or decreased immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment; and/or the decrease in the level of or decreased immunosuppressive activity of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or in a peripheral immune cell compartment can, for example, be determined by comparing to a control. The control can be the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; the level of or immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment; and/or the level of or activity of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or in a peripheral immune cell compartment from a sample from the subject prior to administration of the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens. By way of another example, the control can be the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; the level of or immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment; and/or the level of or activity of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or in a peripheral immune cell compartment from a subject administered the effective amount of the pharmaceutical composition comprising the RNAi component, but not the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens; or from a subject administered the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, but not the effective amount of the pharmaceutical composition comprising the RNAi component. A person skilled in the art will understand the proper control for determining an increase in the level of or activation of HBV-specific T cells, B cells, or NK cells in the liver; the level of or activation of NK cells, T cells, or B cells in the peripheral immune cell compartment; a decrease in the level of or immunosuppressive activity of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or peripheral immune cell compartment; and/or a decrease in the level of or activity of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or in a peripheral immune cell compartment in the subject.
[0338] Activation of HBV-specific NK cells can be measured by any means that the person of average skill in the art may find appropriate, for example, by interferon gamma production (e.g., interferon gamma ELISPOT). Activation of HBV-specific B cells can be measured by any means that the person of average skill in the art may find appropriate, for example, by anti-HBs antibody production (e.g., anti-HBs ELISPOT). The immunosuppressive activity of MDSCs can be measured by any means that the person of average skill in the art may find appropriate, for example, by arginase expression.
[0339] In some embodiments, decreasing the viral replication in the subject results in a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 lU/mL, more particularly is lower than 15 lU/mL, more particularly is lower than 10 lU/mL. [0340] In some embodiments, the targeted hepatocytes comprise cccDNA. In some embodiments, the targeted hepatocytes comprise integrated HBV DNA.
[0341] In some embodiments, the RNAi component comprises: (i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and (ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO:111.
[0342] In some embodiments, the first RNAi agent comprises SEQ ID NO:97 and SEQ ID NO: 106. In some embodiments, the first RNAi agent comprises SEQ ID NO:98 and SEQ ID NO: 106. In some embodiments, the first RNAi agent comprises SEQ ID NO:99 and SEQ ID NO: 107. In some embodiments, the first RNAi agent comprises SEQ ID NO:93 and SEQ ID NO: 102, 103, or 105. In some embodiments, the first RNAi agent comprises SEQ ID NO:94 and SEQ ID NO: 102, 103, or 105. In some embodiments, the first RNAi agent comprises SEQ ID NO:95 and SEQ ID NO: 102, 103, or 105. In some embodiments, the first RNAi agent comprises SEQ ID NO:96 and SEQ ID NO: 104. In some embodiments, the second RNAi agent comprises SEQ ID NO: 101 and SEQ ID NO: 111. In some embodiments, the second RNAi agent comprises SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110.
[0343] In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:97 and SEQ ID NO: 106 and a second RNAi agent comprising SEQ ID NO: 101 and SEQ ID NO: 111. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:98 and SEQ ID NO: 106 and a second RNAi agent comprising SEQ ID NO: 101 and SEQ ID NO: 111. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:99 and SEQ ID NO: 107 and a second RNAi agent comprising SEQ ID NO: 101 and SEQ ID NO: 111. [0344] In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:93 and SEQ ID NO: 102, 103, or 105 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:94 and SEQ ID NO: 102, 103, or 105 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:95 and SEQ ID NO: 102, 103, or 105 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110. In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:96 and SEQ ID NO: 104 and a second RNAi agent comprising SEQ ID NO: 100 and SEQ ID NO: 108, 109, or 110.
[0345] In some embodiments, the RNAi component comprises a first RNAi agent comprising SEQ ID NO:94 and SEQ ID NO: 103 or SEQ ID NO: 105 and the second RNAi agent comprising SEQ ID NO: 108 or SEQ ID NO: 110 and SEQ ID NO: 100.
[0346] In some embodiments, the two HBV RNAi agents are administered in a ratio of about 1:1, 2:1, 3:1, 4:1 or 5:1. In some embodiments, the two HBV RNAi agents are administered in a ratio of about 2: 1.
[0347] In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 25-75 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1: 1, about 4:1, about 5: 1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 35-40 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1:1, about 4:1, about 5:1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50-125 mg per dose administration and in the ratio of about 2: 1, about 3:1, about 1:1, about 4:1, about 5:1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 75-150 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1:1, about 4:1, about 5 : 1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 100-200 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1:1, about 4:1, about 5:1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 150-250 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1:1, about 4:1, about 5 : 1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 200-300 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1:1, about 4:1, about 5:1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 300-400 mg per dose administration and in the ratio of about 2:1, about 3:1, about 1:1, about 4:1, about 5 : 1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50-100 mg per dose administration and in the ratio of about 2: 1, about 3:1, about 1:1, about 4:1, about 5:1 or about 1:2. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 25-400 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 25-75 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 35-40 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50-125 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 75-150 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 100-200 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 125-225 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 150-250 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 200-300 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 300-400 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 100 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 25 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 35 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 40 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 50 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 75 mg per dose administration and in the ratio of about 2:1. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 200 mg per dose administration and in the ratio of about 2: 1.
[0348] In some embodiments, the first RNAi agent is administered in an amount of about 3-650 mg per dose administration, and the second RNAi agent is administered in an amount of about 2-325 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 15-150 mg per dose administration, and the second RNAi agent is administered in an amount of about 5-75 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 35-265 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 50-75 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 15-75 mg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 20-125 mg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 25-50 mg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 5-40 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 17 mg per dose administration, and the second RNAi agent is administered in an amount of about 8 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 23 mg per dose administration, and the second RNAi agent is administered in an amount of about 12 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 27 mg per dose administration, and the second RNAi agent is administered in an amount of about 13 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 33 mg per dose administration, and the second RNAi agent is administered in an amount of about 17 mg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 67 mg per dose administration, and the second RNAi agent is administered in an amount of about 33 mg per dose administration.
[0349] In some embodiments, two RNAi agents are administered at a combined dose of 25-400 mg per dose administration. In an embodiment, two RNAi agents are administered at a combined dose of 25-400 mg, and the first RNAi agent is administered with the second RNAi agent at a ratio of 1 : 1. In an embodiment, the dose of each of the first and second RNAi agents is in an amount of about 12 mg for a combined dose of about 25 mg. In an embodiment, the dose of each of the first and second RNAi agents is in an amount of about 17 mg for a combined dose of about 35 mg. In an embodiment, the dose of each of the first and second RNAi agents is in an amount of about 20 mg for a combined dose of about 40 mg. In an embodiment, the dose of each of the first and second RNAi agents is in an amount of about 25 mg for a combined dose of about 50 mg. In an embodiment, the dose of each of the first and second RNAi agents is in an amount of about 50 mg for a combined dose of about 100 mg. In an embodiment, the dose of each of the first and second RNAi agents is in an amount of about 100 mg for a combined dose of about 200 mg. In an embodiment, the dose of each of the first and second RNAi agents is in an amount of about 150 mg for a combined dose of about 300 mg. In an embodiment, the dose of each of the first and second RNAi agents is in an amount of about 200 mg for a combined dose of about 400 mg.
[0350] In an embodiment, two RNAi agents are administered at a combined dose of 25- 400 mg per dose, and the first RNAi agent is administered with the second RNAi agent at a ratio of 2: 1. In an embodiment, the dose of the first RNAi agent is in an amount of about 16 mg, and the dose of the second RNAi agent is in an amount of about 8 mg for a combined dose of about 25 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 24 mg, and the dose of the second RNAi agent is in an amount of about 12 mg for a combined dose of about 35 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 27 mg, and the dose of the second RNAi agent is in an amount of about 13 mg for a combined dose of about 40 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 33 mg, and the dose of the second RNAi agent is in an amount of about 17 mg for a combined dose of about 50 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 65 mg, and the dose of the second RNAi agent is in an amount of about 35 mg for a combined dose of about 100 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 133 mg, and the dose of the second RNAi agent is in an amount of about 67 mg for a combined dose of about 200 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 200 mg, and the dose of the second RNAi agent is in an amount of about 100 mg for a combined dose of about 300 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 270 mg, and the dose of the second RNAi agent is in an amount of about 135 mg for a combined dose of about 400 mg.
[0351] In an embodiment, two RNAi agents are administered at a combined dose of 25- 400 mg per dose, the first RNAi agent is administered with the second RNAi agent at a ratio of 3 : 1. In an embodiment, the dose of the first RNAi agent is in an amount of about 18 mg, and the dose of the second RNAi agent is in an amount of about 6 mg for a combined dose of about 25 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 27 mg, and the dose of the second RNAi agent is in an amount of about 9 mg for a combined dose of about 35 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 30 mg, and the dose of the second RNAi agent is in an amount of about 10 mg for a combined dose of about 40 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 36 mg, and the dose of the second RNAi agent is in an amount of about 12 mg for a combined dose of about 50 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 75 mg, and the dose of the second RNAi agent is in an amount of about 25 mg for a combined dose of about 100 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 150 mg, and the dose of the second RNAi agent is in an amount of about 50 mg for a combined dose of about 200 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 225 mg, and the dose of the second RNAi agent is in an amount of about 75 mg for a combined dose of about 300 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 300 mg, and the dose of the second RNAi agent is in an amount of about 100 mg for a combined dose of about 400 mg.
[0352] In some embodiments, the first RNAi agent and the second RNAi agent are administered in a combined amount of about 25-400 mg per dose administration. In some embodiments, the first RNAi agent and the second RNAi agent are administered in a combined amount of about 25-50 mg, 50-75 mg, 75-100 mg, 100-125 mg, 125-150 mg, 150- 175 mg, 175-200 mg, 200-225 mg, 225-250 mg, 250-275 mg, 275-300 mg, 300-325 mg, 325- 350 mg, 350-375 mg, 375-400 mg, 25-75 mg, 50-100 mg, 100-150 mg, 150-200 mg, 200-250 mg, 250-300 mg, 300-350 mg, 350-400 mg, 25-100 mg, 50-150 mg, 100-200 mg, 150-250 mg, 200-300 mg, 300-400 mg, 25-200 mg, or 200-400 mg per dose administration. In some embodiments, the first RNAi agent to the second RNAi agent are administered in a combined amount of about 25 mg, about 50 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, or about 400 mg per dose administration. In some embodiments, the first RNAi agent and the second RNAi agent are administered in a combined amount of about 50 mg, about 75 mg, about 100 mg, or about 125 mg per dose administration. In some embodiments, the first RNAi agent and the second RNAi agent are administered in a combined amount of about 25 mg, about 35 mg, about 40 mg, or about 200 mg per dose administration.
[0353] In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 1-10 mg/kg per dose administration. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 1-5 mg/kg per dose administration. In some embodiments, the two HBV RNAi agents are administered in a combined amount of about 1-1.5 mg/kg, about 1.5-2.0 mg/kg, about 2.0-2.5 mg/kg, about 2.5-3.0 mg/kg, about 3.0-3.5 mg/kg, about 3.5-4.0 mg/kg, about 4.0-4.5 mg/kg, about 4.5-5.0 mg/kg, about 5.0-5.5 mg/kg, about 5.5-6.0 mg/kg, about 6.0-6.5 mg/kg, about 6.5-7.0 mg/kg, about 7.0-7.5 mg/kg, about 7.5-8.0 mg/kg, about 8.0-8.5 mg/kg, about 8.5-9.0 mg/kg, about 9.0-9.5 mg/kg, about 9.5-10 mg/kg, about 1-2.5 mg/kg, about 2.5-5.0 mg/kg, about 5.0-7.5 mg/kg, about 7.5-10 mg/kg, about 1-5.0 mg/kg, or about 5.0-10 mg/kg per dose administration.
[0354] In some embodiments, the first RNAi agent is administered in an amount of about 0.6-7 mg/kg per dose administration, and the second RNAi agent is administered in an amount of about 0.3-5 mg/kg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 0.5-2.5 mg/kg per dose administration. In some embodiments, the second RNAi agent is administered in an amount of about 0.3- 1.5 mg/kg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 0.6-5 mg/kg per dose administration. In some embodiments, the first RNAi agent is administered in an amount of about 1-2.5 mg/kg per dose administration. [0355] In some embodiments, the two RNAi agents are administered in about 1-18 week intervals. In some embodiments, the two RNAi agents are administered in about 1-week intervals, about 2-week intervals, about 3-week intervals, about 4-week intervals, about 5- week intervals, about 6-week intervals, about 7-week intervals, about 8-week intervals, about 9-week intervals, about 10-week intervals, about 11-week intervals, about 12-week intervals, about 13 -week intervals, about 14-week intervals, about 15 -week intervals, about 16-week intervals, about 17-week intervals, or about 18-week intervals. In some embodiments, the two RNAi agents are administered in about 1-6 month intervals. In some embodiments, the two RNAi agents are administered in about 1 -month intervals, about 2-month intervals, about 3-month intervals, about 4-month intervals, about 5-month intervals, or about 6-month intervals. In some embodiments, the two RNAi agents are administered in about 4-week intervals or 1 -month intervals. In some embodiments, the two RNAi agents are administered once per month.
[0356] In some embodiments, the first RNAi agent and the second RNAi agent are administered for a duration of about 1-12 months. In some embodiments, the first RNAi agent and the second RNAi agent are administered for a duration of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months or at least about 12 months. In some embodiments, the first RNAi agent and the second RNAi agent are administered for a duration of about 1-18 weeks. In some embodiments, the first RNAi agent and the second RNAi agent are administered for a duration of at least about 1 week, at least about 5 weeks, at least about 10 weeks, at least about 15 weeks, at least about 20 weeks, at least about 25 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at least about 50 weeks, at least about 55 weeks, at least about 60 weeks, at least about 65 weeks, at least about 70 weeks, at least about 75 weeks, at least about 80 weeks, at least about 90 weeks, or at least 96 weeks.
[0357] In some embodiments, the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg per dose administration. In an embodiment, the first RNAi agent and the second RNAi agent are administered at a combined dose of 25- 400 mg, and the first RNAi agent is administered with the second RNAi agent at a ratio of 1: 1. In an embodiment, the dose of the first RNAi agent is administered with the second RNAi agent is in an amount of about 12 mg for a combined dose of about 25 mg. In an embodiment, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 17 mg for a combined dose of about 35 mg. In an embodiment, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 20 mg for a combined dose of about 40 mg. In an embodiment, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 25 mg for a combined dose of about 50 mg. In an embodiment, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 50 mg for a combined dose of about 100 mg. In an embodiment, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 100 mg for a combined dose of about 200 mg. In an embodiment, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 150 mg for a combined dose of about 300 mg. In an embodiment, the dose of each of the first RNAi agent and the second RNAi agent is in an amount of about 200 mg for a combined dose of about 400 mg. [0358] In an embodiment, the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg per dose, and the second RNAi agent is administered with the first RNAi agent at a ratio of 1 :2. In an embodiment, the dose of the first RNAi agent is in an amount of about 16 mg, and the dose of the second RNAi agent is in an amount of about 8 mg for a combined dose of about 25 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 12 mg, and the dose of the first RNAi agent is in an amount of about 24 mg for a combined dose of about 35 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 27 mg, and the dose of the second RNAi agent is in an amount of about 13 mg for a combined dose of about 40 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 33 mg, and the dose of the second RNAi agent is in an amount of about 17 mg for a combined dose of about 50 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 35 mg, and the dose of the first RNAi agent is in an amount of about 65 mg for a combined dose of about 100 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 67 mg, and the dose of the first RNAi agent is in an amount of about 133 mg for a combined dose of about 200 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 100 mg, and the dose of the first RNAi agent is in an amount of about 200 mg for a combined dose of about 300 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 135 mg, and the dose of the first RNAi agent is in an amount of about 270 mg for a combined dose of about 400 mg.
[0359] In an embodiment, the first RNAi agent and the second RNAi agent are administered at a combined dose of 25-400 mg per dose, the second RNAi agent is administered with the first RNAi agent at a ratio of 1 :3. In an embodiment, the dose of the first RNAi agent is in an amount of about 18 mg, and the dose of the second RNAi agent is in an amount of about 6 mg for a combined dose of about 25 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 9 mg, and the dose of the first RNAi agent is in an amount of about 27 mg for a combined dose of about 35 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 30 mg, and the dose of the second RNAi agent is in an amount of about 10 mg for a combined dose of about 40 mg. In an embodiment, the dose of the first RNAi agent is in an amount of about 36 mg, and the dose of the second RNAi agent is in an amount of about 12 mg for a combined dose of about 50 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 25 mg, and the dose of the first RNAi agent is in an amount of about 75 mg for a combined dose of about 100 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 50 mg, and the dose of the first RNAi agent is in an amount of about 150 mg for a combined dose of about 200 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 75 mg, and the dose of the first RNAi agent is in an amount of about 225 mg for a combined dose of about 300 mg. In an embodiment, the dose of the second RNAi agent is in an amount of about 100 mg, and the dose of the first RNAi agent is in an amount of about 300 mg for a combined dose of about 400 mg. [0360] In some embodiments, about 1 mg/kg (mpk) of the first RNAi agent and about 1 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 1.5 mg/kg of the first RNAi agent and about 1.5 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 2.0 mg/kg of the first RNAi agent and about 1.0 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 3.0 mg/kg of the first RNAi agent and about 1.0 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 3.2 mg/kg of the first RNAi agent and about 0.8 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 2.7 mg/kg of the first RNAi agent and about 1.3 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 4.0 mg/kg of the first RNAi agent and about 1.0 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about 3.3 mg/kg of the first RNAi agent and about 1.7 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, between about 0.05 and about 5 mg/kg of the first RNAi agent and between about 0.05 and about 5 mg/kg of the second RNAi agent are administered to a subject in need thereof. In some embodiments, about the first RNAi agent and about the second RNAi agent are administered separately (e.g., in separate injections). In some embodiments, the respective dose of the first RNAi agent and the respective dose of the second RNAi agent are administered together (e.g., in the same injection). In some embodiments, the respective dose of the first RNAi agent and the respective dose of the second RNAi agent are prepared in a single pharmaceutical composition.
[0361] In some embodiments, the RNAi component is administered to the subject once monthly in a dose of about 40-350 mg, such as about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg, more particularly 200 mg.
[0362] In another aspect, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end, is as follows:
(1) a polynucleotide sequence encoding a first hepatitis B virus (HBV) antigen,
(2) a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and
(3) a polynucleotide sequence encoding a second HBV antigen, wherein the first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
[0363] As general guidance, an effective amount when used with reference to a nucleic acid molecule, vector, or RNA replicon can range from about 10 pg of nucleic acid molecule, vector, or RNA replicon to about 300 pg of nucleic acid molecule, vector, or RNA replicon. Preferably, an effective amount of a nucleic acid molecule, vector, or RNA replicon is about 10 pg to about 300 pg, more particularly about 20 pg to about 200 pg. An effective amount can be from one nucleic acid molecule, vector, or RNA replicon, or from multiple nucleic acid molecules, vectors, or RNA replicons. An effective amount can be administered in a single composition, or in multiple compositions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compositions (e.g., tablets, capsules or injectables, or any composition adapted to intradermal delivery, e.g., to intradermal delivery using an intradermal delivery patch), wherein the administration of the multiple capsules or injections collectively provides a subject with an effective amount. It is also possible to administer an effective amount to a subject, and subsequently administer another dose of an effective amount to the same subject, in a so- called prime-boost regimen. This general concept of a prime-boost regimen is well known to the skilled person in the vaccine field. Further booster administrations can optionally be added to the regimen, as needed.
[0364] An immunogenic combination comprising two vectors, e.g., a first vector encoding a first HBV antigen and second vector encoding a second HBV antigen can be administered to a subject by mixing both vectors and delivering the mixture to a single anatomic site. Alternatively, two separate immunizations each delivering a single expression vector can be performed. In such embodiments, whether both vectors are administered in a single immunization as a mixture of in two separate immunizations, the first vector and the second vector can be administered in a ratio of 10:1 to 1:10, by weight, such as 10:1, 9:1, 8:1, 7:1, 6:1, 5: 1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, by weight. Preferably, the first and second vectors are administered in a ratio of 1:1, by weight.
[0365] In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose in the range of about 10 pg to about 300 pg, more particularly abouut 20 pg to about 200 pg. In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered for a duration of at least about 1 week, at least about 5 weeks, at least about 10 weeks, at least about 15 weeks, at least about 20 weeks, at least about 25 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at least about 50 weeks, at least about 55 weeks. In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered for a duration of about 12 weeks or of about 24 weeks. In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered once every 3 weeks, once every month (Q4W), once every six weeks (Q6W), or once every eight weeks (Q8W).
[0366] In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is formulated for subcutaneous, intradermal injection, or intramuscular injection, more preferably intramuscular injection. In some embodiments, the nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens is formulated in in a liquid form, such as suspensions, solutions, emulsions, or syrups, or may be lyophilized. In some embodiments, the RNAi component is formulated for subcutaneous injection. In some embodiments, the RNAi component is formulated in in a liquid form, such as suspensions, solutions, emulsions, or may be lyophilized.
[0367] In some embodiments, the RNAi component and the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens are administered simultaneously or intermittently. In some embodiments, the RNAi component and the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens are administered and formulated separately and administered with different dosing frequencies. In some embodiments, the RNAi component and the nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens are formulated as one or separate compositions. In some embodiments, the RNAi component is formulated as a solution and administered once per month or once every four weeks via subcutaneous injection. In some embodiments, the nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens is formulated as an intramuscular injection and administered in up to five (5) doses, preferably at least three doses.
[0368] In some embodiments, the RNAi component is administered in the amount of about 50-250 mg once a month via subcutaneous injection while the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20-200 pg in the form of a liquid solution at least six (6) months after the administration of the RNAi component has started, wherein said dose is administered up five (5) timese. In some embodiments, the RNAi component is administered in the amount of about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200 mg. In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg, wherein said dose is administered up to five (5) times. In some embodiments, the RNAi component is administered in the amount of about 100 mg once a month or once every four weeks via subcutaneous injection while the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg in the form of a liquid solution at least six (6) months after the administration of the RNAi component has started, wherein said dose is administered up to five (5) times.
[0369] In some embodiments, the RNAi component is administered in the amount of about 25-200 mg once a month or once every four weeks via subcutaneous injection while the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg in the form of a liquid solution at least six (6) months after the administration of the RNAi component has started, wherein said dose is administered up to five (5) times. In some embodiments, the RNAi component is administered in the amount of about 35 mg, about 40 mg, about 50 mg, about 100 mg, or about 200 mg. In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg in the form of a liquid solution, wherein said dose is administered up to five (5) times. In some embodiments, the RNAi component is administered in the amount of about 40 mg once a month or once every four weeks via subcutaneous injection while the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg in the form of a liquid solution at least six (6) months after the administration of the RNAi component has started, wherein said dose is administered up to five (5) times. In some embodiments, the RNAi component is administered in the amount of about 100 mg once a month or once every four weeks via subcutaneous injection while the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg in the form of a liquid solution at least six (6) months after the administration of the RNAi component has started, wherein said dose is administered up to five (5) times. In some embodiments, the RNAi component is administered in the amount of about 200 mg once a month or once every four weeks via subcutaneous injection while the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered at a dose of about 20 pg to about 200 pg in the form of a liquid solution at least six (6) months after the administration of the RNAi component has started, wherein said dose is administered up to five (5) times.
[0370] In some embodiments, a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, such as any of those described herein, is administered to the subject once or twice at a dose of about 20 pg to about 200 pg.
[0371] In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject about six (6) months after the administration of the RNAi component has started. [0372] In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject about six (6) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject up to five (5) times over the course of six (6) months in total.
[0373] In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject about three (3) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject up to three (3) times over the course of three (3) months in total.
[0374] In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject about six (6) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject up to three (3) times over the course of about three (3) months in total. [0375] In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject about eight (8) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject up to three (3) times over the course of about three (3) months in total.
[0376] In some embodiments, the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject about three (3) months after the administration of the RNAi component has started, wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is administered to the subject for about three (3) months in total, wherein the administration of the RNAi component continues, e.g., for about six (6) months, after the administration of the up to three (3) doses of the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens has been stopped.
[0377] Methods according to embodiments of the application further comprises administering to the subject in need thereof another immunogenic agent (such as another HBV antigen or other antigen) or another anti-HBV agent (such as a nucleoside analog or other anti-HBV agent) in combination with a pharmaceutical composition of the application. For example, another anti-HBV agent or immunogenic agent can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PDl, anti- PDL1, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/oror TLR8 agonists), RIG-1 agonists, IL- 15 superagonists (Aitor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant, IL-7-hyFc; CAR-T which bind HBV env (S-CAR cells); capsid assembly modulators; cccDNA inhibitors, HBV polymerase inhibitors (e.g., entecavir and tenofovir). The one or other anti-HBV active agents can be, for example, a small molecule, an antibody or antigen binding fragment thereof, a polypeptide, protein, or nucleic acid.
[0378] In some embodiments, the method further comprises administering a nucleoside analog. In some embodiments, the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof. In some embodiments, the nucleoside analog is entecavir and it is administered in a daily dose in the amount of about 0.01-5 mg, about 0.01-0.05 mg, about 0.05-0.1 mg, about 0.1-0.5 mg, about 0.5-1 mg, about 1-2 mg, about 2-3 mg, about 3-4 mg or about 4-5 mg. In some embodiments, the nucleoside analog is entecavir and it is administered in a daily dose in the amount of about 0.5 mg. In some embodiments, the nucleoside analog is tenofovir disoproxil fumarate and it is administered in a daily dose in the amount of about 100-500 mg, about 100-150 mg, about 150-200 mg, about 200-250 mg, about 250-300 mg, 300-400 mg, about 400-500 mg. In some embodiments, the nucleoside analog is tenofovir disoproxil fumarate and it is administered in a daily dose in the amount of about 300 mg In some embodiments, the nucleoside analog is tenofovir alafenamide and it is administered in a daily dose in the amount of about 5-100 mg, about 5-25 mg, about 25-50 mg, about 50-75 or about 75-100 mg. In some embodiments, the nucleoside analog is tenofovir alafenamide and it is administered in a daily dose in the amount of about 25 mg. In some embodiments, the nucleoside analog is lamivudine and it is administered in a daily dose in the amount of about 50-600 mg, about SO- SOO mg, about 100-300 mg, about 100-500 mg, about 150-400 mg, about 200-350, or about 250-300 mg. In some embodiments, the nucleoside analog is lamivudine and it is administered in a daily dose in the amount of 100 mg, 150 mg, or 300 mg. In some embodiments, the nucleoside analog is telbivudine and it is administered in a daily dose in the amount of about 300-800 mg, about 400-700 mg, about 300-600 mg, about 300-400 mg, about 400-500 mg, or about 500-600 mg. In some embodiments, the nucleoside analog is telbivudine and it is administered in a daily dose in the amount of 600 mg. In some embodiments, the patients have been exposed to the nucleoside analog prior to the combination therapy. In some embodiments, the patients have been administered the nucleoside analog for at least 1 month, at least 3 months, at least 6 months, or at least 1 year prior to receiving the combination therapy.
[0379] In some embodiments, the other agent is a nucleic acid polymer (NAP). The NAP can, for example, be selected from REP2139 or REP2165. REP2139 has a sequence of (A,5'MeC)20 with each linkage being phosphorothioated and every ribose being 2’0 methylated (which is disclosed as SEQ ID NOTO in WO2016/04525, the content of which is incorporated herein by reference in its entirety). REP2165 has a sequence of (A,5'MeC)20 with each linkage being phosphorothioated, every rbose being 2’0 methylated except adenosines at positions 11, 21, and 31, where riboses are 2’OH (which is disclosed as SEQ ID NO: 13 in WO2016/04525).
[0380] The NAP can also be other exemplary nucleic acid polymers, which include, but are not limited to, REP2006, REP2031, REP2055, STOPS™ (S-antigen transport-inhibiting oligonucleotide polymers), and those disclosed in Patent Application Publication Nos. WO200424919; WO201221985; and WO202097342 and U.S. Patent Nos. 7,358,068; 8,008,269; 8,008,270; and 8,067,385, the content of each is incorporated herein by reference in its entirety.
[0381] In some embodiments, the patients are screened for HBeAg status prior to administration of the combination therapy. In some embodiments, the patients are HBeAg positive. In some embodiments, the patients are HBeAg negative. In some embodiment, the patients are screened for immune tolerance prior to administration of the combination therapy.
[0382] In some embodiments, the HBsAg level in the patient is reduced by at least about logio 0.5, about logio 0.75, about logio 1, about logio 1.25, about logio 1.5, about logio 1.75, about logio 2 or about logio 2.5 from base line on Day 1. In some embodiments, the HBeAg level in the patient is reduced by at least about logio 0.5, about logio 0.75, about logio 1, about logio 1.25, about logio 1.5, about logio 1.75, about logio 2 or about logio 2.5 from base line on Day 1. In some embodiments, the HBcrAg level in the patient is reduced by at least about logio 0.5, about logio 0.75, about logio 1, about logio 1.25, about logio 1.5, about logio 1.75, about logio 2 or about logio 2.5 from base line on Day 1. In some embodiments, the HBV DNA level in the patient is reduced by at least about logio 0.5, about logio 1, about logio 1.5, about logio 2, about logio 3, about logio 4, about logio 5 or about logio 7.5 from base line on Day 1. In some embodiments, the HBV RNA level in the patient is reduced by at least about logio 0.5, about logio 0.75, about logio 1, about logio 1.25, about logio 1.5, about logio 1.75, about logio 2 or about logio 2.5 from base line on Day 1.
[0383] The application also relates to an effective amount of an RNAi component and a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens, optionally a nucleoside analog, each of which as that described herein, in the manufacture of a medicament for treating a Hepatitis B viral (HBV) infection in a subject; enhancing an immune response in a subject with an HBV infection; decreasing viral replication in a subject with HBV; decreasing the expression of one or more HBV polypeptide(s), more particularly of one or more polypeptide(s) selected from HBsAg and HBeAg, in a subject in need thereof; and/or increasing the targeted killing of hepatocytes comprising integrated viral DNA and/or extrachromosomal viral DNA in a subject with a HBV infection. In some embodiments, the RNAi component is for once monthly or once every four weeks administration to a subject in a dose of about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg, more particularly 200 mg; the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens is for administration to a subject at a dose of about 20-200 p.g, wherein said dose is administered up to five (5) times. In some embodiments, the medicament is for administering to a subject infected by HBV, in particular, a subject having a chronic HBV infection.
EMBODIMENTS
[0384] Embodiment 1 is a combination for use in treating a Hepatitis B Virus (HBV) infection, more particularly chronic HBV infection (CHB) with or without viral co-infection, and/or for treating chronic HDV infection, in a subject in need thereof, comprising:
(a) an effective amount of a pharmaceutical composition comprising an RNAi component having:
(i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
(ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; and
(b) an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
(1) a polynucleotide sequence encoding a first hepatitis B virus (HBV) antigen,
(2) a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and
(3) a polynucleotide sequence encoding a second HBV antigen, wherein the first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen. [0385] Embodiment 2 is a combination for use in enhancing an immune response in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection, comprising: (a) an effective amount of a pharmaceutical composition comprising an RNAi component having:
(i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
(ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; and
(b) an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
(1) a polynucleotide sequence encoding a first hepatitis B virus (HBV) antigen,
(2) a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and
(3) a polynucleotide sequence encoding a second HBV antigen, wherein the first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
[0386] Embodiment 3 is a combination for use in decreasing viral replication in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection, comprising:
(a) an effective amount of a pharmaceutical composition comprising an RNAi component having:
(i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and (ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; and
(b) an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
(1) a polynucleotide sequence encoding a first hepatitis B virus (HBV) antigen,
(2) a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and
(3) a polynucleotide sequence encoding a second HBV antigen, wherein the first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen. [0387] Embodiment 4 is a combination for use in decreasing the expression of one or more Hepatitis B Virus (HBV) polypeptide(s), more particularly of one or more polypeptide(s) selected from HBsAg and HBeAg, in a subject in need thereof, comprising:
(a) an effective amount of a pharmaceutical composition comprising an RNAi component having:
(i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
(ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; and
(b) an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
(1) a polynucleotide sequence encoding a first hepatitis B virus (HBV) antigen, (2) a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and
(3) a polynucleotide sequence encoding a second HBV antigen, wherein the first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
[0388] Embodiment 5 is a combination for use in increasing the targeted killing of hepatocytes containing integrated viral DNA or extrachromosomal viral DNA in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection, comprising:
(a) an effective amount of a pharmaceutical composition comprising an RNAi component having:
(i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
(ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; and
(b) an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
(1) a polynucleotide sequence encoding a first hepatitis B virus (HBV) antigen,
(2) a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and
(3) a polynucleotide sequence encoding a second HBV antigen, wherein the first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen. [0389] Embodiment 6 is the combination for use of any one of embodiments 1-5, wherein the HBV infection is a chronic HBV infection.
[0390] Embodiment 7 is the combination for use of any one of embodiments 1 -6, wherein the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens decrease the immune tolerogenicity of the subject to HBV, more particularly the immune tolerogenicity of the liver of the subject to HBV.
[0391] Embodiment 8 is the combination for use of any one of embodiments 1-7, wherein the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence encoding one or more HBV antigens are administered to the subject at least until the subject meets at least one of, at least two of, at least three of, at least four of, or the five following features: i. a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 lU/mL, more particularly is lower than 15 lU/mL, more particularly is lower than 10 lU/ml; ii. a serum ALT concentration lower than 3 times the upper normal limit, or lower than 129 U/L if the subject is a male subject, or lower than 108 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 120 U/L if the subj ect is a male subj ect or lower than 105 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 90 U/L if the subject is a male subject or lower than 57 U/L if the subject is a female subject; iii. a HBeAg-negative serum; iv. a serum HBsAg level of 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly of 10 lU/mL or lower; and v. HBs seroconversion.
[0392] Embodiment 9 is the combination for use of embodiment 8, wherein at least one of, at least two of, at least three of, at least four of, or the five features i., ii., iii., iv., and v. are still met 6 months after the end of the treatment.
[0393] Embodiment 10 is the combination for use of embodiment 8, wherein the serum HBsAg level is 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly of 10 lU/mL or lower. [0394] Embodiment 11 is the combination for use of any one of embodiments 1-10, wherein the effective amount of the pharmaceutical composition comprising the RNAi component is administered to the subject at least until the serum HBsAg level of the subject decreases down to 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly down to 10 lU/mL or lower.
[0395] Embodiment 12 is the combination for use of any one of embodiments 1-11, wherein the effective amount of the nucleic acid molecule comprising a non-naturally occurring polynucleotide is administered to the subject after the serum HBsAg level of the patient has decreased down to 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly to 10 lU/mL or lower.
[0396] Embodiment 13 is the combination for use of any one of embodiments 8-12, wherein the serum HBsAg level in the subject is reduced at a faster rate, is reduced at a greater level, and/or is reduced for a greater time period than in a subject in which only an effective amount of the pharmaceutical composition comprising the RNAi component is administered or an effective amount of the nucleic acid molecule comprising a non-naturally occurring polynucleotide is administered.
[0397] Embodiment 14 is the combination for use of embodiment 2, wherein enhancing an immune response in the subject results in an increase in an innate immune system response and/or an adaptive immune system response.
[0398] Embodiment 15 is the combination for use of embodiment 14, wherein the increase in the innate immune system response results in an increase in the expression of one or several from among an interferon stimulated gene (ISG), an interferon gamma-induced protein 10 (IP10), an interferon alpha (IFNoc), an interleukin 12, or an interleukin-6 (IL-6). [0399] Embodiment 16 is the combination for use of embodiment 14, wherein the increase in the adaptive immune system response results in an increase in HBV-specific T cell responses and/or an increased activation of the HBV-specific T cells.
[0400] Embodiment 17 is the combination for use of embodiment 2, wherein enhancing an immune response in the subject results in an increase in the immunocompetence of the subject.
[0401] Embodiment 18 is the combination for use of embodiment 17, wherein the increase in the immunocompetence of the subject results in an increase of HBV-specific T cells, B cells, or NK cells in the liver and/or an increased activation of HBV-specific T cells, B cells, or NK cells in the liver. [0402] Embodiment 19 is the combination for use of embodiment 17 or 18, wherein the increase in the immunocompetence of the subject results in an increase in NK cells, T cells, or B cells in a peripheral immune cell compartment and/or an increased activation of NK cells, T cells, or B cells in a peripheral immune cell compartment.
[0403] Embodiment 20 is the combination for use of any one of embodiments 17-19, wherein the increase in the immunocompetence of the subject results in a decrease of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or in a peripheral immune cell compartment and/or a decreased immunosuppressive activity of MDSCs in the liver and/or in a peripheral immune cell compartment, and/or a decrease of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or peripheral immune cell compartment and/or a decreased immunosuppressive activity of Treg in the liver and/or a peripheral immune cell compartment.
[0404] Embodiment 21 is the combination for use of embodiment 3, wherein decreasing viral replication in the subject results in a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 lU/mL, more particularly is lower than 15 lU/mL, more particularly is lower than 10 lU/mL.
[0405] Embodiment 22 is the combination for use of embodiment 5, wherein the targeted hepatocytes comprise cccDNA.
[0406] Embodiment 23 is the combination for use of embodiment 5 or 22, wherein the targeted hepatocytes comprise integrated HBV DNA.
[0407] Embodiment 24 is the combination for use of any one of embodiments 1-23, wherein the first or the second RNAi agent comprises at least one modified nucleotide and/or at least one modified intemucleoside linkage.
[0408] Embodiment 25 is the combination for use of any one of embodiments 1-24, wherein at least 90% of the nucleotides in the first and the second RNAi agents are modified nucleotides.
[0409] Embodiment 26 is the combination for use of any one of embodiments 1-25, wherein the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent.
[0410] Embodiment 27 is the combination for use of embodiment 26, wherein the targeting ligand comprises N-acetyl-galactosamine.
[0411] Embodiment 28 is the combination for use of embodiment 27, wherein the targeting ligand is selected from the group consisting of (NAGI 3), (NAG13)s, (NAGI 8), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), and (NAG39)s.
[0412] Embodiment 29 is the combination for use of embodiment 28, wherein the targeting ligand is (NAG25), (NAG25)s, (NAG31), (NAG31)s, (NAG37), or (NAG37)s. [0413] Embodiment 30 is the combination for use of any one of embodiments 26-29, wherein the targeting ligand is conjugated to the sense strand of the first or the second RNAi agent.
[0414] Embodiment 31 is the combination for use of embodiment 30, wherein the targeting ligand is conjugated to the 5’ terminus of the sense stand of the first or the second RNAi agent.
[0415] Embodiment 32 is the combination for use of any one of embodiments 1-31, wherein the first and the second RNAi agents independently comprise a duplex selected from the group consisting of:
(a) an antisense strand comprising SEQ ID NO:93 and a sense strand comprising SEQ ID NO: 102;
(b) an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 103;
(c) an antisense strand comprising SEQ ID NO:95 and a sense strand comprising SEQ ID NO: 103;
(d) an antisense strand comprising SEQ ID NO:96 and a sense strand comprising SEQ ID NO: 104;
(e) an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 108;
(f) an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 109;
(g) an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 104; and
(h) an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 110.
[0416] Embodiment 33 is the combination for use of any one of embodiments 1-32, wherein the first and the second RNAi agents are each independently conjugated to a targeting ligand comprising N-acetyl-galactosamine, and the first and the second RNAi agents independently comprise a duplex selected from the group consisting of:
(a) an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 103;
(b) an antisense strand comprising SEQ ID NO:96 and a sense strand comprising SEQ ID NO: 104;
(c) an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 108;
(d) an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 105; and
(e) an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 110.
[0417] Embodiment 34 is the combination for use of any one of embodiments 1-33, wherein the ratio of the first RNAi agent to the second RNAi agent by weight is in the range of about 1:2 to about 5:1.
[0418] Embodiment 35 is the combination for use of any one of embodiments 1-34, wherein one of the first or second HBV antigens is an HBV core or an HBV pol antigen. [0419] Embodiment 36 is the combination for use of any one of embodiments 1-35, wherein the non-naturally occurring polynucleotide sequence further comprises, ordered from the 5’ - to 3 ’-end:
(4) a second IRES element or a polynucleotide sequence encoding a second autoprotease peptide operably linked to the 3’ end of the polynucleotide sequence encoding the second HBV antigen, and
(5) a polynucleotide sequence encoding a third HBV antigen independently selected from the group consisting of an HBV core antigen, an HBV pol antigen, and an HBV surface antigen.
[0420] Embodiment 37 is the combination for use of embodiment 36, wherein the non- naturally occurring polynucleotide sequence further comprises, ordered from the 5’- to 3’- end:
(6) a third IRES element or a polynucleotide sequence encoding a third autoprotease peptide operably linked to the 3’ end of the polynucleotide sequence encoding the third HBV antigen, and (7) a polynucleotide sequence encoding a fourth HBV antigen independently selected from the group consisting of an HBV core antigen, an HBV pol antigen, and an HBV surface antigen.
[0421] Embodiment 38 is the combination for use of embodiment 37, wherein each of the first, second, third and fourth HBV antigens is different from each other.
[0422] Embodiment 39 is the combination for use of embodiment 37 or 38, wherein each of the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
(i) a first HBV Pre-S 1 antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: i;
(ii) a second HBV Pre-S 1 antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 3;
(iii) an HBV PreS2.S antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 5;
(iv) an HBV core antigen comprising, preferably consisting of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7; and
(v) an HBV pol antigen comprising, preferably consisting of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 9, preferably, each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a signal peptide, and the HBV PreS2.S antigen comprises an internal signal peptide.
[0423] Embodiment 40 is the combination for use of embodiment 39, wherein the HBV core antigen comprises, preferably consists of, an amino acid sequence that is at least 98% identical to at least one of SEQ ID NOs: 84, 85, or 86, such as at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 84, 85, or 86.
[0424] Embodiment 41 is the combination for use of embodiment 39 or 40, wherein the last five C-terminal amino acids of the HBV core antigen comprise a VVR amino acid sequence, more particularly a VVRR (SEQ ID NO: 91) amino acid sequence, more particularly a VVRRR (SEQ ID NO: 92) amino acid sequence.
[0425] Embodiment 42 is the combination for use of embodiment 39, wherein each of the
HBV surface antigen, the HBV core antigen and the HBV pol antigen comprises:
(i) a consensus sequence for HBV genotypes A, B, C and D; and/or one or more epitopes for HLA-A*11:01, HLA-A*24:02, HLA-A*02:01, HLA-A*A2402, HLA-A*A0101, or HLA-B*40:01.
[0426] Embodiment 43 is the combination for use of embodiment 42, wherein each of the
HBV surface antigens, the HBV core antigen and the HBV pol antigen comprises one or more epitopes for HL A- A* 11:01.
[0427] Embodiment 44 is the combination for use of embodiment 39, wherein each of the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
(i) the first HBV Pre-S 1 antigen consisting of the amino acid sequence of SEQ ID NO: i;
(ii) the second HBV Pre-S 1 antigen consisting of the amino acid sequence of SEQ ID NO: 3;
(iii) the HBV PreS2.S antigen consisting of the amino acid sequence of SEQ ID NO: 5;
(iv) the HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86; and
(v) the HBV pol antigen consisting of the amino acid sequence of SEQ ID NO: 9; preferably, each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a signal peptide, such as the signal peptide comprising the amino acid sequence of SEQ ID NO: 77. [0428] Embodiment 45 is the combination for use of any one of embodiments 37-44, wherein each of the polynucleotide sequences encoding the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
(i) a polynucleotide sequence encoding the first HBV Pre-S 1 antigen having a sequence that is at least 90% identical to SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2;
(ii) a polynucleotide sequence encoding the second HBV Pre-S 1 antigen having a sequence that is at least 90% identical to SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4;
(iii) a polynucleotide sequence encoding the HBV PreS2.S antigen having a sequence that is at least 90% identical to SEQ ID NO: 6, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 6;
(iv) a polynucleotide sequence encoding the HBV core antigen having a sequence that is at least 90% identical to SEQ ID NO: 8, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 8; and
(v) the polynucleotide sequence encoding the HBV pol antigen having a sequence that is at least 90% identical to SEQ ID NO: 10, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 10; preferably, the polynucleotide sequence encoding each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a polynucleotide sequence encoding a signal peptide, and the HBV PreS2.S antigen comprises an internal signal peptide.
[0429] Embodiment 46 is the combination for use of embodiment 45, wherein each of the polynucleotide sequences encoding the first, second, third and fourth HBV antigens is independently selected from the group consisting of: (i) a polynucleotide sequence encoding the first HBV Pre-S 1 antigen consisting of the sequence of SEQ ID NO: 2;
(ii) a polynucleotide sequence encoding the second HBV Pre-S 1 antigen consisting of the sequence of SEQ ID NO: 4;
(iii) a polynucleotide sequence encoding the HBV PreS2.S antigen consisting of the sequence of SEQ ID NO: 6;
(iv) a polynucleotide sequence encoding the HBV core antigen consisting of the sequence of any one of SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89; and
(v) a polynucleotide sequence encoding the HBV pol antigen consisting of the sequence of SEQ ID NO: 10; preferably, the polynucleotide sequence encoding each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a polynucleotide encoding a signal peptide, such as the polynucleotide comprising the sequence of SEQ ID NO: 90.
[0430] Embodiment 47 is the combination for use of any one of embodiments 37-46, wherein each of the first, second and third autoprotease peptides independently comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2 A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof, preferably, each of the first, second and third autoprotease peptides comprise the peptide sequence of P2A, such as a P2A sequence of SEQ ID NO: 11.
[0431] Embodiment 48 is the combination for use of any one of embodiments 37-47, wherein each of the first, second and third IRES is derived from encephalomyocarditis virus (EMCV) or Enterovirus 71 (EV71), preferably each of the first, second and third IRES comprises the polynucleotide sequence of SEQ ID NO: 13 or 14.
[0432] Embodiment 49 is the combination for use of embodiment 1, wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprises, ordered from the 5’ - to 3 ’-end:
(1) a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5; (2) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV Pre-Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(3) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(4) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO:
11, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(5) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(6) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- S1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(7) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(8) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(9) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- S1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9; (10) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- S1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(11) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(12) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(13) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- S1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(14) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- S1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(15) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(16) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(17) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5;
(18) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5;
(19) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(20) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(21) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5; or
(22) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, preferably, the polynucleotide sequence encoding each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a polynucleotide sequence encoding a signal peptide, such as the signal peptide comprising the amino acid sequence of SEQ ID NO: 77.
[0433] Embodiment 50 is the combination for use of embodiment 49, wherein the non- naturally occurring polynucleotide sequence comprises a sequence of any one of SEQ ID NOs: 15 to 54.
[0434] Embodiment 51 is the combination for use of any one of embodiments 1-50, wherein a vector comprises the non-naturally occurring polynucleotide sequence.
[0435] Embodiment 52 is the combination for use of embodiment 51, wherein the vector is a DNA plasmid, a DNA viral vector, or an RNA viral vector.
[0436] Embodiment 53 is the combination for use of any one of embodiment 1-50, wherein an RNA replicon comprises the non-naturally polynucleotide sequence, and wherein the RNA replicon comprises, ordered from the 5’ - to 3 ’-end:
(1) a 5’ untranslated region (5’-UTR) required for nonstructural protein-mediated amplification of an RNA virus;
(2) a polynucleotide sequence encoding at least one, preferably all, of non-structural proteins of the RNA virus;
(3) a subgenomic promoter of the RNA virus;
(4) the non-naturally occurring polynucleotide sequence; and
(5) a 3’ untranslated region (3’-UTR) required for nonstructural protein-mediated amplification of the RNA virus.
[0437] Embodiment 54 is the combination for use of any one of embodiments 1-50, wherein an RNA replicon comprises the non-naturally polynucleotide sequence, and wherein the RNA replicon comprises, ordered from the 5’ - to 3 ’-end:
(1) an alphavirus 5’ untranslated region (5’-UTR);
(2) a 5’ replication sequence of an alphavirus non-structural gene nspl;
(3) a downstream loop (DLP) motif of a virus species;
(4) a polynucleotide sequence encoding a fourth autoprotease peptide;
(5) a polynucleotide sequence encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4;
(6) an alphavirus subgenomic promoter;
(7) the non-naturally occurring polynucleotide sequence;
(8) an alphavirus 3' untranslated region (3' UTR); and (9) optionally, a poly adenosine sequence.
[0438] Embodiment 55 is the combination for use of embodiment 54, wherein the DLP motif is from a virus species selected from the group consisting of Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MTDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (U AV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (B ABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), and Buggy Creek virus.
[0439] Embodiment 56 is the combination for use of embodiment 55, wherein the fourth autoprotease peptide is selected from the group consisting of porcine tesehovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof, preferably, the fourth autoprotease peptide comprises the peptide sequence of P2A.
[0440] Embodiment 57 is the combination for use of any one of embodiments 1-50, wherein an RNA replicon comprises the non-naturally polynucleotide sequence, and wherein the RNA replicon comprises, ordered from the 5’ - to 3 ’-end:
(1) a 5’-UTR having the polynucleotide sequence of SEQ ID NO: 55;
(2) a 5’ replication sequence having the polynucleotide sequence of SEQ ID NO: 56;
(3) a DLP motif comprising the polynucleotide sequence of SEQ ID NO: 57;
(4) a polynucleotide sequence encoding a P2A sequence of SEQ ID NO: 11;
(5) polynucleotide sequences encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4, having the nucleic acid sequences of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively;
(6) a subgenomic promoter having polynucleotide sequence of SEQ ID NO: 62;
(7) the non-naturally occurring polynucleotide sequence; and
(8) a 3' UTR having the polynucleotide sequence of SEQ ID NO: 63.
[0441] Embodiment 58 is the combination for use of embodiment 57, wherein:
(i) the polynucleotide sequence encoding the P2A sequence comprises SEQ ID NO: 12, (ii) the non-naturally occurring polynucleotide sequence comprises the polynucleotide sequence of any one of SEQ ID NOs: 15 to 54, and
(iii)the RNA replicon further comprises a poly adenosine sequence, preferably the poly adenosine sequence has the sequence of SEQ ID NO: 64, at the 3’-end of the replicon.
[0442] Embodiment 59 is the combination for use of any one of embodiments 53-58, wherein the RNA replicon comprises the polynucleotide sequence of any one of SEQ ID NOs: 65 to 72.
[0443] Embodiment 60 is the combination for use of any one of embodiments 53-58, wherein a nucleic acid molecule comprises a DNA sequence encoding the RNA replicon, preferably wherein the nucleic acid molecule further comprises a T7 promoter operably linked to the 5 ’-end of the DNA sequence, more preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO: 73.
[0444] Embodiment 61 is the combination for use of any one of embodiments 1-60, wherein a pharmaceutical composition comprises the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence, the vector, or the RNA replicon, and a pharmaceutically acceptable carrier.
[0445] Embodiment 62 is the combination for use of embodiment 61, wherein the pharmaceutical composition further comprises:
(1) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9; or
(2) a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85 or 86.
[0446] Embodiment 63 is the combination for use of any one of embodiments 1-62, wherein the RNAi component is administered to the subject once monthly (Q4W) in a dose of about 40-1000 mg, more particularly of about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg, more particularly 200 mg.
[0447] Embodiment 64 is the combination for use of any one of embodiments 1-63, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject at a concentration of about 10-300 mg/mL, more particularly of about 20-200 μg/mL in a dose, wherein said dose is administered up to five (5) times.
[0448] Embodiment 65 is the combination for use of any one of embodiments 1-64, wherein the RNAi component is administered to the subject via intravenous or subcutaneous injection.
[0449] Embodiment 66 is the combination for use of any one of embodiments 1-65, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject via intramuscular injection.
[0450] Embodiment 67 is the combination for use of any one of embodiments 1-66, wherein the RNAi component is administered simultaneously or sequentially with the nucleic acid molecule comprising the non-naturally occurring polynucleotide.
[0451] Embodiment 68 is the combination for use of any one of embodiments 1-66, wherein the RNAi component is administered separately from the nucleic acid molecule comprising the non-naturally occurring polynucleotide.
[0452] Embodiment 69 is the combination for use of embodiment 68, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about six (6) months after the administration of the RNAi component has started. [0453] Embodiment 70 is the combination for use of embodiment 68 or 69, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about six (6) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to five (5) times over the course of about six (6) months in total.
[0454] Embodiment 71 is the combination for use of embodiment 68 or 69, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about three (3) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to three (3) times over the course of about three (3) months in total. [0455] Embodiment 72 is the combination for use of embodiment 68 or 69, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about six (6) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to three (3) times over the course of about three (3) months in total.
[0456] Embodiment 73 is the combination for use of embodiment 68 or 69, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about eight (8) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to three (3) times over the course of about three (3) months in total.
[0457] Embodiment 74 is the combination for use of embodiment 68 or 69, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about three (3) months after the administration of the RNAi component has started, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to three (3) times over the course of about three (3) months in total, and wherein the administration of the RNAi component continues, e.g., for about six (6) months, after the administration of the nucleic acid molecule comprising the non-naturally occurring polynucleotide has been stopped.
[0458] Embodiment 75 is the combination for use of any one of embodiments 1-74 further comprising administering to the subject a nucleoside analog or a nucleotide analog. [0459] Embodiment 76 is the combination for use of embodiment 75, wherein the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof.
[0460] Embodiment 77 is the combination for use of embodiment 76, wherein entecavir is administered to the subject in a daily dose of about 0.1-5 mg.
[0461] Embodiment 78 is the combination for use of embodiment 76, wherein tenofovir is administered to the subject in a daily dose of about 5-50 mg of tenofovir alafenamide or about 200-500 mg of tenofovir disoproxil fumarate.
[0462] Embodiment 79 is the combination for use of embodiment 76, wherein lamivudine is administered to the subject in a daily dose of about 100 mg, about 150 mg or about 300 mg. [0463] Embodiment 80 is the combination for use of embodiment 76, wherein telbivudine is administered to the subject in a daily dose of about 600 mg.
[0464] Embodiment 81 is the combination for use of any one of embodiments 1-80, wherein the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the compound of formula (I) is administered to the subject for 10-96 weeks, more particularly 12-72 weeks, more particularly 12-60 weeks, more particularly 12-52 weeks, more particularly 48 weeks.
[0465] Embodiment 82 is the combination for use of embodiment 81, further comprising administration of a nucleoside analog or a nucleotide analog, such as entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof to the subject, and wherein the administration of the nucleoside or nucleotide analog is optionally being continued once the administration of the effective amount the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising the non- naturally occurring polynucleotide stops.
[0466] Embodiment 83 is an RNAi component for use in combination with a nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence in the treatment of a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without a viral co-infection, and/or in the treatment of a chronic Hepatitis D Virus (HDV) infection, wherein the RNAi component and the nucleic acid molecule comprising the non- naturally occurring polynucleotide sequence are as defined in any one of embodiments 1-82, and wherein the RNAi component optionally is indicated for simultaneous, sequential, or separate administration with the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence.
[0467] Embodiment 84 is a nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence for use in combination with an RNAi component in the treatment of a Hepatits B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without a viral co-infection, and/or in the treatment of a chronic Hepatitis D Virus (HDV) infection, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence and the RNAi component are as defined in any one of embodiments 1-82, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is indicated for simultaneous, sequential, or separate administration with the RNAi component. [0468] Embodiment 85 is an effective amount of an RNAi component and a nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence in the manufacture of a medicament for inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof, wherein the RNAi component and the nucleic acid molecule comprising the non- naturally occurring polynucleotide sequence are as defined in any one of embodiments 1-82.
EXAMPLES
[0469] The following examples are offered to illustrate but not to limit the invention. One of skill in the art will recognize that the following procedures may be modified using methods known to one of ordinary skill in the art.
[0470] Example 1: Efficacy and Immunogenicity of the self-replicating RNA HBV therapeutic vaccine together with a liver-specific HBV targeted siRNA
[0471] In this study mice are inoculated with AAV -HBV (1011 viral genomes/mouse) to establish chronic infection. At 6 weeks post infection, HBsAg levels are measured and animals with serum HBsAg levels reflective of those seen in CHB patients (103 - 104 lU/mL) are distributed into a 1st cohort to be monitored for efficacy and a 2nd cohort to examine immunogenicity .
[0472] Both cohorts are given 3 subcutaneous administrations of siRNA (3 mg/kg) at 3- week intervals starting 6 weeks post infection with AAV -HBV. The siRNA is a mixture of a first RNAi agent and a second RNAi agent as described in Table 3 (e.g., the SEQ ID NO: 94 and SEQ ID NO: 103 or SEQ ID NO: 105 duplex in a 2: 1 molar miture with the SEQ ID NO: 100 and SEQ ID NO: 108 or SEQ ID NO: 110 duplex), which are each independently conjugated to (NAG37)s or (NAG25)s, e.g., on the 5’ end of their respective sense strands, which may further carry an invAb at the 5’ or 3’ end of the sense strand.
[0473] The HBV replicon therapeutic vaccine is provided:
- as a mixture of two bigenic constructs, wherein one of the two bigenic constructs provides the Core coding sequence (e.g. SEQ ID NO: 8) and the Pol coding sequence (e.g. SEQ ID NO: 10) linked by a P2A or IRES coding sequence (e.g. SEQ ID NO: 12, 13 or 14), and wherein the other of the two bigenic constructs provides the PreS2.S coding sequence (e.g., SEQ ID NO: 6) and the preSl coding sequence (e.g., SEQ ID NO: 4) linked by a P2A or IRES coding sequence (e.g., SEQ ID NO: 12, 13 or 14), e.g., the mixture of the bigenic construct of SEQ ID NO: 68 and of the bigenic construct of SEQ ID NO: 65 or 66; or the mixture of the bigenic construct of SEQ ID NO: 67 and of the bigenic construct of SEQ ID NO: 66 or 65; or
- as a tetracistronic construct providing the Core, Pol, preS2.S and PreSl coding sequences (SEQ ID NO: 8, 10, 6 and 4) linked together by P2A or IRES coding sequences (e.g. SEQ ID NO: 12, 13 or 14) (e.g., the tetracistronic organization shown in FIG. 2B), e.g., the tetracistronic construct of SEQ ID NO: 70, 71, 69 or 72.
[0474] The construct(s) is(are) optionally formulated in LNPs e.g., as described in US2014255472A1). Two 10 microgram doses of the therapeutic vaccine are given via intramuscular injection 6 weeks apart with an optional 3rd dose administered if HBsAg levels are observed to rebound. The first dose of replicon vaccine is administered the same day as the last dose of siRNA treatment. Serum is collected at the time points indicated in the study design in FIGs. 1 A-1C in order to monitor serum cytokines as well as HBV DNA, HBsAg andHBeAg levels.
[0475] All animals from cohort 2, which is setup to examine immunogenicity, are sacrificed 10 days post second administration of replicon vaccine and splenocyte and intra- hepatic immune cells are harvested. To evaluate induction of HBV specific T cell responses, the frequency of IFNy producing cells and polyfunctional CD4 and CD8 T cells in response to stimulation with overlapping 15mer peptide pools covering HBV core, polymerase, PreS2S and PreSl domains is measured by IFNγELIspot and intracellular cytokine staining (IFNγ, TNFα and IL-2) by flow cytometry, respectively. Humoral responses to HBsAg are measured by anti-HBsAg ELISA.
[0476] EXAMPLE 2: Therapeutic efficacy and immunogenicity of combination siRNA and replicon therapeutic vaccine in AAV-HBV mouse model.
[0477] The study design of example 1 has been implemented with the tetra-3 construct as follows. The therapeutic efficacy of siRNA targeting HBsAg in combination with HBV tetra-3 replicon TxVx was evaluated in the AAV-HBV mouse model. Two cohorts (immunogenicity cohort and efficacy cohort) of C57/BL6 mice were injected with 1.0x1011 genomic equivalents of AAV vector expressing 1.2 genomes of HBV genotype D by IV injection. 43 (immunogenicity cohort) and 50 days (efficacy cohort) after inoculation with AAV-HBV, 60 mice/cohort with clinically relevant serum HBsAg concentrations (103-104 lU/mL; Autobio HBsAg CLIA) were randomized into 6 groups (Table 4; n=10 mice/group) based on serum HBsAg, serum HBV DNA and mouse weight. 63 (immunogenicity cohort) and 50 days (efficacy cohort) after randomization, mice were administered three Q3W doses of siRNA (3 mg/kg) or vehicle and two Q6W doses of replicon (10 pg; formulated in ATX-2 LNP) or vehicle as listed in Table 4. The immunogenicity cohort was sacrificed two weeks after the final replicon dose and spleen and liver were harvested for assessment of HBV- specific, IFN-y+ T-cell responses (ELISpot; Mabtech) while the efficacy cohort was monitored for serum HBsAg levels for 14 weeks following the last replicon dose.
[0478] Table 4 Treatment groups and dosing schedule for AAV-HBV mouse efficacy study
Figure imgf000212_0001
[0479] HBV tetra-3 is the following construct: a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: r 86, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3; and where HBV siRNA is the 2: 1 (molar) mixture of a S trigger (SEQ ID NOs: 94 and 105) and X trigger (SEQ ID NOs: 100 and 110) with GalNAc moiety being (NAG)25s [where the sense strand of each trigger carries the GalNAc moiety at the 5’ end and may further carry an invAb at the 5’ end and the 3’ end, e.g., with (NAG25)s(invAb)sguggacuuCfUfCfucaauuuucus(invAb) as the sense strand for the S trigger and with (NAG25)s(invAb)scgcuguagGfCfAfuaaauugguas(invAb) as the sense strand for the X trigger],
[0480] Two weeks after the 2nd replicon dose, HBsAg serum concentrations were lower in mice that received HBV siRNA + HBV tetra-3 replicon (group 1) compared to mice that received HBV siRNA + control replicon. When analyses combined both efficacy and immunogenicity cohorts to increase the statistical power, serum HBsAg concentrations were significantly lower in HBV siRNA + HBV tetra-3 replicon mice (n=18; 1.97 log10 lU/mL) compared to HBV siRNA + control replicon mice (n=20; 2.21 log10 lU/mL; Mann- Whitney P value, P=0.044), despite similar serum HBsAg concentrations immediately prior to dosing siRNA (wk 0; Mann- Whitney P value, P=0.2395) and immediately prior to the first replicon dose (wk 6; Mann- Whitney P value, P=0.8696; FIGs.. 3A-3C).
[0481] Indeed, two weeks after the 2nd replicon dose, 5/18 HBV siRNA + HBV tetra-3 replicon treated mice had serum HBsAg concentrations below the limit of quantification (0.398 log10 lU/mL) compared to 0/20 HBV siRNA + control replicon treated mice (Fisher’s exact test; P=0.0171; FIG. 3C), despite similar number of mice with serum HBsAg concentrations below the limit of quantification immediately prior to replicon dosing (3/18 HBV siRNA + HBV tetra-3 replicon mice vs 3/20 HBV siRNA + control replicon; Fisher’s exact test, P=1.0; FIG. 3B). Similar trends were observed in the individual immunogenicity and efficacy cohorts (FIGs. 3D-3F).
[0482] Analyses beyond 2 weeks after the 2nd replicon dose were limited to the efficacy cohort since the immunogenicity cohort was sacrificed 2 weeks after the 2nd replicon dose to assess spleen and liver HBV-specific T cell responses. HBsAg serum concentrations were significantly lower in the HBV siRNA + HBV tetra-3 replicon treated mice compared to the HBV siRNA + control replicon treated mice at numerous timepoints 2 weeks after the 2nd replicon dose, however HBV siRNA + HBV tetra-3 replicon treated mice also had significantly lower baseline HBsAg serum concentrations (immediately prior to siRNA dosing) than the HBV siRNA + control replicon treated mice (P=0.0155; FIG. 4A), due to fluctuations in serum HBsAg concentrations between the time the mice were randomized and the start of siRNA treatment. To account for the different HBsAg serum concentrations at baseline, each mouse’s serum HBsAg concentration was normalized to their baseline HBsAg concentration. Delta HBsAg concentrations were not significantly different in HBV siRNA + HBV tetra-3 replicon treated mice and HBV siRNA + control replicon treated mice at any timepoint after initiation of treatment (FIG. 4B).
[0483] To determine if HBV siRNA + HBV tetra-3 replicon combination therapy enhanced HBV-specific T cell responses compared to monotherapy, the immunogenicity cohort was sacrificed, and spleens were harvested two weeks after the second replicon dose. Splenocytes were isolated by mechanical digestion and HBV-specific IFNγ T cell responses were determined by ELISpot. Briefly, 200k cells/well were cultured in the presence of 2 ug/mL of overlapping peptides (15mers overlapping by 9 amino acids) covering pol, core, preSl and preS2.S for 18-24 hrs and ELISpot plates were processed according to the manufacturer’s instructions (Mabtech). As shown in FIG. 5, HBV-specific IFNy T cells were only detected from mice that received HBV tetra-3 replicon. HBV-specific IFNy T cell responses were significantly higher in mice that received HBV siRNA + HBV tetra-3 replicon (mean ± SD, 1172 ± 398 SFU/106 cells, Mann-Whitney test, P=0.0115) or AAT siRNA + HBV tetra-3 replicon (mean ± SD, 1375 ± 530 SFU/106 cells, Mann- Whitney test, P=0.0052) compared to mice that received siRNA vehicle + replicon (mean ± SD, 598 ± 491 SFU/106 cells).
[0484] The combination of the siRNA with the replicon led to a lower HBsAg serum concentrations two weeks after the last replicon dose compared to the combination of the HBV siRNA with the control replicon or compared to the combination of the control siRNA with the HBV replicon.

Claims

CLAIMS We Claim:
1. A combination for use in treating a Hepatitis B Virus (HBV) infection, more particularly chronic HBV infection (CHB) with or without viral co-infection, and/or for treating chronic HDV infection, in a subject in need thereof, comprising:
(a) an effective amount of a pharmaceutical composition comprising an RNAi component having:
(i) a first RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and
(ii) a second RNAi agent comprising: an antisense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 100 and SEQ ID NO: 101, and a sense strand comprising a nucleotide sequence of any one of the following: SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111; and
(b) an effective amount of a nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprising, ordered from the 5’- to 3 ’-end:
(1) a polynucleotide sequence encoding a first hepatitis B virus (HBV) antigen,
(2) a first internal ribosome entry sequence (IRES) element or a polynucleotide sequence encoding a first autoprotease peptide, and
(3) a polynucleotide sequence encoding a second HBV antigen, wherein the first HBV antigen and the second HBV antigen are each independently selected from the group consisting of an HBV core antigen, an HBV polymerase (pol) antigen, and an HBV surface antigen, and at least one of the first and second HBV antigens is an HBV surface antigen, preferably an HBV Pre-S 1 antigen or an HBV PreS2.S antigen.
2. The combination for the use of claim 1, wherein the combination enhances an immune response in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection.
3. The combination for the use of claim 1, wherein the combination decreases viral replication in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection.
4. The combination for the use of claim 1, wherein the combination decreases the expression of one or more Hepatitis B Virus (HBV) polypeptide(s), more particularly of one or more polypeptide(s) selected from HBsAg and HBeAg, in a subject in need thereof.
5. The combination for the use of claim 1, wherein the combination increases the targeted killing of hepatocytes containing integrated viral DNA or extrachromosomal viral DNA in a subject with a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without viral co-infection.
6. The combination for the use of any one of claims 1-5, wherein the HBV infection is a chronic HBV infection.
7. The combination for the use of any one of claims 1-6, wherein the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens decrease the immune tolerogenicity of the subject to HBV, more particularly the immune tolerogenicity of the liver of the subject to HBV.
8. The combination for the use of any one of claims 1-7, wherein the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising a non- naturally occurring polynucleotide sequence encoding one or more HBV antigens are administered to the subject at least until the subject meets at least one of, at least two of, at least three of, at least four of, or the five following features: i. a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 lU/mL, more particularly is lower than 15 lU/mL, more particularly is lower than 10 lU/ml; ii. a serum ALT concentration lower than 3 times the upper normal limit, or lower than 129 U/L if the subject is a male subject, or lower than 108 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 120 U/L if the subj ect is a male subj ect or lower than 105 U/L if the subject is a female subject, more particularly a serum ALT concentration lower than 90 U/L if the subject is a male subject or lower than 57 U/L if the subject is a female subject; iii. a HBeAg-negative serum; iv. a serum HBsAg level of 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly of 10 lU/mL or lower; and v. HBs seroconversion.
9. The combination for the use of claim 8, wherein at least one of, at least two of, at least three of, at least four of, or the five features i., ii., iii. , iv., and v. are still met 6 months after the end of the treatment.
10. The combination for the use of claim 8, wherein the serum HBsAg level is 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly of 10 lU/mL or lower.
11. The combination for the use of any one of claims 1-10, wherein the effective amount of the pharmaceutical composition comprising the RNAi component is administered to the subject at least until the serum HBsAg level of the subject decreases down to 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly down to 10 lU/mL or lower.
12. The combination for the use of any one of claims 1-11, wherein the effective amount of the nucleic acid molecule comprising a non-naturally occurring polynucleotide is administered to the subject after the serum HBsAg level of the patient has decreased down to 1000 lU/mL or lower, more particularly 300 lU/mL or lower, more particularly 100 lU/mL or lower, more particularly to 10 lU/mL or lower.
13. The combination for the use of any one of claims 8-12, wherein the serum HBsAg level in the subject is reduced at a faster rate, is reduced at a greater level, and/or is reduced for a greater time period than in a subject in which only an effective amount of the pharmaceutical composition comprising the RNAi component is administered or an effective amount of the nucleic acid molecule comprising a non-naturally occurring polynucleotide is administered.
14. The combination for the use of claim 2, wherein enhancing an immune response in the subject results in an increase in an innate immune system response and/or an adaptive immune system response.
15. The combination for the use of claim 14, wherein the increase in the innate immune system response results in an increase in the expression of one or several from among an interferon stimulated gene (ISG), an interferon gamma-induced protein 10 (IP 10), an interferon alpha (IFNa), an interleukin 12, or an interleukin-6 (IL-6).
16. The combination for the use of claim 14, wherein the increase in the adaptive immune system response results in an increase in HBV-specific T cell responses and/or an increased activation of the HBV-specific T cells.
17. The combination for the use of claim 2, wherein enhancing an immune response in the subject results in an increase in the immunocompetence of the subject.
18. The combination for the use of claim 17, wherein the increase in the immunocompetence of the subject results in an increase of HBV-specific T cells, B cells, or NK cells in the liver and/or an increased activation of HBV-specific T cells, B cells, or NK cells in the liver.
19. The combination for the use of claim 17 or 18, wherein the increase in the immunocompetence of the subject results in an increase in NK cells, T cells, or B cells in a peripheral immune cell compartment and/or an increased activation of NK cells, T cells, or B cells in a peripheral immune cell compartment.
20. The combination for the use of any one of claims 17-19, wherein the increase in the immunocompetence of the subject results in a decrease of Myeloid Derived Suppressive Cells (MDSCs) in the liver and/or in a peripheral immune cell compartment and/or a decreased immunosuppressive activity of MDSCs in the liver and/or in a peripheral immune cell compartment, and/or a decrease of regulatory T cells (Treg) and/or regulatory T cells in the liver and/or peripheral immune cell compartment and/or a decreased immunosuppressive activity of Treg in the liver and/or a peripheral immune cell compartment.
21. The combination for the use of claim 3, wherein decreasing viral replication in the subject results in a serum HBV DNA lower than the lower limit of quantification (LLoQ) or is lower than 20 lU/mL, more particularly is lower than 15 lU/mL, more particularly is lower than 10 lU/mL.
22. The combination for the use of claim 5, wherein the targeted hepatocytes comprise cccDNA.
23. The combination for the use of claim 5 or 22, wherein the targeted hepatocytes comprise integrated HBV DNA.
24. The combination for the use of any one of claims 1-23, wherein the first or the second RNAi agent comprises at least one modified nucleotide and/or at least one modified intemucleoside linkage.
25. The combination for the use of any one of claims 1-24, wherein at least 90% of the nucleotides in the first and the second RNAi agents are modified nucleotides.
26. The combination for the use of any one of claims 1-25, wherein the first or the second RNAi agent further comprises a targeting ligand that is conjugated to the first or the second RNAi agent.
217
27. The combination for the use of claim 26, wherein the targeting ligand comprises N- acetyl-galactosamine.
28. The combination for the use of claim 27, wherein the targeting ligand is selected from the group consisting of (NAGI 3), (NAG13)s, (NAGI 8), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), and (NAG39)s.
29. The combination for the use of claim 28, wherein the targeting ligand is (NAG25), (NAG25)s, (NAG31), (NAG31)s, (NAG37), or (NAG37)s.
30. The combination for the use of any one of claims 26-29, wherein the targeting ligand is conjugated to the sense strand of the first or the second RNAi agent.
31. The combination for the use of claim 30, wherein the targeting ligand is conjugated to the 5’ terminus of the sense stand of the first or the second RNAi agent.
32. The combination for the use of any one of claims 1-31, wherein the first and the second RNAi agents independently comprise a duplex selected from the group consisting of:
(a) an antisense strand comprising SEQ ID NO:93 and a sense strand comprising SEQ ID NO: 102;
(b) an antisense strand comprising SEQ ID NO: 94 and a sense strand comprising SEQ ID NO: 103;
(c) an antisense strand comprising SEQ ID NO:95 and a sense strand comprising SEQ ID NO: 103;
(d) an antisense strand comprising SEQ ID NO: 96 and a sense strand comprising SEQ ID NO: 104;
(e) an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 108;
(1) an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 109;
(g) an antisense strand comprising SEQ ID NO: 94 and a sense strand comprising SEQ ID NO: 104; and
(h) an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 110.
33. The combination for the use of any one of claims 1-32, wherein the first and the second RNAi agents are each independently conjugated to a targeting ligand comprising N-
218 acetyl-galactosamine, and the first and the second RNAi agents independently comprise a duplex selected from the group consisting of:
(a) an antisense strand comprising SEQ ID NO:94 and a sense strand comprising SEQ ID NO: 103;
(b) an antisense strand comprising SEQ ID NO: 96 and a sense strand comprising SEQ ID NO: 104;
(c) an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 108;
(d) an antisense strand comprising SEQ ID NO: 94 and a sense strand comprising SEQ ID NO: 105; and
(e) an antisense strand comprising SEQ ID NO: 100 and a sense strand comprising SEQ ID NO: 110.
34. The combination for the use of any one of claims 1-33, wherein one of the first or second HBV antigens is an HBV core or an HBV pol antigen.
35. The combination for the use of any one of claims 1-34, wherein the non-naturally occurring polynucleotide sequence further comprises, ordered from the 5’ - to 3 ’-end:
(4) a second IRES element or a polynucleotide sequence encoding a second autoprotease peptide operably linked to the 3’ end of the polynucleotide sequence encoding the second HBV antigen, and
(5) a polynucleotide sequence encoding a third HBV antigen independently selected from the group consisting of an HBV core antigen, an HBV pol antigen, and an HBV surface antigen.
36. The combination for the use of claim 35, wherein the non-naturally occurring polynucleotide sequence further comprises, ordered from the 5’ - to 3 ’-end:
(6) a third IRES element or a polynucleotide sequence encoding a third autoprotease peptide operably linked to the 3’ end of the polynucleotide sequence encoding the third HBV antigen, and
(7) a polynucleotide sequence encoding a fourth HBV antigen independently selected from the group consisting of an HBV core antigen, an HBV pol antigen, and an HBV surface antigen.
37. The combination for the use of claim 36, wherein each of the first, second, third and fourth HBV antigens is different from each other.
38. The combination for the use of claim 36 or 37, wherein each of the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
219 (i) a first HBV Pre-S 1 antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: i;
(ii) a second HBV Pre-S 1 antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 3;
(iii) an HBV PreS2.S antigen comprising, preferably consisting of, an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to the amino acid sequence of SEQ ID NO: 5;
(iv) an HBV core antigen comprising, preferably consisting of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 7; and
(v) an HBV pol antigen comprising, preferably consisting of, an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 9, preferably, each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a signal peptide, and the HBV PreS2.S antigen comprises an internal signal peptide.
39. The combination for the use of claim 38, wherein the HBV core antigen comprises, preferably consists of, an amino acid sequence that is at least 98% identical to at least one of SEQ ID NOs: 84, 85, or 86, such as at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 84, 85, or 86.
40. The combination for the use of claim 38 or 39, wherein the last five C-terminal amino acids of the HBV core antigen comprise a VVR amino acid sequence, more particularly a
220 VVRR (SEQ ID NO: 91) amino acid sequence, more particularly a VVRRR (SEQ ID NO: 92) amino acid sequence.
41. The combination for the use of claim 38, wherein each of the HBV surface antigen, the HBV core antigen and the HBV pol antigen comprises:
(ii) a consensus sequence for HBV genotypes A, B, C and D; and/or one or more epitopes for HLA-A*11:01, HLA-A*24:02, HLA-A*02:01, HLA-A*A2402, HLA-A*A0101, or HLA-B*40:01.
42. The combination for the use of claim 41, wherein each of the HBV surface antigens, the HBV core antigen and the HBV pol antigen comprises one or more epitopes for HLA- A*ll:01.
43. The combination for the use of claim 38, wherein each of the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
(vi) the first HBV Pre-S 1 antigen consisting of the amino acid sequence of SEQ ID NO: i;
(vii)the second HBV Pre-S 1 antigen consisting of the amino acid sequence of SEQ ID NO: 3;
(viii) the HBV PreS2.S antigen consisting of the amino acid sequence of SEQ ID NO: 5;
(ix) the HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86; and
(x) the HBV pol antigen consisting of the amino acid sequence of SEQ ID NO: 9; preferably, each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a signal peptide, such as the signal peptide comprising the amino acid sequence of SEQ ID NO: 77.
44. The combination for the use of any one of claims 36-43, wherein each of the polynucleotide sequences encoding the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
(i) a polynucleotide sequence encoding the first HBV Pre-S 1 antigen having a sequence that is at least 90% identical to SEQ ID NO: 2, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2;
(ii) a polynucleotide sequence encoding the second HBV Pre-S 1 antigen having a sequence that is at least 90% identical to SEQ ID NO: 4, such as at least 90%, 91%,
221 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4;
(iii) a polynucleotide sequence encoding the HBV PreS2.S antigen having a sequence that is at least 90% identical to SEQ ID NO: 6, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 6;
(iv) a polynucleotide sequence encoding the HBV core antigen having a sequence that is at least 90% identical to SEQ ID NO: 8, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 8; and
(v) the polynucleotide sequence encoding the HBV pol antigen having a sequence that is at least 90% identical to SEQ ID NO: 10, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 10; preferably, the polynucleotide sequence encoding each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a polynucleotide sequence encoding a signal peptide, and the HBV PreS2.S antigen comprises an internal signal peptide.
45. The combination for the use of claim 44, wherein each of the polynucleotide sequences encoding the first, second, third and fourth HBV antigens is independently selected from the group consisting of:
(i) a polynucleotide sequence encoding the first HBV Pre-S 1 antigen consisting of the sequence of SEQ ID NO: 2;
(ii) a polynucleotide sequence encoding the second HBV Pre-S 1 antigen consisting of the sequence of SEQ ID NO: 4;
(iii) a polynucleotide sequence encoding the HBV PreS2.S antigen consisting of the sequence of SEQ ID NO: 6;
(iv) a polynucleotide sequence encoding the HBV core antigen consisting of the sequence of any one of SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89; and
(v) a polynucleotide sequence encoding the HBV pol antigen consisting of the sequence of SEQ ID NO: 10;
222 preferably, the polynucleotide sequence encoding each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a polynucleotide encoding a signal peptide, such as the polynucleotide comprising the sequence of SEQ ID NO: 90.
46. The combination for the use of any one of claims 36-45, wherein each of the first, second and third autoprotease peptides independently comprises a peptide sequence selected from the group consisting of porcine teschovirus-1 2 A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof, preferably, each of the first, second and third autoprotease peptides comprise the peptide sequence of P2A, such as a P2A sequence of SEQ ID NO: 11.
47. The combination for the use of any one of claims 36-46, wherein each of the first, second and third IRES is derived from encephalomyocarditis virus (EMCV) or Enterovirus 71 (EV71), preferably each of the first, second and third IRES comprises the polynucleotide sequence of SEQ ID NO: 13 or 14.
48. The combination for the use of claim 1, wherein the nucleic acid molecule comprising a non-naturally occurring polynucleotide sequence comprises, ordered from the 5’- to 3 ’-end:
(1) a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5;
(2) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV Pre-Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(3) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(4) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO:
11, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(5) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(6) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- Sl antigen having the amino acid sequence of SEQ ID NO: 1 or 3, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(7) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(8) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(9) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- S1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(10) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- S1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 13, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(11) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(12) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV Pre-S 1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3;
(13) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- S1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(14) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV Pre- S1 antigen having the amino acid sequence of SEQ ID NO: 1 or 3, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86; (15) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(16) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, an IRES having the polynucleotide sequence of SEQ ID NO: 14, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(17) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5;
(18) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5;
(19) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86;
(20) a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9;
(21) a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5; or
(22) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85, or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, a polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11, and a polynucleotide sequence encoding an HBV PreS2.S antigen having the amino acid sequence of SEQ ID NO: 5, preferably, the polynucleotide sequence encoding each of the first and second HBV Pre-S 1 antigens, the HBV core antigen and the HBV pol antigen is independently operably linked to a polynucleotide sequence encoding a signal peptide, such as the signal peptide comprising the amino acid sequence of SEQ ID NO: 77.
49. The combination for the use of claim 48, wherein the non-naturally occurring polynucleotide sequence comprises a sequence of any one of SEQ ID NOs: 15 to 54.
50. The combination for the use of any one of claims 1-49, wherein a vector comprises the non-naturally occurring polynucleotide sequence.
51. The combination for the use of claim 50, wherein the vector is a DNA plasmid, a DNA viral vector, or an RNA viral vector.
52. The combination for the use of any one of claims 1-49, wherein an RNA replicon comprises the non-naturally polynucleotide sequence, and wherein the RNA replicon comprises, ordered from the 5’- to 3’-end:
(1) a 5’ untranslated region (5’-UTR) required for nonstructural protein-mediated amplification of an RNA virus;
(2) a polynucleotide sequence encoding at least one, preferably all, of non-structural proteins of the RNA virus;
(3) a subgenomic promoter of the RNA virus;
(4) the non-naturally occurring polynucleotide sequence; and
(5) a 3’ untranslated region (3’-UTR) required for nonstructural protein-mediated amplification of the RNA virus.
53. The combination for the use of any one of claims 1-49, wherein an RNA replicon comprises the non-naturally polynucleotide sequence, and wherein the RNA replicon comprises, ordered from the 5’- to 3’-end:
(1) an alphavirus 5’ untranslated region (5’-UTR);
(2) a 5’ replication sequence of an alphavirus non-structural gene nspl;
(3) a downstream loop (DLP) motif of a virus species;
(4) a polynucleotide sequence encoding a fourth autoprotease peptide;
(5) a polynucleotide sequence encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4;
(6) an alphavirus subgenomic promoter;
(7) the non-naturally occurring polynucleotide sequence;
(8) an alphavirus 3' untranslated region (3' UTR); and
(9) optionally, a poly adenosine sequence.
54. The combination for the use of claim 53, wherein the DLP motif is from a virus species selected from the group consisting of Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MTDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (U AV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), and Buggy Creek virus.
229
55. The combination for the use of claim 54, wherein the fourth autoprotease peptide is selected from the group consisting of porcine tesehovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof, preferably, the fourth autoprotease peptide comprises the peptide sequence of P2A.
56. The combination for the use of any one of claims 1-49, wherein an RNA replicon comprises the non-naturally polynucleotide sequence, and wherein the RNA replicon comprises, ordered from the 5’- to 3’-end:
(1) a 5’-UTR having the polynucleotide sequence of SEQ ID NO: 55;
(2) a 5’ replication sequence having the polynucleotide sequence of SEQ ID NO: 56;
(3) a DLP motif comprising the polynucleotide sequence of SEQ ID NO: 57;
(4) a polynucleotide sequence encoding a P2A sequence of SEQ ID NO: 11;
(5) polynucleotide sequences encoding alphavirus non-structural proteins nspl, nsp2, nsp3 and nsp4, having the nucleic acid sequences of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively;
(6) a subgenomic promoter having polynucleotide sequence of SEQ ID NO: 62;
(7) the non-naturally occurring polynucleotide sequence; and
(8) a 3' UTR having the polynucleotide sequence of SEQ ID NO: 63.
57. The combination for the use of claim 56, wherein:
(iv)the polynucleotide sequence encoding the P2A sequence comprises SEQ ID NO: 12,
(v) the non-naturally occurring polynucleotide sequence comprises the polynucleotide sequence of any one of SEQ ID NOs: 15 to 54, and
(vi)the RNA replicon further comprises a poly adenosine sequence, preferably the poly adenosine sequence has the sequence of SEQ ID NO: 64, at the 3’-end of the replicon.
58. The combination for the use of any one of claims 52-57, wherein the RNA replicon comprises the polynucleotide sequence of any one of SEQ ID NOs: 65 to 72.
59. The combination for the use of any one of claims 52-57, wherein a nucleic acid molecule comprises a DNA sequence encoding the RNA replicon, preferably wherein the nucleic acid molecule further comprises a T7 promoter operably linked to the 5 ’-end of the DNA sequence, more preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO: 73.
230
60. The combination for the use of any one of claims 1-59, wherein a pharmaceutical composition comprises the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence, the vector, or the RNA replicon, and a pharmaceutically acceptable carrier.
61. The combination for the use of claim 60, wherein the pharmaceutical composition further comprises:
(1) a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85 or 86, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9; or
(2) a polynucleotide sequence encoding an HBV polymerase antigen having, preferably consisting of, the amino acid sequence of SEQ ID NO: 9, a polynucleotide sequence encoding a P2A amino acid sequence of SEQ ID NO: 11 or an IRES having the polynucleotide sequence of SEQ ID NO: 13 or 14, and a polynucleotide sequence encoding an HBV core antigen having the amino acid sequence of any one of SEQ ID NOs: 84, 85 or 86.
62. The combination for the use of any one of claims 1-61, wherein the RNAi component is administered to the subject once monthly (Q4W) in a dose of about 40-1000 mg, more particularly of about 40-250 mg, more particularly 40-200 mg, more particularly 100 mg or 200 mg, more particularly 200 mg.
63. The combination for the use of any one of claims 1-62, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject at a concentration of about 10-300 mg/mL, more particularly of about 20-200 pg/mL in a dose, wherein said dose is administered up to five (5) times.
64. The combination for the use of any one of claims 1-63, wherein the RNAi component is administered to the subject via intravenous or subcutaneous injection.
65. The combination for the use of any one of claims 1-64, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject via intramuscular injection.
66. The combination for the use of any one of claims 1-65, wherein the RNAi component is administered simultaneously or sequentially with the nucleic acid molecule comprising the non-naturally occurring polynucleotide.
67. The combination for the use of any one of claims 1-65, wherein the RNAi component is administered separately from the nucleic acid molecule comprising the non-naturally occurring polynucleotide.
68. The combination for the use of claim 67, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about six (6) months after the administration of the RNAi component has started.
69. The combination for the use of claim 67 or 68, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about six (6) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to five (5) times over the course of about six (6) months in total.
70. The combination for the use of claim 67 or 68, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about three (3) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to three (3) times over the course of about three (3) months in total.
71. The combination for the use of claim 67 or 68, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about six (6) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to three (3) times over the course of about three (3) months in total.
72. The combination for the use of claim 67 or 68, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about eight (8) months after the administration of the RNAi component has started, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to three (3) times over the course of about three (3) months in total.
73. The combination for the use of claim 67 or 68, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject about three (3) months after the administration of the RNAi component has started, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is administered to the subject up to three (3) times over the course of about three (3) months in total, and wherein the administration of the RNAi component continues, e.g., for about six (6) months, after the administration of the nucleic acid molecule comprising the non-naturally occurring polynucleotide has been stopped.
74. The combination for the use of any one of claims 1-73 further comprising administering to the subject a nucleoside analog or a nucleotide analog.
75. The combination for the use of claim 74, wherein the nucleoside analog is entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof.
76. The combination for the use of claim 75, wherein entecavir is administered to the subject in a daily dose of about 0.1-5 mg.
77. The combination for the use of claim 75, wherein tenofovir is administered to the subject in a daily dose of about 5-50 mg of tenofovir alafenamide or about 200-500 mg of tenofovir disoproxil fumarate.
78. The combination for the use of claim 75, wherein lamivudine is administered to the subject in a daily dose of about 100 mg, about 150 mg or about 300 mg.
79. The combination for the use of claim 75, wherein telbivudine is administered to the subject in a daily dose of about 600 mg.
80. The combination for the use of any one of claims 1-79, wherein the effective amount of the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the compound of formula (I) is administered to the subject for 10-96 weeks, more particularly 12-72 weeks, more particularly 12-60 weeks, more particularly 12-52 weeks, more particularly 48 weeks.
81. The combination for the use of claim 80, further comprising administration of a nucleoside analog or a nucleotide analog, such as entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, telbivudine, or a combination thereof to the subject, and wherein the administration of the nucleoside or nucleotide analog is optionally being continued once the administration of the effective amount the pharmaceutical composition comprising the RNAi component and the effective amount of the pharmaceutical composition comprising the nucleic acid molecule comprising the non-naturally occurring polynucleotide stops.
82. An RNAi component for use in combination with a nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence in the treatment of a Hepatitis B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without a viral co-infection, and/or in the treatment of a chronic Hepatitis D Virus (HDV) infection, wherein
233 the RNAi component and the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence are as defined in any one of claims 1-81, and wherein the RNAi component optionally is indicated for simultaneous, sequential, or separate administration with the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence.
83. A nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence for use in combination with an RNAi component in the treatment of a Hepatits B Virus (HBV) infection, more particularly a chronic HBV infection (CHB) with or without a viral co-infection, and/or in the treatment of a chronic Hepatitis D Virus (HDV) infection, wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence and the RNAi component are as defined in any one of claims 1-81, and wherein the nucleic acid molecule comprising the non-naturally occurring polynucleotide is indicated for simultaneous, sequential, or separate administration with the RNAi component.
84. An effective amount of an RNAi component and a nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence in the manufacture of a medicament for inhibiting the expression of a Hepatitis B Virus gene in a subject in need thereof, wherein the RNAi component and the nucleic acid molecule comprising the non-naturally occurring polynucleotide sequence are as defined in any one of claims 1-81.
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