HBV vaccines and methods of treating HBV

Abstract

The present invention provides HBV immunogenic polypeptides useful for eliciting an immune response against HBV, polynucleotides encoding such polypeptides, vectors expressing such immunogenic polypeptides; pharmaceutical and immunogenic compositions and kits comprising such polypeptides, polynucleotides or vectors, as well as methods for treating and / or preventing HBV.

Description

[0001] This application is a divisional application of the invention patent application with the application date of September 28, 2020, application number 202080068039.0, and invention name “HBV vaccine and method for treating HBV”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 908,494, filed on September 30, 2019, which is hereby incorporated by reference in its entirety for all purposes.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which is submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on September 4, 2020, is named 1324_PF_SL.txt, and is 296,675 bytes in size. Background Art

[0006] There have been many attempts to use vaccination to treat patients with chronic hepatitis B virus (HBV) infection to improve the rate of HBV surface antigen (sAg) loss, which is the main marker of functional cure. Such attempts have included the use of recombinant proteins (Dikici et al., J Gastroenterol Hepatol. (2003) 18(2):218-22; Pol et al., J Hepatol. (2001) 34(6):917-21; Vandepapeliere et al., Vaccine (2007) 25(51):8585-97; Yalcin et al., J Clin Gastroenterol. (2003) 37(4):330-5; Al-Mahtab, Hepatol Int. (2013) 7(4):981-9; Hoa et al., Antimicrob Agents Chemother. (2009) 53(12):5134-40; and Yalcin et al., Infection. (2003) 31(4):221-5), recombinant DNA (Mancini-Bourgine et al., Hepatology. (2004) 40(4):874-82; Yang et al., World J Gastroenterol. (2017) 23(2):306-17; Yang et al., J Viral Hepat. (2012) 19(8):581-93; Yoon et al., Liver Int. (2015) 35(3):805-15; Cavenaugh et al., PLoS One. (2011) 6(2):e14626; and Godon et al., Mol. Ther. (2014) 22(3): 675-84), dendritic cells (Luo et al., Vaccine. (2010) 28(13): 2497-504; and Wei et al., Int Immunopharmacol. (2015) 27(2): 238-43), yeast vectors (Gane et al., J Hepatol. (2019) Epub 2019 / 07 / 16.doi:10.1016 / j.jhep.2019.06.028. PubMed PMID: 31306680), and some viral vectors (Cavenaugh et al., supra; and Zoulim et al., Hum Vaccin Immunother. (2019) Epub 2019 2019 / 08 / 03.doi:10.1080 / 21645515.2019.1651141.PubMed PMID:31373537) vaccination.Despite these many attempts, to date, no therapeutic vaccination approach has demonstrated consistent benefit in chronic HBV infection (CHB).Flaws in previous vaccine approaches may explain the failure of previous vaccine approaches.

[0007] Such deficiencies include limitations in antigen design and the vaccine technology used. The optimal antigen would contain highly conserved portions of the HBV protein and exclude poorly conserved regions, because highly conserved regions can induce responses to epitopes that are identical in the vaccine antigen and in the virus present in the treated patient, whereas poorly conserved regions can elicit immunodominant T cell responses to epitopes that are not present in the patient's infectious viral strain (Swadling et al., Vaccines (Basel). (2016) 4(3). Epub 2016 / 08 / 05. doi: 10.3390 / vaccines4030027. PubMed PMID: 27490575). However, some existing vaccines are designed using antigens that do not meet these criteria (Yalcin et al., J Clin Gastroenterol. (2003) 37(4):330-5; Hoa et al., supra; Yalcin et al., Infection. (2003) 31(4):221-5; Mancini-Bourgine et al., supra; Yang et al., J Viral Hepat. (2012) 19(8):581-93; Cavenaugh et al., supra; Godon et al., supra; Gane et al., supra; and Obeng-Adjei et al., Cancer Gene Ther. (2013) 20(12):652-62). In addition, many existing vaccines fail to induce virus-specific CD4 + T cells, CD8 + The complete combination of T cell and antibody responses (Dikici et al., supra; Pol et al., supra; Vandepapeliere et al., supra; Yalcin et al., J Clin Gastroenterol. (2003) 37(4):330-5; Al-mahaster, supra; Hoa et al., supra; Yalcin et al., Infection. (2003) 31(4):221-5; Mancini-Bourgine et al., supra; Yang et al., J Viral Hepat. (2012) 19(8):581-93; Gane et al., supra; and Zoulim et al., supra). These immune components are particularly important for the treatment of chronic HBV infection because CD8 +T cells have been shown to be the primary effector cells responsible for viral clearance during acute HBV infection in chimpanzees (Thimme et al., J Virol. (2003) 77(1): 68-76). In addition, antibodies that bind to HBV surface antigen (HBsAg) promote HBsAg clearance and prevent the spread of residual HBV. In addition, a high-magnitude immune response may be necessary to achieve a therapeutic effect, but many existing CHB vaccines fail to induce such a strong effector response (Mancini-Bourgine et al., supra; Yang et al., J Viral Hepat. (2012) 19(8): 581-93; Cavenaugh et al., supra; Gane et al., supra; and Zoulim et al., supra). Finally, some existing CHB vaccine antigens are not sufficiently stable in delivery vehicles to enable commercial-scale vaccine manufacturing. Summary of the Invention

[0008] In one aspect, a truncated hepatitis B virus (HBV) polymerase polypeptide is provided, for example, capable of inducing or eliciting an immune response in a human body when administered. In some embodiments, the truncated HBV polymerase polypeptide comprises an inactivated reverse transcriptase domain and an inactivated RNase H, and does not comprise all terminal protein (TP) domains and all or part of the spacer domain. In some embodiments, the length of the polypeptide is not longer than 600 amino acids, for example, a length of not longer than 595, 590, 585, 580, 575, 570, 565, 560, 555, 550, 545, 540 or 535 amino acids. In some embodiments, the reverse transcriptase domain does not comprise a YMDD motif (SEQ ID NO: 97) and the RNase H domain does not comprise an AELL motif (SEQ ID NO: 98). In some embodiments, the YMDD motif (SEQ ID NO: 97) in the reverse transcriptase domain is mutated to YMHD (SEQ ID NO: 99) and wherein the AELL motif (SEQ ID NO: 98) in the RNase H domain is mutated to AHLL (SEQ ID NO: 100). In some embodiments, the polypeptide is from HBV genotype A, B, C, or D. In some embodiments, (a) the polypeptide is from HBV genotype B and does not comprise a polypeptide sequence of SEQ ID NO:50, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:50 (e.g., the sequence is removed or deleted or not included); or (b) the polypeptide is from HBV genotype D and does not comprise a polypeptide sequence of SEQ ID NO:51, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:51. In some embodiments, the truncated HBV polymerase polypeptide comprises or consists of: any one of SEQ ID NOs: 13-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 13-14.

[0009] On the other hand, HBV polymerase deletion mutant polypeptides are provided. In some embodiments, the HBV polymerase deletion mutant polypeptide comprises a terminal protein (TP) domain, an inactivated reverse transcriptase domain, and an inactivated RNase H from the N-terminus to the C-terminus, wherein the mutant polypeptide does not comprise all or part of the spacer domain. In some embodiments, the length of the polypeptide is not longer than 800 amino acids, for example, a length of not longer than 795, 790, 785, 780, 775, 770, 765, 760, 755, 750, 745, 740, 735, 730, 725, 720, 715, 710, or 705 amino acids. In some embodiments, the reverse transcriptase domain does not comprise a YMDD motif (SEQ ID NO: 97) and the RNase H domain does not comprise an AELL motif (SEQ ID NO: 98). In some embodiments, the YMDD motif (SEQ ID NO: 97) in the reverse transcriptase domain is mutated to YMHD (SEQ ID NO: 99) and wherein the AELL motif (SEQ ID NO: 98) in the RNase H domain is mutated to AHLL (SEQ ID NO: 100). In some embodiments, the polypeptide is from HBV genotype A, B, C, or D. In some embodiments, (a) the polypeptide is from HBV genotype A and does not comprise SEQ ID NO: 42 or 46, or a polypeptide having a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 42 or 46; (b) the polypeptide is from HBV genotype B and does not comprise SEQ ID NO: 43 or 47, or a polypeptide having a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 43 or 47; (c) the polypeptide is from HBV genotype C and does not comprise SEQ ID NO: 44 or 48, or a polypeptide having a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: NO:44 or 48, or a polypeptide having a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:44 or 48; or (d) a polypeptide from HBV genotype D and does not comprise SEQ ID NO:45 or 49, or a polypeptide having a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:45 or 49.In some embodiments, the HBV polymerase deletion mutant polypeptide comprises or consists of any one of SEQ ID NO: 5-12, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 5-12. In some embodiments, the HBV polymerase deletion mutant polypeptide further comprises an HBV core polypeptide (e.g., a fusion protein comprising an HBV core polypeptide). In some embodiments, the HBV polymerase deletion mutant polypeptide comprises an HBV core polypeptide and an HBV polymerase deletion mutant polypeptide from N-terminus to C-terminus, as described herein. In some embodiments, the HBV polymerase deletion mutant polypeptide comprises or consists of: any one of SEQ ID NO:19-26, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:19-26.

[0010] In another aspect, an HBV core-sAg fusion protein is provided. In some embodiments, the core-sAg fusion protein comprises, from N-terminus to C-terminus, an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide. In various embodiments, the core polypeptide is from HBV genotype B or C, and the sAg polypeptide is from HBV genotype C. In some embodiments, the core polypeptide is from HBV genotype D and the sAg polypeptide is from HBV genotype D. In some embodiments, the core-sAg fusion protein comprises: (a) a core polypeptide comprising or consisting of SEQ ID NO: 65, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 65, and a sAg polypeptide comprising or consisting of SEQ ID NO: 3, or a sAg polypeptide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: NO:3 or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3; or (b) a core polypeptide comprising or consisting of SEQ ID NO:66, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:66, and a sAg polypeptide comprising or consisting of SEQ ID NO:4, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4 NO: 4 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the present invention. In some embodiments, the core polypeptide comprises a serine (S) residue at the amino acid position corresponding to position 12 and an asparagine (N) residue at the amino acid position corresponding to position 67, wherein the position numbering is with reference to SEQ ID NO: 65 or SEQ ID NO: 66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at the amino acid position corresponding to position 68, wherein the position numbering is with reference to SEQ ID NO: 3 or SEQ ID NO: 4.In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 53, an isoleucine (I) residue at an amino acid position corresponding to position 68, a threonine (T) residue at an amino acid position corresponding to position 125, a proline (P) residue at an amino acid position corresponding to position 127, a phenylalanine (F) residue at an amino acid position corresponding to position 161, a tyrosine (Y) residue at an amino acid position corresponding to position 200, a serine (S) residue at an amino acid position corresponding to position 210, and a leucine (L) residue at an amino acid position corresponding to position 213, wherein position numbers are with reference to SEQ ID NO: 3 or SEQ ID NO: 4. In various embodiments, the sAg polypeptide does not comprise a pre-S1 polypeptide. In various embodiments, the sAg polypeptide does not comprise a pre-S2 polypeptide. In some embodiments, the sAg polypeptide does not comprise an HBV pre-S2 polypeptide, the sAg polypeptide comprising or consisting of any one of SEQ ID NOs: 79-83, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 79-83. In some embodiments, the sAg polypeptide does not comprise both an HBV pre-S1 polypeptide and an HBV pre-S2 polypeptide. In some embodiments, the sAg polypeptide does not comprise an HBV pre-S1-pre-S2 polypeptide, and the sAg polypeptide comprises or consists of any one of SEQ ID NOs: 84-88, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 84-88. In various embodiments, the core-sAg fusion protein comprises a cleavable linker operably linked to and located between the HBV core polypeptide and the HBV sAg polypeptide. In some embodiments, the cleavable linker is a 2A cleavable peptide. In some embodiments, the cleavable linker is a 2A cleavable peptide selected from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and thosea asigna virus (T2A). In some embodiments, the cleavable linker is a porcine teschovirus-1 (P2A) linker.In some embodiments, the cleavable linker comprises or consists of the amino acid sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO:56), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:57), QCTNYALLKLAGDVESNPGP (SEQ ID NO:58), or EGRGSLLTCGDVEENPGP (SEQ ID NO:59), or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO:56), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:57), QCTNYALLKLAGDVESNPGP (SEQ ID NO:58), or EGRGSLLTCGDVEENPGP (SEQ ID NO:59). In some embodiments, the cleavable linker comprises or consists of the amino acid sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 56), or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 56). In some embodiments, the core-sAg fusion protein comprises a flexible linker and / or a furin recognition / cleavage site operably linked to the N-terminus of the cleavable linker and the C-terminus of the HBV core polypeptide and located therebetween. In some embodiments, the furin recognition / cleavage site comprises or consists of an amino acid sequence selected from the group consisting of RAKR (SEQ ID NO: 60), REKR (SEQ ID NO: 61), and RRKR (SEQ ID NO: 62). In some embodiments, the flexible linker comprises a polyglycine or polyalanine sequence. In some embodiments, the flexible linker comprises or consists of a polyglycine or polyalanine sequence selected from the group consisting of AA, AAA, AAY, GG, GGG, GGS, GSG, and GGGS (SEQ ID NO: 63). In some embodiments, the core-sAg fusion protein is no longer than 450 amino acids, e.g., no longer than 445, 440, 435, 430, 425, 420, 415, or 410 amino acids.In some embodiments, the core-sAg fusion protein comprises or consists of: an amino acid sequence of any one of SEQ ID NOs: 38-41 (e.g., SEQ ID NO: 41), or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41 (e.g., SEQ ID NO: 41). In some embodiments, the fusion polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 12, an asparagine (N) residue at the amino acid position corresponding to position 67, a valine (V) residue at the amino acid position corresponding to position 74, a phenylalanine (F) residue at the amino acid position corresponding to position 97, a threonine (T) residue at the amino acid position corresponding to position 249, a threonine (T) residue at the amino acid position corresponding to position 250, a serine (S) residue at the amino acid position corresponding to position 317, a serine (S) residue at the amino acid position corresponding to position 318, an arginine (R) residue at the amino acid position corresponding to position 326, a tyrosine (Y) residue at the amino acid position corresponding to position 338, a glycine (G) residue at the amino acid position corresponding to position 363, and an alanine (A) residue at the amino acid position corresponding to position 372, wherein positions are numbered with reference to SEQ ID NO: 41. In various embodiments, the core-sAg fusion polypeptide does not comprise an amino sequence from an HBV protein or a fragment thereof selected from the group consisting of: X, pre-core, pre-S1, and pre-S2.

[0011] With respect to immunogenic HBV polypeptides, in some embodiments, the truncated HBV polymerase polypeptide, HBV polymerase deletion mutant polypeptide, or core-sAg fusion protein as described herein further comprises an N-terminal signal peptide or leader sequence. In various embodiments, the signal peptide or leader sequence is derived from a source protein selected from a serum protein, a cytokine, a chemokine, a chaperone protein, an invariant protein, and a protein that directs a protein to the lysosomal compartment. In various embodiments, the signal peptide or leader sequence is from a source protein selected from the group consisting of colony stimulating factor 2 (CSF2, GM-CSF), tissue plasminogen activator (PLAT, t-PA), CC motif chemokine ligand 7 (CCL7, MCP-3), CXC motif chemokine ligand 10 (CXCL10, IP-10), catenin beta 1 (CTNNB1), CD74 (p33; DHLAG; HLADG; Ia-GAMMA, invariant chain), serum albumin (ALB), polyubiquitin B / C (UBB / UBC), calreticulin (CALR), vesicular stomatitis virus G protein (VSV-G), lysosomal associated membrane protein 1 (LAMP-1), and lysosomal associated membrane protein 2 (LAMP-2). In some embodiments, the signal peptide or leader sequence is selected from any one of SEQ ID NOs: 67-78, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 67-78. In various embodiments, the truncated HBV polymerase polypeptide, HBV polymerase deletion mutant polypeptide and / or core-sAg fusion protein as described herein can be recombinantly produced or chemically synthesized. In various embodiments, the truncated HBV polymerase polypeptide, HBV polymerase deletion mutant polypeptide and / or core-sAg fusion protein as described herein can induce, promote or stimulate an immune response in a human (e.g., expansion and / or activation of CD8+ and / or CD4+ T cells; production of antibodies that bind to and / or neutralize one or more of HBV polymerase, HBV core and HBV sAg). In various embodiments, the truncated HBV polymerase polypeptides, HBV polymerase deletion mutant polypeptides and / or core-sAg fusion proteins as described herein are capable of inducing, promoting or stimulating an immune response against HBV in a human body (e.g., they prevent HBV infection, delay the progression of HBV infection, inhibit and / or reverse HBV infection). In various embodiments, the truncated HBV polymerase polypeptides, HBV polymerase deletion mutant polypeptides and / or core-sAg fusion proteins as described herein are capable of inducing, promoting or stimulating the proliferation and / or activation of one or more cell types selected from monocyte-derived dendritic cells (DCs), CD8+ T cells and CD4+ T cells.

[0012] On the other hand, polynucleotides encoding immunogenic HBV polypeptides as described herein are provided. For example, polynucleotides encoding one or more of the truncated HBV polymerase polypeptides, HBV polymerase deletion mutant polypeptides, or core-sAg fusion proteins as described herein are provided. In some embodiments, the polynucleotides include cDNA, mRNA, self-amplifying RNA (SAM), self-replicating RNA, or self-amplifying replicon RNA (RepRNA). In some embodiments, the polynucleotides include self-replicating or self-amplifying alphavirus replicons. In some embodiments, the polynucleotide comprises or consists of SEQ ID NOs: 27-37 (e.g., SEQ ID NO: 37) and any one of 89-94 (e.g., SEQ ID NOs: 29, 89, 90, or 92), or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 27-37 (e.g., SEQ ID NO: 37) and any one of 89-94 (e.g., SEQ ID NOs: 29, 89, 90, or 92).

[0013] In another aspect, provided is a lipid nanoparticle (LNP) comprising one or more of the polynucleotides encoding the immunogenic HBV polypeptides described herein.

[0014] On the other hand, there is provided an expression cassette comprising one or more of the polynucleotides encoding immunogenic HBV polypeptides as described herein, these polynucleotides being operably connected to one or more regulatory sequences. In some embodiments, the polynucleotides are operably connected to a constitutive promoter and are under its control. In some embodiments, the promoter is selected from cytomegalovirus major immediate early promoter (CMV), the CMV enhancer fused to chicken beta-actin promoter (CAG), human elongation factor -1 alpha (HEF-1 alpha), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor -1 alpha (CHEF-1 alpha) and phosphoglycerate kinase (PGK).

[0015] On the other hand, one or more of the polynucleotides encoding immunogenic HBV polypeptides as described herein, or one or more expression cassettes comprising such polynucleotides are provided. In various embodiments, the vector is a plasmid vector, a bacterial vector or a viral vector. In some embodiments, the vector is a viral vector. In various embodiments, the viral vector is a DNA virus or an RNA virus. In some embodiments, the viral vector is from a virus selected from adenovirus, adeno-associated virus, arenavirus, alphavirus, poxvirus, cytomegalovirus, rhabdovirus, vesicular stomatitis virus, flavivirus, Maraba virus and vaccinia virus. In some embodiments, the viral vector is from a virus selected from the taxonomic family of Adenoviridae, Arenaviridae, Herpesviridae (e.g., cytomegalovirus), Poxviridae (e.g., vaccinia virus, e.g., modified vaccinia Ankara (MVA)), Flaviviridae (e.g., yellow fever virus), Rhabdoviridae (e.g., vesiculovirus, e.g., Maraba vesiculovirus), Togaviridae (e.g., alphavirus). In some embodiments, the viral vector is an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV), Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Lujo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV). In some embodiments, the viral vector is an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV) or Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)). In some embodiments, the viral vector is a human adenovirus or a simian adenovirus (eg, a chimpanzee adenovirus, a gorilla adenovirus, or a rhesus macaque adenovirus).In some embodiments, the viral vector is an adenovirus vector selected from the group consisting of adenovirus serotype 5 (Ad5), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), chimpanzee adenovirus (e.g., ChAdOx1, ChAdOx2, ChAd3 (AdC3), ChAd5 (AdC5), ChAd6 (AdC6), ChAd7 (AdC7), ChAd8 (AdC8), , ChAd9(AdC9), ChAd10(AdC10), ChAd11(AdC11), ChAd17(AdC17), ChAd16(AdC16), ChAd19(AdC19), ChAd20(A dC20), ChAd22(AdC22), ChAd24(AdC24), ChAdY25, ChAd26(AdC26), ChAd28(AdC28), ChAd30(AdC30), ChAd31(A dC31), ChAd37(AdC37), ChAd38(AdC38), ChAd43(AdC43), ChAd44(AdC44), ChAd55(AdC55), ChAd63(AdC63), C hAdV63, ChAd68(AdC68), ChAd73(AdC73), ChAd82(AdC82), ChAd83(AdC83), ChAd143(AdC143), ChAd144(AdC14 4), ChAd145 (AdC145), ChAd147 (AdC147), gorilla adenovirus (e.g., GC44, GC45, GC46), and rhesus macaque adenovirus (e.g., RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65, RhAd66). In some embodiments, the viral vector is replication-defective, replication-deficient, replication-attenuated, or replication-competent. In some embodiments, the viral vector is a replication-defective arenavirus having a bi-segmented genome. In some embodiments, the viral vector is a replication-attenuated arenavirus having a tri-segmented genome.

[0016] In another aspect, an arenavirus vector is provided. In one embodiment, an arenavirus vector is provided, comprising a polynucleotide encoding an HBV core-sAg fusion polypeptide comprising or consisting of any one of SEQ ID NOs: 38-41 (e.g., SEQ ID NO: 41), or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-41 (e.g., SEQ ID NO: 41), and wherein the sAg polypeptide does not comprise an HBV pre-S1 polypeptide and / or an HBV pre-S2 polypeptide. In some embodiments, the core polypeptide comprises a serine (S) residue at the amino acid position corresponding to position 12 and an asparagine (N) residue at the amino acid position corresponding to position 67, wherein position numbering is with reference to SEQ ID NO: 65 or SEQ ID NO: 66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at the amino acid position corresponding to position 68, wherein positions are numbered with reference to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 53, an isoleucine (I) residue at an amino acid position corresponding to position 68, a threonine (T) residue at an amino acid position corresponding to position 125, a proline (P) residue at an amino acid position corresponding to position 127, a phenylalanine (F) residue at an amino acid position corresponding to position 161, a tyrosine (Y) residue at an amino acid position corresponding to position 200, a serine (S) residue at an amino acid position corresponding to position 210, and a leucine (L) residue at an amino acid position corresponding to position 213, wherein positions are numbered with reference to SEQ ID NO: 3 or SEQ ID NO: 4.In some embodiments, the core-sAg fusion polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 12, an asparagine (N) residue at the amino acid position corresponding to position 67, a valine (V) residue at the amino acid position corresponding to position 74, a phenylalanine (F) residue at the amino acid position corresponding to position 97, a threonine (T) residue at the amino acid position corresponding to position 249, a threonine (T) residue at the amino acid position corresponding to position 250, a serine (S) residue at the amino acid position corresponding to position 317, a serine (S) residue at the amino acid position corresponding to position 318, an arginine (R) residue at the amino acid position corresponding to position 326, a tyrosine (Y) residue at the amino acid position corresponding to position 338, a glycine (G) residue at the amino acid position corresponding to position 363, and an alanine (A) residue at the amino acid position corresponding to position 372, wherein positions are numbered with reference to SEQ ID NO: 41. In some embodiments, the polynucleotide comprises or consists of any one of SEQ ID NOs: 33-37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 33-37. In some embodiments, the polynucleotide comprises or consists of SEQ ID NO: 37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. In some embodiments, the arenavirus vector has a bi-segmented genome and further comprises a polynucleotide encoding a truncated HBV polymerase comprising or consisting of any one of SEQ ID NOs: 13-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 13-14, and wherein the truncated HBV polymerase does not comprise all of the HBV polymerase terminal protein (TP) domain and does not comprise all or part of the HBV polymerase spacer domain.In some embodiments, the truncated HBV polymerase does not comprise a polypeptide sequence of SEQ ID NO:50 or SEQ ID NO:51, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to either SEQ ID NO:50 or SEQ ID NO:51. In some embodiments, the polynucleotide comprises or consists of any one of SEQ ID NOs: 29 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, or 92), or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 29 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, or 92). In some embodiments, the arenavirus vector is a lymphocytic choriomeningitis mammalian arenavirus (LCMV) vector and the polynucleotide comprises or consists of SEQ ID NO: 29, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 29. In some embodiments, the arenavirus vector is a Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)) and the polynucleotide comprises or consists of SEQ ID NO: 90, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 90.

[0017] Also provided is an arenavirus vector comprising a polynucleotide encoding a truncated HBV polymerase comprising or consisting of: any one of SEQ ID NOs: 13-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 13-14, and wherein the truncated HBV polymerase does not comprise all of the HBV polymerase terminal protein (TP) domain and does not comprise all or part of the HBV polymerase spacer domain. In some embodiments, the truncated HBV polymerase does not comprise a polypeptide sequence of SEQ ID NO:50 or SEQ ID NO:51, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to either SEQ ID NO:50 or SEQ ID NO:51. In some embodiments, the polynucleotide comprises or consists of any one of SEQ ID NOs: 29 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, or 92), or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 29 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, or 92). In some embodiments, the arenavirus vector is a lymphocytic choriomeningitis mammalian arenavirus (LCMV) vector and the polynucleotide comprises or consists of SEQ ID NO: 29, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 29. In some embodiments, the arenavirus vector is a Cali mammalian arenavirus vector and the polynucleotide comprises or consists of SEQ ID NO: 90, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 90. In some embodiments, the arenavirus vector is replication defective, replication-deficient, or replication-incompetent.

[0018] On the other hand, a host cell is provided, which comprises one or more polynucleotides encoding one or more immunogenic HBV polypeptides as described herein, or one or more vectors comprising such polynucleotides. In some embodiments, the one or more polynucleotides encoding one or more immunogenic HBV polypeptides as described herein are not integrated into the host cell genome, for example, are free. In some embodiments, the one or more polynucleotides are integrated into the host cell genome. In some embodiments, the host cell is a mammalian cell, such as a human cell. In various embodiments, the host cell can be in vitro or in vivo.

[0019] On the other hand, there is provided an immunogenic composition comprising one or more of the immunogenic HBV polypeptides as described herein. In some embodiments, the immunogenic composition comprises one or more (e.g., two or more) of the truncated HBV polymerase polypeptides as described herein, one or more (e.g., two or more) of the HBV polymerase deletion mutant polypeptides and / or one or more (e.g., two or more) of the core-sAg fusion proteins. In some embodiments, the immunogenic composition comprises one or more (e.g., two or more) of the truncated HBV polymerase polypeptides as described herein, one or more (e.g., two or more) of the HBV polymerase deletion mutant polypeptides and / or one or more (e.g., two or more) of the core-sAg fusion proteins. In some embodiments, the immunogenic composition comprises one or more (e.g., two or more) of the vectors, which comprise one or more (e.g., two or more) of the truncated HBV polymerase polypeptides as described herein, one or more (e.g., two or more) of the HBV polymerase deletion mutant polypeptides and / or one or more (e.g., two or more) of the core-sAg fusion proteins. The immunogenic composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises one or more polynucleotides in the form of DNA, cDNA, mRNA, or self-replicating RNA. In various embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a truncated HBV polymerase polypeptide or an HBV polymerase deletion mutant polypeptide as described herein; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein.In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding an HBV polymerase polypeptide mutant comprising or consisting of: any one of SEQ ID NOs: 5-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 5-14; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising or consisting of: any one of SEQ ID NOs: 38-41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41. An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NOs:38-41. In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding an HBV polymerase polypeptide mutant comprising or consisting of any one of SEQ ID NOs: 13-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 13-14; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising or consisting of any one of SEQ ID NOs: 38-41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41. An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NOs:38-41.In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding an HBV polymerase polypeptide mutant comprising or consisting of SEQ ID NO: 13, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising or consisting of SEQ ID NO: 41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41. NO: 41 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the present invention. In some embodiments, the core polypeptide comprises a serine (S) residue at the amino acid position corresponding to position 12 and an asparagine (N) residue at the amino acid position corresponding to position 67, wherein the position numbering is with reference to SEQ ID NO: 65 or SEQ ID NO: 66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at the amino acid position corresponding to position 68, wherein the position numbering is with reference to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 53, an isoleucine (I) residue at the amino acid position corresponding to position 68, a threonine (T) residue at the amino acid position corresponding to position 125, a proline (P) residue at the amino acid position corresponding to position 127, a phenylalanine (F) residue at the amino acid position corresponding to position 161, a tyrosine (Y) residue at the amino acid position corresponding to position 200, a serine (S) residue at the amino acid position corresponding to position 210, and a leucine (L) residue at the amino acid position corresponding to position 213, wherein the positions are numbered with reference to SEQ ID NO: 3 or SEQ ID NO: 4.In some embodiments, the core-sAg fusion polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 12, an asparagine (N) residue at the amino acid position corresponding to position 67, a valine (V) residue at the amino acid position corresponding to position 74, a phenylalanine (F) residue at the amino acid position corresponding to position 97, a threonine (T) residue at the amino acid position corresponding to position 249, a threonine (T) residue at the amino acid position corresponding to position 250, a serine (S) residue at the amino acid position corresponding to position 317, a serine (S) residue at the amino acid position corresponding to position 318, an arginine (R) residue at the amino acid position corresponding to position 326, a tyrosine (Y) residue at the amino acid position corresponding to position 338, a glycine (G) residue at the amino acid position corresponding to position 363, and an alanine (A) residue at the amino acid position corresponding to position 372, wherein positions are numbered with reference to SEQ ID NO: 41. In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any one of SEQ ID NOs: 27-32 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, or 92), or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 27-32 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, or 92); and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of any one of SEQ ID NOs: 33-37, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 33-37. A nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NOs:33-37.In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 29 or 90, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29 or 90; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. NO: 37 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of the above. In various embodiments, the first viral expression vector and the second viral expression vector are independently from a taxonomic family selected from the family Adenoviridae, Arenaviridae, Herpesviridae (e.g., cytomegalovirus), Poxviridae (e.g., vaccinia virus, e.g., Modified Vaccinia Ankara (MVA)), Flaviviridae (e.g., yellow fever virus), Rhabdoviridae (e.g., vesiculovirus, e.g., Maraba vesiculovirus), Togaviridae (e.g., alphavirus). In various embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition may be from the same taxonomic family or different taxonomic families. In some embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition are from the family Arenaviridae. In some embodiments, the first and second viral expression vectors in the immunogenic composition are independently derived from an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV), Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV). In some embodiments, the first and second viral expression vectors are independently derived from an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV) or Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)). In some embodiments, the first and second viral expression vectors are replication-defective or replication-defective. In some embodiments, the first and second viral expression vectors are replication-attenuated.In some embodiments, the immunogenic composition comprises a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: (a) the first LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 29, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29; and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. A nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NO:37. In some embodiments, the immunogenic composition comprises a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: (a) the first Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO:90, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:90; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO:37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:37. NO:37 any one of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleic acid sequence. In various embodiments, in the immunogenic composition with 1:10 to 10:1, for example 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1 or 1:1 ratio provides the first viral expression vector and the second viral expression vector. In some embodiments, the immunogenic composition comprises per milliliter at about 10. 3 to about 10 12 The concentration of virus is in the range of about 10 focus-forming units (FFU) or plaque-forming units (PFU) or infectious units (IU) or virus particles (vp) per ml (e.g., about 10 per ml). 4to about 10 7 FFU or PFU or IU or vp of virus, for example, about 10 per ml 3 to about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some embodiments, the immunogenic composition is a composition comprising a first viral expression vector and a second viral expression vector containing at least one viral FFU or PFU or IU or vp. In some embodiments, the immunogenic composition further comprises one or more adjuvants, detergents, micelle formers, and oils. In various embodiments, the immunogenic composition is formulated for administration via a route selected from intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., buccal, intranasal, rectal, vaginal). In some embodiments, the immunogenic composition is an aqueous solution or suspension, for example, formulated into a liquid. In some embodiments, the immunogenic composition is lyophilized.

[0020] On the other hand, a kit is provided. In various embodiments, the kit includes one or more (e.g., two or more) overall doses of one or more (e.g., two or more) of one or more (e.g., two or more) of truncated HBV polymerase polypeptides as described herein, one or more (e.g., two or more) of HBV polymerase deletion mutant polypeptides, and / or one or more (e.g., two or more) of core-sAg fusion proteins. In some embodiments, the kit includes one or more (e.g., two or more) overall doses of one or more (e.g., two or more) ... In various embodiments, the kit includes one or more (e.g., two or more) overall doses of one or more (e.g., two or more) immunogenic compositions as described herein. In some embodiments, the one or more overall doses in the kit are in a single container. In some embodiments, the one or more overall doses in the kit are in two or more separate containers. In some embodiments, the kit includes one or more containers selected from vials, ampoules, and preloaded syringes. In some embodiments, the kit includes one or more containers that hold one or more polypeptides, one or more polynucleotides, one or more vectors, or one or more immunogenic compositions in an aqueous solution or suspension or as a lyophilized formulation. In various embodiments, the one or more overall doses may be the same or different. In some embodiments, the kit includes one or more overall doses of one or more viral vectors as described herein, wherein the overall dose is about 10 3 to about 10 12 The viral focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or virus particles (vp) range is about 10 4 to about 10 7 Virus FFU or PFU or IU or vp, for example about 10 3 to about 10 4 , 10 5 , 10 6 , 10 7, 10 8 , 10 9 , 10 10 , 10 11 or 10 12In some embodiments, the kit includes one or more polynucleotides encoding at least two immunogenic polypeptides, or one or more vectors expressing at least two immunogenic polypeptides, or an immunogenic composition comprising at least two immunogenic polypeptides, the immunogenic polypeptides comprising: (a) an HBV polymerase polypeptide mutant comprising or consisting of any one of SEQ ID NOs: 5-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 5-14; and (b) an HBV core-sAg fusion protein comprising or consisting of any one of SEQ ID NOs: 38-41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41. An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NOs:38-41. In some embodiments, the kit includes one or more polynucleotides encoding at least two immunogenic polypeptides, or one or more vectors expressing at least two immunogenic polypeptides, or an immunogenic composition comprising at least two immunogenic polypeptides, wherein the immunogenic polypeptides comprise: (a) an HBV polymerase polypeptide mutant comprising or consisting of any one of SEQ ID NOs: 13-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 13-14; and (b) an HBV core-sAg fusion protein comprising or consisting of any one of SEQ ID NOs: 38-41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41. NO:38-41 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence. In some embodiments, the kit includes one or more polynucleotides encoding at least two immunogenic polypeptides, or one or more vectors expressing at least two immunogenic polypeptides, or an immunogenic composition comprising at least two immunogenic polypeptides, the immunogenic polypeptides comprising: (a) an HBV polymerase polypeptide mutant comprising or consisting of SEQ ID NO:13,or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13; and (b) an HBV core-sAg fusion protein comprising or consisting of SEQ ID NO: 41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41. In some embodiments, the core polypeptide comprises a serine (S) residue at the amino acid position corresponding to position 12 and an asparagine (N) residue at the amino acid position corresponding to position 67, wherein position numbering is with reference to SEQ ID NO: 65 or SEQ ID NO: 66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at the amino acid position corresponding to position 68, wherein position numbering is with reference to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 53, an isoleucine (I) residue at the amino acid position corresponding to position 68, a threonine (T) residue at the amino acid position corresponding to position 125, a proline (P) residue at the amino acid position corresponding to position 127, a phenylalanine (F) residue at the amino acid position corresponding to position 161, a tyrosine (Y) residue at the amino acid position corresponding to position 200, a serine (S) residue at the amino acid position corresponding to position 210, and a leucine (L) residue at the amino acid position corresponding to position 213, wherein the positions are numbered with reference to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the core-sAg fusion polypeptide comprises a serine (S) residue at the amino acid position corresponding to position 12, an asparagine (N) residue at the amino acid position corresponding to position 67, a valine (V) residue at the amino acid position corresponding to position 74, a phenylalanine (F) residue at the amino acid position corresponding to position 97, a threonine (T) residue at the amino acid position corresponding to position 249, a threonine (T) residue at the amino acid position corresponding to position 250, a serine (S) residue at the amino acid position corresponding to position 317, a serine (S) residue at the amino acid position corresponding to position 318, an arginine (R) residue at the amino acid position corresponding to position 326, a tyrosine (Y) residue at the amino acid position corresponding to position 338,one or more of a glycine (G) residue at the amino acid position corresponding to position 363 and an alanine (A) residue at the amino acid position corresponding to position 372, wherein the positions are numbered with reference to SEQ ID NO: 41. In some embodiments, the kit comprises a first vector and a second vector encoding a first immunogenic polypeptide and a second immunogenic polypeptide, respectively, the first immunogenic polypeptide and the second immunogenic polypeptide, respectively comprising: (a) an HBV polymerase polypeptide mutant comprising or consisting of SEQ ID NO: 13, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13; and (b) an HBV core-sAg fusion protein comprising or consisting of SEQ ID NO: 41, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13. NO:41 is an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence. In some embodiments, the kit comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of: any one of SEQ ID NOs: 27-32 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, or 92), or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 27-32 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, or 92); and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of: any one of SEQ ID NOs: 33-37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 33-37 NO:33-37 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NO:33-37. In some embodiments, the kit includes a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO:29 or 90, or a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:33-37.(a) the first viral expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37. In some embodiments, the kit comprises one or more total doses of an immunogenic composition comprising a first viral expression vector and a second viral expression vector as described herein, wherein the first viral expression vector and the second viral expression vector comprise replication-deficient or replication-defective Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)). In some embodiments, the kit comprises one or more total doses of an immunogenic composition comprising a first viral expression vector and a second viral expression vector as described herein, wherein the first viral expression vector and the second viral expression vector comprise replication-deficient or replication-defective lymphocytic choriomeningitis mammalian arenavirus (LCMV). In some embodiments, the kit comprises (a) one or more total doses of an immunogenic composition as described herein, wherein the first viral expression vector and the second viral expression vector are from the Adenoviridae family; and (b) one or more total doses of an immunogenic composition as described herein, wherein the first viral expression vector and the second viral expression vector are from the Poxviridae family (e.g., vaccinia virus, e.g., modified vaccinia Ankara (MVA)). In some embodiments, the kit comprises (a) one or more total doses of an immunogenic composition as described herein, wherein the first viral expression vector and the second viral expression vector are from the Arenaviridae family; and (b) one or more total doses of an immunogenic composition as described herein, wherein the first viral expression vector and the second viral expression vector are from the Adenoviridae family. In some embodiments, the kit comprises a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: (a) the first LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of: SEQ ID NO: 29, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO:37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:37. In some embodiments, the kit includes a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: (a) the first Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO:90, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:90; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO:37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:37. NO:37 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequence of nucleic acid sequence. In various embodiments, test kit also includes one or more additional therapeutic agents of one or more overall doses. In some embodiments, test kit also includes one or more agonists or activators of one or more Toll-like receptors (TLR). In some embodiments, test kit also includes one or more TLR agonists or activators selected from TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR7 agonist, TLR8 agonist and TLR9 agonist. In some embodiments, the kit further comprises a TLR7 agonist selected from GS 9620 (vesatolimod), R848 (Resiquimod), DS-0509, LHC-165, and TMX-101 (imiquimod). In some embodiments, the kit further comprises a TLR8 agonist selected from GS-9688, R848 (Resiquimod), and NKTR-262 (dual TLR7 / TLR8 agonist). In some embodiments, the kit further comprises one or more interleukin receptor agonists selected from the interleukin receptors of IL-2, IL-7, IL-12, and IL-15. In some embodiments, the kit further comprises one or more interleukin receptor agonists selected from the interleukin receptors of IL-2, IL-7, IL-12, and IL-15.One or more cytokines of IL-15 and their variants. In some embodiments, the kit also includes one or more innate immune activators. In some embodiments, the kit also includes one or more innate immune activators, including agonists selected from the following receptors: fms-related tyrosine kinase 3 (FLT3), interferon gene stimulator (STING) receptor, DExD / H-box helicase 58 (DDX58; also known as RIG-I), oligomeric domain protein 2 (NOD2) containing nucleotide binding. In some embodiments, the kit also includes one or more overall doses of GS-3583 and / or GS-9992. In some embodiments, the kit also includes one or more antagonists or inhibitors of inhibitory immune checkpoint proteins or receptors and / or one or more activators or agonists of stimulating immune checkpoint proteins or receptors. In some embodiments, the kit further comprises one or more immune checkpoint proteins or receptors selected from the group consisting of: CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain-containing 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244 (SLAMF4); CD276 (B7H3); V-set domain-containing T cell activation inhibitor 1 (VTCN1, B7H4); V-set immunomodulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR-associated 2 (HHLA2, B7H7); inducible T cell costimulator (ICOS, CD278); inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFR SF9 (CD137), TNFSF9 (CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF); TNFRSF18 (GITR),TNFSF18 (GITRL); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155); PVR-related immunoglobulin domain-containing (PVRIG, CD112R); with Ig and ITIM structures T cell immunoreceptor containing 1 domain (TIGIT); T cell immunoglobulin and mucin domain-containing 4 (TIMD4; TIM4); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); lymphocyte activation 3 (LAG3, CD223); signal transducer lymphocyte activation molecule family member 1 (SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, CD319); UL ULBP1; UL16 binding protein 2 (ULBP2); UL16 binding protein 3 (ULBP3); retinoic acid early transcript 1E (RAET1E; ULBP4); retinoic acid early transcript 1G (RAET1G; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6); lymphocyte activation 3 (CD223); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); killer cell lectin-like receptor K1 ( KLRK1, NKG2D, CD314); killer cell lectin-like receptor C2 (KLRC2, CD159c, NKG2C); killer cell lectin-like receptor C3 (KLRC3, NKG2E); killer cell lectin-like receptor C4 (KLRC4, NKG2F); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); killer cell immunoglobulin-like receptor,Three Ig domains and long cytoplasmic tail 1 (KIR3DL1); Killer cell lectin-like receptor D1 (KLRD1); and SLAM family member 7 (SLAMF7). In some embodiments, the kit also includes one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the kit further comprises one or more T cell inhibitory immune checkpoint proteins or receptors selected from the group consisting of: CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4); V-set immunomodulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA) ); PVR-associated immunoglobulin domain-containing (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1). In some embodiments, the kit further comprises one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors. In some embodiments, the kit further comprises one or more T cell stimulatory immune checkpoint proteins or receptors selected from the following: CD27, CD70; CD40, CD40LG; inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR),TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). In some embodiments, the kit also includes one or more total doses of AGEN-2373 and / or AGEN-1223. In some embodiments, the kit also includes one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the kit further comprises one or more NK cell inhibitory immune checkpoint proteins or receptors selected from the group consisting of killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); and killer cell lectin-like receptor D1 (KLRD1, CD94). In some embodiments, the kit further comprises one or more agonists or activators of one or more NK cell stimulatory immune checkpoint proteins or receptors. In some embodiments, the kit further comprises one or more NK cell stimulatory immune checkpoint proteins or receptors selected from the group consisting of: CD16, CD226 (DNAM-1); killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314); and SLAM family member 7 (SLAMF7). In some embodiments, the kit further comprises one or more protein inhibitors of PD-L1 (CD274), PD-1 (PDCD1), and / or CTLA4. In some embodiments, the kit further comprises one or more protein inhibitors of CTLA4 selected from the group consisting of ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884, BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044, CG-0161, ATOR-1144, PBI-5D3H5, FPT-155 (CTLA4 / PD-L1 / CD28),PF-06936308 (PD-1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), MEDI-5752 (CTLA4 / PD-1), XmAb-20717 (PD-1 / CTLA4) and AK-104 (CTLA4 / PD-1). In some embodiments, the kit further comprises one or more protein inhibitors of PD-L1 (CD274) or PD-1 (PDCD1) selected from the group consisting of zimberelimab (AB122), pembrolizumab, nivolumab, cemiplimab, pidilizumab, AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, and PD-L1 (CD274). izumab), avelumab, ASC22, durvalumab, BMS-936559, CK-301, PF-06801591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055), AK-103 (HX-008), AK-105, CS-1003, HLX-10, MGA-012, BI-754091, AGEN-2034, JS-001 (toripalimab) ), JNJ-63723283, genolimzumab (CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR-1210 (camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306 (MSB0010718C), CX-072, CBT-502, TSR-042 (dostarlimab), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308 (sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-013 (PD-1 / LAG-3),FS-118(LAG-3 / PD-L1)MGD-019(PD-1 / CTLA4), KN-046(PD-1 / CTLA4), MEDI-5752(CTLA4 / PD-1), RO-7121661(PD-1 / TIM-3), XmAb-20717(PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), M7824 (PD-L1 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM3 / PDL1) and INBRX-105 (4-1BB / PDL1). In some embodiments, the kit further comprises one or more small molecule inhibitors of CD274 (PDL1, PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1), and / or CTLA4. In some embodiments, the kit further comprises one or more small molecule inhibitors of CD274 or PDCD1 selected from GS-4224, GS-4416, INCB086550, and MAX10181. In some embodiments, the kit further comprises BPI-002, a small molecule inhibitor of CTLA4. In some embodiments, the kit further comprises one or more antiviral agents. In some embodiments, the kit further comprises one or more antiviral agents selected from the group consisting of lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF or ) and ledipasvir + sofosbuvir In some embodiments, the kit further comprises one or more therapeutic agents selected from the following: HBV antigen inhibitors (e.g., HBV core antigen (HBcAg) inhibitors, HBV surface antigen (HBsAg) inhibitors, HBx inhibitors, HBV E antigen inhibitors), anti-HBV antigen antibodies, inhibitory nucleic acids targeting HBV (e.g., antisense oligonucleotides, short interfering RNA (siRNA), DNA-guided RNA interference (ddRNAi)), gene editors targeting HBV (e.g., CRISPR-Cas (e.g., Cas9, Cas12, Cascade, Cas13), zinc finger nucleases, homing endonucleases, homing meganucleases (e.g., ARCUS), synthetic nucleases, TALENs), covalently closed circular DNA (cccDNA) inhibitors, and HBsAg secretion or assembly inhibitors and HBV viral entry inhibitors.

[0021] On the other hand, a method for eliciting an immune response against human hepatitis B virus (HBV) in a subject in need thereof is provided. Also provided is a method for treating or preventing human hepatitis B virus (HBV) in a subject in need thereof. In some embodiments, the method includes administering a therapeutically effective amount of one or more immunogenic compositions as described herein to the subject. In some embodiments, the method requires administering one or more immunogenic compositions, which comprise a mixture containing a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a truncated HBV polymerase polypeptide or an HBV polymerase deletion mutant polypeptide as described herein; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein as described herein. In some embodiments, the method requires administering to the subject a therapeutically effective amount of one or more immunogenic compositions, the one or more immunogenic compositions comprising a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding an HBV polymerase polypeptide mutant comprising or consisting of any one of SEQ ID NOs: 5-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 5-14; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising or consisting of any one of SEQ ID NOs: 38-41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41. An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NOs:38-41.In some embodiments, the method requires administering to the subject a therapeutically effective amount of one or more immunogenic compositions, the one or more immunogenic compositions comprising a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding an HBV polymerase polypeptide mutant comprising or consisting of any one of SEQ ID NOs: 13-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 13-14; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising or consisting of any one of SEQ ID NOs: 38-41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41. An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NOs:38-41. In some embodiments, the method requires administering to the subject a therapeutically effective amount of one or more immunogenic compositions, the one or more immunogenic compositions comprising a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding an HBV polymerase polypeptide mutant comprising or consisting of SEQ ID NO: 13, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising or consisting of SEQ ID NO: 41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41. NO: 41 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence. In some embodiments, the core polypeptide comprises a serine (S) residue at the amino acid position corresponding to position 12 and an asparagine (N) residue at the amino acid position corresponding to position 67, wherein positions are numbered with reference to SEQ ID NO: 65 or SEQ ID NO: 66.In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at the amino acid position corresponding to position 68, wherein positions are numbered with reference to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 53, an isoleucine (I) residue at an amino acid position corresponding to position 68, a threonine (T) residue at an amino acid position corresponding to position 125, a proline (P) residue at an amino acid position corresponding to position 127, a phenylalanine (F) residue at an amino acid position corresponding to position 161, a tyrosine (Y) residue at an amino acid position corresponding to position 200, a serine (S) residue at an amino acid position corresponding to position 210, and a leucine (L) residue at an amino acid position corresponding to position 213, wherein positions are numbered with reference to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the core-sAg fusion polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 12, an asparagine (N) residue at the amino acid position corresponding to position 67, a valine (V) residue at the amino acid position corresponding to position 74, a phenylalanine (F) residue at the amino acid position corresponding to position 97, a threonine (T) residue at the amino acid position corresponding to position 249, a threonine (T) residue at the amino acid position corresponding to position 250, a serine (S) residue at the amino acid position corresponding to position 317, a serine (S) residue at the amino acid position corresponding to position 318, an arginine (R) residue at the amino acid position corresponding to position 326, a tyrosine (Y) residue at the amino acid position corresponding to position 338, a glycine (G) residue at the amino acid position corresponding to position 363, and an alanine (A) residue at the amino acid position corresponding to position 372, wherein positions are numbered with reference to SEQ ID NO: 41.In some embodiments, the method entails administering to the subject a therapeutically effective amount of one or more immunogenic compositions comprising a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of: a nucleic acid sequence of any one of SEQ ID NOs: 27-32 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, or 92), or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 27-32 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, or 92); and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of: a nucleic acid sequence of SEQ ID NOs: NO:33-37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:33-37. In some embodiments, the method comprises administering to a subject a therapeutically effective amount of one or more immunogenic compositions comprising a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 29 or 90, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29 or 90; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. NO: 37 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of NO: 37. In some embodiments of these methods, the first viral expression vector and the second viral expression vector are from the Arenaviridae family.In some embodiments of these methods, the first viral expression vector and the second viral expression vector are derived from an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV), Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV). In some embodiments of these methods, the first viral expression vector and the second viral expression vector are derived from an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV) or Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)). In some embodiments of these methods, the first viral expression vector and the second viral expression vector are replication-defective or replication-defective. In some embodiments of these methods, the first viral expression vector and the second viral expression vector are replication attenuated. In some embodiments, the method comprises administering to a subject a therapeutically effective amount of one or more immunogenic compositions comprising a mixture comprising a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: (a) the first LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of: SEQ ID NO: 29, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29; and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of: SEQ ID NO: 37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. NO:37 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence.In some embodiments, the method comprises administering to a subject a therapeutically effective amount of one or more immunogenic compositions comprising a mixture comprising a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: (a) the first Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO:90, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:90; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO:37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:37. NO:37 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequence of nucleic acid sequence. In some embodiments of these methods, the subject has been infected with HBV virus, suspected HBV virus, or has the risk of infecting HBV virus. In some embodiments of these methods, the subject is asymptomatic. In some embodiments of these methods, the subject has been chronically infected with HBV. In some embodiments of these methods, the subject exhibits or experiences one or more symptoms selected from liver failure, liver cancer, liver fibrosis and cirrhosis. In some embodiments of these methods, the subject has been acutely infected with HBV. In some embodiments of these methods, the subject exhibits or experiences one or more symptoms selected from jaundice, a network of swollen blood vessels visible on the skin, dark (e.g., orange or brown) urine, light-colored stools, fever, persistent fatigue, discomfort, abdominal pain, abdominal fluid, loss of appetite, nausea, and vomiting. In some embodiments of these methods, the subject is co-infected with hepatitis D virus (HDV). In some embodiments of these methods, the composition is administered via a route selected from intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., buccal, intranasal, rectal, vaginal). In some embodiments, the method requires administering about 10 to the subject. 3 to about 10 12 Virus focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or virus particles (vp), for example, about 10 per administration 4 to about 10 7 Virus FFU or PFU or IU or vp, for example about 10 3 to about 10 4 , 10 5 , 10 6 , 107 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some embodiments of these methods, the one or more compositions are administered multiple times. In some embodiments, the method requires intravenous or intramuscular administration of about 10 viral FFU or PFU or IU or vp every other week (Q2W) or every month (Q4W). 6 to about 10 8 ) and ledipasvir + sofosbuvir In some embodiments, the method includes co-administering to the subject one or more therapeutic agents selected from the following: HBV antigen inhibitors (e.g., HBV core antigen (HBcAg) inhibitors, HBV surface antigen (HBsAg) inhibitors, HBx inhibitors, HBV E antigen inhibitors), anti-HBV antigen antibodies, inhibitory nucleic acids targeting HBV (e.g., antisense oligonucleotides, short interfering RNA (siRNA), DNA-guided RNA interference (ddRNAi)), gene editors targeting HBV (e.g., CRISPR-Cas (e.g., Cas9, Cas12, Cascade, Cas13), zinc finger nucleases, homing endonucleases, homing meganucleases (e.g., ARCUS), synthetic nucleases, TALENs), covalently closed circular DNA (cccDNA) inhibitors, and HBsAg secretion or assembly inhibitors and HBV viral entry inhibitors. In some embodiments, the method activates CD8+ T cells and / or CD4+ T cells targeting one or more HBV polypeptide epitopes in the subject. In some embodiments, the method triggers the production of antibodies that bind to one or more HBV polypeptides in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The immunogenicity of adenoviral vectors expressing HBsAg from genotypes (GT) A, B, C, and D in DO mice is shown. Five to seven-week-old diverse outbred (DO) mice (n=8 per group) were injected intramuscularly with 1×10 8 Adenoviral particles (vp) encoding the consensus HBsAg sequences of HBV genotypes (GT)-A, B, C, and D (SEQ ID NOs: 1-4, respectively) were injected. Splenocytes were harvested 14 days after injection, and T cell responses were assessed by interferon (IFN)-γ ELISPOT (BD Mouse IFN-γ ELISPOT Kit, Catalog No. 551083). Each symbol corresponds to an individual mouse, whose response to overlapping peptide pools corresponding to GT-A, B, C, and D HBsAg was assessed.

[0023] Figure 2Schematic diagrams of each Pol-containing antigen design are shown. Each Pol domain is indicated individually (TP, terminal protein; RT, reverse transcriptase; RNH, RNAse H). The approximate position of the D to H mutation in the YMDD motif (SEQ ID NO: 97) in RT and the approximate position of the E to H mutation in the AELL motif (SEQ ID NO: 98) in RNH are indicated below the RT and RNH domains. The name of each construct is shown on the left, and the amino acid size range of the GT-A, B, C, and D constructs is shown on the right. "YMHD" and "AHLL" are disclosed as SEQ ID NOs: 99 and 100, respectively.

[0024] Figure 3 The immunogenicity of adenoviral vectors expressing core-Pol fusion protein in C57BL / 6 mice was shown. Six to eight-week-old C57BL / 6 mice (n=5 per group) were injected with 1×10 8 Adenovirus virus particles (vp) encoding core-Pol fusion variants of SEQ ID NOs: 15-26. The genotype of each antigen is shown above each graph, and the antigen name is shown on the horizontal axis (Mut: Core-Pol mut ,Δ1: Core-Pol Δ1 , Δ3: Core-Pol Δ3 ). On day 14 after injection, splenocytes were harvested and T cell responses were assessed by IFN-γ ELISPOT (BD Mouse IFN-γ ELISPOT Kit, catalog number 551083) using overlapping peptide pools corresponding to GT-D core and Pol. The bar graph shows the stacked geometric mean responses for each group. SFU: spot forming unit.

[0025] Figure 4A-4B The immunogenicity of adenoviral vectors expressing core-pol fusion protein in DO mice was shown. DO mice aged five to seven weeks (n=8 per group) were injected intramuscularly with 1×10 8 Adenovirus virus particles (vp) encoding GT-A core-Pol Δ3 , or GT-B, C or D core-Pol Δ1 . On day 14 after injection, splenocytes were harvested and T cell responses were evaluated by IFN-γ ELISPOT (BD Mouse IFN-γ ELISPOT Kit, catalog number 551083) responses to overlapping peptide pools corresponding to GT-A and D core and Pol. Statistical comparisons between responses to peptides of different genotypes in mice receiving the same vaccine were evaluated using the Wilcoxon signed-rank test. Statistical comparisons between mice receiving different vaccines were evaluated using the Mann-Whitney test. (A) Response to Pol peptide. (B) Response to core peptide. SFU: spot forming unit.

[0026] Figure 5 The immunogenicity of adenoviral vectors expressing Pol was shown. Six to eight-week-old C57BL / 6 mice (n=5 per group) were injected with 1×10 8 Adenovirus virus particles (vp) expressing SEQ ID NO: 8.12, 13, 14, or the full-length unmodified GT-D Pol sequence (GT-D Pol Ctrl ) Pol antigen variant. On day 14 after injection, splenocytes were harvested and T cell responses were assessed by IFN-γ ELISPOT (BD Mouse IFN-γ ELISPOT Kit, Catalog No. 551083) using overlapping peptide pools corresponding to GT-D pol. SFU: spot-forming unit.

[0027] Figure 6 The study design for evaluating the efficacy of Ad5 and vaccinia vectors expressing HBV in an AAV mouse model of CHB (AAV-HBV) is shown. 12 Six to eight-week-old C57BL / 6 mice were transduced with 100 copies of the AAV-HBV genome. On day -7, mice were randomly assigned to treatment groups based on serum HBsAg levels. 50 μl of adenovirus type 5 primary vaccine expressing HBV antigens was administered intramuscularly on day 0, and 50 μl of vaccinia booster vaccine expressing the same HBV antigens was administered intramuscularly on day 32. From day 46 to 67, mice were given anti-PD-1 (anti-CD279) monoclonal antibody RMP1-14 or isotype control mAb. Blood samples were collected on days -7, 14, 27, 46, 60, 67, and 88 for viral antigen testing. Splenocytes were harvested on day 88 and IFN-γ ELISPOT was evaluated.

[0028] Figure 7 The immunogenicity of Ad5 prime-vaccinia boost vaccination in AAV-HBV mice is shown. Figure 6 In the studies shown, splenocytes were harvested on day 88. T cell responses to HBsAg and Pol were evaluated by IFN-γ ELISPOT (BD Mouse IFN-γ ELISPOT Kit, catalog number 551083) using overlapping peptide pools corresponding to GT-D sAg and Pol. The dotted line indicates the highest signal in the HBsAg ELISPOT observed in mice receiving the control vaccine. mAb: administered monoclonal antibody. Iso: isotype control. αPD-1: anti-PD-1. Vax: indicates whether the vaccine contained HBV antigen or control (Ctrl) antigen. SFU: spot forming unit.

[0029] Figure 8The effect of Ad5 prime-vaccinia boost vaccination expressing HBV combined with PD-1 blockade in AAV-HBV mice is shown. Figure 6 Serum HBeAg levels in the studies shown. The dashed line indicates the limit of detection. Ad: adenovirus 5 vector. Vac: vaccinia vector. Ctrl: control antigen. Isotype: isotype control antibody. αPD-1: anti-mouse PD-1 antibody.

[0030] Figures 9A-9C An overview of the arenavirus vector platform presented in the Examples provided herein is shown. (A) Schematic diagram of the phylogenetic tree of the arenavirus family (Anaviridae). In the Examples provided herein, lymphocytic choriomeningitis mammalian arenavirus (LCMV) from the Old World (OW) clade (NCBI: txid11623) and Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)) from the New World (NW) clade (NCBI: txid2169993) were selected for the generation of HBV antigen encoding vectors. See, e.g., Buchmeier et al., 2001, "Arenaviridae: The Viruses and Their Replication," Fields Virology, Vol. 2, 1635-1668. Arenavirus taxonomy has been recently reviewed, e.g., in Radoshitzky et al., Arch Virol. (2015) 160(7): 1851-74. Phylogenetic information for the Arenaviridae family is also available at the Viral Pathogen Resource website at viprbrc.org. (B) Schematic diagram of a replication-defective arenaviral vector with a bi-segmented genome described in WO2009083210, and (C) a replicating, attenuated arenaviral vector with a tri-segmented genome described in WO2016075250 and WO2017198726. The replication-defective arenaviral vector with a bi-segmented genome described in WO2009083210 and used in the Examples provided herein encodes three of the four viral proteins (L, Z, and NP) and an open reading frame for insertion of a heterologous polynucleotide encoding, for example, an antigen. Replication-defective arenaviral vectors with a bi-segmented genome can only proliferate when the viral GP is delivered in trans. The replicating attenuated arenavirus vectors with a three-segmented genome described in WO2016075250 and WO2017198726 have an artificial replication of the S segment of the genome, encoding all four viral proteins (L, Z, NP and GP) and having two open reading frames for the insertion of one or two heterologous polynucleotides, e.g., encoding one or two antigens.

[0031] Figure 10 The immunogenicity of Pol antigen in a replication-incompetent lymphocytic choriomeningitis mammalian arenavirus (LCMV) vector was shown. Six to eight-week-old C57BL / 6 mice (n=6 per group) were injected intravenously with 1×10 6 Focus forming units (FFU) expressing pol antigen variants GT-D and GT-B Pol Δ1 (SEQ ID NO: 6 and 8), Pol Δ3 (SEQ ID NO: 10 and 12) and Pol 300 The cells were injected with a replication-incompetent LCMV vector (SEQ ID NOs: 13 and 14), or culture medium as a negative control. On day 7 post-injection, splenocytes were harvested and T cell responses were assessed by IFN-γ ELISPOT (BD Mouse IFN-γ ELISPOT Kit, Catalog No. 551083) using a pool of Pol overlapping peptides corresponding to the immunizing antigen genotype in each group. SFU: spot-forming unit.

[0032] Figure 11 The immunogenicity of LCMV vector expressing core-HBsAg fusion protein in C57BL / 6 mice is shown. Six to eight-week-old C57BL / 6 mice (n=6 per group) were injected with 1×10 6 The LCMV vector or the simulation immunity of the core-HBsAg fusion variant of expression SEQ ID NO:38-41 of each focus forming unit (FFU) were used as negative controls. At the 7th day after injection, splenocytes were harvested and T cells were evaluated by IFN-γ ELISPOT (BD mouse IFN-γ ELISPOT kit, catalog number (Cat. No.) 551083) using the core and HBsAg overlapping peptide pools corresponding to the immune antigen genotype in each group. SFU: spot forming unit.

[0033] Figure 12 Figure 2 shows the antibody response to HBsAg obtained in mice administered a replication-incompetent LCMV vector expressing the core-sAg fusion protein. Six to eight-week-old C57BL / 6 (left) or Balb / C (right) mice (n=5 per group) were injected with 1×10 6 The 17th day after injection, serum was collected and tested for anti-HBsAg antibodies by ELISA (International Immunodiagnostics). The dotted line indicates the lower limit of detection of 11mIU / ml. *P<0.05 by Mann-Whitney test.

[0034] Figure 13The effect of nucleotide sequence modification on T cell immunogenicity of core-P2A-sAg fusion protein is shown. Six to eight-week-old C57BL / 6 mice (n=6 per group) were injected with 1×10 6 The cells were then injected with 100 focus-forming units (FFU) of GT-D Core-P2A-sAg (SEQ ID NO: 36) or GT-DiCore-P2A-sAg (SEQ ID NO: 37) without replication-competent LCMV vectors or mock immunization as negative controls. At day 7 post-injection, splenocytes were harvested and T cell responses were evaluated by IFN-γ ELISPOT (BD Mouse IFN-γ ELISPOT Kit, Catalog No. 551083) using a pool of core and sAg overlapping peptides. Statistical analysis was performed using the Mann-Whitney test.

[0035] Figures 14A-14B The results show that the expression of GT-B / C Core-P2A-sAg or GT-DiCore-P2A-sAg in multi-line outbred mice was significantly correlated with the expression of GT-B / C Core-P2A-sAg or GT-DiCore-P2A-sAg. Figure 14A ) and GT-B Pol Δ3 or GT-B Pol 300 ( Figure 14B ) were used to perform the primary / boost vaccination. As described in Table 9, two doses of each vaccine were administered to animals on days 0 and 28. Splenocytes were harvested on day 42, and T cell responses to HBV antigens were measured by IFN-γ ELISPOT using sAg, core, and polymerase peptide pools from various viral genotypes as indicated. Data are expressed as background (no peptide) subtracted values. Statistical analysis was performed using the Mann-Whitney test. ns: statistically not significant; *p<0.0332.

[0036] Figures 15A-15B The results show that the expression of GT-D iCore-P2A-sAg in multi-line outbred mice was significantly correlated with the expression of GT-D iCore-P2A-sAg ( Figure 15A ) or GT-B Pol 300 ( Figure 15B Figure 3. Breadth of HBV-specific T cell responses generated by prime / boost vaccination with replication-incompetent LCMV (VV1) vectors. IFN-γ ELISPOT was performed using peptides from the same viral genotype (filled circles) or different viral genotypes (open circles).

[0037] Figures 16A-16C Shown are the effects of serotypes encoding GT-D iCore-P2A-sAg and GT-B Pol in C57BL / 6 mice when delivered as single vectors or as a co-formulated mixture. 300The immunogenicity of the primary / boost vaccination was determined using a replication-incompetent LCMV (VV1) vector. Animals were administered two doses of the vector on days 0 and 21 as described in Table 10. Splenocytes were harvested on day 28 and HBV-specific T cell responses were measured by IFN-γ ELISPOT using core (16A), sAg (16B), and Pol (16C) peptide pools.

[0038] Figures 17A-17F The expression of GT-D iCore-P2A-sAg and GT-BPol in cynomolgus monkeys is shown. 300 The immunogenicity of repeated vaccinations with a replication-incompetent LCMV vector was evaluated. A group of animals was also vaccinated with Ad5 and a vaccinia vector encoding the same HBV antigen. Vectors were administered to animals as described in Table 11. 17A: Group 1; 17B: Group 2; 17C: Group 3; 17D: Group 4; 17E: Group 5; 17F: Group 6. T cell responses to HBV antigens were assessed by IFN-γ ELISPOT using sAg, core, and Pol peptide pools at the indicated time points. Data are expressed as total HBV-specific T cell responses, defined as the sum of IFN-γ ELISPOT values ​​obtained after stimulation with sAg, core, and polymerase peptide pools.

[0039] Figures 18A-18F The results of the experiments in cynomolgus monkeys using the vector encoding GT-DiCore-P2A-sAg and GT-B Pol are shown as shown in FIG17 and Table 11. 300 Immunogenicity of repeated vaccinations with replication-incompetent LCMV vectors. Figures 18A-18F Focus is on IFN-γ ELISPOT obtained after stimulation with the core peptide pool. 18A: Group 1; 18B: Group 2; 18C: Group 3; 18D: Group 4; 18E: Group 5; 18F: Group 6.

[0040] Figures 19A-19F The results of the experiments in cynomolgus monkeys using the vector encoding GT-DiCore-P2A-sAg and GT-B Pol are shown as shown in FIG17 and Table 11. 300 Immunogenicity of repeated vaccinations with replication-incompetent LCMV vectors. Figures 19A-19F Focus on IFN-γ ELISPOT obtained after stimulation with sAg peptide pools. 19A: Group 1; 19B: Group 2; 19C: Group 3; 19D: Group 4; 19E: Group 5; 19F: Group 6.

[0041] Figures 20A-20F The immunogenicity of repeated vaccinations in cynomolgus monkeys with replication-incompetent LCMV vectors encoding GT-DiCore-P2A-sAg and GT-B Pol300 is shown as described in FIG17 and Table 11. Figures 20A-20F Focus is on IFN-γ ELISPOT obtained after stimulation with Pol peptide pool. 20A: Group 1; 20B: Group 2; 20C: Group 3; 20D: Group 4; 20E: Group 5; 20F: Group 6.

[0042] Figures 21A-21B The frequencies of peripheral HBV-specific IFN-γ CD8+ T cells (A) and CD4+ T cells (B) at week 14 are shown for cynomolgus monkeys from groups 1, 2, and week 6 as described in Table 11. Data were obtained from PBMCs harvested at week 14 and restimulated with HBV sAg, core, and polymerase peptide pools. Intracellular IFN-γ of CD4+ and CD8+ T subsets was then analyzed by flow cytometry.

[0043] Figures 22A-22C The antibody response to HBsAg in cynomolgus macaques from Group 1 (22A), Group 2 (22B) and Group 6 (22C) as described in Table 11 is shown. Serum samples were collected at the indicated time points and quantified by ELISA for anti-HBsAg antibodies. The dotted line indicates the lower limit of quantitation of the assay (5 mIU / mL).

[0044] Figure 23 Figure 2 shows the evaluation of GT-D iCore-P2A-sAg and GT-B Pol encoding alone or in combination with the immunomodulators anti-PD1, anti-CTLA4, anti-CD137, and FLT3L-Fc fusion in an AAV-HBV mouse model. 300 Study design of the immunogenicity of a replication-incompetent LCMV vector (HBV vaccine). 11Six to ten-week-old C57BL / 6 mice were transduced with 10 copies of the AAV-HBV genome. On day -11, mice were randomly assigned to treatment groups based on serum HBsAg levels. 200 μl of a replication-incompetent LCMV vector encoding GT-D iCore-P2A-sAg and GT-B Pol300 was administered intravenously (iv) on days 0, 21, and 42. Mice were given 200 μl intraperitoneally: i) saline solution on days 0, 7, 14, 21, 28, 35, 42, 49, and 56; ii) anti-PD-1 monoclonal antibody RMP1-14 on days 42, 46, 49, 53, 56, and 60; iii) lipopolysaccharide (LPS) on days 0, 4, 7, 11, 14, 1 iv) anti-CTLA-4 monoclonal antibody clone 9D9 on days 0, 21, 25, 28, 32, 35, 39, 42, 46, 49, and 53; iv) anti-CD137 monoclonal antibody clone mAb8 (IgG2b) on days 0, 21, and 42; v) FLT3L-Fc fusion protein on days -7, 14, and 35. Asterisks depict the dose of each immunomodulator. Splenocytes were harvested on day 105 and IFN-γ ELISPOT assessed using sAg, core, and pol peptide pools. A group of C57BL / 6 mice that did not receive AAV-HBV but were administered replication-incompetent LCMV vector alone served as IFN-γ ELISPOT positive controls.

[0045] Figures 24A-24C As shown in Table 12 and Figure 23 Immunogenicity of repeated vaccinations in AAV-HBV mice with a replication-incompetent LCMV vector encoding GT-D iCore-P2A-sAg and GT-B Pol300 as described in [ 15 ]. Splenocytes were harvested on day 105 and assessed for IFN-γ ELISPOT using sAg (24A), core (24B), and polymerase (24C) peptide pools. Statistical analysis was performed using the Mann-Whitney test. ns: statistically not significant; *p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001.

[0046] Figure 25 The expression of GT-B Pol in C57BL / 6 mice is shown. 300 ori or GT-B Pol 300The immunogenicity of the primary-boost vaccination was evaluated using a replication-incompetent PICV (VV2) vector of dint. Two doses of the vaccine were administered to the animals on days 0 and 21. Splenocytes were harvested on day 28, and HBV polymerase-specific T cell responses were measured by IFN-γ ELISPOT using a Pol peptide pool. Data are presented as background (no peptide) subtracted values. Statistical analysis was performed using the Mann-Whitney test. **p<0.0021.

[0047] Figures 26A-26C The results show that the expression of GT-D iCore-P2A-sAg or GT-B Pol in C57BL / 6 mice was significantly correlated with the expression of GT-D iCore-P2A-sAg or GT-B Pol 300 The immunogenicity of homologous and heterologous primary immunization / boost vaccination was carried out with LCMV (VV1) and PICV (VV2) vectors without replication ability. As described in Table 15, 2 doses of vector were administered to animals at day 0 and day 21. Splenocytes were harvested at day 28, and HBV-specific T cell responses were measured by IFN-γ ELISPOT using sAg (26A), core (26B) and polymerase (26C) peptide pools. Data are expressed as background (no peptide) deduction values.

[0048] Figure 27 Figure 2 shows the antibody response to HBsAg in C57BL / 6 mice using homologous (VV1 / VV1) or heterologous (VV2 / VV1) prime / boost vaccinations to administer replication-incompetent LCMV and PICV vectors encoding GT-DiCore-P2A-sAg at day 0 and day 21. Serum samples were collected on day 28 and quantified by ELISA for anti-HBsAg antibodies. The dotted line indicates the lower limit of quantification of the assay (20mIU / mL). Statistical analysis was performed using the Mann-Whitney test. **p<0.0021.

[0049] Figures 28A-28C The immunogenicity of homologous and heterologous prime / boost vaccinations in C57BL / 6 mice using replication-attenuated LCMV (TT1) and PICV (TT2) vectors encoding GT-D core-P2A-sAg and GT-B Pol300 is shown. Animals were administered two doses of vector on days 0 and 21 as described in Table 16. Splenocytes were harvested on day 28, and HBV-specific T cell responses were measured by IFN-γ ELISPOT using sAg (28A), core (28B), and polymerase (28C) peptide pools.

[0050] Figure 29 The expression of GT-D core-P2A-sAg and GT-B Pol in cynomolgus monkeys is shown. 300Immunogenicity of homologous and heterologous primary / boost vaccinations using replication-deficient LCMV (VV1) and PICV (VV2) vectors. Animals were administered 2 doses of vector, once at week 0 and once at week 4. PBMCs were harvested at week 6 and HBV-specific T cell responses were measured by IFN-γ ELISPOT using sAg, core, and polymerase peptide pools. Data are expressed as total HBV-specific T cell responses, defined as the sum of IFN-γ ELISPOT values ​​obtained after stimulation with sAg, core, and polymerase peptide pools. The lower limit of quantification (LLOQ) ELISPOT (dashed line) is defined as 200 IFN-γ+SFU / 10 6 PBMCs were analyzed statistically using the Mann-Whitney test.

[0051] Figure 30 Weekly administration of sAg encoding GT-D core-P2A-sAg and GT-BPol in cynomolgus monkeys is shown. 300 Immunogenicity of homologous and heterologous primary / boost vaccinations with replication-deficient LCMV (VV1) and PICV (VV2) vectors. Animals were administered four doses of vector at weeks 0, 1, 2, and 3. PBMCs were harvested at week 4 and HBV-specific T cell responses were measured by IFN-γ ELISPOT using sAg, core, and polymerase peptide pools. Data are expressed as total HBV-specific T cell responses, defined as the sum of IFN-γ ELISPOT values ​​obtained after stimulation with sAg, core, and polymerase peptide pools. The lower limit of quantification (LLOQ) ELISPOT (dashed line) was defined as 200 IFN-γ+SFU / 10 6 PBMCs. DETAILED DESCRIPTION

[0052] 1. Introduction

[0053] The present invention provides polypeptides that can be used to induce a protective immune response against one or more hepatitis B virus (HBV) antigens in the human body. The immunogenic polypeptides described herein are capable of eliciting a preventive and / or therapeutic immune response against one or more hepatitis B virus (HBV) antigens in the human body. Typically, the immunogenic polypeptides described herein contain highly conserved portions of the HBV protein to induce responses against the same epitopes in the vaccine antigen and in the infectious HBV present in the patient, while also excluding regions with poor conservation, thereby avoiding inducing immunodominant T cell responses targeting epitopes that are not present in the patient's infectious HBV strain. The immunogenic polypeptides described herein also induce CD4+ and CD8+ T cell responses to promote the clearance of infected cells, and additionally induce anti-sAg antibody responses that promote sAg clearance, thereby reducing or eliminating the spread of residual virus if sterilized virus clearance is not fully achieved. In addition, the immunogenic polypeptides described herein are proven to be immunogenic when delivered using a vaccine technology that can induce the desired response in the human body, and are stable in the delivery vector through sufficient rounds of vector replication to enable commercial-scale vaccine manufacturing. Immunogenic polypeptides can be used in a variety of vector systems known to induce CD4+ and CD8+ T cell and antibody responses in humans and other non-human primates. In certain embodiments, immunogenic polypeptides are expressed from arenavirus vectors that can be repeatedly administered without inducing anti-vector antibodies, thereby overcoming many limitations of previous viral vector technologies and offering the potential for enhanced therapeutic benefit through repeated administration.

[0054] 2. Polypeptides that can be used to promote immune responses against hepatitis B virus (HBV)

[0055] Immunogenic polypeptides that can be used to promote, induce, and / or elicit an immunogenic response against one or more hepatitis B virus (HBV) antigens are provided. In various embodiments, the immunogenic polypeptides comprise polypeptides and / or fragments encoded by the HBV polymerase (Pol) gene, and fusion polypeptides comprising, from N-terminus to C-terminus, variants and / or fragments of polypeptides encoded by the HBV core gene and variants and / or fragments of polypeptides encoded by the surface antigen (sAg) gene. The immunogenic polypeptides may contain amino acid sequences based on consensus sequences or near-consensus sequences from HBV A, B, C, or D genotypes, as well as combinations thereof. Typically, the immunogenic polypeptides described herein do not comprise sequences of HBV X protein (HBx), pre-core, pre-S1, pre-S2, or fragments thereof.

[0056] In various embodiments, the immunogenic polypeptides described herein and / or polynucleotides encoding such polypeptides are provided in an isolated form. This means that the purity of such polypeptides or polynucleotides is generally at least 50% by weight of interfering proteins, cells, and other contaminants produced by their production or purification, but does not exclude the possibility of combining the agent with an excess of a pharmaceutically acceptable carrier or other vehicle intended to promote its use. As described herein, when applied to polypeptides or polynucleotides, the term "isolated" means that the polypeptide or polynucleotide is substantially free of cellular components associated with it in its native state. It can, for example, be in a homogeneous state and can be in a dry state or in an aqueous solution. Purity and homogeneity can be determined using known methods, such as analytical chemistry techniques, such as polyacrylamide gel electrophoresis, column chromatography, thin-layer chromatography, or high-performance liquid chromatography (HPLC) analysis. Proteins that are the main species present in the preparation are substantially purified. "Isolated" or "purified" polypeptides or polynucleotides are substantially free of other cellular materials or culture media when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. In various embodiments, the purified polypeptide and / or polynucleotide is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (w / w) isolated, purified or free of interfering proteins and contaminants from production or purification. Typically, the pharmaceutical agent is the major macromolecular species remaining after its purification.

[0057] HBV polymerase polypeptide variants

[0058] In various embodiments, truncated and / or internally deleted mutant hepatitis B virus (HBV) polymerase polypeptides are provided.

[0059] Wild-type HBV polymerase has four domains arranged in tandem from the N-terminus to the C-terminus in a single polypeptide: a terminal protein (TP) domain (amino acid residues 1 to 177) that is conserved across hepatotropic viruses, a spacer region (amino acid residues 178 to 335) that connects the TP to the reverse transcriptase (RT) domain (amino acid residues 336 to 678; including the NCBI conserved domain pfam00078 or cd01645), and a C-terminal RNase H (RH) domain (amino acid residues 679 to 832). See, e.g., Lanford et al., J. Virol. (1999) 73(3):1885–93; et al., J Virol. (2014) 88(5):2584-99 and Jones et al., J Virol. (2014) 88(3):1564-72. In the HBV polymerase variants described herein, all or part of the spacer region has been deleted or removed. In the HBV polymerase truncation mutants, the entire TP domain has been deleted or removed.

[0060] Typically, in the HBV polymerase protein mutants described herein, the enzymatic domains, i.e., the reverse transcriptase and RNase H domains, are inactivated. In various embodiments, the reverse transcriptase domain does not contain a YMDD motif (SEQ ID NO: 97). In some embodiments, the YMDD motif (SEQ ID NO: 97) in the reverse transcriptase domain is changed to a YMHD (SEQ ID NO: 99). In some embodiments, the RNase H domain does not contain an AELL motif (SEQ ID NO: 98). In some embodiments, the AELL motif (SEQ ID NO: 98) in the RNase H domain is changed to an AHLL (SEQ ID NO: 100).

[0061] Truncated polymerase mutants

[0062] In some embodiments, the truncated HBV polymerase polypeptide comprises an inactivated reverse transcriptase domain and an inactivated RNase H, wherein the polypeptide does not comprise all terminal protein (TP) domains and does not comprise all or part of the spacer domain (i.e., the terminal protein (TP) domain and all or part of the spacer domain are removed, excised or excluded). In the truncated HBV polymerase polypeptides described herein, all TP domains and all or part of the spacer domain or region are deleted or removed. For example, in some embodiments, the N-terminal 300 amino acids of a natural or wild-type HBV polymerase are deleted or removed from the truncated HBV polymerase polypeptides described herein. In various embodiments, the inactivated reverse transcriptase domain and the inactivated RNase H can be directly fused or operably linked or connected via a linker as described herein. In some embodiments, the truncated HBV polymerase polypeptide is no longer than 600 amino acids, for example, no longer than 595, 590, 585, 580, 575, 570, 565, 560, 555, 550, 545, 540, or 535 amino acids. In some embodiments, the truncated HBV polymerase polypeptide comprises the C-terminal 528, 529, 530, 531, 532, 533, 534, or 535 amino acids of a native or wild-type HBV polymerase.

[0063] In some embodiments, the truncated HBV polymerase polypeptide comprises amino acid residues 300-832, 301-832, 302-832, 303-832, 304-832, 305-832, 306-832, 307-832, 308-832, 309-832, 310-832, 311-832, 312-832, 313-832, 314-832, 315-832, 316-832, 317-832, 318-832, 319-832, 320-832, 321-832, 322-832, 323-832, 324-832, 325-832, 326-832, 327-832, 328-832, 329-832 or 336-832. As used herein, the numbering of a given amino acid polymer or nucleic acid polymer "corresponds to," "corresponds to," or "is relative to" the numbering of a selected or reference amino acid polymer or nucleic acid polymer when the position of any given polymer component (e.g., amino acid, nucleotide, also collectively referred to as "residue") is designated by reference to the same or equivalent position in a selected amino acid or nucleic acid polymer (e.g., based on an optimal alignment or consensus sequence), rather than by the actual numerical position of the component in the given polymer. In various embodiments, the truncated HBV polymerase polypeptide comprises an amino acid sequence corresponding to amino acid residues 300-832. In such embodiments, the N-terminus corresponds to amino acid position 300 of the prototype genotype D pol protein. The N-terminal six amino acid residues of this sequence are SARSQS (SEQ ID NO:95) in the genotype D pol antigen and SSRSQS (SEQ ID NO:96) in the genotype B pol antigen. Literature reports indicate that this N-terminal start site allows for function of the RT domain (see, e.g., Lanford et al., supra) and in vitro expression of the truncated protein (see, e.g., et al., supra).

[0064] In some embodiments, the truncated HBV polymerase polypeptide is from HBV genotype B and comprises or consists of an amino acid sequence of SEQ ID NO: 13, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the truncated HBV polymerase polypeptide is from HBV genotype B and does not comprise (i.e., the sequence is excluded, excised, or removed; the sequence is not included) a polypeptide sequence of SEQ ID NO: 50, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 50.

[0065] In some embodiments, the truncated HBV polymerase polypeptide is from HBV genotype D and comprises or consists of an amino acid sequence of SEQ ID NO: 14, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the truncated HBV polymerase polypeptide is from HBV genotype D and does not comprise a polypeptide sequence of SEQ ID NO: 51, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 51.

[0066] Modifications can be made in the structure of the polypeptides described herein and the polynucleotides encoding such polypeptides, and these modifications still result in functional molecules encoding variants or derivative polypeptides having desired (e.g., immunogenic) characteristics. When it is desired to change the amino acid sequence of a polypeptide to produce an equivalent or even improved variant or portion of a polypeptide described herein, one skilled in the art will typically change one or more of the codons encoding the DNA sequence.

[0067] For example, certain amino acids can be substituted for other amino acids in a protein structure without significantly losing its ability to bind to other polypeptides (e.g., antigens) or cells. Since it is the binding ability and properties of a protein that define the biological functional activity of that protein, certain amino acid sequence substitutions can be made in a protein sequence, and certainly in its underlying DNA coding sequence, while still obtaining a protein with similar properties. It is therefore contemplated that various changes can be made in the polypeptide sequences of the disclosed polypeptides or the corresponding DNA sequences encoding such polypeptides without significantly losing their biological utility or activity.

[0068] As used herein, "substitution" means that one or more amino acids or nucleotides are replaced by different amino acids or nucleotides, respectively.

[0069] In many cases, a polypeptide variant will contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid with similar properties, such that one skilled in the art of peptide chemistry would expect that the secondary structure and hydrophilicity of the polypeptide would be substantially unchanged.

[0070] As used herein, " identity " means the percentage ratio of the nucleotide or amino acid residues in the corresponding position that is identical in two or more sequences when the sequence is aligned so that sequence matching is maximized, i.e., considering room and insertion. The sequence is usually compared for maximum correspondence within a specified region, and for example, a length of at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or more amino acids or nucleotides, and can be up to the full length of a reference polypeptide or polynucleotide sequence. For sequence comparison, usually a sequence serves as a reference sequence, and a test sequence is compared with the reference sequence. When using a sequence comparison algorithm, test sequence and reference sequence are input into a computer program, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Otherwise, standard parameters can be used. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters.

[0071] When comparing polynucleotide and polypeptide sequences, the two sequences are considered "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions (typically 30 to about 75, 40 to about 50) or over the full length of a sequence, wherein the sequence can be compared to a reference sequence after optimal alignment with a reference sequence having the same number of contiguous positions.

[0072] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene bioinformatics software package (DNASTAR, Inc., Madison, WI) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM (1989) CABIOS 5: 151-153; Myers, EW and Muller W. (1988) CABIOS 4: 11-17; Robinson, ED (1971) Comb. Theor 77:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4: 406-425; Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJand Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0073] Alternatively, optimal alignment of sequences for comparison can be performed by the local identity algorithm (Smith and Waterman (1981) Add. APL. Math 2:482), the identity alignment algorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48:443), the search similarity method (Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444), computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or inspection.

[0074] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 methods, which are described in Altschul et al. (1977) Nucl. Acids Res. 25: 3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein to determine percent sequence identity for the polynucleotides and polypeptides described herein. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0075] In an illustrative example, for nucleotide sequences, the cumulative score can be calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). Extension of the word hits in each direction is stopped when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11 and an expectation (E) of 10 as defaults, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) for alignments, (B) of 50, an expectation (E) of 10, M = 5, N = -4, and a comparison of both chains.

[0076] For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score goes to zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0077] In one method, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions (e.g., at least 50 positions, at least 100 positions, or over the entire length of a reference sequence), wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less (usually 5% to 15%, or 10% to 12%) additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences (not comprising additions or deletions). The percentage is calculated by determining the number of positions where the identical nucleic acid base or amino acid residue occurs in the two sequences to generate the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to generate the percentage of sequence identity.

[0078] As used herein, the term polypeptide "variant" is a polypeptide that generally differs from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be generated synthetically, for example, by modifying one or more of the above-described polypeptide sequences described herein and evaluating one or more biological activities of the polypeptides as described herein and / or using any of a variety of techniques well known in the art. The term "variant" may also refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0079] Exemplary HBV polymerase truncation mutants for promoting, inducing or eliciting an immunogenic response, e.g., to a polymerase antigen expressed by HBV are provided in Table A. Exemplary N-terminal sequence fragments that are deleted or removed from, and therefore not included in, the HBV polymerase truncation mutants described herein are provided in Table B.

[0080]

[0081]

[0082]

[0083] In some embodiments, the truncated HBV polymerase polypeptide does not comprise an amino acid sequence from another HBV protein, or a fragment thereof. In some embodiments, the truncated HBV polymerase polypeptide does not comprise an amino acid sequence from an HBV protein, or a fragment thereof, selected from the group consisting of: pre-core, core, X, and envelope (e.g., small, medium, or large surface antigen (sAg)).

[0084] Internal deletion polymerase mutant

[0085] Further provided is an HBV polymerase internal deletion mutant polypeptide. In various embodiments, the HBV polymerase internal deletion mutant polypeptide comprises a terminal protein (TP) domain, an inactivated reverse transcriptase domain, and an inactivated RNase H from the N-terminus to the C-terminus, wherein the mutant polypeptide does not comprise all or part of the spacer domain (i.e., all or part of the spacer domain or region is deleted or removed). In various embodiments, the length of the HBV polymerase deletion mutant polypeptide is not longer than 800 amino acids, for example, a length of not longer than 795, 790, 785, 780, 775, 770, 765, 760, 755, 750, 745, 740, 735, 730, 725, 720, 715, 710 or 705 amino acids. In some embodiments, the HBV polymerase internal deletion mutant polypeptide comprises, from N-terminus to C-terminus: a terminal protein (TP) domain and amino acid residues 300-832, 301-832, 302-832, 303-832, 304-832, 305-832, 306-832, 307-832, 308-832, 309-832, 310-832, 311-832, 312 In various embodiments, the terminal protein (TP) domain, the inactivated reverse transcriptase domain, and the inactivated RNase H domain can independently be directly fused or operably linked or connected via a linker, e.g., as described herein, e.g., as provided in Table J.

[0086] In some embodiments, the HBV polymerase internal deletion mutant polypeptide is from HBV genotype A and comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 5 and 9, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5 and 9. In some embodiments, the HBV polymerase internal deletion mutant polypeptide is from HBV genotype A and does not comprise SEQ ID NOs: 42 or 46, or a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 42 or 46.

[0087] In some embodiments, the HBV polymerase internal deletion mutant polypeptide is from HBV genotype B and comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 6 and 10, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 6 and 10. In some embodiments, the HBV polymerase internal deletion mutant polypeptide is from HBV genotype B and does not comprise SEQ ID NOs: 43 or 47, or a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 43 or 47.

[0088] In some embodiments, the HBV polymerase internal deletion mutant polypeptide is from HBV genotype C and comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 8 and 11, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 8 and 11. In some embodiments, the HBV polymerase internal deletion mutant polypeptide is from HBV genotype C and does not comprise SEQ ID NOs: 44 or 48, or a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 44 or 48.

[0089] In some embodiments, the HBV polymerase internal deletion mutant polypeptide is from HBV genotype D and comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 9 and 12, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 9 and 12. In some embodiments, the HBV polymerase internal deletion mutant polypeptide is from HBV genotype D and does not comprise SEQ ID NOs: 45 or 49, or a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 45 or 49.

[0090] In some embodiments, the HBV polymerase internal deletion mutant polypeptide does not comprise an amino acid sequence or fragment thereof from another HBV protein. In some embodiments, the HBV polymerase internal deletion mutant polypeptide does not comprise an amino acid sequence or fragment thereof from an HBV protein selected from the group consisting of: pre-core, core, X, and envelope (e.g., small, medium, or large surface antigen (sAg)).

[0091] Exemplary HBV polymerase internal deletion mutants for promoting, inducing or eliciting an immunogenic response, e.g., to a polymerase antigen expressed by HBV, are provided in Tables C and E. Exemplary internal amino acid sequence fragments, e.g., corresponding to all or part of the HBV polymerase spacer region, that are deleted or removed from, and therefore not included in, the HBV polymerase internal deletion mutants described herein are provided in Tables D and F.

[0092] Core-polymerase fusion polypeptide

[0093] In various embodiments, the truncated and internally deleted HBV polymerase polypeptide variants described herein are fused to the HBV core polypeptide. The core polypeptide may be located at the N-terminus or C-terminus of the HBV polymerase. Further provided are fusion polypeptides comprising, from N-terminus to C-terminus, an HBV core polypeptide as described herein and a truncated or internally deleted HBV polymerase polypeptide mutant. In some embodiments, the core-Pol fusion polypeptide comprises an HBV polymerase deletion mutant polypeptide as described herein, which comprises, from N-terminus to C-terminus, an HBV core polypeptide as described herein and an internally deleted HBV polymerase polypeptide mutant.

[0094] In some embodiments, the Core-Pol fusion polypeptide comprises or consists of any one of SEQ ID NOs: 19-26, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 19-26.

[0095] In some embodiments, the HBV core-polymerase internal deletion mutant fusion protein does not comprise an amino sequence from an HBV protein selected from the group consisting of: X, pre-core, and envelope (eg, small, medium, or large surface antigen (sAg)), or a fragment thereof.

[0096] Exemplary core-polymerase fusion proteins for promoting, inducing or eliciting an immunogenic response, eg, to core and / or polymerase antigens expressed by HBV are provided in Table G.

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] core-sAg fusion protein

[0109] Also provided are fusion proteins consisting of an N-terminal portion comprising an HBV core polypeptide or an immunogenic fragment thereof and a C-terminal portion comprising an HBV small surface antigen or an immunogenic fragment thereof. In various embodiments, the HBV core polypeptide or a fragment thereof and the HBV small surface antigen (sAg) or a fragment thereof are directly fused or contiguous. In some embodiments, the HBV core polypeptide or a fragment thereof and the HBV small surface antigen or a fragment thereof are connected via a linker.

[0110] HBV core polypeptide or immunogenic fragment thereof

[0111] In various embodiments, the HBV core polypeptide or immunogenic fragment thereof of the core-sAg fusion protein can independently be from HBV genotype A, B / C, or D. Exemplary HBV core polypeptide amino acid sequences that can be used in the core-sAg fusion proteins described herein are provided in Table H.

[0112]

[0113] In some embodiments, the core polypeptide in the core-sAg fusion polypeptide comprises or consists of any one of SEQ ID NOs: 64-66, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 64-66. In some embodiments, the core polypeptide comprises a serine (S) residue at the amino acid position corresponding to position 12 and an asparagine (N) residue at the amino acid position corresponding to position 67, wherein position numbering is with reference to SEQ ID NO: 65 or SEQ ID NO: 66.

[0114] HBV small surface antigen or its immunogenic fragment

[0115] In various embodiments, the HBV sAg polypeptide or immunogenic fragment thereof of the core-sAg fusion protein can independently be from HBV genotype A, B, C, or D. Exemplary HBV sAg polypeptide amino acid sequences that can be used in the core-sAg fusion proteins described herein are provided in Table 1 in Example 1 below.

[0116] In some embodiments, the sAg polypeptide in the core-sAg fusion polypeptide comprises or consists of any one of SEQ ID NOs: 1-4, or a polypeptide having the same or similar sequence as SEQ ID NOs: NOs: 1-4 that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NOs: 1-4, such as comprising one or more of a serine (S) residue at the amino acid position corresponding to position 53, an isoleucine (I) residue at the amino acid position corresponding to position 68, a threonine (T) residue at the amino acid position corresponding to position 125, a proline (P) residue at the amino acid position corresponding to position 127, a phenylalanine (F) residue at the amino acid position corresponding to position 161, a tyrosine (Y) residue at the amino acid position corresponding to position 200, a serine (S) residue at the amino acid position corresponding to position 210, and a leucine (L) residue at the amino acid position corresponding to position 213.

[0117] Regarding the core-sAg fusion protein, the HBV core polypeptide and the HBV sAg polypeptide may be from the same or different HBV genotypes. In some embodiments, the core-sAg fusion protein comprises, from N-terminus to C-terminus, an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, wherein:

[0118] The core polypeptide is derived from HBV genotype A and the sAg polypeptide is derived from HBV genotype A;

[0119] The core polypeptide is from HBV genotype B or C and the sAg polypeptide is from HBV genotype B;

[0120] The core polypeptide is from HBV genotype B or C and the sAg polypeptide is from HBV genotype C;

[0121] The core polypeptide is derived from HBV genotype D and the sAg polypeptide is derived from HBV genotype D;

[0122] The core polypeptide is derived from HBV genotype A and the sAg polypeptide is derived from HBV genotype B;

[0123] The core polypeptide is derived from HBV genotype A and the sAg polypeptide is derived from HBV genotype C;

[0124] The core polypeptide is derived from HBV genotype A and the sAg polypeptide is derived from HBV genotype D;

[0125] The core polypeptide is from HBV genotype B or C and the sAg polypeptide is from HBV genotype A;

[0126] The core polypeptide is from HBV genotype B or C and the sAg polypeptide is from HBV genotype D;

[0127] The core polypeptide is derived from HBV genotype D and the sAg polypeptide is derived from HBV genotype A;

[0128] The core polypeptide is from HBV genotype D and the sAg polypeptide is from HBV genotype B; or

[0129] • The core polypeptide is from HBV genotype D and the sAg polypeptide is from HBV genotype C.

[0130] In some embodiments, the core-sAg fusion protein comprises, from N-terminus to C-terminus, an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, wherein:

[0131] The core polypeptide is from HBV genotype B or C and the sAg polypeptide is from HBV genotype C; or

[0132] • The core polypeptide is from HBV genotype D and the sAg polypeptide is from HBV genotype D.

[0133] In some embodiments, the core-sAg fusion protein comprises, from N-terminus to C-terminus, (i) an HBV core polypeptide comprising or consisting of SEQ ID NO: 65, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 65; and (ii) an HBV small surface antigen (sAg) polypeptide comprising or consisting of SEQ ID NO: 3, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3. NO:3 is an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence.

[0134] In some embodiments, the core-sAg fusion protein comprises, from N-terminus to C-terminus, (i) an HBV core polypeptide comprising or consisting of SEQ ID NO: 66, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 66; and (ii) an HBV small surface antigen (sAg) polypeptide comprising or consisting of SEQ ID NO: 4, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4. NO:4 is an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence.

[0135] In various embodiments, the core-sAg fusion proteins described herein comprise HBV small surface antigen isotypes but do not comprise HBV medium surface antigen isotypes or HBV large surface antigen isotypes. Thus, in some embodiments, the core-sAg fusion proteins described herein do not comprise HBV pre-S1 polypeptide. In some embodiments, the core-sAg fusion proteins described herein do not comprise HBV pre-S2 polypeptide. In some embodiments, the core-sAg fusion proteins described herein do not comprise both HBV pre-S1 polypeptide and HBV pre-S2 polypeptide.

[0136] Exemplary HBV pre-S2 polypeptides that are not included in the core-sAg fusion proteins described herein are provided below:

[0137] MQWNST[A / T]FHQ[T / A]LQDPRVR[A / G]LYFP[A / G]GGSS[L / S]G[A / T][V / I]NPV[L / P]TT[A / V]S[P / H][L / I]SSIF[S / A]RIGDP[A / V][L / M / P / T]N (SEQ ID NO: 79).

[0138] Exemplary HBV pre-S2 consensus polypeptides from HBV genotype A that are not included in the core-sAg fusion proteins described herein are provided below:

[0139] MQWNSTAFHQALQDPRVRGLYFPAGGSSSGTVNPAPNIASHISSISARTGDPVTN (SEQ ID NO:80).

[0140] Exemplary HBV pre-S2 consensus polypeptides from HBV genotype B that are not included in the core-sAg fusion proteins described herein are provided below:

[0141] MQWNSTTFHQTLQDPRVRALYFPAGGSSSGTVSPAQNTVSAISSILSKTGDPVPN (SEQ ID NO:81).

[0142] Exemplary HBV pre-S2 consensus polypeptides from HBV genotype C that are not included in the core-sAg fusion proteins described herein are provided below:

[0143] MQWNSTTFHQALLDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRTGDPAPN (SEQ ID NO:82).

[0144] Exemplary HBV pre-S2 consensus polypeptides from HBV genotype D that are not included in the core-sAg fusion proteins described herein are provided below:

[0145] MQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRIGDPALN (SEQ ID NO:83).

[0146] In some embodiments, the core-sAg fusion protein described herein does not comprise the HBV pre-S2 polypeptide, and comprises or consists of: any one of SEQ ID NOs:79-83, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs:79-83.

[0147] Exemplary HBV pre-S1-pre-S2 polypeptides that are not included in the core-sAg fusion proteins described herein are provided below:

[0148] MGQNLSTSNPLGFFPDHQL[D / A]PAFRANT[A / G / R]NPDWDFNPNKDTWPDANKVGAGAFGGLGFTPPHGGLLGWSPQAQGI[I / L]QT[L / V]PANPPPAS[T / A]NRQ[S / T]GRQPTPLSPPLR [N / D]THPQAMQWNST[A / T]FHQ[T / A]LQDPRVR[A / G]LYFP[A / G]GGSS[L / S]G[A / T][V / I]NPV[L / P]TT[A / V]S[P / H][L / I]SSIF[S / A]RIGDP[A / V][L / M / P / T]N(SEQ ID NO:84).

[0149] Exemplary HBV pre-S1-pre-S2 consensus polypeptides from HBV genotype A that are not included in the core-sAg fusion proteins described herein are provided below:

[0150] MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVSTIPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFHQALQDPRVRGLYFPAGGSSSGTVNPAPNIASHISSISARTGDPVTN (SEQ ID NO: 85).

[0151] Exemplary HBV pre-S1-pre-S2 consensus polypeptides from HBV genotype B that are not included in the core-sAg fusion proteins described herein are provided below:

[0152] MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFKANSENPDWDLNPHKDNWPDANKVGVGAFGPGFTPPHGGLLGWSPQAQGLLTTVPAAPPPASTNRQSGRQPTPLSPPLRDTHPQAMQWNSTTFHQTLQDPRVRALYFPAGGSSSGTVSPAQNTVSAISSILSKTGDPVPN (SEQ ID NO: 86).

[0153] Exemplary HBV pre-S1-pre-S2 consensus polypeptides from HBV genotype C that are not included in the core-sAg fusion proteins described herein are provided below:

[0154] MGGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVGAGAFGPGFTPPHGGLLGWSPQAQGILTTVPAAPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTTFHQALLDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRTGDPAPN (SEQ ID NO: 87).

[0155] Exemplary HBV pre-S1-pre-S2 consensus polypeptides from HBV genotype D that are not included in the core-sAg fusion proteins described herein are provided below:

[0156] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQSGRQPTPLSPPLRNTHPQAMQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRIGDPALN (SEQ ID NO: 88).

[0157] In some embodiments, the core-sAg fusion protein described herein does not comprise an HBV pre-S1-pre-S2 polypeptide, which comprises or consists of: any one of SEQ ID NOs: 84-88, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 84-88.

[0158] Optional polypeptide linker

[0159] As appropriate, the HBV core polypeptide and the HBV sAg polypeptide in the core-sAg fusion protein may be directly adjacent or fused, or may be joined, connected or linked by one or more peptide linkers. In various embodiments, the one or more peptide linkers are selected from one or more of a polyalanine linker, a polyglycine linker, a cleavable linker, a flexible linker, a rigid linker, and a combination thereof, for example, within a linker or within a full-length fusion polypeptide. Exemplary fusion protein linkers that can be used in the fusion polypeptide of the present invention to connect the HBV core polypeptide and the HBV sAg polypeptide are described, for example, in Chen et al., Adv Drug Deliv Rev. (2013) 65(10): 1357–1369. In some embodiments, the polyalanine linker comprises or consists of 2 or 3 consecutive alanine residues, for example, AA, AAA, AAY, or AAX, where X is any amino acid (e.g., A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y). In some embodiments, a polyglycine linker is used, such as GG, GGG, GGS, GSG, or GGGS (SEQ ID NO: 63). In some embodiments, the cleavable linker is selected from a 2A cleavable peptide. Exemplary 2A cleavable peptides that can be used to link HBV core polypeptide and HBV sAg polypeptide are described, for example, in Donnelly et al., J. Gen. Virol (2001), 82, 1027–1041 and Chng et al., mAbs (2015) 7: 2, 403–412. Exemplary 2A cleavable peptides that can be used to link the HBV core polypeptide and the HBV sAg polypeptide include, but are not limited to, 2A cleavage sequences (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and truncatula virus (T2A)), optionally in combination with furin recognition / cleavage sequences (e.g., RAKR (SEQ ID NO: 60), REKR (SEQ ID NO: 61), and RRKR (SEQ ID NO: 62). In certain embodiments, the furin recognition / cleavage sequences (e.g., RAKR (SEQ ID NO: 60), REKR (SEQ ID NO: 61), and RRKR (SEQ ID NO: 62)) are combined or fused in a single linker with a 2A cleavage peptide (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and truncatula virus (T2A)). See, e.g., Chng et al., mAbs (2015) 7:2, 403-412. In some embodiments, the linker comprises a porcine teschovirus-1 (P2A) linker.In various embodiments, the 2A cleavable linker comprises or consists of the amino acid sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO:56), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:57), QCTNYALLKLAGDVESNPGP (SEQ ID NO:58), or EGRGSLLTCGDVEENPGP (SEQ ID NO:59), or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO:56), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:57), QCTNYALLKLAGDVESNPGP (SEQ ID NO:58), or EGRGSLLTCGDVEENPGP (SEQ ID NO:59). In various embodiments, the 2A cleavable linker comprises or consists of the amino acid sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 56), or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 56). Optionally, in certain embodiments, the furin recognition / cleavage sequence may be located at the N-terminus or C-terminus of the 2A linker. In some embodiments, the cleavable linker comprises or consists of a furin recognition / cleavage site selected from RAKR (SEQ ID NO: 60), REKR (SEQ ID NO: 61), and RRKR (SEQ ID NO: 62). Exemplary linkers that can be used to link or connect the HBV core polypeptide and the HBV sAg polypeptide are provided in Table J.

[0160]

[0161]

[0162]

[0163] In some embodiments, the core-sAg fusion protein is no longer than 450 amino acids in length, eg, no longer than 445, 440, 435, 430, 425, 420, 415, or 410 amino acids in length.

[0164] In some embodiments, the core-sAg fusion protein does not comprise an amino sequence from an HBV protein or a fragment thereof selected from the group consisting of: X, pre-core, pre-S1, pre-S2, and polymerase.

[0165] In some embodiments, the core-sAg fusion protein comprises or consists of: an amino acid sequence of any one of SEQ ID NOs: 38-41 (e.g., SEQ ID NO: 41), or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41 (SEQ ID NO: 41). In some embodiments, the fusion polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 12, an asparagine (N) residue at the amino acid position corresponding to position 67, a valine (V) residue at the amino acid position corresponding to position 74, a phenylalanine (F) residue at the amino acid position corresponding to position 97, a threonine (T) residue at the amino acid position corresponding to position 249, a threonine (T) residue at the amino acid position corresponding to position 250, a serine (S) residue at the amino acid position corresponding to position 317, a serine (S) residue at the amino acid position corresponding to position 318, an arginine (R) residue at the amino acid position corresponding to position 326, a tyrosine (Y) residue at the amino acid position corresponding to position 338, a glycine (G) residue at the amino acid position corresponding to position 363, and an alanine (A) residue at the amino acid position corresponding to position 372, wherein positions are numbered with reference to SEQ ID NO: 41.

[0166] Exemplary core-sAg fusion proteins, eg, for promoting, inducing or eliciting an immunogenic response, eg, to core and / or small surface antigens expressed by HBV, are provided in Table K.

[0167]

[0168]

[0169] Signal or leader sequence

[0170] In various embodiments, the immunogenic polypeptides described herein comprise a signal sequence or signal peptide, e.g., to direct intracellular transport of the polypeptide to the proteasome or lysosomal compartment. In various embodiments, the immunogenic polypeptides comprise a signal sequence at the N-terminus and / or the C-terminus. In some embodiments, the immunogenic polypeptides comprise an N-terminal signal peptide or leader sequence. In various embodiments, the signal peptide or leader sequence is from a source protein selected from serum proteins, cytokines, chemokines, chaperones, invariant proteins, and proteins that direct proteins to the lysosomal compartment. In some embodiments, the signal peptide or leader sequence is from a source protein selected from the group consisting of colony-stimulating factor 2 (CSF2, GM-CSF), tissue-type plasminogen activator (PLAT, t-PA), CC motif chemokine ligand 7 (CCL7, MCP-3), CXC motif chemokine ligand 10 (CXCL10, IP-10), catenin beta 1 (CTNNB1), CD74 (p33; DHLAG; HLADG; Ia-GAMMA, invariant chain), serum albumin (ALB), polyubiquitin B / C (UBB / UBC), calreticulin (CALR), vesicular stomatitis virus G protein (VSV-G), lysosomal associated membrane protein 1 (LAMP-1), and lysosomal associated membrane protein 2 (LAMP-2). In various embodiments, the signal peptide or leader sequence is selected from any one of SEQ ID NOs: 67-76, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 67-76. In certain embodiments, the immunogenic polypeptide comprises an N-terminal and a C-terminal signal sequence, respectively, from LAMP-1, for example, SEQ ID NOs: 77 and 78, respectively. Exemplary signal sequences that can be used in immunogenic polypeptides of the invention are provided in Table L.

[0171]

[0172]

[0173] Also provided are methods for preparing the immunogenic polypeptides described herein. In some embodiments, the method comprises constructing the immunogenic polypeptide using peptide synthesis. In some embodiments, the method comprises constructing a polynucleotide encoding each of the bivalent antigenic polypeptides using synthetic or recombinant DNA techniques and expressing the polypeptide from an expression vector. In some embodiments, the method may further comprise inserting the polynucleotide into one or more vectors and expressing the encoded polypeptide in a cell. This can be performed using known recombinant techniques.

[0174] 3. Polynucleotides encoding immunogenic polypeptides

[0175] Provided are polynucleotides encoding immunogenic polypeptides as described herein, vectors comprising such polynucleotides, and host cells (e.g., human cells, mammalian cells, yeast cells, plant cells, insect cells, bacterial cells, such as E. coli) comprising such polynucleotides or expression vectors. Provided herein are polynucleotides comprising a nucleotide sequence encoding any one of the immunogenic polypeptides provided herein, and expression cassettes and vectors comprising such polynucleotide sequences, such as expression vectors for efficient expression in host cells (e.g., mammalian cells). In various embodiments, the polynucleotides are DNA, cDNA, mRNA, self-amplifying RNA (SAM), self-replicating RNA, or self-amplifying replicon RNA (RepRNA). In some embodiments, the polynucleotides comprise or are expressed by alphavirus self-replicating or self-amplifying replicon RNA (RepRNA). Self-replicating RNA and self-amplifying replicon RNA as vaccine delivery modes are described, for example, in Tews et al., Methods Mol Biol. (2017) 1499: 15-35; Démoulins et al., Methods Mol Biol. (2017) 1499: 37-75; Englezou et al., Mol Ther Nucleic Acids. (2018) 12: 118-134; McCollough et al., Vaccines (Basel). (2014) 2(4): 735-54; and McCollough et al., Mol Ther Nucleic Acids. (2014) 3: e173.

[0176] The terms "polynucleotide" and "nucleic acid molecule" refer interchangeably to polymeric forms of nucleotides, and include sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers thereof. As used herein, the term "nucleic acid molecule" is interchangeable with the term "polynucleotide." In some embodiments, nucleotides refer to ribonucleotides, deoxyribonucleotides, or modified forms of any type of nucleotide, and combinations thereof. The term also includes, but is not limited to, single-stranded and double-stranded forms of DNA. In addition, a polynucleotide, such as a cDNA or mRNA, may include one or both of naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds. As will be readily understood by those skilled in the art, nucleic acid molecules may be chemically or biochemically modified, or may contain non-natural or derived nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more of the naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged bonds (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), side chain moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralens, etc.), chelating agents, alkylates, and modified bonds (e.g., α-anomeric nucleic acids, etc.). The above terms are also intended to include any topological conformation, including single-stranded conformation, double-stranded conformation, partially double-stranded conformation, triple-stranded conformation, hairpin conformation, circular conformation, and padlock conformation. Unless otherwise indicated, reference to a nucleic acid sequence encompasses its complementary sequence. Therefore, reference to a nucleic acid molecule with a specific sequence is understood to encompass its complementary chain and its complementary sequence. The term also includes codon-biased polynucleotides for improving expression in desired viral expression vectors or host cells.

[0177] As used herein, "substitution" means that one or more amino acids or nucleotides are replaced by different amino acids or nucleotides, respectively.

[0178] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. An "isolated nucleic acid encoding an immunogenic polypeptide" refers to one or more nucleic acid molecules encoding such an immunogenic polypeptide, including such nucleic acid molecules in a single vector or multiple separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0179] As used herein, the term polynucleotide "variant" is a polynucleotide that typically differs from the polynucleotides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be generated synthetically, for example, by modifying one or more of the polynucleotide sequences described herein and evaluating one or more biological activities of the encoded polypeptides as described herein and / or using any of a variety of techniques well known in the art.

[0180] In some embodiments, the nucleic acid molecule has a codon preference to enhance expression in a desired host cell (e.g., a human cell, a mammalian cell, a yeast cell, a plant cell, an insect cell, or a bacterial cell, such as an E. coli cell). Thus, polynucleotides encoding immunogenic polypeptides as described herein are provided, wherein the polynucleotides have a codon preference, comprise a replacement heterologous signal sequence, and / or eliminate an mRNA instability element. Methods for generating codon-biased nucleic acids can be performed by adapting the methods described in, for example, U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498. Preferred codon usage for expressing immunogenic polypeptides from a desired viral expression vector and / or in a desired host cell is provided, for example, at kazusa.or.jp / codon / and genscript.com / tools / codon-frequency-table.

[0181] In some embodiments, the polynucleotide encoding the immunogenic polypeptide as described herein has a nucleic acid sequence that is at least 80%, at least 85%, 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% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27-37 and 89-94 as provided in Table 24.

[0182] Optionally, in certain embodiments, the 3' end of a polynucleotide encoding one or more of the immunogenic polypeptides described herein comprises one or more tandem stop codons, such as two or more tandem TAG ("amber"), TAA ("ochre"), or TGA ("opal" or "umber") stop codons. The multiple tandem stop codons may be the same or different.

[0183] Also provided is an expression cassette comprising a polynucleotide encoding an immunogenic polypeptide as described herein, the polynucleotide being operably linked to one or more regulatory sequences. In some embodiments, the polynucleotide is operably linked to and under the control of a constitutive promoter. In some embodiments, the promoter is selected from the group consisting of the cytomegalovirus major immediate early promoter (CMV), a CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK).

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198] 4. Vectors and Host Cells

[0199] Also provided are vectors comprising one or more polynucleotides encoding one or more of the immunogenic polypeptides as described herein, or expression cassettes comprising such polynucleotides. The vector can be of any type, such as a recombinant vector, such as an expression vector. The vector includes, but is not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs), as well as vectors derived from bacteriophages or plants or animals (including humans) viruses. The vector may include a replication origin recognized by the proposed host cell, and, with respect to expression vectors, includes a promoter and other regulatory regions recognized by the host cell. In another embodiment, the vector includes one or more polynucleotides encoding one or more immunogenic polypeptides of the present disclosure, which are operably linked to a promoter and optional other regulatory elements. Certain vectors are capable of autonomous replication in the host into which they are introduced (for example, vectors with a bacterial replication origin can replicate in bacteria). Other vectors can be integrated into the host's genome when introduced into the host, thereby replicating together with the host genome. The vector includes, but is not limited to, those suitable for recombinant production of the immunogenic polypeptides disclosed herein.

[0200] As used herein, the term "vector" refers to a nucleic acid molecule that is capable of propagating another nucleic acid to which it is attached. The term includes vectors that are self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which the vector has been introduced. Some vectors are suitable for delivering the nucleic acid molecules or polynucleotides of the present application. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively attached. Such vectors are referred to herein as expression vectors.

[0201] The term "operably linked" refers to two or more nucleic acid or polypeptide sequence elements that are typically physically linked and in a functional relationship with each other. For example, in the context of nucleic acid sequence elements, a promoter is operably linked to a coding sequence if the promoter is capable of initiating or regulating the transcription or expression of the coding sequence, in which case the coding sequence is understood to be "under the control of the promoter."

[0202] The choice of vector depends on the subsequent recombination process and the host used. The introduction of the vector into the host cell can be achieved by, in particular, calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofectamine transfection, electroporation, viral infection, or via administration to a subject as described herein. The vector can replicate autonomously or can replicate together with the chromosome into which it is integrated. In certain embodiments, the vector contains one or more selection markers. The selection of markers can depend on the host cell selected. These include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, zeocin, the thymidine kinase gene (HSV-TK) from herpes simplex virus, and the dihydrofolate reductase gene (dhfr) from mouse. The present invention also encompasses vectors comprising one or more nucleic acid molecules encoding the immunogenic polypeptides described herein, which are operably linked to one or more nucleic acid molecules encoding proteins or peptides that can be used to separate the immunogenic polypeptides. These proteins or peptides include, but are not limited to, glutathione-S-transferase, maltose binding protein, metal-binding polyhistidine, green fluorescent protein, luciferase, and beta-galactosidase.

[0203] In other embodiments, the vector used is pcDNA TM 3.1+ (ThermoFisher, MA).

[0204] In some embodiments, the vector is a viral vector. Depending on the circumstances, the viral vector can be a DNA virus or an RNA virus, including a self-replicating RNA virus. Self-replicating RNA viruses include alphaviruses and are described, for example, in Lundstrom, Molecules. (2018) 23 (12). pii: E3310 (PMID: 30551668); and Ljungberg et al., Expert Rev Vaccines. (2015) 14 (2): 177-94. In various embodiments, the viral vector is from a virus selected from the group consisting of adenovirus, adeno-associated virus, arenavirus, alphavirus, self-replicating alphavirus, poxvirus, cytomegalovirus, rhabdovirus, vesicular stomatitis virus, flavivirus, Maraba virus, and vaccinia virus. In some embodiments, the viral vector is from a virus family selected from the group consisting of: Adenoviridae (e.g., adenovirus, adeno-associated virus), Arenaviridae (e.g., lymphocytic choriomeningitis mammalian arenavirus or Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus (PICV)), Poxviridae (e.g., vaccinia virus), Herpesviridae (e.g., cytomegalovirus, herpes virus, such as HSV-1), Parvoviridae (e.g., Parvovirus H1), Poxviridae (e.g., vaccinia virus, such as modified vaccinia Ankara (MVA)), Flaviviridae (e.g., yellow fever virus), Reovirus (e.g., reovirus), Retroviridae (e.g., lentivirus), Picornaviridae (e.g., Coxsackievirus, Seneca Valley Virus, Poliovirus), Paramyxoviridae (e.g., measles virus, Newcastle disease virus), Virus (NDV)), Rhabdoviridae (e.g., vesiculoviruses, including Maraba vesiculovirus and vesicular stomatitis virus (VSV)), Togaviridae (VSV) (e.g., alphaviruses, such as self-replicating alphaviruses; Sindbis virus), Enteroviridae (e.g., Echovirus). Exemplary modified vaccinia virus vectors for expressing immunogenic polypeptides of the invention are described, for example, in WO2019 / 134049.

[0205] In some embodiments, the viral expression vector is an arenavirus vector selected from the group consisting of: lymphocytic choriomeningitis mammalian arenavirus (LCMV) (NCBI: txid11623), Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde virus) (NCBI: txid2169993), Guanarito virus (GTOV) (NCBI: txid45219), Argentinian mammalian arenavirus (also known as Junin virus (JUNV)) (NCBI: txid2169993), In some embodiments, the viral expression vector is an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV) or Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)). Exemplary arenavirus vectors that can be used as delivery and expression vehicles for the immunogenic polypeptides described herein are described, for example, in WO 2009 / 083210, WO 2015 / 183895, WO 2016 / 075250, WO 2017 / 198726, and U.S. Patents 9,943,585 and 10,342,861, which are hereby incorporated by reference in their entirety for all purposes.

[0206] In some embodiments, the viral expression vector is an adenoviral vector, e.g., from a human adenovirus or a simian adenovirus (e.g., a chimpanzee adenovirus, a gorilla adenovirus, or a rhesus monkey adenovirus). In various embodiments, the adenoviral vector is selected from adenovirus serotype 5 (Ad5), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), chimpanzee adenovirus (e.g., ChAdOx1, ChAdOx2, ChAd3 (AdC3), ChAd5 (AdC5), ChAd6 (AdC6), ChAd7 (AdC7), ChAd8 (AdC8), ChAd9 ( AdC9), ChAd10(AdC10), ChAd11(AdC11), ChAd17(AdC17), ChAd16(AdC16), ChAd19(AdC19), ChAd20(AdC20), ChAd22(AdC22), ChAd24(AdC24), ChAdY25, ChAd26(AdC26), ChAd28(AdC28), ChAd30(AdC30), ChAd31(AdC31) , ChAd37(AdC37), ChAd38(AdC38), ChAd43(AdC43), ChAd44(AdC44), ChAd55(AdC55), ChAd63(AdC63), ChAdV 63. ChAd68(AdC68), ChAd73(AdC73), ChAd82(AdC82), ChAd83(AdC83), ChAd143(AdC143), ChAd144(AdC144) , ChAd145 (AdC145), ChAd147 (AdC147)), gorilla adenovirus (e.g., GC44, GC45, GC46), and rhesus macaque adenovirus (e.g., RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65, RhAd66).Exemplary chimpanzee, gorilla and rhesus monkey adenoviral vectors that can be used as delivery and expression vehicles for the immunogenic polypeptides described herein are described, for example, in WO2012 / 172277 (ChAdOx1), WO2017 / 221031 (ChAdOx2), WO2019 / 076880; WO2019 / 076877; Andrabi et al. (2019) Cell Reports 27:2426–2441, Guo et al., Hum Vaccin Immunother. (2018) 14(7):1679-1685; Abbink et al., J Virol. (2015) 89(3):1512-22; and Abbink et al., J Virol. (2018) 92(6).pii:e01924-17.

[0207] In various embodiments, the viral expression vector is unable to replicate (i.e., replication-defective or replication-defective), for example, has reduced or diminished replication capacity compared to a wild-type viral vector (i.e., replication-attenuated), or is replication-competent. In various embodiments, the viral expression vector is a replication-defective or replication-defective arenavirus vector having a bi-segmented genome, for example, as described in WO 2009 / 083210 and WO 2017 / 076988. In various embodiments, the viral expression vector is a replication-attenuated arenavirus vector having a tri-segmented genome, for example, as described in WO 2016 / 075250, WO 2017 / 076988, and WO 2017 / 198726.

[0208] Also provided are host cells comprising one or more polynucleotides encoding one or more of the immunogenic polypeptides as described herein or one or more vectors expressing the immunogenic polypeptides. Any of a variety of host cells can be used. In one embodiment, the host cell is a prokaryotic cell, such as E. coli. In another embodiment, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, a mammalian cell, such as a cell line based on Chinese hamster ovary (CHO) or CHO derived cells (e.g., CHO-S, CHO DG44, ExpiCHO, TM 、 ZFN-modified GS- / - CHO cell lines, CHO-K1, CHO-K1a), COS cells, BHK cells, NSO cells or Bowes melanoma cells. Examples of human host cells include HeLa, 911, AT1080, A549 and HEK293 (e.g., HEK293E, HEK293F, HEK293H, HEK293T, Expi293 TMAdditionally, the immunogenic polypeptides can be expressed in yeast cells such as Pichia pastoris (see, e.g., Powers et al., J Immunol Methods. 251: 123-35 (2001)), Hansenula, or Saccharomyces cerevisiae.

[0209] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0210] As the case may be, host cells may be stably or transiently transfected with one or more polynucleotides encoding one or more immunogenic polypeptides as described herein. As the case may be, host cells may be infected with one or more vectors expressing one or more immunogenic polypeptides as described herein. In some embodiments, host cells can be infected and propagated by one or more replication-attenuated or replication-competent vectors expressing one or more immunogenic polypeptides as described herein. Exemplary cells that can be used to infect and / or propagate viral vectors include, but are not limited to, BHK-21, A549, Vero, and HEK293 (e.g., HEK293E, HEK293F, HEK293H, HEK293T, Expi293 TM ) cells. In certain embodiments, the host cell expresses a coxsackievirus and adenovirus receptor (CAR), such as MDCK, Caco-2, or Calu-3 host cells. In certain embodiments, the polynucleotide is integrated into the genome of the host cell.

[0211] 5. Pharmaceutical compositions / immunogenic compositions

[0212] Provided are pharmaceutical compositions or immunogenic compositions comprising one or more of the immunogenic HBV polypeptides described herein, or polynucleotides encoding one or more of the immunogenic HBV polypeptides described herein, or viral expression vectors comprising one or more of such polynucleotides, and a pharmaceutically acceptable diluent, carrier, or excipient. "Pharmaceutically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the U.S. Food and Drug Administration as acceptable for use in humans or livestock.

[0213] Generally, pharmaceutical compositions as described herein are immunogenic. In certain embodiments, pharmaceutical compositions include one or more (such as two or more, three or more) immunogenic HBV polypeptides or one or more (such as two or more, three or more) polynucleotides encoding immunogenic HBV polypeptides of a therapeutically effective amount, or one or more (such as two or more, three or more) polynucleotides containing one or more (such as two or more, three or more) polynucleotides encoding immunogenic HBV polypeptides of a therapeutically effective amount, or one or more (such as two or more, three or more) polynucleotides containing one or more (such as two or more, three or more) polynucleotides encoding immunogenic HBV polypeptides of a viral expression vector.

[0214] Various pharmaceutically acceptable diluents, carriers, and excipients, as well as techniques for preparing and using pharmaceutical compositions, will be known to those skilled in the art in light of the present disclosure. Exemplary pharmaceutical compositions and pharmaceutically acceptable diluents, carriers, and excipients are also described, for example, in Loyd V. Allen Jr (ed.), "Remington: The Science and Practice of Pharmacy," 22nd ed., 2012, Pharmaceutical Press; Brunton, Knollman and Hilal-Dandan, "Goodman and Gilman's The Pharmacological Basis of Therapeutics," 13th ed., 2017, McGraw-Hill Education / Medical; McNally and Hastedt (eds.), "Protein Formulation and Delivery," 2nd ed., 2007, CRC Press; Banga, "Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems," 3rd ed., 2015, CRC Press; Lars Hovgaard, Frokjaer and van de Weert (eds.), "Pharmaceutical Formulation Development of Peptides and Proteins," 2nd ed., 2012, CRC Press Press; Carpenter and Manning (Editors), "Rational Design of Stable Protein Formulations: Theory and Practice," 2002, Springer (Pharmaceutical Biotechnology (Book13)); Meyer (Editor), "Therapeutic Protein Drug Products: Practical Approaches to Formulation in the Laboratory, Manufacturing, and the Clinic," 2012, Woodhead Publishing.

[0215] In certain embodiments, polynucleotide or carrier are configured to lipid nanoparticles.For example, in some embodiments in which immunogenic HBV polypeptides are expressed by self-replication or self-amplification RNA molecules, self-replication or self-amplification RNA can be configured to lipid nanoparticles (LNP). As used herein, term " lipid nanoparticles" refers to one or more spherical nanoparticles having an average diameter between about 10 nanometers and about 1000 nanometers and including the solid lipid core matrix that can dissolve lipophilic molecules. In certain embodiments, lipid cores are stabilized by surfactants (for example, emulsifiers), and one or more of the following items can be included: triglycerides (for example, tristearin), diglycerides (for example, glyceryl behenate), monoglycerides (for example, glyceryl monostearate), fatty acids (for example, stearic acid), steroids (for example, cholesterol) and waxes (for example, cetyl palmitate), including their combinations. Lipid nanoparticles are described, for example, in Petrilli et al., Curr Pharm Biotechnol. 15:847-55, 2014; and U.S. Pat. Nos. 6,217,912, 6,881,421, 7,402,573, 7,404,969, 7,550,441, 7,727,969, 8,003,621, 8,691,750, 8,871,509, 9,017,726, 9,173,853, 9,220,779, 9,227,917, and 9,278,130, each of which is incorporated by reference in its entirety. In one embodiment, the self-replicating or self-amplifying RNA molecules encoding one or more of the immunogenic HBV polypeptides described herein are formulated or condensed into a polyethyleneimine (PEI)-polyplex delivery vehicle, for example, as described in Démoulins et al., Nanomedicine. (2016) April; 12(3): 711-722 and Démoulins et al., J Control Release. (2017) November 28; 266: 256-271, which can be a nanoparticle.

[0216] In embodiments where the immunogenic HBV polypeptide is expressed by a viral expression vector, the viral expression vector can be formulated for a desired route of administration, e.g., as an isotonic, pharmaceutically acceptable aqueous solution or suspension suitable for intravenous, intramuscular, subcutaneous, or intradermal administration. In some embodiments, the viral expression vector can be formulated for mucosal, e.g., buccal, intranasal, intravaginal, or rectal delivery. Exemplary formulations of viral expression vectors that can be used in the pharmaceutical compositions and methods described herein are described, for example, in Manfredsson and Benskey, eds., “Viral Vectors for Gene Therapy: Methods and Protocols (Methods in Molecular Biology),” 2019, Book 1937 in Methods in Molecular Biology Series, Humana Press; WO 2017 / 013169 (formulation of Adenoviral vectors in an aqueous mixture or freeze-dried composition in the presence of amorphous sugar and low salt concentration); and Kumru et al., J Pharm Sci. (2018) November; 107(11): 2764-2774 (aqueous formulations buffered in Trisand containing proline, lactose, and mannitol as stabilizing additives). Formulations of arenavirus vectors are described, for example, in WO 2009 / 083210, WO 2016075250, and WO 2017 / 198726. In certain embodiments, viral expression vectors are delivered via microneedle-mediated delivery, for example, as described in Zaric et al., Expert Opin Drug Deliv. (2017) October; 14(10): 1177-1187.

[0217] In some embodiments, each carrier, diluent or excipient is "acceptable" in the sense that it is compatible with the other ingredients of the pharmaceutical composition and not injurious to the subject. Typically, a pharmaceutically acceptable carrier is an aqueous pH buffer. Some examples of materials that can be used as pharmaceutically acceptable carriers, diluents or excipients include: water; buffers, e.g., buffers having a pKa in the range of about 6.0 to about 8.0, e.g., physiologically acceptable buffers, e.g., selected from phosphate, carbonate, bicarbonate, citrate, maleate, glycine-glycine, HEPES, HEPPSO, HEPPS, imidazole, BICINE, TRICINE, Tris and BIS-Tris; sugars, such as lactose, trehalose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered Tragacanth gum; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Hank's solution, Ringer's solution; ethanol; phosphate buffered saline; amino acids (e.g., charged amino acids, including but not limited to aspartic acid, asparagine, glutamic acid, glutamine, histidine, arginine, lysine); and other nontoxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers, and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as colorants, release agents, coating agents, sweeteners, flavoring agents, and fragrances, preservatives, and antioxidants may also be present in the composition.

[0218] In a specific formulation, an arenavirus vector described herein (e.g., LCMV or a Pichinde mammalian arenavirus vector (PICV)) is formulated in an isotonic aqueous solution comprising a biocompatible buffer (e.g., HEPES and NaCl) at a neutral or near-neutral pH with a pKa in the range of about 6.0 to about 8.0 and a nonionic surfactant (e.g., PLURONIC F68 (also known as poloxamer 188). In a specific formulation, an arenavirus vector described herein (e.g., LCMV or a Pichinde mammalian arenavirus vector) is formulated in an isotonic aqueous solution comprising a HEPES buffer at pH 7.4, NaCl, and PLURONIC F68 (also known as poloxamer 188). Schleiss et al. (Clin Vaccine Immunol. 2017 Jan 5;24(1):e00300-16) describe the formulation of LCMV vectors in a diluent of 25 mM HEPES, 150 mM NaCl, 0.01% PLURONIC F68 at pH 7.4, which can be used to formulate the arenavirus vectors described herein. Sorbitol was added to a final concentration of 10% before freezing below -60°C.

[0219] The preparation and delivery method of the pharmaceutical composition will generally be adjusted according to the position to be treated and the disease. Exemplary preparations include, but are not limited to, those suitable for parenteral administration (e.g., intravenous, intraarterial, intramuscular or subcutaneous administration), including preparations encapsulated in micelles, liposomes or drug release capsules (incorporated into the active agent in the biocompatible coating designed for slow release); ingestible preparations; preparations for topical use, such as creams, ointments and gels; and other preparations, such as inhalants, aerosols and sprays. In some embodiments, the pharmaceutical composition is formulated for parenteral (e.g., intravenous, subcutaneous or oral) administration. In some embodiments, the pharmaceutical composition is formulated for mucosal (e.g., buccal, intranasal, rectal and / or vaginal) administration.

[0220] In certain embodiments, the pharmaceutical composition is sterile. In certain embodiments, the pharmaceutical composition has a pH in the range of 4.5 to 8.5, 4.5 to 6.5, 6.5 to 8.5, 6.0 to 8.0, 6.5 to 8.5, or about 5.0, about 5.5, about 6.0, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.5, about 8.0 or about 8.5. In one embodiment, the pharmaceutical composition has an osmotic concentration in the range of 240-260 or 250-330 mOsmol / L. In certain embodiments, the pharmaceutical composition is isotonic or nearly isotonic.

[0221] In some embodiments, the pharmaceutical composition is a liquid or solid. In some embodiments, the pharmaceutical composition comprises an aqueous solution or suspension. In some embodiments, the pharmaceutical composition is lyophilized or a frozen liquid.

[0222] In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents, eg, a second therapeutic agent, or a second therapeutic agent and a third therapeutic agent, for use in combination therapy as described herein.

[0223] In certain embodiments, the pharmaceutical composition further comprises an adjuvant. Exemplary adjuvants that can be co-formulated or co-administered with the immunogenic HBV polypeptides described herein, the polynucleotides encoding such immunogenic HBV polypeptides, and the vectors expressing such immunogenic HBV polypeptides include, but are not limited to, cytokines, chemokines, immune co-stimulatory molecules, toll-like receptor agonists, or inhibitors of immunosuppressive pathways, as described herein and described in Li et al., Curr Issues Mol Biol. (2017) 22: 17-40. Other adjuvants that can be co-formulated or co-administered with the immunogenic HBV polypeptides described herein, the polynucleotides encoding such immunogenic HBV polypeptides, and the vectors expressing such immunogenic HBV polypeptides include, but are not limited to, mineral salts (e.g., aluminum salts (e.g., alum), calcium phosphate, incomplete Freund's adjuvant), lipid particles (e.g., MF59, cochleates, virus-like particles), microparticles (e.g., virosomes, polylactic acid (PLA), poly [lactide-

[0015] Adjuvants that can be co-formulated or co-administered with the immunogenic HBV polypeptides described herein, polynucleotides encoding such immunogenic HBV polypeptides, and vectors expressing such immunogenic HBV polypeptides are generally described in Apostólico et al., J Immunol Res. (2016) 2016: 1459394.

[0224] In some embodiments, the pharmaceutical composition or immunogenic composition comprises a mixture of two or more immunogenic HBV polypeptides, two or more polynucleotides encoding such immunogenic HBV polypeptides, or two or more vectors expressing such immunogenic HBV polypeptides. In some embodiments, the pharmaceutical composition comprises two or more immunogenic HBV polypeptides, two or more polynucleotides encoding such immunogenic HBV polypeptides, or two or more vectors expressing such immunogenic HBV polypeptides.

[0225] In various embodiments, the immunogenic composition comprises one or more polynucleotides encoding two immunogenic polypeptides, or one or more vectors capable of expressing two immunogenic polypeptides, comprising: (a) an HBV polymerase polypeptide mutant comprising or consisting of any one of SEQ ID NOs: 5-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 5-14; and (b) an HBV core-sAg fusion protein comprising or consisting of any one of SEQ ID NOs: 38-41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41. The invention relates to an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of ID NOs: 38-41.

[0226] In various embodiments, the immunogenic composition comprises one or more polynucleotides encoding two immunogenic polypeptides, or one or more vectors capable of expressing two immunogenic polypeptides, comprising: (a) an HBV polymerase polypeptide mutant comprising or consisting of any one of SEQ ID NOs: 13-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 13-14; and (b) an HBV core-sAg fusion protein comprising or consisting of any one of SEQ ID NOs: 38-41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41. The invention relates to an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of ID NOs: 38-41.

[0227] In various embodiments, the immunogenic composition comprises one or more polynucleotides encoding two immunogenic polypeptides, or one or more vectors capable of expressing two immunogenic polypeptides, comprising: (a) an HBV polymerase polypeptide mutant comprising or consisting of SEQ ID NO: 13, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13; and (b) an HBV core-sAg fusion protein comprising or consisting of SEQ ID NO: 41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41. NO:41 is an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence.

[0228] With respect to the core-sAg fusion polypeptide in the immunogenic composition, in some embodiments, the core polypeptide comprises a serine (S) residue at the amino acid position corresponding to position 12 and an asparagine (N) residue at the amino acid position corresponding to position 67, wherein position numbering is with reference to SEQ ID NO: 65 or SEQ ID NO: 66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at the amino acid position corresponding to position 68, wherein position numbering is with reference to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 53, an isoleucine (I) residue at the amino acid position corresponding to position 68, a threonine (T) residue at the amino acid position corresponding to position 125, a proline (P) residue at the amino acid position corresponding to position 127, a phenylalanine (F) residue at the amino acid position corresponding to position 161, a tyrosine (Y) residue at the amino acid position corresponding to position 200, a serine (S) residue at the amino acid position corresponding to position 210, and a leucine (L) residue at the amino acid position corresponding to position 213, wherein the positions are numbered with reference to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the core-sAg fusion polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 12, an asparagine (N) residue at the amino acid position corresponding to position 67, a valine (V) residue at the amino acid position corresponding to position 74, a phenylalanine (F) residue at the amino acid position corresponding to position 97, a threonine (T) residue at the amino acid position corresponding to position 249, a threonine (T) residue at the amino acid position corresponding to position 250, a serine (S) residue at the amino acid position corresponding to position 317, a serine (S) residue at the amino acid position corresponding to position 318, an arginine (R) residue at the amino acid position corresponding to position 326, a tyrosine (Y) residue at the amino acid position corresponding to position 338, a glycine (G) residue at the amino acid position corresponding to position 363, and an alanine (A) residue at the amino acid position corresponding to position 372, wherein positions are numbered with reference to SEQ ID NO: 41.

[0229] In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of: a nucleic acid sequence of any one of SEQ ID NOs: 27-32 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, and 92), or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 27-32 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, and 92); and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of: any one of SEQ ID NOs: 33-37, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 33-37. A nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NOs:33-37.

[0230] In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 29 or 90, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, 89, 90, or 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. A nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NO:37.

[0231] In some embodiments, the immunogenic composition comprises a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: (a) the first LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 29, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29; and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. A nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NO:37.

[0232] In some embodiments, the immunogenic composition comprises a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: (a) the first Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO:90, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:90; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO:37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:37. A nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of NO:37.

[0233] As appropriate or as needed, the HBV polymerase polypeptide mutant and HBV core-sAg fusion protein can be provided in an immunogenic composition at a ratio ranging from 1:10 to 10:1 (e.g., 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1). In various embodiments, the ratio can be measured in units of plaque forming units (PFU), focus forming units (FFU), infectious units (IU), or viral particles (vp).

[0234] In various embodiments, the one or more polynucleotides is DNA, cDNA, mRNA, or self-replicating RNA.

[0235] In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a truncated HBV polymerase polypeptide or an HBV polymerase deletion mutant polypeptide as described herein; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein as described herein. Depending on the circumstances or needs, the first viral expression vector and the second viral expression vector can be provided in a ratio ranging from 1:10 to 10:1 (e.g., 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1).

[0236] In some embodiments, the immunogenic composition comprises about 10 3 to about 10 12 The concentration of virus is in the range of about 10 focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or virus particles (vp) 4 to about 10 7 FFU or PFU of virus, for example, about 10 3 to about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 Each of the first viral expression vector and the second viral expression vector (100 viral FFU or PFU or IU or vp).

[0237] In various embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition are independently from a taxonomic family selected from the family Adenoviridae, Arenaviridae, Herpesviridae (e.g., cytomegalovirus), Poxviridae (e.g., vaccinia virus, e.g., modified vaccinia Ankara (MVA)), Flaviviridae (e.g., yellow fever virus), Rhabdoviridae (e.g., vesiculovirus, e.g., Maraba vesiculovirus), Togaviridae (e.g., alphavirus). In various embodiments, the first viral expression vector and the second viral expression vector can be from the same taxonomic family or from different taxonomic families. For example, in some embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition are both from the family Adenoviridae, Arenaviridae, or Poxviridae (e.g., vaccinia virus, e.g., modified vaccinia Ankara (MVA)).

[0238] In some embodiments, the first and second viral expression vectors in the immunogenic composition are from the Arenaviridae family. In some embodiments, the first and second viral expression vectors are from an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV), Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV). In some embodiments, the first and second viral expression vectors are from an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV) or Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)).

[0239] In various embodiments, the first and second viral expression vectors in the immunogenic composition are replication defective or replication-deficient. In some embodiments, the first and second viral expression vectors in the immunogenic composition are replication-attenuated.

[0240] 6. Treatment Methods

[0241] Also provided is a method for eliciting an immune response against human hepatitis B virus (HBV) in a subject in need thereof. Also provided is a method for treating or preventing human hepatitis B virus (HBV) in a subject in need thereof. Also provided is a method for suppressing HBV replication in an infected individual. Also provided is a method for reducing the viral load associated with HBV infection. In various embodiments, the method includes administering an effective amount of an immunogenic composition as described herein to the subject. In various embodiments, a "subject" or "individual" is a human, a marmot, a Peking duck, a mouse, or a non-human primate (e.g., a chimpanzee).

[0242] As used herein, "treatment" or "treat" or "treating" refers to an approach for obtaining a beneficial or desired result. For the purposes of this disclosure, a beneficial or desired result includes, but is not limited to, alleviation of symptoms and / or reduction in the extent of symptoms, delay in progression, and / or prevention of worsening of symptoms associated with a disease or condition. "Treatment" or "treating" may include one or more of the following: a) inhibiting a disease or condition (e.g., reducing one or more symptoms caused by the disease or condition, and / or reducing the extent of the disease or condition); b) slowing or preventing the development of one or more symptoms associated with a disease or condition (e.g., stabilizing the disease or condition, delaying worsening or progression of the disease or condition); and c) alleviating a disease or condition, e.g., causing regression of clinical symptoms, improving the disease state, delaying progression of the disease, improving quality of life, and / or prolonging survival.

[0243] As used herein, "delay" refers to the progression of a disease or condition, meaning to slow, hinder, slow, retard, stabilize, and / or postpone the progression of the disease or condition. The delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. It will be apparent to one skilled in the art that a sufficient or significant delay can actually encompass prevention, as the individual does not develop the disease or condition.

[0244] As used herein, "prevention" or "prevention" or "preventing" refers to a scheme that prevents the onset of a disease or disorder so that the clinical symptoms of the disease do not develop. Therefore, "prevention" involves administering therapy (e.g., administering a therapeutic substance) to a subject before signs of disease can be detected in the subject (e.g., administering a therapeutic substance to the subject in the absence of a detectable infectious agent (e.g., virus) in the subject). The subject can be an individual at risk of developing a disease or disorder, such as an individual with one or more risk factors known to be associated with the development or onset of a disease or disorder. Therefore, in certain embodiments, the term "prevention of HBV infection" refers to administering an anti-HBV therapeutic substance to a subject without detectable HBV infection. It should be understood that the subject for anti-HBV preventive therapy can be an individual at risk of being infected with the HBV virus. It should also be understood that prevention does not require a 100% success rate. In some cases, prevention can be understood as reducing the risk of infection, rather than completely eliminating the occurrence of infection.

[0245] As used herein, "therapeutically effective amount" or "effective amount" refers to an amount that effectively elicits a desired biological or medical response, including an amount of an immunogenic composition that is sufficient to achieve such treatment of the disease when administered to a subject for the treatment of a disease. The effective amount will vary depending on the immunogenic composition, the disease and severity of the subject to be treated, as well as the age, weight, etc. The effective amount may include a range of amounts. The effective amount may be one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired therapeutic endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and if a desired or beneficial result can be achieved or realized in combination with one or more other agents, then a single agent may be considered to be administered in an effective amount. The appropriate dosage of any co-administered compound may optionally be reduced due to the combined action of the compounds (e.g., additive or synergistic effect).

[0246] In various embodiments, the immunogenic composition administered comprises a mixture containing a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a truncated HBV polymerase polypeptide as described herein or an HBV polymerase deletion mutant polypeptide as described herein; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein as described herein.

[0247] In various embodiments, the immunogenic composition administered comprises a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding an HBV polymerase polypeptide mutant comprising or consisting of: any one of SEQ ID NOs: 5-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 5-14; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising or consisting of: any one of SEQ ID NOs: 38-41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41. The present invention also provides the amino acid sequence of any one of NO:38-41 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequence. Such immunogenic compositions can be used in the initial exemption composition and / or booster composition.

[0248] In various embodiments, the immunogenic composition administered comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding an HBV polymerase polypeptide mutant comprising or consisting of: any one of SEQ ID NOs: 13-14, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 13-14; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising or consisting of: any one of SEQ ID NOs: 38-41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41. The present invention also provides the amino acid sequence of any one of NO:38-41 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequence. Such immunogenic compositions can be used in the initial exemption composition and / or booster composition.

[0249] With respect to the core-sAg fusion polypeptide in the administered immunogenic composition, in some embodiments, the core polypeptide comprises a serine (S) residue at the amino acid position corresponding to position 12 and an asparagine (N) residue at the amino acid position corresponding to position 67, wherein position numbering is with reference to SEQ ID NO: 65 or SEQ ID NO: 66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at the amino acid position corresponding to position 68, wherein position numbering is with reference to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 53, an isoleucine (I) residue at the amino acid position corresponding to position 68, a threonine (T) residue at the amino acid position corresponding to position 125, a proline (P) residue at the amino acid position corresponding to position 127, a phenylalanine (F) residue at the amino acid position corresponding to position 161, a tyrosine (Y) residue at the amino acid position corresponding to position 200, a serine (S) residue at the amino acid position corresponding to position 210, and a leucine (L) residue at the amino acid position corresponding to position 213, wherein the positions are numbered with reference to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the core-sAg fusion polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 12, an asparagine (N) residue at the amino acid position corresponding to position 67, a valine (V) residue at the amino acid position corresponding to position 74, a phenylalanine (F) residue at the amino acid position corresponding to position 97, a threonine (T) residue at the amino acid position corresponding to position 249, a threonine (T) residue at the amino acid position corresponding to position 250, a serine (S) residue at the amino acid position corresponding to position 317, a serine (S) residue at the amino acid position corresponding to position 318, an arginine (R) residue at the amino acid position corresponding to position 326, a tyrosine (Y) residue at the amino acid position corresponding to position 338, a glycine (G) residue at the amino acid position corresponding to position 363, and an alanine (A) residue at the amino acid position corresponding to position 372, wherein positions are numbered with reference to SEQ ID NO: 41.

[0250] In various embodiments, the immunogenic composition administered comprises a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding an HBV polymerase polypeptide mutant comprising or consisting of SEQ ID NO: 13, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising or consisting of SEQ ID NO: 41, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41. NO:41 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of NO:41. Such immunogenic compositions can be used in primary immunity compositions and / or booster compositions.

[0251] In various embodiments, the immunogenic composition administered comprises a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of: a nucleic acid sequence of any one of SEQ ID NOs: 27-32 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, and 92), or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 27-32 and 89-94 (e.g., SEQ ID NOs: 29, 89, 90, and 92); and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of: NO: 33-37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 33-37. Such immunogenic compositions can be administered in a priming composition and / or a boosting composition.

[0252] In various embodiments, the immunogenic composition administered comprises a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 29, 89, 90, or 92, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, 89, 90, or 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. NO:37 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleic acid sequence. Such immunogenic compositions can be used in primary immunity compositions and / or booster compositions.

[0253] In various embodiments, the first viral expression vector and the second viral expression vector in the administered immunogenic composition are independently from a taxonomic family selected from the family Adenoviridae, Arenaviridae, Herpesviridae (e.g., cytomegalovirus), Poxviridae (e.g., vaccinia virus, e.g., modified vaccinia Ankara (MVA)), Flaviviridae (e.g., yellow fever virus), Rhabdoviridae (e.g., vesiculovirus, e.g., Maraba vesiculovirus), Togaviridae (e.g., alphavirus), as described above and herein. In various embodiments, the first viral expression vector and the second viral expression vector can be from the same taxonomic family or from different taxonomic families. For example, in some embodiments, the first viral expression vector and the second viral expression vector in the administered immunogenic composition are both from the family Adenoviridae, Arenaviridae, or Poxviridae (e.g., vaccinia virus, e.g., modified vaccinia Ankara (MVA)).

[0254] In some embodiments, the first viral expression vector and the second viral expression vector are from the Arenaviridae family. In some embodiments, the first viral expression vector and the second viral expression vector in the administered immunogenic composition are from an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV), Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV). In some embodiments, the first viral expression vector and the second viral expression vector in the administered immunogenic composition are from an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV) or Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus (PICV)).

[0255] In various embodiments, the first and second viral expression vectors in the administered immunogenic composition are replication-defective or replication-deficient. In some embodiments, the first and second viral expression vectors in the administered immunogenic composition are replication-attenuated.

[0256] In some embodiments, the immunogenic composition administered comprises a mixture comprising a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: (a) the first LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 29, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29; and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO: 37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. NO:37 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleic acid sequence. Such immunogenic compositions can be used in primary immunity compositions and / or booster compositions.

[0257] In some embodiments, the immunogenic composition administered comprises a mixture comprising a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: (a) the first Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO:90, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:90; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of SEQ ID NO:37, or a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:37. NO:37 at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleic acid sequence. Such immunogenic compositions can be used in primary immunity compositions and / or booster compositions.

[0258] In various embodiments, the subject is infected with HBV virus, suspected of being infected with HBV virus, or has the risk of being infected with HBV virus. As used herein, "risky individual" refers to an individual with the risk of developing a disease to be treated. The individual of "risky" may or may not have a detectable disease or disease, and may or may not show a detectable disease before the treatment of the method described herein. "Risky" means that the individual has one or more so-called risk factors, which are relevant to the development of the disease or disease and are measurable parameters known in the art. The probability of an individual developing a disease or disease with one or more of these risk factors is higher than that without these risk factors. In various embodiments, the subject is chronically infected with HBV, for example, infected with HBV for longer than 6 months. Typically, the individual suffers from chronic hepatitis B infection, although the people who treat acute HBV infection are within the scope of the present disclosure. Therefore, in some embodiments, the subject is acutely infected with HBV. In some embodiments, the subject is co-infected with hepatitis D virus (HDV).

[0259] In various embodiments, the subject can be asymptomatic. In some embodiments, the subject experiences or shows symptoms related to HBV infection. The symptoms of HBV may include, for example, jaundice, a swollen vascular network visible on the skin, dark (for example, orange or brown) urine, light-colored stool, fever, lasting fatigue, discomfort, abdominal pain, abdominal fluid, loss of appetite, nausea and vomiting. Chronic HBV infection can cause one or more symptoms, including, for example, liver failure, liver cancer, liver fibrosis and cirrhosis. One or more administrations of immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, carriers comprising such polypeptides or polynucleotides, LNPs and immunogenic compositions can prevent, delay, alleviate, mitigate, suppress, reverse or eliminate one or more symptoms related to or caused by HBV infection.

[0260] In some embodiments, the immunogenic composition is administered via a route selected from intravenous, intramuscular, intradermal, subcutaneous, and mucosal (eg, buccal, intranasal, rectal, vaginal).

[0261] In some embodiments, the dosage of the immunogenic composition administered comprises about 10 3 to about 10 12 The concentration of virus is in the range of about 10 focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or virus particles (vp) 4 to about 10 7 FFU or PFU of virus, for example, about 10 3 to about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some embodiments, the method requires intravenous or intramuscular administration of about 10 viral FFU or PFU or IU or vp) of the first viral expression vector and the second viral expression vector. 6 to about 10 8 Virus FFU or PFU or IU or vp / administration.

[0262] In various embodiments, the method includes a primary immunization-boosting regimen. In some embodiments, the primary immunization-boosting regimen needs to be administered at the first time point with the primary immunization composition and administered at one or more subsequent time points with one or more boosting compositions. Depending on the circumstances, the method may need to repeat the primary immunization-boosting regimen once or multiple iterations. In various embodiments, the primary immunization composition and the one or more boosting compositions are administered at intervals of at least 1 week and at most at least 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. Depending on the circumstances, the dosage or frequency of administration of the immunogenic composition can be adjusted during treatment according to the judgment of the physician administering the composition. As appropriate, the subject can be treated with multiple administrations for at least about 2 to 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, or longer, or until sAg is no longer detected in the subject's serum or plasma.

[0263] In some embodiments, after one or more administrations of one or more immunogenic polypeptides as described herein, or one or more polynucleotides encoding one or more immunogenic polypeptides as described herein, or one or more vectors expressing one or more immunogenic polypeptides as described herein, optionally with one or more additional therapeutic agents as described herein, the subject does not exhibit symptoms of HBV in the absence of antiviral treatment for at least 6 months, at least 1 year, at least 2 years, at least 3 years, or longer. In some embodiments, after one or more administrations of one or more immunogenic polypeptides as described herein, or one or more polynucleotides encoding one or more immunogenic polypeptides as described herein, or one or more vectors expressing one or more immunogenic polypeptides as described herein, optionally with one or more additional therapeutic agents as described herein, sAg is no longer detectable in the serum or plasma of the subject in the absence of antiviral treatment for at least 6 months, e.g., at least 1 year, at least 2 years, at least 3 years, or longer.

[0264] Depending on the circumstances or needs, the initial immunization composition and the booster composition may comprise the same immunogenic composition or different immunogenic compositions. In various embodiments, the initial immunization composition and the booster composition comprise the same one or more polypeptides and the same expression vector (e.g., viral expression vector). In some embodiments, the initial immunization composition and the booster composition comprise different polypeptides and / or different expression vectors (e.g., viral expression vector). For example, in some embodiments, the initial immunization composition and the booster composition comprise the same one or more polypeptides and different expression vectors (e.g., viral vectors from different virus species within a taxonomic family, viral vectors from different taxonomic families, viral vectors with different replication capabilities). In some embodiments, the initial immunization composition and the booster composition comprise different immunogenic polypeptides and the same expression vector (e.g., viral expression vector).

[0265] In some embodiments, the method comprises priming with a priming composition comprising one or more viral expression vectors and boosting with a boosting composition comprising one or more viral expression vectors. In some embodiments, the prime-boost regimen comprises:

[0266] a) priming with a priming composition comprising one or more viral expression vectors and boosting with a boosting composition comprising one or more polynucleotides, wherein the one or more polynucleotides comprise DNA, cDNA, mRNA, or self-replicating RNA;

[0267] b) priming with a priming composition comprising one or more polynucleotides, wherein the one or more polynucleotides comprise DNA, cDNA, mRNA or self-replicating RNA, and boosting with a boosting composition comprising one or more viral expression vectors;

[0268] c) priming with a priming composition comprising one or more viral expression vectors and boosting with a boosting composition comprising one or more viral expression vectors, wherein the one or more viral expression vectors in the priming composition and the one or more viral expression vectors in the boosting composition are from the same, related, or unrelated taxonomic families;

[0269] d) priming with a priming composition comprising one or more replication-defective viral expression vectors and boosting with a boosting composition comprising one or more replication-defective viral expression vectors, wherein the one or more replication-defective viral expression vectors in the priming composition and the one or more replication-defective viral expression vectors in the boosting composition are from the same, related, or unrelated taxonomic families;

[0270] e) priming with a priming composition comprising one or more replication-attenuated viral expression vectors and boosting with a boosting composition comprising one or more replication-attenuated viral expression vectors, wherein the one or more replication-attenuated viral expression vectors in the priming composition and the one or more replication-attenuated viral expression vectors in the boosting composition are from the same, related, or unrelated taxonomic families;

[0271] f) priming with a priming composition comprising one or more replication-defective viral expression vectors and boosting with a boosting composition comprising one or more replication-attenuated viral expression vectors;

[0272] g) priming with a priming composition comprising one or more replication-attenuated viral expression vectors and boosting with a boosting composition comprising one or more replication-defective viral expression vectors;

[0273] h) priming with a priming composition comprising one or more lymphocytic choriomeningitis mammalian arenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more picinder mammalian arenavirus (PICV) viral expression vectors;

[0274] i) priming with a priming composition comprising one or more Pichin's mammalian arenavirus (PICV) viral expression vectors and boosting with a boosting composition comprising one or more lymphocytic choriomeningitis mammalian arenavirus (LCMV) viral expression vectors;

[0275] j) priming with a priming composition comprising one or more replication-defective Pichinde mammalian arenavirus (PICV) viral expression vectors and boosting with a boosting composition comprising one or more replication-defective lymphocytic choriomeningitis mammalian arenavirus (LCMV) viral expression vectors;

[0276] k) priming with a priming composition comprising one or more replication-defective lymphocytic choriomeningitis mammalian arenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more replication-defective picinder mammalian arenavirus (PICV) viral expression vectors;

[0277] 1) priming with a priming composition comprising one or more arenavirus expression vectors and boosting with a boosting composition comprising one or more adenovirus expression vectors;

[0278] m) priming with a priming composition comprising one or more adenovirus viral expression vectors and boosting with a boosting composition comprising one or more arenavirus viral expression vectors;

[0279] n) priming with a priming composition comprising one or more poxvirus expression vectors and boosting with a boosting composition comprising one or more arenavirus expression vectors;

[0280] o) priming with a priming composition comprising one or more arenavirus expression vectors and boosting with a boosting composition comprising one or more poxvirus expression vectors;

[0281] p) priming with a priming composition comprising one or more poxvirus expression vectors and boosting with a boosting composition comprising one or more adenovirus expression vectors; or

[0282] q) priming with a priming composition comprising one or more adenoviral expression vectors and boosting with a boosting composition comprising one or more poxviral expression vectors.

[0283] In some embodiments, the method comprises priming with a priming composition comprising one or more viral expression vectors and boosting with a boosting composition comprising one or more viral expression vectors. In some embodiments, the prime-boost regimen comprises:

[0284] a) priming with a priming composition comprising one or more lymphocytic choriomeningitis mammalian arenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more picinder mammalian arenavirus (PICV) viral expression vectors;

[0285] b) priming with a priming composition comprising one or more Pichinde mammalian arenavirus (PICV) viral expression vectors and boosting with a boosting composition comprising one or more lymphocytic choriomeningitis mammalian arenavirus (LCMV) viral expression vectors;

[0286] c) priming with a priming composition comprising one or more replication-defective Pichin's mammalian arenavirus (PICV) viral expression vectors and boosting with a boosting composition comprising one or more replication-defective lymphocytic choriomeningitis mammalian arenavirus (LCMV) viral expression vectors; or

[0287] d) priming with a priming composition comprising one or more replication-defective lymphocytic choriomeningitis mammalian arenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more replication-defective picinder mammalian arenavirus (PICV) viral expression vectors.

[0288] In various embodiments, the priming composition and the boosting composition comprise an immunogenic composition as described herein.

[0289] In some embodiments, the subject is not receiving antiviral therapy or antiviral therapy is discontinued prior to administration of the one or more immunogenic compositions. In some embodiments, antiviral therapy is discontinued after one or more administrations of the composition.

[0290] In some embodiments, the treatment method activates CD8+ T cells that target one or more HBV polypeptide epitopes in the subject. In some embodiments, the treatment method elicits the production of antibodies that bind to one or more HBV polypeptides in the subject.

[0291] 7. Combination therapy

[0292] In certain embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions are combined or co-administered with one, two, three, four, or more additional therapeutic agents. In certain embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions are combined or co-administered with two additional therapeutic agents. In certain embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions are combined or co-administered with three additional therapeutic agents. In certain embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions are combined or co-administered with four additional therapeutic agents. The one, two, three, four, or more additional therapeutic agents can be different therapeutic agents selected from the same class of therapeutic agents, and / or they can be selected from different classes of therapeutic agents.

[0293] As used herein, "co-administration" refers to administering a unit dose of an immunogenic polypeptide as described herein, a polynucleotide encoding such a polypeptide, a carrier comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition before or after administering a unit dose of one or more additional therapeutic agents. For example, an immunogenic composition disclosed herein is administered within seconds, minutes, or hours of administering one or more additional therapeutic agents. For example, in some embodiments, a unit dose of the immunogenic composition of the present disclosure is administered first, followed by administration of a unit dose of one or more additional therapeutic agents within seconds or minutes. Alternatively, in other embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed by administration of a unit dose of the immunogenic composition of the present disclosure within seconds or minutes. In some embodiments, a unit dose of an immunogenic polypeptide as described herein, a polynucleotide encoding such a polypeptide, a carrier comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition is administered first, followed by administration of a unit dose of one or more additional therapeutic agents after a few hours (e.g., 1-12 hours). In other embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed several hours (e.g., 1-12 hours) later by administration of a unit dose of an immunogenic polypeptide, polynucleotide encoding such polypeptide, vector comprising such polypeptide or polynucleotide, LNP, and immunogenic composition as described herein.

[0294] Co-administration of immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein with one or more additional therapeutic agents generally refers to the simultaneous or sequential administration of an immunogenic composition disclosed herein and one or more additional therapeutic agents such that a therapeutically effective amount of each agent is present in the patient's body.

[0295] In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more additional therapeutic agents as described herein, with the components of the composition being administered as a simultaneous or sequential regimen. When administered sequentially, the combination can be administered in two or more administrations.

[0296] In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one, two, three, four, or more additional therapeutic agents selected from the group consisting of: HBV combination drugs, HBV vaccines, HBV DNA polymerase inhibitors, immunomodulators, toll-like receptor (TLR) modulators, interferon alpha receptor ligands, hyaluronidase inhibitors, HBV antigen inhibitors (e.g., HBV core antigen (HBcAg) inhibitors, HBV surface antigen (HBsAg) inhibitors, HBx inhibitors, HBV E antigen inhibitors), anti-HBV antigen antibodies, inhibitory nucleic acids targeting HBV (e.g., antisense oligonucleotides, short interfering RNA (siRNA), DNA-guided RNA interference (ddRNAi)), HBsAg secretion or assembly inhibitors, HBV viral entry inhibitors, immune checkpoint inhibitors, cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibitors, cyclophilin inhibitors, endonuclease modulators, ribonucleotide reductase inhibitors, covalently closed circular DNA (cccDNA) inhibitors, farnesoid X receptor (FXR) agonists, STING agonists, anti-HBV antibodies, CCR2 chemokine antagonists, thymosin agonists , cytokines, nuclear protein regulators, retinoic acid-inducible gene 1 stimulators, NOD2 stimulators, phosphatidylinositol 3-kinase (PI3K) inhibitors, indoleamine-2,3-dioxygenase (IDO) pathway inhibitors, ZCCHC14 inhibitors, tertiary lymphoid aggregate inducers, nucleic acid polymers (e.g., NAP and STOPS), PD-1 inhibitors, PD-L1 inhibitors, recombinant thymosin α-1, Bruton's tyrosine kinase (BTK) inhibitors, lysine demethylase (KDM) inhibitors, HBV replication inhibitors, arginase inhibitors, gene therapy and cell therapy, gene editing, cell therapy, TCR-T cell therapy and other HBV drugs.

[0297] In certain embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein can be combined with chemotherapeutic agents, immunomodulatory agents, immunotherapeutic agents, therapeutic antibodies, therapeutic vaccines, bispecific antibodies, and "antibody-like" therapeutic proteins (such as Anti-pMHC TCR-like antibodies, Fab derivatives), antibody-drug conjugates (ADCs), gene modifiers or gene editors targeting HBV (e.g., CRISPR-Cas (e.g., Cas9, Cas12, Cascade, Cas13), zinc finger nucleases, homing endonucleases, homing meganucleases (e.g., ARCUS), synthetic nucleases, TALENs), cell therapies (e.g., T-cells, NK cells, macrophages with chimeric antigen receptors (CAR)), and TCR-T (engineered T cell receptors), or any combination thereof, are combined or co-administered.

[0298] In certain embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one, two, three, four, or more additional therapeutic agents, such as β-dioxygenase (IDO) inhibitors, apolipoprotein A1 regulators, arginase inhibitors, B and T attenuation factor inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, CCR2 chemokine antagonists, CD137 inhibitors, CD160 inhibitors, CD305 inhibitors, CD4 agonists and modulators, compounds targeting hepatitis B core antigen (HBcAg), core protein allosteric modulators, covalently closed circular DNA (cccDNA) inhibitors, cyclophilin inhibitors, cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibitors, DNA polymerase inhibitors, endonuclease regulators, epigenetic modifiers, farnesoid X receptor (FXR) agonists, HBV DNA polymerase inhibitors, HBV replication inhibitors, HBVRNase inhibitors, HBV virus entry inhibitors, HBx inhibitors, hepatitis B large envelope protein modulators, hepatitis B large envelope protein stimulators, hepatitis B structural protein modulators, hepatitis B surface antigen (HBsAg) inhibitors, hepatitis B surface antigen (HBsAg) secretion or assembly inhibitors, hepatitis B virus E antigen inhibitors, hepatitis B virus replication inhibitors, hepatitis virus structural protein inhibitors, HIV-1 reverse transcriptase inhibitors, hyaluronidase inhibitors, inhibitor of apoptosis protein family protein (IAP) inhibitors, IL-2 agonists, IL-7 agonists, immunomodulators, indoleamine-2 inhibitors, Ribonucleotide reductase inhibitors, interleukin-2 ligands, ipi4 inhibitors, lysine demethylase inhibitors, histone demethylase inhibitors, KDM1 inhibitors, KDM5 inhibitors, killer cell lectin-like receptor subfamily G member 1 inhibitors, lymphocyte activation gene 3 inhibitors, lymphotoxin beta receptor activators, Axl modulators, B7-H3 modulators, B7-H4 modulators, CD160 modulators, CD161 modulators, CD27 modulators, CD47 modulators, CD70 modulators, GITR modulators, HEVEM modulators, ICOS modulators, Me r regulators, NKG2A regulators, NKG2D regulators, OX40 regulators, SIRPα regulators, TIGIT regulators, Tim-4 regulators, Tyro regulators, Na+-taurocholate co-transporting polypeptide (NTCP) inhibitors, natural killer cell receptor 2B4 inhibitors, NOD2 gene stimulators, nuclear protein inhibitors, nuclear protein regulators, OX-40 receptor agonists, PD-1 inhibitors, PD-L1 inhibitors, peptidyl prolyl isomerase inhibitors, phosphatidylinositol-3 kinase (PI3K) inhibitors, retinoic acid-inducible gene 1 stimulators, reverse transcriptase inhibitors, ribonuclease inhibitors, RNA DNA polymerase inhibitors, SLC10A1 gene inhibitors, SMAC mimetics, Src tyrosine kinase inhibitors, stimulators of interferon genes (STING) agonists, stimulators of NOD1, T cell surface glycoprotein CD28 inhibitors, T cell surface glycoprotein CD8 regulators, thymosin agonists, thymosin α1 ligands, Tim-3 inhibitors, TLR-3 agonists, TLR-7 agonists, TLR-9 agonists, TLR9 agonists or gene stimulators, toll-like receptor (TLR) modulators, viral ribonucleotide reductase inhibitors, and combinations thereof.

[0299] Antiviral drugs that inhibit HBV

[0300] In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more antiviral agents. In some embodiments, the one or more antiviral agents are selected from the group consisting of lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), tenofovir disoproxil fumarate (TDF), tenofovir disoproxil fumarate, and emtricitabine. Tenofovir alafenamide (TAF or VEMLIDY) and ledipasvir and sofosbuvir

[0301] Other HBV drugs

[0302] Examples of other drugs for treating HBV that can be combined or co-administered include α-hydroxytropolone, amdosovir, antroquinol, β-hydroxycytidine, ARB-199, CCC-0975, ccc-R08, efcitabine, ezetimibe, cyclosporine A, gentioside (gentiopicroside), HH-003, hepalatide, JNJ-56136379, nitazoxanide, birenapam, NJK14047, NOV-205 (molixan, BAM-205), oligonucleotides, mivotifen, feron, GST-HG-131, levamisole, Ka Shu Ning, alloferon, WS-007, Y-101 (Ti Fen Tai), rSIFN-co, PEG-IIFNm, KW-3, BP-Inter-014, caryophyllin, HepB-nRNA, cTP-5(rTP-5), HSK-II-2, HEISCO-106-1, HEISCO-106, Hepbarna, IBPB-006IA, Hepuyinfen, DasKloster 0014-01, ISA-204, Jiangantai (Ganxikang), MIV-210, OB-AI-004, PF-06, picroside, DasKloster-0039, hepulantai, IMB-2613, NCO-48 fumarate, XTYW-001, SFA-001, TCM-800B, reduced glutathione, RO-6864018, ENOB-HB-01, RG-7834, QL-007 sofosbuvir, ledipasvir, UB-551, PA-1010, HPN-BV1, STSG-0002 and ZH-2N, as well as US20150210682 (Roche), US2016 / 01223 44 (Roche), WO2015173164, WO2016023877, US2015252057A (Roche), WO16128335A1 (Roche), WO16120186A1 (Roche), US2016237090A (Roche), WO16107833A1 (Roche), WO16107832A1 (Roche), US2016176899A (Roche), WO16102438A1 (Roche), WO16012470A1 (Roche), US2016220586A (Roche) and US2015031687A (Roche).

[0303] Examples of combination drugs for treating HBV that can be combined or co-administered include tenofovir disoproxil fumarate and emtricitabine Ledipasvir and sofosbuvir ABX-203 (NASVAC), lamivudine and PEG-IFNα; adefovir and PEG-IFNα; and INO-1800 (INO-9112 and RG7944).

[0304] HBV vaccine

[0305] In various embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs and immunogenic compositions are combined or co-administered with one or more HBV vaccines. HBV vaccines that can be combined or co-administered (e.g., in a primary immunization-boosting prevention regimen) include preventive and therapeutic vaccines. Examples of HBV preventive vaccines include Vaxelis, Hexaxim, Heplisav, Mosquirix, DTwP-HBV vaccine, Bio-Hep-B, D / T / P / HBV / M (LBVP-0101; LBVW-0101), DTwP-Hepb-Hib-IPV vaccine, HeberpentaL, DTwP-HepB-Hib, V-419, CVI-HBV-001, Tetrabhay, Hepatitis B preventive vaccine (Advax Super D), Hepatrol-07, GSK-223192A, ENGERIX Recombinant hepatitis B vaccine (intramuscular injection, Kangtai Biological Products), recombinant hepatitis B vaccine (Hansenual polymorpha yeast, intramuscular injection, Hualan Biological Engineering), recombinant hepatitis B surface antigen vaccine, Bimmugen, CARG-101, Euforavac, Eutravac, anrix-DTaP-IPV-Hep B, HBAI-20, Infanrix-DTaP-IPV-Hep B-Hib, Pentabio Vaksin DTP-HB-Hib, Comvac 4, Twinrix, Euvax-B, Tritanrix HB, Infanrix Hep B, Comvax, DTP-Hib-HBV vaccine, DTP-HBV vaccine, Yi Tai, Heberbiovac HB, Trivac HB, GerVax, DTwP-Hep B-Hib vaccine, Bilive, Hepavax-Gene, SUPERVAX, Comvac5, Shanvac-B, Hebsulin, Recombivax HB, Revac B mcf, Revac B+, Fendrix, DTwP-HepB-Hib, DNA-001, Shan5, Shan6, rhHBsAG vaccine, HBI pentavalent vaccine, LBVD, Infanrix HeXa, YS-HBV-001, IR-101H, TVAX-008, and DTaP-rHB-Hib vaccine.

[0306] Examples of HBV therapeutic vaccines that can be combined or co-administered (e.g., in a prime-boost regimen) include HBsAG-HBIG complex, ARB-1598, Bio-Hep-B, abi-HB (intravenous), ABX-203 (NASVAC), Tetrabhay, GX-110E, GS-4774, peptide vaccine (εPA-44), Hepatrol-07, NASVAC (NASTERAP), IMP-321, BEVAC, Revac B mcf, Revac B+, MGN-1333, KW-2, CVI-HBV-002, AltraHepB, VGX-6200, FP-02, FP-02.2 (HepTcell), NU-500, HBVax, im / TriGrid / antigen vaccine, Mega-CD40L-adjuvant vaccine, HepB-v, RG7944 (INO-1800), recombinant VLP-based therapeutic vaccine (HBV infection, VLP Biotech), AdTG-17909, AdTG-17910, AdTG-18202, ChronVac-B, TG-1050, VVX-001, GSK-3528869A (ChAd155-hli-HBV+MVA-HBV+Hbc-HBs / AS01B-4), VBI-2601, VTP-300 (ChAdOx1-SIi-HBV-CPmut-TPA-Ssh prime and MVA-SIi-HBV-CPmut-TPA-Ssh boost), Lm HBV, and BM32 (Tulaeva et al., EBioMedicine (2020) 102953). HBV arenavirus vaccines are described, for example, in WO2017076988 and WO2017198726.

[0307] HBV DNA polymerase inhibitors

[0308] In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more polymerase inhibitors. Examples of HBV DNA polymerase inhibitors that can be combined or co-administered include adefovir, Emtricitabine Tenofovir disoproxil fumarate Tenofovir alafenamide, tenofovir, tenofovir disoproxil, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir disoproxil fumarate, tenofovir disoproxil fumarate, tenofovir octadecyloxyethyl ester, CMX-157, tenofovir exalidex, besifovir, entecavir Entecavir maleate, telbivudine Feloxivir, pradefovir, clavudine, ribavirin, lamivudine Phosphorus azide, famciclovir, fusolin, metcapavir, SNC-019754, FMCA, AGX-1009, AR-II-04-26, HIP-1302, tenofovir disoproxil aspartate, tenofovir disoproxil orotate, AiB-001, and HS-10234.

[0309] Immunomodulators

[0310] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more immunomodulatory agents (e.g., immune checkpoint inhibitors, tumor necrosis factor (TNF) receptor superfamily (TNFRSF) agonists, immunostimulatory agents, e.g., TLR agonists). Examples of immunomodulators that can be combined or co-administered include retasimid, imidol hydrochloride, ingaron, dermaVir, hydroxychloroquine, proleukin, hydroxyurea, mycophenolate mofetil (MPA) and its ester derivative mycophenolate mofetil (MMF), JNJ-440, WF-10, AB-452, ribavirin, IL-12, INO-9112, polymer polyethyleneimine (PEI), Gepon, VGV-1, MOR-22, CRV-431, JNJ-0535, TG-1050, ABI-H2158, BMS-936559, GS-9688, RO-7011785 and corresponding prodrugs RO-702053, RG-7854, RO-6871765, AIC-649 and IR-103.

[0311] Toll-like receptor (TLR) agonists

[0312] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more agonists or stimulators of toll-like receptors (TLRs). In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with agonists of TLRs (e.g., TLR1 (NCBI gene ID: 7096), TLR2 (NCBI gene ID: 7097), TLR3 (NCBI gene ID: 7098), TLR4 (NCBI gene ID: 7099), TLR5 (NCBI gene ID: 7100), TLR6 (NCBI gene ID: 10333), TLR7 (NCBI gene ID: 51284), TLR8 (NCBI gene ID: 51311), TLR9 (NCBI gene ID: 54106), and / or TLR10 (NCBI gene ID: 81793), TLR11, TLR12, and TLR13).

[0313] Examples of TLR3 agonists that can be combined or co-administered include resalimod, polyICLC, Apoxxim, IPH-33, MCT-465, MCT-475, and ND-1.1.

[0314] Examples of TLR4 agonists that may be combined or co-administered include G-100 and GSK-1795091.

[0315] Exemplary TLR7 agonists that can be combined or co-administered include, but are not limited to, AL-034, DSP-0509, GS-9620 (visamod), LHC-165, TMX-101 (imiquimod), GSK-2245035, resiquimod, DSR-6434, DSP-3025, IMO-4200, MCT-465, telratolimod (MEDI-9197), 3M-051, SB-9922, 3M-052, Limtop, TMX-30X, TMX-202, RG-7863, RG-7854, RG-7795, RO-7011785 and the corresponding prodrug RO-702053, and US20100143301 (Gilead Sciences), US20110098248 (Gilead Sciences), andUS20090047249 (Gilead Sciences), US20140045849 (Janssen), US20140073642 (Janssen), WO2014 / 056953 (Janssen), WO2014 / 076221 (Ja nssen), WO2014 / 128189(Janssen), US20140350031(Janssen), WO2014 / 023813(Janssen), US20080234251(Array Biopharma), US20080306050 (Array Biopharma), US20100029585 (Ventirx Pharma), US20110092485 (Ventirx Pharma), US20110118235 (Ventirx Pharma), US20120082658 (Ventirx Pharma), US20120219615 (Ventirx Pharma), US20140066432 (Ventirx Pharma), US20140088085 (Ventirx Pharma), US20140275167 (Novira Therapeutics) and US20130251673 (Novira Therapeutics).

[0316] Exemplary dual TLR7 / TLR8 agonists that may be combined or co-administered are NKTR-262, teramod, and BDB-001.

[0317] Exemplary TLR8 agonists that can be co-administered include, but are not limited to, E-6887, IMO-4200, IMO-8400, IMO-9200, MCT-465, teramod (MEDI-9197), motolimod, resiquimod, selgantolimod (GS-9688), HRS-9950, VTX-1463, VTX-763, 3M-051, 3M-052, SBT6050, and US2016289229 (Gilead Sciences), US20140045849(Janssen), US20140073642(Janssen), WO2014 / 056953(Janssen), WO2014 / 076221(Ja nssen), WO2014 / 128189(Janssen), US20140350031(Janssen), WO2014 / 023813(Janssen), US20080234251(Array Biopharma), US20080306050 (Array Biopharma), US20100029585 (Ventirx Pharma), US20110092485 (Ventirx Pharma), US20110118235 (Ventirx Pharma), US20120082658 (Ventirx Pharma), US20120219615 (Ventirx Pharma), US20140066432 (Ventirx Pharma), US20140088085 (Ventirx Pharma), US20140275167 (Novira Therapeutics) and US20130251673 (Novira Therapeutics), US Patent No. 9670205 (Gilead Sciences, Inc.), US20160289229 (Gilead Sciences, Inc.), WO2017 / 048727 (Gilead Sciences, Inc.), US20180065938 (Gilead Sciences, Inc.) and US20180086755 (Gilead Sciences, Inc.).

[0318] Exemplary TLR9 agonists that can be combined or co-administered include, but are not limited to, AST-008, cobitolimod, CMP-001, IMO-2055, IMO-2125, S-540956, litenimod, MGN-1601, BB-001, BB-006, IMO-3100, IMO-8400, IR-103, IMO-9200, agatolimod, DIMS-9054, DV-1079, DV-1179, AZD-1419, lefitolimod (MGN-1703), CYT-003, CYT-003-QbG10, tilsotolimod, and PUL-042.

[0319] Additional examples of TLR7, TLR8, and TLR9 modulators that can be combined or co-administered include WO2017047769 (Teika Seiyaku), WO2015014815 (Janssen), WO2018045150 (Gilead Sciences Inc), WO2018045144 (Gilead Sciences Inc), WO2015162075 (Roche), WO2017034986 (University of Kansas), WO2018095426 (Jiangsu Hengrui Medicine Co Ltd), WO2016091698 (Roche), WO2016075661 (GlaxoSmithKline Biologicals), WO2016180743 (Roche), WO2018089695 (Dynavax Technologies), WO2016055553 (Roche), WO2015168279 (Novartis), WO2016107536 (Medshine Discovery), WO2018086593 (Livo (Shanghai) Pharmaceutical), WO2017106607 (Merck), WO2017061532 (Sumitomo Dainippon Pharma), WO2016023511 (ChiaTai Tianqing Pharmaceutical), WO2017076346 (Chia Tai Tianqing Pharmaceutical), WO2017046112 (Roche), WO2018078149 (Roche), WO2017040233 (3M Co), WO2016141092 (Gilead Sciences), WO2018049089 (BristolMyers Squibb), WO2015057655 (Eisai Co Ltd), WO2017001307 (Roche), WO2018005586 (BristolMyers Squibb), WO201704023 (3MCo), WO2017163264 (Council of Scientific and Industrial Research (India)), WO2018046460 (GlaxoSmithKlineBiologicals), WO2018047081 (Novartis), WO2016142250 (Roche), WO2015168269 (Novartis), WO201804163 (Roche), WO2018038877 (3M Co), WO2015057659 (Eisai Co) Ltd), WO2017202704 (Roche), WO2018026620 (Bristol Myers Squibb), WO2016029077 (Janus Biotherapeutics), WO201803143 (Merck), WO2016096778 (Roche), WO2017190669 (Shanghai De Novo Pharmatech), US09884866 (University of Minnesota), WO2017219931 (Sichuan KelunBiotech Biopharmaceutical), WO2018002319 (Janssen Sciences), WO2017216054 (Roche), WO2017202703 (Roche), WO2017184735 (IFM Therapeutics), WO2017184746 (IFM Therapeutics), WO2015088045 (Takeda Pharmaceutical), WO2017038909 (Takeda Pharmaceutical), WO2015095780 (University of Kansas), and WO2015023958 (University of Kansas).

[0320] In certain embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with a TLR7, TLR8, or TLR9 agonist.

[0321] Interferon α receptor ligand

[0322] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more interferon alpha receptor ligands. Examples of interferon alpha receptor ligands that can be combined or co-administered include interferon alpha-2b (INTRON ), pegylated interferon α-2a Pegylated interferon α-1b, interferon α-1b Veldona, Infradure, Roferon-A, YPEG-interferon alpha-2a (YPEG-rhIFNα-2a), P-1101, Algeron, Alfarona, Ingaron (interferon gamma), rSIFN-co (recombinant super compound interferon), Ypeg interferon alpha-2b (YPEG-rhIFNα-2b), MOR-22, pegylated interferon alpha-2b Bioferon, Novaferon, Inmutag (Inferon), Interferon α-n1 Interferon beta-1a Shaferon, interferon alpha-2b (Axxo), Alfaferone, interferon alpha-2b (BioGeneric Pharma), interferon-alpha 2 (CJ), Laferonum, VIPEG, BLAUFERON-A, BLAUFERON-B, Intermax alpha, Realdiron, Lanstion, Pegaferon, PDferon-B, interferon alpha-2b (IFN, Laboratorios Bioprofarma), alpha interferon a 2b, Kalferon, Pegnano, Feronsure, PegiHep, interferon alpha 2b (Zydus-Cadila), interferon alpha 2a, Optipeg A, Realfa 2B, Reliferon, interferon alpha-2b (Amega), interferon alpha-2b (Virchow), ropeg interferon alpha-2b, rHSA-IFN alpha-2a (recombinant human serum albumin interferon alpha 2a fusion protein), PEG-IFN-α, rHSA-IFN alpha 2b, recombinant human interferon alpha-(1b,2a,2b), pegylated interferon alpha-2b (Amega), pegylated interferon alpha-2a, Reaferon-EC, Proquiferon, Uniferon, Urifron, interferon alpha-2b (Changchun Institute of Biological Products), Anterferon, Shanferon, Layfferon, Shang Sheng Lei Tai, INTEFEN, SINOGEN, Fukangtai, Pegstat, rHSA-IFN alpha-2b, SFR-9216, and Interapo (Interapa).

[0323] Hyaluronidase inhibitors

[0324] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more hyaluronidase inhibitors. Examples of hyaluronidase inhibitors that can be combined or co-administered include astodrimers.

[0325] Hepatitis B surface antigen (HBsAg) inhibitors

[0326] In various embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs and immunogenic compositions are combined or co-administered with one or more HBsAg inhibitors. Examples of HBsAg inhibitors that can be combined or co-administered include AK-074, HBF-0259, GP-605, PBHBV-001, PBHBV-2-15, PBHBV-2-1, REP-9AC, REP-9C, REP-9, REP-2139, REP-2139-Ca, REP-2055, REP-2163, REP-2165, REP-2053, REP-2031 and REP-006 and REP-9AC'. Examples of HBsAg secretion inhibitors that can be combined or co-administered include BM601, GST-HG-131, AB-452 and ALG-010093.

[0327] Cyclophilin inhibitors

[0328] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more cyclophilin inhibitors. Examples of cyclophilin inhibitors that can be combined or co-administered include CPI-431-32, EDP-494, OCB-030, SCY-635, NVP-015, NVP-018, NVP-019, STG-175, and compounds disclosed in US8513184 (Gilead Sciences), US20140030221 (Gilead Sciences), US20130344030 (Gilead Sciences), and US20130344029 (Gilead Sciences).

[0329] HBV viral entry inhibitors

[0330] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more HBV viral entry inhibitors. Examples of HBV viral entry inhibitors that can be combined or co-administered include bulevirtide (Hepcludex; myrlidex B).

[0331] Inhibitory nucleic acids

[0332] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more inhibitory nucleic acids (e.g., antisense oligonucleotides, short interfering RNA (siRNA), DNA-guided RNA interference (ddRNAi)) that specifically target HBV polynucleotides. In some embodiments, HBV polynucleotides encode HBV proteins (i.e., coding regions located within the HBV genome).

[0333] Antisense oligonucleotides targeting viral mRNA

[0334] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more antisense oligonucleotides. Examples of antisense oligonucleotides targeting viral mRNA that can be combined or co-administered include ISIS-HBVRx, IONIS-HBVRx, IONIS-HBV-LRx, IONIS-GSK6-LRx, GSK-3389404, BNC-1701, and RG-6004.

[0335] Short interfering RNA (siRNA)

[0336] In various embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions are combined or co-administered with one or more siRNAs specifically targeting HBV polynucleotides. Examples of siRNAs specifically targeting HBV polynucleotides that can be combined or co-administered include TKM-HBV (TKM-HepB), ALN-HBV, SR-008, HepB-nRNA, ARC-520, ARC-521, ARB-1740, ARB-1467, AB-729, DCR-HBVS, RG-6084 (PD-L1), RG-6217, ALN-HBV-02, JNJ-3989 (ARO-HBV), STSG-0002, LUNAR-HBV, and DCR-HBVS (DCR-S219).

[0337] DNA-guided RNA interference (ddRNAi)

[0338] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more ddRNAi that specifically target HBV polynucleotides. Examples of ddRNAi that specifically target HBV polynucleotides that can be combined or co-administered include BB-HB-331.

[0339] Endonuclease modulators

[0340] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more endonuclease modulators. Examples of endonuclease modulators that can be combined or co-administered include PGN-514.

[0341] Ribonucleotide reductase inhibitors

[0342] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more ribonucleotide reductase inhibitors. Examples of ribonucleotide reductase inhibitors that can be combined or co-administered include Trimidox.

[0343] Non-nucleoside reverse transcriptase inhibitors (NNRTIs)

[0344] In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more NNRTIs. Examples of NNRTIs that can be combined or co-administered include compounds disclosed in WO2018118826 (Merck), WO2018080903 (Merck), WO2018119013 (Merck), WO2017100108 (Idenix), WO2017027434 (Merck), WO2017007701 (Merck), and WO2008005555 (Gilead).

[0345] HBV replication inhibitors

[0346] In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more HBV replication inhibitors. Examples of HBV replication inhibitors that can be combined or co-administered include GP-31502, isothiafludine, IQP-HBV, RM-5038, and Xingantie.

[0347] Covalently closed circular DNA (cccDNA) inhibitors

[0348] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more cccDNA inhibitors. Examples of cccDNA inhibitors that can be combined or co-administered include BSBI-25, ccc-R08, and CHR-101.

[0349] Farnesoid X receptor (FXR) agonists

[0350] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, carriers comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more FXR agonists. Examples of FXR agonists that can be combined or co-administered include EYP-001, cilofexor (GS-9674), EDP-305, MET-409, Tropifexor, AKN-083, RDX-023, BWD-100, LMB-763, INV-3, NTX-023-1, EP-024297, and GS-8670.

[0351] Anti-HBV antibodies

[0352] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more antibodies that specifically bind to HBV antigens, including HBV peptides present in major histocompatibility molecules (MHC) molecules (pMHC). Examples of HBV antibodies targeting surface antigens of hepatitis B virus that can be combined or co-administered include lenvervimab (GC-1102), XTL-17, XTL-19, KN-003, IV Hepabulin SN, and fully human monoclonal antibody therapy (hepatitis B virus infection, Humabs BioMed). Antibodies targeting HBV X protein (HBx) that can be combined or co-administered are described, for example, in Kornyeyev et al., J Virol. 2019 Jul 30; 93(16). pii: e00248-19.

[0353] Examples of HBV antibodies (including monoclonal antibodies and polyclonal antibodies) that can be combined or co-administered include Zutectra, Shangsheng Gandi, Uman Big (hepatitis B hyperimmune), Omri-Hep-B, Nabi-HB, Hepatect CP, HepaGam B, igantibe, Niuliva, CT-P24, hepatitis B immune globulin (intravenous, pH 4, HBV infection, Shanghai RAAS blood products) and Fovepta (BT-088).

[0354] An example of a fully human monoclonal HBV antibody that can be combined or co-administered includes HBC-34.

[0355] Antibodies against HBV viral peptide / major histocompatibility complex (MHC) class I (pMHC) complexes that can be combined or co-administered are described, for example, in Sastry et al., J Virol. 2011 Mar;85(5):1935-42 and WO2011062562.

[0356] CCR2 chemokine antagonists

[0357] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more CCR2 chemokine antagonists. Examples of CCR2 chemokine antagonists that can be combined or co-administered include propagermanium.

[0358] Thymosin agonists

[0359] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more thymosin agonists (e.g., recombinant thymosin alpha-1). Examples of thymosin agonists that can be combined or co-administered include Thymalfasin and recombinant thymosin alpha 1 (GeneScience). Examples of recombinant thymosin alpha-1 include NL-004 and PEGylated thymosin alpha-1.

[0360] Interleukin receptor agonists (eg, cytokines)

[0361] In some embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more interleukin receptor agonists selected from the group consisting of IL-2, IL-7, IL-12, and IL- 15. In some embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more cytokines selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-21, IL-24, and variants thereof. Examples of IL-2 receptor agonists that can be combined or co-administered include interleukins (aldesleukin, IL-2); celmoleukin; pegylated IL-2 (e.g., NKTR-214); modified variants of IL-2 (e.g., THOR-707), bepegylated aldesleukin, AIC-284, ALKS-4230, CUI-101, and Neo-2 / 15. Examples of IL-15 receptor agonists that can be combined or co-administered include, but are not limited to, ALT-803, NKTR-255, and hetIL-15, interleukin-15 / Fc fusion protein, AM-0015, NIZ-985, SO-C101, IL-15 (pegylated IL-15), P-22339, and IL-15-PD-1 fusion protein N-809. Examples of IL-7 receptor agonists that may be combined or co-administered include CYT-107.

[0362] Nuclear protein regulators

[0363] In various embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs and immunogenic compositions are combined or co-administered with one or more nucleoprotein regulators. Nucleoprotein regulators can be HBV core protein or capsid protein inhibitors. Examples of nucleoprotein regulators that can be combined or co-administered include GS-4882, AB-423, AB-836, AT-130, ALG-001075, ALG-001024, ALG-000184, EDP-514, GLS4, NVR-1221, NVR-3778, AL-3778, BAY 41-4109, morphothiadine mesylate, and morphothiadine mesylate. mesilate), ARB-168786, ARB-880, ARB-1820, GST-HG-141, JNJ-379, JNJ-632, RG-7907, GST-HG-141, HEC-72702, KL-060332, AB-5 06, ABI-H0731, ABI-H3733, JNJ-440, AK-0605, HRS-5091, VNRX-9945, ABI-H2158, CB-HBV-001, AK-0605, SOC-10, SOC-11 and DVR-23.

[0364] Examples of capsid inhibitors that can be combined or co-administered include ALG-000184, ABI-H0731, NVR 3-778, and US2018161307 (Gilead Sciences), US20140275167 (Novira Therapeutics), US20130251673 (Novira Therapeutics), US20140343032 (Roche), WO2014037480 (Roche), US20130267517 (Roche), WO2014131847 (Janssen), WO2014033176 (Janssen), WO2014033170 (Janssen), WO2014033167 (Janssen), WO2015 / 059212 (Janssen), WO2015118057 (Janssen), WO2015011281(Janssen), WO2014184365(Janssen), WO2014184350(Janssen), WO2014161888(Janssen), WO2013096744(Nov ira), US20150225355(Novira), US20140178337(Novira), US20150315159(Novira), US20150197533(Novira), US2015027465 2(Novira), US20150259324(Novira), US20150132258(Novira), US9181288(Novira), WO2014184350(Janssen), WO20131441 29(Roche), WO2017198744(Roche), US20170334882(Novira), US20170334898(Roche), WO2017202798(Roche), WO2017214395 (Enanta), WO2018001944 (Roche), WO2018001952 (Roche), WO2018005881 (Novira), WO2018005883 (Novira), WO2018011100 ( Roche), WO2018011160 (Roche), WO2018011162 (Roche), WO2018011163 (Roche), WO2018036941 (Roche), WO2018043747 (KyotoUniv), US20180065929 (Janssen), WO2016168619 (Indiana University), WO2016195982 (The PennState Foundation), WO2017001655 (Janssen), WO2017048950 (Assembly Biosciences), WO2017048954 (Assembly Biosciences), WO2017048962 (Assembly Biosciences), US20170121328 (Novira), and US20170121329 (Novira).

[0365] Examples of transcript inhibitors that can be combined or co-administered include compounds disclosed in WO2017013046 (Roche), WO2017016960 (Roche), WO2017017042 (Roche), WO2017017043 (Roche), WO2017061466 (Toyoma chemicals), WO2016177655 (Roche), WO2016161268 (Enanta), WO2017001853 (Redex Pharma), WO2017211791 (Roche), WO2017216685 (Novartis), WO2017216686 (Novartis), WO2018019297 (Ginkgo Pharma), WO2018022282 (Newave Pharma), US20180030053 (Novartis), WO2018045911 (Zhejiang Pharma).

[0366] Innate immunity activators

[0367] In some embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions are combined or co-administered with one or more innate immune activators. In various embodiments, the one or more innate immune activators include agonists of receptors selected from the group consisting of fms-related tyrosine kinase 3 (FLT3), stimulator of interferon genes (STING) receptor, DExD / H-box helicase 58 (DDX58; also known as RIG-I), nucleotide-bound oligomeric domain protein 2 (NOD2). In some embodiments, the method requires the co-administration of GS-3583 and / or GS-9992. In some embodiments, the method requires the combination or co-administration of a FLT3 agonist, such as GS-3583 or CDX-301.

[0368] STING agonists, RIG-I and NOD2 modulators

[0369] In some embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with stimulator of interferon response cGAMP interactor 1 (STING or STING1; NCBI gene ID: 340061) agonists. In some embodiments, the STING / STING1 agonist or activator is selected from the group consisting of ADU-S100 (MIW-815), SB-11285, MK-1454, SR-8291, AdVCA0848, STINGVAX, GSK-532, SYN-STING, MSA-1, SR-8291, 5,6-dimethylxanthone-4-acetic acid (DMXAA), cyclic GAMP (cGAMP), and cyclic diAMP. Examples of STING agonists that can be combined or co-administered include compounds disclosed in WO 2018065360 (Biolog Life Science Institute Forschungslabor und Biochemica-Vertrieb GmbH, Germany), WO 2018009466 (AduroBiotech), WO 2017186711 (InvivoGen), WO 2017161349 (Immune Sensor), WO 2017106740 (Aduro Biotech), US 20170158724 (Glaxo Smithkline), WO 2017075477 (AduroBiotech), US 20170044206 (Merck), WO 2014179760 (University of California), WO2018098203 (Janssen), WO2018118665 (Merck), WO2018118664 (Merck), WO2018100558 (Takeda), WO2018067423 (Merck), WO2018060323 (Boehringer).

[0370] In some embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with DExD / H-box helicase 58 (DDX58; also known as retinoic acid-inducible gene 1 (RIG-I), RIG1, RIGI, RLR-1, SGMRT2; NCBI gene ID: 23586). Exemplary RIG-I agonists that can be combined or co-administered include inarigivir soproxil (SB-9200; GS-9992); SB-40, SB-44, ORI-7246, ORI-9350, ORI-7537, ORI-9020, ORI-9198, ORI-7170, and RGT-100.

[0371] In some embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with nucleotide-binding oligomerization domain-containing protein 2 (NOD2; NCBI Gene ID: 64127) agonists such as inagvir-piroxetine (SB-9200; GS-9992) and IR-103.

[0372] Phosphatidylinositol 3-kinase (PI3K) inhibitors

[0373] In some embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with an inhibitor of: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit, such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA, CLAPO, CLOVE, CWS5, MCAP, MCM, MCMTC, PI3K, PI3K-α, p110-α; NCBI Gene ID: 5290); phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA, CLAPO, CLOVE, CWS5, MCAP, MCM, MCMTC, PI3K, PI3K-α, p110-α; NCBI Gene ID: 5290); In some embodiments, the PI3K inhibitor is a pan-PI3K inhibitor. Examples of PI3K inhibitors include, but are not limited to, ACP-319, AEZA-129, AMG-319, AS252424, AZD8186, BAY 1082439, BEZ235, bimiralisib (PQR309), buparlisib (BKM120), BYL719 (alpelisib), carboxyamidotriazole orotate (CTO), CH5132799, CLR-457, CLR-1401, copanlisib (BAY 80-6946), DS-7423, duvelisib (IPI-145), fimepinostat (CUDC-907), gedatolisib (PF-05212384), GDC-0032, GDC-0084 (RG7666), GDC-0077, pictilisib (GDC-0941), GDC-0980, GSK2636771, GSK2269577, idelalisib INCB040093, INCB50465, IPI-443, IPI-549, KAR4141, LY294002, LY3023414, (neratinib), nemiralisib (GSK2269557), omipalisib (GSK2126458, GSK458), OXY111A, panulisib (P7170, AK151761), PA799, perifosine (KRX-0401), pilarisib (SAR245408; XL147), puquitinib mesylate mesylate (XC-302), SAR260301, seletalisib (UCB-5857), serabelisib (INK-1117, MLN-1117, TAK-117), SF1126, sonolisib (PX-866), RG7604, rigosertib sodium sodium) (ON-01910 sodium), RP5090, tenalisib (RP6530), RV-1729, SRX3177, taselisib, TG100115, umbralisib (TGR-1202), TGX221, voxtalisib (SAR245409), VS-5584, WX-037, X-339, X-414, XL499, XL756, wortmannin, ZSTK474, and WO 2005 / 113556 (ICOS), WO 2013 / 052699 (Gilead Calistoga), WO 2013 / 116562 (Gilead Calistoga), WO 2014 / 100765 (Gilead Calistoga), WO Compounds described in WO 2014 / 100767 (Gilead Calistoga) and WO 2014 / 201409 (Gilead Sciences).

[0374] Immune checkpoint modulators

[0375] In some embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, carriers comprising such polypeptides or polynucleotides, LNPs and immunogenic compositions are combined or co-administered with one or more blockers or inhibitors of inhibitory immune checkpoint proteins or receptors and / or with one or more stimulators, activators or agonists of one or more stimulatory immune checkpoint proteins or receptors. The blocking or suppression of inhibitory immune checkpoints can positively regulate T cells or NK cell activation and prevent immune escape of infected cells. Stimulating the activation or stimulation of immune checkpoints can enhance the effect of immune checkpoint inhibitors in infectious treatments. In various embodiments, immune checkpoint proteins or receptors regulate T cell responses (e.g., reviewed in Xu et al., J Exp Clin Cancer Res. (2018) 37:110). In various embodiments, immune checkpoint proteins or receptors modulate NK cell responses (e.g., reviewed in Davis et al., Semin Immunol. (2017) 31:64–75 and Chiossone et al., Nat Rev Immunol. (2018) 18(11):671-688).

[0376] Examples of immune checkpoint proteins or receptors include, but are not limited to, CD27 (NCBI gene ID: 939); CD70 (NCBI gene ID: 970); CD40 (NCBI gene ID: 958); CD40LG (NCBI gene ID: 959); CD47 (NCBI gene ID: 961); CD48 (SLAMF2; NCBI gene ID: 962); transmembrane and immunoglobulin domain-containing 2 (TMIGD2, CD28H; NCBI gene ID: 126259); CD84 (LY9B, SLAMF5; NCBI gene ID: 8832); CD96 (NCBI gene ID: 10225); CD160 (NCBI gene ID: 11126); MS 4A1 (CD20; NCBI Gene ID: 931); CD244 (SLAMF4; NCBI Gene ID: 51744); CD276 (B7H3; NCBI Gene ID: 80381); V-set domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4; NCBI Gene ID: 79679); V-set immunomodulatory receptor (VSIR, B7H5, VISTA; NCBI Gene ID: 64115); immunoglobulin superfamily member 11 (IGSF11, VSIG3; NCBI Gene ID: 152404); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6; NCBI Gene ID: 374383); HERV-H LTR-associated 2 (HHLA2, B7H7; NCBI gene ID: 11148); inducible T cell costimulator (ICOS, CD278; NCBI gene ID: 29851); inducible T cell costimulator ligand (ICOSLG, B7H2; NCBI gene ID: 23308); TNF receptor superfamily member 4 (TNFRSF4, OX40; NCBI gene ID: 7293); TNF superfamily member 4 (TNFSF4, OX40L; NCBI gene ID: 7292); TNFRSF8 (CD30; NCBI gene ID: 943); TNFSF8 (C D30L; NCBI Gene ID: 944); TNFRSF10A (CD261, DR4, TRAILR1; NCBI Gene ID: 8797); TNFRSF9 (CD137; NCBI Gene ID: 3604); TNFSF9 (CD137L; NCBI Gene ID: 8744); TNFRSF10B (CD262, DR5, TRAILR2; NCBI Gene ID: 8795); TNFRSF10 (TRAIL; NCBI Gene ID: 8743); TNFRSF14 (HVEM, CD270; NCBI Gene ID: 8764);TNFSF14 (HVEML; NCBI Gene ID: 8740); CD272 (B and T lymphocyte-associated (BTLA); NCBI Gene ID: 151888); TNFRSF17 (BCMA, CD269; NCBI Gene ID: 608); TNFSF13B (BAFF; NCBI Gene ID: 10673); TNFRSF18 (GITR; NCBI Gene ID: 8784); TNFSF18 (GITRL; NCBI Gene ID: 8995); MHC class I polypeptide-related sequence A (MICA; NCBI Gene ID: 100507436); MHC Class I polypeptide-related sequence B (MICB; NCBI Gene ID: 4277); CD274 (CD274, PDL1, PD-L1; NCBI Gene ID: 29126); programmed cell death 1 (PDCD1, PD1, PD-1; NCBI Gene ID: 5133); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152; NCBI Gene ID: 1493); CD80 (B7-1; NCBI Gene ID: 941); CD28 (NCBI Gene ID: 940); nectin cell adhesion molecule 2 (NECTIN2, CD112; NCBI gene ID: 5819); CD226 (DNAM-1; NCBI gene ID: 10666); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155; NCBI gene ID: 5817); PVR-related immunoglobulin domain-containing (PVRIG, CD112R; NCBI gene ID: 79037); T cell immunoreceptor with Ig and ITIM domains (T IGIT; NCBI Gene ID: 201633); T cell immunoglobulin and mucin domain-containing 4 (TIMD4; TIM4; NCBI Gene ID: 91937); Hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3; NCBI Gene ID: 84868); Galectin 9 (LGALS9; NCBI Gene ID: 3965); Lymphocyte activation 3 (LAG3, CD223; NCBI Gene ID: 3902); Signal transducer lymphocyte activation molecule family SLAM family member 1 (SLAMF1, SLAM, CD150; NCBI gene ID: 6504); lymphocyte antigen 9 (LY9, CD229, SLAMF3; NCBI gene ID: 4063); SLAM family member 6 (SLAMF6, CD352; NCBI gene ID: 114836); SLAM family member 7 (SLAMF7, CD319; NCBI gene ID: 57823); UL16 binding protein 1 (ULBP1; NCBI gene ID: 80329);UL16 binding protein 2 (ULBP2; NCBI Gene ID: 80328); UL16 binding protein 3 (ULBP3; NCBI Gene ID: 79465); retinoic acid early transcript 1E (RAET1E; ULBP4; NCBI Gene ID: 135250); retinoic acid early transcript 1G (RAET1G; ULBP5; NCBI Gene ID: 353091); retinoic acid early transcript 1L (RAET1L; ULBP6; NCBI Gene ID: 353091); I gene ID: 154064); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A; NCBI gene ID: 3821); killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314; NCBI gene ID: 22914); killer cell lectin-like receptor C2 (KLRC2, CD159c, NKG2C; NCBI gene ID: 3822); killer cell lectin-like receptor C3 (KLRC3, NKG2E; NCBI Gene ID: 3823); Killer cell lectin-like receptor C4 (KLRC4, NKG2F; NCBI Gene ID: 8302); Killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1; NCBI Gene ID: 3802); Killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2; NCBI Gene ID: 3803); Killer cell immunoglobulin-like receptor , two Ig domains and long cytoplasmic tail 3 (KIR2DL3; NCBI Gene ID: 3804); Killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1, KIR, CD158E1; NCBI Gene ID: 3811) (e.g., lirelumab (IPH2102 / BMS-986015), IPH-4102); and Killer cell lectin-like receptor D1 (KLRD1; NCBI Gene ID: 3824).

[0377] In some embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors. Exemplary T cell inhibitory immune checkpoint proteins or receptors include, but are not limited to, CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4); V-set immunomodulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); PVR-associated immunoglobulin domains (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1). In various embodiments, the agents as described herein are combined with one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors.Exemplary T cell stimulatory immune checkpoint proteins or receptors include, but are not limited to, CD27, CD70; CD40, CD40LG; inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); CD244 (2B4, SLAMF4), poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). See, e.g., Xu et al., J Exp Clin Cancer Res. (2018) 37:110.

[0378] In some embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. Exemplary NK cell inhibitory immune checkpoint proteins or receptors include, but are not limited to, killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); and killer cell lectin-like receptor D1 (KLRD1, CD94).

[0379] In some embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions are combined or co-administered with one or more agonists or activators of one or more NK cell stimulating immune checkpoint proteins or receptors. Exemplary NK cell stimulating immune checkpoint proteins or receptors include, but are not limited to, CD16, CD226 (DNAM-1); CD244 (2B4, SLAMF4); Killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314); SLAM family member 7 (SLAMF7). See, for example, Davis et al., Semin Immunol. (2017) 31: 64–75; Fang et al., Semin Immunol. (2017) 31: 37-54; and Chiossone et al., Nat Rev Immunol. (2018) 18 (11): 671-688.

[0380] Inhibitors of cytotoxic T lymphocyte-associated protein 4 (CTLA4)

[0381] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more inhibitors of cytotoxic T lymphocyte-associated protein 4 (CTLA4) (CD152; NCBI gene ID: 1493). Examples of co-administered CTLA4 inhibitors include, but are not limited to, ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884, AGEN2041, BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, JHL-1155, KN-044, CG-0161, ATOR-1144, PBI-5D3H5, BPI-0 02, belatacept, PSI-001, PRS-010, JHL-1155, and the multispecific inhibitors FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), MEDI-5752 (CTLA4 / PD-1), XmAb-20717 (PD-1 / CTLA4), and AK-104 (CTLA4 / PD-1).

[0382] Inhibitors of PD-L1 (CD274) or PD-1 (PDCD1; CD279)

[0383] In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more inhibitors of programmed cell death 1 ligand 1 (PD-L1; CD274; NCBI Gene ID: 29126) or programmed cell death 1 (PD-1; PDCD1; CD279; NCBI Gene ID: 5133). Examples of inhibitors of PD-L1 (CD274) or PD-1 (PDCD1) that can be combined or co-administered include, but are not limited to, sepalizumab (AB122), pembrolizumab, nivolumab, cimetuzumab, pidilizumab, AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab (MSB0010718C), ASC22, durvalumab, ALN-PDL, BMS-936559, CK-301, PF-06801591, BG B-108, BGB-A317 (tislelizumab), GLS-010 (WBP-3055), AK-103 (HX-008), GB-226, AK-105, CS-1003, HLX-10, MGA-012, BI-754091, PDR-001, AGEN-2034, JS-001 (toripalimab), JNJ-63723283, genoluzumab (CBT-501), LZM-009, BCD-100, LY-3300054, SHR- 1201, SHR-1210 (camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306, RO-6084 (PD-L1 antisense oligonucleotide), STI-1110, GX-P2, RG-7446, mDX-400, CX-072, CBT-502, TSR-042 (dotalizumab), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155), ME DI-0680, envafolimab (KN-035), KD-033, KY-1003, IBI-308 (sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, MSB-0010718C, GS-4224, GS-4416, INCB086550, MAX10181,As well as the bispecific inhibitors FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-013 (PD-1 / LAG-3), FS-118 (LAG-3 / PD-L1) MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), MEDI-5752 (CTLA4 / PD-1), RO-7121661 (PD-1 / TIM-3), XmAb-20717 (PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), M7824 (PD-L1 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM3 / PDL1), GNS-1480 (epidermal growth factor receptor antagonist; programmed cell death ligand 1 inhibitor), M-7824 (PD-L1 / TGF-β bifunctional fusion protein) and INBRX-105 (4-1BB / PDL1).

[0384] Examples of PD-1 inhibitors that can be combined or co-administered also include compounds disclosed in the following patents: WO2017112730 (Incyte Corp), WO2017087777 (Incyte Corp), WO2017017624, WO2014151634 (BristolMyers Squibb Co), WO201317322 (BristolMyers Squibb Co), WO2018119286 (Incyte Corp), WO2018119266 (Incyte Corp), WO2018119263 (Incyte Corp), WO2018119236 (Incyte Corp), WO2018119221 (Incyte Corp), WO2018118848 (BristolMyers Squibb Co), WO20161266460 (BristolMyers Squibb Co), Co), WO2017087678 (BristolMyers Squibb Co), WO2016149351 (BristolMyers Squibb Co), WO2015033299 (Aurigene Discovery Technologies Ltd), WO2015179615 (Eisai Co Ltd; Eisai Research Institute), WO2017066227 (BristolMyers Squibb Co), WO2016142886 (Aurigene Discovery Technologies Ltd), WO2016142852 (Aurigene Discovery Technologies Ltd), WO2016142835 (Aurigene Discovery Technologies Ltd; Individual), WO2016142833 (Aurigene Discovery Technologies Ltd), WO2018085750 (BristolMyers Squibb Co), WO2015033303 (Aurigene Discovery Technologies Ltd), WO2017205464 (Incyte Corp), WO2016019232 (3M Co; Individual;Texas A&MUniversity System)、WO2015160641(BristolMyers Squibb Co)、WO2017079669(Incyte Corp)、WO2015033301(AurigeneDiscoveryTechnologies Ltd)、WO2015034820(BristolMyers Squibb Co)、WO2018073754(Aurigene Discovery Technologies Ltd)、WO2016077518(BristolMyers Squibb Co)、WO2016057624(BristolMyers Squibb Co)、WO2018044783(Incyte Corp)、WO2016100608(BristolMyers Squibb Co)、WO2016100285(BristolMyers Squibb Co)、WO2016039749(BristolMyersSquibb Co)、WO2015019284(Cambridge Enterprise Ltd)、WO2016142894(Aurigene Discovery Technologies Ltd)、WO2015134605(BristolMyers Squibb Co)、WO2018051255(Aurigene DiscoveryTechnologies Ltd)、WO2018051254(AurigeneDiscovery Technologies Ltd)、WO2017222976(Incyte Corp)、WO2017070089(IncyteCorp)、WO2018044963(BristolMyers Squibb Co)、WO2013144704(AurigeneDiscoveryTechnologies Ltd)、WO2018013789(Incyte Corp)、WO2017176608(BristolMyers SquibbCo)、WO2018009505(BristolMyersSquibb Co)、WO2011161699(Aurigene DiscoveryTechnologies Ltd)、WO2015119944(Incyte Corp;Merck Sharp & Dohme Corp), WO2017192961 (Incyte Corp), WO2017106634 (Incyte Corp), WO2013132317 (Aurigene Discovery Technologies Ltd), WO2012168944 (Aurigene Discovery Technologies Ltd), WO2015036927 (Aurigene Discovery Technologies Ltd) Ltd), WO2015044900 (Aurigene Discovery Technologies Ltd), WO2018026971 (Arising International). ;

[0385] In various embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, carriers comprising such polypeptides or polynucleotides, LNPs and immunogenic compositions are combined or co-administered with one or more proteins (e.g., antibodies or fragments thereof, or antibody mimetics) inhibitors of PD-L1 (CD274), PD-1 (PDCD1) or CTLA4. In some embodiments, one or more immune checkpoint inhibitors include organic small molecule inhibitors of PD-L1 (CD274), PD-1 (PDCD1) or CTLA4. In some embodiments, the small molecule inhibitors of CD274 or PDCD1 are selected from the group consisting of GS-4224, GS-4416, INCB086550 and MAX10181. Additional examples of small molecule PD-L1 inhibitors include those disclosed in U.S. Publication Nos. US2018305315 (Gilead Sciences), US2020017471 (Gilead Sciences) and US2019270727 (Gilead Sciences). In some embodiments, small molecule inhibitors of CTLA4 include BPI-002.

[0386] Inhibitors of T-cell immunoreceptor with Ig and ITIM domains (TIGIT)

[0387] In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more inhibitors of the T cell immunoreceptor with Ig and ITIM domains (TIGIT) (NCBI gene ID: 201633). Exemplary anti-TIGIT antibodies that can be combined or co-administered include etigilimab, BMS-986207, tiragolumab (also known as MTIG-7192A; RG-6058; RO 7092284), AGEN1307, AGEN1327, AGEN1777, COM-902, IBI-939, AB154, MG1131, and EOS884448 (EOS-448).

[0388] TNF receptor superfamily (TNFRSF) member agonists or activators

[0389] In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more agonists of one or more TNF receptor superfamily (TNFRSF) members, such as agonists of one or more of the following: TNFRSF1A (NCBI Gene ID: 7132), TNFRSF1B (NCBI Gene ID: 7133), TNFRSF4 (OX40, CD134; NCBI Gene ID: 7293), TNFRSF5 (CD40; N TNFRSF6 (FAS, NCBI gene ID: 355), TNFRSF7 (CD27, NCBI gene ID: 939), TNFRSF8 (CD30, NCBI gene ID: 943), TNFRSF9 (4-1BB, CD137, NCBI gene ID: 3604), TNFRSF10A (CD261, DR4, TRAILR1, NCBI gene ID: 8797), TNFRSF10B (CD262, DR5, TRAILR2, NCBI gene ID: 8795), TNFRSF10C (CD263, TRAILR3, NCBI gene ID: 8794), TNFRSF10D (CD264, TRAILR4, NCBI gene ID: 8793), TNFRSF11A (CD265, RANK, NCBI gene ID: 8792), TNFRSF11B (NCBI gene ID: 4982), TNFRSF12A (CD266, NCBI gene ID: 51330), TNFRSF13B (CD267, NCBI gene ID: 23495), TNFRSF13C (CD268, NCBI gene ID: 23496), and TNFRSF14A (CD269, NCBI gene ID: 23507). D268, NCBI gene ID: 115650), TNFRSF16 (NGFR, CD271, NCBI gene ID: 4804), TNFRSF17 (BCMA, CD269, NCBI gene ID: 608), TNFRSF18 (GITR, CD357, NCBI gene ID: 8784), TNFRSF19 (NCBI gene ID: 55504), TNFRSF21 (CD358, DR6, NCBI gene ID: 27242) and TNFRSF25 (DR3, NCBI gene ID: 8718).

[0390] Exemplary anti-TNFRSF4 (OX40) antibodies that may be combined or co-administered include, but are not limited to, MEDI6469, MEDI6383, MEDI0562 (tavolizumab), MOXR0916, PF-04518600, RG-7888, GSK-3174998, INCAGN1949, BMS-986178, GBR-8383, ABBV-368, and those described in WO2016179517, WO2017096179, WO2017096182, WO2017096281, and WO2018089628.

[0391] Exemplary anti-TNFRSF5 (CD40) antibodies that may be combined or co-administered include, but are not limited to, RG7876, SEA-CD40, APX-005M, and ABBV-428.

[0392] In some embodiments, the anti-TNFRSF7 (CD27) antibody varilutumab (CDX-1127) is administered in combination or co-administered.

[0393] Exemplary anti-TNFRSF9 (4-1BB, CD137) antibodies that may be combined or co-administered include, but are not limited to, usurelumab, utolumab (PF-05082566), AGEN-2373, and ADG-106.

[0394] Exemplary anti-TNFRSF18 (GITR) antibodies that can be combined or co-administered include, but are not limited to, MEDI1873, FPA-154, INCAGN-1876, TRX-518, BMS-986156, MK-1248, GWN-323, and those described in WO2017096179, WO2017096276, WO2017096189, and WO2018089628. In some embodiments, antibodies or fragments thereof that co-target TNFRSF4 (OX40) and TNFRSF18 (GITR) are co-administered. Such antibodies are described, for example, in WO2017096179 and WO2018089628.

[0395] Indoleamine-pyrrole-2,3-dioxygenase (IDO1) inhibitors

[0396] In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions as described herein are combined or co-administered with one or more inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1; NCBI Gene ID: 3620). Examples of IDO1 inhibitors that can be combined or co-administered include, but are not limited to, BLV-0801, epacadostat, resminostat, F-001287, GBV-1012, GBV-1028, GDC-0919, indoximod, NKTR-218, NLG-919-based vaccines, PF-06840003, pyranonaphthoquinone derivatives (SN-35837), SBLK-200802, BMS-986205 and shIDO-ST, EOS-200271, KHK-2455, LY-3381916, and US20100015178 (Incyte), US2016137652 (Flexus Biosciences, Inc.), WO2014073738 (Flexus Biosciences, Inc.) and WO2015188085 (Flexus Biosciences, Inc.).

[0397] LAG-3 and TIM-3 inhibitors

[0398] In certain embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions are combined or co-administered with anti-TIM-3 antibodies such as TSR-022, LY-3321367, MBG-453, and INCAGN-2390. In certain embodiments, immunogenic polypeptides as described herein, polynucleotides encoding such polypeptides, vectors comprising such polypeptides or polynucleotides, LNPs, and immunogenic compositions are combined or co-administered with anti-LAG-3 (lymphocyte activation) antibodies such as relatlima...

Claims

1. An immunogenic composition comprising a first viral expression vector and a second viral expression vector, wherein: a) the first viral expression vector comprises a polynucleotide encoding a truncated HBV polymerase polypeptide, the truncated HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, wherein the polypeptide is no longer than 600 amino acids and does not comprise all of the terminal protein (TP) domain and does not comprise all or part of the spacer domain, wherein the polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 13-14; and b) the second viral expression vector comprises a polynucleotide encoding the core-sAg fusion protein, wherein the core-sAg fusion protein comprises, from N-terminus to C-terminus, a HBV core polypeptide and a HBV small surface antigen (sAg) polypeptide, wherein the length of the fusion protein is no longer than 450 amino acids, and wherein: i) the core polypeptide is from HBV genotype B or C and the sAg polypeptide is from HBV genotype C, wherein the fusion protein comprises or consists of: an amino acid sequence of any one of SEQ ID NOs: 38-39, or an amino acid sequence that is at least 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the full length of any one of SEQ ID NOs: 38-39; or ii) the core polypeptide is from HBV genotype D and the sAg polypeptide is from HBV genotype D, wherein the fusion protein comprises or consists of: an amino acid sequence of any one of SEQ ID NOs: 40-41, or an amino acid sequence that is at least 97%, 98% or 99% identical to the full length of any one of SEQ ID NOs: 40-41, wherein the first viral expression vector and the second viral expression vector are from the Arenaviridae family, and wherein the immunogenic composition is formulated in a physiologically acceptable buffer with a pKa in the range of about 6.0 to about 8.

0.

2. The immunogenic composition according to claim 1, comprising a first viral expression vector and a second viral expression vector, wherein: a) the first viral expression vector comprises a polynucleotide encoding an HBV polymerase polypeptide mutant, wherein the HBV polymerase polypeptide mutant comprises or consists of the amino acid sequence of SEQ ID NO: 13; and b) The second viral expression vector comprises a polynucleotide encoding the core-sAg fusion protein, and the core-sAg fusion protein comprises or consists of: the amino acid sequence of SEQ ID NO:41, or an amino acid sequence that is at least 97%, 98% or 99% identical to the full length of SEQ ID NO:

41.

3. The immunogenic composition according to claim 1 or claim 2, comprising a first viral expression vector and a second viral expression vector, wherein: a) the first viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NOs: 29, 89, 90 and 92, or a nucleic acid sequence that is at least 99% identical to the full length of any one of SEQ ID NOs: 29, 89, 90 and 92; and b) the second viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO:37, or a nucleic acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the full length of SEQ ID NO:

37.

4. The immunogenic composition of claim 1 or claim 2, wherein the first viral expression vector and the second viral expression vector are independently derived from an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV), Cali mammalian arenavirus, Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV).

5. The immunogenic composition of claim 1 or claim 2, wherein the first viral expression vector and the second viral expression vector are from an arenavirus vector selected from lymphocytic choriomeningitis mammalian arenavirus (LCMV) or Californium mammalian arenavirus.

6. An immunogenic composition comprising a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: a) the first LCMV arenavirus expression vector comprises a polynucleotide encoding a truncated HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, wherein the polypeptide is no longer than 600 amino acids in length and does not comprise all of the terminal protein (TP) domain and does not comprise all or part of the spacer domain, wherein the polynucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 29, or a nucleic acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the full length of SEQ ID NO: 29; and b) the second LCMV arenavirus expression vector comprises a polynucleotide encoding a core-sAg fusion protein, wherein the core-sAg fusion protein comprises, from N-terminus to C-terminus, a HBV genotype D core polypeptide and a HBV genotype D small surface antigen (sAg) polypeptide, wherein the length of the fusion protein is no longer than 450 amino acids, wherein the polynucleotide comprises or consists of the following: a nucleic acid sequence of SEQ ID NO: 37, or a nucleic acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the full length of SEQ ID NO: 37, wherein the immunogenic composition is formulated in a physiologically acceptable buffer having a pKa in the range of about 6.0 to about 8.

0.

7. An immunogenic composition comprising a first Pichinde Arenavirus expression vector and a second Pichinde Arenavirus expression vector, wherein: a) the first Pichinde arenavirus expression vector comprises a polynucleotide encoding a truncated HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, wherein the polypeptide is no longer than 600 amino acids and does not comprise all of the terminal protein (TP) domain and does not comprise all or part of the spacer domain, wherein the polynucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 90, or a nucleic acid sequence that is at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the full length of SEQ ID NO: 90; and b) the second Pichinde arenavirus expression vector comprises a polynucleotide encoding a core-sAg fusion protein, wherein the core-sAg fusion protein comprises, from N-terminus to C-terminus, a HBV genotype D core polypeptide and a HBV genotype D small surface antigen (sAg) polypeptide, wherein the length of the fusion protein is no longer than 450 amino acids, wherein the polynucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 37, or a nucleic acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the full length of SEQ ID NO: 37, wherein the immunogenic composition is formulated in a physiologically acceptable buffer having a pKa in the range of about 6.0 to about 8.

0.

8. The immunogenic composition of claim 6 or claim 7, wherein the first viral expression vector and the second viral expression vector are provided in a ratio ranging from 1:10 to 10:

1.

9. The immunogenic composition according to claim 6 or claim 7, comprising about 10 3 to about 10 12 Each of the first viral expression vector and the second viral expression vector is within the range of viral focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or viral particles (vp).

10. The immunogenic composition of any one of claims 1, 2, 6 and 7, formulated for administration via a route selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous and mucosal.

11. The immunogenic composition of any one of claims 1, 2, 6 and 7, wherein the physiologically acceptable buffer is selected from phosphate, carbonate, bicarbonate, citrate, maleate, glycine-glycine, HEPES, HEPPSO, HEPPS, imidazole, BICINE, TRICINE, Tris and BIS-Tris.

12. The immunogenic composition of any one of claims 1, 2, 6 and 7, wherein the physiologically acceptable buffer comprises amino acids selected from the group consisting of aspartic acid, asparagine, glutamic acid, glutamine, histidine, arginine and lysine.

13. The immunogenic composition of claim 11, further comprising a sugar selected from the group consisting of lactose, trehalose, glucose and sucrose.

14. The immunogenic composition of claim 11, further comprising cellulose or a derivative thereof.

15. The immunogenic composition of claim 14, comprising a cellulose derivative selected from the group consisting of sodium carboxymethylcellulose, ethylcellulose and cellulose acetate.

16. The immunogenic composition of claim 11, further comprising a polyol.

17. The immunogenic composition of claim 16, wherein the polyol is selected from the group consisting of glycerol, sorbitol, mannitol and polyethylene glycol.

18. The immunogenic composition of any one of claims 1, 2, 6 and 7, formulated in HEPES, NaCl and a nonionic surfactant at neutral or near neutral pH.

19. The immunogenic composition of any one of claims 1, 2, 6 and 7, formulated in an isotonic aqueous solution.

20. The immunogenic composition according to any one of claims 1, 2, 6 and 7, formulated in an isotonic aqueous solution comprising HEPES buffer at pH 7.4, NaCl and poloxamer 188.

21. The immunogenic composition of any one of claims 1, 2, 6 and 7, formulated in an isotonic aqueous solution of 25 mM HEPES buffer, pH 7.4, 150 mM NaCl and 0.01% Poloxamer 188.

22. The immunogenic composition of claim 20, further comprising 10% sorbitol.

23. The immunogenic composition of any one of claims 1, 2, 6 and 7, wherein the composition is lyophilized.

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