HBV vaccines and methods for treating hbv

Abstract

To provide HBV vaccines and methods for treating HBV.SOLUTION: Polypeptides are provided which are useful to elicit a protective immune response against one or more hepatitis B virus (HBV) antigens in a human. The immunogenic polypeptides described in this specification can elicit preventative and / or therapeutic immune responses in a human against one or more hepatitis B virus (HBV) antigens. Generally, the immunogenic polypeptides described in this specification contain highly conserved portions of HBV proteins in order to induce responses against epitopes that are identical in the vaccine antigen and in the infecting HBV present in the patient, while also excluding poorly conserved regions, thereby avoiding eliciting immunodominant T cell responses targeting epitopes that are not present in the infecting HBV strain of the patient.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Application No. 62 / 908,494, filed on September 30, 2019, under 35 U.S.C. § 119(e), and the entire disclosure thereof is incorporated herein by reference for all purposes.

[0002] (Sequence Listing) This application includes a sequence listing that was submitted electronically in ASCII format, and the entire disclosure thereof is incorporated herein by reference. The ASCII copy, created on September 4, 2020, is named 1324_PF_SL.txt and is 296,675 bytes in size.

Background Art

[0003] To improve the rate of disappearance of hepatitis B virus (HBV) surface antigen (sAg), a major marker of functional cure, numerous attempts have been made to treat patients with chronic hepatitis B virus (HBV) infection using vaccination. Such attempts have included 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 several viral vectors (Cavenaugh, et al., supra; and Zoulim, et al., including vaccination using Hum Vaccin Immunother. (2019) Epub 2019 / 08 / 03. doi:10.1080 / 21645515.2019.1651141. PubMed PMID:31373537). Despite these numerous attempts, to date, there has been no therapeutic vaccination approach that has shown consistent benefits in chronic hepatitis B virus (CHB) infection. The drawbacks of previous vaccination approaches can clarify the failure of previous vaccination approaches. Such drawbacks include limitations in antigen design and the vaccine technology used. The optimal antigen would include highly conserved portions of the HBV protein and exclude poorly conserved regions, as highly conserved regions can induce responses to epitopes that are identical in both the vaccine antigen and the virus present in the treated patients, while poorly conserved regions can elicit immunodominant T cell responses to epitopes not present in the patient's infecting virus strain (Swadling, et al., Vaccines (Basel). (2016) 4(3). Epub 2016 / 08 / 05. doi:10.3390 / vaccines4030027. PubMed PMID:27490575). However, some of the previous vaccines used antigen designs that did 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). Furthermore, many of the previous vaccines were virus-specific CD4 + T cells, CD8 +Failed to induce a complete combination of T cells 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-Mahtab, 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 cure of chronic HBV infection, and CD8 + T cells have been shown to be the major effector cells responsible for viral clearance in acute HBV infection in chimpanzees (Thimme, et al., J Virol. (2003) 77(1):68-76). In addition, antibodies that bind to hepatitis B virus surface antigen (HBsAg) promote HBsAg clearance and prevent the spread of residual HBV. Furthermore, although a high level of immune response is thought to be required to achieve a therapeutic effect, many previous CHB vaccines have not been able to induce such a strong 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 previous CHB vaccine antigens are not sufficiently stable in delivery vectors to enable vaccine production on a commercial scale.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non - Patent Document 2

Non - Patent Document 3

Summary of the Invention

Means for Solving the Problems

[0005] In one aspect, for example, a truncated hepatitis B virus (HBV) polymerase polypeptide that can induce or elicit an immune response in humans upon administration is provided. In some embodiments, the truncated HBV polymerase polypeptide comprises an inactivated reverse transcriptase domain and an inactivated RNase H, and does not include all of the terminal protein (TP) domain and all or part of the spacer domain. In some embodiments, the polypeptide is 600 amino acids or less in length, for example, 595, 590, 585, 580, 575, 570, 565, 560, 555, 550, 545, 540, or 535 amino acids or less. In some embodiments, the reverse transcriptase domain does not contain the YMDD motif (SEQ ID NO: 97), and the RNase H domain does not contain the AELL motif (SEQ ID NO: 98). In some embodiments, the YMDD motif (SEQ ID NO: 97) within the reverse transcriptase domain is mutated to YMHD (SEQ ID NO: 99), and the AELL motif (SEQ ID NO: 98) within the RNase H domain is mutated to AHLL (SEQ ID NO: 100). In some embodiments, the polypeptide is derived from HBV genotype A, B, C, or D. In some embodiments, (a) the polypeptide is derived from HBV genotype B and does not include the polypeptide sequence of SEQ ID NO: 50 (for example, the sequence is removed or deleted or not included), 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, or (b) the polypeptide is derived from HBV genotype D and does not include the 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 an amino acid sequence of any one of SEQ ID NOs: 13-14, 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: 13-14.

[0006] In another aspect, an HBV polymerase deletion mutant polypeptide is provided. In some embodiments, the HBV polymerase deletion mutant polypeptide comprises, in order from the N-terminus to the C-terminus, a terminal protein (TP) domain, an inactivated reverse transcriptase domain, and an inactivated RNase H, and the mutant polypeptide does not include all or part of the spacer domain. In some embodiments, the polypeptide is 800 amino acids or less in length, for example, 795, 790, 785, 780, 775, 770, 765, 760, 755, 750, 745, 740, 735, 730, 725, 720, 715, 710, or 705 amino acids or less. In some embodiments, the reverse transcriptase domain does not include the YMDD motif (SEQ ID NO: 97), and the RNase H domain does not include the AELL motif (SEQ ID NO: 98). In some embodiments, the YMDD motif (SEQ ID NO: 97) within the reverse transcriptase domain is mutated to YMHD (SEQ ID NO: 99), and the AELL motif (SEQ ID NO: 98) within the RNase H domain is mutated to AHLL (SEQ ID NO: 100). In some embodiments, the polypeptide is derived from HBV genotype A, B, C, or D.In some embodiments, (a) the polypeptide is derived from HBV genotype A and does not contain a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 42 or 46, or any one of SEQ ID NO: 42 or 46; (b) the polypeptide is derived from HBV genotype B and does not contain a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 43 or 47, or any one of SEQ ID NO: 43 or 47; (c) the polypeptide is derived from HBV genotype C and does not contain a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 44 or 48, or any one of SEQ ID NO: 44 or 48; or (d) the polypeptide is derived from HBV genotype D and does not contain a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide of SEQ ID NO: 45 or 49, or any one of SEQ ID NO: 45 or 49. In some embodiments, the truncated HBV polymerase deletion mutant polypeptide comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 5-12, 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: 5-12. In some embodiments, the HBV polymerase deletion mutant polypeptide further comprises an HBV core polypeptide (e.g., is a fusion protein comprising the HBV core polypeptide). In some embodiments, the HBV polymerase deletion mutant polypeptide comprises the HBV core polypeptide and the HBV polymerase deletion mutant polypeptide as described herein, in order from the N-terminus to the C-terminus.In some embodiments, the truncated HBV polymerase deletion mutant polypeptide comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 19-26, 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: 19-26.

[0007] In a further aspect, an HBV core-sAg fusion protein is provided. In some embodiments, the core-sAg fusion protein comprises an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, in order from the N-terminus to the C-terminus. 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 the amino acid sequence of SEQ ID NO: 65, 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: 65, and an sAg polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 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: 3, or (b) a core polypeptide comprising the amino acid sequence of SEQ ID NO: 66, 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: 66, and an sAg polypeptide comprising the amino acid sequence of SEQ ID NO: 4, 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: 4. 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, where the position numbers are based on 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, where the position number is based on 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, where the position number is based on SEQ ID NO: 3 or SEQ ID NO: 4. In various embodiments, the sAg polypeptide does not comprise the pre-S1 polypeptide. In various embodiments, the sAg polypeptide does not comprise the pre-S2 polypeptide. In some embodiments, the sAg polypeptide does not comprise an HBV pre-S2 polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 79-83, 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: 79-83. In some embodiments, the sAg polypeptide does not comprise both the HBV pre-S1 polypeptide and the HBV pre-S2 polypeptide. In some embodiments, the sAg polypeptide does not comprise an HBV pre-S1-pre-S2 polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 84-88, 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: 84-88.In various embodiments, the core-sAg fusion protein is operably linked to an HBV core polypeptide and an HBV sAg polypeptide and includes a cleavable linker positioned between the HBV core polypeptide and the HBV sAg polypeptide. In some embodiments, the cleavable linker is a 2A cleavable linker peptide. In some embodiments, the cleavable linker is a 2A cleavable linker 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 is operably linked and positioned at the N-terminus of the cleavable linker and the C-terminus of the HBV core polypeptide and includes a flexible linker and / or a furin recognition / cleavage site.In some embodiments, the furin recognition / cleavage site comprises or consists of an amino acid sequence selected from 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 sequence or a polyalanine sequence. In some embodiments, the flexible linker comprises or consists of a polyglycine sequence or a polyalanine sequence (SEQ ID NO: 63) selected from AA, AAA, AAY, GG, GGG, GGS, GSG, and GGGS. In some embodiments, the core-sAg fusion protein is 450 amino acids or less in length, for example, 445, 440, 435, 430, 425, 420, 415, or 410 amino acids or less. In some embodiments, the core-sAg fusion protein comprises or consists of any one of SEQ ID NOs: 38-41, for example, the amino acid sequence of SEQ ID NO: 41, or any one of SEQ ID NOs: 38-41, for example, a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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, and the position numbers are based on SEQ ID NO: 41. In various embodiments, the core-sAg fusion polypeptide does not comprise an amino sequence or a fragment thereof derived from an HBV protein selected from the group consisting of X, pre-core, pre-S1, and pre-S2.

[0008] Regarding immunogenic HBV polypeptides, in some embodiments, an HBV polymerase polypeptide, an HBV polymerase deletion mutant polypeptide, or a 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 serum proteins, cytokines, chemokines, chaperone proteins, invariant proteins, and proteins that direct proteins to the lysosomal compartment. In various embodiments, the signal peptide or leader sequence is derived from a source protein selected from colony stimulating factor 2 (CSF2, GM-CSF), tissue-type plasminogen activator (PLAT, t-PA), C-C motif chemokine ligand 7 (CCL7, MCP-3), C-X-C 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 membrane protein 1 (LAMP-1), and lysosomal membrane protein 2 (LAMP-2). In some embodiments, the signal peptide or leader sequence is selected from the amino acid sequence of any one of SEQ ID NOs: 67-78, or a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 67-78. In various embodiments, a truncated HBV polymerase polypeptide, an HBV polymerase deletion mutant polypeptide, and / or a core-sAg fusion protein as described herein can be recombinantly produced or chemically synthesized.In various embodiments, a truncated HBV polymerase polypeptide, an HBV polymerase deletion mutant polypeptide, and / or a core-sAg fusion protein as described herein can induce, enhance, 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, a truncated HBV polymerase polypeptide, an HBV polymerase deletion mutant polypeptide, and / or a core-sAg fusion protein as described herein can induce, enhance, or stimulate an immune response against HBV in a human (e.g., prevent HBV infection, delay progression of HBV infection, block HBV infection, and / or recover from HBV infection). In various embodiments, a truncated HBV polymerase polypeptide, an HBV polymerase deletion mutant polypeptide, and / or a core-sAg fusion protein as described herein can induce, enhance, or stimulate 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.

[0009] In a further aspect, there is provided a polynucleotide encoding an immunogenic HBV polypeptide as described herein. For example, there is provided a polynucleotide encoding one or more of a truncated HBV polymerase polypeptide, an HBV polymerase deletion mutant polypeptide, or a core-sAg fusion protein as described herein. In some embodiments, the polynucleotide comprises cDNA, mRNA, self-amplifying RNA (SAM), self-replicating RNA, or self-amplifying replicon RNA (RepRNA). In some embodiments, the polynucleotide comprises a self-replicating or self-amplifying alphavirus replicon. In some embodiments, the polynucleotide comprises a nucleic acid sequence of any one of SEQ ID NOs: 27-37, such as SEQ ID NO: 37 and SEQ ID NOs: 89-94, such as 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 any one of SEQ ID NOs: 27-37, such as SEQ ID NO: 37 and SEQ ID NOs: 89-94, such as SEQ ID NO: 29, 89, 90, or 92, or consists of such a sequence.

[0010] In another aspect, there is provided a lipid nanoparticle (LNP) comprising one or more of the polynucleotides encoding an immunogenic HBV polypeptide as described herein.

[0011] In another aspect, there is provided an expression cassette comprising one or more of the polynucleotides encoding an immunogenic HBV polypeptide as described herein operably linked to one or more regulatory sequences. In some embodiments, the polynucleotide is operably linked to a constitutive promoter and is under the control of the constitutive promoter. In some embodiments, the promoter is fused with the cytomegalovirus major immediate-early (CMV), chicken beta-actin promoter (CAG) It is selected from the following: CMV enhancer, human elongation factor-1α (HEF-1α), murine cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK).

[0012] In another aspect, provided is a composition comprising one or more of the polynucleotides encoding an immunogenic HBV polypeptide as described herein, or one or more expression cassettes comprising such polynucleotides. In some embodiments, the vector is a plasmid vector, a bacterial vector, or a viral vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a DNA virus or an RNA virus. In some embodiments, the viral vector is derived from a virus selected from the group consisting of 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 derived from a virus belonging to a taxonomic family selected from the group consisting 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), and Togaviridae (e.g., alphavirus). In some embodiments, the viral vector is an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammarenavirus (LCMV), Cali mammarenavirus (also known as Pichinde mammarenavirus 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 mammarenavirus (LCMV) or Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)).In some embodiments, the viral vector is a human adenovirus or a simian adenovirus (e.g., chimpanzee adenovirus, gorilla adenovirus, or rhesus adenovirus). In some embodiments, the viral vector is 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), ChAdV63, 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 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-deficient, replication-deleted, replication-attenuated, or replication-competent. In some embodiments, the viral vector is a replication-deficient arenavirus having a two-segmented genome.In some embodiments, the viral vector is a replication-attenuated arenavirus having a segmented genome.

[0013] In a further aspect, an arenavirus vector is provided. In one embodiment, the arenavirus vector comprises a polynucleotide encoding an HBV core-sAg fusion protein comprising any one of SEQ ID NOs: 38 to 41, for example, the amino acid sequence of 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 to 41, for example, SEQ ID NO: 41, and the sAg polypeptide does not include an HBV preS1 polypeptide and / or an HBV preS2 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, and the position numbers are based on 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, and the position numbers are based on 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, and the position numbers are based on 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, and the position numbers are based on SEQ ID NO: 41. In some embodiments, the polynucleotide comprises or consists of a nucleic acid sequence 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 the nucleic acid sequence 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 two-segmented genome and further comprises a polynucleotide encoding a truncated HBV polymerase comprising the amino acid sequence of any one of SEQ ID NOs: 13-14, 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: 13-14. The truncated HBV polymerase does not include all of the HBV polymerase terminal protein (TP) domain and does not include all or part of the HBV polymerase spacer domain. In some embodiments, the truncated HBV polymerase does not include the 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 any one of SEQ ID NO: 50 or SEQ ID NO: 51. In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence of any one of SEQ ID NOs: 29 and 89-94, such as the nucleic acid sequence 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 any one of SEQ ID NOs: 29 and 89-94, such as the nucleic acid sequence of SEQ ID NO: 29, 89, 90, or 92. In some embodiments, the arenavirus vector is a lymphocytic choriomeningitis mammarenavirus (LCMV) vector, and the polynucleotide comprises or consists of the nucleic acid sequence 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.In some embodiments, the arena virus vector is a Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)) vector, and the polynucleotide comprises or consists of a nucleic acid sequence 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.

[0014] An arenavirus vector is further provided that comprises a polynucleotide encoding a truncated HBV polymerase that comprises any one amino acid sequence of SEQ ID NOs: 13-14, 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: 13-14. The truncated HBV polymerase does not include all of the HBV polymerase terminal protein (TP) domain and does not include all or part of the HBV polymerase spacer domain. In some embodiments, the truncated HBV polymerase does not include the 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 any one of SEQ ID NO: 50 or SEQ ID NO: 51. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs: 29 and 89-94, such as the nucleic acid sequence 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 any one of SEQ ID NOs: 29 and 89-94, such as SEQ ID NO: 29, 89, 90, or 92. In some embodiments, the arenavirus vector is a lymphocytic choriomeningitis mammarenavirus (LCMV) vector, and the polynucleotide comprises the nucleic acid sequence 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.In some embodiments, the arenavirus vector is a Caly murine arenavirus vector, and the polynucleotide comprises or consists of a nucleic acid sequence 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-deficient, replication-deleted, or replication-incompetent.

[0015] In a further aspect, there is provided a host cell comprising 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 episomal. 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, for example, a human cell. In various embodiments, the host cell can be in vitro or in vivo.

[0016] In another aspect, 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, such as two or more, of the truncated HBV polymerase polypeptides as described herein, one or more, such as two or more, of the HBV polymerase deletion mutant polypeptides, and / or one or more, such as two or more, of the core-sAg fusion proteins. In some embodiments, the immunogenic composition comprises one or more, such as two or more, polynucleotides encoding one or more, such as two or more, of the truncated HBV polymerase polypeptides as described herein, one or more, such as two or more, of the HBV polymerase deletion mutant polypeptides, and / or one or more, such as two or more, of the core-sAg fusion proteins. In some embodiments, the immunogenic composition comprises one or more, such as two or more, vectors comprising one or more, such as two or more, polynucleotides encoding one or more, such as two or more, of the truncated HBV polymerase polypeptides as described herein, one or more, such as two or more, of the HBV polymerase deletion mutant polypeptides, and / or one or more, such as 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 as described.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 variant comprising an amino acid sequence of any one of SEQ ID NOs: 5-14, 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: 5-14, or consisting of such a sequence; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 38-41, 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 any one of SEQ ID NOs: 38-41, or consisting of such a sequence. 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 variant comprising an amino acid sequence of any one of SEQ ID NOs: 13-14, 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: 13-14, or consisting of such a sequence; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 38-41, 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 any one of SEQ ID NOs: 38-41, or consisting of such a sequence.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 variant comprising the 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; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising the amino acid sequence 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: 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, where the position numbers are based on 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, where the position number is based on 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, where the position numbers are based on 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, and the position numbers are based on 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 nucleic acid sequence of any one of SEQ ID NOs: 27-32 and 89-94, for example, the nucleic acid sequence of SEQ ID NO: 29, 89, 90, or 92, or a polynucleotide comprising or consisting of 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, for example, SEQ ID NO: 29, 89, 90, or 92, and (b) the second viral expression vector comprises a nucleic acid sequence of any one of SEQ ID NOs: 33-37, or a polynucleotide comprising or consisting of 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: 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 the nucleic acid sequence of SEQ ID NO: 29 or 90, 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: 29 or 90, or consisting of such a sequence; and (b) the second viral expression vector comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 37, 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 NO: 37, or consisting of such a sequence. In various embodiments, the first viral expression vector and the second viral expression vector are independent of 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 can 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 viral expression vector and the second viral expression vector in the immunogenic composition are independent of arenavirus vectors selected from lymphocytic choriomeningitis mammarenavirus (LCMV), Cali mammarenavirus (also known as Pichinde mammarenavirus 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 first viral expression vector and the second viral expression vector are independent of arenavirus vectors selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)). In some embodiments, the first viral expression vector and the second viral expression vector are replication-deficient or replication-deleted. In some embodiments, the first viral expression vector and the second viral expression vector 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 nucleic acid sequence of SEQ ID NO: 29, 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: 29, or consists of such a sequence polynucleotide. comprising do, (b) the second LCMV arena virus expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of SEQ ID NO: 37, 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 NO: 37. In some embodiments, the immunogenic composition comprises a first pichinde arena virus expression vector and a second pichinde arena virus expression vector, (a) the first pichinde arena virus expression vector comprises a nucleic acid sequence of SEQ ID NO: 90, or a polynucleotide comprising or consisting of 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: 90, (b) the second pichinde arena virus expression vector comprises a nucleic acid sequence of SEQ ID NO: 37, or a polynucleotide comprising or consisting of 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 NO: 37. In various embodiments, the first virus expression vector and the second virus expression vector are provided in the immunogenic composition in a ratio in the range of 1:10 to 10:1, such as, for example, in the range of 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 some embodiments, the immunogenic composition comprises each of the first virus expression vector and the second virus expression vector at about 10 3 ~ about 10 12 virus focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or virus particles (vp) per milliliter, such as, for example, about 10 4 ~ about 10 7 virus FFU or PFU or IU or vp per milliliter, such as, for example, about 10 3 ~ about 10 4, 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 It is included within the range of viral FFU or PFU or IU or vp. In some embodiments, the immunogenic composition further comprises one or more of an adjuvant, a detergent, a micelle-forming agent, and an oil. In various embodiments, the immunogenic composition is formulated for administration via a route selected from intravenous, intramuscular, intradermal, subcutaneous, and intramucosal (e.g., buccal, intranasal, rectal, vaginal). In some embodiments, the immunogenic composition is an aqueous solution or suspension, e.g., formulated as a liquid. In some embodiments, the immunogenic composition is lyophilized.

[0017] In a further aspect, a kit is provided. In various embodiments, the kit 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 one or more, e.g., two or more, unit doses. In some embodiments, the kit comprises one or more, e.g., two or more, unit doses of one or more, e.g., two or more, polynucleotides encoding 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 kit comprises one or more, e.g., two or more, unit doses of one or more, e.g., two or more, vectors comprising one or more, e.g., two or more, polynucleotides encoding 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 various embodiments, the kit comprises one or more, e.g., two or more, unit doses of one or more, e.g., two or more, immunogenic compositions as described herein. In some embodiments, one or more of the unit doses within the kit are in a single container. In some embodiments, one or more of the unit doses within the kit are in two or more separate containers. In some embodiments, the kit comprises one or more containers selected from vials, ampules, and pre-filled syringes. In some embodiments, the kit comprises one or more containers containing 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 preparation. In various embodiments, the one or more unit doses may be the same or different.In some embodiments, the kit comprises one or more unit doses of one or more viral vectors, as described herein, and the unit dose is about 10. 3 ~ about 10 12 in the range of viral focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or viral particles (vp), e.g., about 10 4 ~ about 10 7 viral FFU or PFU or IU or vp, e.g., about 10 3 ~ about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12It is in the range of viral FFU or PFU or IU or vp. In some embodiments, the kit comprises 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 amino acid sequence of any one of SEQ ID NOs: 5-14, or a sequence comprising or consisting of 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: 5-14, which is an HBV polymerase polypeptide variant; and (b) an amino acid sequence of any one of SEQ ID NOs: 38-41, or a sequence comprising or consisting of 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: 38-41, which is an HBV core-sAg fusion protein.In some embodiments, the kit comprises one or more polynucleotides encoding at least two immunogenic polypeptides, 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 variant comprising the amino acid sequence of any one of SEQ ID NOs: 13-14, or a sequence comprising or consisting of 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: 13-14; and (b) an HBV core-sAg fusion protein comprising the amino acid sequence of any one of SEQ ID NOs: 38-41, or a sequence comprising or consisting of 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: 38-41. In some embodiments, the kit comprises one or more polynucleotides encoding at least two immunogenic polypeptides, 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 variant comprising the amino acid sequence of SEQ ID NO: 13, or a sequence comprising or consisting of 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; and (b) an HBV core-sAg fusion protein comprising the amino acid sequence of SEQ ID NO: 41, or a sequence comprising or consisting of 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: 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, and the position numbers are based on 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, and the position numbers are based on 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, and the position numbers are based on 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, and the position numbers are based on 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, wherein the first immunogenic polypeptide and the second immunogenic polypeptide each comprise (a) an HBV polymerase polypeptide variant comprising the 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, or consisting of such a sequence, and (b) an HBV core-sAg fusion protein comprising the amino acid sequence 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: 41, or consisting of such a 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 any one of the nucleic acid sequences of SEQ ID NOs: 27-32 and 89-94, for example, the nucleic acid sequence of SEQ ID NO: 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 the nucleic acid sequences of SEQ ID NOs: 27-32 and 89-94, for example, the nucleic acid sequence of SEQ ID NO: 29, 89, 90, or 92, or consisting of such a sequence, and (b) the second viral expression vector comprises a polynucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 33-37, 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 the nucleic acid sequences of SEQ ID NOs: 33-37, or consisting of such a 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 the nucleic acid sequence of SEQ ID NO: 29 or 90, 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: 29 or 90, or consisting of such a sequence; and (b) the second viral expression vector comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 37, 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: 37, or consisting of such a sequence. In some embodiments, the kit comprises one or more unit doses of an immunogenic composition comprising the first viral expression vector and the second viral expression vector as described herein, and the first viral expression vector and the second viral expression vector comprise a replication-deficient or replication-deleted Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)). In some embodiments, the kit comprises one or more unit doses of an immunogenic composition comprising the first viral expression vector and the second viral expression vector as described herein, and the first viral expression vect. The first and second viral expression vectors include a replication-deficient or replication-deleted lymphocytic choriomeningitis mammarenavirus (LCMV). In some embodiments, the kit comprises: (a) one or more unit doses of an immunogenic composition as described herein, wherein the first and second viral expression vectors are from the family Adenoviridae, and (b) one or more unit doses of an immunogenic composition as described herein, wherein the first and second viral expression vectors are from the family Poxviridae (e.g., vaccinia virus, e.g., modified vaccinia Ankara (MVA)). In some embodiments, the kit comprises: (a) one or more unit doses of an immunogenic composition as described herein, wherein the first and second viral expression vectors are from the family Arenaviridae, and (b) one or more unit doses of an immunogenic composition as described herein, wherein the first and second viral expression vectors are from the family Adenoviridae. 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 the nucleic acid sequence of SEQ ID NO: 29, 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: 29, or consisting of such a sequence; and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 37, 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: 37, or consisting of such a sequence.In some embodiments, the kit 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 the nucleic acid sequence of SEQ ID NO: 90, 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: 90, or consisting of such a sequence; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 37, 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: 37, or consisting of such a sequence. In various embodiments, the kit further comprises one or more unit doses of one or more additional therapeutic agents. In some embodiments, the kit further comprises one or more agonists or activators of one or more Toll-like receptors (TLRs). In some embodiments, the kit further comprises one or more TLR agonists or activators selected from TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR7 agonists, TLR8 agonists, and TLR9 agonists. In some embodiments, the kit further comprises a TLR7 agonist selected from GS9620 (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 of interleukin receptors selected from IL-2, IL-7, IL-12, and IL-15. In some embodiments, the kit further comprises one or more cytokines selected from IL-2, IL-7, IL-12, IL-15, and variants thereof.In some embodiments, the kit further comprises one or more innate immune activating factors. In some embodiments, the kit further comprises one or more innate immune activating factors comprising an agonist of a receptor selected from fms-related tyrosine kinase 3 (FLT3), stimulator of interferon genes (STING) receptor, DExD / H box helicase 58 (DDX58, also known as RIG-I), nucleotide-binding oligomerization domain-containing 2 (NOD2). In some embodiments, the kit further comprises one or more unit doses of GS-3583 and / or GS-9992. In some embodiments, the kit further comprises one or more antagonists or inhibitors of an inhibitory immune checkpoint protein or receptor, and / or one or more activating factors or agonists of a stimulatory immune checkpoint protein or receptor. In some embodiments, the kit comprises CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane domain and immunoglobulin domain-containing 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244 (S. LAMF4); CD276 (B7H3); V-set domain-containing T cell activation inhibitor 1 (VTCN1, B7H4); V-set immunoregulatory receptor (VSIR, B7H5, VISTA); Immunoglobulin superfamily member 11 (IGSF11, VSIG3); Natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR-related 2 (HHLA2, B7H7); Inducible T cell co-stimulatory molecule (ICOS, CD278); Inducible T cell co-stimulatory molecule ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (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); T cell immunoreceptor with Ig and ITIM domains (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); Signaling 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); UL16 binding protein 1 (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 one or more immune checkpoint proteins or receptors selected from SLAM family member 7 (SLAMF7). In some embodiments, the kit further comprises one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the kit comprises 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 T cell activation inhibitor 1 (VTCN1,B7H4); V-set immunoregulatory receptor (VSIR, B7H5, VISTA); Immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte associated (BTLA)); PVR-related 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), and further comprises one or more T cell inhibitory immune checkpoint proteins or receptors selected therefrom. 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 comprises CD27, CD70; CD40, CD40LG; Inducible T cell co-stimulatory molecule (ICOS, CD278); Inducible T cell co-stimulatory molecule 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 (Poliovirus receptor: PVR) cell adhesion molecule (PVR,It further comprises one or more T cell-stimulating immune checkpoint proteins or receptors selected from CD155). In some embodiments, the kit further comprises one or more unit doses of AGEN-2373 and / or AGEN-1223. In some embodiments, the kit further comprises one or more blockers or inhibitors of one or more NK cell-inhibitory immune checkpoint proteins or receptors. In some embodiments, the kit comprises 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), and further comprises one or more NK cell-inhibitory immune checkpoint proteins or receptors selected therefrom. In some embodiments, the kit further comprises one or more agonists or activators of one or more NK cell-stimulating immune checkpoint proteins or receptors. In some embodiments, the kit further comprises one or more NK cell-stimulating immune checkpoint proteins or receptors selected from 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 proteinaceous inhibitors of PD-L1 (CD274), PD-1 (PDCD1), and / or CTLA4. In some embodiments, the kit comprises 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,It further comprises one or more proteinaceous inhibitors of CTLA4 selected from 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 comprises zimberelimab (AB122), pembrolizumab, nivolumab, semiprimab, pidilizumab, AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, 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),One or more proteinaceous inhibitors of PD-L1 (CD274) or PD-1 (PDCD1) selected from 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) Further includes. In some embodiments, the kit further includes 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 includes 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 includes a small molecule inhibitor of CTLA4, BPI-002. In some embodiments, the kit further includes one or more antiviral drugs. In some embodiments, the kit further includes one or more antiviral drugs selected from lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), tenofovir disoproxil fumarate (TDF), tenofovir disoproxyl fumarate (TDF), tenofovir alafenamide (TAF or VEMLIDY™), and ledipasvir + sofosbuvir (HARVONI™). In some embodiments, the kit further includes one or more therapeutic agents selected from 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-directed 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, TALEN), covalently closed circular DNA (cccDNA) inhibitors, and HBsAg secretion or assembly inhibitors, and HBV virus entry inhibitors.

[0018] In a further aspect, there is provided a method for inducing an immune response against hepatitis B virus (HBV) in a subject in need thereof. Also provided is a method for treating or preventing hepatitis B virus (HBV) in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of one or more immunogenic compositions as described herein. In some embodiments, the method involves administering 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 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 involves 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 encoding an HBV polymerase polypeptide variant comprising an amino acid sequence of any one of SEQ ID NOs: 5-14, 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: 5-14, or consisting of such a sequence, and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 38-41, 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 any one of SEQ ID NOs: 38-41, or consisting of such a sequence.In some embodiments, the method involves 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 encoding an HBV polymerase polypeptide variant comprising an amino acid sequence of any one of SEQ ID NOs: 13-14, 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: 13-14, or consisting of such a sequence; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 38-41, 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 any one of SEQ ID NOs: 38-41, or consisting of such a sequence. In some embodiments, the method involves 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 encoding an HBV polymerase polypeptide variant comprising the 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, or consisting of such a sequence; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising the amino acid sequence 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: 41, or consisting of such a sequence.In some embodiments, the core polypeptide includes 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, and the position numbers are based on SEQ ID NO: 65 or SEQ ID NO: 66. In some embodiments, the sAg polypeptide includes an isoleucine (I) residue at the amino acid position corresponding to position 68, and the position numbers are based on SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sAg polypeptide includes 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, and the position numbers are based on SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the core-sAg fusion polypeptide includes 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, and the position numbers are based on SEQ ID NO: 41.In some embodiments, the method involves 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 a sequence that is identical to any one of SEQ ID NOs: 27-32 and 89-94, such as the nucleic acid sequence of SEQ ID NO: 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, such as the nucleic acid sequence of SEQ ID NO: 29, 89, 90, or 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any one of SEQ ID NOs: 33-37, 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: 33-37. In some embodiments, the method involves 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 the nucleic acid sequence of SEQ ID NO: 29 or 90, 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 the nucleic acid sequence of SEQ ID NO: 29 or 90; 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 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 nucleic acid sequence of SEQ ID NO: 37. In some embodiments of the method, the first viral expression vector and the second viral expression vector are derived from the family Arenaviridae.In some embodiments of the method, the first viral expression vector and the second viral expression vector are derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV), Cali mammarenavirus (also known as Pichinde mammarenavirus 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 of the method, the first viral expression vector and the second viral expression vector are derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)). In some embodiments of the method, the first viral expression vector and the second viral expression vector are replication-deficient or replication-deleted. In some embodiments of the method, the first viral expression vector and the second viral expression vector are replication-attenuated. In some embodiments, the method involves 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 the nucleotide sequence of SEQ ID NO: 29, 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: 29, or consisting of such a sequence, and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 37, 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: 37, or consisting of such a sequence.In some embodiments, the method involves 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 the nucleotide sequence of SEQ ID NO: 90, 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: 90, or consisting of such a sequence; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 37, 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: 37, or consisting of such a sequence. Some of the methods. In embodiments, the subject is infected with HBV, suspected of being infected with HBV, or at risk of infection with HBV. In some embodiments of the method, the subject is asymptomatic. In some embodiments of the method, the subject is chronically infected with HBV. In some embodiments of the method, the subject exhibits or has experienced one or more conditions selected from liver failure, liver cancer, liver fibrosis, and cirrhosis. In some embodiments of the method, the subject is acutely infected with HBV. In some embodiments of the method, the subject exhibits or has experienced one or more symptoms selected from jaundice, a visible network of dilated blood vessels in the skin, dark (e.g., orange or brown) urine, light-colored feces, fever, persistent fatigue, malaise, abdominal pain, ascites, loss of appetite, nausea, and vomiting. In some embodiments of the method, the subject is co-infected with hepatitis D virus (HDV). In some embodiments of the method, 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 involves administering to the subject from about 10 3 ~ about 10 12Viral focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or viral particles (vp), e.g., about 10 4 ~ about 10 7 viral FFU or PFU or IU or vp, e.g., about 10 3 ~ about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 viral FFU or PFU or IU or vp. Some embodiments of the method involve administration of one or more compositions multiple times. In some embodiments, the method is about 10 per administration on a biweekly (Q2W) or monthly (Q4W) schedule 6 ~ about 10 8It involves administering the virus FFU or PFU or IU or vp intravenously or intramuscularly. In some embodiments, the method involves administering one or more immunogenic compositions multiple times over a period of at least about 2 weeks, 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 more, or until sAg is no longer detectable in the subject's serum or plasma. In some embodiments, the method includes a prime-boost regimen that includes administering a priming composition at an initial time point and administering one or more boosting compositions at one or more subsequent time points. Optionally, the method may involve repeating the prime-boost regimen one or more times. In some embodiments of the method, the administrations of the priming composition and the one or more boosting compositions are spaced 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 apart. In some embodiments of the method, the priming composition and the boosting composition may contain the same immunogenic composition or different immunogenic compositions. In some embodiments of the method, the priming composition and the boosting composition contain the same one or more polypeptides and the same viral expression vector. In some embodiments of the method, the priming composition and the boosting composition contain different polypeptides and / or different viral expression vectors. In some embodiments, the method involves priming with a priming composition containing one or more (e.g., first and second) viral expression vectors and boosting with a boosting composition containing one or more (e.g., third and fourth) viral expression vectors. In various embodiments, the prime-boost regimen is (a) priming with a priming composition containing one or more viral expression vectors and boosting with a boosting composition containing one or more polynucleotides, wherein the one or more polynucleotides include DNA, cDNA, mRNA, or self-replicating RNA,(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; (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; (d) priming with a priming composition comprising one or more replication-deficient viral expression vectors, and boosting with a boosting composition comprising one or more replication-deficient viral expression vectors, wherein the one or more replication-deficient viral expression vectors in the priming composition and the one or more replication-deficient viral expression vectors in the boosting composition are from the same, related, or unrelated taxonomic families; (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; (f) priming with a priming composition comprising one or more replication-deficient viral expression vectors, and boosting with a boosting composition comprising one or more replication-attenuated viral expression vectors; (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-deficient viral expression vectors; (h) priming with a priming composition comprising one or more lymphocytic choriomeningitis mammarenavirus (LCMV) viral expression vectors, and boosting with a boosting composition comprising one or more picornavirus encephalomyelitis virus (PICV) viral expression vectors,(i) priming with a priming composition comprising one or more Pichinde mammarenavirus (PICV) virus expression vectors and boosting with a boosting composition comprising one or more Lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors, (j) priming with a priming composition comprising one or more replication-deficient Pichinde mammarenavirus (PICV) virus expression vectors and boosting with a boosting composition comprising one or more replication-deficient Lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors, (k) priming with a priming composition comprising one or more replication-deficient Lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors and boosting with a boosting composition comprising one or more replication-deficient Pichinde mammarenavirus (PICV) virus expression vectors, (l) priming with a priming composition comprising one or more arenavirus virus expression vectors and boosting with a boosting composition comprising one or more adenovirus virus expression vectors, (m) priming with a priming composition comprising one or more adenovirus virus expression vectors and boosting with a boosting composition comprising one or more arenavirus virus expression vectors, (n) priming with a priming composition comprising one or more poxvirus virus expression vectors and boosting with a boosting composition comprising one or more arenavirus virus expression vectors, (o) priming with a priming composition comprising one or more arenavirus virus expression vectors and boosting with a boosting composition comprising one or more poxvirus virus expression vectors, (p) priming with a priming composition comprising one or more poxvirus virus expression vectors and boosting with a boosting composition comprising one or more adenovirus virus expression vectors, or (q) priming with a priming composition comprising one or more adenovirus virus expression vectors and boosting with a boosting composition comprising one or more poxvirus virus expression vectors, comprising. In some embodiments, the method is,(a) Priming with a priming composition comprising one or more lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors and boosting with a boosting composition comprising one or more pichinde mammarenavirus (PICV) virus expression vectors; (b) Priming with a priming composition comprising one or more pichinde mammarenavirus (PICV) virus expression vectors and boosting with a boosting composition comprising one or more lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors; (c) Priming with a priming composition comprising one or more replication-deficient pichinde mammarenavirus (PICV) virus expression vectors and boosting with a boosting composition comprising one or more replication-deficient lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors; or, (d) Priming with a priming composition comprising one or more replication-deficient lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors and boosting with a boosting composition comprising one or more replication-deficient pichinde mammarenavirus (PICV) virus expression vectors, which involves a prime-boost regimen. In some embodiments, the priming composition and the boosting composition comprise an immunogenic composition as described herein. In some embodiments, the subject has not received antiviral therapy or the antiviral therapy is discontinued prior to administration of one or more immunogenic compositions. In some embodiments of the method, the antiviral therapy is discontinued after one or more administrations of one or more immunogenic compositions. In some embodiments, the method further comprises administering to the subject one or more additional therapeutic agents, such as two, three, four, or more additional therapeutic agents. In some embodiments, the method comprises co-administering one or more agonists or activators of one or more Toll-like receptors (TLRs). In some embodiments, the TLR agonist or activator is a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist,comprising co-administering one or more TLR agonists or activators selected from TLR9 agonists. In some embodiments, the method involves co-administering a TLR7 agonist selected from GS-9620 (bevacutamod), R848 (resiquimod), DS-0509, LHC-165, and TMX-101 (imiquimod). In some embodiments, the method involves co-administering a TLR8 agonist selected from GS-9688, R848 (resiquimod), and NKTR-262 (dual TLR7 / TLR8 agonist). In some embodiments, the method involves co-administering one or more interleukin receptor agonists of interleukin receptors selected from IL-2, IL-7, IL-12, and IL-15. In some embodiments, the method involves IL-2, IL-7, IL-12, IL-15, and those, involves co-administering one or more cytokines selected from variants thereof. In some embodiments, the method involves co-administering one or more innate immune activating factors. In some embodiments, the method involves co-administering one or more innate immune activating factors including an agonist of a receptor selected from fms-related tyrosine kinase 3 (FLT3), stimulator of interferon genes (STING) receptor, DExD / H box helicase 58 (DDX58, also known as RIG-I), nucleotide-binding oligomerization domain-containing 2 (NOD2). In some embodiments, the method involves co-administering GS-3583 and / or GS-9992. In some embodiments, the method involves co-administering one or more antagonists or inhibitors of an inhibitory immune checkpoint protein or receptor, and / or one or more activating factors or agonists of a stimulatory immune checkpoint protein or receptor. In some embodiments, the method involves CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane domain 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 immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR-related 2 (HHLA2, B7H7); inducible T cell co-stimulatory molecule (ICOS, CD278); inducible T cell co-stimulatory molecule ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (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); T cell immunoreceptor with Ig and ITIM domains (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); Signaling 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); UL16 binding protein 1 (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 co-administering one or more immune checkpoint proteins or receptors selected from SLAM family member 7 (SLAMF7). In some embodiments, the method involves co-administering one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the method includes 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 T cell activation inhibitor 1 (VTCN1, B7H4); V-set immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte associated (BTLA)); PVR-related 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);Comprising co-administering one or more T cell inhibitory immune checkpoint proteins or receptors selected from 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 method comprises co-administering one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors. In some embodiments, the method comprises co-administering one or more T cell stimulatory immune checkpoint proteins or receptors selected from CD27, CD70; CD40, CD40LG; inducible T cell co-stimulatory molecule (ICOS, CD278); inducible T cell co-stimulatory molecule 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 (Poliovirus receptor: PVR) cell adhesion molecule (PVR, CD155). In some embodiments, the method comprises co-administering AGEN-2373 and / or AGEN-1223. In some embodiments, the method comprises co-administering one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the method comprises 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);Comprising co-administering one or more NK cell inhibitory immune checkpoint proteins or receptors selected from 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 method comprises co-administering one or more agonists or activators of one or more NK cell stimulatory immune checkpoint proteins or receptors. In some embodiments, the method is CD16, CD226 (DNAM-1); killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314);and co-administering one or more NK cell-stimulating immune checkpoint proteins or receptors selected from the SLAM family member 7 (SLAMF7). In some embodiments, the method involves co-administering one or more proteinaceous inhibitors of PD-L1 (CD274), PD-1 (PDCD1), or CTLA4. In some embodiments, the method involves co-administering one or more protein inhibitors of CTLA4 selected from 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 method involves co-administering zimberelimab (AB122), pembrolizumab, nivolumab, semiprimab, pidilizumab, AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, 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 (tripalimumab), JNJ-63723283, genolimuzumab (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), and involves co-administering one or more proteinaceous inhibitors of PD-L1 (CD274) or PD-1 (PDCD1) selected therefrom. In some embodiments, the method involves co-administering one or more small molecule inhibitors of CD274 (PDL1, PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1), or CTLA4. In some embodiments, the method involves co-administering one or more small molecule inhibitors of CD274 or PDCD1 selected from GS-4224, GS-4416, INCB086550, and MAX10181. In some embodiments, the method involves co-administering BPI-002 (a small molecule inhibitor of CTLA4). In some embodiments, the method includes co-administering to the subject one or more antiviral agents.In some embodiments, the method comprises co-administering to the subject one or more antiviral agents selected from lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), tenofovir disoproxil fumarate (TDF), tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF or VEMLIDY™), and ledipasvir + sofosbuvir (HARVONI™). In some embodiments, the method comprises co-administering to the subject one or more therapeutic agents selected from 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-directed RNA interference (ddRNAi)), gene editors targeting HBV (e.g., CRISPR-Cas (e.g., Cas9, Cas12, Cascade, Cas13), zinc finger nucleases, homing endonucleases, homing megonucleases (e.g., ARCUS), synthetic nucleases, TALEN), covalently closed circular DNA (cccDNA) inhibitors, and HBsAg secretion or assembly inhibitors, and HBV virus 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 induces the production of antibodies that bind to one or more HBV polypeptides in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

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Modes for Carrying Out the Invention

[0049] 1. Introduction There is provided a polypeptide useful for inducing a protective immune response against one or more hepatitis B virus (HBV) antigens in humans. The immunogenic polypeptides described herein are capable of inducing a prophylactic and / or therapeutic immune response in humans against one or more hepatitis B virus (HBV) antigens. Generally, the immunogenic polypeptides described herein contain highly conserved portions of HBV proteins to induce a response against epitopes that are identical in the vaccine antigen and in the infectious HBV present in the patient, while excluding regions of insufficient conservation, thereby avoiding inducing immunodominant T cell responses that target epitopes not present in the patient's infectious HBV strain. The immunogenic polypeptides described herein further induce both CD4+ and CD8+ T cell responses to promote the elimination of infected cells, and in addition, induce an anti-sAg antibody response to promote sAg clearance, thereby reducing or eliminating the spread of residual virus if sterilizing virus clearance is not fully achieved. Further, the immunogenic polypeptides described herein are immunogenic when delivered using vaccine technologies capable of inducing the desired response in humans, and have been demonstrated to be stable in the delivery vector through a sufficient number of vector replications to enable vaccine production on a commercial scale. The immunogenic polypeptides can be used in a variety of vectors known to induce CD4+ and CD8+ T cells, as well as antibody responses in humans and other non-human primates. In certain embodiments, the immunogenic polypeptide is expressed from an arenavirus vector that can be repeatedly administered without inducing anti-vector antibodies, thereby overcoming many of the limitations of previous viral vector technologies and offering the potential to enhance the therapeutic effect with repeated administration.

[0050] 2. Polypeptides useful for promoting an immune response against hepatitis B virus (HBV) There is provided an immunogenic polypeptide useful for promoting, inducing, and / or eliciting an immunogenic response against one or more hepatitis B virus (HBV) antigens. In various embodiments, the immunogenic polypeptide comprises, in an N-terminal to C-terminal order, a variant and / or fragment of a polypeptide encoded by the HBV polymerase (Pol) gene, a variant and / or fragment of a polypeptide encoded by the HBV core gene, and a variant and / or fragment of a polypeptide encoded by the surface antigen (sAg) gene, and a fusion polypeptide comprising the same. The immunogenic polypeptide may comprise an amino acid sequence based on a consensus sequence or a near-consensus sequence derived from HBV genotype A, B, C, or D, and combinations thereof. Generally, the immunogenic polypeptides described herein do not comprise the sequence of the HBV X protein (HBx), precore, preS1, preS2, or fragments thereof.

[0051] In various embodiments, the immunogenic polypeptides described herein, and / or polynucleotides encoding such polypeptides, are provided in isolated form. This means that such polypeptides or polynucleotides are at least 50% w / w pure with respect to interfering proteins, cells, and other contaminants resulting from their production or purification, but does not exclude the possibility that the agent is combined with an excess of pharmaceutically acceptable carrier or other vehicle for the purpose of facilitating its use. The term "isolated", as applied to a polypeptide or polynucleotide as described herein, indicates that the polypeptide or polynucleotide is substantially free of cellular components associated with its natural state. It can be, for example, in a homogeneous state and can be either in a dry agent or in an aqueous solution. Purity and homogeneity can be determined using known methods, such as analytical chemistry techniques like polyacrylamide gel electrophoresis, column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis. Proteins that are the predominant species present in a preparation are substantially purified. An "isolated" or "purified" polypeptide or polynucleotide substantially lacks other cellular material or culture medium when produced by recombinant techniques, or precursor chemicals or other chemicals when chemically synthesized. In various embodiments, the purified polypeptide and / or polynucleotide is separated from, purified of, or free of interfering proteins and contaminants associated with production or purification by at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (w / w). In many cases, the agent is the major macromolecular species remaining after its purification.

[0052] HBV polymerase polypeptide variant In various embodiments, truncated and / or internally deleted hepatitis B virus (HBV) polymerase polypeptides are provided.

[0053] Wild-type HBV polymerase has four domains tandemly arranged in a single polypeptide from the N-terminus to the C-terminus: a terminal protein (TP) domain conserved throughout the Hepadnaviridae family (amino acid residues 1-177), a spacer region (amino acid residues 178-335), a domain where TP binds to reverse transcriptase (RT) (amino acid residues 336-678, including NCBI conserved domain pfam00078 or cd01645), and a C-terminal RNase H (RH) domain (amino acid residues 679-832). See, for example, Lanford, et al., J. Virol. (1999) 73(3):1885-93; Voros, 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 HBV polymerase truncated variants, the entire TP domain has been deleted or removed.

[0054] Generally, the enzyme domains, namely, the reverse transcriptase and RNase H domains, are inactivated in the HBV polymerase protein variants described herein. In various embodiments, the reverse transcriptase domain does not include the YMDD motif (SEQ ID NO: 97). In some embodiments, the YMDD motif (SEQ ID NO: 97) within the reverse transcriptase domain is changed to YMHD (SEQ ID NO: 99). In some embodiments, the RNase H domain does not include the AELL motif (SEQ ID NO: 98). In some embodiments, the AELL motif (SEQ ID NO: 98) within the RNase H domain is changed to AHLL (SEQ ID NO: 100).

[0055] Truncated polymerase variants In some embodiments, the truncated HBV polymerase polypeptide comprises an inactivated reverse transcriptase domain and an inactivated RNase H, and the polypeptide does not include all of the terminal protein (TP) domain and does not include all or a portion of the spacer domain (i.e., all or a portion of the terminal protein (TP) domain and the spacer domain are removed, excised, or excluded). In the truncated HBV polymerase polypeptides described herein, all of the TP domain and all or a portion of the spacer domain or region are deleted or removed. For example, in some embodiments, the N-terminal 300 amino acids of the native 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 may be directly fused via a linker or operably linked or connected as described herein. In some embodiments, the truncated HBV polymerase polypeptide is 600 amino acids or less in length, e.g., 595, 590, 585, 580, 575, 570, 565, 560, 555, 550, 545, 540, or 535 amino acids or less. In some embodiments, the truncated HBV polymerase polypeptide comprises the C-terminal 528, 529, 530, 531, 532, 533, 534, or 535 amino acids of the native or wild-type HBV polymerase.

[0056] In some embodiments, the truncated HBV polymerase polypeptide comprises an amino acid sequence corresponding to 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, 325-832, 326-832, 327-832, 328-832, 329-832, 330-832, 331-832, 332-832, 333-832, 334-832, 335-832, or 336-832 of the native or wild-type HBV polymerase. As used herein, the numbering of a given amino acid polymer or nucleic acid polymer refers to the position of any given polymer component (e.g., an amino acid, nucleotide, commonly also referred to as a "residue") not as the actual numerical position of the component in a given polymer, but rather 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), the numbering of the selected or referenced amino acid polymer or nucleic acid polymer, "corresponds to", "corresponding to", or "relative to". 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 6 amino acid residues of this sequence are SARSQS (SEQ ID NO: 95) of the genotype D Pol antigen and SSRSQS (SEQ ID NO: 96) of the genotype B Pol antigen. Document reports have shown that this N-terminal start site enables the function of the RT domain (see, e.g., Lanford, et al., supra) and the expression of the truncated protein in vitro (see, e.g., Voros, et al., supra).

[0057] In some embodiments, the truncated HBV polymerase polypeptide is derived from HBV genotype B and comprises or consists of the nucleic 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 derived from HBV genotype B and does not include (i.e., the sequence is excluded, excised, or removed; the sequence is not included) the polypeptide sequence of SEQ ID NO: 50 and also does not include any 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.

[0058] In some embodiments, the truncated HBV polymerase polypeptide is derived from HBV genotype D and comprises or consists of the nucleic acid sequence of SEQ ID NO: 14, 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: 14. In some embodiments, the truncated HBV polymerase polypeptide is derived from HBV genotype D and does not include the polypeptide sequence of SEQ ID NO: 51 and also does not include any 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.

[0059] Modifications can be made to the structure of the polypeptides described herein and the polynucleotides encoding such polypeptides such that a functional molecule encoding a variant or derivative polypeptide having desirable (e.g., immunogenic) characteristics can be obtained. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent or even improved variant or portion of a polypeptide described herein, one of ordinary skill in the art will typically change one or more of the codons of the encoding DNA sequence.

[0060] For example, a particular amino acid can be substituted with another amino acid in the protein structure without apparently losing its ability to bind to other polypeptides (e.g., antigens) or cells. Since this is the binding ability and property of the protein that defines the biological functional activity of the protein, specific amino acid sequence substitutions can be made in the protein sequence and, of course, the underlying DNA coding sequence, and yet a protein with similar characteristics can be obtained. Therefore, it is considered that various changes can be made to the polypeptide sequence of the disclosed polypeptide, or the corresponding DNA sequence encoding such a polypeptide, without apparently losing their biological usefulness or activity.

[0061] As used herein, "substitution" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively.

[0062] In many cases, polypeptide variants contain one or more conservative substitutions. "Conservative substitution" refers to the substitution of an amino acid with another amino acid having similar properties, such that one skilled in the art of peptide chemistry would expect that the secondary structure and hydropathicity properties of the polypeptide would not substantially change.

[0063] As used herein, "identity" means the percentage of identical nucleotides or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Sequences are generally aligned to maximize correspondence in a specified region, e.g., a region of at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or more amino acids or nucleotides in length, and the maximum length can be the full length of the reference polypeptide or polynucleotide sequence. For sequence comparison, typically one sequence acts as a reference sequence and that sequence is compared to a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer program, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Otherwise, standard parameters can be used. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence(s) relative to the reference sequence based on the specified program parameters.

[0064] When comparing polynucleotide and polypeptide sequences, two sequences are said to be "identical" if the sequences of nucleotides or amino acids in the two sequences are the same when the two sequences are aligned for maximum correspondence as described below. The comparison between two sequences is typically performed by comparing 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, usually 30 to about 75, 40 to about 50, or the full length of the sequence, and the sequence can be compared to a reference sequence at the same number of contiguous positions after the two sequences are optimally aligned.

[0065] Optimal alignment of the arrays for comparison may be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc. (Madison, WI)) with default parameters. This program implements several alignment schemes described in the following references: Dayhoff, "M.O." (1978) "A model of evolutionary change in proteins-Matrices for detecting distant relationships.", In Dayhoff, M.O. (ed.), "Atlas of Protein Sequence and Structure", National Biomedical Research Foundation (Washington DC), Volume 5, Supplement 3, pp. 345-358; Hein J. (1990), "Unified Approach to Alignment and Phylogenes", pp. 626-645, "Methods in Enzymology", Volume 183, Academic Press, Inc. (San Diego, CA); Higgins, D.G. and Sharp, P.M. (1989), "CABIOS" Volume 5: pp. 151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E.D. (1971), "Comb.Theor" Volume 77: p. 105; Santou, N. Nes, M. (1987) "Mol.Biol.Evol." Volume 4: pp. 406-425; Sneath, P.H.A. and Sokal, R.R. (1973) "Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy", Freeman Press (San Francisco, CA); Wilbur, W.J. and Lipman, D.J. (1983) "Proc.Natl.Acad.,Sci.USA" Volume 80: pp. 726-730.

[0066] Alternatively, the optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981), "Add. APL. Math" Vol. 2: p. 482, by the identity alignment algorithm of Needleman and Wunsch (1970), "J. Mol. Biol." Vol. 48: p. 443, by the similarity search of Pearson and Lipman (1988) "Proc. Natl. Acad. Sci. USA" Vol. 85: p. 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr. (Madison, WI)), or by inspection.

[0067] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described, respectively, in: Altschul et al., (1977) "Nucl. Acids Res." Vol. 25: pp. 3389 - 3402 and Altschul et al., (1990) "J. Mol. Biol." Vol. 215: pp. 403 - 410. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein to determine the percent sequence identity of the polynucleotides and polypeptides described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0068] In an exemplary case, for a nucleotide sequence, the cumulative score can be calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatched residue; always <0). The extension of a word hit in each direction stops when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of alignments of one or more negative-score residues; 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 value (E) of 10 as defaults, and BLOSUM62 is the scoring matrix (see the following: Henikoff and Henikoff (1989) "Proc. Natl. Acad. Sci. USA" Vol. 89: p. 10915) alignment, (B) 50, expectation value (E) 10, M = 5, N = -4, and comparison of both strands as defaults.

[0069] For an amino acid sequence, the cumulative score can be calculated using a scoring matrix. The extension of a word hit in each direction stops when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of alignments of one or more negative-score residues; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0070] In one approach, the "percentage of sequence identity" is determined by comparing two sequences optimally aligned over a comparison window that covers at least 20 positions, such as at least 50 positions, at least 100 positions, or the full length of the reference sequence. The portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) of 20% or less, usually 5 - 15%, or 10 - 12%, as compared to the reference sequence (without additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing this number of matching positions by the total number of positions in the reference sequence (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0071] As used herein, the term "polypeptide variant" refers to a polypeptide specifically disclosed herein and one or more substitutions, deletions, additions, and / or insertions, which are typically different polypeptides. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the polypeptide sequences described herein, by evaluating one or more biological activities of the polypeptides described herein, and / or using any of several techniques well known in the art. The term "variant" may also refer to any naturally occurring or genetically engineered molecule that contains one or more nucleotide or amino acid mutations.

[0072] Table A provides exemplary HBV polymerase truncated mutants for use in promoting, inducing, or eliciting an immunogenic response against, for example, the polymerase antigen expressed by HBV. Table B provides exemplary N-terminal sequence segments that are deleted or removed from the HBV polymerase truncated mutants described herein and are thus not included in the HBV polymerase truncated mutants.

Table A

Table B

[0073] In some embodiments, the truncated HBV polymerase polypeptide does not contain an amino acid sequence or a fragment thereof from another HBV protein. In some embodiments, the truncated HBV polymerase polypeptide does not contain an amino acid sequence or a fragment thereof from an HBV protein selected from the group consisting of precore, core, X, and envelope (e.g., small, medium, or large surface antigen (sAg)).

[0074] Internal deletion polymerase mutant Furthermore, an HBV polymerase deletion mutant polypeptide is provided. In various embodiments, the HBV polymerase internal deletion mutant polypeptide, in order from the N-terminus to the C-terminus, includes a terminal protein (TP) domain, an inactivated reverse transcriptase domain, and an inactivated RNase H, and the mutant polypeptide does not include 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 HBV polymerase deletion mutant polypeptide is 800 amino acids or less in length, for example, 795, 790, 785, 780, 775, 770, 765, 760, 755, 750, 745, 740, 735, 730, 725, 720, 715, 710, or 705 amino acids or less. In some embodiments, the HBV polymerase internal deletion mutant polypeptide, in order from the N-terminus to the C-terminus, includes a terminal protein (TP) domain and an amino acid sequence corresponding to 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, 325-832, 326-832, 327-832, 328-832, 329-832, 330-832, 331-832, 332-832, 333-832, 334-832, 335-832, or 336-832 of the native or wild-type HBV polymerase. In various embodiments, for example, as described herein, for example, as provided in Table J, the terminal protein (TP) domain, the inactivated reverse transcriptase domain, and the inactivated RNase H can be directly fused via a linker or operably linked or connected.

[0075] In some embodiments, the HBV polymerase internal deletion mutant polypeptide comprises or consists of an amino acid sequence of any one of SEQ ID NO: 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 NO: 5 and 9. In some embodiments, the HBV polymerase internal deletion mutant polypeptide is derived from HBV genotype A and does not comprise a polypeptide of SEQ ID NO: 42 or 46, 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: 42 and 46.

[0076] In some embodiments, the HBV polymerase internal deletion mutant polypeptide is derived from HBV genotype B and comprises or consists of an amino acid sequence of any one of SEQ ID NO: 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 NO: 6 and 10. In some embodiments, the HBV polymerase internal deletion mutant polypeptide is derived from HBV genotype B and does not comprise a polypeptide of SEQ ID NO: 43 or 47, 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: 43 and 47.

[0077] In some embodiments, the HBV polymerase internal deletion mutant polypeptide is derived from HBV genotype C and comprises or consists of the 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 derived from HBV genotype C and does not comprise a polypeptide of SEQ ID NO: 44 or 48, 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: 44 and 48.

[0078] In some embodiments, the HBV polymerase internal deletion mutant polypeptide is derived from HBV genotype D and comprises or consists of the 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 derived from HBV genotype D and does not comprise a polypeptide of SEQ ID NO: 45 or 49, 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: 45 and 49.

[0079] In some embodiments, the HBV polymerase internal deletion mutant polypeptide does not comprise an amino acid sequence or a fragment thereof derived from another HBV protein. In some embodiments, the HBV polymerase internal deletion mutant polypeptide does not comprise an amino acid sequence or a fragment thereof derived from an HBV protein selected from the group consisting of precore, core, X, and envelope (e.g., small, medium, or large surface antigen (sAg)).

[0080] Tables C and E provide exemplary HBV polymerase internal deletion mutants for use in promoting, inducing, or eliciting an immunogenic response against, for example, polymerase antigens expressed by HBV. Tables D and F provide exemplary internal amino acid sequence segments that are deleted or removed from the HBV polymerase internal deletion mutants described herein and thus are not included in the HBV polymerase internal deletion mutants, corresponding to all or part of the HBV polymerase spacer region.

[0081] Core - polymerase fusion polypeptide In various embodiments, the truncated and internal deletion HBV polymerase polypeptide variants described herein are fused to an HBV core polypeptide. The core polypeptide can be positioned at either the N - terminus or the C - terminus of the HBV polymerase. As described herein, fusion polypeptides are further provided that include, in order from the N - terminus to the C - terminus, an HBV core polypeptide and a truncated or internal deletion HBV polymerase polypeptide variant. In some embodiments, the core - Pol fusion polypeptide includes an HBV polymerase deletion mutant polypeptide as described herein and includes, in order from the N - terminus to the C - terminus, an HBV core polypeptide and an internal deletion HBV polymerase polypeptide variant as described herein.

[0082] In some embodiments, the core - Pol fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 19 - 26, 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: 19 - 26.

[0083] In some embodiments, the HBV core-polymerase internal deletion mutant fusion protein does not contain an amino acid sequence or a fragment thereof derived from an HBV protein selected from the group consisting of X, precore, core, and envelope (e.g., small, medium, or large surface antigen (sAg)).

[0084] Table G provides exemplary core-polymerase fusion proteins for use in promoting, inducing, or eliciting an immunogenic response against, for example, the core and / or polymerase antigens expressed by HBV. [Table C-1] [Table C-2] [Table D] [Table E-1] [Table E-2] [Table F] [Table G-1] [Table G-2] [Table G-3] [Table G-4] [Table G-5] [Table G-6]

[0085] Core-sAg fusion protein There is further provided a fusion protein comprising 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 fragment thereof and the HBV small surface antigen (sAg) or fragment thereof are directly fused or abut. In some embodiments, the HBV core polypeptide or fragment thereof and the HBV small surface antigen (sAg) or fragment thereof are connected via a linker.

[0086] HBV core polypeptide, or an immunogenic fragment thereof In various embodiments, the HBV core polypeptide or an immunogenic fragment thereof of the core-sAg fusion protein can be independent of HBV genotypes A, B / C or D. Table H provides exemplary HBV core polypeptide amino acid sequences that can be used in the core-sAg fusion proteins described herein.

Table H

[0087] In some embodiments, the core polypeptide in the core-sAg1 fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 64-66, 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: 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, and the position numbers are based on SEQ ID NO: 65 or SEQ ID NO: 66.

[0088] HBV small surface antigen, or an immunogenic fragment thereof In various embodiments, the HBV sAg polypeptide or an immunogenic fragment thereof of the core-sAg fusion protein can independently be derived from HBV genotype A, B, C, or D. In Example 1 below, Table 1 provides exemplary HBV sAg polypeptide amino acid sequences that can be used in the core-sAg fusion proteins described herein.

[0089] In some embodiments, the sAg polypeptide in the core-sAg fusion polypeptide is the amino acid sequence of any one of SEQ ID NOs: 1-4, 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: 1-4, for example, a sequence containing or consisting of one or more of the serine (S) residue at the amino acid position corresponding to position 53, the isoleucine (I) residue at the amino acid position corresponding to position 68, the threonine (T) residue at the amino acid position corresponding to position 125, the proline (P) residue at the amino acid position corresponding to position 127, the phenylalanine (F) residue at the amino acid position corresponding to position 161, the tyrosine (Y) residue at the amino acid position corresponding to position 200, a certain serine (S) residue at the amino acid position corresponding to position 210, and the leucine (L) residue at the amino acid position corresponding to position 213.

[0090] With respect to the core-sAg fusion protein, the HBV core polypeptide and the HBV sAg polypeptide can be derived from the same or different HBV genotypes. In some embodiments, the core-sAg fusion protein comprises, in order from the N-terminus to the C-terminus, an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, · the core polypeptide is derived from HBV genotype A and the sAg polypeptide is derived from HBV genotype A, · the core polypeptide is derived from HBV genotype B or C and the sAg polypeptide is derived from HBV genotype B, · The core polypeptide is derived from HBV genotype B or C, and the sAg polypeptide is derived from HBV genotype C. · The core polypeptide is derived from HBV genotype D, and the sAg polypeptide is derived from HBV genotype D. · The core polypeptide is derived from HBV genotype A, and the sAg polypeptide is derived from HBV genotype B. · The core polypeptide is derived from HBV genotype A, and the sAg polypeptide is derived from HBV genotype C. · The core polypeptide is derived from HBV genotype A, and the sAg polypeptide is derived from HBV genotype D. · The core polypeptide is derived from HBV genotype B or C, and the sAg polypeptide is derived from HBV genotype A. · The core polypeptide is derived from HBV genotype B or C, and the sAg polypeptide is derived from HBV genotype D. · The core polypeptide is derived from HBV genotype D, and the sAg polypeptide is derived from HBV genotype A. · The core polypeptide is derived from HBV genotype D, and the sAg polypeptide is derived from HBV genotype B, or · The core polypeptide is derived from HBV genotype D, and the sAg polypeptide is derived from HBV genotype C.

[0091] In some embodiments, the core-sAg fusion protein comprises an HBV core polypeptide and an HBV small surface antigen (sAg) polypeptide, in order from the N-terminus to the C-terminus. · The core polypeptide is derived from HBV genotype B or C, and the sAg polypeptide is derived from HBV genotype C, or · The core polypeptide is derived from HBV genotype D, and the sAg polypeptide is derived from HBV genotype D.

[0092] In some embodiments, the core-sAg fusion protein, in the order from the N-terminus to the C-terminus, comprises (i) an HBV core polypeptide having the amino acid sequence of SEQ ID NO: 65, or a sequence comprising or consisting of 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: 65, and (ii) an HBV small surface antigen (sAg) polypeptide having the amino acid sequence of SEQ ID NO: 3, or a sequence comprising or consisting of 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: 3.

[0093] In some embodiments, the core-sAg fusion protein, in the order from the N-terminus to the C-terminus, comprises (i) an HBV core polypeptide having the amino acid sequence of SEQ ID NO: 66, or a sequence comprising or consisting of 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: 66, and (ii) an HBV small surface antigen (sAg) polypeptide having the amino acid sequence of SEQ ID NO: 4, or a sequence comprising or consisting of 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: 4.

[0094] In various embodiments, the core-sAg fusion proteins described herein include HBV small surface antigen isoforms, but do not include HBV medium surface antigen isoforms or HBV large surface antigen isoforms. Thus, in some embodiments, the core-sAg fusion proteins described herein do not include the HBV preS1 polypeptide. In some embodiments, the core-sAg fusion proteins described herein do not include the HBV preS2 polypeptide. In some embodiments, the core-sAg fusion proteins described herein do not include both the HBV preS1 polypeptide and the HBV preS2 polypeptide.

[0095] Exemplary HBV preS2 polypeptides not included in the core-sAg fusion proteins described herein are shown below.

Chemical Formula

[0096] Exemplary HBV preS2 consensus polypeptides from HBV genotype A that are not included in the core-sAg fusion proteins described herein are shown below. MQWNSTAFHQALQDPRVRGLYFPAGGSSSGTVNPAPNIASHISSISARTGDPVTN (SEQ ID NO: 80).

[0097] Exemplary HBV preS2 consensus polypeptides from HBV genotype B that are not included in the core-sAg fusion proteins described herein are shown below. MQWNSTTFHQTLQDPRVRALYFPAGGSSSGTVSPAQNTVSAISSILSKTGDPVPN (SEQ ID NO: 81).

[0098] Exemplary HBV preS2 consensus polypeptides from HBV genotype C that are not included in the core-sAg fusion proteins described herein are shown below. MQWNSTTFHQALLDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRTGDPAPN (SEQ ID NO: 82).

[0099] Exemplary HBV preS2 consensus polypeptides derived from HBV genotype D that are not included in the core-sAg fusion proteins described herein are shown below. MQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRIGDPALN (SEQ ID NO: 83).

[0100] In some embodiments, the core-sAg fusion proteins described herein do not include an HBV preS2 polypeptide having an amino acid sequence of any one of SEQ ID NOs: 79-83, 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: 79-83.

[0101] Exemplary HBV preS1-preS2 polypeptides that are not included in the core-sAg fusion proteins described herein are shown below.

Chemical formula

[0102] Exemplary HBV preS1-preS2 consensus polypeptides derived from HBV genotype A that are not included in the core-sAg fusion proteins described herein are shown below. MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVSTIPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFHQALQDPRVRGLYFPAGGSSSGTVNPAPNIASHISSISARTGDPVTN (SEQ ID NO: 85).

[0103] An exemplary HBV preS1-preS2 consensus polypeptide derived from HBV genotype B that is not included in the core-sAg fusion protein described herein is shown below. MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFKANSENPDWDLNPHKDNWPDANKVGVGAFGPGFTPPHGGLLGWSPQAQGLLTTVPAAPPPASTNRQSGRQPTPLSPPLRDTHPQAMQWNSTTFHQTLQDPRVRALYFPAGGSSSGTVSPAQNTVSAISSILSKTGDPVPN (SEQ ID NO: 86).

[0104] An exemplary HBV preS1-preS2 consensus polypeptide derived from HBV genotype C that is not included in the core-sAg fusion protein described herein is shown below. MGGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVGAGAFGPGFTPPHGGLLGWSPQAQGILTTVPAAPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTTFHQALLDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRTGDPAPN (SEQ ID NO: 87).

[0105] An exemplary HBV preS1-preS2 consensus polypeptide derived from HBV genotype D that is not included in the core-sAg fusion protein described herein is shown below. MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQSGRQPTPLSPPLRNTHPQAMQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRIGDPALN (SEQ ID NO: 88).

[0106] In some embodiments, the core-sAg fusion protein described herein does not include an HBV preS1-preS2 polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 84 to 88, 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: 84 to 88 or consisting of such a sequence.

[0107] Optional polypeptide linker Optionally, the HBV core polypeptide and the HBV sAg polypeptide in the core-sAg fusion protein can be in direct abutment or fused, or can be joined, connected, or linked by one or more peptide linkers. In various embodiments, the one or more peptide linkers are selected from, for example, one or more of a polyalanine linker, a polyglycine linker, a cleavable linker, a flexible linker, a rigid linker, and combinations thereof, within the linker or within the full-length fusion polypeptide. The HBV core polypeptide and the HBV Exemplary fusion protein linkers that can be used in this fusion polypeptide for connecting the 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, such as 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, such as GG, GGG, GGS, GSG, or GGGS (SEQ ID NO: 63) is used. In some embodiments, the cleavable linker is selected from 2A cleavable polypeptides. Exemplary 2A cleavable polypeptides that can be used to connect the HBV core polypeptide and the 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 polypeptides that can be used to link the HBV core polypeptide and the HBV sAg polypeptide include, in an optional combination with a furin recognition / cleavage sequence (e.g., RAKR (SEQ ID NO: 60), REKR (SEQ ID NO: 61), and RRKR (SEQ ID NO: 62)), 2A cleavage sequences (e.g., foot - and - mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus - 1 (P2A), and Thosea asigna virus (T2A)), but are not limited thereto. In certain embodiments, the furin recognition / cleavage sequence (e.g., RAKR (SEQ ID NO: 60), REKR (SEQ ID NO: 61), and RRKR (SEQ ID NO: 62)) is combined or fused with a 2A - cleavable polypeptide (e.g., foot - and - mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus - 1 (P2A), and Thosea asigna virus (T2A)) by a single linker. See, for example, Chng, et al., mAbs (2015)7:2,403 - 412. In some embodiments, the linker includes a porcine teschovirus - 1 (P2A) linker. In various embodiments, the 2A - cleavable linker includes 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 and including or consisting of 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 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 can be positioned at either the N-terminus or the 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). Table J provides exemplary linkers that can be used to link or connect the HBV core polypeptide and the HBV sAg polypeptide.

Table J

[0108] In some embodiments, the core-sAg fusion protein is 450 amino acids or less in length, such as 445, 440, 435, 430, 425, 420, 415, or 410 amino acids or less.

[0109] In some embodiments, the core-sAg fusion protein does not contain an amino acid sequence or a fragment thereof derived from an HBV protein selected from the group consisting of X, precore, preS1, preS2, and polymerase.

[0110] In some embodiments, the core-sAg fusion protein comprises or consists of any one of SEQ ID NOs: 38-41, for example, the amino acid sequence of SEQ ID NO: 41, or any one of SEQ ID NOs: 38-41, a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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, where the position numbers are based on SEQ ID NO: 41.

[0111] Table K provides exemplary core-sAg fusion proteins for use, for example, in promoting, inducing, or eliciting an immunogenic response against a core and / or small surface antigen expressed by HBV.

Table K

[0112] Signal or leader sequence In various embodiments, the immunogenic polypeptides described herein include, for example, a signal sequence or signal peptide to direct intracellular transport of the polypeptide to the proteasome or lysosomal compartment. In various embodiments, the immunogenic polypeptide includes a signal sequence at the N-terminus and / or C-terminus. In some embodiments, the immunogenic polypeptide includes 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 serum proteins, cytokines, chemokines, chaperone proteins, invariant proteins, and proteins that direct proteins to the lysosomal compartment. In some embodiments, the signal peptide or leader sequence is derived from a source protein selected from colony stimulating factor 2 (CSF2, GM-CSF), tissue-type plasminogen activator (PLAT, t-PA), C-C motif chemokine ligand 7 (CCL7, MCP-3), C-X-C 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 membrane protein 1 (LAMP-1), and lysosomal membrane protein 2 (LAMP-2). In various embodiments, the signal peptide or leader sequence is selected from the amino acid sequence of any one of SEQ ID NOs: 67-76, or a 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 includes N-terminal and C-terminal signal sequences from LAMP-1, for example, SEQ ID NOs: 77 and 78, respectively. Table L provides exemplary signal sequences that can be used with the present immunogenic polypeptides.

Table L-1

Table L-2

[0113] Furthermore, provided is a method for producing the immunogenic polypeptides described herein. In some implementations, the method includes constructing an immunogenic polypeptide using peptide synthesis. In some implementations, the method includes constructing a polynucleotide that encodes each of the polypeptides of a bivalent antigen and expressing the polypeptide from an expression vector using synthetic or recombinant DNA techniques. In some implementations, the method may further include inserting the polynucleotide into one or more vectors and expressing the encoded polypeptide intracellularly. This can be done using known recombinant techniques.

[0114] 3. Polynucleotide encoding an immunogenic polypeptide Provided herein are polynucleotides encoding the immunogenic polypeptides described herein, vectors containing such polynucleotides, and host cells (e.g., human cells, mammalian cells, yeast cells, plant cells, insect cells, bacterial cells such as E. coli) containing such polynucleotides or expression vectors. As used herein, provided are polynucleotides containing nucleotide sequences encoding any of the immunogenic polypeptides provided herein, as well as expression cassettes and vectors containing such nucleotide sequences, e.g., expression vectors for their efficient expression in host cells such as mammalian cells. In various embodiments, the polynucleotide includes DNA, cDNA, mRNA, self-amplifying RNA (SAM), self-replicating RNA, or self-amplifying replicon RNA (RepRNA). In some embodiments, the polynucleotide includes or is expressed from an alphavirus self-replicating or self-amplifying RNA replicon RNA (RepRNA). Self-replicating RNA and self-amplifying replicon RNA as modes of vaccine delivery are described, for example, in Tews, et al., Methods Mol Biol. (2017) 1499:15-35; Demoulins, 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.

[0115] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides, including RNA, cDNA, genomic DNA, and both sense and antisense strands of the above-mentioned synthetic and mixed polymers. As used herein, the term nucleic acid molecule may be interchangeable with the term polynucleotide. In some embodiments, a nucleotide refers to a ribonucleotide, a deoxynucleotide, or a modified form of either 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 cDNA or mRNA, may contain either or both natural and modified nucleotides joined together by natural and / or non-natural nucleotide linkages. A nucleic acid molecule may be chemically or biochemically modified, or contain non-natural or derivatized nucleotide bases, as will be readily understood by those of skill in the art. Such modifications include, for example, labeling, methylation, substitution of one or more of the natural nucleotides with an analog, internucleotide modifications such as, for example, uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylating agents, and modified linkages (e.g., α-anomeric nucleic acids, etc.). The above terms are also intended to include any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations. References to nucleic acid sequences include the complement thereof unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to include the complementary strand having its complementary sequence. The term also includes codon-biased polynucleotides for improved expression in a desired viral expression vector or host cell.

[0116] As used herein, "substitution" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively.

[0117] "Isolated" nucleic acid refers to a nucleic acid molecule separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. "Isolated nucleic acid encoding an immunogenic polypeptide" refers to one or more discrete molecules encoding such an immunogenic polypeptide, such nucleic acid molecule(s) in a single vector or multiple separate vectors, and such nucleic acid molecule(s) present at one or more locations within a host cell.

[0118] "Polynucleotide variant", as the term is used herein, is a polynucleotide that is specifically disclosed herein and that has one or more substitutions, deletions, additions, and / or insertions and is typically a different polynucleotide. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the polynucleotide sequences described herein, by assessing one or more biological activities of the encoded polypeptides described herein, and / or using any of several techniques well known in the art.

[0119] In some embodiments, the nucleic acid molecule is codon-biased to enhance expression in a desired host cell, such as a human cell, mammalian cell, yeast cell, plant cell, insect cell, or bacterial cell, such as an Escherichia coli (E. coli) cell. Accordingly, provided are polynucleotides encoding the immunogenic polypeptides described herein, wherein the polynucleotides are codon-biased, contain a replacement heterologous signal sequence, and / or have eliminated mRNA instability elements. Methods for generating codon-biased nucleic acids can be carried out, for example, by adapting the methods described in U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498. Preferred codon usage for expression of the 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.

[0120] In some embodiments, the polynucleotide encoding an immunogenic polypeptide as described herein 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 C.

[0121] Optionally, in certain embodiments, the 3' end of the polynucleotide encoding one or more of the immunogenic polypeptides described herein contains one or more tandem stop codons, such as two or more tandem TAG ("amber"), TAA ("ochre"), or TGA ("opal" or "amber") stop codons. The multiple tandem stop codons may be the same or different.

[0122] As described herein, there is further provided an expression cassette comprising a polynucleotide encoding an immunogenic polypeptide operably linked to one or more control sequences. In some embodiments, the polynucleotide is operably linked to a constitutive promoter and is under the control of the constitutive promoter. In some embodiments, the promoter is selected from the cytomegalovirus major immediate-early (CMV), CMV enhancer fused with the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), murine cytomegalovirus (murine CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK).

Table M-1

Table M-2

Table M-3

Table M-4

Table M-5

Table M-6

Table M-7

Table M-8

Table M-9

Table M-10

Table M-11

Table M-12

Table M-13

Table M-14

Table M-15

[0123] 4. Vector and Host Cell Further provided are vectors comprising one or more polynucleotides encoding one or more of the immunogenic polypeptides described herein, or expression cassettes comprising such polynucleotides. The vector can be of any type, for example, a recombinant vector such as an expression vector. Examples of vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), bacteriophages, or vectors derived from plant or animal (including human) viruses. The vector can include an origin of replication recognized by the proposed host cell, and in the case of an expression vector, can include a promoter and other regulatory regions recognized by the host cell. In additional embodiments, the vector comprises one or more polynucleotides encoding one or more of the immunogenic polypeptides of the present disclosure operably linked to a promoter and optional additional regulatory elements. Certain vectors can replicate autonomously in the host into which they are introduced (for example, a vector having a bacterial origin of replication can replicate within bacteria). Other vectors can integrate into the host genome upon introduction into the host and thereby be replicated with the host genome. Examples of vectors include, but are not limited to, vectors suitable for the recombinant production of the immunogenic polypeptides disclosed herein.

[0124] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of the introduced host cell. Some vectors are suitable for delivering the nucleic acid molecules or polynucleotides of the present application. Certain vectors are capable of inducing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as expression vectors.

[0125] The term "operably linked" refers to two or more nucleic acid sequence or polypeptide sequence elements that are usually physically linked and in a functional relationship with each other. By way of example, in the context of a nucleic acid sequence element, 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 should be understood to be "under the control of the promoter".

[0126] The selection of vectors depends on the recombinant procedures to be followed and the host to be used. The introduction of vectors into host cells can be achieved, inter alia, by calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofectamine transfection, electroporation, viral infection, or, as described herein, via administration to a subject. Vectors can replicate autonomously or can replicate together with the chromosomes into which they are integrated. In certain embodiments, the vectors contain one or more selectable markers. The choice of marker may depend on the host cell to be selected. These include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, zeocin, the herpes simplex virus-derived thymidine kinase gene (HSV-TK), and the mouse-derived dihydrofolate reductase gene (dhfr). Also within the scope of the present disclosure are vectors comprising one or more nucleic acid molecules encoding an immunogenic polypeptide as described herein, operably linked to one or more nucleic acid molecules encoding a protein or peptide that can be used to isolate the immunogenic polypeptide. These proteins or peptides include, but are not limited to, glutathione-S-transferase, maltose-binding protein, metal-binding polyhistidine, green fluorescent protein, luciferase, and β-galactosidase.

[0127] In other embodiments, the vector used is pcDNA™ 3.1+ (ThermoFisher, MA).

[0128] In some embodiments, the vector is a viral vector. Optionally, the viral vector can be a DNA virus or an RNA virus, including self-replicating RNA viruses. Examples of self-replicating RNA viruses include alphaviruses, as 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 derived 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 derived from a virus family selected from the group consisting of Adenoviridae (e.g., adenovirus, adeno-associated virus), Arenaviridae (e.g., lymphocytic choriomeningitis mammarenavirus, Cali mammarenavirus (also known as Pichinde mammarenavirus (PI_CV))), Poxviridae (e.g., vaccinia virus), Herpesviridae (e.g., cytomegalovirus, herpesvirus, e.g., HSV-1), Parvoviridae (e.g., parvovirus H1), Poxviridae (e.g., vaccinia virus, e.g., modified vaccinia Ankara (MVA)), Flaviviridae (e.g., yellow fever virus), Reoviridae (e.g., reovirus), Retroviridae (e.g., lentivirus), Picornaviridae (e.g., coxsackievirus, Seneca Valley virus, poliovirus), Paramyxoviridae (e.g., measles virus, Newcastle disease virus (NDV)), Rhabdoviridae (e.g., vesiculovirus including Maraba vesiculovirus and vesicular stomatitis virus (VSV)), Togaviridae (e.g., alphavirus, e.g., self-replicating alphavirus; Sindbis virus), Enteroviridae (e.g., echovirus).Exemplary modified vaccinia virus vectors used to express the immunogenic polypeptide are described, for example, in International Publication No. WO 2019 / 134049.

[0129] In some embodiments, the viral expression vector is an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) (NCBI:txid11623), Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus) (NCBI:txid2169993), Guanarito virus (GTOV) (NCBI:txid45219), Argentine mammarenavirus (also known as Junin virus (JUNV)) (NCBI:txid2169991), Lassa virus (LASV) (NCBI:txid11620), Lujo virus (LUJV) (NCBI:txid649188), Machupo virus (MACV) (NCBI:txid11628), Brazilian mammarenavirus (also known as Sabiá virus (SABV)) (NCBI:txid2169992), and Whitewater Arroyo virus (WWAV) (NCBI:txid46919). In some embodiments, the viral expression vector is an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Cali mammarenavirus (also known as Pichinde mammarenavirus 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 International Publication Nos. WO 2009 / 083210; WO 2015 / 183895; WO 2016 / 075250; WO 2017 / 198726; and U.S. Pat. Nos. 9,943,585 and 10,342,861, which are hereby incorporated by reference in their entirety for all purposes.

[0130] In some embodiments, the viral expression vector is an adenoviral vector derived from a human adenovirus or a simian adenovirus (e.g., chimpanzee adenovirus, gorilla adenovirus, or rhesus 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), ChAdV63, 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 adenovirus (e.g., RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65, RhAd66).Exemplary chimpanzee, gorilla, and rhesus adenovirus vectors that can be used as delivery and expression vehicles for the immunogenic polypeptides described herein are described, for example, in International Publication Nos. 2012 / 172277 (ChAdOx1), 2017 / 221031 (ChAdOx2), 2019 / 076880; 2019,076877; Andrabi et al., (2019) Cell Reports. It is described in 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.

[0131] In various embodiments, the viral expression vector may be unable to replicate (i.e., replication - defective or replication - deleted), for example, having a reduced or decreased ability to replicate compared to a wild - type viral vector (i.e., replication - attenuated), or may have the ability to replicate. In various embodiments, the viral expression vector is a replication - defective or replication - deleted arenavirus vector having a two - segmented genome, as described, for example, in International Publication Nos. 2009 / 083210 and 2017 / 076988. In various embodiments, the viral expression vector is a replication - attenuated arenavirus vector having a three - segmented genome, as described, for example, in International Publication Nos. 2016 / 075250, 2017 / 076988, and 2017 / 198726.

[0132] As described herein, there is further provided a host cell comprising one or more polynucleotides encoding one or more of an immunogenic polypeptide or one or more of the vectors expressing the immunogenic polypeptide. Any of a variety of host cells can be used. In one embodiment, the host cell is a prokaryotic cell, such as Escherichia coli (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 Chinese hamster ovary (CHO) system or a cell line derived from CHO (e.g., CHO-S, CHO DG44, ExpiCHO™, CHOZN® ZFN-modified GS- / - CHO cell line, CHO-K1, CHO-K1a), a COS cell, a BHK cell, an NSO cell, or a Bowes melanoma cell. Examples of human host cells include, inter alia, HeLa, 911, AT1080, A549, and HEK293 (e.g., HEK293E, HEK293F, HEK293H, HEK293T, Expi293™). In addition, the immunogenic polypeptide can be expressed in yeast cells such as Pichia (see, e.g., Powers et al., J Immunol Methods. 251:123-35 (2001)), Hanseula, or Saccharomyces.

[0133] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and progeny derived therefrom regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parental cell and may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected in the originally transformed cell are included herein.

[0134] Optionally, the host cell can be stably or transiently transfected with one or more polynucleotides encoding one or more immunogenic polypeptides, as described herein. Optionally, the host cell can be infected with one or more vectors expressing one or more immunogenic polypeptides, as described herein. In some embodiments, the host cell can be infected with and propagate one or more replication-defective vectors or replicable vectors expressing one or more immunogenic polypeptides, as described herein. Exemplary cells useful for infecting with and / or propagating viral vectors include, but are not limited to, BHK-21, A549, Vero, and HEK 293 (e.g., HEK293E, HEK293F, HEK293H, HEK293T, Expi293™) cells. In certain embodiments, host cells such as, for example, MDCK, Caco-2, or Calu-3 host cells express coxsackievirus and adenovirus receptor (CAR). In certain embodiments, the polynucleotide is integrated into the genome of the host cell.

[0135] 5. Pharmaceutical Composition / Immunogenic Composition Provided is a pharmaceutical composition or immunogenic composition comprising one or more of the immunogenic HBV polypeptides as described herein, or one or more polynucleotides encoding one or more of the immunogenic HBV polypeptides as described herein, or a viral expression vector comprising one or more of such polynucleotides, and a pharmaceutically acceptable diluent, carrier, or excipient. "Pharmaceutically acceptable excipients" include any adjuvant, carrier, excipient, lubricant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent approved by the U.S. Food and Drug Administration as acceptable for use in humans or livestock.

[0136] Generally, the pharmaceutical compositions described herein are immunogenic. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more (e.g., two or more, three or more) immunogenic HBV polypeptides, or one or more (e.g., two or more, three or more) polynucleotides encoding one or more (e.g., two or more, three or more) of the immunogenic HBV polypeptides, or one or more (e.g., two or more, three or more) viral expression vectors containing one or more (e.g., two or more, three or more) of the polynucleotides encoding one or more of the immunogenic HBV polypeptides.

[0137] A variety of pharmaceutically acceptable diluents, carriers, and excipients, as well as techniques for the preparation and use of pharmaceutical compositions, are known to those of skill in the art in light of the present disclosure. Exemplary pharmaceutical compositions, as well as 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 Edition (2012), Pharmaceutical Press; Brunton, Knollman, and Hilal-Dandan, "Goodman and Gilman’s The Pharmacological Basis of Therapeutics," 13th Edition (2017), McGraw-Hill Education / Medical; McNally and Hastedt (eds.), "Protein Formulation and Delivery," 2nd Edition (2007), CRC Press; Banga, "Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems," 3rd Edition (2015), CRC Press; Lars Hovgaard, Frokjaer, and van de Weert (eds.), "Pharmaceutical Formulation Development of Peptides and Proteins," 2nd Edition (2012), CRC Press; Carpenter and Manning (eds.), "Rational Design of Stable Protein Formulations: Theory and Practice" (2002), Springer ("Pharmaceutical Biotechnology" (Volume 13)); Meyer (ed.), "Therapeutic Protein Drug Products: Practical Approaches to Formulation in the Laboratory, Manufacturing, and the Clinic" (2012), Woodhead Publishing.

[0138] In certain embodiments, the polynucleotide or vector is formulated into lipid nanoparticles. For example, in some embodiments where an immunogenic HBV polypeptide is expressed from a self-replicating or self-amplifying RNA molecule, the self-replicating or self-amplifying RNA can be formulated into lipid nanoparticles (LNPs). As used herein, the term "lipid nanoparticles" refers to one or more spherical nanoparticles having an average diameter of about 10 to about 1000 nanometers and including a solid lipid core matrix that can solubilize lipophilic molecules. In certain embodiments, the lipid core is stabilized by a surfactant (e.g., an emulsifier) and can include one or more of triglycerides (e.g., tristearin), diglycerides (e.g., glyceryl behenate), monoglycerides (e.g., glycerol monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate) that include combinations thereof. Lipid nanoparticles are described, for example, in Petrilli et al., Curr Pharm Biotechnol. 15:847-55, 2014; and U.S. Patent 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, a self-replicating or self-amplifying RNA molecule encoding one or more of the immunogenic HBV polypeptides described herein is formulated or condensed into a polyethyleneimine (PEI)-polyplex delivery vehicle, such as described in Demoulins, et al., Nanomedicine. (2016) Apr;12(3):711-722 and Demoulins, et al., J Control Release. (2017) Nov 28;266:256-271, and these can be nanoparticles.

[0139] In embodiments where the immunogenic HBV polypeptide is expressed from a viral expression vector, the viral expression vector can be formulated as an isotonic pharmaceutically acceptable aqueous solution or suspension suitable for the desired route of administration, e.g., intravenous, intramuscular, subcutaneous, or intradermal administration. In some embodiments, the viral expression vector can be formulated for mucosal use, e.g., for oral, nasal, vaginal, 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; International Publication No. 2017 / 013169 (formulation of adenoviral vectors in aqueous mixtures or lyophilized compositions in the presence of amorphous sugars and low salt concentrations); and Kumru, et al., J Pharm Sci. (2018) Nov; 107(11):2764 - 2774 (aqueous formulation buffered with Tris and containing proline, lactose, and mannitol as stabilizing additives). Formulations of arenaviral vectors are described, for example, in International Publication Nos. 2009 / 083210; 2016075250 and 2017 / 198726. In certain embodiments, the viral expression vector is delivered via microneedle-mediated delivery, as described, for example, in Zaric, et al., Expert Opin Drug Deliv. (2017) Oct; 14(10):1177 - 1187.

[0140] In some embodiments, each carrier, diluent, or excipient is "acceptable" in the sense that it is compatible with the other components of the pharmaceutical composition and not harmful to the subject. In many cases, a pharmaceutically acceptable carrier is an aqueous pH-buffered solution. Some examples of materials that can serve as pharmaceutically acceptable carriers, diluents, or excipients include the following: water; buffers such as buffers having a pKa in the range of about 6.0 to about 8.0, such as phosphates, carbonates, bicarbonates, citrates, maleates, glycine-glycine, HEPES, HEPPSO, HEPPS, imidazole, bicine, tricine, tris, and bis-tris, for example, physiologically acceptable buffers; sugars such as lactose, trehalose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Hank's solution, Ringer's solution, ethyl alcohol; phosphate buffer; amino acids (e.g., charged amino acids including, but not limited to, aspartic acid, asparagine, glutamic acid, glutamine, histidine, arginine, lysine); and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents and fragrances, preservatives and antioxidants can also be present in the composition.

[0141] In one particular formulation, the arenavirus vector described herein (e.g., LCMV or Pichinde mammarenavirus vector (PICV)) is formulated in an isotonic aqueous solution comprising a biologically compatible buffer (e.g., HEPES and NaCl) having a pKa in the range of about 6.0 to about 8.0 at neutral or near-neutral pH, and a nonionic surfactant (e.g., PLURONIC® F68 (also known as poloxamer 188)). In one particular formulation, the arenavirus vector described herein (e.g., LCMV or Pichinde mammarenavirus vector) is formulated in an isotonic aqueous solution comprising HEPES buffer, NaCl, and PLURONIC® F68 (also known as poloxamer 188) at pH 7.4. Schleiss, et al. (Clin Vaccine Immunol. 2017 Jan 5;24(1):e00300-16) describes an LCMV that formulates an LCMV vector in a diluent of 25 mM HEPES, 150 mM NaCl, 0.01% PLURONIC® F68 (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.

[0142] Formulation and delivery methods of pharmaceutical compositions generally are adapted according to the site and disease being treated. Exemplary formulations include those suitable for parenteral administration, such as intravenous, intraarterial, intramuscular, or subcutaneous administration; formulations encapsulated in micelles, liposomes, or drug release capsules (active pharmaceutical agent incorporated within a biocompatible coating designed for sustained release); ingestible formulations; formulations for topical use such as creams, ointments, and gels; and other formulations such as inhalants, aerosols, and sprays, but are not limited thereto. In some embodiments, the pharmaceutical composition is formulated for parenteral administration, such as intravenous administration, subcutaneous administration, or oral administration. In some embodiments, the pharmaceutical composition is formulated for mucosal administration, such as buccal, intranasal, rectal, or vaginal administration.

[0143] 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 a pH of 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 osmolality in the range of 240 to 260 or 250 to 330 mOsmol / L. In certain embodiments, the pharmaceutical composition is isotonic or substantially isotonic.

[0144] In some embodiments, the pharmaceutical composition is 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 cryogenic fluid.

[0145] In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents, such as a second therapeutic agent, or second and third therapeutic agents, for use in combination therapy as described herein.

[0146] 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, polynucleotides encoding such immunogenic HBV polypeptides, and vectors expressing such immunogenic HBV polypeptides include, but are not limited to, cytokines, chemokines, immunostimulatory molecules, Toll-like receptor agonists, or inhibitors of immunosuppressive pathways, as described herein and 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, polynucleotides encoding such immunogenic HBV polypeptides, and vectors expressing such immunogenic HBV polypeptides include mineral salts (e.g., aluminum salts (e.g., alum), calcium phosphate, incomplete Freund's adjuvant), lipid particles (e.g., MF59, cochleate, virus-like particles), microparticles (e.g., virosomes, poly(lactic acid) (PLA), poly[lactide-co-glycolide] (PLG)), immunopotentiators (e.g., dsRNA: poly(I:C), poly-IC:LC, monophosphoryl lipid A (MPL), LPS, flagellin, imidazoquinoline: imiquimod (R837), resiquimod (848), CpG oligodeoxynucleotides (ODN), muramyl dipeptide (MDP), saponin (QS-21)), and mucosal adjuvants (e.g., cholera toxin (CT), heat-labile enterotoxin (LTK3 and LTR72), chitosan), but are not limited to these. 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 summarized in Apostolico, et al., J Immunol Res. (2016) 2016:1459394.

[0147] 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.

[0148] 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, wherein the immunogenic polypeptides comprise (a) an amino acid sequence of any one of SEQ ID NOs: 5-14, or a sequence comprising or consisting of 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: 5-14, which is an HBV polymerase polypeptide variant, and (b) an amino acid sequence of any one of SEQ ID NOs: 38-41, or a sequence comprising or consisting of 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: 38-41, which is an HBV core-sAg fusion protein.

[0149] 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, and the immunogenic polypeptides comprise (a) an amino acid sequence of any one of SEQ ID NOs: 13-14, or a sequence comprising or consisting of 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: 13-14, which is an HBV polymerase polypeptide variant, and (b) an amino acid sequence of any one of SEQ ID NOs: 38-41, or a sequence comprising or consisting of 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: 38-41, which is an HBV core-sAg fusion protein.

[0150] 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, and the immunogenic polypeptides comprise (a) the amino acid sequence of SEQ ID NO: 13, or a sequence comprising or consisting of 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, which is an HBV polymerase polypeptide variant, and (b) the amino acid sequence of SEQ ID NO: 41, or a sequence comprising or consisting of 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: 41, which is an HBV core-sAg fusion protein.

[0151] Regarding 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, and the position numbers are based on 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, and the position numbers are based on 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, and the position numbers are based on 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, and the position numbers are based on SEQ ID NO: 41.

[0152] 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 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, such as the nucleic acid sequences of SEQ ID NOs: 29, 89, 90, and 92, or any one of SEQ ID NOs: 27-32 and 89-94, such as the nucleic acid sequences of SEQ ID NOs: 29, 89, 90, and 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of 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: 33-37 or any one of SEQ ID NOs: 33-37.

[0153] 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 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 nucleic acid sequence of SEQ ID NO: 29 or 90, or any one of SEQ ID NOs: 29, 89, 90, or 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of 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 the nucleic acid sequence of SEQ ID NO: 37 or any one of SEQ ID NO: 37.

[0154] 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 the nucleic acid sequence of SEQ ID NO: 29, 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: 29, or consisting of such a sequence; and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 37, 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 NO: 37, or consisting of such a sequence.

[0155] 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 the nucleic acid sequence of SEQ ID NO: 90, 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: 90, or consisting of such a sequence; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 37, 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 NO: 37, or consisting of such a sequence.

[0156] Optionally or as desired, the HBV polymerase polypeptide variant and the HBV core-sAg fusion protein can be provided in the immunogenic composition in a ratio in the range of 1:10 to 10:1, such as, for example, in a ratio in the range of 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 virus particles (vp).

[0157] In various embodiments, the one or more polynucleotides are DNA, cDNA, mRNA, or self-replicating RNA.

[0158] 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. Optionally or as desired, the first viral expression vector and the second viral expression vector can be provided in a ratio in the range of 1:10 to 10:1, such as, for example, in a ratio in the range of 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.

[0159] In some embodiments, the immunogenic composition comprises each of the first viral expression vector and the second viral expression vector at about 10 3 ~ about 10 12 virus focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or virus particles (vp) per milliliter, such as, for example, about 10 4 ~ about 10 7 virus FFU or PFU, such as, for example, about 10 3 ~ about 10 4 , 105 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 is included in the range of viral FFU or PFU or IU or vp.

[0160] In various embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition are independent of the taxonomic family selected from the 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, both the first viral expression vector and the second viral expression vector in the immunogenic composition are from the Adenoviridae, Arenaviridae, or Poxviridae (e.g., vaccinia virus, e.g., modified vaccinia Ankara (MVA)).

[0161] In some embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition are derived from the Arenaviridae family. In some embodiments, the first viral expression vector and the second viral expression vector are derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV), Cali mammarenavirus (also known as Pichinde mammarenavirus 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 first viral expression vector and the second viral expression vector are derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)).

[0162] In various embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition are replication-deficient or replication-deleted. In some embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition are replication-attenuated.

[0163] 6. Method of treatment Furthermore, a method for inducing an immune response against hepatitis B virus (HBV) in a subject in need thereof is provided. Also provided is a method for treating or preventing hepatitis B virus (HBV) in a subject in need thereof. Also provided is a method for inhibiting HBV replication in an infected individual. Furthermore, a method for reducing the viral load associated with HBV infection is provided. In various embodiments, the method comprises administering to the subject an effective amount of the immunogenic composition as described herein. In various embodiments, the "subject" or "individual" is a human, woodchuck, Pekin duck, mouse, or non-human primate (e.g., chimpanzee).

[0164] As used herein, "treatment" or "treating" or "treatment" refers to an approach for obtaining a beneficial or desired result. For the purposes of the present disclosure, beneficial or desired results include, but are not limited to, alleviation of symptoms and / or reduction in the degree of symptoms, delay in progression, and / or prevention of worsening of symptoms associated with a disease or condition. "Treatment" or "treatment" may include one or more of the following: a) inhibiting a disease or condition (e.g., reducing one or more symptoms resulting from the disease or condition and / or reducing the degree of the disease or condition), b) delaying or arresting the onset of one or more symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, delaying the worsening or progression of the disease or condition), and c) alleviating the disease or condition, e.g., causing regression of clinical symptoms, improving the disease state, delaying the progression of the disease, enhancing the quality of life, and / or prolonging the survival period.

[0165] As used herein, "delay" means deferring, interfering with, delaying, preventing, stabilizing, and / or postponing the onset of a disease or condition. This delay can be of various durations depending on the disease being treated and / or the medical history of the individual. As will be apparent to those skilled in the art, a sufficient or significant delay can, in effect, encompass prevention in that the individual does not develop the disease or condition.

[0166] As used herein, "prevent", "prevention", or "preventing" refers to a regimen that protects against the onset of a disease or disorder such that the clinical symptoms of the disease do not develop. Thus, "prevention" relates to performing (e.g., administering a therapeutic substance to a subject) a treatment on a subject before signs of the disease are detectable in the subject (e.g., administering a therapeutic substance to a subject in the absence of a detectable infectious pathogen (e.g., a virus) in the subject). The subject may be an individual at risk of developing a disease or disorder, such as an individual having one or more risk factors known to be associated with the onset or development of the disease or disorder. Thus, in certain embodiments, the term "preventing HIV infection" refers to administering an anti-HIV therapeutic substance to a subject who does not have detectable HIV infection. It is understood that the subject of anti-HBV prophylactic therapy may be an individual at risk of contracting the HBV virus. It is also understood that prevention does not require a 100% success rate. In some cases, prevention may be understood as reducing the risk of infection, but not as a complete elimination of the occurrence of infection.

[0167] As used herein, "therapeutically effective amount" or "effective amount" refers to an amount of an immunogenic composition effective to induce a desired biological or medical response, including an amount sufficient to effect such treatment of a disease when administered to a subject for treating the disease. The effective amount will vary depending on the immunogenic composition, the disease and its severity, and the age, weight, etc. of the subject being treated. The effective amount can 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. The effective amount can be considered in the context of administering one or more therapeutic agents, and a desired or beneficial result may be achieved by administering in combination with one or more other agents, or if achieved, a single agent may be considered to be administered in an effective amount. The appropriate dose of any co-administered compound may be reduced due to the combined action (e.g., additive or synergistic effect) of the compounds.

[0168] In various embodiments, the immunogenic composition to be 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 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.

[0169] In various embodiments, the immunogenic composition to be administered comprises a mixture of 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 variant comprising an amino acid sequence of any one of SEQ ID NOs: 5-14, 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: 5-14, or consisting of such a sequence, and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 38-41, 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 any one of SEQ ID NOs: 38-41, or consisting of such a sequence. Such an immunogenic composition can be administered in a priming composition and / or a boosting composition.

[0170] In various embodiments, the immunogenic composition to be 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 variant comprising any one of the amino acid sequences of SEQ ID NOs: 13-14, 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: 13-14, or consisting of such a sequence; and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising any one of the amino acid sequences of SEQ ID NOs: 38-41, 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 any one of SEQ ID NOs: 38-41, or consisting of such a sequence. Such immunogenic compositions can be administered in a priming composition and / or a boosting composition.

[0171] Regarding 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, and the position numbers are based on 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, and the position numbers are based on 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, and the position numbers are based on 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, and the position numbers are based on SEQ ID NO: 41.

[0172] In various embodiments, the immunogenic composition to be 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 variant comprising the 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, or consisting of such a sequence, and (b) the second viral expression vector comprises a polynucleotide encoding a core-sAg fusion protein comprising the amino acid sequence 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: 41, or consisting of such a sequence. Such immunogenic compositions can be administered in a priming composition and / or a boosting composition.

[0173] In various embodiments, the immunogenic composition to be 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 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, such as the nucleotide sequences of SEQ ID NOs: 29, 89, 90, and 92, or any one of SEQ ID NOs: 27-32 and 89-94, such as the nucleotide sequences of SEQ ID NOs: 29, 89, 90, and 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of 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: 33-37 or any one of SEQ ID NOs: 33-37. Such immunogenic compositions can be administered in a priming composition and / or a boosting composition.

[0174] In various embodiments, the immunogenic composition to be 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 nucleic acid sequence of SEQ ID NO: 29, 89, 90, or 92, or a polynucleotide comprising or consisting of 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: 29, 89, 90, or 92, and (b) the second viral expression vector comprises a nucleic acid sequence of SEQ ID NO: 37, or a polynucleotide comprising or consisting of 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: 37. Such immunogenic compositions can be administered in a priming composition and / or a boosting composition.

[0175] In various embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition to be administered are independent of the taxonomic family selected from the family Adenoviridae, Arenaviridae, Herpesviridae (e.g., Cytomegalovirus), Poxviridae (e.g., Vaccinia virus such as 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, both the first viral expression vector and the second viral expression vector in the immunogenic composition to be administered are from the family Adenoviridae, Arenaviridae, or Poxviridae (e.g., Vaccinia virus, e.g., Modified Vaccinia Ankara (MVA)).

[0176] In some embodiments, the first viral expression vector and the second viral expression vector are derived from the family Arenaviridae. In some embodiments, the first viral expression vector and the second viral expression vector in the administered immunogenic composition are derived from an arenavirus vector selected from Lymphocytic choriomeningitis mammarenavirus (LCMV), Cali mammarenavirus (also known as Pichinde mammarenavirus 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 first viral expression vector and the second viral expression vector in the administered immunogenic composition are derived from an arenavirus vector selected from Lymphocytic choriomeningitis mammarenavirus (LCMV) or Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)).

[0177] In various embodiments, the first viral expression vector and the second viral expression vector in the administered immunogenic composition are replication-deficient or replication-deleted. In some embodiments, the first viral expression vector and the second viral expression vector in the administered immunogenic composition are replication-attenuated.

[0178] In various embodiments, the immunogenic composition to be 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 the nucleotide sequence of SEQ ID NO: 29, 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: 29, or consisting of such a sequence; and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 37, 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: 37, or consisting of such a sequence. Such immunogenic compositions can be administered in a priming composition and / or a boosting composition.

[0179] In various embodiments, the immunogenic composition to be 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 the nucleotide sequence of SEQ ID NO: 90, 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: 90, or consisting of such a sequence; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 37, 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: 37, or consisting of such a sequence. Such immunogenic compositions can be administered in a priming composition and / or a boosting composition.

[0180] In various embodiments, the subject is infected with HBV, suspected of being infected with HBV, or at risk of being infected with HBV. As used herein, an "at-risk individual" refers to an individual at risk of developing the condition to be treated. An "at-risk" individual may or may not have a detectable disease or condition and may or may not exhibit a detectable disease prior to treatment with the methods described herein. "At risk" means that the individual has one or more so-called risk factors, which are measurable parameters that correlate with the development of a disease or condition and are known in the art. An individual having one or more of these risk factors has a higher probability of developing a disease or condition than an individual not having these risk factors. In various embodiments, the subject is chronically infected with HBV, for example, infected with HBV for more than six months. Typically, the individual has a chronic hepatitis B infection, but treating people who are acutely infected with HBV is within the scope of the present disclosure. Thus, in some embodiments, the subject is acutely infected with HBV. In some embodiments, the subject is coinfected with hepatitis D virus (HDV).

[0181] In various embodiments, the subject may be asymptomatic. In some embodiments, the subject experiences or presents with symptoms associated with HBV infection. Symptoms of HBV can include, for example, jaundice, a visible network of dilated blood vessels in the skin, dark (e.g., orange or brown) urine, light-colored stools, fever, persistent fatigue, malaise, abdominal pain, ascites, loss of appetite, nausea, and vomiting. Chronic infection with HBV can result in one or more symptoms including, for example, liver failure, liver cancer, liver fibrosis, and cirrhosis. One or more administrations of an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein can prevent, delay, alleviate, reduce, inhibit, reverse, or eliminate one or more symptoms associated with or caused by HBV infection.

[0182] In some embodiments, the immunogenic composition is administered via a route selected from intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., buccal, intranasal, rectal, intravaginal).

[0183] In some embodiments, the dosage of the immunogenic composition is about 10 3 to about 10 12 viral focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or viral particles (vp) per milliliter of each of the first viral expression vector and the second viral expression vector, e.g., about 10 4 to about 10 7 viral FFU or PFU, e.g., about 10 3 to about 10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、10 11 、or 10 12 viral FFU or PFU or IU or vp. In some embodiments, the method involves administering about 10 6 to about 10 8 viral FFU or PFU or IU or vp intravenously or intramuscularly per administration at biweekly (Q2W) or monthly (Q4W) intervals.

[0184] In various embodiments, the method includes a prime-boost regimen. In some embodiments, the prime-boost regimen involves administering a priming composition at an initial time point and one or more boosting compositions at one or more subsequent time points. Optionally, the method may involve repeating the prime-boost regimen one or more times. In various embodiments, the administration of the priming composition and the one or more boosting compositions are spaced at intervals of at least one week, up to at least two weeks, three weeks, one month, two months, three months, four months, five months, or six months. Optionally, the dosage or frequency of administration of the immunogenic composition may be adjusted during the course of treatment based on the judgment of the administering physician. Optionally, the subject may be treated by multiple administrations over a period of at least about two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, twenty-four months, or more, or until sAg can no longer be detected in the subject's serum or plasma.

[0185] In some embodiments, after 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 are administered one or more times, optionally together with one or more additional therapeutic agents as described herein, the subject is free of HBV symptoms for at least 6 months, at least 1 year, at least 2 years, at least 3 years, or more without antiviral treatment. In some embodiments, after 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 are administered one or more times, optionally together with one or more additional therapeutic agents as described herein, sAg becomes undetectable in the serum or plasma of the subject for at least 6 months, e.g., at least 1 year, at least 2 years, at least 3 years, or more without antiviral treatment.

[0186] Optionally or as desired, the priming composition and the boosting composition can comprise the same immunogenic composition or different immunogenic compositions. In various embodiments, the priming composition and the boosting composition comprise the same one or more polypeptides and the same viral expression vector (e.g., a viral expression vector). In some embodiments, the priming composition and the boosting composition comprise different polypeptides and / or different viral expression vectors (e.g., a viral expression vector). For example, in some embodiments, the priming composition and the boosting composition comprise the same one or more polypeptides and different expression vectors (e.g., viral vectors from different viral species within a classification family, viral vectors from different classification families, viral vectors having different replication capabilities). In some embodiments, the priming composition and the boosting composition comprise different immunogenic polypeptides and the same expression vector (e.g., a viral expression vector).

[0187] 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 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, 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, 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, d) priming with a priming composition comprising one or more replication-deficient viral expression vectors and boosting with a boosting composition comprising one or more replication-deficient viral expression vectors, wherein the one or more replication-deficient viral expression vectors in the priming composition and the one or more replication-deficient viral expression vectors in the boosting composition are from the same, related, or unrelated taxonomic families, e) Priming with a priming composition comprising one or more replication-attenuated virus expression vectors and boosting with a boosting composition comprising one or more replication-attenuated virus expression vectors, wherein the one or more replication-attenuated virus expression vectors in the priming composition and the one or more replication-attenuated virus expression vectors in the boosting composition are from the same, related, or unrelated taxonomic families, f) Priming with a priming composition comprising one or more replication-deficient virus expression vectors and boosting with a boosting composition comprising one or more replication-attenuated virus expression vectors, g) Priming with a priming composition comprising one or more replication-attenuated virus expression vectors and boosting with a boosting composition comprising one or more replication-deficient virus expression vectors, h) Priming with a priming composition comprising one or more lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors and boosting with a boosting composition comprising one or more Pichinde mammarenavirus (PICV) virus expression vectors, i) Priming with a priming composition comprising one or more Pichinde mammarenavirus (PICV) virus expression vectors and boosting with a boosting composition comprising one or more lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors, j) Priming with a priming composition comprising one or more replication-deficient Pichinde mammarenavirus (PICV) virus expression vectors and boosting with a boosting composition comprising one or more replication-deficient lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors, k) Priming with a priming composition comprising one or more replication-deficient lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors and boosting with a boosting composition comprising one or more replication-deficient Pichinde mammarenavirus (PICV) virus expression vectors, l) priming with a priming composition comprising one or more arenavirus virus expression vectors and boosting with a boosting composition comprising one or more adenovirus virus expression vectors, m) priming with a priming composition comprising one or more adenovirus virus expression vectors and boosting with a boosting composition comprising one or more arenavirus virus expression vectors, n) priming with a priming composition comprising one or more poxvirus virus expression vectors and boosting with a boosting composition comprising one or more arenavirus virus expression vectors, o) priming with a priming composition comprising one or more arenavirus virus expression vectors and boosting with a boosting composition comprising one or more poxvirus virus expression vectors, p) priming with a priming composition comprising one or more poxvirus virus expression vectors and boosting with a boosting composition comprising one or more adenovirus virus expression vectors, or q) priming with a priming composition comprising one or more adenovirus virus expression vectors and boosting with a boosting composition comprising one or more poxvirus virus expression vectors,

[0188] 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 is a) priming with a priming composition comprising one or more lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors and boosting with a boosting composition comprising one or more pichinde mammarenavirus (PICV) virus expression vectors, b) priming with a priming composition comprising one or more Pichinde mammarenavirus (PICV) virus expression vectors, and boosting with a boosting composition comprising one or more Lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors, c) priming with a priming composition comprising one or more replication-deficient Pichinde mammarenavirus (PICV) virus expression vectors, and boosting with a boosting composition comprising one or more replication-deficient Lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors, or d) priming with a priming composition comprising one or more replication-deficient Lymphocytic choriomeningitis mammarenavirus (LCMV) virus expression vectors, and boosting with a boosting composition comprising one or more replication-deficient Pichinde mammarenavirus (PICV) virus expression vectors,

[0189] In various embodiments, the priming composition and the boosting composition comprise an immunogenic composition as described herein.

[0190] In some embodiments, the subject has not received antiviral therapy or the antiviral therapy is discontinued prior to administration of one or more of the immunogenic compositions. In some embodiments, the antiviral therapy is discontinued after one or more administrations of the composition.

[0191] 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 induces the production of antibodies that bind to one or more HBV polypeptides in the subject.

[0192] 7. Combination Therapy In certain embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein are combined with or co-administered with one, two, three, four, or more additional therapeutic agents. In certain embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein are combined with or co-administered with two additional therapeutic agents. In certain embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector, an LNP, and an immunogenic composition comprising a polypeptide or polynucleotide as described herein are combined with or co-administered with three additional therapeutic agents. In certain embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein are combined with or co-administered with four additional therapeutic agents. The one, two, three, four, or more additional therapeutic agents may be different therapeutic agents selected from the same class of therapeutic agents and / or may be selected from different classes of therapeutic agents.

[0193] As used herein, "co - administration" refers to the administration of a unit dose of an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and a unit dose of an immunogenic composition as described herein, either before or after the administration of a unit dose of one or more additional therapeutic agents. For example, it is the administration of an immunogenic composition disclosed herein within seconds, minutes, or hours of the administration of one or more additional therapeutic agents. For example, in some embodiments, a unit dose of the immunogenic composition disclosed herein is administered first, followed by the 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 the administration of a unit dose of the immunogenic composition of the present disclosure within seconds or minutes. In some embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein are administered first, followed by the administration of a unit dose of one or more additional therapeutic agents several hours (e.g., 1 - 12 hours) later. In other embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed by the administration of a unit dose of an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein several hours (e.g., 1 - 12 hours) later.

[0194] Co - administration of an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as disclosed herein, and one or more additional therapeutic agents generally refers to the simultaneous or sequential administration of the 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.

[0195] In various embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein are combined with or co-administered with one or more additional therapeutic agents as described herein, and the components of the composition are administered as simultaneous or sequential regimens. When administered sequentially, the combination may be administered in more than one administration.

[0196] In various embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector containing such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein are combined with or co-administered with one, two, three, four, or more additional therapeutic agents selected from HBV formulations, 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-directed RNA interference (ddRNAi)), HBsAg secretion or assembly inhibitors, HBV virus 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, nucleoprotein modulators, retinoic acid-inducible gene 1 stimulators, NOD2 stimulators, phosphatidylinositol 3-kinase (PI3K) inhibitors, indoleamine-pyrrole-2,3-dioxygenase (IDO) pathway inhibitors, ZCCHC14 inhibitors, inducers of tertiary lymphoid aggregates, 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 editors, cell therapy, TCR-T cell therapy, and other HBV drugs.

[0197] In certain embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector, LNP, and an immunogenic composition as described herein can be combined with or co-administered with a chemotherapeutic agent, an immunomodulatory agent, an immunotherapeutic agent, an antibody drug, a therapeutic vaccine, a bispecific antibody, and “antibody-like” therapeutic proteins (DARPins®, anti-pMHC TCR-like antibodies, DARTs®, Duobodies®, Bites®, XmAbs®, TandAbs®, Fab derivatives, etc.), antibody-drug conjugates (ADCs), gene modifying agents or 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, TALEN), cell therapies (e.g., T cells, NK cells, macrophages having a chimeric antigen receptor (CAR), and TCR-T (genetically engineered T cell receptor)), one or more of which or any combination thereof.

[0198] In certain embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein are combined with one, two, three, four, or more therapeutic agents, such as 3-dioxygenase (IDO) inhibitors, apolipoprotein A1 modulators, arginase inhibitors, B and T lymphocyte attenuation 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 modulators, epigenetic modulators, farnesoid X receptor (FXR) agonists, HBV DNA polymerase inhibitors, HBV replication inhibitors, HBVRNAse inhibitor, HBV virus entry inhibitor, HBx inhibitor, hepatitis B large envelope protein modulator, hepatitis B large envelope protein stimulator, hepatitis B structural protein modulator, hepatitis B surface antigen (HBsAg) inhibitor, hepatitis B surface antigen (HBsAg) secretion or assembly inhibitor, hepatitis B virus e antigen inhibitor, hepatitis B virus replication inhibitor, hepatitis virus structural protein inhibitor, HIV-1 reverse transcriptase inhibitor, hyaluronidase inhibitor, inhibitor of inhibitor of apoptosis protein family protein (IAP), IL-2 agonist, IL-7 agonist, immunomodulator, indoleamine-2 inhibitor, ribonucleotide reductase inhibitor, interleukin-2 ligand, ipi4 inhibitor, lysine demethylase inhibitor, histone demethylase inhibitor, KDM1 inhibitor, KDM5 inhibitor, killer cell lectin-like receptor subfamily G member 1 inhibitor, lymphocyte activation gene 3 inhibitor, lymphotoxin β receptor activator, modulator of Axl, modulator of B7-H3, modulator of B7-H4, modulator of CD160, modulator of CD161, modulator of CD27, modulator of CD47, modulator of CD70, modulator of GITR, modulator of IHEVEM, modulator of ICOS, modulator of Mer, modulator of NKG2A, modulator of NKG2D, modulator of OX40, modulator of SIRPalpha, modulator of TIGIT, modulator of Tim-4, modulator of Tyro, Na+-taurocholate cotransporting polypeptide (NTCP) inhibitor, natural killer cell receptor 2B4 inhibitor, NOD2 gene stimulator, nucleoprotein inhibitor, nucleoprotein modulator, OX-40 receptor agonist, PD-1 inhibitor, PD-L1 inhibitor, peptidylprolyl isomerase inhibitor, phosphatidylinositol-3 kinase (PI3K) inhibitor, retinoic acid-inducible gene 1 stimulator, reverse transcriptase inhibitor, ribonuclease inhibitor, RNADNA polymerase inhibitor, SLC10A1 gene inhibitor, SMAC mimetic, Src tyrosine kinase inhibitor, stimulator of interferon gene (STING) agonist, stimulator of NOD1, inhibitor of T cell surface glycoprotein CD28, modulator of T cell surface glycoprotein CD8, thymosin agonist, thymosin α1 ligand, Tim-3 inhibitor, TLR-3 agonist, TLR-7 agonist, TLR-9 agonist, TLR9 agonist or gene stimulator, Toll-like receptor (TLR) modulator, viral ribonucleotide reductase inhibitor, and combinations thereof are combined with or co-administered with.

[0199] HBV inhibitory antiviral drug In various embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector, LNP, and an immunogenic composition as described herein are combined with or co-administered with one or more antiviral drugs. In some embodiments, the one or more antiviral drugs 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 (TRUVADA®), tenofovir alafenamide (TAF or VEMLIDY®), and ledipasvir and sofosbuvir (HARVONI®).

[0200] Other HBV drugs Examples of other drugs for HBV treatment that can be combined or co-administered include α-hydroxy tropolone, amdoxovir, anthraquinonol, β-hydroxy cytosine nucleoside, ARB-199, CCC-0975, ccc-R08, elvucitabine, ezetimibe, cyclosporine A, gentiopicrin (gentiopicroside), HH-003, heparotide, JNJ-56136379, nitazoxanide, velipanib, NJK14047, NOV-205 (molixan, BAM-205), oligonucleotide, miboleret, feron, GST-HG-131, levamisole, Ka Shu Ning, alophron, WS-007, Y-101 (Ti Fen Tai), rSIFN-co, PEG-IIFNm, KW-3, BP-Inter-014, oleanolic acid, HepB-nRNA, cTP-5 (rTP-5), HSK-II-2, HEISCO-106-1, HEISCO-106, Hepbarna, IBPB-006IA, Hepuyinfen, DasKloster0014-01, ISA-204, Jiangantai (Ganxikang), MIV-210, OB-AI-004, PF-06, Picroside, DasKloster-0039, Hepranta, 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, and the compounds disclosed in U.S. Patent Application Publication No. 20150210682 (Roche), No. 2016 / 0122344 (Roche), International Publication No. 2015173164, No. 2016023877, U.S. Patent Application Publication No. 2015252057(A) (Roche), International Publication No. 16128335(A1) (Roche), No. 16120186(A1) (Roche), U.S. Patent Application Publication No. 2016237090(A) (Roche), International Publication No. 16107833(A1) (Roche), No. 16107832(A1) (Roche), U.S. Patent Application Publication No. 2016176899(A) (Roche), International Publication No. 16102438(A1) (Roche), No. 16012470(A1) (Roche), U.S. Patent Application Publication No. 2016220586(A) (Roche), and No. 2015031687(A) (Roche) are included.

[0201] Examples of formulations for the treatment of HBV that can be combined or co-administered include tenofovir disoproxil fumarate and emtricitabine (TRUVADA®), ledipasvir, and sofosbuvir (HARVONI®); ABX-203 (NASVAC), lamivudine and PEG-IFNα; adefovir and PEG-IFNα; and INO-1800 (INO-9112 and RG7944).

[0202] HBV Vaccine In various embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein are combined with or co-administered with one or more HBV vaccines. HBV vaccines that can be combined or co-administered (e.g., prime-boost prophylactic regimens) include both prophylactic 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, Heberpenta L, DTwP-HepB-Hib, V-419, CVI-HBV-001, Tetrabhay, Hepatitis B preventive vaccine (Advax Super D), Hepatrol-07, GSK-223192A, ENGERIX B (registered trademark), recombinant hepatitis B vaccine (intramuscular, Kangtai Biological Products), recombinant hepatitis B vaccine (Hansenual polymorpha yeast, intramuscular, 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.

[0203] Examples of HBV vaccines that can be combined or co-administered (e.g., in a prime-boost treatment 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 adjuvanted 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 arena virus vaccines are described, for example, in International Publication Nos. WO 2017 / 076988 and WO 2017 / 198726.

[0204] HBV DNA polymerase inhibitor In various embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein are combined with or co-administered with one or more polymerase inhibitors. Examples of HBV DNA polymerase inhibitors that can be combined or co-administered include adefovir (HEPSERA®), emtricitabine (EMTRIVA®), tenofovir disoproxil fumarate (VIREAD®), tenofovir alafenamide, tenofovir, tenofovir disoproxil, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir dipivoxil, tenofovir dipivoxil fumarate, tenofovir octadecyl oxyethyl ester, CMX-157, tenofovir exalidex, bicifovir, entecavir (BARACLUDE®), entecavir maleate, telbivudine (TYZEKA®), filoscirivol, pradefovir, clevudine, ribavirin, lamivudine (EPIVIR-HBV®), phosphazide, famciclovir, fosolin, metacavir, SNC-019754, FMCA, AGX-1009, AR-II-04-26, HIP-1302, tenofovir disoproxil aspartate, tenofovir disoproxil orotate, AiB-001, and HS-10234.

[0205] Immunomodulator In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions as described herein are combined with or co-administered with one or more immunomodulators (e.g., immune checkpoint inhibitors, tumor necrosis factor (TNF) receptor superfamily (TNFRSF) agonists, immunostimulatory factors such as Toll-like receptor (TLR) agonists). Examples of immunomodulators that can be combined with or co-administered include linterferon modulator, imidazole hydrochloride, ingaron, dermaVir, plaquenil (hydroxychloroquine), proleukin, hydroxyurea, mycophenolate mofetil (MPA) and its ester derivative mycophenolate mofetil (MMF), JNJ-440, WF-10, AB-452, ribavirin, IL-12, INO-9112, polymeric polyethyleneimine (PEI), Gepon, VGV-1, MOR-22, CRV-431, JNJ-0535, TG-1050, ABI-H2158, BMS-936559, GS-9688, RO-7011785, and the corresponding prodrug RO-702053, RG-7854, RO-6871765, AIC-649, and IR-103.

[0206] Toll-like receptor (TLR) agonist In various embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein are combined with or co-administered with one or more agonists or stimulators of Toll-like receptors (TLRs). In various embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein are combined with or co-administered with agonists of TLRs, such as 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 agonists of TLR13.

[0207] Examples of TLR3 agonists that can be combined or co-administered include lintatrimod, poly-ICLC, RIBOXXON®, Apoxxim, RIBOXXIM®, IPH-33, MCT-465, MCT-475, and ND-1.1.

[0208] Examples of TLR4 agonists that can be combined or co-administered include G-100 and GSK-1795091.

[0209] Exemplary TLR7 agonists that can be combined or co-administered include AL-034, DSP-0509, GS-9620 (vesatolimod), 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, as well as those disclosed in U.S. Patent Application Publication Nos. 20100143301 (Gilead Sciences), 20110098248 (Gilead Sciences), 20090047249 (Gilead Sciences), 20140045849 (Janssen), 20140073642 (Janssen), International Publication Nos. 2014 / 056953 (Janssen), 2014 / 076221 (Janssen), 2014 / 128189 (Janssen), U.S. Patent Application Publication No. 20140350031 (Janssen), International Publication No. 2014 / 023813 (Janssen), U.S. Patent Application Publication Nos. 20080234251 (Array Biopharma), 20080306050 (Array Biopharma), 20100029585 (Ventirx Pharma), 20110092485 (Ventirx Pharma), 20110118235 (Ventirx Pharma), 20120082658 (Ventirx Pharma), 20120219615 (Ventirx Pharma), 20140066432 (Ventirx Pharma), 20140088085 (Ventirx Pharma), 20140275167 (Novira Therapeutics), and those disclosed in 20130251673 (Novira Therapeutics), but are not limited thereto.

[0210] Exemplary dual TLR7 / TLR8 agonists that can be combined or co-administered are NKTR-262, telratolimod, and BDB-001.

[0211] Exemplary TLR8 agonists that can be co-administered include E-6887, IMO-4200, IMO-8400, IMO-9200, MCT-465, telratolimod (MEDI-9197), motolimod, resiquimod, selgantolimod (GS-9688), HRS-9950, VTX-1463, VTX-763, 3M-051, 3M-052, SBT6050, and the compounds disclosed in U.S. Patent Application Publication No. 2016 / 289229 (Gilead Sciences), No. 2014 / 0045849 (Janssen), No. 2014 / 0073642 (Janssen), International Publication No. 2014 / 056953 (Janssen), No. 2014 / 076221 (Janssen), No. 2014 / 128189 (Janssen), U.S. Patent Application Publication No. 2014 / 0350031 (Janssen), International Publication No. 2014 / 023813 (Janssen), U.S. Patent Application Publication No. 2008 / 0234251 (Array Biopharma), No. 2008 / 0306050 (Array Biopharma), No. 2010 / 0029585 (Ventirx Pharma), No. 2011 / 0092485 (Ventirx Pharma), No. 2011 / 0118235 (Ventirx Pharma), No. 2012 / 0082658 (Ventirx Pharma), No. 2012 / 0219615 (Ventirx Pharma), No. 2014 / 0066432 (Ventirx Pharma), No. 2014 / 0088085 (Ventirx Pharma), No. 2014 / 0275167 (Novira Therapeutics), and No. 2013 / 0251673 (Novira Therapeutics), U.S. Patent No. 9,670,205 (Gilead Sciences, Inc.), U.S. Patent Application Publication No. 2016 / 0289229 (Gilead Sciences, Inc.), International Publication No. 2017 / 048727 (Gilead Sciences, Inc.), U.S. Patent Application Publication No. 2018 / 0065938 (Gilead Sciences, Inc.), and No. 2018 / 0086755 (Gilead Sciences, Inc.), or are not limited thereto.

[0212] 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, ritonimod, MGN-1601, BB-001, BB-006, IMO-3100, IMO-8400, IR-103, IMO-9200, agatrimod, DIMS-9054, DV-1079, DV-1179, AZD-1419, leftirimod (MGN-1703), CYT-003, CYT-003-QbG10, chiltrimod, and PUL-042.

[0213] Additional examples of TLR7, TLR8, and TLR9 modulators that can be combined or co-administered include International Publication Nos. WO 2017 / 047769 (Teika Seiyaku), WO 2015 / 014815 (Janssen), WO 2018 / 045150 (Gilead Sciences Inc), WO 2018 / 045144 (Gilead Sciences Inc), WO 2015 / 162075 (Roche), WO 2017 / 034986 (University of Kansas), WO 2018 / 095426 (Jiangsu Hengrui Medicine Co Ltd), WO 2016 / 091698 (Roche), WO 2016 / 075661 (GlaxoSmithKline Biologicals), WO 2016 / 180743 (Roche), WO 2018 / 089695 (Dynavax Technologies), WO 2016 / 055553 (Roche), WO 2015 / 168279 (Novartis), WO 2016 / 107536 (Medshine Discovery), WO 2018 / 086593 (Livo (Shanghai) Pharmaceutical), WO 2017 / 106607 (Merck), WO 2017 / 061532 (Sumitomo Dainippon Pharma), WO 2016 / 023511 (Chia Tai Tianqing Pharmaceutical), WO 2017 / 076346 (Chia Tai Tianqing Pharmaceutical), WO 2017 / 046112 (Roche), WO 2018 / 078149 (Roche), WO 2017 / 040233 (3M Co), WO 2016 / 141092 (Gilead Sciences), WO 2018 / 049089 (Bristol Myers Squibb), WO 2015 / 057655 (Eisai Co Ltd), WO 2017 / 001307 (Roche), WO 2018 / 005586 (Bristol Myers Squibb), WO 2017 / 04023 (3M Co), WO 2017 / 163264 (Council of Scientific and IndustrialResearch (India)), No. 2018046460 (GlaxoSmithKline Biologicals), No. 2018047081 (Novartis), No. 2016142250 (Roche), No. 2015168269 (Novartis), No. 201804163 (Roche), No. 2018038877 (3M Co), No. 2015057659 (Eisai Co Ltd), No. 2017202704 (Roche), No. 2018026620 (Bristol Myers Squibb), No. 2016029077 (Janus Biotherapeutics), No. 201803143 (Merck), No. 2016096778 (Roche), No. 2017190669 (Shanghai De Novo Pharmatech), US Patent No. 09884866 (University Of Minnesota), International Publication No. 2017219931 (Sichuan KelunBiotech Biopharmaceutical), No. 2018002319 (Janssen Sciences), No. 2017216054 (Roche), No. 2017202703 (Roche), No. 2017184735 (IFM Therapeutics), No. 2017184746 (IFM Therapeutics), No. 2015088045 (Takeda Pharmaceutical), No. 2017038909 (Takeda Pharmaceutical), No. 2015095780 (University of Kansas), No. 2015023958 (University of Kansas). Compounds disclosed in these are included.

[0214] In certain embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector, LNP, and an immunogenic composition as described herein are combined with or co-administered with a TLR7, TLR8, or TLR9 agonist.

[0215] Interferon α receptor ligand In various embodiments, an immunogenic polypeptide, a polynucleotide encoding such a polypeptide, a vector comprising such a polypeptide or polynucleotide, an LNP, and an immunogenic composition as described herein are combined with or co-administered with one or more interferon alpha receptor ligands.Examples of interferon α receptor ligands that can be combined or co-administered include interferon α-2b (INTRON A®), pegylated interferon α-2a (PEGASYS®), pegylated interferon α-1b, interferon α1b (HAPGEN®), Veldona, Infradure, Roferon-A, YPEG-interferon α-2a (YPEG-rhIFNalpha-2a), P-1101, Algeron, Alfarona, Ingaron (interferon γ), rSIFN-co (recombinant super-compound interferon), YPEG interferon α-2b (YPEG-rhIFNalpha-2b), MOR-22, peginterferon α-2b (PEG-INTRON®), Bioferon, Novaferon, Inmutag (Inferon), MULTIFERON®, interferon α-n1 (HUMOFERON®), interferon β-1a (AVONEX®), Shaferon, interferon α-2b (Axxo), Alfaferone, interferon α-2b (BioGeneric Pharma), interferon-α2 (CJ), Laferonum, VIPEG, BLAUFERON-A, BLAUFERON-B, Intermax Alpha, Realdiron, Lanstion, Pegaferon, PDferon-B, interferon α-2b (IFN, Laboratorios Bioprofarma), alphainterferona 2b, Kalferon, Pegnano, Feronsure, PegiHep, interferon α 2b (Zydus-Cadila), interferon α 2a, Optipeg A, Realfa 2B, Reliferon, interferon α-2b (Amega), interferon α-2b (Virchow), lopeginterferon α-2b, rHSA-IFN α-2a (recombinant human serum albumin interferon α2a fusion protein), PEG-IFN-α, rHSA-IFN. α 2b, recombinant human interferon α-(1b, 2a, 2b), peginterferon α-2b (Amega), peginterferon α-2a, Reaferon-EC, Proquiferon, Uniferon, Urifron, inter...

Claims

[Claim 1] The invention as depicted in the drawings.

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