Immunogenic composition against hepatitis b virus

Abstract

The purpose of the present invention is to provide an immunogenic composition containing a new HBV polypeptide or polynucleotides encoding the polypeptide. The immunogenic composition contains at least one polypeptide selected from among polypeptides a) through c) or polynucleotides encoding the polypeptide, and can be used for treating or preventing HBV infection. a) an HBV surface protein polypeptide including a specific amino acid substitution; b) an HBV polymerase polypeptide including an inactivated reverse transcriptase domain and an inactivated ribonuclease H domain, and including a specific amino acid substitution; and c) an HBV core protein polypeptide including a specific amino acid sequence.

Description

Immunogenic composition against hepatitis B virus

[0001] The present invention relates to an immunogenic composition comprising a novel HBV (hepatitis B virus) polypeptide. More specifically, it relates to an immunogenic composition comprising one or more polypeptides selected from novel HBp (HBV polymerase) polypeptide, HBs (HBV surface protein) polypeptide, and HBc (HBV core protein) polypeptide (an immunogenic composition comprising the polypeptide of the present invention). Furthermore, the present invention relates to an immunogenic composition comprising one or more polynucleotides selected from novel polynucleotides encoding HBp polypeptide, polynucleotides encoding HBs polypeptide, and polynucleotides encoding HBc polypeptide (an immunogenic composition comprising the polynucleotide of the present invention). The "immunogenic composition comprising the polypeptide of the present invention" and the "immunogenic composition comprising the polynucleotide of the present invention" are collectively referred to as the "immunogenic composition of the present invention."

[0002] HBV infection affects approximately 250 million people worldwide and can cause complications such as liver cancer, cirrhosis, and acute hepatitis. While the disappearance of serum HBsAg (HBV surface protein antigen) and the production of anti-HBsAg antibodies are known to suppress the progression and onset of HBV complications, current treatments such as nucleoside analogs and interferon preparations have the drawback of low HBsAg disappearance rates and a high likelihood of hepatitis recurrence after treatment discontinuation. Furthermore, in patients who have become HBsAg-negative, CTLs (cytotoxic T cells) are induced in response to multiple antigens, suggesting that simultaneous immune induction against multiple antigens may be important for HBV treatment (Non-Patent Literature 1).

[0003] HBV is an enveloped DNA virus belonging to the Hepadnaviridae family, which infects and replicates in liver cells and other tissues. The HBV virion has an outer lipoprotein shell formed by HBsAg (HBV surface protein antigen) and an icosahedral capsid formed by HBcAg (HBV core antigen) which encapsulates the rcDNA (incomplete double-stranded DNA) genome. HBV DNA mainly encodes HBp, HBsAg, HBcAg, and HBeAg.

[0004] Patent documents 1 to 4 disclose various HBp sequences. Example 1 of Patent Document 1 discloses an HBp polypeptide in which a YMNN mutation (SEQ ID NO: 384) in the YMDD motif (SEQ ID NO: 382) within the reverse transcriptase (RTase) domain and an NADD mutation (SEQ ID NO: 395) in the DEDD motif (SEQ ID NO: 389) within the ribonuclease H (RNaseH) domain are introduced into an HBp consensus sequence designed based on HBV genotypes B, C, and D.

[0079] states that RNaseH function can be reduced by substituting, for example, one or more aspartate residues (D) of the DEDD motif (SEQ ID NO: 389) with asparagine residues (N) and / or a glutamate residue (E) with a glutamine residue (Q). Examples 4 and 5 describe the results of immunizing mice or cynomolgus monkeys with an adenovirus vector expressing an HBp and HBc fusion protein (core Pol fusion) and confirming the T cell response. However, Patent Document 1 does not disclose the results of evaluating the immune-inducing ability of HBp polypeptide alone. Example 2 of Patent Document 2 discloses an HBp polypeptide containing an inactive RTase domain and an inactive RNaseH domain derived from HBV genotype A, B, C, or D. Specifically, it has a YMHD mutation (SEQ ID NO: 383) in the YMDD motif (SEQ ID NO: 382) within the RTase domain and an AHLL mutation (SEQ ID NO: 388) within the RNaseH domain, and a Pol that does not contain part of the spacer domain. Δ１ and Pol Δ３ Furthermore, Pol has the YMHD mutation (SEQ ID NO: 383) and the AHLL mutation (SEQ ID NO: 388), and the entire length of the terminal domain and most of the spacer domain have been removed.３００ This is disclosed (Table 2). Furthermore, since consensus sequences that do not exist in nature carry the risk of being inefficiently expressed or insufficiently treating T cell epitopes, it is described that the naturally occurring HBp sequence closest to the consensus was identified for each genotype, and that because the spacer domain sequence varies greatly among HBV strains and genotypes, an HBp polypeptide with a deletion in the spacer domain was generated. Example 2.2 of Patent Document 3 describes a mutant polymerase polypeptide (Poly) comprising two internal deletions and four amino acid substitutions in the RNaseH domain. ＊ The results of evaluating mice after administering an adenovirus expressing ) are described. Example 3 of Patent Document 4 describes the results of evaluating mice after administering a DNA plasmid encoding a full-length HBp polypeptide.

[0005] Example 3 of Patent Document 5 describes the results of immunizing mice with plasmids expressing either S-HBs of HBs only or polypeptides in which an IgE reader sequence and a Furin sequence are added to the full length of HBs, and evaluating the results.

[0006] Examples 1 of Patent Document 5 and Patent Document 6 describe DNA (composition pM core) encoding a polypeptide to which an IgE reader has been added, to which an IgE reader has been added, to a consensus HBc polypeptide designed with epitope sequences derived from HBV genotypes A, B, C, D, and E. The results of administering the pM core to mice and evaluating the T cell response are described.

[0007] Patent Document 5 and Non-Patent Document 2 disclose a mixed vaccine containing multiple HBV antigens. Examples 5-6 of Patent Document 5 describe the preparation of a DNA vaccine (HBs-HBc DNA vaccine cocktail, pLHBs / pMCore) by combining a cocktail containing a plasmid encoding full-length HBs (L-HBs) and a cocktail containing a plasmid encoding an HBc polypeptide, and the results of evaluating the vaccine after immunizing mice or rhesus monkeys with it. The cocktail containing the plasmid encoding L-HBs includes a plasmid encoding the L-HBs consensus sequence of HBs derived from genotype A (pLHBs-A) and a plasmid encoding the L-HBs consensus sequence of HBs derived from genotype C (pLHBs-C), thus containing two plasmids derived from different genotypes. Non-Patent Document 2 describes the results of evaluating the immunogenicity of a preparation containing a mixture of three different LNPs, each containing mRNA encoding HBp, HBc, or M-HBs (PreS2 and S-HBs), after administering it to mice. Furthermore, when mice were administered a formulation in which all three mRNAs were encapsulated in a single LNP and evaluated, a strong immune response was observed in naive C57BL / 6 and BALB / c mice, while a lower immune response was observed in AAV-HBV transduced C57BL / 6 mice, an HBV infection model. It was also noted that no change in ALT levels was observed between vaccinated and unvaccinated mice, and since clearance of infected hepatocytes was not achieved, further investigation, including combination therapy, is necessary.

[0008] International Publication No. 2019 / 123250, International Publication No. 2021 / 067181, International Publication No. 2013 / 007772, International Publication No. 2020 / 255055, International Publication No. 2014 / 047286, International Publication No. 2012 / 109668, International Publication No. 2017 / 081082

[0009] J Hepatol. 2022 Jul;77(1):42-54Vaccines(2024)Feb 25;12(3):237

[0010] The object of the present invention is to provide a novel HBV polypeptide, or a polynucleotide encoding the HBV polypeptide, that is immunogenic against HBV and can be used as a therapeutic and / or prophylactic agent for HBV infection.

[0011] The present invention relates to the following: (1) an immunogenic composition comprising one or more polypeptides selected from a) to c) below. a) An HBV surface protein polypeptide (also referred to as "the HBs polypeptide of the present invention") comprising a K residue at the amino acid position corresponding to position 56, an A residue at the amino acid position corresponding to position 59, an L residue at the amino acid position corresponding to position 107, a T residue at the amino acid position corresponding to position 114, a T residue at the amino acid position corresponding to position 129, an A residue at the amino acid position corresponding to position 153, an N residue at the amino acid position corresponding to position 155, and a V residue at the amino acid position corresponding to position 171 of the amino acid sequence of SEQ ID NO: 351; b) An HBV polymerase polypeptide (also referred to as "the HBp polypeptide of the present invention") comprising an inactivated reverse transcriptase domain and an inactivated ribonuclease H domain, and comprising an N residue at the amino acid position corresponding to position 124, an M residue at the amino acid position corresponding to position 145, a Y residue at the amino acid position corresponding to position 151, a Y residue at the amino acid position corresponding to position 221, and an S residue at the amino acid position corresponding to position 332 of the amino acid sequence of SEQ ID NO: 65; and c) An HBV core protein polypeptide (also referred to as "the HBc polypeptide of the present invention") comprising the amino acid sequence described in any of SEQ ID NOs. 368 to 380. (2) The immunogenic composition according to (1), wherein the HBV surface protein polypeptide of (a) is a polypeptide comprising the amino acid sequence described in any of SEQ ID NOs. 353, 358 to 361 and 365 to 367. (3-1) The immunogenic composition according to (1) or (2), wherein the HBV surface protein polypeptide of (a) is a polypeptide to which a signal peptide comprising or consisting of the amino acid sequence described in any of SEQ ID NOs. 55 to 57, 124, 125, 135 and 137 is linked. (3-2) The immunogenic composition according to any of (1) to (3-1), wherein the HBV surface protein polypeptide of (a) is further a polypeptide to which a functional sequence comprising or consisting of the amino acid sequence described in SEQ ID NOs. 142 or 381 is linked. The immunogenic composition according to any one of (1) to (3-2), wherein the HBV polymerase polypeptide in (4)b) is a polypeptide including a part of the terminal domain or the entire length.The immunogenic composition according to any one of (1) to (4), wherein the HBV polymerase polypeptide in (5)b) is a polypeptide containing part or the entire spacer domain. The immunogenic composition according to any one of (1) to (5), wherein the HBV polymerase polypeptide in (6)b) is a polypeptide containing an N residue at the amino acid position corresponding to position 205 and an N residue at the amino acid position corresponding to position 206 of the amino acid sequence of SEQ ID NO: 65. The immunogenic composition according to any one of (1) to (6), wherein the HBV polymerase polypeptide in (7-1)b) is a polypeptide containing an N residue at the amino acid position corresponding to position 354 and a Q residue at the amino acid position corresponding to position 383 of the amino acid sequence of SEQ ID NO: 65. The immunogenic composition according to any one of (1) to (7-1), wherein the HBV polymerase polypeptide of (7-2)b) is a polypeptide comprising one or more amino acid residues from among the amino acid positions corresponding to position 38, 53, 91, 263, 317, 452, and 472 of the amino acid sequence of SEQ ID NO: 65. The immunogenic composition according to any one of (1) to (7-2), wherein the HBV polymerase polypeptide of (8)b) is a polypeptide comprising an amino acid sequence having the amino acid sequence described in any one of SEQ ID NOs: 36, 37, 40-49, and 400-402. The immunogenic composition according to any one of (1) to (8), wherein the HBV polymerase polypeptide of (9-1)b) is a polypeptide to which a signal peptide consisting of any of the amino acid sequences described in SEQ ID NOs. 55-57, 124, 125, 135, and 137 is linked. The immunogenic composition according to any one of (1) to (9-1), wherein the HBV polymerase polypeptide of (9-2)b) is a polypeptide to which a functional sequence consisting of or containing the amino acid sequence described in SEQ ID NOs. 142 or 381 is linked.An immunogenic composition according to any one of (1) to (9-2), wherein the HBV core protein polypeptide of (10-1)c) is a polypeptide to which a signal peptide comprising or consisting of any of the amino acid sequences described in SEQ ID NOs. 55-57, 124, 125, 135, and 137 is linked. An immunogenic composition according to any one of (1) to (10-1), wherein the HBV core protein polypeptide of (10-2)c) is further an HBV core protein polypeptide to which a functional sequence comprising or consisting of the amino acid sequence described in SEQ ID NOs. 142 or 381 is linked. An immunogenic composition comprising (11) a polynucleotide (also referred to as "the polynucleotide of the present invention") containing an open reading frame encoding the polypeptide described in any one of (1) to (10-2) (also referred to as "the polypeptide of the present invention"). An immunogenic composition according to (11), wherein the polynucleotide is mRNA. (12-2) The immunogenic composition according to (12-1), wherein the mRNA is an mRNA comprising a 5'Cap structure, a 5'UTR, an open reading frame encoding a polypeptide described in any of (1) to (10-2), a 3'UTR, and Poly(A) (also referred to as "the mRNA of the present invention"; "the mRNA of the present invention" is included in "the polynucleotide of the present invention"). (12-3) The mRNA is 5-methyluridine, pseudouridine, N1-methylpseudridine, 5-methoxyuridine, 2-thiouridine, 6-methyladenosine, 2-aminoadenosine, inosine, 5-methylcytidine, N1-ethylpseudridine, 4-thiouridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-di An immunogenic composition according to (12-1) or (12-2), comprising hydropseudridine, 2-thiodihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-azauridine, dihydropseudridine, 2'-O-methyluridine, and modified bases selected from combinations thereof.(12-4) The immunogenic composition according to (12-3), wherein the modified base is selected from 5-methyluridine, N1-methylpseudridine, 5-methylcytidine, and combinations thereof. (12-5) The immunogenic composition according to any one of (12-1) to (12-4), wherein the mRNA comprises a 5' Cap structure which is Cap1 or Cap2. (12-6) The immunogenic composition according to (12-5), wherein the 5' Cap structure is Cap1. (12-7) The immunogenic composition according to any one of (12-1) to (12-6), wherein the mRNA comprises 60 to 160 mers of Poly(A). (12-8) The immunogenic composition according to (12-7), wherein the Poly(A) is 80 mers. (12-9) An immunogenic composition according to any one of (12-1) to (12-8), wherein the mRNA comprises a 5'UTR which is a polynucleotide consisting of a base sequence identical to or having 90% or more identity with the base sequence described in any of SEQ ID NOs. 154, 155 and 217 to 224. (12-10) An immunogenic composition according to any one of (12-1) to (12-9), wherein the mRNA comprises a 3'UTR which is a polynucleotide consisting of a base sequence identical to or having 90% or more identity with the base sequence described in any of SEQ ID NOs. 225, 226, 227 or 232. (13-1) An immunogenic composition according to any one of (11) to (12-10), comprising one or more polynucleotides selected from A) to C) below.A) A polynucleotide in which the open reading frame contains or comprises a nucleotide sequence described in any of SEQ ID NOs. 305, 312-322, and 326-328, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence described in any of SEQ ID NOs. 305, 312-322, and 326-328; B) A polynucleotide in which the open reading frame contains or comprises a nucleotide sequence described in any of SEQ ID NOs. 280, 281, 284-297, and 403-406, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence described in any of SEQ ID NOs. 280, 281, 284-297, and 403-406; and C) A polynucleotide in which the open reading frame contains or comprises a nucleotide sequence described in any of SEQ ID NOs. 329-347, or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence described in any of SEQ ID NOs. 329-347. The immunogenic composition according to (13-1), wherein (13-2)A) is mRNA defined by any of HBs14, 39-45, 52, 61-63 and 67-72, 74, 75. (The specific structure of the mRNA is shown in Table 9 of Example 3.) The immunogenic composition according to (13-1) or (13-2), wherein (13-3)B) is mRNA defined by any of HBp12, 13, 16-19, 26, 32-41, 43, 49-51, 55, 58, 60, and 65-70. (The specific structure of the mRNA is described in Tables 8-1 and 8-2 of Example 3.) (13-4) C) is an mRNA defined by any of HBc6, 8, 9, 12, 19, 20, 22, 23, 25, 26, 31-38, 41, 42, 53-69 and 71, 72, as described in any of (13-1) to (13-3). (The specific structure of the mRNA is described in Table 10 of Example 3.) (14) An immunogenic composition according to any of (1) to (13-4) that is a therapeutic and / or prophylactic agent for HBV infection.

[0012] (15) A method for inducing an immune response to HBV, comprising administering to a subject in need a therapeutically effective amount of the immunogenic composition described in any of (1) to (13-4). (16) The immunogenic composition described in any of (1) to (13-4), used to induce an immune response to HBV in the treatment and / or prevention of HBV infection.

[0013] (17) A method for treating and / or preventing HBV infection, comprising administering an immunogenic composition according to any one of (1) to (13-4). (18) An immunogenic composition according to any one of (1) to (13-4) for manufacturing a therapeutic and / or prophylactic agent for HBV infection. (19) An immunogenic composition according to any one of (1) to (13-4) for the treatment and / or prevention of HBV infection. (20) Use of an immunogenic composition according to any one of (1) to (13-4) for the treatment and / or prevention of HBV infection.

[0014] The immunogenic composition of the present invention is useful as a therapeutic and / or prophylactic agent for HBV infection because it exhibits excellent immunogenicity against different HBV antigens.

[0015] Structure of HBp polypeptide. Structure of HBs polypeptide. Structure of HBc polypeptide. Evaluation of HBp-specific immune cells induced by HBp mRNA administration. Evaluation of HBp-specific immune cells induced by HBp mRNA administration. Evaluation of HBp-specific immune cells induced by HBp mRNA administration. Evaluation of HBp-specific immune cells induced by HBp mRNA administration. Evaluation of HBs-specific immune cells induced by HBs mRNA administration. Evaluation of anti-L-HBs antibody induction ability by HBs mRNA administration. Evaluation of HBV neutralizing activity induced by HBs mRNA administration. Evaluation of HBc-specific immune cells induced by HBc mRNA administration. Detection of HBc antigen-specific cytotoxic T cells. Detection of HBs antigen-specific cytotoxic T cells. Evaluation of anti-L-HBs antibody induction ability by HBs mRNA administration.

[0016] The present invention will now be described with reference to embodiments. Throughout this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified. Therefore, singular articles (for example, "a," "an," "the," etc. in English) should be understood to include the concept of their plural form unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In case of any conflict, this specification (including definitions) shall prevail.

[0017] The term "consists of" means having only the constituent elements. The term "includes" means not being limited to the constituent elements and not excluding any elements not listed.

[0018] A polypeptide is a polymer of amino acid residues (natural or unnatural) linked by peptide bonds, and refers to a protein or peptide of any size, structure, or function. If the encoded polypeptide is larger than about 50 amino acids, it is called a protein. If the encoded polypeptide is smaller than about 50 amino acids, it is called a peptide, and if a polypeptide is a peptide, it is at least about 2, 3, 4, or 5 amino acid residues long. Therefore, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, the aforementioned fragments and other equivalents, variants, and analogs. A polypeptide may be a single molecule or a multimolecular complex such as a dimer, trimer, or tetramer.

[0019] The natural HBp polypeptide has four functional domains: a terminal domain, a spacer domain, an RTase domain, and an RNaseH domain (Figure 1). For example, the amino acid sequence of the HBp polypeptide derived from the HBV D genotype is described in NCBI YP_009173866.1. The terminal domain is the region consisting of amino acids from positions 1 to 177, the spacer domain is the region consisting of amino acids from positions 178 to 335, the RTase domain is the region consisting of amino acids from positions 336 to 679, and the RNaseH domain is the region consisting of amino acids from positions 680 to 832.

[0020] The HBp polypeptide of the present invention comprises an inactivated RT-ase domain and an inactivated RNaseH domain, and is an HBp polypeptide comprising an N residue at the amino acid position corresponding to position 124, an M residue at the amino acid position corresponding to position 145, a Y residue at the amino acid position corresponding to position 151, a Y residue at the amino acid position corresponding to position 221, and an S residue at the amino acid position corresponding to position 332 of the amino acid sequence of SEQ ID NO: 65 (see (1) b) above). The amino acid sequence of SEQ ID NO: 65 is the reference sequence of an HBp polypeptide consisting of an RT-ase domain and an RNaseH domain. Amino acids other than the specified amino acid residues may be amino acids derived from the natural HBp polypeptide or amino acids derived from multiple genotypes of HBV. Preferably, the HBp polypeptide of the present invention is a polypeptide having mutations in the HBp consensus sequence derived from HBV A, B, C, and D genotypes that inactivate the RTase domain and the RNaseH domain, as well as an N residue at the amino acid position corresponding to position 124, an M residue at the amino acid position corresponding to position 145, a Y residue at the amino acid position corresponding to position 151, a Y residue at the amino acid position corresponding to position 221, and an S residue at the amino acid position corresponding to position 332 of the amino acid sequence of SEQ ID NO: 65.

[0021] As one embodiment, the HBP polypeptide of the present invention is a polypeptide having a mutation that inactivates the RTase domain and the RNaseH domain in the HBP consensus sequence derived from HBV genotypes A, B, C, and D, the amino acid residues described in b) of (1) above, and the A residue at the amino acid position corresponding to position 38 of the amino acid sequence of SEQ ID NO: 65, the N residue at the amino acid position corresponding to position 53, the I residue at the amino acid position corresponding to position 91, the D residue at the amino acid position corresponding to position 263, the S residue at the amino acid position corresponding to position 317, the S residue at the amino acid position corresponding to position 452, and the D residue at the amino acid position corresponding to position 472.

[0022] As one embodiment, the HBP polypeptide of the present invention is a polypeptide having a mutation that inactivates the RTase domain and the RNaseH domain in the HBP consensus sequence derived from HBV genotypes A, B, C, and D, the amino acid residues described in b) of (1) above, and the A residue at the amino acid position corresponding to position 38 of the amino acid sequence of SEQ ID NO: 65, the I residue at the amino acid position corresponding to position 91, the D residue at the amino acid position corresponding to position 263, and the S residue at the amino acid position corresponding to position 452.

[0023] As one embodiment, the HBP polypeptide of the present invention is a polypeptide having a mutation that inactivates the RTase domain and the RNaseH domain in the HBP consensus sequence derived from HBV genotypes A, B, C, and D, the amino acid residues described in b) of (1) above, and the N residue at the amino acid position corresponding to position 53 of the amino acid sequence of SEQ ID NO: 65, and the D residue at the amino acid position corresponding to position 472.

[0024] The HBp polypeptide of the present invention comprises an inactivated RTase domain and an inactivated RNaseH domain. In one embodiment, the YMDD motif (SEQ ID NO: 382) in the RTase domain may have one or two aspartic acid residues in the motif substituted with histidine residues, asparagine residues, etc., and may be substituted with YMHD (SEQ ID NO: 383), YMNN (SEQ ID NO: 384), YMND (SEQ ID NO: 385), or YMDN (SEQ ID NO: 386). Preferred mutations for inactivating the RTase domain of the HBp polypeptide of the present invention are the N residues at the amino acid positions corresponding to position 205 and 206 of SEQ ID NO: 65. In one embodiment, the AELL motif (SEQ ID NO: 387) in the RNaseH domain may be substituted with AHLL (SEQ ID NO: 388). In addition, the Mg in the RNaseH domain... ２＋ The DEDD motif (SEQ ID NO: 389), which is necessary for coordination, may have one or two aspartic acid residues within the motif substituted with histidine or asparagine residues, and / or a glutamic acid residue substituted with glutamine. Preferably, the mutations that inactivate the RTase domain of the HBp polypeptide of the present invention are the N residue at the amino acid position corresponding to position 354 and the Q residue at the amino acid position corresponding to position 383 of SEQ ID NO: 65.

[0025] As one embodiment, the Hbp polypeptide of the present invention may include an inactivated RTase domain, an inactivated RNaseH domain, and a part or the entire length of the terminal domain. The "entire length" of the terminal domain refers to a polypeptide corresponding to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 58. The "part" of the terminal domain refers to a polypeptide corresponding to a polypeptide consisting of the amino acid sequence at positions 1 to 50, 1 to 100, 1 to 150, 50 to 100, or 50 to 150 of the amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 58. The terminal domain contained in the Hbp polypeptide of the present invention is preferably a polypeptide consisting of a part or the entire length of the amino acid sequence of SEQ ID NO: 58, more preferably a polypeptide consisting of the amino acid sequence at positions 1 to 20, 1 to 62, 1 to 116, 1 to 133, 1 to 167, or the entire length of the amino acid sequence of SEQ ID NO: 58, and even more preferably a polypeptide consisting of the entire length of the amino acid sequence of SEQ ID NO: 58.

[0026] As one embodiment, the Hbp polypeptide of the present invention may include an inactivated RTase domain, an inactivated RNaseH domain, and a part or the entire length of the spacer domain. The "entire length" of the spacer domain refers to a polypeptide corresponding to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 64. The "part" of the spacer domain refers to a polypeptide corresponding to a polypeptide consisting of the amino acid sequence at positions 1 to 50, 1 to 100, 1 to 150, 50 to 100, or 50 to 150 of the amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 64. The spacer domain contained in the Hbp polypeptide of the present invention is preferably a polypeptide consisting of the entire length of the amino acid sequence of SEQ ID NO: 64.

[0027] As one embodiment, the Hbp polypeptide of the present invention may include an inactivated RTase domain, an inactivated RNaseH domain, a part or the entire length of the terminal domain, and a part or the entire length of the spacer domain.

[0028] In one embodiment, the HBp polypeptide of the present invention may include an inactivated RTase domain containing a YMNN motif (SEQ ID NO: 384), an inactivated RNaseH domain containing an NQDD motif (SEQ ID NO: 407), a portion or the entire length of the terminal domain, and a portion or the entire length of the spacer domain.

[0029] As one embodiment, the HBp polypeptide of the present invention is a polypeptide having an HBp consensus sequence derived from HBV A, B, C, and D genotypes, an inactivated RTase domain containing a YMNN motif (SEQ ID NO: 384), an inactivated RNaseH domain containing an NQDD motif (SEQ ID NO: 407), the amino acid residues described in (1) b) above, and the full length of the terminal domain or a portion corresponding to positions 1 to 20 of SEQ ID NO: 58.

[0030] In one embodiment, the HBp polypeptide of the present invention is a polypeptide having an HBp consensus sequence derived from HBV A, B, C, and D genotypes, an inactivated RTase domain containing a YMNN motif (SEQ ID NO: 384), an inactivated RNaseH domain containing an NQDD motif (SEQ ID NO: 407), the amino acid residues described in (1) b) above, the full length of the terminal domain, and the full length of the spacer domain.

[0031] As one embodiment, the HBp polypeptide of the present invention is a polypeptide having an HBp consensus sequence derived from HBV A, B, C, and D genotypes, a mutation that inactivates the RTase domain and the RNaseH domain, the amino acid residues described in (1) b) above, an A residue at the amino acid position corresponding to position 38 of the amino acid sequence of SEQ ID NO: 65, an N residue at the amino acid position corresponding to position 53, an I residue at the amino acid position corresponding to position 91, a D residue at the amino acid position corresponding to position 263, an S residue at the amino acid position corresponding to position 317, an S residue at the amino acid position corresponding to position 452, and a D residue at the amino acid position corresponding to position 472, as well as the full length of the terminal domain or a portion corresponding to positions 1-20, 1-62, 1-116, 1-133, or 1-167 of SEQ ID NO: 58.

[0032] As one embodiment, the HBp polypeptide of the present invention is a polypeptide having mutations that inactivate the RTase domain and RNaseH domain in the HBp consensus sequence derived from HBV A, B, C and D genotypes, the amino acid residues described in (1) b) above, an A residue at the amino acid position corresponding to position 38 of the amino acid sequence of SEQ ID NO: 65, an N residue at the amino acid position corresponding to position 53, an I residue at the amino acid position corresponding to position 91, a D residue at the amino acid position corresponding to position 263, an S residue at the amino acid position corresponding to position 317, an S residue at the amino acid position corresponding to position 452, and a D residue at the amino acid position corresponding to position 472, the full length of the terminal domain, and the full length of the spacer domain.

[0033] In one embodiment, the HBp polypeptide of the present invention may be a polypeptide comprising an inactivated RTase domain containing YMNN (SEQ ID NO: 384), an inactivated RNaseH domain containing an NQDD motif (SEQ ID NO: 407), and the amino acid residue described in (1) b) above, and comprising an amino acid sequence described in any of SEQ ID NOs: 36, 37, 40-49 and 400-402, or an amino acid sequence having 80%, 85%, 90%, or 95% or more identity with the amino acid sequence described in any of SEQ ID NOs: 36, 37, 40-49 and 400-402. Preferably, it is a polypeptide comprising an amino acid sequence described in any of SEQ ID NOs: 36, 37, 40-49 and 400-402.

[0034] Natural HBs polypeptides have three functional domains: Pre-S1, Pre-S2, and S-HBs (S region) (Figure 2). HBs polypeptides containing all three functional domains are also called L-HBs polypeptides, HBs polypeptides containing PreS2 and S-HBs are also called M-HBs polypeptides, and HBp polypeptides containing S-HBs are also called S-HBs polypeptides. For example, the amino acid sequence of L-HBs polypeptide derived from the HBV D genotype is described in NCBI YP_009173869.1. Pre-S1 corresponds to the region consisting of amino acids from positions 1 to 108, Pre-S2 to the region consisting of amino acids from positions 109 to 163, and S-HBs to the region consisting of amino acids from positions 164 to 389.

[0035] The HBs polypeptide of the present invention is an HBs polypeptide comprising a K residue at the amino acid position corresponding to position 56 of the amino acid sequence of SEQ ID NO: 351, an A residue at the amino acid position corresponding to position 59, an L residue at the amino acid position corresponding to position 107, a T residue at the amino acid position corresponding to position 114, a T residue at the amino acid position corresponding to position 129, an A residue at the amino acid position corresponding to position 153, an N residue at the amino acid position corresponding to position 155, and a V residue at the amino acid position corresponding to position 171 (as described in (1) above). The amino acid sequence of SEQ ID NO: 351 is the reference sequence of the entire HBs polypeptide. The K residue at position 56, the A residue at position 59, and the L residue at position 107 of the amino acid sequence of SEQ ID NO: 351 are located within Pre-S1, while the T residue at position 114, the T residue at position 129, the A residue at position 153, the N residue at position 155, and the V residue at position 171 are located within Pre-S2. Amino acids other than those specified may be amino acids derived from the natural HBs polypeptide or amino acids derived from multiple genotypes of HBV. Preferably, the HBs polypeptide of the present invention is a polypeptide having an HBs consensus sequence derived from HBV A, B, C, and D genotypes, with a K residue at the amino acid position corresponding to position 56 of the amino acid sequence of SEQ ID NO: 351, an A residue at the amino acid position corresponding to position 59, an L residue at the amino acid position corresponding to position 107, a T residue at the amino acid position corresponding to position 114, a T residue at the amino acid position corresponding to position 129, an A residue at the amino acid position corresponding to position 153, an N residue at the amino acid position corresponding to position 155, and a V residue at the amino acid position corresponding to position 171.

[0036] In one embodiment, the HBs polypeptide of the present invention is a polypeptide that does not contain amino acids corresponding to positions 1 to 11 of the amino acid sequence of SEQ ID NO: 351.

[0037] In one embodiment, the HBs polypeptide of the present invention may be a polypeptide comprising an amino acid sequence that is 80%, 85%, 90%, or 95% or more identical to the amino acid sequence described in any of SEQ ID NOs: 353, 358-361, and 365-367, or an amino acid sequence that is 80%, 85%, 90%, or 95% or more identical to the amino acid sequence described in any of SEQ ID NOs: 353, 358-361, and 365-367. Preferably, it is a polypeptide comprising the amino acid sequence described in any of SEQ ID NOs: 353, 358-361, and 365-367.

[0038] In one embodiment, the HBs polypeptide of the present invention may contain the amino acid residues described in (1) a) above, and may also contain a T residue at the amino acid position corresponding to position 316 of the amino acid sequence of SEQ ID NO: 351, a Y residue at the amino acid position corresponding to position 334, and an S residue at the amino acid position corresponding to position 380.

[0039] As one embodiment, the HBs polypeptide of the present invention is a polypeptide that contains the amino acid residues described in (1) a) above in the HBs consensus sequence derived from HBV A, B, C, and D genotypes, but does not contain the amino acids corresponding to positions 1 to 11 of the amino acid sequence of SEQ ID NO: 351.

[0040] As one embodiment, the HBs polypeptide of the present invention is a polypeptide comprising an HBs consensus sequence derived from HBV A, B, C, and D genotypes, the amino acid residues described in (1) a) above, as well as a T residue at the amino acid position corresponding to position 316, a Y residue at the amino acid position corresponding to position 334, and an S residue at the amino acid position corresponding to position 380 of the amino acid sequence of SEQ ID NO: 351.

[0041] In one embodiment, the HBs polypeptide of the present invention is a polypeptide comprising an HBs consensus sequence derived from HBV A, B, C, and D genotypes, the amino acid residue described in (1) a) above, and a T residue at the amino acid position corresponding to position 316 and a Y residue at the amino acid position corresponding to position 334 of the amino acid sequence of SEQ ID NO: 351.

[0042] In one embodiment, the HBs polypeptide of the present invention is a polypeptide comprising an HBs consensus sequence derived from HBV A, B, C, and D genotypes, the amino acid residue described in (1) a) above, and an S residue at the amino acid position corresponding to position 380 of the amino acid sequence of SEQ ID NO: 351.

[0043] As one embodiment, the HBs polypeptide of the present invention is a polypeptide that includes the amino acid residues described in (1) a) above, as well as a T residue at the amino acid position corresponding to position 316, a Y residue at the amino acid position corresponding to position 334, and an S residue at the amino acid position corresponding to position 380 of the amino acid sequence of SEQ ID NO: 351, in the HBs consensus sequence derived from HBV A, B, C, and D genotypes, and does not include the amino acids corresponding to positions 1 to 11 of the amino acid sequence of SEQ ID NO: 351.

[0044] In one embodiment, the HBs polypeptide of the present invention is a polypeptide that includes the amino acid residues described in (1) a) above, as well as a T residue at the amino acid position corresponding to position 316 and a Y residue at the amino acid position corresponding to position 334 of the amino acid sequence of SEQ ID NO: 351, in the HBs consensus sequence derived from HBV A, B, C and D genotypes, but does not include the amino acids corresponding to positions 1 to 11 of the amino acid sequence of SEQ ID NO: 351.

[0045] As one embodiment, the HBs polypeptide of the present invention is a polypeptide that includes the amino acid residues described in (1) a) above, and an S residue at the amino acid position corresponding to position 380 of the amino acid sequence of SEQ ID NO: 351, in the HBs consensus sequence derived from HBV A, B, C, and D genotypes, but does not include the amino acids corresponding to positions 1 to 11 of the amino acid sequence of SEQ ID NO: 351.

[0046] The natural HBc polypeptide has two functional domains: a precore domain and an N-terminal domain (NTD) and a C-terminal domain (CTD) (Figure 3). For example, the amino acid sequence of the HBc polypeptide derived from the HBV D genotype is described in NCBI YP_009173868.3. The NTD is the region consisting of amino acids from positions 1 to 149, and the CTD is the region consisting of amino acids from positions 150 to 183.

[0047] Examples of the HBc polypeptide of the present invention include polypeptides comprising an amino acid sequence described in any of SEQ ID NOs. 368 to 380, or an amino acid sequence having 80%, 85%, 90%, or 95% or more identity with the amino acid sequence described in any of SEQ ID NOs. 368 to 380. More preferably, the polypeptide comprises an amino acid sequence described in any of SEQ ID NOs. 368 to 380.

[0048] The HBp polypeptide, HBs polypeptide, or HBc polypeptide (polypeptide of the present invention) may each have a functional sequence linked to it.

[0049] A "functional sequence" refers to a polypeptide of 3 to 60 amino acids located at the N-terminus, central, or C-terminus of a protein or its peptide fragment, which can determine the secretory pathway and localization of the protein or its peptide fragment. Functional sequences are synonymous with heterologous peptides. Functional sequences are classified into N-terminal functional sequences, central functional sequences, and C-terminal functional sequences depending on their position within the protein or its peptide fragment. Examples of functional sequences include signal peptides, linker peptides, immune-activating helper epitopes, TLR-activating peptides, and multimerization domain peptides. Examples of signal peptides include secretory signal peptides, nuclear localization signal peptides, nuclear localization signal peptides, ubiquitinated signal peptides, endoplasmic reticulum-targeting signal peptides, cell membrane localization signal peptides, Golgi apparatus localization signal peptides, endosomal localization signal peptides, cytoskeleton localization signal peptides, and cytoplasmic localization signal peptides.

[0050] "Secretory signal peptides (SSPs)" refer to short peptides of about 15 to 30 amino acids that are located at the N-terminus of a protein or a peptide fragment thereof, promote the transport of the protein or peptide fragment into the endoplasmic reticulum, and have a secretory function. Secretory signal peptides may be operably linked (fused) to the protein or the peptide fragment thereof, or they may be linked to a polypeptide from which methionine has been removed from the N-terminus of the protein or the peptide fragment thereof.

[0051] Examples of secretion signal peptides linked to the polypeptide of the present invention include polypeptides consisting of amino acid sequences described in any of SEQ ID NOs: 1 to 1115 and 1728 in Patent Document 7, and SEQ ID NOs: 55 to 57 and 123 to 140 in this specification, and polypeptides consisting of amino acid sequences in which one or more amino acids are deleted, substituted, inserted, or added, and which have secretion signal activity. Here, "addition" means that one or more amino acids are bound to the N-terminus or C-terminus of the amino acid sequence. "One or more" refers to 1 to 10, preferably 1 to 5.

[0052] Preferably, the signal peptide linked to the polypeptide of the present invention is a secretory signal peptide having secretory signal activity, which consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in any of the amino acid sequences described in SEQ ID NOs. 55-57, 124, 125, 135, and 137. More preferably, it is a secretory signal peptide consisting of an amino acid sequence described in any of SEQ ID NOs. 55-57 and 135.

[0053] In one embodiment, the functional sequence linked to the polypeptide of the present invention is preferably a peptide consisting of the amino acid sequence (MITD) described in SEQ ID NO: 142 or the amino acid sequence (KDEL) described in SEQ ID NO: 381.

[0054] The polypeptide of the present invention may have an arbitrary tag attached to its C-terminus. Examples of tags include His tags and FLAG tags.

[0055] "Immunogenicity" refers to the property of stimulating the immune system of an individual to which an immunogenic substance is administered, thereby inducing immune responses such as the production of antibodies against a target antigen and cellular immunity. "Immunogenic composition" refers to a composition that, when administered to an individual, elicits an immune response from that individual.

[0056] A "consensus sequence" refers to a non-natural polypeptide sequence or corresponding polynucleotide sequence constructed based on the alignment of multiple strains of the HBV gene using known alignment tools. For example, the consensus sequence may be the most frequently occurring amino acid residue found at each position in the sequence alignment, or a sequence further modified from that sequence may be used as the consensus sequence. The HBV consensus sequence can be used to induce broad immunity against multiple subtypes or serotypes of HBV.

[0057] "The Z residue at the amino acid position corresponding to position Y of the amino acid sequence of Sequence ID X" refers to the Z residue corresponding to the amino acid position Y in Sequence ID X, with the amino acid sequence represented by Sequence ID X being the reference sequence. Those skilled in the art can understand the shift in amino acid positions due to differences in HBV genotypes through appropriate sequence alignment.

[0058] "Identity" refers to the percentage of bases or amino acids that match the same base or amino acid in the reference sequence at that sequence position. Two or more sequences are considered identical if they have bases or amino acids of the same length and order. When evaluating identity, the sequences being compared are assumed to have the same length, i.e., the length of the longest sequence among those being compared. This means that a first sequence consisting of nine bases or amino acids is 90% identical to a second sequence consisting of ten bases or amino acids that includes the first sequence. The identity of amino acid sequences or base sequences can be determined using the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90:5873-7) or software such as CrystalW2 provided by the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI).

[0059] In one embodiment, the immunogenic composition of the present invention may contain a polynucleotide encoding the polypeptide of the present invention (the polynucleotide of the present invention).

[0060] "Polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogues, or mixtures thereof. This includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid (DNA) and triple-stranded, double-stranded, and single-stranded ribonucleic acid (RNA). It also includes polynucleotides in modified and unmodified forms, as well as cyclic polynucleotides. Examples include polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides such as tRNA, rRNA, hRNA, siRNA, and mRNA containing D-ribose, other types of polynucleotides that are N-glycosides or C-glycosides of purine or pyrimidine bases, and other polymers containing non-nucleotide backbones. Polymers include polyamides such as peptide nucleic acid PNA, polymorpholinopolymers, and other synthetic sequence-specific nucleic acid polymers. mRNA includes IVT mRNA, self-replicating RNA, and replicon RNA. Self-replicating RNA and replicon RNA include ssRNA viruses such as positive-strand ssRNA viruses.

[0061] The polynucleotide of the present invention is preferably a ribonucleotide or a deoxyribonucleotide, and more preferably mRNA. Here, the polynucleotide of the present invention means a non-natural polynucleotide, and the mRNA of the present invention means a non-natural mRNA. A non-natural mRNA means mRNA that differs from the wild-type sequence found in nature by at least one nucleotide. The mRNA is produced by existing gene modification technology. In one embodiment, the non-natural mRNA contains at least one modified base.

[0062] The polynucleotide of the present invention may contain at least one chemical modification. Any chemical modification of polynucleotides known in the art can be used in the polynucleotide of the present invention. Examples include modified phosphate groups, modified bases, and sugar modifications.

[0063] Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramides, alkyl or aryl phosphonates, and phosphotryesters. In phosphorothioates, both unbound oxygen atoms are substituted with sulfur. The phosphate linker may also be modified by substituting the bound oxygen atoms with nitrogen (bridged phosphoramide), sulfur (bridged phosphorothioate), and carbon (bridged methylene phosphonate).

[0064] In one embodiment, the immunogenic composition comprising a polynucleotide encoding the polypeptide of the present invention may be mRNA encoding the polypeptide of the present invention. The mRNA may be mRNA (mRNA of the present invention) consisting of a 5' Cap structure, a 5' UTR, an open reading frame encoding the polypeptide of the present invention, a 3' UTR, and Poly(A).

[0065] Examples of mRNA modification bases of the present invention include 5-methyluridine, pseudouridine (Ψ), N1-methylpseudridine (m1Ψ), 5-methoxyuridine, 2-thiouridine, 6-methyladenosine, 2-aminoadenosine, inosine, 5-methylcytidine, N1-ethylpseudridine, 4'-thiouridine, 2-thio-1-methyl-1-deazapseudridine, 2-thio-1-methylpseudridine, 2-thio-5-azapseudridine, 2-thio-dihydropseudridine, 2-thio-dihydropseudridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-azapseudridine, and dihydropseudridine. Furthermore, International Publication Nos. 2001 / 79502, 2002 / 98443, 2009 / 127230, US20100249219, 2012 / 019168, 2012 / 138453, 2013 / 143698, 2015 / 089511, US20150056253, and 2015 Modified bases described in publications such as 077123, 2016 / 209966, 2017 / 070613, US20180126003, 2020 / 243002, 2021 / 198258, 2021 / 251453, 2022 / 233880, and 2023 / 006999 are also available. The polynucleotide of the present invention may contain multiple types of modified bases.Preferably, the mRNA modification bases of the present invention are 5-methyluridine, pseudouridine, N1-methylpseudridine (m1Ψ), 5-methoxyuridine, 2-thiouridine, 6-methyladenosine, 2-aminoadenosine, inosine, 5-methylcytidine, N1-ethylpseudridine, 4-thiouridine, 2-thio-1-methyl-1-deazapseudridine, 2-thio-1-methylpseudridine, 2-thio-5-azapseudridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-azapseudridine, dihydropseudridine, 2'-O-methyluridine, or combinations thereof. More preferably, the modified base is selected from 5-methyluridine, 5-methylcytidine, and N1-methylpseudridine, or a combination thereof. Particularly preferred is N1-methylpseudridine.

[0066] Examples of the sugar modification include nucleosides having a substituent at the 2'-position of the sugar and / or nucleosides having a cross-linked structure between the 4'-position and the 2'-position of the sugar. Examples of the substituent of the 2'-hydroxy group of the sugar include an oxy or deoxy substituent. Examples of the "oxy" modification include an alkoxy group or an aryloxy (-OR, for example, R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), polyethylene glycol (PEG), and aminoalkoxy. Examples of the "deoxy" modification include hydrogen and amino, halogen (for example, fluorine, etc.). Examples of the amino include alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, and amino acid. The amino may be bonded to the sugar via a linker, and the linker contains one or more of atoms C, N, and O. Examples of the cross-linked structure include BNA (Bridged Nucleic Acid). Examples of BNA include AmNA (Amido-bridged nucleic acid, see International Publication No. WO2011 / 052436), TrNA (Triazole-Bridged Nucleic Acid, see International Publication No. WO2014 / 126229), 4'-(CH ２ )n-O-2' (n is an integer from 1 to 4), 4'-(CH ２ )m-C(=O)-NR １ -2' (m is an integer from 0 to 4, R １ is a hydrogen atom or an alkyl). Specific examples and their preparation methods are described in International Publication Nos. WO98 / 39352, WO2003 / 068795, WO2005 / 021570, WO2011 / 052436, and WO2013 / 052523, etc.

[0067] Modifications of nucleotides known in this field and methods of modification are disclosed in the following patent documents, for example: International Publication No. 98 / 39352, International Publication No. 99 / 014226, International Publication No. 2000 / 056748, International Publication No. 2003 / 068795, International Publication No. 2004 / 016749, International Publication No. 2005 / 021570, International Publication No. 2005 / 083124, International Publication No. 2007 / 143315, International Publication No. 2009 / 071680, International Publication No. 2011 / 052436, International Publication No. 2014 / 112463, International Publication No. 2014 / 126229, etc.

[0068] "5'UTR" is synonymous with "5'-untranslated region" and refers to the mRNA region located between the 5'Cap structure and the start codon on the mRNA translated by the ribosome. Typically, it refers to the region from the nucleotide adjacent to the 3' end of the 5'Cap structure to the nucleotide adjacent to the 5' end of the start codon on the open reading frame (ORF). The 5'UTR may include gene expression regulatory elements or protein binding regions. Examples of regulatory elements include ribosome binding sites, miRNA binding sites, or 5'-terminal oligopyrimidine tracts.

[0069] The 5'UTR of the mRNA of the present invention may be a polynucleotide consisting of a base sequence that is identical to any of the base sequences described in SEQ ID NOs. 154 to 224, or a base sequence having 80%, 85%, 90%, or 95% or more identity. Preferably, it is a polynucleotide that is identical to the base sequence described in any of SEQ ID NOs. 154, 155, and 217 to 224.

[0070] "3'UTR" is synonymous with "3'-untranslated region" and refers to the mRNA region located downstream (3' side) of the stop codon on mRNA translated by ribosomes. The 3'UTR may include gene expression regulatory elements, protein binding regions, or PolyA addition signal sequences. Examples of regulatory elements include ribosome binding sites and miRNA binding sites.

[0071] The 3' UTR of the mRNA of the present invention may be a polynucleotide consisting of a base sequence that is identical to any of the base sequences described in SEQ ID NOs. 225 to 273, or has 80%, 85%, 90%, or 95% or more of that identity. Preferably, it is a polynucleotide identical to the base sequence described in SEQ ID NOs. 225, 226, 227, or 232. The 3' UTR of the mRNA of the present invention may contain a short base sequence, such as a restriction enzyme cleavage sequence derived from a cloning site like NheI, at its 3' end.

[0072] "Poly(A)," also known as the PolyA tail or 3'-PolyA tail, refers to a chain of adenosine nucleotides approximately 10 to 500 mers long located directly downstream (i.e., on the 3' side) of the mRNA's 3' UTR. The Poly(A) tailing reaction for adding Poly(A) does not always yield an adenine nucleotide of the exact desired length, which can vary by about 10 mers from the desired length. For example, if Poly(A) is described as 80 mers in this specification, then Poly(A) of about 60 to 100 mers, preferably about 70 to 90 mers, 75 mers, 76 mers, 77 mers, 78 mers, 79 mers, 80 mers, 81 mers, 82 mers, 83 mers, 84 mers, or 85 mers is within the range of mRNA of the present invention.

[0073] In one embodiment, the mRNA of the present invention may have an amino-terminal enhancer of split (AES) sequence, a mitochondrial code 12S ribosomal RNA sequence, an A30LA70 sequence, etc., inserted between ORF and Poly(A). In another embodiment, the mRNA of the present invention may have a mask structure of nucleotides other than adenyl(A) residues, INVERTED-dT, 2'-modified RNA, etc., inserted 3'-terminally to Poly(A). The phosphate diester portion of Poly(A) may have a phosphorothioate, phosphorodithioate, phosphoroserenate, or phosphorodiserenate bond for the purpose of improving mRNA stability. In another embodiment, the mRNA of the present invention may have Poly(A) and / or a histone stem loop at the 3' terminal, or may be present in combination with other elements such as a purine-rich region (hitone downstream element, or HDE).

[0074] The Poly(A) of the mRNA of the present invention is preferably an adenosine nucleotide of 20 to 250 mer, more preferably 60 to 160 mer, and particularly preferably 75 to 85 mer or 80 mer.

[0075] An "Open Reading Frame (ORF)" refers to the base sequence from a start codon to a stop codon. The stop codon may contain one or more, and may contain any number of UAA, UAG, or UGA codons. The ORF may have all convertible codons converted and may be optimized using an optimization algorithm so that the mRNA forms a stable secondary structure. Codon optimization tools, algorithms, and services are known in the art and include GeneArt's services (Life Technologies), DNA2.0 (Menlo Park CA), ViennaRNA Package 2.0 (University of Vienna), and LinearDesign (Baidu).

[0076] Examples of ORF base sequences for the polynucleotides of the present invention include base sequences in which the expression level of the protein encoded by the ORF is increased by increasing the G / C content or optimizing the codons without changing the amino acid sequence from the wild-type sequence. In some embodiments of the mRNA of the present invention, the G / C content of the ORF is increased by, for example, 5% or more, 10% or more, 15% or more, or 20% or more compared to the G / C content of the ORF of wild-type HBV mRNA.

[0077] The ORF of the HBp polynucleotide of the present invention encodes the HBp polypeptide of the present invention. Examples include polynucleotides consisting of a base sequence that is identical to, or has 80%, 85%, 90%, or 95% or more identity with, any of the base sequences described in SEQ ID NOs. 280, 281, 284-297, and 403-406. The ORF of the HBs polynucleotide of the present invention encodes the HBs polypeptide of the present invention. Examples include polynucleotides consisting of a base sequence that is identical to, or has 80%, 85%, 90%, or 95% or more identity with, any of the base sequences described in SEQ ID NOs. 305, 312-322, and 326-328. The ORF of the HBc polynucleotide of the present invention encodes the HBc polypeptide of the present invention. Examples include polynucleotides consisting of a base sequence that is identical to, or has 80%, 85%, 90%, or 95% or more identity with, any of the base sequences described in SEQ ID NOs. 329-347.

[0078] Examples of HBp mRNA for the present invention include HBp12, 13, 16-19, 26, 32-41, 43, 49-51, 55, 58, 60, 65-70, etc., as described in the examples. Examples of HBs mRNA for the present invention include HBs14, 39-45, 52, 61-63, 67-72, 74, 75, etc., as described in the examples. Examples of HBc mRNA for the present invention include HBc6, 8, 9, 12, 19, 20, 22, 23, 25, 26, 31-38, 41, 42, 53-69, 71, 72, etc., as described in the examples.

[0079] 5'Cap refers to both native 5'Cap and modified 5'Cap. Native 5'Cap refers to the ribose-guanosine residue (m7G) with a methylated 7' position of the guanine base, which is present in all eukaryotes. m7G is linked to the 5'-terminal nucleotide of mRNA via a 5'-5'-triphosphate bond (hereinafter referred to as ppp). 5'Cap contributes to the efficient translation of mRNA-encoded proteins by protecting mRNA from degradation by exonucleases, promoting the transport of mRNA from the nucleus to the cytoplasm, and playing a crucial role in the assembly of the translation initiation complex. Modified 5'Cap refers to 5'Cap that has undergone some chemical modification to the native 5'Cap, resulting in a 5'Cap that has equivalent or superior function to the native 5'Cap in terms of protein translation.

[0080] Examples of modified 5'Cap include modified guanosine (hereinafter referred to as [G]), and structures described in International Publication Nos. 2017 / 066793, 2023 / 007019, CN116987137, and 2023 / 246860. Examples of nucleosides in [G] include guanosine with methylation at position 7 of guanine and position 3' of ribose (m7G-3'OMe), guanosine with methylation at position 7 of guanine residue and position 2' of ribose (m7G-2'OMe), and other guanine modifications and sugar modifications. Examples of guanine modifications include hypoxanthine, N1-methyl-guanine, 7-deaza-guanine, and 8-oxo-guanine. Examples of sugar-modified riboses include riboses modified at the 2' and / or 3' positions, such as 2'-fluororibose, 2'-aminoribose, and 2'-azidribose, BNA (Bridged Nucleic Acid) with a cross-linking structure, and modified riboses described in International Publication No. 2014 / 081507.

[0081] The natural 5'Cap, m7G, and the modified 5'Caps, m7G-3'OMe and m7G-2'OMe, are specifically represented by the following structures.

[0082] In this specification, the "5' Cap structure" refers to the 5'-[G]pppN structure located at the 5' end of mRNA. １ N ２ N ３ (N １ , N ２ , N ３ This means that is any nucleoside. 5' Cap structure is Cap0, Cap1 (N １ This is a structure in which the 2' position is methylated, and 5'-[m7G]-ppp-N １ mN ２ N ３ It is expressed as follows: ), Cap2(N １ And the 2' position of N2 is methylated, 5'-[m7G]-ppp-N １ mN ２ mN ３ It is expressed as follows: ), Cap3(N １ , N ２ and N ３ This is a structure in which the 2' position is methylated, and 5'-[m7G]-ppp-N １ mN ２ mN ３ It is represented as m.) and others. Specifically, Cap1 is represented by the following structure. Base in the chemical formula refers to any base. For example, depending on the capping method, it can take the form of GGG, AGG, AUA, etc. Furthermore, it has been confirmed that the expression levels of mRNA having a 5'Cap structure represented as 5'-[m7G]-ppp-GmGG, 5'-[m7G]-ppp-AmGG, or 5'-[m7G]-ppp-AmUA are equivalent in HEK293 cells and in mice. Cap2 is represented by the following structure. In the chemical formula, Base refers to any base. Cap3 is represented by the following structure. In the chemical formula, Base refers to any base.

[0083] The 5'-5'-triphosphate bond (ppp) linking the 5'Cap to the 5'-terminal nucleotide of mRNA can be a modified phosphate group such as phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or arylphosphonate, or phosphotriester.

[0084] The 5' Cap structure of the mRNA in the present invention is preferably Cap1 or Cap2, and particularly preferably Cap1. The 5' Cap structure of the mRNA in one embodiment of the present invention is 5'-[G]pppN, depending on the reagent used for capping. １ N ２ N ３ N inside １ N ２ N ３ This can be GGG, AGG, AGA, AUG, GAA, or AUA. Therefore, the mRNA base sequence in one embodiment of the present invention begins with 5'-GGGG, 5'-GAGG, 5'-GAGA, 5'-GAUG, 5'-GGAA, or 5'-GAAUA, followed by a 5'UTR sequence.

[0085] The mRNA of the present invention may have an internal ribosome entry site (IRES) instead of a 5' Cap structure. Preferred IRESs include those derived from picornavirus, plague virus, poliovirus, encephalomyocarditis virus, foot-and-mouth disease virus, hepatitis C virus, swine cholera virus, mouse leukemia virus, simian immunodeficiency virus, and cricket paralysis virus.

[0086] The polynucleotides of the present invention can be synthesized by conventional methods. For example, they can be synthesized by chemical synthesis, enzymatic synthesis generally referred to as in vitro transcription (IVT), or enzymatic or chemical cleavage of longer precursors, but are not limited to these methods. For example, see this specification and Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984, and Herdewijn, P. This is disclosed in (ed.) Oligoncleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.; Totowa, N.J.; Humana Press, 2005), etc.

[0087] The mRNA of the present invention can be synthesized by conventional methods. For example, the mRNA of the present invention can be synthesized by performing IVT based on a template DNA encoding the mRNA sequence of the present invention. When synthesizing mRNA by IVT, template DNA is required. Template DNA can be a plasmid linearized with restriction enzymes, or a DNA fragment produced by PCR. The template DNA of the mRNA of the present invention includes, from the 5′ end, an RNA polymerase promoter sequence (e.g., T7 promoter sequence, SP6 promoter sequence, T3 promoter sequence, etc.), a 5′-UTR, an ORF region, and a 3′-UTR. The template DNA may further include a portion corresponding to Poly(A), and by including a portion corresponding to Poly(A) in the DNA template, the portion corresponding to Poly(A) is synthesized in the RNA synthesis reaction by RNA polymerase. Alternatively, as a method for adding Poly(A) to mRNA, the 3′ end of the mRNA can be polyadenylated after IVT using an enzyme such as E. coli Poly(A) polymerase.

[0088] The RNA polymerase added to the IVT reaction solution can be appropriately selected depending on the promoter sequence of the template DNA. For example, if the template DNA has a T7 promoter sequence, it is preferable to use a T7 RNA polymerase that recognizes that sequence. If the transcription template has an SP6 or T3 promoter sequence, it is preferable to use an SP6 RNA polymerase or a T3 RNA polymerase, respectively.

[0089] The polynucleotides of the present invention may have known ligands attached to them. Ligands known in the art can be used to enable tracking of the polynucleotides of the present invention, to improve the pharmacokinetics or pharmacodynamics of the polynucleotides of the present invention, to improve the stability or binding affinity of the polynucleotides of the present invention, to improve the intracellular dynamics of the polynucleotides of the present invention, including intracellular uptake, or to be components of transport carriers (liposomes, lipid nanoparticles (LNPs), polymers, micelles, virus particles, etc.) consisting of one or more types of molecules. Examples include nucleic acids, reporter molecules, lipids (fatty acids, fatty acid chains, cholesterol, phospholipids, etc.), sugars (N-acetylgalactosamine, etc.), vitamins, peptides (membrane-permeable peptides, cell-targeting peptides, receptor-binding peptides, endosome escape-promoting peptides, RGD peptides, peptides with high affinity for blood components, tissue-targeting peptides, etc.), PEG (polyethylene glycol), dyes, fluorescent molecules, etc.

[0090] In one embodiment, the immunogenic composition containing the polypeptide of the present invention may be a composition containing one or more of the following: a) the HBs polypeptide of the present invention, b) the HBp polypeptide of the present invention, and c) the HBc polypeptide of the present invention. For example, it may be an immunogenic composition containing any of a), b), or c), an immunogenic composition containing a) and b), a) and c), or an immunogenic composition containing a), b), and c). Preferably, the immunogenic composition containing the polypeptide of the present invention is a composition containing a) the HBs polypeptide of the present invention, b) the HBp polypeptide of the present invention, and c) the HBc polypeptide of the present invention. When the immunogenic composition containing the polypeptide of the present invention includes multiple types of polypeptides and transport carriers, each polypeptide may be encapsulated in the same transport carrier, each polypeptide may be encapsulated in its respective transport carrier and then mixed, or a fusion of each polypeptide may be encapsulated in a transport carrier.

[0091] In one embodiment, the immunogenic composition containing the polynucleotide of the present invention may be a composition comprising one or more of the following: A) a polynucleotide encoding the HBs polypeptide of the present invention, B) a polynucleotide encoding the HBp polypeptide of the present invention, and C) a polynucleotide encoding the HBc polypeptide of the present invention. For example, the immunogenic composition may comprise any of A), B), or C), or A) and B), A) and C), or B) and C), or A), B), and C). Preferably, the immunogenic composition containing the polynucleotide of the present invention is a composition comprising A) a polynucleotide encoding the HBs polypeptide of the present invention, B) a polynucleotide encoding the HBp polypeptide of the present invention, and C) a polynucleotide encoding the HBc polypeptide of the present invention. When the immunogenic composition containing the polynucleotide of the present invention comprises multiple types of polynucleotides and transport carriers, each polynucleotide may be encapsulated in the same transport carrier, each polynucleotide may be encapsulated in its respective transport carrier and then mixed, or a fusion of each polynucleotide may be encapsulated in a transport carrier.

[0092] In one embodiment, the immunogenic composition of the present invention may further contain an adjuvant. Any adjuvant known in the art can be used as the adjuvant.

[0093] Preferred adjuvants include those containing squalene, tocopherol, and polysorbate 80. Specifically, A-910823, a squalene-based adjuvant, is an example. Details of A-910823 are described in Reference 1: Front Immunol. 2023 Feb 20;14:1116238.

[0094] Preferred adjuvants include an adjuvant comprising an aluminum salt (Alum) and an oligodeoxynucleotide (ODN2006), or an adjuvant comprising an aluminum salt (Alum) and an oligodeoxynucleotide (CpG1018).

[0095] The polypeptide of the present invention can be produced by known genetic engineering techniques. For example, it may be expressed using a host such as cultured cells derived from mammals or insects, or methods well known to those skilled in the art can be used.

[0096] In addition to the modification method and ligand attachment method described above, any method of administration and formulation known in the art can be used for the immunogenic composition of the present invention. For example, transport carriers consisting of a single molecule or multiple molecules (liposomes, LNPs, polymers, micelles, virus particles, etc.) can be used. Other options include polysomes, lipoplexes, etc.

[0097] The dosage form of the immunogenic composition of the present invention can be selected as appropriate. It may be a liquid formulation, a powder formulation, or a lyophilized formulation.

[0098] The immunogenic composition of the present invention can be administered via any route as appropriate, and may be administered orally or parenterally. Parenteral administration methods include intramuscular, intraperitoneal, or subcutaneous injection, intravenous injection or drip infusion, pulmonary administration by aspiration or inhalation, subdural administration, and intraventricular administration. Intramuscular injection is preferred as the parenteral administration method.

[0099] When administering the immunogenic composition of the present invention topically, formulations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders can be used. Examples of compositions for oral administration include powders, granules, suspensions or solutions dissolved in water or a non-aqueous medium, capsules, powders, and tablets. Examples of compositions for parenteral, subdural, or intraventricular administration include sterile aqueous solutions containing buffers, diluents, and other suitable additives.

[0100] Depending on the purpose and application, the immunogenic composition of the present invention may contain, as appropriate, buffering agents, isotonic agents, analgesics, preservatives, antibacterial agents, antioxidants, pH adjusters, dispersants, fragrances, colorants, defoaming agents, and the like.

[0101] The dosage of the immunogenic composition of the present invention is appropriately selected depending on the type of active ingredient, route of administration, target population, patient's age, weight, sex, symptoms, and other conditions.

[0102] The immunogenic composition of the present invention may be administered as a combination agent in combination with other agents for the purposes of (1) complementing and / or enhancing the therapeutic and / or prophylactic effects of the immunogenic composition of the present invention; (2) improving the kinetics, absorption, and dosage of the immunogenic composition of the present invention; and / or (3) reducing the side effects of the immunogenic composition of the present invention.

[0103] The immunogenic composition of the present invention can be used as a therapeutic and / or prophylactic agent for HBV infection. It is particularly useful as a therapeutic and / or prophylactic agent for chronic HBV infection.

[0104] HBV infection refers to diseases caused by HBV infection, such as hepatitis B (including acute hepatitis, chronic hepatitis, or fulminant hepatitis), hepatitis D (including acute hepatitis, chronic hepatitis, or fulminant hepatitis), delayed-onset liver failure, cirrhosis, and liver cancer.

[0105] The immunogenic compositions of the present invention possess any or all of the following excellent characteristics: a) They promote, induce, and / or trigger an immunogenic response to various genotypes of HBV (particularly genotypes A, B, C, and D). b) They promote, induce, and / or trigger an immunogenic response even when multiple types of immunogenic compositions are combined. c) They promote, induce, and / or trigger an immune response at low doses due to improved immunogenicity. d) They promote, induce, and / or trigger an immune response with two or three low-frequency doses due to improved immunogenicity. e) They neutralize the infectivity of HBV and / or inhibit and / or eliminate HBV infection through an immunogenic response to HBV.

[0106] "Immune response" refers to the reaction of immune system cells such as B cells, T cells, and monocytes to stimulation. An immune response may also be a B cell response that produces specific antibodies such as antigen-specific neutralizing antibodies, and CD4 ＋ Response and / or CD8 ＋ This may also include T cell responses such as a response. "Induction of immune response" means induction, promotion, or stimulation of an immune response. The immunogenic composition of the present invention can induce an excellent immune response and can therefore prevent HBV infection in humans, delay the progression of HBV infection, block HBV infection, and / or recover from HBV infection. The ability to induce an immune response can be measured, for example, by the method described in this embodiment.

[0107] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0108] Example 1: Obtaining Consensus Sequences (1-1) Consensus sequences of HBp, HBs, and HBc polypeptides were obtained based on the amino acid sequence alignment of antigens of genotypes A, B, C, and D obtained from the Hepatitis B Virus Database (HBVdb). A consensus sequence of a portion of the HBc polypeptide was obtained based on the amino acid sequence alignment of genotypes A, B, C, D, E, F, G, and H.

[0109] (1-2) The obtained HBp consensus sequence was modified by introducing D205N and D206N mutations (i.e., YMNN mutations (SEQ ID NO: 384) of the YMDD motif (SEQ ID NO: 382) that inactivate the RTase domain, and D354N and E383Q mutations (i.e., NQDD mutations (SEQ ID NO: 407) of the DEDD motif (SEQ ID NO: 389) that inactivate the RNaseH domain), and IgE was added to the N-terminus to obtain P-HBp5. The amino acid positions correspond to the amino acid sequence of SEQ ID NO: 65. P-HBp5 was modified by signal peptide conversion, amino acid substitution, and deletion of some domains to obtain the P-HBp sequences shown in Table 1 (see also Figure 1). In Table 1, plus (+) or minus (-) indicates the presence or absence of a domain. In the terminal domain column, [FULL] refers to the full-length (177 amino acid) terminal domain polypeptide, and [20aa] refers to the polypeptide consisting of the 20 amino acids at the N-terminus of the terminal domain. The same applies to others. Note that HBp polypeptides other than P-HBp40 and P-HBp41 have a His tag (SEQ ID NO: 77) added to their C-terminus. Methionine (M) is omitted from the N-terminus of the sequences listed in the Full length column. In Table 2, amino acids substituted from the consensus sequence P-1, which consists of the RTase and RNaseH domains of P-HBp5, other than the signal peptide, are shown underlined and in bold, and the amino acid positions correspond to SEQ ID NO: 65. Among the polypeptides listed in Table 1, polypeptides in which the RTase & RNaseH domain pattern is P-2, P-3, P-6, or P-7 are included in the HBp polypeptides of the present invention.

[0110]

[0111]

[0112] (1-3) P-HBs8 was obtained by adding IgE to the N-terminus of the consensus P-HBs sequence obtained by the method described in (1-1). The P-HBs sequences shown in Table 3 were designed by converting the signal peptide, substituting amino acids, deleting some domains, etc., on P-HBs8 (see also Figure 2). Note that a His tag (SEQ ID NO: 77) is added to the C-terminus of each HBs polypeptide. Methionine (M) is omitted from the N-terminus of the sequences listed in the Full length column. In Table 4, amino acids other than the signal peptide that were substituted from the consensus sequence S-1 consisting of the full length of P-HBs8 are shown underlined and in bold, and the amino acid positions correspond to SEQ ID NO: 351. The plus (+) or minus (-) in Table 4 means that the amino acids corresponding to positions 1 to 11 of the amino acid sequence of SEQ ID NO: 351 are present or absent. Among the polypeptides listed in Table 3, polypeptides having a full length of S-3, 8-11, or 15-17 are included in the HBs polypeptides of the present invention.

[0113]

[0114]

[0115] (1-4) P-HBc8 was obtained by adding IgE to the N-terminus of the consensus P-HBc sequence obtained by the method described in (1-1). The P-HBc sequences shown in Table 5 were designed by converting the signal peptide, substituting amino acids, deleting some domains, etc., on P-HBc8 (see also Figure 3). Note that a His tag (SEQ ID NO: 77) is added to the C-terminus of each HBc polypeptide. Methionine (M) is omitted from the N-terminus of the sequences listed in the Full length column. All polypeptides listed in Table 5 are included in the HBc polypeptides of the present invention.

[0116] Tables 6 and 7 show the correspondence between signal peptides or functional sequences and their sequence numbers.

[0117]

[0118]

[0119] Example 2 mRNA Synthesis (2-1) mRNA encoding the consensus sequence described in Example 1 was synthesized by the method shown below. Plasmid DNA was amplified and purified by PCR to prepare template DNA for use in IVT. A DNA fragment containing a sequence in which the T7 promoter sequence, 5'UTR sequence, ORF, and 3'UTR sequence are sequentially linked was introduced into the plasmid. DNA was amplified using 10 μM sense primers (SEQ ID NOs. 348, 350, or 390) and 10 μM antisense primers (SEQ ID NOs. 349 or 391). After the reaction, the template DNA was purified. mRNA synthesis from the template DNA was performed by the method of (2-2) or (2-3).

[0120] (2-2) The template DNA obtained in (2-1), 75 mM ATP, 75 mM UTP, 75 mM GTP, 75 mM CTP or 100 mM 5-Methyl-CTP, and 100 mM N1-Methylpseudo-UTP or 100 mM 5-Methyl-UTP, nucleotide-free water, 10X reaction buffer, and Enzyme mix T7 RNA Polymerase (Thermo Fisher catalog #AMB13345) were mixed and incubated at 37°C for 2 hours. TURBO® DNase (Thermo Fisher catalog #AM2238) was mixed and incubated at 37°C for 15 minutes. A 7.5 M LiCl solution was mixed in and left to stand overnight at -20°C. After centrifugation, the supernatant was discarded. The residue obtained by ethanol precipitation was dissolved in nucleotide-free water. A portion of the resulting solution was mixed with nucleotide-free water, 10 mM GTP, 20 mM SAM, 10X Capping Buffer, Vaccina Capping Enzyme (all from New England Biolab catalogo #M2080), and mRNA Cap2'-O-Methyltransferase (New England Biolab), and incubated at 37°C for 2 hours. A 7.5 M LiCl solution was added, and the mixture was left to stand overnight at -20°C. After centrifugation, the supernatant was discarded. The residue obtained by ethanol precipitation was dissolved in nuclease-free water. The resulting solution was then mixed with 10X Antarctic Phosphatase Reaction Buffer (New England Biolab) and Antarctic Phosphatase (New England Biolab). After incubation at 37°C for 30 minutes, the target mRNA was obtained by generating it according to the included manual using the Monarch® RNA Cleanup Kit (New England Biolab). The obtained mRNA was analyzed using TapeStation (Agilent) to confirm that it was of the desired length.

[0121] (2-3) The template DNA obtained in (2-1), 75 mM ATP, 75 mM UTP, 75 mM GTP, 75 mM CTP or 100 mM 5-Methyl-CTP, 100 mM N1-Methylpseudo-UTP or 100 mM 5-Methyl-UTP, CleanCap® AG (TriLink) or CleanCap® AU (TriLink), Nuclear-free water, 10X Reaction Buffer, and Enzyme mix T7 RNA Polymerase were mixed and incubated at 37°C for 2 hours. TURBO® DNase was mixed and incubated at 37°C for 15 minutes. A 7.5 M LiCl solution was mixed and left to stand overnight at -20°C. After centrifugation, the supernatant was discarded. The residue obtained by ethanol precipitation was dissolved in nucleotide-free water. A portion of the resulting solution was mixed with 10X Antarctic Phosphatase Reaction Buffer and Antarctic Phosphatase, incubated at 37°C for 30 minutes, and then the target mRNA was obtained by generating it according to the included manual using the Monarch® RNA Cleanup Kit. The obtained mRNA was analyzed using TapeStation and confirmed to be of the target length.

[0122] Example 3 mRNA Synthesis mRNAs containing 5'UTR, ORF, 3'UTR, Poly(A), and modified bases as described in Tables 8-1 to 8-10 were synthesized using the method of Example 2. The Polypeptide column in the tables refers to the encoded polypeptide and corresponds to Tables 1, 3, and 5. For Tables 8-1, 8-2, and 9, the mRNAs encoding "HBp polypeptide of the present invention" in Table 1 and "HBs polypeptide of the present invention" in Table 3 are included in the mRNA of the present invention (also referred to as "HBp mRNA of the present invention" and "HBs mRNA of the present invention," respectively). All mRNAs in Table 10 are the mRNA of the present invention (also referred to as "HBc mRNA of the present invention"). The 5' Cap structure of each mRNA is Cap1, and is either 5'-[m7G]-ppp-GmGG, 5'-[m7G]-ppp-AmGG, or 5'-[m7G]-ppp-AmUA. Here, Gm indicates guanosine with methylated 2' position. Specifically, it is represented by the following structure. In some mRNA sequences, all uridine bases in the sequence are replaced with N1-methylpseudridine (m1Ψ) bases or 5-methyluridine (5MeU) bases, and in some sequences, all cytidine bases in the sequence are further replaced with 5-methylcytidine (5MeC) bases. For example, HBp5 is an mRNA consisting of a Cap1 structure, a 5' UTR represented by SEQ ID NO: 155, an ORF represented by SEQ ID NO: 274, a 3' UTR represented by SEQ ID NO: 226, and approximately 80 mer of Poly(A) in the 5' to 3' direction.

[0123]

[0124]

[0125]

[0126]

[0127] Example 4 Preparation of mRNA-encapsulated nucleic acid lipid particles using mRNA Cationic lipids described in International Publication No. 2022 / 168884 (hereinafter referred to as Lipid), distearoylphosphatidylcholine (1,2-Distearoyl-sn-glycero-3-phosphacholine: hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (Cholesterol: hereinafter referred to as Chol, NIPPON FINE CHEMICAL CO., LTD.), and polyethylene glycol with a molecular weight of approximately 2000, 1,2-Dimyristoyl-sn-glycerol methoxypolyethylene glycol (1,2-Dimyristoyl-sn-Glycero-3-Methoxypolyethylene Glycol: hereinafter referred to as PEG-DMG, NOF A lipid solution was obtained by dissolving CORPORATION in ethanol. Meanwhile, one or more mRNAs from among HBp mRNA, HBs mRNA, and HBc mRNA synthesized in Example 3 were prepared to 292 μg / mL in acetic acid / sodium acetate buffer (pH 4.0, Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain an mRNA solution containing one type of mRNA or an mRNA mixed solution containing one or more types of mRNA. The above lipid solution and mRNA solution were mixed in NanoAssemblr® IGNITE in a volume ratio of 1:3. ＴＭ The nucleic acid lipid particle dispersion was mixed in a microfluidic channel using Precision Nanosystems Inc. to obtain a dispersion of nucleic acid lipid particles. The dispersion of nucleic acid lipid particles was dialyzed for 3 hours with approximately 300 times the volume of phosphate-buffered saline (pH 7.5, Thermo Fisher Scientific), and then dialyzed again for 12-18 hours with approximately 300 times the volume of phosphate-buffered saline (Slide-A-Lyzer). ＴＭ Ethanol was removed by Dialesis Cassette G2 (MWCO: 10 kD, Spectra / Po), the solution was concentrated using Amicon (Amicon® Ultra-15 PLHK Ultracel-PL membrane, MWCO 100 kDa, Merck), and the concentration was adjusted with phosphate-buffered saline to obtain a dispersion of mRNA-encapsulated nucleic acid lipid particles (RNA vaccine and RNA mixed vaccine).

[0128] Example 5 Evaluation of the ability to induce cellular immunity C57BL / 6 mice (7 weeks old, female) were inoculated twice at 3-week intervals into the hind limb thigh muscle with an RNA vaccine containing 25 μg of HBp mRNA, an RNA vaccine containing HBs mRNA, or an RNA vaccine containing HBc mRNA per mouse. Two weeks after the second inoculation, the mice were sacrificed and their spleens were collected. The spleens were homogenized in phosphate-buffered saline containing 1% fetal bovine serum, and splenocytes were collected. The splenocytes were passed through a 100 μm cell strainer and suspended in RPMI-1640 medium containing 10% fetal bovine serum. Splenocytes were placed in 96-well ELISPOT plates provided with the mouse IFNγ ELISPOT kit (3321-4APW-10, MABTECH) at a rate of 10 per well. ５ The cells were seeded at a specific cell density. The seeded splenocytes were stimulated by adding an antigen stimulant at a concentration of 60 μg / mL. The antigen stimulant was a solution prepared by dissolving partial peptides of HBV antigen derived from genotype A, genotype B, genotype C, genotype D, genotypes B and C, or genotypes A, B, C, and D in dimethyl sulfoxide. These stimulants are denoted as GtA, GtB, GtC, GtD, GtB / C, or Pep, respectively. Each partial peptide consists of 15 amino acids. The plates were kept in a humidified environment under 5% CO2. ２ The cells were cultured overnight at 37°C. After culturing, the produced IFNγ was detected according to the attached protocol. The generated spots were counted using an ELISPOT analyzer (ImmunoSpot S6 Micro, CTL). The number of spots in the wells to which dimethyl sulfoxide was added instead of the stimulant was subtracted from the number of spots in the wells to which the stimulant was added, and this value was expressed as the number of IFNγ-producing cells. Table 11 shows the origin of the partial peptides contained in the antigen stimulants used in the examples. The HBp, HBs, or HBc antigen stimulants are solutions in which 120, 53, or 25 partial peptides are dissolved in dimethyl sulfoxide, respectively. The antigen stimulants used in each example are shown in the figure.

[0129]

[0130] Example 6 Evaluation of HBp-specific immune cells induced by HBp mRNA administration (6-1) The immunogenicity of the RNA vaccine containing the HBp mRNA described in Example 3 was evaluated using the method described in Example 5. HBp12, HBp13, and HBp16 contained in the HBp mRNA of the present invention showed higher immunogenicity against various HBp antigens than HBp5, HBp14, and HBp15, which are reference examples (Figure 4). From these results, five amino acid substitutions (essential amino acid substitutions of HBp) corresponding to 124M, 145M, 151Y, 221Y, and 332S of SEQ ID NO: 65, which contribute to improving the immunogenicity of the HBp vaccine, were identified.

[0131] Using the same method as in (6-2) and (6-1), the HBp mRNA of the present invention showed excellent immunogenicity against various HBp antigens, regardless of the deletion or length of deletions in the terminal domain and spacer domain, and even when the encoded signal peptide was substituted (Figures 5 and 6). Note that all HBp mRNAs shown in Figures 5 and 6 are included in the HBp mRNA of the present invention. Using the same method, the HBp mRNA of the present invention showed excellent immunogenicity against various HBp antigens, regardless of the presence or absence of the encoded signal peptide. Therefore, it was shown that the HBp mRNA of the present invention has higher immunogenicity against multiple different genotypes of HBp antigens.

[0132] Based on evaluations similar to those in (6-3) and (6-1), HBp16 and HBp43, which are included in the HBp mRNA of the present invention and have essential amino acid substitutions of HBp, showed higher immunogenicity against various HBp antigens than HBp5 and HBp44-48 (Figure 7).

[0133] Example 7 Evaluation of HBs-specific immune cells induced by HBs mRNA administration The immunogenicity of the RNA vaccine containing the HBs mRNA described in Example 3 was evaluated using the method described in Example 5. HBs14, which encodes P-HBs with eight amino acid substitutions (essential amino acid substitutions for HBs) corresponding to 56K, 59A, 107L, 114T, 129T, 153A, 155N, and 171V in the amino acid sequence of SEQ ID NO: 351, showed higher immunogenicity against various HBs antigens than HBs14-18, which encodes P-HBs without amino acid substitutions (Figure 8). Note that all mRNAs other than HBs14 shown in Figure 7 are for reference only. Thus, it was shown that the HBs mRNA of the present invention has higher immunogenicity against multiple different genotypes of HBs antigens.

[0134] Example 8 Evaluation of anti-L-HBs antibody induction ability by HBs mRNA administration C57BL / 6 mice (7 weeks old, female) were inoculated with an RNA vaccine containing 25 μg of HBs mRNA per mouse three times at 3-week intervals into the hind limb thigh muscle. Blood was collected from the abdominal vena cava of the mice two weeks after the third inoculation. After the blood was allowed to stand at room temperature for 1 hour, it was centrifuged to obtain serum. Mouse serum diluted in 50 mM Tris-HCl buffer was added to a 384-well plate immobilized with goat anti-mouse IgG Fcγ fragment-specific antibody (115-005-008, Jackson ImmunoResearch), and the plate was shaken at room temperature for 2 hours. After removing the serum dilution, EZ-Link ＴＭL-HBs protein (BCL-AGC-01, Vehicle) labeled with Sulfo-NHS-LC-Biotin (21335, Thermo Scientific) and DELFIA® Eu-N1 Streptavidin (1244-360, Revvity) were added to a plate and left to stand overnight at 4°C. After removing the added solution, DELFIA® Enhancement Solution (4001-0010, Revvity) was added to the plate and shaken for 10 minutes. The fluorescence intensity of the wells was measured using a plate reader (ARVO X4, Perkin Elmer) according to the attached instructions. The mean and standard deviation of the fluorescence intensity in wells to which 50 mM Tris-HCl buffer was added instead of serum were calculated, and the value obtained by adding three times the standard deviation to this mean was set as the cutoff value (A). The measurement data from wells to which mouse serum was added was plotted on a logarithmic graph with serum dilution ratio on the X axis and fluorescence intensity on the Y axis, and the serum dilution ratio (B) that falls within the linear range was determined. The antibody titer was calculated by dividing the fluorescence intensity value at dilution ratio (B) by (A) and multiplying by (B). If the fluorescence intensity at dilution ratio (B) was lower than (A), the antibody titer of that mouse individual was taken as (B). HBs14, which encodes P-HBs with essential amino acid substitutions introduced, showed a higher antibody titer than HBs8, 15-18, which encodes P-HBs without essential amino acid substitutions (Figure 9). Note that all mRNAs other than HBs14 shown in Figure 8 are for reference only.

[0135] Example 9 Evaluation of HBV Neutralizing Activity To evaluate the HBV neutralizing activity of anti-L-HBs antibodies induced in mice by HBs mRNA administration, an in vitro infection evaluation system was created to infect cultured hepatocytes with HBV. If the anti-L-HBs antibodies contained in mouse serum have the effect of neutralizing HBV, the addition of serum inhibits HBV infection of hepatocytes, and the amount of HBV DNA in the culture medium decreases. Using this evaluation system, the neutralizing activity of mouse serum evaluated in Example 8 was measured. Mouse serum was diluted with PXB-cells medium (PPC-M100, Phoenix Bio). The diluted mouse serum and HBV infection source C_AT (GenBank #AB246345, Phoenix Bio, PPC-BC) were mixed and added to a fresh hepatocyte plate (PPC-P96, Phoenix Bio). The source of this HBV infection was donated by Professor Yasuhito Tanaka, Visiting Professor of Virology, Graduate School of Medicine, Nagoya City University. The plate was subjected to a humidified environment with 5% CO2. ２ The cultures were incubated at 37°C. Five days after the addition of serum and HBV infection source, all the culture medium was removed and new medium was added. The plates were incubated again. Ten days after the addition of serum and HBV infection source, all the culture medium was collected. The HBV DNA concentration in the collected medium was measured by quantitative PCR. The mean DNA concentration in the wells to which unvaccinated mouse serum was added was defined as 0% inhibition, and the DNA concentration at the detection limit of quantitative PCR was defined as 100% inhibition. The DNA concentration value (A) corresponding to 50% inhibition was calculated. The data for the wells to which mouse serum was added were plotted on a graph with the serum dilution ratio and the measured DNA concentration, and regression analysis was performed to calculate the serum dilution ratio that showed (A). This value was defined as the neutralizing activity titer. For mouse serum where the DNA concentration exceeded (A) at all dilution stages, the neutralizing activity titer was defined as the minimum dilution ratio. For mouse serum where the DNA concentration fell below (A) at all dilution stages, the neutralizing activity titer was defined as the maximum dilution ratio. HBs14, which encodes P-HBs with essential amino acid substitutions introduced, showed higher HBV neutralizing activity induced in mice than HBs8, 15-18, which encode P-HBs without essential amino acid substitutions (Figure 10). Note that all mRNAs other than HBs14 shown in Figure 10 are for reference only.

[0136] Example 10 Evaluation of HBc-specific immune cells by HBc mRNA administration The immunogenicity of the RNA vaccine containing the HBc mRNA described in Example 3 was evaluated using the method described in Example 5. HBc8, 31, and 42 contained in the HBc mRNA of the present invention all showed high immunogenicity against various HBc antigens (Figure 11).

[0137] Example 11 Detection of HBc antigen-specific cytotoxic T cells C57BL / 6 mice (7 weeks old, female) were inoculated twice at 3-week intervals into the hind limb thigh muscle with an RNA vaccine containing 25 μg of HBc mRNA per mouse. One week after the second inoculation, blood was collected from the mice using a heparin-containing capillary. Erythrocyte lysis buffer (555899, BD) was added to the collected blood and allowed to stand for 3 minutes, after which the buffer and erythrocytes were removed by centrifugation. The remaining cells were suspended in phosphate-buffered saline containing 1% fetal bovine serum. The cell suspension was stained at room temperature for 1 hour using PE-labeled HBV core antigen tetramer (TB-M537-1, MBL) and the dead cell staining dye eFluor® 780 (65-0865-18, Invitrogen). After staining, the cells were washed once with phosphate-buffered saline containing 1% fetal bovine serum. The washed cells were stained at room temperature for 15 minutes using Alexa Fluor® 647-labeled anti-CD8 antibody (K0227-A64, MBL), Brilliant Violet® 650-labeled anti-CD4 antibody (100469, BioLegend), and Brilliant Violet® 785-labeled anti-CD3 antibody (100355, BioLegend). After staining, the cells were washed once. The cells were passed through a 70 μm cell strainer and analyzed using a NovoCyte flow cytometer system (Agilent Technologies). The analytical data were analyzed using NovoExpress software (Agilent Technologies). The number of HBc antigen-specific cytotoxic T cells induced by the vaccine is measured using CD3 ＋ CD8 in T cells ＋ HBc Tetramer ＋The results are shown as a percentage of cells. All HBc mRNAs strongly induced HBc antigen-specific cytotoxic T cells (Figure 12). Note that all mRNAs shown in Figure 12 are included in the HBc mRNA of the present invention. Using a similar method, it was confirmed that HBc53-61, which have a UTR sequence different from that of HBc6, HBc62-63, which have modified bases of HBc54, and HBc64-66, which have a modified 5' Cap structure of HBc54, also induce highly antigen-specific cytotoxic T cells.

[0138] Example 12 Evaluation of the immune-inducing ability of the mixed vaccine C57BL / 6 mice (7 weeks old, female) were administered a total of 25 μg or 50 μg of the mixed vaccine per mouse to the thigh muscle of the hind limb twice at 3-week intervals. The mixed vaccine refers to the following RNA mixed vaccines or mixtures prepared by the method of Example 4: ・RNA mixed vaccine containing two or more types of HBp mRNA, HBs mRNA and HBc mRNA of the present invention ・A mixture containing two or more types of RNA vaccines containing HBp mRNA, RNA vaccines containing HBs mRNA and RNA vaccines containing HBc mRNA of the present invention ・A mixture containing an RNA mixed vaccine containing two or more types of HBp mRNA, HBs mRNA and HBc mRNA and an RNA vaccine containing one or more types of mRNA from HBp mRNA, HBs mRNA and HBc mRNA of the present invention The immune-inducing ability of the mixed vaccine was evaluated by the same method as in Examples 5, 8, 9 or 11. The HBp mRNA of the present invention refers to any of the mRNAs of HBp12, 13, 16-19, 26, 32-41, 43, 49-51, 55, 58, 60, and 65-70. The HBs mRNA of the present invention refers to any of the mRNAs of HBs14, 39-45, 52, 61-63, 67-72, 74, and 75. The HBc mRNA of the present invention refers to any of the mRNAs listed in Table 10. Note that the 3' end may or may not contain an His tag.

[0139] Example 13 Evaluation of HBs-specific immune cells induced by HBs mRNA administration The immunogenicity of the RNA vaccine containing the HBs mRNA described in Example 3 was evaluated using the method described in Example 11, but with BV421-labeled HBs antigen tetramer (TB-5110-4, MBL) instead of PE-labeled HBV core antigen tetramer (TB-M537-1, MBL). The HBs mRNA of the present invention strongly induced HBs antigen-specific cytotoxic T cells even when the encoded signal peptide was substituted or when the signal peptide was absent (Figure 13). Note that all HBs shown in Figure 13 are included in the HBs mRNA of the present invention.

[0140] Example 14 Evaluation of anti-L-HBs antibody induction ability by HBs mRNA administration The antibody induction ability of the HBs mRNA of the present invention, which encodes an HBs polypeptide containing the amino acid residues described in (1) a) above and amino acid substitutions in the S-HBs domain, was evaluated by the method described in Example 8. The HBs mRNA of the present invention showed high antibody titers even when encoding a polypeptide in which the amino acids in the S-HBs domain were substituted (Figure 14). Note that all HBs shown in Figure 14 are included in the HBs mRNA of the present invention.

[0141] The immunogenic composition of the present invention has the ability to induce immunity against HBV antigen, and is therefore very useful as a pharmaceutical for the treatment and / or prevention of HBV infection.

Claims

1. An immunogenic composition comprising one or more polypeptides selected from a) to c) below: a) an HBV surface protein polypeptide comprising a K residue at the amino acid position corresponding to position 56, an A residue at the amino acid position corresponding to position 59, an L residue at the amino acid position corresponding to position 107, a T residue at the amino acid position corresponding to position 114, a T residue at the amino acid position corresponding to position 129, an A residue at the amino acid position corresponding to position 153, an N residue at the amino acid position corresponding to position 155, and a V residue at the amino acid position corresponding to position 171 of the amino acid sequence of SEQ ID NO: 351; b) an HBV polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated ribonuclease H domain, and comprising an N residue at the amino acid position corresponding to position 124, an M residue at the amino acid position corresponding to position 145, a Y residue at the amino acid position corresponding to position 151, a Y residue at the amino acid position corresponding to position 221, and an S residue at the amino acid position corresponding to position 332 of the amino acid sequence of SEQ ID NO: 65; and c) An HBV core protein polypeptide comprising an amino acid sequence described in any of SEQ ID NOs. 368 to 380, or an amino acid sequence having 90% or more identity with an amino acid sequence described in any of SEQ ID NOs. 368 to 380.

2. The immunogenic composition according to claim 1, wherein the HBV surface protein polypeptide in a) is a polypeptide comprising an amino acid sequence described in any of SEQ ID NOs. 353, 358-361, and 365-367, or an amino acid sequence having 90% or more identity with the amino acid sequence described in any of SEQ ID NOs. 353, 358-361, and 365-367.

3. The immunogenic composition according to claim 1 or 2, wherein the HBV surface protein polypeptide in a) is a polypeptide to which a signal peptide consisting of any of the amino acid sequences described in SEQ ID NOs. 55-57, 124, 125, 135, and 137 is linked.

4. The immunogenic composition according to any one of claims 1 to 3, wherein the HBV polymerase polypeptide in b) is a polypeptide comprising a part of the terminal domain or the entire length.

5. The immunogenic composition according to any one of claims 1 to 4, wherein the HBV polymerase polypeptide in b) is a polypeptide comprising a part or the entire spacer domain.

6. The immunogenic composition according to any one of claims 1 to 5, wherein the HBV polymerase polypeptide in b) is a polypeptide comprising an N residue at the amino acid position corresponding to position 205 and an N residue at the amino acid position corresponding to position 206 of the amino acid sequence of SEQ ID NO:

65.

7. The immunogenic composition according to any one of claims 1 to 6, wherein the HBV polymerase polypeptide in b) is a polypeptide comprising an N residue at the amino acid position corresponding to position 354 and a Q residue at the amino acid position corresponding to position 383 of the amino acid sequence of SEQ ID NO:

65.

8. The immunogenic composition according to any one of claims 1 to 7, wherein the HBV polymerase polypeptide in b) is a polypeptide comprising an amino acid sequence described in any of SEQ ID NOs. 36, 37, 40-49 and 400-402 or an amino acid sequence having 90% or more identity with the amino acid sequence described in any of SEQ ID NOs. 36, 37, 40-49 and 400-402.

9. The immunogenic composition according to any one of claims 1 to 8, wherein the HBV polymerase polypeptide in b) is a polypeptide to which a signal peptide consisting of any of the amino acid sequences described in SEQ ID NOs. 55-57, 124, 125, 135, and 137 is linked.

10. The immunogenic composition according to any one of claims 1 to 9, wherein the HBV core protein polypeptide in c) is a polypeptide to which a signal peptide consisting of any of the amino acid sequences described in SEQ ID NOs. 55-57, 124, 125, 135, and 137 is linked.

11. An immunogenic composition comprising a polynucleotide having an open reading frame encoding a polypeptide according to any one of claims 1 to 10.

12. The immunogenic composition according to claim 11, wherein the polynucleotide is mRNA.

13. An immunogenic composition according to claim 11 or 12, comprising one or more polynucleotides selected from A) to C) below: A) A polynucleotide whose open reading frame comprises a nucleotide sequence described in any of SEQ ID NOs. 305, 312-322 and 326-328 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence described in any of SEQ ID NOs. 305, 312-322 and 326-328; B) A polynucleotide whose open reading frame comprises a nucleotide sequence described in any of SEQ ID NOs. 280, 281, 284-297 and 403-406 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence described in any of SEQ ID NOs. 280, 281, 284-297 and 403-406; and C) A polynucleotide whose open reading frame comprises a nucleotide sequence described in any of SEQ ID NOs. 329-347 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence described in any of SEQ ID NOs. 329-347.

14. An immunogenic composition according to any one of claims 1 to 13, which is a therapeutic agent and / or prophylactic agent for HBV infection.

15. A method for inducing an immune response to HBV, comprising administering to a subject in need a therapeutically effective amount of the immunogenic composition according to any one of claims 1 to 13.

16. An immunogenic composition according to any one of claims 1 to 13, used to induce an immune response to HBV in the treatment and / or prevention of HBV infection.

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