Oligonucleotides for use in modulating the immune response to hepatitis B virus infection

Abstract

This specification provides vaccine compositions comprising oligonucleotides and hepatitis B virus antigens, either present in the same composition or in separate compositions, that are useful for the prevention or treatment of hepatitis B virus infection in a target. The vaccine compositions may exert a sustained immunomodulatory effect, resulting in a reduction in HBV antigen levels in the infected target. Methods for preparing and using the vaccines are also provided herein.

Description

[Technical Field]

[0001] (Related applications) This application claims priority and benefits of PCT application number PCT / CN2023 / 090098, filed on 23 April 2023, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Hepatitis B is a viral infection that primarily attacks the liver and can cause both acute and chronic disease. The hepatitis B virus (HBV) is transmitted parenterally through contaminants such as blood and blood products and contaminated needles, sexually, and vertically from an infected or carrier mother to her offspring. The World Health Organization estimates that more than 2 billion people worldwide are infected, and that approximately 296 million people were living with chronic hepatitis B infection in 2019. In 2019, hepatitis B accounted for an estimated 820,000 deaths, primarily from cirrhosis and hepatocellular carcinoma (primary liver cancer). HBV is a leading cause of death globally (World Health Organization Fact Sheet, Hepatitis B, June 24, 2022).

[0003] HBV is a double-stranded hepatic-tropic virus that infects only humans and non-human primates. Viral replication occurs primarily in the liver, and to a lesser extent in the kidneys, pancreas, bone marrow, and spleen (Hepatitis B Virus Biology). (Microbiol Mol Biol Rev.64:2000;51-68.). Prophylactic vaccines are administered to healthy individuals to prevent the disease. Several prophylactic vaccines have been developed to prevent hepatitis B infection, but for example, Engerix-B (SmithKline Beecham), Recombivax HB (Merck), Heplisav-B (Dynavax), and GenHevac B (Sanofi Pasteur) face certain limitations, such as their ability to induce a sufficient immune response. The development of therapeutic vaccines to treat hepatitis B virus infection has been even more challenging. Generally, the goal of therapeutic vaccines is to stimulate and destroy the patient's own adaptive immune system against specific viral antigens. In such situations, the goal would be, for example, to improve the responsiveness of hepatitis B-specific T cells in chronically impaired hepatitis B virus-infected patients. To date, HBV vaccines have not been shown to have therapeutic potential to reduce, eliminate, or cure hepatitis B virus infection because they do not induce a sufficient immune response in HBV patients.

[0004] Effective prevention and long-term treatment of hepatitis B virus (HBV) infection using vaccines remains a challenge and an unmet need. This specification provides compositions and methods to address this need. [Overview of the Initiative]

[0005] In one embodiment, a hepatitis B virus (HBV) vaccine composition comprising at least one oligonucleotide and at least one HBV antigen is provided herein.

[0006] In another aspect, provided herein is a method of treating or preventing HBV infection, or an HBV-related disease, disorder, or condition in a subject, the method comprising administering to the subject a vaccine composition comprising at least one oligonucleotide and at least one HBV antigen.

[0007] In yet another aspect, provided herein is a method of treating or preventing HBV infection, or an HBV-related disease, disorder, or condition in a subject with a vaccine treatment, the method comprising administering to the subject at least one oligonucleotide and at least one hepatitis B virus (HBV) antigen.

[0008] In other aspects, kits and pharmaceutical compositions are provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] [Figure 1] A legend for nucleoside modifications at each position of the sequences shown in FIG. 2 is provided. The column titled "Example" provides non-limiting examples for each type of nucleoside modification. [Figure 2-1] A table of exemplary modified oligonucleotides of the present disclosure is shown. Modifications at each position of the modified oligonucleotide sequences are read using the legend of FIG. 1. AUS1010 to AUS1714 (SEQ ID NOs: 11 to SEQ ID NOs: 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NO: 10), also referred to as AUS1138, is a reference modified oligonucleotide sequence. [Figure 2-2] A table of exemplary modified oligonucleotides of the present disclosure is shown. Modifications at each position of the modified oligonucleotide sequences are read using the legend of FIG. 1. AUS1010 to AUS1714 (SEQ ID NOs: 11 to SEQ ID NOs: 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NO: 10), also referred to as AUS1138, is a reference modified oligonucleotide sequence. [Figure 2-3]A table of exemplary modified oligonucleotides of this disclosure is shown. The modifications at each position in the modified oligonucleotide sequence are read using the legend in Figure 1. AUS1010 to AUS1714 (SEQ ID NOs. 11 to 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NOs. 10), also known as AUS1138, is a reference modified oligonucleotide sequence. [Figure 2-4] A table of exemplary modified oligonucleotides of this disclosure is shown. The modifications at each position in the modified oligonucleotide sequence are read using the legend in Figure 1. AUS1010 to AUS1714 (SEQ ID NOs. 11 to 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NOs. 10), also known as AUS1138, is a reference modified oligonucleotide sequence. [Figure 2-5] A table of exemplary modified oligonucleotides of this disclosure is shown. The modifications at each position in the modified oligonucleotide sequence are read using the legend in Figure 1. AUS1010 to AUS1714 (SEQ ID NOs. 11 to 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NOs. 10), also known as AUS1138, is a reference modified oligonucleotide sequence. [Figure 2-6] A table of exemplary modified oligonucleotides of this disclosure is shown. The modifications at each position in the modified oligonucleotide sequence are read using the legend in Figure 1. AUS1010 to AUS1714 (SEQ ID NOs. 11 to 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NOs. 10), also known as AUS1138, is a reference modified oligonucleotide sequence. [Figure 2-7] A table of exemplary modified oligonucleotides of this disclosure is shown. The modifications at each position in the modified oligonucleotide sequence are read using the legend in Figure 1. AUS1010 to AUS1714 (SEQ ID NOs. 11 to 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NOs. 10), also known as AUS1138, is a reference modified oligonucleotide sequence. [Figure 2-8]A table of exemplary modified oligonucleotides of this disclosure is shown. The modifications at each position in the modified oligonucleotide sequence are read using the legend in Figure 1. AUS1010 to AUS1714 (SEQ ID NOs. 11 to 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NOs. 10), also known as AUS1138, is a reference modified oligonucleotide sequence. [Figure 2-9] A table of exemplary modified oligonucleotides of this disclosure is shown. The modifications at each position in the modified oligonucleotide sequence are read using the legend in Figure 1. AUS1010 to AUS1714 (SEQ ID NOs. 11 to 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NOs. 10), also known as AUS1138, is a reference modified oligonucleotide sequence. [Figure 2-10] A table of exemplary modified oligonucleotides of this disclosure is shown. The modifications at each position in the modified oligonucleotide sequence are read using the legend in Figure 1. AUS1010 to AUS1714 (SEQ ID NOs. 11 to 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NOs. 10), also known as AUS1138, is a reference modified oligonucleotide sequence. [Figure 2-11] A table of exemplary modified oligonucleotides of this disclosure is shown. The modifications at each position in the modified oligonucleotide sequence are read using the legend in Figure 1. AUS1010 to AUS1714 (SEQ ID NOs. 11 to 666) represent modified oligonucleotide sequences. AUS1233 (SEQ ID NOs. 10), also known as AUS1138, is a reference modified oligonucleotide sequence. [Figure 3] The immunoscavenging effect of the selected oligonucloethides of this disclosure is shown. The figure shows the levels of hepatitis B surface antigen protein (HBsAg) in HDI-HBV mice (HBV animal model) after administration of the selected oligonucleotides of this disclosure. [Figure 4]This figure shows the immunoscavenging effect of the selected oligonucloethides of this disclosure. This figure shows serum hepatitis B surface antigen protein antibody (HBsAb) levels in HDI-HBV mice after administration of the selected oligonucleotides of this disclosure. [Figure 5] The immunoscavenging effects of the selected oligonucloethides of this disclosure are shown. The figure shows serum HBsAg levels in HDI-HBV mice after administration of the selected oligonucleotides of DilcoCure. [Figure 6] The immunoscavenging effects of the selected oligonucloethides of this disclosure are shown. The figure shows serum HBsAb levels in HDI-HBV mice after administration of the selected oligonucleotides of this disclosure. [Figure 7] This figure shows the immunoscavenging effects of the selected oligonucleotides of this disclosure. This figure shows that normal animals showed an increase in serum HBsAb levels after administration of the selected oligonucleotides of this disclosure. The results showed that the immunoscavenging activity of the tested oligonucleotides was similar to or better than that of other known adjuvants in normal mice. [Figure 8] The present disclosure shows that administration of the oligonucleotide in combination with HBsAg resulted in a significantly stronger and more sustained decrease in serum HBsAg levels and induced much higher levels of HBsAb compared to HBsAg antigen alone. [Figure 9] This figure shows the effect of the oligonucleotides of this disclosure on serum HBsAg and HBsAb levels in HDI-HBV mice that received only a vaccine dose and no prior therapeutic dose. The data indicate that inclusion of prior therapeutic doses is optional. [Figure 10-1] This disclosure demonstrates the beneficial effects of the oligonucleotides on serum HBsAg and HBsAb levels, T cell response, and HBV mRNA and HBV DNA expression levels in the liver. [Figure 10-2] This disclosure demonstrates the beneficial effects of the oligonucleotides on serum HBsAg and HBsAb levels, T cell response, and HBV mRNA and HBV DNA expression levels in the liver. [Figure 11] This figure shows the immunoenhancing effect of the oligonucleotides of this disclosure in a primate model for inducing an anti-HBsAg antibody response in monkeys. The results indicate that the subcutaneous administration route can activate the immune response more quickly and produce higher levels of HBsAb. [Figure 12] This disclosure demonstrates that the serum HBsAb induction kinetics in mice after administration of the oligonucleotides described herein differ from those of the known adjuvant CpG1018. [Figure 13] This disclosure demonstrates the effect of a preliminary mixture of oligonucleotides and HBsAg versus separate, adjacent administrations at HBsAb levels. The results show that both methods ultimately produce equivalent levels of HBsAb. [Figure 14] This study demonstrates that the immunoenhancing activity of oligonucleotides is independent of their complementarity to a portion of the HBV genome nucleic acid sequence. [Figure 15] Figure 15 shows a graph illustrating the effect of oligonucleotide AUS1476 and / or aram-adjuvant-added recombinant miniature HBsAg administration on HBsAg levels in AAV-HBV transgenic mice. In Figure 15, aram-adjuvant-added recombinant miniature HBsAg is referred to as the "vaccine." Physiological saline was administered as a control. Data are shown as mean ± standard deviation. Arrows along the x-axis indicate the days on which mice were administered AUS1476 or aram-adjuvant-added recombinant miniature HBsAg. The limit of quantification (LOQ) at 1 IU / mL is indicated by a dotted line. [Figure 16] Figure 16 shows the effect of oligonucleotide AUS1476 and / or aram-adjuvanted recombinant miniature HBsAg administration on HBsAb levels in AAV-HBV transgenic mice. In Figure 16, aram-adjuvanted recombinant miniature HBsAg is referred to as the "vaccine." Physiological saline was administered as a control. The bars represent the geometric mean of each sample. The limit of quantification (LOQ) of 5 mIU / mL is indicated by the dotted line. [Modes for carrying out the invention]

[0010] It should be understood that both the general description above and the detailed description below are illustrative and descriptive only and do not limit the claimed invention. In this specification, the use of the singular includes the plural unless otherwise specifically stated. Where used herein, the use of “or” means “and / or” unless otherwise specifically stated. Furthermore, the use of the term “including,” as well as other forms such as “includes” and “included,” is not limiting. Also, terms such as “element” or “component” include both elements and components containing one unit, and elements and components containing two or more subunits, unless otherwise specifically stated.

[0011] Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. All documents or parts of documents cited herein, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference, both in part and in whole, of the documents considered herein.

[0012] I. Vaccines containing oligonucleotides This specification provides oligonucleotides that, when administered in combination with an antigen (e.g., a viral antigen, e.g., HBV antigen), exhibit an immunoenhancing effect useful as a vaccine. In some embodiments, use is in a prophylactic situation, and the vaccine is a prophylactic vaccine administered before the onset of infection. In other embodiments, use is in a therapeutic situation, and the vaccine is used as a therapeutic vaccine administered after the onset of infection. Thus, in some embodiments, the oligonucleotide / HBV antigen vaccine of this disclosure is a prophylactic vaccine, and in other embodiments, the vaccine is a therapeutic vaccine.

[0013] In relation to therapeutic vaccines, the inventors have surprisingly found that the oligonucleotides of the present disclosure exhibit immunoenhancing effects and can be used as vaccines for the treatment of infection (e.g., viral infections) when administered in combination with an antigen, for example, in combination with HBV antigen, for the treatment of HBV infection in a subject. In this regard, the inventors have found that the oligonucleotides of the present disclosure have adjuvant-like properties and, when administered in combination with HBV antigen, result in a sustained reduction in viral load, as assessed by a decrease in HBV antigen levels, and are accompanied by an increase in HBV-specific antibodies, a decrease in HBV DNA, a decrease in HBV mRNA, and / or an increase in the HBV-specific T cell immune response.

[0014] As intended herein, the vaccines of this disclosure comprise at least one oligonucleotide and at least one antigen of this disclosure. Similarly, the HBV vaccines of this disclosure comprise at least one oligonucleotide and at least one HBV antigen of this disclosure. In some embodiments, the at least one oligonucleotide and at least one HBV antigen are present in a single composition and administered together in a single composition. In other embodiments, the at least one oligonucleotide and at least one HBV antigen are present in separate compositions and mixed together before administration. In yet another embodiment, the at least one oligonucleotide and at least one HBV antigen are present in separate compositions and administered separately, for example, sequentially or simultaneously, for example, to the same site or different sites. For clarity, it should be noted that the term “vaccine” as used herein refers to both examples of single compositions and examples of separate compositions.

[0015] a. oligonucleotides The vaccine of this disclosure comprises at least one oligonucleotide and at least one HBV antigen, and the two components can be administered in separate compositions or together in a single composition.

[0016] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is a double-stranded oligonucleotide or has a double-stranded portion. In some embodiments, the oligonucleotide is a chain-like oligonucleotide that can form a double-stranded secondary structure under certain conditions.

[0017] The length of the oligonucleotides in this disclosure may be about 10 to about 60 nucleic acid base lengths. In some embodiments, the oligonucleotides are about 20 to about 50 nucleic acid base lengths, about 20 to about 40 nucleic acid base lengths, about 20 to about 30 nucleic acid base lengths, about 15 to about 45 nucleic acid base lengths, about 25 to about 50 nucleic acid base lengths, about 10 to about 30 nucleic acid base lengths, about 14 to about 30 nucleic acid base lengths, about 16 to about 24 nucleic acid base lengths, about 18 to about 22 nucleic acid base lengths, about 19 to about 21 nucleic acid base lengths, or even about 18 to about 20 nucleic acid base lengths. In some embodiments, the oligonucleotides have nucleic acid base lengths of approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 nucleic acid base lengths.

[0018] In some embodiments, the oligonucleotides target the HBV sequence and are complementary to a portion of the nucleic acid sequence of the HBV genome, as exemplified, for example, in Examples 1-9 and 11. However, in yet other embodiments, the oligonucleotides are not complementary to a portion of the nucleic acid sequence of the HBV genome and do not need to be complementary to achieve the desired prophylactic and / or therapeutic benefits, as exemplified, for example, in Example 10, as shown herein.

[0019] In some embodiments, the oligonucleotide targets an HBV sequence and is complementary to a portion of the HBV genome nucleic acid sequence. The oligonucleotide and the target nucleic acid are complementary if a sufficient number of nucleic acid bases of the oligonucleotide can hydrogen bond with the corresponding nucleic acid bases of the target nucleic acid. As intended herein, the oligonucleotide does not have to be complementary to the entire stretch of the target nucleic acid, and there may be complementary regions. In some embodiments, the oligonucleotide is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% complementary to one or more portions of the HBV genome nucleic acid sequence, and the complementarity percentage is determined over the entire length of the target oligonucleotide, and the term “complementary” as used herein encompasses all of the enumerated ranges.

[0020] The oligonucleotides of this disclosure may exhibit complementarity to the 5'UTR, coding region, 3'UTR, introns, exons, or exon / intron junctions of the HBV genome. Target segments containing start or stop codons are also preferred target segments. Preferred target segments may specifically exclude certain structurally defined regions, such as start or stop codons. Exemplary HBV genomes include HBV genotypes (GT) A, B, C, D, E, F, G, H, I, and J, which are denoted as GT-A, GT-B, GT-C, GT-D, GT-E, GT-F, GT-G, GT-H, GT-I, and GT-J, respectively, or as GTA, GTB, GTC, GTD, GTE, GTF, GTG, GTH, GTI, and GTJ, respectively. Exemplary HBV genome nucleic acid sequences include, but are not limited to, those shown in Table 1.

[0021] [Table 1]

[0022] In some embodiments, the HBV nucleic acid is the sequence described in GENBANK accession number U95551.1 (provided herein as SEQ ID NO: 3). In some embodiments, the oligonucleotide targets the sequence or a portion thereof listed in SEQ ID NO: 3. In some embodiments, the oligonucleotide targets the sequence at positions 1583–1602 in SEQ ID NO: 3.

[0023] Examples of HBV nucleic acid target sequences include, but are not limited to, those shown in Table 2.

[0024] [Table 2-1]

[0025] [Table 2-2]

[0026] [Table 2-3]

[0027] In some embodiments, the HBV target includes sequences of SEQ ID NOs: 1, 4, and 675-838. In some embodiments, the HBV target includes the sequence of SEQ ID NO: 1. In some embodiments, the HBV target includes the sequence of SEQ ID NO: 4.

[0028] In some embodiments, the HBV target comprises the sequence described in SEQ ID NO: 1, or a portion thereof, or a variant thereof. In some embodiments, the oligonucleotide is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% complementary to SEQ ID NO: 1.

[0029] In some embodiments, the HBV target comprises the sequence described in SEQ ID NO: 4, or a portion thereof, or a variant thereof. In some embodiments, the oligonucleotide is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% complementary to SEQ ID NO: 4.

[0030] In some embodiments, the HBV target includes positions 1583–1602 of SEQ ID NO: 3, or a portion thereof, or variants thereof. In some embodiments, the oligonucleotide is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% complementary to positions 1583–1602 of SEQ ID NO: 3.

[0031] In some embodiments, the oligonucleotide has the nucleic acid base sequence GCAGA GGTGA AGCGA AGTGC (SEQ ID NO: 2). A chart showing the nucleic acid base positions of SEQ ID NO: 2 is shown below:

[0032] [Table 3]

[0033] In some embodiments, the modified oligonucleotide of 20 linked nucleosides has the nucleic acid base sequence GTGAA GCGAA GTGCA CACGG (SEQ ID NO: 5). A chart showing the nucleic acid base positions of SEQ ID NO: 5 is shown below:

[0034] [Table 4]

[0035] The oligonucleotides of this disclosure may comprise linked nucleosides, such as linked deoxynucleosides, linked ribonucleosides, and linked deoxyribonucleosides. Thus, the oligonucleotides of this disclosure may comprise deoxyribonucleotides, ribonucleotides, or mixtures of both. In some embodiments, the oligonucleotides are modified oligonucleotides, for example, comprising one or more sugar modifications, one or more modified nucleoside bonds, and / or one or more base modifications. The characteristics of the modified oligonucleotides of this disclosure are discussed in more detail below.

[0036] In some embodiments, an oligonucleotide is conjugated to a moiety or conjugate called a conjugated oligonucleotide. The characteristics of the conjugated oligonucleotides of this disclosure are discussed in more detail below. The conjugated oligonucleotide may be modified or unmodified.

[0037] In some embodiments, the oligonucleotides of the present disclosure include oligonucleotides comprising any one sequence of SEQ ID NOs: 10-666, or one, two, three, four, five, six, seven, eight, nine, or ten sequence modifications thereof. As used herein, the term “sequence modification” refers to sequence identity (e.g., modification from A to T).

[0038] In some embodiments, the oligonucleotides of the present disclosure include oligonucleotides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of the sequences from SEQ ID NOs: 10 to 666.

[0039] In some embodiments, the vaccine comprises only one unique oligonucleotide, and for clarity, the vaccine may comprise copies of the same unique oligonucleotide. In some embodiments, the vaccine comprises multiple different oligonucleotides, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more different oligonucleotides of the present disclosure.

[0040] b. Characteristics of modified oligonucleotides In some embodiments, the oligonucleotide is a modified oligonucleotide. As intended herein, modification of an oligonucleotide includes substitution or alteration of nucleoside bonds, sugar moieties, and nucleic acid bases. Modified oligonucleotides often confer desirable properties such as enhanced cellular uptake, increased affinity to nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. Chemically modified nucleosides can also be used to increase their binding affinity to target nucleic acids.

[0041] In some embodiments, the modified oligonucleotides of the Disclosure comprise one or more modified nucleoside bonds. In some embodiments, the modified oligonucleotides of the Disclosure comprise one or more modified nucleoside bonds, the modified nucleoside bonds being phosphorothioate nucleoside bonds. In some embodiments, the phosphorothioate modification comprises a mixture of Sp and Rp stereoisomers. In some embodiments, the phosphorothioate modification comprises an Sp stereoisomer. In some embodiments, the phosphorothioate modification comprises an Rp stereoisomer.

[0042] In some embodiments, the modified oligonucleotides of the Disclosure comprise one or more sugar modifications. In some embodiments, the modified oligonucleotides of the Disclosure comprise one or more linked nucleosides comprising a 2' sugar modification (sugar modification at the C2 position). In exemplary embodiments, the modified oligonucleotides of the present disclosure include one or more modifications to a 2'-deoxynucleoside, such as 2'-O-ethyl modification, 2'-O-(2-methoxyethyl) modification, 2'-fluoro modification, 2'-amino modification, 2'-O-propyl modification, 2'-O-butyl modification, 2'-O-cyclopropylmethyl modification, 2'-O-(2-hydroxyethyl) modification, 2'-O-[2-(methylamino)-2-oxyethyl] modification, 2'-O-2-[2-(N,N-dimethylamino)ethoxy]ethyl] modification, 2'-O-(2-dimethylaminoethyl) modification, 2'-O-(3-aminopropyl) modification, 2'-O-(2-aminopropyl) modification, and 2'-O-(2-hydroxyisopropyl) modification. The nucleoside comprises one or more nucleosides including the following modifications: 2'-O-(2-methoxyisopropyl), 2'-O-(2-dimethylaminoisopropyl), 2'-O-(2-aminobutyl), 2'-O-(2-hydroxybutyl), 2'-O-(2-methoxybutyl), 2'-O-(2-dimethylaminobutyl), 2'-O-(2-aminocyclopropylmethyl), 2'-O-(2-hydroxycyclopropylmethyl), 2'-O-(2-methoxycyclopropylmethyl), 2'-O-(2-dimethylaminocyclopropylmethyl), 2'-O-[(2-guanidinium)ethyl], and / or 2'-fluoro-arabino(2'-fluoro-ANA).

[0043] In some embodiments, the modified oligonucleotide comprises one or more bicyclic sugar-modified nucleosides. In some embodiments, the modified oligonucleotide of the present disclosure comprises one or more crosslinked nucleic acids, wherein the 2'-oxygen is linked to the 4'-carbon of ribose via a crosslinking carbon to form a crosslinked nucleic acid (BNA), for example via a 2',4'-methylene crosslink to form a locked nucleic acid (LNA), or crosslinked by, for example, a 2',4'-restricted 2'-O-ethyl modification ((R)-cET or (S)-cET), or for example, a 2',4'-restricted 2'-O-methoxyethyl modification ((R)-cMOE or (S)-cMOE).

[0044] In some embodiments, the modified oligonucleotide comprises one or more carbocyclic LNAs (cLNAs) in which the 2'-oxygen atom in the LNA is replaced by a carbon atom. In some exemplary embodiments, the modified oligonucleotide of the present disclosure comprises one or more metanocarbabicyclo[3.1.0]hexane sugars in either a North C2'-exo type (N-MC), a South C3'-exo type (S-MC), or a 2'-F-NMC. In some embodiments, the modified oligonucleotide comprises (R)-Me-cLNA, or (S)-Me-cLNA, or F-LNA, or methylene-cLNA, as shown below.

[0045] [ka]

[0046] In some embodiments, the modified oligonucleotide comprises one or more α-L-LNA, bcDNA, or tcDNA as shown below.

[0047] [ka]

[0048] In some embodiments, the modified oligonucleotides of the Disclosure include one or more morpholino modifications in which a morpholino group is present instead of a ribose sugar. In some embodiments, the modified oligonucleotides of the Disclosure include phosphorodiamidate morpholino modifications in which the sugar-phosphate backbone is replaced by a phosphorodiamidate morpholino moiety. In some embodiments, the modified oligonucleotides of the Disclosure include one or more peptide nucleic acid (PNA) modifications in which the sugar-phosphate backbone or sugar-phosphorothioate backbone is replaced by a peptide backbone. In some embodiments, the modified oligonucleotides of the Disclosure include one or more glycol nucleic acid (GNA) modifications in which a propylene glycol group is present instead of a ribose sugar, which may also be called glycerol nucleic acid. In some embodiments, the modified oligonucleotides of the Disclosure include one or more phosphate analogs E vinylphosphonate (E-VP) and amide backbone linkages as shown below.

[0049] [ka]

[0050] In some embodiments, the modified oligonucleotides of the Disclosure comprise one or more base modifications. In some embodiments, the modified oligonucleotides of the Disclosure comprise one or more nucleosides that are 5-methylcytidine. In some embodiments, the modified oligonucleotides of the Disclosure comprise one or more nucleosides that are N1-methylpsoiduridine.

[0051] In some embodiments, the modified oligonucleotides of the present disclosure include one or more modifications to the 3' and / or 5' ends of the oligonucleotide, such as 3'- and / or 5'-amino modifications.

[0052] In some embodiments, the modified oligonucleotides of the present disclosure comprise a segmented oligonucleotide, creating one or more internal regions (gaps) having multiple nucleotides supporting RNaseH cleavage, which are located between external regions (5' and 3' wing segments) having multiple nucleotides chemically distinct from the nucleoside of the internal region. In the case of oligonucleotides exhibiting complementarity to a target having a segmented motif, the gap segments may hybridize with the target RNA sequence for endonuclease cleavage, while the wing segments contain the modified nucleoside. In some embodiments, the gap regions are distinguished by the type of sugar moiety containing different regions. Types of sugar moieties used to distinguish the gap region include, in some embodiments, β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides may include 2'-MOE and 2'-O-CH3, among those described in the previous paragraph), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides may include those having a restricted ethyl group). In some embodiments, the nucleosides in the wing may include, for example, 2'-MOE and bicyclic sugar moieties, such as a restricted ethyl group or a modified sugar moiety containing LNA. In some embodiments, the wing may include some of the modified and unmodified sugar moieties described in the previous paragraph. It is possible. In some embodiments, the wings may comprise various combinations of a bicyclic sugar moiety such as a 2'-MOE nucleoside, a restricted ethyl nucleoside, or an LNA nucleoside, and a 2'-deoxynucleoside. A single internal region (gap) may be present or may be further segmented by separator segments, thus resulting in discontinuous gap segments, which in some embodiments provide improved activity (e.g., reduction of HBsAg, HBeAg, and / or HBcAg levels in serum or other body fluids or tissues) compared to conventional oligonucleotides with continuous gaps. One or more separator segments directly positioned between gap segments may be beneficial.Examples 1-11 demonstrate the advantages of oligonucleotides, including various segmented oligonucleotides.

[0053] Exemplary modifications of this disclosure include, but are not limited to, those listed in Table 1.1 (Figure 1). Exemplary modified oligonucleotides of this disclosure include, but are not limited to, those illustrated in Table 1.2 (Figure 2, as read in reference to the legend in Figure 1). In some embodiments, the modified oligonucleotide comprises one of the sequences AUS1010–AUS1714.

[0054] c. Conjugate oligonucleotides The oligonucleotides of this disclosure may be further covalently bonded to one or more conjugate groups / molets. In some embodiments, such groups enhance the activity, cell distribution, and / or cell uptake of the resulting vaccine. Exemplary conjugate groups include, but are not limited to, carbohydrate moieties (e.g., N-acetylgalactosamine-containing carbohydrate chains) and lipid moieties (e.g., cholesterol and phospholipids). Other conjugate groups include, but are not limited to, phospholipids, biotin or other affinity tags (e.g., streptavidin), polymers (e.g., polyethylene glycol (PEG), polylysine), cell-permeable peptides (e.g., Tat, penetratin), enzymes (e.g., horseradish peroxidase, alkaline phosphatase), antibodies or antibody fragments (e.g., anti-HER2, anti-EGFR), proteins or peptides (e.g., growth factors, cytokines), metal nanoparticles (e.g., gold nanoparticles), small molecule drugs (e.g., doxorubicin, paclitaxel), phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0055] The oligonucleotides of this disclosure may also be modified to have one or more stabilizing groups generally attached to one or both ends of the oligonucleotide to enhance properties such as nuclease stability. Stabilizing groups include cap structures. These end modifications can protect oligonucleotides having terminal nucleic acids from exonuclease degradation and can aid in intracellular delivery and / or localization. The caps may be present at the 5' end (5' cap), the 3' end (3' cap), or on both ends. Cap structures are well known in the art and include, for example, reverse deoxydecay caps.

[0056] The conjugate oligonucleotides of this disclosure may be modified as described in the previous section.

[0057] Hepatitis dB type hepatitis virus antigen As provided herein, the vaccines of the Disclosure comprise at least one oligonucleotide and at least one HBV antigen, and the two components can be administered in two separate compositions or together in a single composition. In some embodiments, the vaccine comprises a mixture of different HBV antigens, for example, at least two, three, four, or five different HBV antigens.

[0058] In some embodiments, the HBV antigen comprises HBV surface antigen (HBsAg), HBV e antigen (HBeAg), and / or HBV core antigen (HBcAg). In some embodiments, the HBV antigen is human HBV antigen. In some embodiments, the HBV antigen comprises hepatitis B small surface antigen (Small-HBs (S-HBs)). In some embodiments, the HBV antigen comprises hepatitis B medium surface antigen (Middle-HBs (M-HBs)). In some embodiments, the HBV antigen comprises hepatitis B large surface antigen (Large-HBs (L-HBs)). In some embodiments, the HBV antigen comprises a mixture of hepatitis B small and medium surface antigens. In some embodiments, the HBV antigen comprises a mixture of hepatitis B small and large surface antigens. In some embodiments, the HBV antigen comprises a mixture of hepatitis B medium and large surface antigen. In some embodiments, the HBV antigen comprises a mixture of hepatitis B medium and large surface antigen. In some embodiments, the HBV antigen is derived from HBV of the GT-A, GT-B, GT-C, GT-D, GT-E, GT-F, GT-G, GT-H, GT-I, or GT-J genotype.

[0059] HBV antigen can be isolated from patients, recombinantly produced (e.g., in mammalian cells, yeast cells, bacterial cells, insect cells), or synthesized.

[0060] e. Adjuvant As provided herein, the vaccines of this disclosure comprise at least one oligonucleotide and at least one HBV antigen, and may further comprise an adjuvant. If the vaccine is provided as a single composition, the composition may include an adjuvant. If the vaccine is provided as separate compositions, either the oligonucleotide component or the antigen component may comprise an adjuvant, or both may comprise an adjuvant. Exemplary adjuvants include, but are not limited to, aluminum hydroxide, Toll-like receptor 9 (TLR9) agonist adjuvants, or oligonucleotides. An exemplary oligonucleotide adjuvant comprises CpG1018, a 22-mer CpG oligodeoxynucleotide-containing sequence having a modified phosphorothioate skeleton, 5'-tgactgtgaacgttcgagatga-3' (SEQ ID NO: 839) (each lowercase letter represents a 2'-deoxynucleoside as presented in Table 1.1 of Figure 1).

[0061] II. Method a. Preventive measures In one embodiment, the present disclosure provides a method for preventing HBV infection in a subject, comprising administering a prophylactic amount of at least one oligonucleotide described herein together with at least one HBV antigen (i.e., vaccine) or a pharmaceutical composition comprising the same. As stated throughout, the oligonucleotide and the HBV antigen may be present together in a single composition or may be provided separately for such prophylactic use.

[0062] In some embodiments, subjects are at risk of developing HBV-related symptoms. This includes subjects who have one or more risk factors for developing HBV-related symptoms, including sexual exposure to a subject infected with hepatitis B virus, living in the same household as a subject with lifelong hepatitis B virus infection, exposure to human blood infected with hepatitis B virus, injection of illegal drugs, having hemophilia, and visiting areas with a high prevalence of hepatitis B.

[0063] b.Treatment method In another aspect, the Disclosure provides a method for treating a subject having HBV infection or an HBV-related disease, disorder, or condition, comprising administering a therapeutically effective amount of at least one oligonucleotide described herein together with at least one HBV antigen (i.e., vaccine) or a pharmaceutical composition comprising the same. As stated throughout, the oligonucleotide and the HBV antigen may be present together in a single composition or may be provided separately for such prophylactic use.

[0064] In some embodiments, the subjects are human beings suffering from hepatitis B virus infection derived from human hepatitis B virus. In exemplary embodiments, the human hepatitis B virus may be one of the following human geographical genotypes: A (Northwestern Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, South Korea, China, Japan, Polynesia, Vietnam); D (India, Middle East, Mediterranean region); E (Africa); F (Native Americans, Polynesia); G (United States, France); H (Central America), I (Asia, India, and Vietnam), or J (Japan).

[0065] Examples of HBV-related diseases, disorders, or conditions include, but are not limited to, chronic HBV infection, jaundice, liver cancer, hepatitis, hepatic fibrosis, cirrhosis, hepatic failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, hepatitis delta (HDV) infection (e.g., in subjects co-infected with HBV), hepatitis C virus (HCV) infection (e.g., in subjects co-infected with HBV), inflammation, fibrosis, and HBV viremia. HBV-related symptoms or disorders may include any or all of the following: influenza-like illness, weakness, pain, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain in the liver region of the body, clay-colored or gray stools, generalized itching, and dark urine. These indicate HBV-related symptoms or disorders when combined with a positive test for the presence of hepatitis B virus, hepatitis B virus antigen, or antibodies specific to hepatitis B virus antigen.

[0066] In some embodiments, positive indicators of treatment include reduced fatigue, reduced flu-like symptoms, increased appetite, reduced nausea, reduced arthralgia, reduced jaundice, reduced abdominal pain, reduced weakness, reduced weight loss, reduced breast enlargement in men, reduced palmar rash, reduced blood clotting, reduced cirrhosis, reduced spider angiography of the skin, increased absorption of vitamins A and D, reduced tumor growth, reduced tumor volume, reduced headache, reduced fever, reduced diarrhea, reduced pain in the liver region of the body, reduced clay-colored or gray stools, reduced itching, reduced dark urine, and reduced nausea and vomiting, which may indicate inhibition of HBV expression.

[0067] In some embodiments, administration of a therapeutically effective dose of the vaccine of this disclosure is accompanied by monitoring of HBV mRNA levels in the subject's serum or tissue (or other body sample) to determine the subject's response to the vaccine administration. In some embodiments, administration of a therapeutically effective dose of the vaccine of this disclosure is accompanied by monitoring of HBV DNA levels in the subject's serum or tissue (or other body sample) to determine the subject's response to the vaccine administration. In some embodiments, administration of a therapeutically effective dose of the vaccine of this disclosure is accompanied by monitoring of HBV protein levels in the subject's serum or tissue (or other body sample) to determine the subject's response to the vaccine administration. In some embodiments, administration of a therapeutically effective dose of the vaccine of this disclosure is accompanied by monitoring of HBV S antigen (HBsAg) levels in the subject's serum or tissue (or other body sample) to determine the subject's response to the vaccine administration. In some embodiments, administration of a therapeutically effective dose of the vaccine of this disclosure is accompanied by monitoring of HBV E antigen (HBeAg) levels in the subject's serum or tissue (or other body sample) to determine the subject's response to the vaccine administration. The subject's response to the vaccine administration is used by a physician to determine the amount and duration of the therapeutic intervention.

[0068] In some embodiments, administration of the vaccine of the present disclosure results in a reduction of at least 30%, 50%, 65%, 90%, 97%, 99%, 99.7%, 99.9%, 99.97%, or 99.99% of HBV expression, corresponding to a reduction of approximately 0.15, 0.3, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 Log10, respectively, or a range defined by any two of these values.

[0069] In some embodiments, administration of the vaccines of the Disclosure results in a reduction of symptoms associated with HBV-related conditions and a decrease in HBV-related markers in the blood. In some embodiments, administration of the vaccines of the Disclosure reduces HBV RNA levels, HBV DNA levels, HBV protein levels, HBsAg levels, HBcAg levels, and / or HBeAg levels by at least about 30%, 50%, 60%, 68%, 80%, 90%, 97%, 99%, 99.7%, 99.9%, 99.97%, 99.99, or 99.997%, respectively, corresponding to a reduction of about 0.15, 0.3, 0.4, 0.5, 0.7, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 Log10, or a range defined by any two of these values.

[0070] In some embodiments, the vaccines of the Disclosure inhibit HBV mRNA expression in a subject. In some embodiments, the vaccines of the Disclosure inhibit HBV DNA levels in a subject. In some embodiments, the vaccines of the Disclosure inhibit HBV protein and / or antigen levels in a subject. When administered to a subject, the vaccines of the Disclosure can reduce levels of HBV mRNA, DNA, and / or proteins, including but not limited to HBV antigens such as HBsAg and HBeAg. In some embodiments, the vaccines of the Disclosure result in an increase in HBV antibody levels.

[0071] Certain embodiments provide a method for reducing HBV mRNA expression in a subject, comprising administering the oligonucleotide / HBV antigen vaccine of the present disclosure to the subject. In some embodiments, the reduction of HBV mRNA expression in a subject prevents, improves or treats HBV-related disease, disorder, or condition. In some embodiments, the reduction of HBV mRNA expression in a subject improves or treats HBV infection. In some embodiments, the reduction of HBV mRNA expression in a subject prevents, improves or treats liver disease. In some embodiments, administration of the vaccine of the present disclosure reduces HBV RNA levels by at least about 30%, 50%, 60%, 68%, 80%, 90%, 97%, 99%, 99.7%, 99.9%, 99.97%, 99.99, or 99.997%, corresponding to reductions of about 0.15, 0.3, 0.4, 0.5, 0.7, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 Log10, respectively, or a range defined by any two of these values.

[0072] Certain embodiments provide a method for reducing HBV protein levels in a subject, comprising administering the subject an oligonucleotide / HBV antigen vaccine of the Disclosure. In some embodiments, reducing HBV protein levels in a subject prevents, improves, or treats HBV-related diseases, disorders, or conditions. In some embodiments, reducing HBV protein levels in a subject improves or treats HBV infection. In some embodiments, reducing HBV protein levels in a subject prevents, improves, or treats liver disease. In some embodiments, administration of the vaccine of the present disclosure reduces HBV protein levels by at least about 30%, 50%, 60%, 68%, 80%, 90%, 97%, 99%, 99.7%, 99.9%, 99.97%, 99.99, or 99.997%, corresponding to reductions of about 0.15, 0.3, 0.4, 0.5, 0.7, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 Log10, respectively, or a range defined by any two of these values.

[0073] Certain embodiments provide a method for reducing the quality level of HBV DNA in a subject, comprising administering the subject an oligonucleotide / HBV antigen vaccine of the Disclosure. In some embodiments, the reduction in the quality level of HBV DNA in the subject prevents, improves or treats an HBV-related disease, disorder, or condition. In some embodiments, administration of the vaccine of the Disclosure reduces the HBV DNA level by at least about 30%, 50%, 60%, 68%, 80%, 90%, 97%, 99%, 99.7%, 99.9%, 99.97%, 99.99, or 99.997%, corresponding to a range defined by about 0.15, 0.3, 0.4, 0.5, 0.7, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 Log10 reduction, or any two of these values.

[0074] Certain embodiments provide a method for reducing one or more HBV antigen levels in a subject, comprising administering the oligonucleotide / HBV antigen vaccine of the Disclosure to the subject. In some embodiments, the antigens are HBsAg, HBcAg, and / or HBeAg. In some embodiments, the reduction of HBV antigen levels in the subject prevents, improves, or treats HBV-related diseases, disorders, or conditions. In some embodiments, the reduction of HBV antigen levels in the subject prevents, improves, or treats liver disease. In some embodiments, administration of the vaccine of the present disclosure reduces HBV antigen levels by at least about 30%, 50%, 60%, 68%, 80%, 90%, 97%, 99%, 99.7%, 99.9%, 99.97%, 99.99, or 99.997%, corresponding to reductions of about 0.15, 0.3, 0.4, 0.5, 0.7, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 Log10, respectively, or a range defined by any two of these values. In some embodiments, administration of the vaccine of the present disclosure reduces HBsAg levels by at least about 30%, 50%, 60%, 68%, 80%, 90%, 97%, 99%, 99.7%, 99.9%, 99.97%, 99.99, or 99.997%, corresponding to reductions of about 0.15, 0.3, 0.4, 0.5, 0.7, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 Log10, respectively, or a range defined by any two of these values.

[0075] Certain embodiments provide a method for increasing the level of circulating antibodies against HBV protein and / or HBV antigen in a subject, comprising administering the oligonucleotide / HBV antigen vaccine of the Disclosure to the subject. In some embodiments, the antibodies are against HBsAg, HBcAg, and / or HBeAg. In some embodiments, the increase in the level of antibodies against HBV antigen in the subject prevents, improves, or treats HBV-related disease, disorder, or condition. In some embodiments, administration of the vaccine of the Disclosure increases the antibodies against HBV protein and / or HBV antigen by at least about 1.25 times, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 250 times, 500 times, 1000 times, 10000 times, and even 1,000,000 times, or by any two of these values.

[0076] Certain embodiments provide a method for increasing the T cell response to HBV antigen in a subject, comprising administering the oligonucleotide / HBV antigen vaccine of the present disclosure to the subject. In some embodiments, the increase in the T cell response in the subject prevents, improves or treats HBV-related disease, disorder, or condition. In some embodiments, the increase in the T cell response in the subject prevents, improves or treats liver disease in the subject. In some embodiments, the increase in the T cell response in the subject is by at least about 1.25 times, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 250 times, 500 times, 1000 times, 10000 times, and even 1,000,000 times, or by a range defined by any two of these values.

[0077] In some embodiments, when HBsAg is monitored as a determinant of serological conversion, serum HBsAg can be sufficiently reduced to reduce the amount of HBV antigen, which is defined as undetectable, to induce serological conversion.

[0078] In some embodiments, the amount of HBV antigen is undetectable for at least one, two, three, four, five, six months, or longer after treatment. In some embodiments, administration of the oligonucleotide / HBV antigen vaccine of this disclosure to a subject results in functional cure (HBsAg is undetectable for six months or longer). In some embodiments, administration of the oligonucleotide / HBV antigen vaccine of this disclosure to a subject results in true cure (e.g., clearance of HBV DNA / mRNA and HBV antigen).

[0079] Certain embodiments provide a method for treating a subject having HBV infection, HBV-related disease, disorder, or condition, comprising: a) identifying the subject having HBV infection, HBV-related disease, disorder, or condition; and b) administering a therapeutically effective dose of the oligonucleotide / HBV antigen vaccine of the Disclosure to the subject. In some embodiments, the therapeutically effective dose of the oligonucleotide / HBV antigen vaccine of the Disclosure administered to the subject treats or reduces the HBV infection, or HBV-related disease, disorder, or condition, or symptoms thereof, in the subject. In some embodiments, the HBV-related disease, disorder, or condition is liver disease. In some embodiments, the related disease, disorder, or condition is chronic HBV infection, jaundice, liver cancer such as hepatocellular carcinoma, hepatitis, hepatic fibrosis, cirrhosis, hepatic failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, transplant-related liver disease, or any combination thereof.

[0080] It should be noted that the reduction in HBV nucleic acid levels or expression during treatment can be assayed using various methods known in the art. For example, target nucleic acid levels can be quantified by, for instance, Northern blotting, competitive polymerase chain reaction (PCR), or quantitative real-time PCR. RNA analysis can be performed on whole cellular RNA or poly(A)+ mRNA. Methods for RNA isolation are well known in the art. Northern blotting is also common practice in the art. Quantitative real-time PCR can be conveniently achieved using commercially available ABI PRISM 7600, 7700, or 7900 Sequence Detection Systems, available from PE-Applied Biosystems, Foster City, CA, and used according to the manufacturer's instructions.

[0081] Similarly, it should be noted that a decrease in HBV antigen levels and / or an increase in HBV antibody levels can be assessed using standard techniques. HBV and antibody protein levels can be assessed or quantified by various methods well known in the art, such as immunoprecipitation, Western blotting, enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (e.g., caspase activity assays), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS). Targeted antibodies can be identified and obtained from various sources, such as the MSRS antibody catalog (Aerie Corporation, Birmingham, MI), or prepared via conventional monoclonal or polyclonal antibody production methods well known in the art.

[0082] c. Target The subjects of this disclosure eligible for the preventive and therapeutic vaccines of this disclosure include all mammals, e.g., humans and non-human primates. In some embodiments, the subject is humans. In some embodiments, the subject is a non-human primate, e.g., crab-eating macaque, rhesus macaque or chimpanzee. In some embodiments, the subject is a rodent, such as a mouse or rat. The subjects may also include domestic pets (e.g., dogs, cats) and livestock / animals (e.g., cattle, goats, sheep, pigs).

[0083] In some embodiments, the subjects are human children aged over 0 to under 13 years of age.

[0084] In some embodiments, the subjects are human adolescents aged between 13 and 18 years of age.

[0085] In some embodiments, the subjects are human adults aged 18 years or older.

[0086] In some embodiments, the subjects are adult humans aged 65 years or older.

[0087] d. Medication and administration This disclosure provides a method comprising administering an effective amount of the oligonucleotide / HBV antigen vaccine or a pharmaceutical composition containing the same to a subject in need.

[0088] Dosage may need to be adjusted by a physician, for example, based on the age of the subject. The vaccines of this disclosure are useful for children, adolescents, adults, and elderly adults, as described above.

[0089] As described herein, various routes of administration are intended, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intra-arterial, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, intranasal, intracranial, intrathecal, intraocular, or intraventricular administration. In exemplary embodiments, the route of administration is subcutaneous. In exemplary embodiments, the route of administration is intravenous. In exemplary embodiments, the route of administration is intradermal. In exemplary embodiments, the route of administration is intramuscular.

[0090] In some embodiments, the oligonucleotide component and HBV component of the vaccine are administered as separate compositions, for example, simultaneously at two adjacent administration sites. In some embodiments, the oligonucleotide component and HBV component of the vaccine are administered as separate compositions, and are administered sequentially at two adjacent administration sites. In some embodiments, the oligonucleotide component and HBV component of the vaccine are administered as separate compositions, and are administered sequentially at two simultaneous administration sites. The sequential administrations may be spaced at least about 1, 2, 3, 5, 10, 15, 20, 25, 30, 45, 60, 90, or even 120 minutes apart, or within a range defined by any two of these values.

[0091] In some embodiments, the oligonucleotide and the HBV antigen vaccine are administered as separate compositions and sequentially, as illustrated in Example 11, for example. In some embodiments, the oligonucleotide is administered before the HBV antigen. In some embodiments, the oligonucleotide is administered multiple times before the HBV antigen. In some embodiments, the multiple doses of the oligonucleotide are administered before the multiple doses of the HBV antigen. In some embodiments, one, two, three, four, five or more doses of the oligonucleotide are administered before one, two, three, four, five or more doses of the HBV antigen. In some embodiments, one, two, three, four, or five doses of the oligonucleotide are administered before one, two, three, four, or five doses of the HBV antigen. In exemplary embodiments, four doses of the oligonucleotide are administered before three doses of the HBV antigen. In exemplary embodiments, three doses of the oligonucleotide are administered before three doses of the HBV antigen vaccine. In exemplary embodiments, three doses of the oligonucleotide are administered before four doses of the HBV antigen vaccine.

[0092] In some embodiments, the oligonucleotide is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days before the HBV antigen vaccine is administered. In some embodiments, the oligonucleotide is administered 1, 2, 3, 4, 5, 6, 7, or 8 weeks before the HBV antigen vaccine is administered.

[0093] In some embodiments, a single dose of the oligonucleotide / HBV antigen vaccine of the present disclosure is administered to the target. In other embodiments, multiple doses are administered to the target. In some embodiments, the administration of the oligonucleotide / HBV antigen vaccine of the present disclosure may include a dosing schedule in which they are administered more frequently first, followed by less frequent administrations. In other embodiments, the administration of the oligonucleotide / HBV antigen vaccine of the present disclosure may include a dosing schedule in which they are administered at different intervals in different doses.

[0094] In some embodiments, the oligonucleotides in the vaccine are approximately 1 mg to 1000 mg, 1 mg to 500 mg, 1 mg to 250 mg, 1 mg to 150 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 10 mg to 1000 mg, 10 mg to 500 mg, 10 mg to 250 mg, 10 mg to 150 mg, 10 mg to 100 mg, 10 mg to 50 mg, 10 mg to 50 mg, 10 mg to 25 mg, 50 mg to 1000 mg, and 50 mg. The drug is administered to the target population in doses ranging from ~500mg, 50mg~250mg, 50mg~150mg, 50mg~100mg, 50mg~75mg, 100mg~1000mg, 100mg~500mg, 100mg~400mg, 100mg~300mg, 100mg~250mg, 100mg~200mg, 100mg~150mg, 200mg~500mg, 200mg~250mg, 300mg~1000mg, or approximately 300mg~500mg.

[0095] In some embodiments, the amount of HBV antigen in the vaccine is approximately 1 μg to 500 μg / dose, approximately 3 μg to 200 μg, approximately 3 μg to 100 μg, approximately 3 μg to 50 μg, approximately 3 μg to 30 μg, approximately 3 μg to 20 μg, approximately 3 μg to 10 μg, approximately 5 μg to 100 μg, approximately 5 μg to 75 μg, approximately 5 μg to 50 μg, approximately 5 μg to 40 μg, approximately 5 μg to The drug is administered to the target population in doses ranging from approximately 30 μg, approximately 5 μg to approximately 20 μg, approximately 5 μg to approximately 10 μg, approximately 10 μg to approximately 100 μg, approximately 10 μg to approximately 75 μg, approximately 10 μg to approximately 50 μg, approximately 10 μg to approximately 40 μg, approximately 10 μg to approximately 30 μg, approximately 10 μg to approximately 20 μg, approximately 20 μg to approximately 100 μg, approximately 50 μg to approximately 200 μg, or approximately 100 μg to approximately 500 μg.

[0096] In some embodiments, the oligonucleotide / HBV antigen vaccine or a pharmaceutical composition comprising the same of the Disclosure is administered to a subject daily, every two days, every three days, every four days, every five days, every six days, every seven days, every eight days, every nine days, every ten days, every eleven days, every twelve days, every thirteen days, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, every twelve weeks, every thirteen weeks, every two months, every three months, or every four months, or any combination thereof. Note that the intervals between administrations do not have to be equal, and the Disclosure is intended to vary the intervals between administrations.

[0097] In some embodiments, the oligonucleotide / HBV antigen vaccine of the Disclosure or a pharmaceutical composition comprising the same is administered to a subject over a period of at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least one year, at least 1.5 years, at least two years, at least three years, at least four years, or at least five years.

[0098] In relation to therapeutic vaccines, in some embodiments, one or more pretreatments may precede the administration of the oligonucleotide / HBV antigen vaccine of this disclosure. While not bound by theory or mechanism, the pretreatments may act to reduce the level of HBV titer, for example, by assaying a decrease in the level of one or more HBV proteins or antigens. However, as shown in the examples, pretreatments are not necessary, but in some embodiments, the variability of the response may be reduced. It should be understood that the treatment may be performed in cycles, and pretreatments may be performed before each cycle.

[0099] Prior treatment may include administration of one or more oligonucleotides of the present disclosure, for example, an oligonucleotide containing any one sequence of SEQ ID NOs. 10 to 666, or a modified oligonucleotide containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequence modifications thereof, or an oligonucleotide containing any one sequence of SEQ ID NOs. 10 to 666, or an oligonucleotide containing at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereof.

[0100] Pre-treatment may include or further include the administration of HBV therapy, such as oligonucleotides, antisense oligonucleotides, siRNA directed at HBV, interferon-gamma, PD-1 inhibitors, PD-L1 inhibitors, or other known HBV agents. In some embodiments, pre-treatment is before the first dose of the vaccine. In some embodiments, pre-treatment is both before the first dose of the vaccine and, if additional treatment cycles are provided, after the first or subsequent doses of the vaccine.

[0101] Table 3 provides exemplary, non-limiting vaccine administration schedules. Prior to each of these exemplary schedules, pretreatment as described herein may be performed. Each of these exemplary schedules may represent one cycle, and the cycle may be optionally repeated at a future time preceded by pretreatment.

[0102] [Table 5]

[0103] In relation to therapeutic vaccines, in some embodiments, the therapeutically effective dose includes a dose at which a maximum log10 serum HBsAg (or other HBV protein) reduction of at least -0.15, at least -0.2, at least -0.3, at least -0.4, at least -0.5, at least -0.7, at least -1.0, at least -1.5, at least -2.0, at least -2.5, at least -3.0, at least -3.5, at least -4.0, or at least -4.5 is observed. In some embodiments, the decrease in maximum log10 serum HBsAg (or other HBV protein) is observed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days after administration of a therapeutically effective dose.

[0104] In relation to therapeutic vaccines, in some embodiments, the therapeutically effective dose of the oligonucleotide / HBV antigen vaccine or a pharmaceutical composition containing the same of the present disclosure includes a dose that reduces the level of HBsAg (or other HBV protein) in the serum of the subject by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.7%, 99.9%, 99.97%, 99.99%, or 99.997%, or any two of these values. In some embodiments, a decrease in serum HBsAg (or other HBV protein) is observed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days after administration of a therapeutically effective dose.

[0105] In relation to therapeutic vaccines, in some embodiments, the oligonucleotide / HBV antigen vaccine of the Disclosure or a pharmaceutical composition comprising the same is administered to a subject until a specific outcome is reached, for example, a decrease in the levels of HBsAg and / or HBeAg in the subject's serum. In some embodiments, administration of the oligonucleotide / HBV antigen vaccine of the Disclosure to a subject is continued until the levels of HBsAg and / or HBeAg in the subject's serum decrease by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.7%, 99.9%, 99.97%, 99.99%, or 99.997%, or by any two of these values, compared to before administration.

[0106] In some embodiments, administration of the oligonucleotide / HBV antigen vaccine of the Disclosure includes administration holidays. Certain periods and frequencies of administration holidays are considered to be within the scope of the Disclosure. For example, the oligonucleotide / HBV antigen vaccine of the Disclosure, or a pharmaceutical composition comprising the same, may be administered to a subject until the level of HBsAg falls below a certain threshold, or until HBV infection is no longer detected in the subject, followed by an administration holiday, and administration is resumed when HBsAg is again detected in the subject's serum.

[0107] e. Combination therapy In some embodiments, the vaccine of this disclosure is referred to as the first agent, and the method comprises administering the first agent and one or more second agents. In some embodiments, the first agent and one or more second agents are administered simultaneously. In some embodiments, the first agent and one or more second agents are administered sequentially or simultaneously. In some embodiments, the first agent and one or more second agents are not administered together.

[0108] In some embodiments, the oligonucleotide, HBV antigen, and second drug are prepared together in a single formulation. In other embodiments, they are prepared separately.

[0109] The second drug of this disclosure may be administered by the same or a different route of administration as the first drug vaccine.

[0110] For clarity, the terms “first” and “second” do not imply an order of administration. Therefore, in some embodiments, the first drug vaccine may be administered first, followed by one or more second drugs of this disclosure. In other embodiments, the first drug vaccine and the second drugs are administered in the reverse order.

[0111] In some embodiments, the second agent is designed to treat the same disease, disorder, or condition as one or more vaccines provided herein. In other embodiments, the second agent is designed to treat a different disease, disorder, or condition from one or more vaccines provided herein. In some embodiments, the second agent is designed to treat an undesirable side effect of one or more vaccines provided herein. In some embodiments, one or more second agents provided herein are administered co-administered with the first drug vaccine to produce a combination effect. In some embodiments, one or more second agents provided herein are administered co-administered with the first drug vaccine to produce a synergistic effect.

[0112] In some embodiments, the disease includes liver disease, and the second agent includes lamivudine, entecavir, terbivudine, tenofovir, adefovir, or tenofovir alafenamide (TAF). In some embodiments, the second agent includes interferon alpha, milkuldex B, amantadine, phanciclovir, prednisone, or more generally, corticosteroids, diuretics, beta-blockers, or combinations thereof. Examples of the second agent may also include, but are not limited to, anti-inflammatory agents, chemotherapeutic agents, or anti-infective agents. In some embodiments, the condition includes liver cancer, and the second agent includes chemotherapeutic agents, such as gemcitabine (Gemzar), oxaliplatin (Eloxatin), cisplatin, doxorubicin, 5-fluorouracil, capecitabine (Xeloda), or mitoxantrone (Novantrone).

[0113] Due to overlapping transmission routes, many people are exposed to both hepatitis B virus (HBV) and hepatitis C virus (HCV), and a smaller proportion are chronically infected with both viruses, particularly in regions such as Asia where HBV is prevalent. Estimates suggest that up to 10% of people with HCV may also have HBV, but perhaps 20% of people with HBV are co-infected with HCV. However, the treatment of hepatitis B or hepatitis B in individuals co-infected with I-IBV-HCV is not well studied. Treatment is complicated by the fact that HCV and HBV appear to inhibit each other's replication (although not all studies have observed this interaction). Therefore, treatment that completely suppresses HBV may allow HCV to reappear, or vice versa. Thus, the vaccine described herein may, conveniently, be used to treat patients infected with both HBV and HCV when administered with an HCV-specific second agent. Exemplary treatment options for hepatitis C (HCV) include interferons, such as interferon alpha-2b, interferon alpha-2a, and interferon alpha-1. Pegylated interferon (interferon conjugated to a polyethylene glycol moiety that improves the pharmacokinetic profile) can be used to achieve lower interferon administration. Combination therapy of interferon alpha-2b (pegylated and non-pegylated) with ribavirin has also been shown to be effective in some patient populations. Other drugs currently under development include HCV RNA replication inhibitors (e.g., ViroPharma's VP50406 series), HCV antisense agents, HCV therapeutic vaccines, HCV protease inhibitors, HCV helicase inhibitors, and HCV antibody therapies (monoclonal or polyclonal). Due to overlapping transmission routes, many people are also exposed to hepatitis D virus (HDV).

[0114] Mai. Pharmaceutical composition This disclosure provides a pharmaceutical composition comprising the vaccine of this disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.

[0115] In some embodiments, the pharmaceutical composition comprises an oligonucleotide of the present disclosure, an HBV antigen, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0116] In other embodiments, one pharmaceutical composition comprises the oligonucleotide of the Disclosure and a pharmaceutically acceptable carrier, diluent, or excipient. A second pharmaceutical composition comprises the HBV antigen of the Disclosure and a pharmaceutically acceptable carrier, diluent, or excipient, and the two pharmaceutical compositions are administered sequentially, simultaneously, or mixed immediately before administration.

[0117] Various routes of administration are possible, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, oral cavity, sublingual, intrapleural, intrathecal, intranasal, and CSF-mediated administration. The pharmaceutical compositions of this disclosure are formulated to be compatible with their intended route of administration.

[0118] Dosage forms for topical or transdermal administration of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In some embodiments, the modified oligonucleotide is mixed under sterile conditions with a pharmaceutically acceptable carrier / diluent / excipient and any necessary preservatives, buffers, or propellants.

[0119] Solutions or suspensions used for parenteral, intradermal, intraperitoneal, or subcutaneous application may contain the following components: sterile diluents, e.g., water for injection, saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates, or phosphates; and agents for adjusting tonicity, e.g., sodium chloride or dextrose. pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral or subcutaneous formulations may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials. These preparations may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the recipient to whom it is intended. Aqueous and non-aqueous sterile suspensions may contain suspending agents and thickeners. The formulations may be presented in unit / dose or multi-dose containers, such as sealed ampoules, syringes, and vials, and may be stored in a freeze-dried state requiring only the addition of a sterile liquid carrier, such as physiological saline or sterile water for injection, immediately before use.

[0120] The pharmaceutical compositions described herein may be manufactured in a commonly known manner, for example, by conventional mixing, dissolution, granulation, sugar-coating, polishing, emulsification, encapsulation, encapsulation, or lyophilization processes. The pharmaceutical compositions may also be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and / or auxiliaries that facilitate the processing of the active agent into a pharmaceutically usable preparation. The appropriate formulation depends on the chosen route of administration.

[0121] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to allow easy passage through the injection needle. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0122] Oral compositions generally contain an inert diluent or a pharmaceutically acceptable food-grade carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active agent may be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, where the drug in the liquid carrier is applied orally and either rinsed in the mouth and spat out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or drugs of similar properties: binders, e.g., microcrystalline cellulose, tragacanth, or gelatin; excipients, e.g., starch or lactose; disintegrants, e.g., alginic acid, Primogel, or corn starch; lubricants, e.g., magnesium stearate or Sterotes; flow enhancers, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose or saccharin; or flavorings, e.g., peppermint, methyl salicylate, or orange flavor.

[0123] When administered by inhalation, the drug is delivered in the form of a suitable nebulizer, such as a gas like carbon dioxide, or an aerosol spray from a pressurized container or dispenser containing a nebulizer.

[0124] Pharmaceutical compositions can be prepared using pharmaceutically acceptable carriers that protect oligonucleotides from rapid elimination from the body, such as controlled-release formulations including implants and microencapsulation delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyoltoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art, and the materials are commercially available. Liposome suspensions (containing liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0125] For ease of administration and uniformity of dosage, it may be desirable to formulate oral or parenteral compositions in dosage unit forms. As used herein, unit dosage forms refer to physically distinct units suitable as unit doses for the target being treated. Each unit contains a predetermined amount of modified oligonucleotide calculated to associate with the required pharmaceutical carrier to produce the desired therapeutic effect. The specifications of the unit dosage forms in this disclosure are determined by and directly depend on the inherent properties of the active agent and the specific therapeutic effect to be achieved.

[0126] The pharmaceutical composition may be included in a container, pack, or dispenser, along with instructions for administration.

[0127] Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic salts of basic residues such as amines, and alkali or organic salts of acidic residues. Pharmaceutically acceptable salts include conventional sodium salts, calcium salts, magnesium salts, or other non-toxic salts, or quaternary ammonium salts of parent compounds formed from, for example, non-toxic inorganic or organic acids.

[0128] Techniques for formulating and administering the disclosed compositions of the present invention can be found in Remington: The Science and Practice of Pharmacy, 19th edition, Mack Publishing Co., Easton, PA (1995).

[0129] The oligonucleotides of this disclosure may be encapsulated within particles or incorporated onto the surface of particles. In some embodiments, the particles are nanoparticles. Exemplary nanoparticles include liposomes, micelles, polymer-based nanoparticles, lipid-polymer-based nanoparticles, and polymer micelles.

[0130] In some embodiments, the nanoparticles include liposomes. Liposomes are spherical vesicles having at least one lipid bilayer and, in some embodiments, an aqueous core. In some embodiments, the lipid bilayer of the liposome may contain phospholipids. An exemplary but non-limiting example of a phospholipid is phosphatidylcholine, but the lipid bilayer may contain additional lipids such as phosphatidylethanolamine. Liposomes may be multilamellar, i.e., consisting of several lamellar phase lipid bilayers, or single-lamellar liposomes having a single lipid bilayer. Liposomes may be fabricated in a specific size range to make them viable targets for phagocytosis. Liposomes may be in the size range of 20 nm to 100 nm, 100 nm to 400 nm, 1 μM or larger, or 200 nm to 3 μM. Examples of lipidoid and lipid-based formulations are provided in U.S. Patent Application Publication No. 20090023673. In other embodiments, one or more lipids are one or more cationic lipids. Those skilled in the art will recognize which liposomes are suitable for encapsulating the modified oligonucleotides described herein.

[0131] In some embodiments, the nanoparticles include micelles. Micelles are aggregates of surfactant molecules. Exemplary micelles include aggregates of amphiphilic polymers, polymers, or copolymers in aqueous solution, where the hydrophilic head portion is in contact with the surrounding solvent, while the hydrophobic tail region is isolated at the center of the micelle.

[0132] In some embodiments, the nanoparticles include polymer-based nanoparticles. These polymer-based nanoparticles include one or more polymers such as polyester, poly(orthoester), poly(ethyleneimine), poly(caprolactone), polyanhydride, poly(acrylic acid), polyglycolide, or poly(urethane). In yet another embodiment, one or more polymers include poly(lactic acid) (PLA) or poly(lactic-co-glycolic acid) (PLGA). In exemplary embodiments, one or more polymers include polyalkylene glycols such as polyethylene glycol (PEG), or polyalkylene oxides such as polyethylene oxide (PEO).

[0133] In some embodiments, the nanoparticles or a portion thereof are degradable. In other embodiments, the lipids and / or polymers of the nanoparticles are degradable.

[0134] In some embodiments, pharmaceutical compositions comprising oligonucleotides and / or HBV antigens are used to prepare pharmaceuticals for the treatment of patients who have or are susceptible to HBV infection and / or HBV-related conditions.

[0135] IV. Kits and manufactured products This disclosure provides kits and products comprising the vaccines described herein, and pharmaceutical compositions comprising them. In some embodiments, at least one oligonucleotide and at least one HBV antigen are provided in the same vial. In some embodiments, at least one oligonucleotide and at least one HBV antigen are provided in two separate vials for bedside mixing or separate administration. In some embodiments, the oligonucleotide comprises a modified oligonucleotide comprising one of the sequences of SEQ ID NOs. 10 to 666, or one, two, three, four, five, six, seven, eight, nine, or ten sequence modifications thereof. In some embodiments, the oligonucleotide comprises one of the sequences of SEQ ID NOs. 10 to 666, or an oligonucleotide comprising at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the oligonucleotide is modified.

[0136] The kit may further include instructions or labels for using the kit to treat, prevent or improve HBV-related diseases, disorders, or conditions.

[0137] Compositions comprising oligonucleotides and / or HBV antigens can be lyophilized before packaging in a kit, or they can be provided in solution or suspension with a pharmaceutically acceptable carrier, diluent, or excipient.

[0138] In some embodiments, the kit further comprises at least one additional agent for treating an HBV-related disease, disorder, or condition described herein.

[0139] V. Definition Unless otherwise specified, the nomenclature, procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well-known and commonly used in the art. Standard techniques may be used for chemical synthesis and chemical analysis. Where permitted, all patents, applications, published applications, and other publications, GENBANK accession numbers, and relevant sequence information available through databases such as the National Center for Biotechnology Information (NCBI), as well as other data referred to throughout the disclosure herein, are incorporated by reference in part with respect to the documents considered herein, and in whole.

[0140] Unless otherwise specified, the following terms have the following meanings:

[0141] "2'-O-methoxyethyl" (also known as 2'-MOE and 2'-O(CH2)2-OCH3) refers to the O-methoxyethyl modification at the 2' position of the furanose ring. 2'-O-methoxyethyl modified sugars are modified sugars.

[0142] "2'-MOE nucleoside" (including 2'-O-methoxyethyl nucleoside) refers to a nucleoside that contains a 2'-MOE modified sugar moiety.

[0143] "5-methylcytosine" refers to cytosine modified with a methyl group attached to the 5th position. 5-methylcytosine is a modified nucleic acid base.

[0144] "Approximately" means within ±10% of the value. For example, if it is stated that "oligonucleotides affected at least approximately 70% inhibition of the target," it means that the target level was inhibited within the range of 60% to 80%.

[0145] "100% complementary" means that each nucleic acid base of the first nucleic acid has a complementary nucleic acid base in the second nucleic acid. In certain embodiments, the first nucleic acid is an oligonucleotide, and the target nucleic acid is the second nucleic acid.

[0146] "Acute hepatitis B infection" occurs when a person exposed to the hepatitis B virus begins to develop the signs and symptoms of viral hepatitis. This period, called the incubation period, averages about 90 days, but can be as short as about 45 days or as long as about 6 months.

[0147] "Animals" refers to humans, or non-human animals including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, primates, monkeys, and chimpanzees, as well as non-human primates.

[0148] An "antibody" refers to a molecule characterized by its ability to react specifically with an antigen in some way, and antibodies and antigens are each defined from the perspective of the other. An antibody is a complete antibody molecule, or a heavy chain, light chain, F ab Region, and F c It can refer to any fragment or region of a domain or area.

[0149] "Base complementarity" refers to the ability of an antisense oligonucleotide to accurately pair (i.e., hybridize) the nucleic acid bases of the corresponding nucleic acid bases in a target nucleic acid, mediated by Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds between the corresponding nucleic acid bases.

[0150] A "bicyclic sugar" refers to a furanose ring modified by a bridge between two non-geminal carbon atoms. Bicyclic sugars are modified sugars.

[0151] "Cap structure" or "terminal cap portion" refers to a chemical modification incorporated into one of the ends of an antisense compound.

[0152] "cEt (constrained ethyl)" or "constrained ethyl" refers to a bicyclic sugar moiety containing a bridge connecting the 4'-carbon and 2'-carbon atoms, and the bridge has the formula: 4'-CH(CH3)-O-2'.

[0153] A "restricted ethyl nucleoside" (also known as a cEt nucleoside) refers to a nucleoside containing a bicyclic sugar moiety that includes a 4'-CH(CH3)-O-2' bridge.

[0154] A "chemically distinct region" refers to a region of an oligonucleotide that is chemically different from another region of the same oligonucleotide. For example, a region containing a 2'-O-methoxyethyl nucleotide is chemically different from a region containing a nucleotide that does not have the 2'-O-methoxyethyl modification.

[0155] Chronic hepatitis B infection occurs when a person initially suffers from an acute infection but is unable to fight off the infection. Whether the disease becomes chronic or fully recovers depends primarily on the age of the infected person.

[0156] "Simultaneous administration" means the administration of two or more pharmaceuticals to an individual. These two or more pharmaceuticals may be in a single pharmaceutical composition or in separate pharmaceutical compositions. Each of the two or more pharmaceuticals may be administered via the same or different route of administration. Simultaneous administration includes parallel administration or sequential administration.

[0157] "Complementarity" refers to the ability of nucleic acid bases of a first nucleic acid and a second nucleic acid to pair.

[0158] "Diluent" refers to a component in a composition that lacks pharmacological activity but is pharmaceutically necessary or desirable. For example, in a drug to be injected, the diluent may be a liquid, such as physiological saline.

[0159] "Dosage unit" means the form in which the drug is provided, for example, a pill, a tablet, or any other dosage unit known in the art.

[0160] "Dose" means a specified amount of a drug to be delivered in a single dose or over a specified period. In certain embodiments, the dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume that cannot be easily contained in a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, the dose may be administered in two or more injections to minimize injection site reactions in the individual. In other embodiments, the drug is administered over a long period or continuously. The dose may be expressed as the amount of the drug per hour, day, week, or month.

[0161] "Duration" refers to the period during which the activity or event persists. In certain embodiments, the treatment period is the period during which the drug dose is administered.

[0162] A "gap" is an internal segment of an oligonucleotide containing one or more linked deoxynucleosides, located directly or indirectly between the 5' wing (W1) and the 3' wing (W2). A gap may be interchangeably referred to as a "gap," "gap region," or "gap segment."

[0163] "HBV" refers to mammalian hepatitis B viruses, including human hepatitis B virus. This term encompasses the geographical genotypes of hepatitis B viruses, particularly human hepatitis B virus, as well as variant strains of the geographical genotypes of hepatitis B viruses.

[0164] "HBV antigen" refers to any hepatitis B virus antigen or protein, including core proteins such as "hepatitis B core antigen" or "HBcAG" and "hepatitis B E antigen" or "HBeAg," and envelope proteins such as "HBV surface antigen" or "HBsAg" or "HBsAG."

[0165] "Hepatitis B E antigen," or "HBeAg," or "HBcAg" is the secreted non-particulate form of the HBV core protein. Because the HBV antigens HBeAg and HBcAg share a primary amino acid sequence, they exhibit cross-reactivity at the T cell level.

[0166] "HBV surface antigen," or "HBsAg," is the envelope protein of infectious HBV virus particles, but it is also secreted as non-infectious particles with higher serum levels than HBV virus particles.

[0167] "Individual" refers to a human or non-human animal selected for treatment or therapy.

[0168] Terms such as "induce," "inhibit," "enhance," "increase," "boost," and "decrease" generally indicate a quantitative difference between two states. Such terms may refer to a statistically significant difference between two states. Such terms apply, for example, to levels of expression and levels of activity. As used herein, the terms "inhibit" or "reduce," or their grammatical variations, refer to a reduction or decrease in activity to a specified level of at least about 5%, about 10%, about 15%, about 25%, about 35%, about 40%, about 50%, about 60%, about 75%, about 80%, about 90%, about 95%, or more. In some embodiments, inhibition or reduction results in little to virtually no detectable activity (at most a small amount, e.g., about 10% or even less than 5%).

[0169] "Intraperitoneal administration" refers to administration by injection or delivery into the peritoneum.

[0170] "Intravenous administration" means administration into a vein.

[0171] "Inhibiting expression or activity" refers to reducing or blocking expression or activity, and does not necessarily mean completely eliminating it.

[0172] "Linked deoxynucleosides" means deoxyribonucleic acid bases (A, G, C, T, U) that are linked by phosphate esters to form nucleotides.

[0173] "Linked nucleosides" means adjacent nucleosides that are linked to each other by internucleoside bonds. Examples of linked nucleosides are linked nucleotides where the linkage includes a phosphate group atom, such as a phosphodiester bond.

[0174] "Locked nucleic acid" or "LNA" or "LNA nucleoside" means a nucleic acid monomer having a bridge connecting two carbon atoms between the 4'-position and the 2'-position of the nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugars include, as depicted below, A) α-L-methyleneoxy (4'-CH2-O-2') LNA, (B) β-D-methyleneoxy (4'-CH2-O-2')-LNA; (C) ethyleneoxy (4'-(CH2)2-O-2') LNA, (D) aminooxy (4'-CH2-O-NI-2') LNA, and (E) oxyamino (4'-CH2-NI-O-2') LNA, but are not limited thereto.

[0175]

Chemical Formula

[0176] As used herein, LNA oligonucleotides include, but are not limited to, oligonucleotides having at least one bridge between the 4'-position and the 2'-position of the sugar, and each bridge is independently -[C(R 1 )(R 2 )]n-, -C(R 1 )=C(R 2 )-, -C(R 1 )=N-, -C(=NR 1 )-, -C(=O)-, -C(=S)-, -O-, -Si(R 1 )2-, -S(=O) x -, and -N(R 1)- comprises one or two to four linking groups independently selected from, where x is 0, 1, or 2, n is 1, 2, 3, or 4, and each R 1 and R 2 These are independently H, protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2~C 12 Alkenyl substitution C2~C 12 Alkenyl, C2~C 12 Alkinyl substitution C2~C 12 Alkinyl, C5~C 20 Aryl substitution C5~C 20 Aryl, heterocyclic group, substituted heterocyclic group, heteroaryl, substituted heteroaryl, C5-C7 alicyclic group, substituted C5-C7 alicyclic group, halogen, OJ 1 , NJ I J 2 SJ 1 N3, COOJ 1 Acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J 1 ) or sulfoxyl (S(=O)-J 1 ) and each J 1 and J 2 These are H, C1~C independently. 12 Alkyl, substituted C1-C 12 Alkyl, C2~C 12 Alkenyl substitution C2~C 12 Alkenyl, C2~C 12 Alkinyl substitution C2~C 12 Alkinyl, C5~C 20 Aryl substitution C5~C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1~C 12 Aminoalkyl, substituted C1-C 12 It is an aminoalkyl group or a protecting group.

[0177] An example of a 4'-2' bridging group included in the definition of LNA is the formula: --[C(R 1 )(R 2 )] n -,-[C(R 1 )(R2 )] n -O-, -C(R 1 )(R 2 )-N(R 1 )-O-, or -C(R 1 )(R 2 )-ON(R 1 )- is one of the following, but is not limited to these. Furthermore, other bridging groups included in the definition of LNA include 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', and 4'-CH2-ON(R 1 )-2' and 4'-CH2-N(R 1 )-O-2'-bridged, and each R 1 and R 2 These are independently H, protecting groups or C1-C 12 It is alkyl.

[0178] Furthermore, the definition of LNA according to the present invention also includes LNA in which the 2'-hydroxyl group of the ribosyl sugar ring is connected to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy(4'-CH2-O-2') bridge and creating a bicyclic sugar moiety. The bridge can also be a methylene(-CH2-) group connecting the 2' oxygen atom and the 4' carbon atom, in which case the term methyleneoxy(4'-CH2-O-2')LNA is used. Moreover, in the case of a bicyclic sugar moiety having an ethylene bridge group at this position, the term ethyleneoxy(4'-CH2CH2-O-2')LNA is used. The isomer of methyleneoxy(4'-CH2-O-2')LNA, α-L-methyleneoxy(4'-CH2-O-2'), is also included in the definition of LNA when used herein.

[0179] "Modified nucleoside linkages" refer to substitutions or modifications to naturally occurring nucleoside links (i.e., phosphodiester nucleoside links).

[0180] "Modified nucleic acid bases" refer to any nucleic acid base other than adenine, cytosine, guanine, thymidine, or uracil. "Unmodified nucleic acid bases" refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0181] "Modified nucleoside" independently refers to a nucleoside that has a modified sugar moiety and / or a modified nucleic acid base.

[0182] "Modified nucleotide" independently refers to a nucleotide that has a modified sugar moiety, a modified nucleoside linkage, or a modified nucleic acid base.

[0183] "Modified oligonucleotide" means an oligonucleotide containing at least one modified nucleoside linkage, a modified sugar, and / or a modified nucleic acid base.

[0184] "Modified sugar" means substitution and / or any alteration of the natural sugar portion.

[0185] A "monomer" refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

[0186] "Motif" refers to the pattern of unmodified and modified nucleosides in antisense oligonucleotides. "Natural sugar moiety" refers to the sugar moiety found in DNA(2'-H) or RNA(2'-OH). "Naturally occurring internucleoside linkage" refers to phosphodiester linkage from 3' to 5'.

[0187] A "nucleic acid base" refers to a nitrogen-containing heterocyclic base portion that can pair with a base of another nucleic acid.

[0188] "Nucleic acid base sequence" refers to the sequence of consecutive nucleic acid bases, independent of any sugars, ligatures, and / or nucleic acid base modifications.

[0189] A "nucleoside" refers to a nucleic acid base bound to a sugar. Nucleosides include deoxynucleosides, such as deoxyribonucleosides.

[0190] A "nucleotide" refers to a nucleoside that has a phosphate group covalently bonded to the sugar portion of the nucleoside.

[0191] "Oligononucleotide" refers to a polymer of nucleosides linked together through nucleoside bonds, where each linked nucleoside may or may not be modified independently of the others.

[0192] "Pareral administration" refers to administration by injection (e.g., bolus injection) or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intraperitoneal administration, or intracranial administration, such as intrathecal or intraventricular administration.

[0193] "Pharmacologically acceptable carrier" means a medium or diluent that does not interfere with the structure of oligonucleotides. Certain such carriers enable the formulation of pharmaceutical compositions as, for example, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral administration by a subject.

[0194] "Pharmacologically acceptable derivatives" include pharmaceutically acceptable salts, conjugates, prodrugs, or isomers of the oligonucleotides described herein.

[0195] "Pharmacologically acceptable salt" means a physiologically and pharmaceutically acceptable salt of an antisense oligonucleotide, i.e., a salt that retains the desired biological activity of the parent oligonucleotide and does not confer any undesirable toxicological effects to it.

[0196] "Pharmaceuticals" refer to substances that provide therapeutic benefits when administered to an individual.

[0197] "Pharmaceutical composition" means a mixture of substances suitable for administration to a target. For example, a pharmaceutical composition may include an antisense oligonucleotide and a sterile aqueous solution. In certain embodiments, the pharmaceutical composition exhibits activity in a free uptake assay in a particular cell line.

[0198] "Phosphothioate linkage" refers to a linkage between nucleosides in which the phosphodiester bond is modified by replacing one of the non-bridged oxygen atoms with a sulfur atom. Phosphothioate linkage is a modified nucleoside linkage.

[0199] "Prevention" or "prevention" refers to delaying or preventing the onset or progression of a condition or disease for a period of several hours to several days, preferably several weeks to several months.

[0200] "Preventive effective dose" refers to the amount of a drug that provides a preventive or preventive benefit to an animal.

[0201] A "ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of the sugar portion. Ribonucleotides can be modified with any of a variety of substituents.

[0202] "Salt" refers to a physiologically and pharmaceutically acceptable salt of an antisense oligonucleotide, that is, a salt that retains the desired biological activity of the parent oligonucleotide without conferring any undesirable toxicological effects to it.

[0203] The term "segment" can be interchangeably referred to as "area" or "part."

[0204] A “separator” in an oligonucleotide of this disclosure is positioned directly between two gap regions in the oligonucleotide, separating the two gap regions. The separator may have one or more nucleosides, which are chemically distinct from the nucleosides containing the gaps. The separator segment may include modified nucleosides to confer properties such as enhanced inhibitory activity, increased binding affinity to target nucleic acids, or reduced in vivo toxicity or resistance to degradation by in vivo nucleases. An oligonucleotide may contain one or more separator segments. Exemplary oligonucleotides of this disclosure may contain one, two, three, four, five, or six separator segments. In some embodiments, the oligonucleotide contains one separator segment. In some embodiments, the oligonucleotide contains two separator segments.

[0205] "Seroconversion" is defined as either the absence of serum HBeAg and the presence of serum HBeAb when monitoring HBeAg as a determinant of seroconversion, or the absence of serum HBsAg when monitoring HBsAg as a determinant of seroconversion, as determined by the currently available detection limits of commercially available ELISA systems.

[0206] "Target nucleic acid," "target RNA," "target RNA transcript," and "nucleic acid target" all refer to nucleic acids that can be targeted by antisense oligonucleotides.

[0207] The "target region" refers to the portion of a target nucleic acid that is targeted by one or more antisense oligonucleotides.

[0208] The "target segment" refers to the nucleotide sequence of the target nucleic acid that is targeted by the antisense oligonucleotide.

[0209] "Therapeutic dose" refers to the amount of a drug that provides a therapeutic benefit to an individual.

[0210] "Treatment" refers to administering a composition to bring about a change or improvement in a disease or condition.

[0211] "Unmodified" nucleic acid bases refer to the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0212] "Unmodified nucleotide" refers to a nucleotide composed of naturally occurring nucleic acid bases, a sugar moiety, and nucleoside linkages. In certain embodiments, the unmodified nucleotide is an RNA nucleotide (i.e., a (3-D-ribonucleoside)) or a DNA nucleotide (i.e., a (3-D-deoxyribonucleoside)).

[0213] A "validated target segment" is defined as at least eight nucleic acid bases (i.e., eight consecutive nucleic acid bases) of the target region targeted by the active oligomeric oligonucleotide.

[0214] A "wing" is a terminal segment of an oligonucleotide modified to confer properties such as enhanced inhibitory activity, improved biological activity, increased binding affinity to target nucleic acids, or reduced in vivo toxicity or resistance to degradation by in vivo nucleases. A wing may be referred to as a "wing," "wing region," or "wing segment." As used herein, a wing comprises at least two linked nucleosides, a subset of which may comprise one or more deoxynucleosides, but the entire wing cannot consist solely of deoxynucleosides. In some embodiments, a wing comprises 2 to 8 linked nucleosides. In some embodiments, a wing comprises 2 to 6 linked nucleosides.

[0215] The oligonucleotides of this disclosure include a 5' wing segment (W1) located at the 5' end of the oligonucleotide, and the residue at the 3' end of W1 is not a deoxynucleoside. The oligonucleotides of this disclosure also include a 3' wing segment (W2) located at the 3' end of the oligonucleotide, and the residue at the 5' end of W2 is not a deoxynucleoside.

[0216] The 5' wing (W1) begins at the 5' end of the oligonucleotide, extends from 5' in the 3' direction, and is directly ligated to the deoxynucleoside of the first gap, thus representing the 3' end of W1 and the 5' end of the first gap (G1).

[0217] The 3' wing (W2) begins at the 3' end of the oligonucleotide, extends from 3' to 5', and is directly ligated to the deoxynucleoside of the gap, thus indicating the 3' end of the gap and the 5' end of W2.

[0218] Listed embodiments The following non-limiting listed embodiments are provided to illustrate embodiments of the present invention.

[0219] Embodiment I-1. A vaccine composition comprising at least one oligonucleotide and at least one hepatitis B virus (HBV) antigen.

[0220] Embodiment I-2. The vaccine composition according to Embodiment I-1, wherein the oligonucleotide is complementary to one or more portions of the nucleic acid sequence of the HBV genome.

[0221] Embodiment I-3. The vaccine composition according to Embodiment I-1, wherein the oligonucleotide is not complementary to a portion of the nucleic acid sequence of the HBV genome.

[0222] Embodiment I-4. A vaccine composition according to any one of Embodiments I-2 to I-3, wherein the HBV genome is the genome of HBV GT-A, GT-B, GT-C, GT-D, GT-E, GT-F, GT-G, GT-H, GT-I, or GT-J.

[0223] Embodiment I-5. A vaccine composition according to any one of Embodiments I-1 to I-4, wherein the oligonucleotide comprises 10 to 60 nucleic acid bases.

[0224] Embodiment I-6. The vaccine composition according to any one of Embodiments I-1 to I-5, wherein the oligonucleotide is a modified oligonucleotide.

[0225] Embodiment I-7. The vaccine composition according to any one of Embodiments I-1 to I-6, wherein the modified oligonucleotide comprises one or more modified sugars, modified bases, modified nucleoside bonds, or a combination thereof.

[0226] Embodiment I-8. The vaccine composition according to any one of Embodiments I-1 to I-6, wherein at least one nucleoside bond is a phosphodiester bond.

[0227] Embodiment I-9. The vaccine composition according to any one of Embodiments I-1 to I-8, wherein at least one nucleoside bond is a modified nucleoside bond.

[0228] Embodiment I-10. The vaccine composition according to Embodiment I-9, wherein the modified nucleoside bond is a phosphorothioate nucleoside bond.

[0229] Embodiment I-11. A vaccine composition according to any one of Embodiments I-1 to I-10, wherein the oligonucleotide comprises a linked deoxynucleoside.

[0230] Embodiment I-12. The vaccine composition according to any one of Embodiments I-1 to I-11, wherein the oligonucleotide comprises at least one nucleoside including modification at the C2' position.

[0231] Embodiment I-13. The vaccine composition according to Embodiment I-12, wherein the oligonucleotide comprises at least one nucleoside including 2'-O-methyl sugar modification, 2'-O-methoxyethyl sugar modification, 2'-fluoro sugar modification and / or 2'-fluoro-arabino nucleic acid (2'-fluoro-ANA) sugar modification.

[0232] Embodiment I-14. The vaccine composition according to any one of Embodiments I-1 to I-13, wherein the oligonucleotide comprises at least one bicyclic sugar-modified nucleoside.

[0233] Embodiment I-15. The vaccine composition according to any one of Embodiments I-1 to I-14, wherein the oligonucleotide comprises at least one nucleoside including a phosphorodiamidate morpholino modification.

[0234] Embodiment I-16. A vaccine composition according to any one of Embodiments I-1 to I-15, wherein the oligonucleotide comprises at least one locked nucleic acid (LNA), peptide nucleic acid, and / or glycol nucleic acid.

[0235] Embodiment I-17. The vaccine composition according to any one of Embodiments I-1 to I-16, wherein the oligonucleotide is selected from SEQ ID NOs: 10 to 666, or is an oligonucleotide having at least one sequence modification.

[0236] Embodiment I-18. The vaccine composition according to any one of Embodiments I-1 to I-17, wherein the oligonucleotide is selected from SEQ ID NOs: 10 to 666, or is an oligonucleotide having at least 70% sequence identity thereto.

[0237] Embodiment I-19. A vaccine composition according to any one of Embodiments I-1 to I-18, comprising at least two different oligonucleotides.

[0238] Embodiment I-20. The vaccine composition according to any one of Embodiments I-1 to I-19, wherein the HBV antigen is human HBV antigen.

[0239] Embodiment I-21. The vaccine composition according to any one of Embodiments I-1 to I-20, wherein the HBV antigen is HBV surface antigen (HBsAg), HBV e antigen (HBeAg), HBV core antigen (HBcAg), or any combination thereof.

[0240] Embodiment I-22. The vaccine composition according to Embodiment I-21, wherein the HBsAg is Small-HBsAg, Medium-HBsAg, Large-HBsAg, or any combination thereof.

[0241] Embodiment I-23. The vaccine composition according to any one of Embodiments I-1 to I-22, wherein the HBV antigen is derived from HBV genotype GT-A, GT-B, GT-C, GT-D, GT-E, GT-F, GT-G, GT-H, GT-I, or GT-J.

[0242] Embodiment I-24. The vaccine composition according to any one of Embodiments I-1 to I-23, wherein the HBV antigen is isolated from a patient, recombinantly produced, or synthesized.

[0243] Embodiment I-25. The vaccine composition according to any one of Embodiments I-1 to I-24, comprising at least two different HBV antigens.

[0244] Embodiment I-26. The vaccine composition according to any one of Embodiments I-1 to I-25, comprising an adjuvant.

[0245] Embodiment I-27. The vaccine composition according to Embodiment I-26, wherein the adjuvant is aluminum hydroxide.

[0246] Embodiment I-28. The vaccine composition according to Embodiment I-26, wherein the adjuvant is a toll-like receptor 9 (TLR9) agonist.

[0247] Embodiment I-29. The vaccine composition according to Embodiment I-26, wherein the adjuvant is an oligonucleotide.

[0248] Embodiment I-30. The vaccine composition according to Embodiment I-27, wherein the oligonucleotide adjuvant comprises CpG1018.

[0249] Embodiment I-31. The vaccine composition according to any one of Embodiments I-1 to I-30, wherein the vaccine composition is a therapeutic vaccine composition.

[0250] Embodiment I-32. The vaccine composition according to any one of Embodiments I-1 to I-30, wherein the vaccine composition is a preventive vaccine composition.

[0251] Embodiment I-33. A pharmaceutical composition comprising the vaccine composition described in any one of Embodiments I-1 to I-32 and a pharmaceutically acceptable carrier, diluent, or excipient.

[0252] Embodiment I-34. A kit comprising the vaccine composition described in any one of Embodiments I-1 to I-32.

[0253] Embodiment I-35. A method for inducing an immune response to the HBV antigen in a subject suffering from HBV infection or an HBV-related disease, disorder, or condition, comprising administering to a subject the vaccine composition described in any one of Embodiments I-1 to I-32 or the pharmaceutical composition described in Embodiment I-33.

[0254] Embodiment I-36. A method for treating or preventing a target HBV infection or HBV-related disease, disorder, or condition by vaccine therapy, comprising administering to a target the vaccine composition described in any one of Embodiments I-1 to I-32, or the pharmaceutical composition described in Embodiment I-33.

[0255] Method for inducing an immune response against a hepatitis B virus (HBV) antigen in a subject suffering from HBV infection, or an HBV-related disease, disorder, or condition, comprising administering an oligonucleotide and an HBV antigen to the subject using vaccine therapy.

[0256] Method for treating or preventing HBV infection, or an HBV-related disease, disorder, or condition in a subject, comprising administering an oligonucleotide and an HBV antigen to the subject using vaccine therapy.

[0257] Method according to any one of Embodiments I-37 to 38, wherein the oligonucleotide and the HBV antigen are administered simultaneously.

[0258] Method according to Embodiment I-39, wherein the oligonucleotide and the HBV antigen are provided as a premix.

[0259] Method according to Embodiment I-39, wherein the oligonucleotide and the HBV antigen are mixed immediately before administration.

[0260] Method according to Embodiment I-39, wherein the oligonucleotide and the HBV antigen are administered simultaneously at different injection sites.

[0261] Method according to Embodiment I-42, wherein the injection sites are adjacent.

[0262] Method according to Embodiment I-39, wherein the oligonucleotide and the HBV antigen are administered simultaneously at the same injection site.

[0263] Method according to any one of Embodiments I-37 to I-38, wherein the oligonucleotide and the HBV antigen are administered sequentially.

[0264] Embodiment I-46. The method according to Embodiment I-45, wherein the oligonucleotide and HBV antigen are administered sequentially to the same injection site.

[0265] Embodiment I-47. The method according to Embodiment I-45, wherein the oligonucleotide and HBV antigen are administered sequentially to different injection sites.

[0266] Embodiment I-48. The method according to Embodiment I-47, wherein the injection sites are adjacent.

[0267] Embodiment I-49. The method according to Embodiment I-45, wherein the oligonucleotide is administered before the HBV antigen.

[0268] Embodiment I-50. The method according to Embodiment I-49, wherein multiple doses of oligonucleotides are administered before the administration of HBV antigen.

[0269] Embodiment I-51. The method according to Embodiment I-50, wherein two, three, four, five, or more doses of oligonucleotides are administered before administering the HBV antigen.

[0270] Embodiment I-52. The method according to Embodiment I-49, wherein a single dose of oligonucleotide is administered before the administration of HBV antigen.

[0271] Embodiment I-53. The method according to any one of Embodiments I-49 to I-52, wherein HBV antigen is administered multiple times after administering an oligonucleotide.

[0272] Embodiment I-54. The method according to any one of Embodiments I-37 to I-53, wherein at least two doses of oligonucleotide and / or HBV antigen are administered.

[0273] Embodiment I-55. The method according to any one of Embodiments I-37 to I-54, wherein the oligonucleotide comprises the oligonucleotide described in any one of Embodiments I-2 to I-18.

[0274] Embodiment I-56. The method according to any one of Embodiments I-37 to I-55, wherein the HBV antigen is HBV surface antigen (HBsAg), HBV e antigen (HBeAg), or HBV core antigen (HBcAg).

[0275] Embodiment I-57. The method according to Embodiment I-56, wherein HBsAg is Small-HBsAg, Middle-HBsAg, Large-HBsAg, or any combination thereof.

[0276] Embodiment I-58. The method according to any one of Embodiments I-37 to I-57, wherein the HBV antigen is derived from HBV GT-A, GT-B, GT-C, GT-D, GT-E, GT-F, GT-G, GT-H, GT-I, or GT-J subtypes.

[0277] Embodiment I-59. The method according to any one of Embodiments I-35 to I-58, wherein the method includes one or more pretreatments.

[0278] Embodiment I-60. The method according to Embodiment I-59, wherein the pretreatment includes administering an oligonucleotide which is the same oligonucleotide as the vaccine composition.

[0279] Embodiment I-61. The method according to Embodiment I-59, wherein the pretreatment includes administering an oligonucleotide different from the oligonucleotide of the vaccine composition.

[0280] Embodiment I-62. The method according to Embodiment I-59, wherein the pretreatment includes administering an HBV treatment selected from the group consisting of siRNA against HBV, interferon-gamma, PD-1 inhibitors, and PD-L1 inhibitors.

[0281] Embodiment I-63. The method according to any one of Embodiments I-35 to I-62, which results in a decrease in serum levels of HBsAg, HBcAg, and / or HBeAg.

[0282] Embodiment I-64. The method according to any one of Embodiments I-35 to I-63, wherein the method results in a reduction of HBV DNA levels in the liver.

[0283] Embodiment I-65. The method according to any one of Embodiments I-35 to I-64, wherein the method results in a reduction of HBV mRNA levels in the liver.

[0284] Embodiment I-66. The method according to any one of Embodiments I-35 to I-65, which results in an increase in serum HBsAb levels.

[0285] Embodiment I-67. The method according to any one of Embodiments I-35 to I-66, which results in an increase in the T cell response.

[0286] Embodiment I-68. The method according to any one of Embodiments I-63 to I-67, which produces a dose-dependent effect.

[0287] Embodiment I-69. The method according to any one of Embodiments I-35 to I-68, wherein the subject is a primate.

[0288] Embodiment I-70. The method of Embodiment I-69, wherein the subject is a human subject.

[0289] Embodiment I-71. Any one of Embodiments I-35 to I-70, wherein the route of administration is subcutaneous, intramuscular, intravenous, or intradermal.

[0290] Embodiment I-72. The method according to any one of Embodiments I-35 to I-71, wherein the subject is suffering from acute HBV infection.

[0291] Embodiment I-73. The method according to any one of Embodiments I-35 to I-71, wherein the subject is suffering from chronic HBV infection.

[0292] Embodiment I-74. The method according to any one of Embodiments I-35 to I-73, wherein the subject is administered approximately 20 mg to approximately 500 mg of oligonucleotide per dose.

[0293] Embodiment I-75. One of Embodiments I-35 to I-74, wherein the subject is administered approximately 1 μg to approximately 500 μg of HBsAg per dose.

[0294] Embodiment I-76. A kit comprising an oligonucleotide of any of the above embodiments and a hepatitis B virus (HBV) antigen.

[0295] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. [Examples]

[0296] Example 1: Immunoenhancing activity of oligonucleotides in HBV carrier mice It should be noted that the oligonucleotides of this disclosure and these examples, as well as their respective AUS numbers, are provided in Tables 1.1 and 1.2, as shown in Figures 1 and 2 of the accompanying drawings. The oligonucleotides of this disclosure were synthesized using a standard solid-phase synthesis protocol as described in International Publication No. 2023131098(A2).

[0297] In an HBV mouse model, the immunoenhancing activity of the oligonucleotide AUS1441 of this disclosure against serum levels of hepatitis B surface antigen protein (HBsAg) and antibody against HBsAg (HBsAb) was evaluated. The HBV mouse model was constructed by hydrodynamic injection (HDI) of an HBV plasmid containing the preS2 / S of the HBV sequence in C57BL / 6 mice. The HBV carrier mice thus prepared are referred to herein as HDI-HBV mice. The HDI-HBV mice were randomly divided into four groups of four animals each. In the initial administration period (pre-treatment period), all animals were administered 60 mg / kg of AUS1463 by subcutaneous injection (SC) to reduce HBsAg levels. During the second administration period (vaccination period), which began 10 days after the administration in the first period, animals were subcutaneously administered 100 μl of an administration solution consisting of one of the following: normal physiological saline (Group A), 3 ug of HBsAg (Group B), 500 ug of AUS1441 and 3 ug of HBsAg (Group C), or 500 ug of AUS1441 (Group D). The start of the second administration period (vaccination period) was counted as day 0. Each group received the corresponding administration solution on day 0, day 14, and day 42.

[0298] The AUS1463 administration solution was prepared by dissolving the AUS1463 powder in saline to a final concentration of 6 mg / mL. The AUS1441 working solution was prepared in physiological saline at a concentration of 10 mg / mL. The HBsAg used in this example is a commercially available natural hepatitis B surface antigen protein isolated and purified from the plasma of chronic hepatitis B patients (purchased from Beijing Biolab Technology Co., Ltd). The HBsAg working solution was prepared in physiological saline at a concentration of 0.06 mg / mL. The HBsAg working solution was mixed with the AUS1441 working solution (group C) in a 1:1 volume ratio for approximately 30 minutes and then administered to the animals. The administration solutions for groups B and D were prepared by diluting the HBsAg and AUS1441 working solutions, respectively, twice with physiological saline.

[0299] Approximately 100 μl of blood sample was collected weekly from the femoral vein of each animal. The blood sample was incubated at 37°C for approximately 30 minutes, and then centrifuged in a pre-cooled (0-4°C) centrifuge to obtain serum sample. The serum sample was stored at -20°C until analysis. Serum HBsAg and HBsAb levels were measured by chemiluminescent immunoassay.

[0300] Figure 3 shows serum HBsAg levels at different time points, normalized to the level on day 0 for each group. The data points are the mean, and the error bars are the standard deviation. Figure 4 shows serum HBsAb levels for each group on days 0, 14, 28, 42, 49, 56, 63, 70, and 77. The boxes represent the geometric mean, the error bars represent the geometric standard deviation, and the dots represent individual HBsAb levels.

[0301] Table 1.3 lists the serum HBsAg and HBsAb levels on day 77. Serum HBsAg levels decreased by more than 2 log in HDI-HBV mice with a dose of 60 mg / kg of AUS1463. In group C (500 μg of AUS 1441 + 3 μg of HBsAg), a sustained decrease in serum HBsAg below the limit of quantification (LOQ of 1 IU / mL for HBsAg) was achieved in all animals (4 / 4), and serum HBsAb levels increased in all animals in this group, peaking at a geometric mean of 567 mIU / mL on day 49. When HBsAg (3 ug) was administered alone (Group B), on day 77, none of the animals in the group had HBsAg levels below the limit of quantification, and HBsAb levels were generally low and fluctuating, with only one of the four animals having an HBsAb level above the limit of quantification (LOQ of 5 mIU / mL for HBsAb). Similarly, when AUS1441 (500 ug) was administered alone (Group D), none of the animals in the group had HBsAg levels below the limit of quantification, and HBsAb levels were below the limit of quantification in all animals in the group (HBsAb LOQ = 5 mIU / mL).

[0302] [Table 6]

[0303] Example 2: Immunoenhancing activity of oligonucleotides in HBV carrier mice The immunoenhancing activity of the oligonucleotides shown in Example 1 is generalizable to other oligonucleotides of the present disclosure, as shown in this example. The immunoenhancing activity of oligonucleotides including AUS1499, AUS1476, AUS1497, AUS1498, AUS1500, and AUS1496 was evaluated in HDI-HBV mice. The animals were randomly divided into eight groups of four animals each. During the initial treatment period (pre-treatment period), all animals were administered three weekly doses of 60 mg / kg of AUS1233 by subcutaneous injection (SC) to reduce HBsAg levels. During the second administration period (vaccination period), which began 21 days after the first dose of the first period, each group of HDI-HBV mice was subcutaneously administered 100 μl of a solution consisting of one of the following: 3 ug of HBsAg (Group 1); a mixture containing 500 ug of AUS1499 and 3 ug of HBsAg (Group 2); a mixture containing 500 ug of AUS1476 and 3 ug of HBsAg (Group 3); or 150 ug of A mixture containing AUS1476 and 3ug of HBsAg (Group 4); a mixture containing 500ug of AUS1497 and 3ug of HBsAg (Group 5); a mixture containing 500ug of AUS1498 and 3ug of HBsAg (Group 6); a mixture containing 500ug of AUS1500 and 3ug of HBsAg (Group 7); or a mixture containing 500ug of AUS1496 and 3ug of HBsAg (Group 8). The start of the second administration period was counted as day 0. The administration solution corresponding to each group was administered on day 0 and day 14.

[0304] An AUS1233 administration solution was prepared by dissolving AUS1233 powder in saline to a concentration of 6 mg / mL. Working solutions for AUS1499, AUS1476, AUS1497, AUS1498, AUS1500, and AUS1496 were each prepared in 10 mg / mL physiological saline. Furthermore, a low-concentration working solution for AUS1476 was prepared in 3 mg / mL saline. The HBsAg used in this example is a commercially available natural hepatitis B surface antigen protein isolated and purified from the plasma of chronic hepatitis B patients (purchased from Beijing Biolab Technology Co., Ltd). The HBsAg working solution was prepared in physiological saline at a concentration of 0.06 mg / mL. The administration solution for Group 1 was prepared by diluting the HBsAg working solution 2-fold with saline. The administration solutions for groups 2, 3, 5, 6, 7, and 8 were prepared by mixing HBsAg working solution with 10 mg / mL of AUS1499, AUS1476, AUS1497, AUS1498, AUS1500, and AUS1496 working solutions, respectively, in a 1:1 volume ratio, approximately 30 minutes before administration to the animals. The administration solution for group 4 was prepared by mixing HBsAg working solution with 3 mg / mL of AUS1476 working solution in a 1:1 volume ratio, approximately 30 minutes before administration to the animals.

[0305] Approximately 100 μl of blood sample was collected weekly from the femoral vein. The blood sample was incubated at 37°C for approximately 30 minutes, and then centrifuged in a pre-cooled (0-4°C) centrifuge to obtain serum sample. The serum sample was stored at -20°C until analysis. HBsAg and HBsAb levels were measured in the serum sample by chemiluminescent immunoassay.

[0306] Serum HBsAg levels at different time points are shown in Figure 5, normalized to the day 0 level for each group, with data points representing the mean. Serum HBsAb levels for each group at days 14, 28, 42, 49, 56, 63, and 70 are shown in Figure 6, with boxes representing the geometric mean and dots representing individual HBsAb levels. Serum HBsAg and HBsAb levels at day 70 are listed in Table 2.1. Serum HBsAg levels decreased by more than 2 log with three weekly subcutaneous administrations of 60 mg / kg of AUS1233 to HDI-HBV mice. When HBsAg (3 ug) was used alone (Group 1), serum HBsAg levels decreased slightly in only one out of four animals, and serum HBsAb exceeded the limit of quantification (LOQ, which is 5 mIU / mL for HBsAb). In groups 3 (500ug AUS1476 + 3ug HBsAg), 5 (500ug AUS1497 + 3ug HBsAg), 7 (500ug AUS1500 + 3ug HBsAg), and 8 (500ug AUS1496 + 3ug HBsAg), a sustained serum HBsAg decrease below the limit of quantification (LOQ = 1 IU / mL for HBsAg) was achieved in all animals (4 / 4), and serum HBsAb levels were boosted in all animals in each of these groups.

[0307] The dose-dependent immunoenhancing effect of oligonucleotides was observed, for example, as shown by the results for groups 3 and 4.

[0308] [Table 7]

[0309] Example 3: Immunoenhancing activity of oligonucleotides in BALB / c mice The immunoenhancing activity of oligonucleotides was investigated in normal mice, and their ability to boost serum HBsAb antibodies was examined. The oligonucleotides used in this example included reference AUS1233, AUS1443, AUS1444, AUS1458, AUS1459, AUS1460, AUS1461, AUS1462, AUS1463, AUS1464, AUS1465, AUS1466, AUS1467, AUS1468, AUS1469, AUS1470, AUS1471, AUS1472, AUS1473, AUS1474, AUS1475, AUS1476, AUS1477, AUS1478, AUS1479, AUS1480, AUS1488, AUS1489, and AUS1490. The animals used were female BALB / c mice. Physiological saline was administered as a negative control. A mixture of HBsAg and either an aluminum adjuvant or CpG1018 (Toll-like receptor 9 (TLR9) agonist adjuvant) was administered as a positive control.

[0310] Working solutions for each oligonucleotide were prepared in physiological saline at a concentration of 3 mg / mL. A CpG1018 working solution was prepared in physiological saline at a concentration of 0.3 mg / mL. An aluminum adjuvant (Alam) was purchased from ThermoFisher Scientific, and its working solution was prepared in physiological saline at a concentration of 0.3 mg / mL. The HBsAg used in this example is a commercially available natural hepatitis B surface antigen protein isolated and purified from the plasma of chronic hepatitis B patients (purchased from Beijing Biolab Technology Co., Ltd). An HBsAg working solution was prepared in physiological saline at a concentration of 0.04 mg / mL.

[0311] Female BALB / c mice were randomly divided into groups of 5 mice each. The administration solution was prepared by mixing each working solution with the HBsAg working solution in a 1:1 volume ratio approximately 30 minutes before administration to the animals. Each animal was subcutaneously administered 100 μl of the administration solution containing 2 ug of HBsAg antigen protein and 150 ug of oligonucleotide or 15 ug of CpG1018 or 150 ug of arum adjuvant on day 0 and day 14.

[0312] Approximately 100 μl of blood samples were collected from the femoral vein of each animal on days 7, 14, 21, 28, 35, and 42 after administration. The blood samples were incubated at 37°C for approximately 30 minutes, and then centrifuged in pre-cooled (0-4°C) centrifugation to obtain serum samples. The serum samples were stored at -20°C until analysis. HBsAb levels were measured in the serum samples by chemiluminescence immunoassay.

[0313] Serum HBsAb levels on day 42 are listed in Table 3.1 and shown in Figure 7, where boxes represent the mean and error bars represent the standard deviation. The results showed that the immunoenhancing activity of the tested oligonucleotides was similar to or better than that of the conventional adjuvants Alum and CpG1018 in BALB / c mice, demonstrating the adjuvant-like properties of the oligonucleotides of this disclosure.

[0314] [Table 8]

[0315] Example 4: Immunoenhancing activity of oligonucleotides in HBV carrier mice The immunoenhancing activity of oligonucleotides AUS1476 versus AUS1233 was evaluated in HDI-HBV mice. Animals were randomly divided into groups of four. During the pre-treatment period, all animals received subcutaneous administration of 60 mg / kg of AUS1233 on days -37, -30, -23, and -16 to reduce HBsAg levels, followed by subcutaneous administration of 60 mg / kg of AUS1441 on day -8. During the second administration period, the vaccine administration period, animals received subcutaneous administration of 100 μl of a solution consisting of either physiological saline (Group 1) or 3 ug of HBsAg (Group 2) on days 0 and 14. A mixture containing 500 ug of AUS1476 and 3 ug of HBsAg (Group 3); a mixture containing 500 ug of AUS1233 and 3 ug of HBsAg (Group 4).

[0316] Working solutions of AUS1476 or AUS1233 were prepared in 10 mg / mL physiological saline. The HBsAg used in this example was a commercially available natural hepatitis B surface antigen protein isolated and purified from the plasma of chronic hepatitis B patients (purchased from Beijing Biolab Technology Co., Ltd). The HBsAg working solution was prepared in physiological saline at a concentration of 0.06 mg / mL. The administration solution for Group 2 was prepared by diluting the HBsAg working solution 2-fold with saline. The administration solutions for Groups 3 and 4 were prepared by mixing the HBsAg working solution with a 10 mg / mL working solution of AUS1476 or AUS1233, respectively, in a 1:1 volume ratio approximately 30 minutes before administration to the animals.

[0317] Approximately 100 μl of blood samples were collected from the femoral vein of each animal on days -37, -30, -23, -16, -8, -1, 7, 14, 21, 28, 35, 42, 49, 56, and 63. The blood samples were incubated at 37°C for approximately 30 minutes, and then centrifuged in a pre-cooled (0-4°C) centrifuge to obtain serum samples. The serum samples were stored in a refrigerator at -20°C until analysis. HBsAg and HBsAb levels were measured in the serum samples by chemiluminescent immunoassay.

[0318] Serum HBsAg levels in HDI-HBV mice are shown in Graph A of Figure 8, and serum HBsAb levels are shown in Graph B of Figure 8. The data points are the mean, and the error bars are the standard deviation.

[0319] Table 4.1 lists the serum levels of HBsAg and HBsAb in HDI-HBV mice at day 63. The combination of AUS1476 and HBsAg (referred to as the AUS1476 vaccine in this example) resulted in a significantly stronger and more sustained decrease in serum HBsAg levels and induced much higher levels of HBsAb compared to HBsAg antigen alone. The combination of AUS1233 and HBsAg (referred to as the AUS1233 vaccine in this example) had a similar effect on HBsAg and HBsAb levels, but its effect was lower than that achieved by the AUS1476 vaccine.

[0320] [Table 9]

[0321] Example 5: Immunoenhancing activity of oligonucleotides in HBV carrier mice The immunoenhancing activity of oligonucleotide AUS1476, which had not been administered prior therapeutic agents, was evaluated in HDI-HBV mice. Animals were subcutaneously administered only physiological saline on days 0 and 7, followed by subcutaneous administration of 100 μl of a mixture containing 500 μg of AUS1476 and 3 μg of HBsAg on days 14 and 28.

[0322] The AUS1476 working solution was prepared in physiological saline at a concentration of 10 mg / mL. The HBsAg protein used in this example was a commercially available recombinant miniature hepatitis B surface antigen protein purchased from Bioforcesci Biomart Co., Ltd., and was produced and purified from incubation medium of genetically modified mammalian Chinese hamster ovary (CHO) cells. The HBsAg(CHO) working solution was prepared in physiological saline at a concentration of 0.06 mg / mL. Approximately 30 minutes before administering to animals to prepare the administration solution, the HBsAg(CHO) working solution was mixed with the AUS1476 working solution in a 1:1 volume ratio.

[0323] Approximately 100 μl of blood samples were collected from the femoral vein of each animal on days 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, and 77. The blood samples were incubated at 37°C for approximately 30 minutes, and then centrifuged in a pre-cooled (0-4°C) centrifuge to obtain serum samples. The serum samples were stored in a -20°C refrigerator until analysis. HBsAg and HBsAb levels in the serum samples were measured by chemiluminescent immunoassay.

[0324] Serum levels of HBsAg and HBsAb in HDI-HBV mice are shown in Figure 9. The data points in Figures 9A and 9B represent the HBsAg or HBsAb levels of individual animals, respectively. Even with high baseline serum HBsAg levels (1044–2570 IU / mL), the AUS1476-HBsAg mixture was still able to reduce serum HBsAg levels to below the limit of quantification (LOQ) in some HDI-HBV mice (3 out of a total of 5 animals), and in these animals, it increased HBsAb levels to 7694 mIU / mL.

[0325] The results here suggest that a pre-treatment dose is optional, especially when baseline surrogate antigen levels are low, prior to the vaccine administration period.

[0326] Example 6: T cell response in HBV carrier mice, and immunoenhancing effects of oligonucleotides on HBV protein, mRNA, and DNA levels. The immunoenhancing activity of the oligonucleotide AUS1476 was evaluated in HDI-HBV mice as assayed by HBsAg antigen and HBsAb levels, HBV mRNA and HBV DNA levels, and T cell response. Animals were administered 40 mg / kg of AUS1476 by subcutaneous injection (SC) on days 0, 7, 21, and 35. Animals were also administered 100 μl of a mixture containing 500 μg of AUS1476 and 3 μg of HBsAg protein on days 14 and 28. A saline control group received saline alone for all treatment periods. This mixture is referred to in this example as the vaccine or "AUS1476-enhanced vaccine."

[0327] An AUS1476 administration solution was prepared in physiological saline at a concentration of 4 mg / mL. An AUS1476 working solution was prepared in physiological saline at a concentration of 10 mg / mL. The HBsAg protein used in this example was a commercially available recombinant miniature hepatitis B surface antigen protein purchased from Bioforcesci Biomart Co., Ltd., and was produced and purified from incubation medium of genetically modified mammalian Chinese hamster ovary (CHO) cells. An HBsAg(CHO) working solution was prepared in physiological saline at a concentration of 0.06 mg / mL. The vaccine administration solution was prepared by mixing the HBsAg(CHO) working solution with the AUS1476 working solution in a 1:1 volume ratio approximately 30 minutes before administration to the animals.

[0328] Approximately 100 μl of blood sample was collected weekly from the femoral vein. The blood sample was incubated at 37°C for approximately 30 minutes, and then centrifuged in a pre-cooled (0-4°C) centrifuge to obtain serum sample. The serum sample was stored at -20°C for further analysis. HBsAg and HBsAb levels were measured in the serum sample by chemiluminescence immunoassay.

[0329] At the end of the 77-day experiment, 30 to 50 mg of liver tissue samples were collected from the animals. Liver tissue DNA samples were prepared using the Total DNA Kit from Omega Bio-tek. The HBV DNA concentration in the samples was determined by qPCR. Liver tissue RNA samples were prepared using the Total RNA Kit from Omega Bio-tek. The HBV RNA concentration in the samples was determined by RT-qPCR.

[0330] Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples (0.8 mL) collected from animals at the end of the experiment on day 77. Furthermore, fresh mouse spleen tissue samples were collected, and splenic T lymphocytes were isolated. The HBV-specific T cell response (CD8+ / IFNy+%) in blood PBMCs and splenic T lymphocytes was determined by flow cytometry using the following procedure: PBMCs and splenic lymphocyte cells (5 × 10⁻¹⁴). 5The cells were cultured in 200 μl of DMEM medium in the presence or absence of a small HBsAg peptide pool (10 ug / mL) in a 96-well round-bottom plate. The peptide pool contained a total of 50 peptides, each with a small HBsAg-specific CTL epitope. Each peptide was 15 amino acids long, with 11 amino acids overlapping between them. After 24 hours of stimulation with the peptide pool, the cells were stained with primary antibodies (anti-mouse CD3-FITC, anti-mouse CD4-PreCP-Cy5.5, anti-mouse CD8-APC, or anti-mouse IFN-γ-PE) and detected by flow cytometry.

[0331] Figure 10 and Table 6.1 show serum HBsAg and HBsAb levels, T cell response, and HBV mRNA and HBV DNA expression levels on day 77. Figure 10, Graph A shows serum HBsAg and HBsAb levels in HDI-HBV mice. Figure 10, Graphs B and C show HBV mRNA and HBV DNA levels in the liver. Figure 10, Graph D shows immune activation of splenic T lymphocytes. Figure 10, Graph E shows immune activation of PBMCs. Data points are the mean, and error bars are the standard deviation. In Figure 10, administration using AUS1476 alone is named "AUS1476 alone". Administration of AUS1476 in combination with HBsAg is named "AUS1476 + vaccine".

[0332] Administration of AUS1476 alone or in combination with 3ug of HBsAg (vaccine composition) or AUS1476 (500ug) resulted in sustained HBsAg clearance in HDI-HBV mice, accompanied by a significant reduction in HBV-positive cells in the liver, as indicated by a strong anti-HBs response (HBsAb) and a substantial decrease in hepatic HBV mRNA and HBV DNA levels. Flow cytometry yielded splenic lymphocytes and PBMC cells, further demonstrating the efficient induction of an HBV-specific T-cell immune response to eliminate HBV-positive hepatocytes.

[0333] [Table 10]

[0334] Example 7: Immunoenhancing activity of oligonucleotides in primates The immunoenhancing activity of the oligonucleotide AUS1476 against serum HBsAb levels was evaluated in rhesus monkeys. Animals were administered an AUS1476-enhanced vaccine (AUS1476 vaccine), consisting of 6 ug of HBsAg and either 10 mg or 40 mg of AUS1476, by subcutaneous injection (SC) on days 0 and 14. As a positive control, a CpG1018 vaccine, consisting of 6 ug of HBsAg and 0.6 mg of CpG1018, was administered to a separate group of animals by intramuscular injection (IM) on days 0 and 14. Serum HBsAb levels were measured in serum samples by chemiluminescent immunoassay.

[0335] The AUS1476 working solution was prepared in physiological saline at concentrations of 8 mg / mL or 32 mg / mL. The CpG1018 working solution was prepared in physiological saline at a concentration of 2.4 mg / mL. The HBsAg protein used in this example was a commercially available recombinant miniature hepatitis B surface antigen protein purchased from Bioforcesci Biomart Co., Ltd., and was produced and purified from incubation medium of genetically modified mammalian Chinese hamster ovary (CHO) cells. The HBsAg working solution was prepared in physiological saline at a concentration of 24 ug / mL.

[0336] Approximately 30 minutes before administration to the animals, the AUS1476 enhanced vaccine (AUS1476 vaccine) administration solution was prepared by mixing 250 μl of diluted HBsAg standard solution with 1250 μl of diluted AUS1476 standard solution. 1.5 mL of the AUS1476 vaccine administration solution was administered subcutaneously to each animal. The CpG1018 vaccine administration solution was prepared approximately 30 minutes before administration to the animals by mixing 250 μl of HBsAg working solution with 250 μl of CpG1018 working solution. 0.5 mL of the CpG1018 vaccine administration solution was administered intramuscularly to each animal.

[0337] Figure 11 shows serum HBsAb (mIU / mL) levels on days 14, 21, 28, and 35. Data points are the mean, and error bars are the standard deviation. Table 7 lists the serum HBsAb (mIU / mL) levels on day 35. 1. The AUS1476-enhanced vaccine induced high and persistent levels of anti-HBs antibodies in a dose-dependent manner, demonstrating that the oligonucleotide compound AUS1476 functioned as an immunoactivator that induced a strong anti-HBsAg antibody response in monkeys. In contrast, the TLR9 adjuvant CpG1018 vaccine induced a much lower and less persistent anti-HBs antibody response.

[0338] [Table 11]

[0339] Example 8: Antibody-Induced Dynamics of Oligonucleotides The immunoenhancing activity of the oligonucleotides of this disclosure against serum HBsAb levels was evaluated in female BALB / c mice. The oligonucleotides used in the study included AUS1476 and AUS1500.

[0340] Working solutions for oligonucleotide compounds were prepared in 3 mg / mL physiological saline. A working solution for CpG1018 was prepared in 0.3 mg / mL physiological saline. Aluminum adjuvant (Alam) was purchased from ThermoFisher Scientific, and its working solution was prepared in 3 mg / mL saline. The HBsAg used in this example was commercially available natural hepatitis B surface antigen protein isolated and purified from the plasma of chronic hepatitis B patients (purchased from Beijing BioLab Technology Co., Ltd). An HBsAg working solution was prepared in physiological saline at a concentration of 0.04 mg / mL.

[0341] The administration solution was prepared approximately 30 minutes before administration to the animals by mixing an equal volume of HBsAg working solution with the working solutions of each oligonucleotide compound or CpG1018 working solution. The administration solution for HBsAg with alum adjuvant was prepared by adding and mixing the aluminum adjuvant working solution to the HBsAg working solution in a 1:1 volume ratio.

[0342] The animals were randomly divided into groups of 5 animals each. 100 μl of the administration solution was subcutaneously administered to each animal group on day 0 and day 14.

[0343] Approximately 100 μl of blood samples were collected from the femoral vein of animals on days 14, 21, 28, 35, and 42 post-administration. The blood samples were incubated at 37°C for approximately 30 minutes, and then centrifuged in a pre-cooled (0-4°C) centrifuge to obtain serum samples. The serum samples were stored at -20°C until analysis. HBsAb levels were measured in the serum samples by chemiluminescence immunoassay.

[0344] Figure 12 shows serum HBsAb (mIU / mL) levels as a function of time. Data points are the mean, and error bars are the standard deviation. Serum HBsAb levels at day 42 are listed in Table 8.1. The results showed that oligonucleotide-enhanced vaccines have different HBsAb induction kinetics than CpG1018 adjuvant-enhanced HBsAg. HBsAb levels increased rapidly after CpG018 adjuvant-enhanced vaccine, peaking 7 days after the second dose of the vaccine, and then plateauing. In contrast, HBsAb levels after oligonucleotide-enhanced vaccine increased steadily over time.

[0345] [Table 12]

[0346] Example 9: Effect of pre-mixing oligonucleotides and HBsAg on immune-enhancing effects The immunoenhancing activity of oligonucleotides administered subcutaneously with HBsAg, either as a pre-mixed drug solution or as two separate injections at adjacent sites, was evaluated in female BALB / c mice. The oligonucleotides used in this study included AUS1233, AUS1441, AUS1462, and AUS1476.

[0347] Female BALB / c mice were randomly divided into groups of 6 mice each. On days 0 and 14, the animals were subcutaneously administered either 100 μl of a pre-mixed administration solution or 50 μl of HBsAg working solution followed by 50 μl of oligonucleotide working solution. In both cases, each dose contained 150 μg of oligonucleotide and 2 μg of HBsAg protein. Physiological saline was administered as a negative control, and HBsAg alone was administered as a positive control. HBsAb levels were measured in serum samples by chemiluminescence immunoassay.

[0348] Working solutions for each oligonucleotide compound were prepared in physiological saline at a concentration of 3 mg / mL. The HBsAg working solution was prepared in physiological saline at a concentration of 0.04 mg / mL. The HBsAg used in this example is a commercially available natural hepatitis B surface antigen protein isolated and purified from the plasma of chronic hepatitis B patients (purchased from Beijing Biolab Technology Co., Ltd). When administering as a pre-mixed mixture, an equal volume of HBsAg working solution was mixed with the working solution for each oligonucleotide to prepare the administration solution approximately 30 minutes before administration to the animals.

[0349] Approximately 100 μl of blood samples were collected from the femoral vein of each animal on days 14, 21, 28, 35, and 42. The blood samples were incubated at 37°C for approximately 30 minutes, and then centrifuged in a pre-cooled (0-4°C) centrifuge to obtain serum samples. The serum samples were stored at -20°C until analysis. Serum HBsAb levels were measured in the serum samples by chemiluminescent immunoassay.

[0350] Figure 13 shows the serum HBsAb (mIU / mL) levels of female BALB / c mice at days 14, 21, 28, 35, and 42. The boxes represent the mean, and the error bars represent the standard deviation. Table 9.1 lists the serum HBsAb (mIU / mL) levels at day 42. The results showed different dynamics of HBsAb production when administered either as a pre-mixed oligonucleotide and HBsAg solution or as separate injections at adjacent injection sites. Initially, HBsAb levels were higher with the pre-mixed solution. However, in subsequent periods, similar HBsAb levels were obtained with either administration method. The results indicate that administering oligonucleotides and antigens as separate injections at adjacent injection sites is a viable alternative to administering them as a pre-mixed solution.

[0351] [Table 13]

[0352] Example 10: Immunoenhancing activity of oligonucleotide compounds in BALB / c mice The immunoenhancing activity of oligonucleotides including AUS1476, AUS1233, AUS1193, AUS1194, and AUS1709 was evaluated in female BALB / c mice. Physiological saline was administered as a negative control. The AUS1709 sequence is not complementary to any part of the HBV genome nucleic acid sequence.

[0353] Working solutions for each oligonucleotide were prepared in physiological saline at a concentration of 6 mg / mL. The HBsAg protein used in this example was a commercially available recombinant miniature hepatitis B surface antigen protein purchased from Bioforcesci Biomart Co., Ltd., and was produced and purified from incubation medium of genetically modified mammalian Chinese hamster ovary (CHO) cells. The HbsAg processing solution was prepared in physiological saline at a concentration of 0.04 mg / mL.

[0354] Female BALB / c mice were randomly divided into groups of 3 to 5 mice each. The administration solution was prepared by mixing equal volumes of each oligonucleotide or positive control working solution with the HBsAg working solution approximately 30 minutes before administration to the animals. Each animal was subcutaneously administered 100 μl of the administration solution containing 2 μg of HBsAg antigen protein and 300 μg of oligonucleotide on days 0 and 14.

[0355] Approximately 100 μl of blood samples were collected from the femoral vein of animals on days 14 and 21 after administration. The blood samples were incubated at 37°C for approximately 30 minutes, and then centrifuged in a pre-cooled (0-4°C) centrifuge to obtain serum samples. The serum samples were stored at -20°C until analysis. HBsAb levels were measured in the serum samples by chemiluminescence immunoassay.

[0356] The serum HBsAb levels on day 21 are listed in Table 1 and shown in Figure 14. The boxes represent the mean, and the error bars represent the standard deviation. The results indicate that the immunoenhancing activity of oligonucleotides is independent of their complementarity to a portion of the HBV genome nucleic acid sequence.

[0357] [Table 14]

[0358] Example 11: Immunoenhancement and antiviral activity of vaccine administration after oligonucleotide administration in an HBV model The immunoenhancing and antiviral activity of recombinant miniature HBsAg containing oligonucleotide AUS1476 and aluminum hydroxide adjuvant (vaccine) was tested when administered alone or sequentially to HBV mice. Physiological saline was administered as a negative control.

[0359] Recombinant adeno-associated virus (rAAV-HBV1.3 mer WT replicon) containing 1.3 copies of the hepatitis B virus genome was used and stored at -70°C before use. AAV-HBV mouse models (rAAV-1.3HBV-GTD carrier mice) were constructed using 4-5 week old male C57BL / 6 mice by hydrodynamic injection of recombinant adeno-associated virus. HBsAg-positive mice were used 57 days after injection. Mice were maintained in a BSL-2+ animal facility under conditions free from specific pathogens.

[0360]

[0001] A 10 mg / mL stock solution was prepared by dissolving the compound in physiological saline, and this was then diluted again with physiological saline to a target concentration of 4.0 mg / mL to prepare the AUS1476 administration solution. The HBV antigen used was a vaccine commercially available from NCPC Genetech Biotechnology (China), which consists of recombinant miniature HBsAg produced in mammalian Chinese hamster ovary (CHO) cells and aluminum hydroxide adjuvant. The concentration of the recombinant miniature HBsAg plus aluminum hydroxide working solution was 20 ug / mL.

[0361] As shown in Table 11.1, 15 male rAAV-1.3HBV-GTD carrier mice were divided into four groups of 3 to 5 mice each. In Group 1, animals were administered physiological saline on days 1, 4, 8, 11, 18, 32, and 46. In Group 2, animals were administered physiological saline on days 1, 4, 8, and 11, followed by intramuscular injection of three doses (3 μg per animal) of recombinant small HBsAg with arum adjuvant. In Group 3, animals were subcutaneously administered 40 mg / kg of AUS1476 on days 1, 4, 8, and 11, followed by three doses of physiological saline on days 18, 32, and 46. In Group 4, animals were subcutaneously administered 40 mg / kg of AUS1476 on days 1, 4, 8, and 11, followed by intramuscular injection of three doses (3 ug per animal) of recombinant HBsAg with arum adjuvant.

[0362] [Table 15]

[0363] Approximately 100 μl of blood samples were collected from the femoral vein on day 1 (before administration) and on days 4, 8, 11, 15, 18, 25, 32, 39, 46, and 52 after administration. The blood samples were placed in a 37°C incubator for approximately 30 minutes, and then centrifuged in a pre-cooled centrifuge (0-4°C) to obtain serum samples. The obtained serum samples were stored in a -20°C refrigerator for further analysis.

[0364]

[0002] Serum hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (HBsAb) levels were measured using a chemiluminescent immunoassay.

[0365] Table 11.2 and Figures 15-16 show the serum levels of HBsAg and HBsAb in AAV-HBV mice. In Figures 15-16, arum-adjuvanted recombinant miniature HBsAg is referred to as the "vaccine."

[0366] [Table 16]

[0367] In the group treated with AUS1476 monotherapy (Group 3), a reduction in serum HBsAg below the lower limit of quantification of 1 IU / mL was achieved in all four animals after four doses of AUS1476, but serum HBsAg levels rebounded to baseline levels by day 52 (Figure 15). In this group, the animals did not produce detectable HBsAb in their serum (Figure 16).

[0368] In the group treated with recombinant small HBsAg monotherapy with alum adjuvant (Group 2), only one out of three animals showed a 1 log10 reduction in HBsAg levels. Serum HBsAb was detectable in this group, but HBsAb levels were generally low and fluctuating. The highest level was only 2867 mIU / mL on day 52.

[0369] In contrast, in the group treated first with AUS1476 followed by recombinant small HBsAg with arum adjuvant (Group 4), a sustained decrease in serum HBsAg below the lower limit of 1 IU / mL was achieved in all five animals tested (Figure 15). Serum HBsAb levels increased in all animals in this group, reaching a maximum of 7796 mIU / mL (geometric mean) on day 52 (Figure 16).

[0370] In summary, administration of the oligonucleotide AUS1476 followed by recombinant miniature HBsAg with arum adjuvant effectively and sustainably reduced serum HBsAg and promoted HBsAb production in AAV-HBV mice. Therefore, sequential administration of oligonucleotides followed by sequential administration of HBV antigen may be effective in the treatment and prevention of HBV infection.

[0371] While the present invention has been described with reference to its specific embodiments, it should be understood by those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications can be made to the spirit and scope of the described invention to adopt specific circumstances, materials, substance compositions, processes, process steps, or procedures. All such modifications are intended to fall within the scope of the appended claims.

[0372] The patents, patent applications, patent application publications, journal articles, and protocols referenced herein are incorporated in their entirety for all purposes.

Claims

1. A vaccine composition comprising at least one oligonucleotide and at least one hepatitis B virus (HBV) antigen.

2. The vaccine composition according to claim 1, wherein the oligonucleotide is complementary to one or more portions of the nucleic acid sequence of the HBV genome.

3. The vaccine composition according to claim 1, wherein the oligonucleotide is not complementary to a portion of the nucleic acid sequence of the HBV genome.

4. The vaccine composition according to any one of claims 2 to 3, wherein the HBV genome is the genome of HBV GT-A, GT-B, GT-C, GT-D, GT-E, GT-F, GT-G, GT-H, GT-I, or GT-J.

5. The vaccine composition according to any one of claims 1 to 4, wherein the oligonucleotide comprises 10 to 60 nucleic acid bases.

6. The vaccine composition according to any one of claims 1 to 5, wherein the oligonucleotide is a modified oligonucleotide.

7. The vaccine composition according to any one of claims 1 to 6, wherein the modified oligonucleotide comprises one or more modified sugars, modified bases, modified nucleoside bonds, or a combination thereof.

8. The vaccine composition according to any one of claims 1 to 6, wherein at least one nucleoside bond is a phosphodiester bond.

9. The vaccine composition according to any one of claims 1 to 8, wherein at least one nucleoside bond is a modified nucleoside bond.

10. The vaccine composition according to claim 9, wherein the modified nucleoside bond is a phosphorothioate nucleoside bond.

11. The vaccine composition according to any one of claims 1 to 10, wherein the oligonucleotide comprises a linked deoxynucleoside.

12. The vaccine composition according to any one of claims 1 to 11, wherein the oligonucleotide comprises at least one nucleoside including modification at the C2' position.

13. The vaccine composition according to claim 12, wherein the oligonucleotide comprises at least one nucleoside including 2'-O-methyl sugar modification, 2'-O-methoxyethyl sugar modification, 2'-fluoro sugar modification and / or 2'-fluoro-arabino nucleic acid (2'-fluoro-ANA) sugar modification.

14. The vaccine composition according to any one of claims 1 to 13, wherein the oligonucleotide comprises at least one bicyclic sugar-modified nucleoside.

15. The vaccine composition according to any one of claims 1 to 14, wherein the oligonucleotide comprises at least one nucleoside having a phosphorodiamidate morpholino modification.

16. The vaccine composition according to any one of claims 1 to 15, wherein the oligonucleotide comprises at least one locked nucleic acid (LNA), peptide nucleic acid and / or glycol nucleic acid.

17. The vaccine composition according to any one of claims 1 to 16, wherein the oligonucleotide is selected from SEQ ID NOs: 10 to 666, or has at least one sequence modification thereto.

18. The vaccine composition according to any one of claims 1 to 17, wherein the oligonucleotide is selected from SEQ ID NOs: 10 to 666, or is an oligonucleotide having at least 70% sequence identity therewith.

19. A vaccine composition according to any one of claims 1 to 18, comprising at least two different oligonucleotides.

20. The vaccine composition according to any one of claims 1 to 19, wherein the HBV antigen is human HBV antigen.

21. The vaccine composition according to any one of claims 1 to 20, wherein the HBV antigen is HBV surface antigen (HBsAg), HBV e antigen (HBeAg), HBV core antigen (HBcAg), or any combination thereof.

22. The vaccine composition according to claim 21, wherein the HBsAg is Small-HBsAg, Medium-HBsAg, Large-HBsAg, or any combination thereof.

23. The vaccine composition according to any one of claims 1 to 22, wherein the HBV antigen is derived from the HBV GT-A, GT-B, GT-C, GT-D, GT-E, GT-F, GT-G, GT-H, GT-I, or GT-J genotype.

24. The vaccine composition according to any one of claims 1 to 23, wherein the HBV antigen is isolated from a patient, recombinantly produced, or synthesized.

25. A vaccine composition according to any one of claims 1 to 24, comprising at least two different HBV antigens.

26. The vaccine composition according to any one of claims 1 to 25, wherein the vaccine composition comprises an adjuvant.

27. The vaccine composition according to claim 26, wherein the adjuvant is aluminum hydroxide.

28. The vaccine composition according to claim 26, wherein the adjuvant is a Toll-like receptor 9 (TLR9) agonist.

29. The vaccine composition according to claim 26, wherein the adjuvant is an oligonucleotide.

30. The vaccine composition according to claim 27, wherein the oligonucleotide adjuvant comprises CpG1018.

31. The vaccine composition according to any one of claims 1 to 30, wherein the vaccine composition is a therapeutic vaccine composition.

32. The vaccine composition according to any one of claims 1 to 30, wherein the vaccine composition is a preventive vaccine composition.

33. A pharmaceutical composition comprising a vaccine composition according to any one of claims 1 to 32 and a pharmaceutically acceptable carrier, diluent, or excipient.

34. A kit comprising the vaccine composition according to any one of claims 1 to 32.

35. A method for inducing an immune response to HBV antigen in a subject suffering from HBV infection or an HBV-related disease, disorder, or condition, comprising administering to a subject the vaccine composition described in any one of claims 1 to 32 or the pharmaceutical composition described in claim 33.

36. A method for treating or preventing a target HBV infection or HBV-related disease, disorder, or condition by vaccine therapy, comprising administering to a target the vaccine composition described in any one of claims 1 to 32, or the pharmaceutical composition described in claim 33.

37. A method for inducing an immune response to HBV antigen in subjects suffering from HBV infection or HBV-related disease, disorder, or condition, using vaccine therapy, comprising administering oligonucleotides and hepatitis B virus (HBV) antigens as the target.

38. A method of treating or preventing HBV infection or HBV-related disease, disorder, or condition in a subject by vaccine therapy, comprising administering oligonucleotides and hepatitis B virus (HBV) antigens to the subject.

39. The method according to any one of claims 37 to 38, wherein the oligonucleotide and the HBV antigen are administered simultaneously.

40. The method according to claim 39, wherein the oligonucleotide and the HBV antigen are provided as a premixture.

41. The method according to claim 39, wherein the oligonucleotide and the HBV antigen are mixed immediately before administration.

42. The method according to claim 39, wherein the oligonucleotide and the HBV antigen are administered simultaneously to different injection sites.

43. The method according to claim 42, wherein the injection sites are adjacent.

44. The method according to claim 39, wherein the oligonucleotide and the HBV antigen are administered simultaneously to the same injection site.

45. The method according to any one of claims 37 to 38, comprising administering the oligonucleotide and the HBV antigen in succession.

46. The method according to claim 45, wherein the oligonucleotide and the HBV antigen are administered sequentially to the same injection site.

47. The method according to claim 45, wherein the oligonucleotide and the HBV antigen are administered sequentially to different injection sites.

48. The method according to claim 47, wherein the injection sites are adjacent.

49. The method according to claim 45, wherein the oligonucleotide is administered before the HBV antigen.

50. The method according to claim 49, wherein the oligonucleotide is administered multiple times before the HBV antigen is administered.

51. The method according to claim 50, wherein two, three, four, five, or more doses of the oligonucleotide are administered before the HBV antigen is administered.

52. The method according to claim 49, wherein a single dose of the oligonucleotide is administered before the HBV antigen is administered.

53. The method according to any one of claims 49 to 52, wherein the HBV antigen is administered multiple times after the oligonucleotide is administered.

54. The method according to any one of claims 37 to 53, comprising administering at least two doses of the oligonucleotide and / or the HBV antigen.

55. The method according to any one of claims 37 to 54, wherein the oligonucleotide comprises the oligonucleotide described in any one of claims 2 to 18.

56. The method according to any one of claims 37 to 55, wherein the HBV antigen is an HBV surface antigen (HBsAg), an HBV e antigen (HBeAg), or an HBV core antigen (HBcAg).

57. The method according to claim 56, wherein the HBsAg is Small-HBsAg, Middle-HBsAg, Large-HBsAg, or any combination thereof.

58. The method according to any one of claims 37 to 57, wherein the HBV antigen is derived from the HBV GT-A, GT-B, GT-C, GT-D, GT-E, GT-F, GT-G, GT-H, GT-I, or GT-J subtype.

59. The method according to any one of claims 35 to 58, wherein the method includes one or more pretreatments.

60. The method according to claim 59, wherein the pretreatment includes administering an oligonucleotide which is the same oligonucleotide as the vaccine composition.

61. The method according to claim 59, wherein the pretreatment includes administering an oligonucleotide different from the oligonucleotide of the vaccine composition.

62. The method according to claim 59, wherein the prior treatment includes administering an HBV treatment selected from the group consisting of siRNA against HBV, interferon-gamma, PD-1 inhibitors, and PD-L1 inhibitors.

63. The method according to any one of claims 35 to 62, wherein the method results in a decrease in the levels of HBsAg, HBcAg and / or HBeAg in the serum.

64. The method according to any one of claims 35 to 63, wherein the method results in a decrease in the level of HBV DNA in the liver.

65. The method according to any one of claims 35 to 64, wherein the method results in a decrease in the level of HBV mRNA in the liver.

66. The method according to any one of claims 35 to 65, wherein the method results in an increase in the HBsAb level in the serum.

67. The method according to any one of claims 35 to 66, wherein the method results in an increase in the T cell response.

68. The method according to any one of claims 63 to 67, wherein the method produces a dose-dependent effect.

69. The method according to any one of claims 35 to 68, wherein the subject is a primate.

70. The method according to claim 69, wherein the subject is a human subject.

71. The method according to any one of claims 35 to 70, wherein the route of administration is subcutaneous, intramuscular, intravenous, or intradermal.

72. The method according to any one of claims 35 to 71, wherein the subject is suffering from acute HBV infection.

73. The method according to any one of claims 35 to 71, wherein the subject is suffering from chronic HBV infection.

74. The method according to any one of claims 35 to 73, wherein the subject is administered about 20 mg to about 500 mg of oligonucleotide per dose.

75. The method according to any one of claims 35 to 74, wherein the subject is administered approximately 1 μg to approximately 500 μg per dose of HBsAg.

76. A kit comprising an oligonucleotide and a hepatitis B virus (HBV) antigen according to any one of claims 1 to 75.

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