EA045279B1 - VACCINES AGAINST HEPATITIS B VIRUS (HBV) AND THEIR USE - Google Patents

Description

Cross references to related applications

This application claims priority to International Patent Application PCT/Ш2017/058142, filed December 19, 2017, and US Provisional Patent Application 62/607,426, filed December 19, 2017, the disclosures of which are incorporated herein by reference in their entirety.

Link to the list of sequences presented in the electronic version

This application contains a sequence listing, which is submitted electronically via EFS-Web as a sequence listing in ASCII format with file name 688097-403 Sequence Listing and creation date December 10, 2018, size 46.6 kB. The sequence listing provided via EFS-Web is part of the specification and is incorporated herein by reference in its entirety.

Field of technology to which the invention relates

Hepatitis B virus (HBV) is a small hepatotropic virus with 3.2 kb DNA that encodes four open reading frames and seven proteins. About two billion people are infected with hepatitis B virus, and about 240 million people suffer from chronic hepatitis B infection (chronic HBV), which is characterized by persistent viral and subviral particles in the blood for more than 6 months (1). Persistent HBV infection results in T cell depletion of HBV-specific CD4+ and CD8+ T cells in the circulation and within the liver through chronic stimulation of HBV-specific T cell receptors by viral peptides and circulating antigens. As a result, the multifunctionality of T cells is reduced (ie, the levels of IL-2, tumor necrosis factor (TNF)-α, IFN-γ are reduced and there is no proliferation).

A safe and effective preventative vaccine against HBV infection has been available since the 1980s and is the mainstay of hepatitis B prevention (3). The World Health Organization recommends vaccination of all infants, and in countries with low or intermediate endemicity of hepatitis B, vaccination of all children and adolescents (<18 years of age), as well as people in certain risk groups. Thanks to vaccination, infection rates around the world have dropped sharply. However, prophylactic vaccines do not cure HBV-mediated infection.

Chronic HBV is currently treated with IFN-α and nucleoside or nucleotide analogues, which do not lead to definitive cure due to the persistence in infected hepatocytes of intracellular viral replicative intermediates called covalently closed circular DNA (cccDNA), which plays a fundamental role in as a template for viral RNAs and, consequently, new virions. It is believed that virus-induced specific T-cell and B-cell responses can effectively eliminate hepatocytes harboring cccDNA. Current therapies targeting HBV polymerase suppress viremia but have limited effect on nuclear cccDNA and associated circulating antigen production. The most radical form of treatment may be to remove HBV cccDNA from the body, which does not occur naturally or as a result of any therapeutic intervention. However, loss of HBV surface antigens (HBsAg) is clinically equivalent to cure, since disease recurrence can only occur in cases of severe immunosuppression, which can then be prevented by prophylactic treatment. Thus, at least from a clinical point of view, loss of HBsAg is associated with the most complete form of restoration of immunity against HBV.

For example, immunomodulation with pegylated interferon (pegIFN)-a has been shown to be more effective than nucleoside or nucleotide therapy in terms of long-term response after the final course of treatment. In addition to its direct antiviral effect, IFN-α has been reported to have epigenetic suppression of cccDNA in cell culture and in humanized mice, resulting in decreased virion and transcript productivity (4). However, such therapy is still fraught with side effects, and the overall response is still quite low, in part because IFN-α has only a weak modulatory effect on HBV-specific T cells. In particular, treatment success rates remain low (<10%) and toxicity remains high. Similarly, direct-acting HBV antivirals, namely the HBV polymerase inhibitors, entecavir and tenofovir, are effective as monotherapy in inducing viral suppression and have a high genetic barrier to the emergence of drug-resistant mutants and subsequent prevention of liver disease progression. However, treatment of chronic hepatitis B, defined by loss of HBsAg or seroconversion, is rarely achieved with such HBV polymerase inhibitors. Therefore, to prevent recurrence of liver disease, theoretically, these antiviral drugs should be administered indefinitely, similar to antiviral therapy directed against human immunodeficiency virus (HIV).

Therapeutic vaccination has the potential to eliminate HBV in chronically infected patients (5). Many strategies have been studied, but to date therapeutic vaccination has not been successful.

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The essence of the invention

Accordingly, there is an unmet medical need for treatment of hepatitis B virus (HBV), particularly chronic HBV, resulting in a well-tolerated treatment with a higher cure rate. The invention makes it possible to satisfy this need by developing immunogenic compositions and methods for inducing an immune response to infection with the hepatitis B virus (HBV). The immunogenic compositions and methods of the invention can be used to induce therapeutic immunity in a subject, such as a subject suffering from chronic HBV infection.

In a General aspect, the invention relates to a non-naturally occurring nucleic acid molecule encoding an HBV antigen, such as a truncated HBV nuclear antigen or an HBV antigen polymerase. The HBV antigen in accordance with an embodiment of the invention is a consensus antigen capable of inducing an immune response (humoral and cellular) in a mammal to at least two HBV genotypes, preferably inducing a T cell response in the mammal to at least genotypes B, C and D HBV, more preferably CD8 T cell response in humans to at least genotypes A, B, C and D of HBV.

In one embodiment, the non-naturally occurring nucleic acid molecule of the invention encodes a truncated HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14, and the non-naturally occurring nucleic acid molecule contains a polynucleotide sequence that is at least 90% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 15. Preferably, the non-naturally occurring nucleic acid molecule contains the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

In one embodiment, the non-naturally occurring nucleic acid molecule of the invention encodes an HBV polymerase antigen comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity Preferably, the HBV polymerase antigen contains the amino acid sequence of SEQ ID NO: 4. More preferably, the non-naturally occurring nucleic acid molecule contains a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16, the most preferably 100% identical to SEQ ID NO: 3 or SEQ ID NO: 16.

In another general aspect, the invention relates to a vector, preferably a DNA plasmid or viral vector, containing a non-naturally occurring nucleic acid molecule of the invention.

In another general aspect, the invention relates to a recombinant host cell containing a non-naturally occurring nucleic acid molecule or vector of the invention.

In another general aspect, the invention relates to a non-naturally occurring polypeptide encoded by a non-naturally occurring nucleic acid molecule of the invention.

In yet another general aspect, the application relates to a composition comprising at least one of a non-naturally occurring nucleic acid molecule, a vector, a recombinant host cell and a non-naturally occurring polypeptide of the invention, and a pharmaceutically acceptable carrier.

In another general aspect, the invention relates to an immunogenic combination, in particular to a kit containing:

(a) a first unnatural nucleic acid molecule comprising a first polynucleotide encoding an HBV polymerase antigen having an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity, and RNase H activity;

(b) a second unnatural nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14; and (c) a pharmaceutically acceptable carrier, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in the same non-naturally occurring nucleic acid molecule or in two different non-naturally occurring nucleic acid molecules .

In some embodiments, an immunogenic combination, particularly a kit, of the invention comprises a first polynucleotide present in a first vector and a second polynucleotide present in a second vector. Preferably, the first vector is different from the second vector. More preferably, the vector is a plasmid vector or a viral vector. More preferably, each of the first vector and the second vector is a DNA plasmid vector.

In one embodiment, the immunogenic combination, in particular the kit, according to the invention contains:

a) the first DNA is a plasmid vector containing a first polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence SEQ ID NO: 4;

b) a second DNA plasmid vector containing a second polynucleotide encoding a truncated HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2 or 14; And

c) a pharmaceutically acceptable carrier, wherein each of the first DNA plasmid vector and the second DNA plasmid vector further comprises an antibiotic resistance gene and an origin of replication, and wherein the first DNA plasmid vector and the second DNA plasmid vector are present in the same compositions or in two different compositions.

In a specific embodiment, the immunogenic combination, in particular the kit, according to the invention contains:

a) a first DNA plasmid vector containing, in the direction from the 3' end to the 5' end, a promoter sequence containing the polynucleotide sequence SEQ ID NO: 7, an enhancer sequence containing the polynucleotide sequence SEQ ID NO: 8, a coding sequence a signal peptide comprising the polynucleotide sequence of SEQ ID NO: 5, a first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 3, and a polyadenylation signal sequence comprising the polynucleotide sequence of SEQ ID NO: 11;

b) a second DNA plasmid vector containing, in the direction from the 3' end to the 5' end, a promoter sequence containing the polynucleotide sequence SEQ ID NO: 7, a regulatory sequence containing the polynucleotide sequence SEQ ID NO: 8, a coding sequence a signal peptide comprising the polynucleotide sequence of SEQ ID NO: 5, a second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 1, and a polyadenylation signal sequence comprising the polynucleotide sequence of SEQ ID NO: 11; And

c) a pharmaceutically acceptable carrier, wherein each of the first DNA plasmid vector and the second DNA plasmid vector further comprises a kanamycin resistance gene having the polynucleotide sequence of SEQ ID NO: 12, and an origin of replication having the polynucleotide sequence of SEQ ID NO: 10, and wherein the first DNA plasmid vector and the second DNA plasmid vector are present in the same composition or in two different compositions.

In other embodiments, an immunogenic combination, in particular a kit, of the invention comprises a first polynucleotide and a second polynucleotide present in the same vector. Preferably the vector is a plasmid vector or a viral vector. More preferably, the vector is an adenoviral vector such as an Ad26 or Ad35 vector.

In one embodiment, the immunogenic combination, in particular a kit, according to the invention contains:

a) a vector containing a first polynucleotide sequence encoding an HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 4, and a second polynucleotide encoding a truncated HBV nuclear antigen consisting of the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 14; And

b) a pharmaceutically acceptable carrier.

In a specific embodiment, the immunogenic combination, in particular the kit, according to the invention contains a viral vector, preferably an adenoviral vector, containing, in the direction from the 5' end to the 3' end, a promoter sequence containing the polynucleotide sequence SEQ ID NO: 17, regulatory a sequence containing the polynucleotide sequence of SEQ ID NO: 23, a signal peptide encoding sequence containing the polynucleotide sequence of SEQ ID NO: 18, a second polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 15, a linker encoding sequence containing the polynucleotide sequence of SEQ ID NO: : 22, a first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 16, and a polyadenylation signal sequence comprising the polynucleotide sequence of SEQ ID NO: 24, and a pharmaceutically acceptable carrier.

Other aspects of the invention relate to methods for producing polynucleotides, vectors, polypeptides and compositions and immunogenic combinations or kits of the invention.

In another general aspect, the invention relates to a method of inducing an immune response to hepatitis B virus (HBV) in a subject in need thereof, comprising administering to the subject an immunogenically effective amount of a composition or immunogenic combination of the invention. Preferably, the method induces in the subject an immune response, such as a humoral antibody response and/or a T cell response, to at least two HBV genotypes. Preferably, the method induces

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T cell response in a subject to at least genotypes B, C, and D of HBV. More preferably, the method induces a CD8 T cell response in a person to at least genotypes A, B, C and D of HBV. In one embodiment, the method further includes administering to the subject another immunogenic agent, preferably another HBV antigen.

In another aspect, the application relates to a method of treating disease caused by hepatitis B virus (HBV) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition or immunogenic combination of the invention. Preferably, the subject has a chronic HBV infection, and the disease caused by HBV is selected from the group consisting of advanced fibrosis, cirrhosis and hepatocellular carcinoma (HCC). In one embodiment, the method further comprises administering to the subject another therapeutic agent, preferably another anti-HBV antigen.

The invention also relates to a composition, immunogenic combination or kit according to the invention for use in inducing an immune response to the hepatitis B virus (HBV); and the use of a composition, immunogenic combination or kit according to the invention in the preparation of a medicament for inducing an immune response to the hepatitis B virus (HBV). The use may further include combination with another immunogenic agent, preferably another HBV antigen. Preferably, the subject suffers from chronic HBV infection.

The invention further provides a composition, immunogenic combination or kit for use in the treatment of an HBV-induced disease in a subject in need thereof; and the use of a composition, immunogenic combination or kit in the preparation of a medicament for treating a disease caused by HBV in a subject in need thereof. The use may further include combination with another therapeutic agent, preferably another anti-HBV antigen. Preferably, the subject suffers from chronic HBV infection, and the disease caused by HBV is selected from the group consisting of advanced fibrosis, cirrhosis and hepatocellular carcinoma (HCC).

Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the accompanying claims.

Brief description of drawings

For a more complete understanding of the foregoing essence of the invention, as well as the following detailed description of the invention, the accompanying drawings are provided. However, it should be understood that the invention is not limited to the specific embodiments shown in the drawings.

On the drawings:

in fig. 1A-1B depict the genome and life cycle of the hepatitis B virus; in fig. 1A is a schematic representation of the hepatitis B virus (HBV) genome; in the native virus, the polymerase protein (Pol) contains a sequence encoding envelope proteins in a different open reading frame; envelope proteins (pre-S1, pre-S2 and S) are in the same open reading frame; in fig. 1B shows the life cycle of the HBV virus;

in fig. 2A-2H show the design and optimization of expression cassettes and DNA plasmids encoding the HBV pol and core antigens described in Example 1; fig. 2A is a schematic representation of an expression strategy in which the coding sequences of the HBV core and pol antigens are fused in frame; fig. 2B is a schematic representation of an expression strategy whereby the coding sequences of both core and pol antigens are expressed from a single plasmid via the FA2 ribosome slip site; fig. 2C is a schematic representation of an expression strategy in which core and pol antigens are expressed from two separate plasmids; in fig. 2D shows a Western blot of core antigen expression in HEK293T cells transfected with a core expression plasmid with and without the post-transcriptional regulatory element WPRE; expression levels were tested in cell lysate (left) and supernatant (above; right) using α-core antibody; in fig. 2E is a Western blot analysis showing a comparison of core expression in HEK293T cells transfected with a plasmid expressing core, including intron/exon sequence derived from the human apolipoprotein A1 precursor (intron AI), T lymphotropic long terminal repeat (LTR) R-U5 untranslated domain human type 1 virus (HTLV-I) (HTLV R) or a sequence consisting of three enhancer sequences (triple enhancer composite sequence) of the HTLV-1 LTR, a synthetic rabbit P-globin intron and a splicing enhancer (triple); the unlabeled band represents purified core protein as a size marker; expression levels were tested in both the lysate (left) and supernatant (above; right); the highest level of core antigen expression was shown by the triple enhancer composite sequence; fig. 2F is a Western blot analysis of core antigen secretion using different signal peptides fused to the N terminus of HBV core antigen; the most efficient protein secretion was observed with cystatin S signal peptide; fig. 2G is a schematic representation of optimized HBV core/pol antigen expression cassettes for each of the three expression strategies illustrated in

- 4 045279 figs. 2A-2C; CMVpr: human CMV-IE promoter; TRE: triple enhancer sequence; SP: cystatin S signal peptide; FA2: FMDV ribosome slip site; pA: BGH polyadenylation signal; fig. 2H is a Western blot analysis of core and pol antigen expression of HBV pDK vectors containing each of the expression cassettes shown in FIG. 2G; lanes 1 and 2: pDK-core; lanes 3 and 4: pDK-pol; lanes 5 and 6: pDK-coreFA2Pol; Lanes 7 and 8: pDK-corepol fusion: the most consistent expression profile of cellular and secreted core and pol antigens was observed when the antigens were encoded by separate vectors;

in fig. 3A-3B are schematic representations of DNA plasmids in accordance with embodiments of the invention; in fig. 3A shows a DNA plasmid encoding an HBV core antigen in accordance with an embodiment of the invention; in fig. 3B shows a DNA plasmid encoding an HBV polymerase (pol) antigen in accordance with an embodiment of the invention; HBV core and pol antigens are expressed under the control of the CMV promoter with an N-terminal cystatin S signal peptide, which is cleaved from the expressed antigen during secretion from the cell; the transcriptional regulatory elements of the plasmid include an enhancer sequence located between the CMV promoter and the polynucleotide sequence encoding the HBV antigen, and a bGH polyadenylation sequence located downstream of the polynucleotide sequence encoding the HBV antigen; the second expression cassette is included in the plasmid in reverse orientation and includes the kanamycin resistance gene under the control of the Ampr (bla) promoter; the origin of replication (pUC) is also included in reverse orientation;

in fig. 4 shows ELISPOT responses in Balb/c mice immunized with different DNA plasmids expressing HBV core antigen or HBV pol antigen, as described in Example 2; peptide pools used to stimulate splenocytes isolated from different groups of vaccinated animals are shown in gray bars; the number of sensitive T cells is shown on the y-axis, expressed as spot-forming cells (SFC) per 106 splenocytes;

in fig. 5 shows ELISPOT responses in Balb/C mice immunized with a combination of DNA plasmids expressing HBV core antigen and HBV pol antigen according to the dose finding study described in Example 3; peptide pools used to stimulate splenocytes isolated from different groups of vaccinated animals are shown in gray bars; the number of sensitive T cells is shown on the y-axis, expressed as spot-forming cells (SFC) per 106 splenocytes;

in fig. 6 shows ELISPOT responses in Balb/c mice immunized with DNA plasmids (pDNA) expressing HBV core antigen and HBV pol antigen according to the immune interference study described in Example 4; Group 1, core pDNA only; Group 2, Pol pDNA only; Group 3, mixed Core and Pol pDNA; Group 4, Core and Pol pDNA, injected separately into different areas; peptide pools used to stimulate splenocytes isolated from different vaccinated groups of animals are shown in gray bars; the number of sensitive T cells is shown on the y-axis, expressed as spot-forming cells (SFC) per 106 splenocytes;

in fig. 7A and 7B show the immunogenicity of a DNA vaccine in accordance with an embodiment of the invention in NHP as described in Example 5; In fig. 7A shows the IFN-γ cytokine response following immunization with DNA plasmids expressing HBV Core and Pol antigens; peptide pools used to stimulate PBMCs isolated from groups of vaccinated animals are shown in gray bars; the number of sensitive T cells is indicated on the y-axis, expressed as spot-forming cells (SFC) per 106 PBMC; in fig. 7B shows the immune response of CD4 and CD8 memory T cells to pools of Core, Pol-1 and Po1-2 peptides measured by flow cytometry; graph shows results obtained at day 76 as % CD4 or CD8 T cell response (IFN-γ, IL-2, and TNF-α) in 3 pools after subtracting background from DMSO-only medium from each pool; the CD4 response is shown on the left and the CD8 response is shown on the right;

in fig. 8A and 8B are a schematic representation of expression cassettes in adenoviral vectors in accordance with embodiments of the invention; In fig. 8A shows an expression cassette for a truncated HBV nuclear antigen, which contains the CMV promoter, an intron (a fragment derived from the human ApoAI gene - base pairs 295-523 with GenBank accession number X01038, carrying the second intron of ApoAI), a human immunoglobulin secretion signal, followed by followed by a sequence encoding the truncated HBV core antigen and the SV40 polyadenylation signal; in fig. 8B shows an expression cassette for a truncated HBV nuclear antigen fusion protein operably linked to an HBV polymerase antigen, which is otherwise identical to the truncated HBV nuclear antigen expression cassette except for the HBV antigen; and in fig. 9 shows ELISPOT responses in F1 mice (C57BL/6xBalb/C) immunized with HBV adenoviral vectors as described in Example 8; Pools of HBV core or polymerase peptides used to stimulate splenocytes isolated from different groups of vaccinated animals are shown in black (core) and gray (pol); Poll and po12 responses were summed; The x-axis shows adenovector dose and experimental groups. The number of sensitive T cells is shown on the y-axis, expressed

- 5 045279 in the form of spot-forming cells (SFC) per 10 splenocytes.

Detailed Description of the Invention

In the prior art section and in the description, various publications, articles and patents are referenced or described; each of these references is incorporated herein by reference in its entirety. A discussion of documents, acts, materials, devices, articles, or the like is included herein to provide context for the present invention. Such discussion is not an admission that each of the matters discussed is part of the prior art pertaining to any disclosed or claimed invention.

Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention relates. Otherwise, certain terms used in this application have the meanings specified in the specification. All patents, published patent applications and publications cited herein are incorporated by reference as if fully set forth herein by reference.

It should be noted that as used in this specification and the accompanying claims, the singular number includes the plural unless the context clearly indicates otherwise.

Unless otherwise specified, the term at least preceding a series of elements should be understood to refer to each element of the series. Those skilled in the art will recognize, or be able to determine by routine experimentation, many equivalent embodiments of the invention to those disclosed herein. Such equivalents are within the scope of the present invention.

In the present specification and in the claims below, unless the context otherwise requires, the word contain and such variants as contains and containing include a specified integer or step or group of integers or steps, without excluding any other integer or step or groups of integers or stages. The term containing, when used herein, may be replaced by the term consisting of or including, or sometimes, when used herein, by the term having.

As used herein, the term consisting of excludes any element, step or ingredient not specified in the claimed element.

As used herein, the term essentially consisting of does not exclude materials or steps that do not materially affect the essential and novel features of the claims. Any of the above terms containing, consisting of, including and having, when used herein in the context of an aspect or embodiment of the application, may be replaced by the term consisting of or essentially consisting of to change the scope of the invention.

As used herein, the term and/or between multiple recited elements should be understood to cover both individual and combined options. For example, when two elements are connected by and/or, the first option refers to the possibility of using the first element without the second. The second option refers to the possibility of using a second element without the first. The third option refers to the possibility of using the first and second elements together. All of these options are within the scope of the stated meaning and therefore satisfy the requirement of the term and/or as used herein. It is clear that the possibility of simultaneous use of more than one of these options corresponds to the specified meaning and, therefore, satisfies the requirement of the term and/or.

Unless otherwise indicated, any numerical values, such as concentration or concentration range, presented herein should in all cases be considered as modified by the term approximately. Thus, the numerical value usually includes ±10% of the specified value. For example, a concentration of 1 mg/ml includes values from 0.9 to 1.1 mg/ml. Likewise, the concentration range from 1 to 10 mg/ml includes values from 0.9 to 11 mg/ml. As used herein, the use of a numeric range explicitly includes all possible subranges, all individual numeric values within that range, including integers within such ranges, and fractional values, unless the context explicitly requires otherwise.

The phrases percentage (%) sequence identity or % identity or % identical when used with reference to an amino acid sequence describe the number of identical amino acid matches of two or more aligned amino acid sequences compared to the number of amino acid residues comprising the total length of the amino acid sequences. In other words, using an alignment of two or more sequences, the percentage of amino acid residues that are the same (e.g., have 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity relative to the entire length of amino acid sequences), can be determined when sequences are compared and aligned to achieve a maximum match, as measured by a comparison algorithm

- 6 045279 sequences known in the art, or by manual alignment and by visual inspection. The sequences that are compared to determine sequence identity may thus differ in amino acid substitution(s), addition(s) or deletion(s). Suitable programs for aligning protein sequences are known to one skilled in the art. The percentage sequence identity of protein sequences can, for example, be determined using programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, for example, using the NCBI BLAST algorithm (Altschul SF, et al. (1997), Nucleic Acids Res. 25:3389 -3402).

As used herein, the terms and phrases in combination, in combination with, co-delivery and are used together in the context of providing a subject with two or more therapeutic treatments or components refers to the simultaneous administration of two or more treatments or components, such as two vectors, e.g. DNA plasmid, or immunogenic combination and adjuvant. Simultaneous administration may mean administration of two components over at least one day. When two components are administered together or administered in combination with, they may be administered in separate compositions sequentially at short intervals, such as 24, 20, 16, 12, 8 or 4 hours, or within 1 hour, or they may be administered in one composition at the same time. Use of the term in combination does not limit the order in which the therapeutic agents or components are administered to a subject. For example, the first therapeutic treatment or first component (eg, the first DNA plasmid encoding an HBV antigen) may be administered before (eg, 5 minutes to one hour before), concurrently or simultaneously with, or after (eg, 5 minutes to one hour before). up to one hour after) provision of a second therapeutic treatment or a second component (eg, a second DNA plasmid encoding an HBV antigen). In some embodiments, a first therapeutic treatment or first component (e.g., a first DNA plasmid encoding an HBV antigen) and a second therapeutic treatment or second component (e.g., a second DNA plasmid encoding an HBV antigen) are provided in the same same compositions. In other embodiments, the first therapeutic treatment or first component (eg, a first DNA plasmid encoding an HBV antigen) and the second therapeutic treatment or second component (eg, a second DNA plasmid encoding an HBV antigen) are provided in different compositions.

As used herein, the term non-naturally occurring nucleic acid or polypeptide refers to a nucleic acid or polypeptide that does not occur in nature. The non-naturally occurring nucleic acid or polypeptide may be synthesized, processed, manufactured and/or otherwise produced under laboratory and/or industrial conditions. In some cases, a non-naturally occurring nucleic acid or polypeptide may comprise a naturally occurring nucleic acid or polypeptide that has been processed, processed, or modified to exhibit properties that were not present in the naturally occurring nucleic acid or polypeptide prior to processing. As used herein, a non-naturally occurring nucleic acid or polypeptide can be a nucleic acid or polypeptide isolated or separated from the natural source in which it was found and lacking covalent bonds to the sequences to which it was linked in natural source. The non-naturally occurring nucleic acid or polypeptide may be produced recombinantly or by other means such as chemical synthesis.

As used herein, the term subject means any animal, preferably a mammal, most preferably a human, that will receive or has received treatment with a method according to an embodiment of the invention. As used herein, the term mammal includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc. , more preferably human.

As used herein, the term operably coupled refers to a connection or adjacent arrangement in which the components so described are in a relationship that allows them to function as intended. For example, a regulatory sequence operably linked to a nucleic acid sequence of interest is capable of directing transcription of the nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest is capable of secreting or translocating the amino acid sequence of interest across a membrane.

To facilitate understanding of the invention, the description is divided into paragraphs or sections or directed to disclose various embodiments of the invention. This division should not be construed as separating the essence of a paragraph or section or embodiments from the essence of another paragraph or section or embodiments. On the contrary, one skilled in the art will understand

- 7 045279 that the description has a broad application and covers all possible intended combinations of various sections, paragraphs and sentences. The discussion of any embodiment is intended to be exemplary only and is not intended to limit the scope of the disclosure, including the claims, to those examples. For example, although embodiments of the HBV vectors of the invention (e.g., DNA plasmid or viral vectors) disclosed herein may contain specific components, including, without limitation, certain promoter sequences, enhancer or regulatory sequences, signal peptides, sequence, encoding HBV antigen, polyadenylation signal sequences, etc., arranged in a particular order, those skilled in the art will appreciate that the concepts disclosed herein can equally be applied to other components arranged in a different order that may used in the HBV vectors of the invention. The invention contemplates the use of any of the suitable components in any combination having any sequence that can be used in the HBV vectors of the invention, whether or not that particular combination is explicitly described.

Hepatitis B virus (HBV).

As used herein, the term hepatitis B virus or HBV refers to a virus of the Hepadnaviridae family. HBV is a small (eg, 3.2 kb) hepatotropic DNA virus that encodes four open reading frames and seven proteins. See fig. 1A. The seven proteins encoded by HBV include small (S), medium (M) and large (L) surface antigen (HBsAg) or envelope proteins (Env), pre-Core protein, core protein, viral polymerase (Pol) and HBx protein. HBV expresses three surface antigens or envelope proteins, L, M and S, of which S is the shortest and L is the largest. The additional domains in the M and L proteins are called Pre-S2 and Pre-S1, respectively. The nuclear protein is a subunit of the viral nucleocapsid. Pol is required for viral DNA synthesis (reverse transcriptase, RNase H, and primer), which occurs in nucleocapsids localized in the cytoplasm of infected hepatocytes. PreCore is a nuclear protein with an N-terminal signal peptide and undergoes proteolytic processing at the N- and C-termini before secretion from infected cells, the so-called hepatitis B e antigen (HBeAg). The HBx protein is required for efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a structural viral protein. All HBV viral proteins have their own mRNA, with the exception of nuclear and polymerase, which share a common mRNA. With the exception of the pre-Core protein, none of the HBV viral proteins undergo post-translational proteolytic processing.

The HBV virion contains the viral envelope, nucleocapsid, and one copy of the partially double-stranded DNA genome. The nucleocapsid contains 120 nuclear protein dimers and is covered by a capsid membrane with embedded viral envelope proteins S, M and L or surface antigenic proteins. After entering the cell, the virus is uncoated, and the capsid-containing relaxed circular DNA (rcDNA) with covalently bound viral polymerase migrates into the nucleus. During this process, nuclear protein phosphorylation causes structural changes, exposing a nuclear localization signal that allows the capsid to interact with so-called importins. These importins mediate the binding of nuclear protein to nuclear pore complexes, during which the capsid is unpacked and the polymerase/rcDNA complex is released into the nucleus. Inside the nucleus, rDNA becomes deproteinized (removal of the polymerase occurs) and, with the help of the host DNA repair mechanism, is converted into a covalently closed circular DNA genome (cccDNA), from which overlapping transcripts encode HBeAg, HBsAg, nuclear protein, viral polymerase and HBx protein . The core protein, viral polymerase, and pregenomic RNA (pgRNA) combine in the cytoplasm and self-assemble into immature capsid particles containing pgRNA, which are further converted into mature capsids with rDNA and function as a common intermediate that is either enveloped or secreted into as infectious viral particles or is transported back into the nucleus to replenish and maintain a stable pool of cccDNA. See fig. 1B.

Today, HBV is divided into four serotypes (adr, adw, ayr, ayw) based on antigenic epitopes present on the envelope proteins, and into eight genotypes (A, B, C, D, E, F, G, and H) on based on the viral genome sequence. HBV genotypes are distributed across different geographic regions. For example, the most common genotypes in Asia are genotypes B and C. Genotype D is dominant in Africa, the Middle East, and India, while genotype A is widespread in northern Europe, sub-Saharan Africa, and West Africa.

HBV antigens.

As used herein, the terms HBV antigen, HBV antigenic polypeptide, HBV antigenic polypeptide, HBV antigenic protein, HBV immunogenic polypeptide, and HBV immunogen refer to a polypeptide capable of inducing an immune response, such as a humoral and/or cell-mediated response in a subject to HBV. The HBV antigen may be an HBV polypeptide, a fragment or epitope thereof, or a combination of several HBV polypeptides, parts thereof, or

- 8 045279 derivatives. The HBV antigen is capable of inducing a protective immune response in a host, such as inducing an immune response to a viral disease or infection and/or producing immunity (ie, vaccinating) in a subject against a viral disease or infection that protects the subject from the viral disease or infection. For example, an HBV antigen may comprise a polypeptide or immunogenic fragment(s) thereof of any HBV protein, such as HBeAg, pre-core protein, HBsAg (S, M, or L proteins), nuclear protein, viral polymerase, or HBx-derived protein from any HBV genotype, for example genotype A, B, C, D, E, F, G and/or H, or a combination thereof.

(1) HBV core antigen.

As used herein, the terms HBV core antigen, HBcAg, and core antigen each refer to an HBV antigen capable of inducing an immune response, such as a humoral and/or cell-mediated response, in a subject to an HBV core protein. The terms nuclear, nuclear polypeptide, and nuclear protein each refer to the nuclear protein of the HBV virus. A full-length nuclear antigen is typically 183 amino acids in length and includes an assembly domain (amino acids 1 to 149) and a nucleic acid binding domain (amino acids 150 to 183). The 34-residue nucleic acid binding domain is required for encapsidation of pregenomic RNA. This domain also functions as a nuclear import (import into the nucleus) signal. It contains 17 arginine residues and is highly basic, consistent with its function. The HBV core protein is dimeric in solution, with the dimers self-assembling into icosahedral capsids. Each nuclear protein dimer has four α-helical bundles flanked by an aspirated domain on both sides. Truncated HBV core proteins lacking the nucleic acid binding domain are also capable of forming capsids.

In an embodiment of the invention, the HBV antigen is a truncated HBV nuclear antigen. As used herein, the term truncated HBV core antigen refers to an HBV antigen that does not contain the full length HBV core protein, but which is capable of inducing an immune response in a subject to the HBV core protein. For example, an HBV core antigen can be modified by removing one or more amino acids from the highly positively charged (arginine-rich) C-terminal nucleic acid binding domain of the core antigen, which typically contains seventeen arginine residues (R). The truncated HBV nuclear antigen of the invention is preferably a truncated C-terminal HBV nuclear protein that does not contain the HBV nuclear protein nuclear import signal, and/or a truncated HBV nuclear protein from which the C-terminal HBV nuclear protein import signal is removed. In one embodiment, the truncated HBV nuclear antigen contains a deletion in the C-terminal nucleic acid binding domain, such as a deletion of 1 to 34 amino acid residues in the C-terminal nucleic acid binding domain, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acid residues, preferably deletion of all 34 amino acid residues. In a preferred embodiment, the truncated HBV nuclear antigen contains a deletion in the C-terminal nucleic acid binding domain, preferably a deletion of all 34 amino acid residues.

The HBV core antigen of the invention may be a consensus sequence derived from several HBV genotypes (eg genotypes A, B, C, D, E, F, G and H). As used herein, the term consensus sequence means an artificial amino acid sequence based on an alignment of the amino acid sequences of homologous proteins, for example, determined by alignment (eg, using Clustal Omega) of the amino acid sequences of homologous proteins. This may be the calculated order of the most frequently occurring amino acid residues found at each position in a sequence alignment, taking into account the HBV antigen sequences (eg, core, pol, etc.) of at least 100 natural HBV isolates. The consensus sequence may not be naturally occurring and may differ from native viral sequences. Consensus sequences can be constructed by aligning multiple HBV antigen sequences obtained from different sources using multiple sequence alignment tools and by selecting the most frequently occurring amino acid at different positions in the alignment. Preferably, the HBV consensus antigen sequence is derived from HBV genotypes B, C and D. The term consensus antigen is used to refer to an antigen having a consensus sequence.

An example of a truncated HBV core antigen of the invention lacks nucleic acid binding function and is capable of inducing an immune response in a mammal against at least two HBV genotypes. Preferably, the truncated HBV core antigen is capable of inducing a T cell response in a mammal to at least HBV genotypes B, C and D. More preferably, the truncated HBV core antigen is capable of inducing a CD8 T cell response in a human subject to at least genotypes A, B, C and D HBV.

Preferably, the HBV core antigen of the invention is a consensus antigen, preferably a consensus antigen derived from HBV genotypes B, C and D, more preferably

- 9 045279 a completely truncated consensus antigen derived from HBV genotypes B, C and D. An example of a truncated HBV consensus core antigen of the invention consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14, e.g., at least 90%, 91%, 92%, 93 %, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14. SEQ ID NO: 2 and SEQ ID NO: 14 are nuclear consensus antigens derived from HBV genotypes B, C and D. SEQ ID NO: 2 and SEQ ID NO: 14 contain a C-terminal 34 amino acid deletion of the highly positively charged (arginine-rich) native nuclear antigen nucleic acid binding domain.

In a specific embodiment, the HBV core antigen is a truncated HBV antigen having the amino acid sequence of SEQ ID NO: 2. In another specific embodiment, the HBV core antigen is a truncated HBV antigen having the amino acid sequence of SEQ ID NO: 14.

(2) HBV polymerase antigen.

As used herein, each of the terms HBV polymerase antigen, HBcAg and HBV pol antigen refers to an HBV antigen capable of inducing an immune response, such as a humoral and/or cell-mediated response in a subject to HBV polymerase. The terms polymerase, polymerase polypeptide, Pol, and pol each refer to the HBV viral DNA polymerase. The HBV viral DNA polymerase has four domains, including, from N-terminal to C-terminal, a terminal protein (TP) domain, which acts as a primer for minus-strand DNA synthesis; a spacer that is not essential for the functions of the polymerase; reverse transcriptase (RT) domain for transcription polymerase; and RNase H domain.

In one embodiment, the HBV antigen comprises an HBV Pol antigen or any immunogenic fragment or combination thereof. The HBV Pol antigen may contain additional modifications that improve the immunogenicity of the antigen, for example, by introducing mutations into the active regions of the polymerase and/or RNase domains to reduce or substantially eliminate certain enzymatic activities.

Preferably, the HBV Pol antigen of the invention does not have reverse transcriptase activity and RNase H activity and is capable of inducing an immune response in a mammal to at least two HBV genotypes. Preferably, the HBV Pol antigen is capable of inducing a T cell response in a mammal to at least HBV genotypes B, C and D. More preferably, the HBV Pol antigen is capable of inducing a CD8 T cell response in humans to at least genotypes A, B, C and D of HBV.

Thus, in some embodiments, the HBV Pol antigen is an inactivated Pol antigen. In one embodiment, the HBV Pol inactivated antigen contains one or more amino acid mutations in the active region of the polymerase domain. In another embodiment, the inactivated HBV Pol antigen contains one or more amino acid mutations in the active region of the RNase H domain. In a preferred embodiment, the inactivated HBV pol antigen contains one or more amino acid mutations in the active region of both the polymerase domain and the RNase H domain. For example, the YXDD motif in the pol polymerase domain of the HBV antigen, which may be required for nucleotide/metal ion binding, can be mutated, for example, by replacing one or more aspartate (D) residues with asparagine (N) residues, thereby eliminating or reducing the metal coordination function , thereby weakening or essentially eliminating reverse transcriptase function. Alternatively, or in addition to mutation of the YXDD motif, the DEDD motif in the RNase H domain of the HBV pol antigen, required for Mg ²⁺ coordination, can be mutated, for example, by replacing one or more aspartate (D) residues with an asparagine (N) residue and/or by replacing the glutamate (E) residue with a glutamine (Q) residue, thereby reducing or substantially eliminating the function of RNase H. In a specific embodiment, the HBV pol antigen is modified by (1) mutating aspartate (D) residues to asparagine (N) residues in the motif YXDD polymerase domain; and (2) mutations of the first aspartate residue (D) to an asparagine residue (N) and the first glutamate residue (E) to a glutamine residue (N) in the DEDD motif of the RNaseH domain, thereby weakening or essentially eliminating both reverse transcriptase and RNase functions H pol antigen.

In a preferred embodiment of the invention, the HBV pol antigen is a consensus antigen, preferably a consensus antigen derived from HBV genotypes B, C and D, more preferably an inactivated consensus antigen derived from HBV genotypes B, C and D. A typical HBV consensus pol antigen of the invention contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 95 ,5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4% , 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 4, preferably at least 98% identical to SEQ ID NO: 4, for example, by at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7 %, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4. SEQ ID NO: 4 is a consensus pol antigen derived from HBV genotypes B, C and D containing

- 10 045279 four mutations located in the active regions of the polymerase and RNase H domains. Specifically, the four mutations include mutation of aspartic acid (D) residues to asparagine (N) residues in the YXDD motif of the polymerase domain; and mutation of the first aspartate residue (D) to an asparagine residue (N) and mutation of the glutamate residue (E) to a glutamine residue (Q) in the DEDD motif of the RNase H domain.

In a specific embodiment, the HBV pol antigen comprises the amino acid sequence of SEQ ID NO: 4. In other embodiments, the HBV pol antigen consists of the amino acid sequence of SEQ ID NO: 4.

(3) Fusion of HBV nuclear antigen and HBV polymerase antigen.

As used herein, the term fusion protein or fusion refers to a single polypeptide chain having at least two polypeptide domains that are not naturally present in a single naturally occurring polypeptide.

In one embodiment, the HBV antigen comprises a fusion protein comprising a truncated HBV nuclear antigen operably linked to an HBV Pol antigen, or an HBV Pol antigen operably linked to a truncated HBV nuclear antigen, preferably via a linker.

As used herein, the term linker refers to a compound or moiety that acts as a molecular bridge to operably link two different molecules, with one portion of the linker operably linked to a first molecule and another portion of the linker operably linked to a second molecule. For example, in a fusion protein comprising a first polypeptide and a second heterologous polypeptide, the linker serves primarily as a spacer between the first and second polypeptides. In one embodiment, the linker consists of amino acids linked to each other by peptide bonds, preferably from 1 to 20 amino acids linked by peptide bonds, the amino acids being selected from 20 naturally occurring amino acids. In one embodiment, from 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine and lysine. Preferably, the linker consists of a majority of sterically unhindered amino acids such as glycine and alanine. Typical linkers are polyglycines, in particular (Gly) ₅ , (Gly)8; poly(G1y-Ala) and polyalanines. One example of suitable linkers, shown in the examples below, is (AlaGly) _n , where n is an integer from 2 to 5.

Preferably, the fusion protein claimed is capable of inducing an immune response in a mammal to nuclear HBV and HBV Pol of at least two HBV genotypes. Preferably, the fusion protein is capable of inducing a T cell response in a mammal to at least genotypes B, C and D of HBV. More preferably, the fusion protein is capable of inducing a T cell response in a human subject to at least genotypes A, B, C and D of HBV.

In one embodiment, the fusion protein comprises a truncated HBV nuclear antigen having an amino acid sequence that is at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 95 .5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4% , 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14, linker and HBV Pol antigen having the amino acid sequence which is at least 90%, for example at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97 ,5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4.

In a preferred embodiment of the invention, the fusion protein comprises a truncated HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 14, a linker comprising (AlaGly)n, where n is an integer from 2 to 5, and an HBV Pol antigen having the amino acid sequence SEQ ID NO: 4. More preferably, the fusion protein in accordance with an embodiment of the invention contains the amino acid sequence of SEQ ID NO: 20.

In an embodiment of the invention, the fusion protein further comprises a signal sequence. Preferably, the signal sequence has the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19. More preferably, the fusion protein contains the amino acid sequence of SEQ ID NO: 21.

Polynucleotides and vectors.

In another general aspect, the invention relates to a non-naturally occurring nucleic acid molecule encoding an HBV antigen of the invention, and a vector containing the non-naturally occurring nucleic acid. The non-naturally occurring nucleic acid molecule may comprise any polynucleotide sequence encoding an HBV antigen as claimed, which can be prepared by methods known in the art in light of the present disclosure. Preferably, the polynucleotide encodes at least one of a truncated HBV nuclear antigen and an HBV polymerase antigen of the invention. The polynucleotide may be in the form of RNA or in the form of DNA, produced recombinantly (eg, cloning) or produced synthetically (eg, chemical synthesis). DNA may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. The DNA may, for example, comprise genomic DNA, cDNA, or combinations thereof. The polynucleotide may also be a DNA/RNA hybrid. The polynucleotides and vectors of the invention can be used to produce a recombinant protein, express a protein in a host cell, or produce viral particles. Preferably the polynucleotide is DNA.

In an embodiment of the invention, the non-naturally occurring nucleic acid molecule comprises a polynucleotide encoding a truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 2 or SEQ ID NO: 14, for example, on at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99 %, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical SEQ ID NO: 2, preferably 98%, 99% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14. In a particular embodiment of the invention, the non-naturally occurring nucleic acid molecule encodes a truncated HBV nuclear antigen consisting of the amino acid sequences SEQ ID NO: 2 or SEQ ID NO: 14.

Examples of polynucleotide sequences of the invention encoding a truncated HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14 include, but are not limited to, a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 15, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5% , 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8 %, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 15, preferably 98, 99 or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 15. Examples of non-naturally occurring nucleic acid molecules acids encoding the truncated HBV nuclear antigen have the polynucleotide sequence SEQ ID NO: 1 or 15.

In an embodiment of the invention, the non-naturally occurring nucleic acid molecule encodes an HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2% , 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4, preferably 100% identical SEQ ID NO: 4. In a specific embodiment, the non-naturally occurring nucleic acid molecule encodes an HBV polymerase antigen consisting of the amino acid sequence of SEQ ID NO: 4.

Examples of polynucleotide sequences as claimed encoding an HBV Pol antigen comprising the amino acid sequence of SEQ ID NO: 4 include, but are not limited to, a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16, e.g. by at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% , 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100 % identical to SEQ ID NO: 3 or SEQ ID NO: 16, preferably 98%, 99% or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 16. Examples of non-naturally occurring nucleic acid molecules encoding pol antigen HBV have the polynucleotide sequence SEQ ID NO: 3 or 16.

In another embodiment, the non-naturally occurring nucleic acid molecule encodes a fusion protein comprising a truncated HBV nuclear antigen operably linked to an HBV Pol antigen, or containing an HBV Pol antigen operably linked to a truncated HBV nuclear antigen. In a specific embodiment, the non-naturally occurring nucleic acid molecule encodes a truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14, for example, to at least 91 %, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1% , 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14, more preferably 100% identical to SEQ ID NO: 14; linker; and an HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5 %, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99 .5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4, preferably 98%, 99% or 100% identical to SEQ ID NO: 4. In a specific embodiment of the invention, the non-naturally occurring nucleic acid molecule encodes a fusion protein containing a truncated HBV nuclear antigen, consisting of the amino acid sequence SEQ ID NO: 14, a linker containing (AlaGly) _n , where n is an integer from 2 to 5 ; and an HBV Pol antigen comprising the amino acid sequence of SEQ ID NO: 4. In a particular embodiment of the invention, the non-naturally occurring nucleic acid molecule encodes a fusion protein comprising the amino acid sequence of SEQ ID NO: 20.

Examples of polynucleotide sequences of the invention encoding a fusion protein include, but are not limited to, a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 15, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99 .2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 15, preferably 98%, 99% or

- 12 045279

100% identical to SEQ ID NO: 1 or SEQ ID NO: 15, operably linked to a linker encoding a sequence that is at least 90% identical to SEQ ID NO: 22, e.g., at least 90%, 91%, 92 %, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99, 2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 22, preferably to 98 %, 99%, or 100% identical to SEQ ID NO: 22, which is further operably linked to a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16, e.g., 90%, 91% identical , 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 16, preferably 98, 99, or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 16. In particular embodiments of the invention, the non-naturally occurring nucleic acid molecule encoding the fusion protein comprises SEQ ID NO: 1 or SEQ ID NO: 15, operably linked to SEQ ID NO: 22, which is further operably linked to SEQ ID NO: 3 or SEQ ID NO: 16.

The invention also relates to a vector containing an isolated polynucleotide encoding an HBV antigen. As used herein, a vector is a nucleic acid molecule used to transfer genetic material to another cell where it can be replicated and/or expressed. Any vector known to those skilled in the art given the present disclosure may be used. Examples of vectors include, but are not limited to, plasmids, viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and artificial chromosomes (eg, YAC). Preferably, the vector is a DNA plasmid. The vector may be a DNA vector or an RNA vector. One skilled in the art can construct the vector of the invention by standard recombinant methods in light of the present disclosure.

The vector of the invention may be an expression vector. As used herein, the term expression vector refers to any type of genetic construct containing a nucleic acid encoding RNA capable of being transcribed. Expression vectors include, but are not limited to, recombinant protein expression vectors, such as a DNA plasmid or viral vector, and vectors for delivering a nucleic acid to a subject for expression in the subject's tissue, such as a DNA plasmid or viral vector. Those skilled in the art will appreciate that the design of an expression vector may depend on factors such as the choice of host cell to be transformed, the desired level of protein expression, etc.

The vectors of the invention may contain multiple regulatory sequences. As used herein, the term regulatory sequence refers to any sequence that allows, promotes or modulates the functional activity of a nucleic acid molecule, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivatives (i.e. e. mRNA) into the host cell or organism. As used herein, the term includes promoters, enhancers, and other expression control elements (eg, polyadenylation signals and elements that affect mRNA stability).

In some embodiments, the vector is a non-viral vector. Examples of non-viral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. Examples of non-viral vectors include, but are not limited to, an RNA replicon, an mRNA replicon, a modified mRNA replicon or self-amplifying mRNA, a closed linear deoxyribonucleic acid, such as a linear covalently closed DNA molecule, such as a linear covalently closed double-stranded DNA molecule. Preferably, the non-viral vector is a DNA plasmid. DNA plasmid, which is used interchangeably with DNA plasmid vector, plasmid DNA or plasmid DNA vector, refers to a double-stranded and usually circular DNA sequence that is capable of autonomous replication in a suitable host cell. DNA plasmids used to express the encoded polynucleotide typically contain an origin of replication, a multiple cloning site, and a selectable marker, which, for example, may be an antibiotic resistance gene. Examples of suitable DNA plasmids that can be used include, but are not limited to, commercially available expression vectors for use in well-known expression systems (including both prokaryotic and eukaryotic systems), such as pSE420 (Invitrogen, San Diego, Calif.), which can be used for the production and/or expression of protein in Escherichia coli; pYES2 (Invitrogen, Thermo Fisher Scientific), which can be used for production and/or expression in strains of the yeast Saccharomyces cerevisiae; the complete baculovirus expression system MAHBAS® (Thermo Fisher Scientific), which can be used for production and/or expression in insect cells; pcDNATM or pcDNA3TM (Life Technologies, Thermo Fisher Scientific), which can be used to express constitutive protein in large quantities in mammalian cells; and pVAX or pVAX-1 (Life Technologies, Thermo Fisher Scientific), which can be used to transiently express protein of interest

- 13 045279 ka in large quantities in most mammalian cells. The backbone of any commercially available DNA plasmid can be modified to optimize protein expression in the host cell, for example by changing the orientation of certain elements (e.g., origin of replication and/or antibiotic resistance cassette), replacing a promoter endogenous to the plasmid (e.g. promoter in an antibiotic resistance cassette), and/or replacement of the polynucleotide sequence encoding transcribed proteins (eg, the coding sequence of an antibiotic resistance gene), using routine methods and readily available starting materials. (See, for example, Sambrook et al., Molecular Cloning the Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)).

Preferably, the DNA plasmid is an expression vector suitable for expression of the protein in mammalian host cells. Expression vectors suitable for expressing the protein in mammalian host cells include, but are not limited to, pcDNATM, pcDNA3TM, pVAX, pVAX-1, ADVAX, NTC8454, etc. Preferably, the expression vector is based on pVAX-1, which can be further modified to optimize protein expression in mammalian cells. pVAX-1 is a commonly used plasmid in DNA vaccines and contains the strong human cytomegalovirus immediate early promoter (CMV-IE) followed by a polyadenylation (pA) sequence derived from bovine growth hormone (bGH). pVAX-1 additionally contains a pUC origin of replication and a kanamycin resistance gene driven by a small prokaryotic promoter that allows propagation of the bacterial plasmid.

The vector of the invention may be a viral vector. Typically, viral vectors are genetically engineered viruses that carry modified viral DNA or RNA that is rendered non-infectious but still contains viral promoters and transgenes, allowing translation of the transgene through the viral promoter. Because viral vectors often lack infectious sequences, they require helper viruses or packaging lines for transfection in large volumes. Examples of viral vectors that can be used include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, smallpox virus vectors, enteric virus vectors, Venezuelan equine encephalitis virus vectors, Semliki forest virus vectors, tobacco mosaic virus vectors, lentiviral vectors, arenavirus vectors, replication-deficient arenavirus vectors or replication-competent arenavirus vectors, two-segmented or three-segmented arenaviruses, infectious arenavirus vectors, nucleic acids that contain an arenovirus genomic segment in which one open reading frame of the genomic segment is removed or functionally inactivated (and replaced by a nucleic acid encoding an antigen HBV as disclosed herein), arenavirus such as lymphocytic choriomeningitis virus (LCMV), e.g. clone 13 strain or MP strain, and arenavirus such as Junin virus, e.g. Candid #1 strain, etc. The vector may also be non-viral.

Preferably, the viral vector is an adenoviral vector, for example a recombinant adenoviral vector. The recombinant adenovirus vector, for example, can be derived from a human adenovirus (HAdV or AdHu) or from a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh or SAdV) or from rhesus macaque adenovirus (rhAd). Preferably, the adenoviral vector is a vector of a recombinant human adenovirus, for example, recombinant human adenovirus serotype 26 or any of the recombinant human adenovirus serotypes 5, 4, 35, 7, 48, etc. In other embodiments, the adenoviral vector is a rhAd vector, such as rhAd51, rhAd52, or rhAd53. A recombinant viral vector useful for use can be prepared by methods known in the art in light of the present disclosure. For example, taking into account the degeneracy of the genetic code, several nucleic acid sequences can be designed that encode the same polypeptide. The polynucleotide encoding the HBV antigen of the invention may optionally be codon optimized to ensure proper expression in a host cell (eg, bacterial or mammalian cells). Codon optimization is a technique widely used in the art, and methods for producing codon-optimized polynucleotides are well known to those skilled in the art in light of the present disclosure.

A vector of the invention, for example a DNA plasmid or a viral vector (especially an adenoviral vector), may contain any regulatory elements to provide the normal function(s) of the vector, including, but not limited to, replication and expression of HBV antigen(s), encoded by the polynucleotide sequence of the vector. Regulatory elements include, but are not limited to, a promoter, enhancer, polyadenylation signal, translation stop codon, ribosome binding element, transcription terminator, selectable markers, origin of replication, etc. The vector may contain one or more expression cassettes. The expression cassette is the part of the vector that directs the cellular machinery to produce RNA and protein. An expression cassette typically contains three components: a promoter sequence, an open reading frame, and a 3' untranslated region (UTR), optionally containing a polyadenylation signal. An open reading frame (ORF) is a reading frame that contains a coding post

- 14 045279 the sequence of the protein of interest (for example, HBV antigen) from the start codon to the stop codon. The expression cassette regulatory elements can be operably linked to a polynucleotide sequence encoding an HBV antigen of interest. As used herein, the term operably linked is to be considered in its broadest reasonable context and refers to the association of polynucleotide elements to provide their functionality. A polynucleotide is operably linked when it is in a functional relationship with another polynucleotide. For example, a promoter is operably linked to a coding sequence if it affects transcription of the coding sequence. Any components suitable for use in the expression cassette disclosed herein can be used in any combination and in any order to create the vectors of the invention.

The vector may contain a promoter sequence, preferably within an expression cassette, to control expression of the HBV antigen of interest. The term promoter is used in its conventional sense and refers to a nucleotide sequence that initiates transcription of an operably linked nucleotide sequence. A promoter is located on the same chain next to the nucleotide sequence that it transcribes. Promoters can be constitutive, inducible or repressive. Promoters can be natural or synthetic. The promoter can be obtained from sources including viruses, bacteria, fungi, plants, insects and animals. The promoter may be a homologous promoter (ie, derived from the same genetic source as the vector) or a heterologous promoter (ie, derived from a different vector or genetic source). For example, if the vector used is a DNA plasmid, the promoter may be endogenous to the plasmid (homologous) or may be derived from other sources (heterologous). Preferably, the promoter is located upstream of the polynucleotide encoding the HBV antigen within the expression cassette.

Examples of promoters that can be used include, but are not limited to, simian virus 40 (SV40) promoter, murine mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter such as the immunodeficiency virus long terminal repeat (LTR) promoter ( BIV), Moloney virus promoter, avian leukemia virus (ALV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter (CMV-IE), Epstein Barr virus (EBV) promoter, or Rous sarcoma virus (RSV) promoter. The promoter may also be the promoter of a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine or human metallothionein. The promoter may also be a tissue-specific promoter, such as a muscle or skin-specific promoter, natural or synthetic.

Preferably, the promoter is a strong eukaryotic promoter, preferably the cytomegalovirus immediate early promoter (CMV-IE). The nucleotide sequence of an exemplary CMV-IE promoter is shown in SEQ ID NO: 7 and SEQ ID NO: 17.

The vector may contain additional polynucleotide sequences that stabilize the expressed transcript, enhance nuclear export of the RNA transcript, and/or improve transcription-translation coupling. Examples of such sequences include polyadenylation signals and enhancer sequences. The polyadenylation signal is typically located downstream of the coding sequence of the protein of interest (eg, HBV antigen) within the vector expression cassette. Enhancer sequences are DNA regulatory sequences that, when bound by transcription factors, enhance transcription of the associated gene. The enhancer sequence is preferably located upstream of the polynucleotide sequence encoding the HBV antigen but downstream of the promoter sequence within the vector expression cassette.

Any polyadenylation signal known to those skilled in the art in light of the present disclosure may be used. For example, the polyadenylation signal may be an SV40 polyadenylation signal (eg, SEQ ID NO: 24), an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. Preferably, the polyadenylation signal is a bovine growth hormone (bGH) polyadenylation signal or an SV40 polyadenylation signal. The nucleotide sequence of the exemplary bGH polyadenylation signal is shown in SEQ ID NO: 11. The nucleotide sequence of the exemplary SV40 polyadenylation signal is shown in SEQ ID NO: 24.

Any enhancer sequence known to those skilled in the art given the present disclosure may be used. For example, the enhancer sequence may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as one of CMV, HA, RSV, or EBV. Examples of specific enhancers include, but are not limited to, the HBV Woodchuck post-transcriptional regulatory element (WPRE), the intron/exon sequence derived from the human apolipoprotein A1 precursor (ApoAI), the R-U5 untranslated domain of the long terminal repeat (LTR) of T virus

- 15 045279 human cell leukemia type 1 (HTLV-1), splicing enhancer, synthetic rabbit β-globin intron, or any combination thereof. Preferably, the enhancer sequence is a composite sequence consisting of three consecutive elements of the HTLV-1 LTR R-U5 untranslated domain, a rabbit β-globin intron and a splicing enhancer, which is referred to herein as a triple enhancer sequence. The nucleotide sequence of an exemplary triple enhancer sequence is shown in SEQ ID NO: 8. Another example of an enhancer sequence is the ApoAI gene fragment shown in SEQ ID NO: 23.

The vector may contain a polynucleotide sequence encoding a signal peptide sequence. Preferably, the polynucleotide sequence encoding the signal peptide sequence is located before the polynucleotide sequence encoding the HBV antigen. Signal peptides typically direct protein localization, facilitate secretion of the protein from the cell in which it is produced, and/or enhance antigen expression and cross-presentation to antigen-presenting cells. The signal peptide may be present at the N-terminus of the HBV antigen when expressed from a vector, but is cleaved off by signal peptidase, for example, when secreted from the cell. The expressed protein from which the signal peptide has been cleaved is often called the mature protein. Any signal peptide known in the art in light of the present disclosure may be used. For example, the signal peptide may be cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as Ig gamma heavy chain signal peptide SPIgG or Ig epsilon heavy chain signal peptide SPIgE.

Preferably, the signal peptide sequence is a cystatin S signal peptide. Exemplary nucleic acid and amino acid sequences of a cystatin S signal peptide are shown in SEQ ID NOs: 5 and 6, respectively. Exemplary nucleic acid and amino acid sequences of the immunoglobulin secretion signal are shown in SEQ ID NO: 18 and 19, respectively.

A vector, such as a DNA plasmid, may also include an origin of replication of bacterial origin and an antibiotic resistance expression cassette for selecting and maintaining the plasmid in bacterial cells, such as E. coli. The origins of replication of bacterial origin and the antibiotic resistance cassette can be located in the vector in the same orientation as the expression cassette encoding the HBV antigen, or in the opposite (reverse) orientation. The origin of replication (ORI) is the sequence at which replication begins, allowing the plasmid to replicate and survive in cells. Examples of ORI suitable for use in the invention include, but are not limited to, ColE1, pMB1, pUC, pSC101, R6K and 15A, preferably pUC. A typical pUC ORI nucleotide sequence is shown in SEQ ID NO: 10.

Expression cassettes for selection and maintenance in bacterial cells typically include a promoter sequence operably linked to an antibiotic resistance gene. Preferably, the promoter sequence operably linked to the antibiotic resistance gene is different from the promoter sequence operably linked to the polynucleotide sequence encoding the protein of interest, such as an HBV antigen. The antibiotic resistance gene can be codon optimized, and the sequence composition of the antibiotic resistance gene is usually selected depending on the codon usage of bacteria, such as E. coli. Any antibiotic resistance gene known to those skilled in the art may be used in connection with the present invention, including, without limitation, the kanamycin resistance gene (Kan ^r ), the ampicillin resistance gene (Amp ^r ), and the tetracycline resistance gene (Tet ^r ). as well as genes that impart resistance to chloramphenicol, bleomycin, spectinomycin, carbenicillin, etc.

Preferably, the antibiotic resistance gene in the antibiotic expression cassette in the vector is a kanamycin resistance gene (Kan ^r ). The sequence of the Kan ^r gene is shown in SEQ ID NO: 13. Preferably, the Kan ^r gene is codon optimized. An exemplary codon optimized nucleic acid sequence of the Kan ^r gene is shown in SEQ ID NO: 12. Kan ^r may be operably linked to a native promoter, or the Kan ^r gene may be linked to a heterologous promoter. In a specific embodiment, the Kan ^r gene is operably linked to the ampicillin resistance gene promoter (Amp ^r ), known as the bla promoter. An exemplary nucleotide sequence of the B1a promoter is shown in SEQ ID NO: 9.

In a specific embodiment, the vector is a DNA plasmid containing an expression cassette comprising a polynucleotide encoding at least one HBV antigen selected from the group consisting of an HBV pol antigen containing an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, for example, at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6% , 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4, and a truncated HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2; higher sequence, functionally related to

- 16 045279 lynucleotide encoding an HBV antigen containing, in the direction from the 5' end to the 3' end, a promoter sequence, preferably the CMV promoter sequence SEQ ID NO: 7, an enhancer sequence, preferably a triple enhancer sequence SEQ ID NO: 8 and a polynucleotide sequence encoding a signal peptide sequence, preferably a cystatin S signal peptide having the amino acid sequence of SEQ ID NO: 6; and a downstream sequence operably linked to a polynucleotide encoding an HBV antigen containing a polyadenylation signal, preferably a bGH polyadenylation signal SEQ ID NO: 11. Such vector further comprises an antibiotic resistance expression cassette comprising a polynucleotide encoding an antibiotic resistance gene, preferably a Kan gene ^r , more preferably a codon-optimized Kan ^r gene that is at least 90% identical to SEQ ID NO: 12, for example at least 90%, 91%, 92%, 93%, 94%, 95%, 95, 5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 12, preferably 100% identical to SEQ ID NO: 12, operably linked to the Amp promoter ^r (bla) SEQ ID NO: 9, located upstream and operably linked to a polynucleotide encoding an antibiotic resistance gene; and an origin of replication, preferably pUC ori of SEQ ID NO: 10. Preferably, the antibiotic resistance cassette and origin of replication are present on the plasmid in the reverse orientation of the HBV antigen expression cassette. Representative DNA plasmids having the above features are shown in FIG. 2A and 2B.

In another specific embodiment of the invention, the vector is a viral vector, preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector, comprising an expression cassette comprising a polynucleotide encoding at least one HBV antigen selected from the group consisting of an HBV antigen pol containing an amino acid sequence , which is at least 98% identical to the sequence of SEQ ID NO: 4, for example, at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99, 4%, 99.5% 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4, and a truncated HBV nuclear antigen consisting of the amino acid sequence SEQ ID NO: 14; an upstream sequence operably linked to a polynucleotide encoding an HBV antigen, containing, in a 5' to 3' direction, a promoter sequence, preferably the CMV promoter sequence SEQ ID NO: 17, an enhancer sequence, preferably an ApoAI gene fragment sequence SEQ ID NO: 23, and a polynucleotide sequence encoding a signal peptide sequence, preferably an immunoglobulin secretion signal, having the amino acid sequence SEQ ID NO: 19; and a downstream sequence operably linked to a polynucleotide encoding an HBV antigen containing a polyadenylation signal, preferably the SV40 polyadenylation signal of SEQ ID NO: 24.

In an embodiment of the invention, a vector, such as a DNA plasmid vector or a viral vector (preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector), encodes an HBV Pol antigen having the amino acid sequence of SEQ ID NO: 4. Preferably, the vector contains a Pol antigen coding sequence HBV that is at least 90% identical to the polynucleotide sequence of SEQ ID NO: 3, for example, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5 %, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6% , 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 3, preferably 100% identical to SEQ ID NO: 3.

In one embodiment, a vector, such as a DNA plasmid vector or a viral vector (preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector), encodes a truncated HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14 Preferably, the vector contains a truncated HBV nuclear antigen coding sequence that is at least 90% identical to the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15, for example, 90%, 91%, 92%, 93%, 94 %, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3 %, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 15, preferably 100 % is identical to SEQ ID NO: 1 or SEQ ID NO: 15.

In yet another embodiment of the invention, a vector, such as a DNA plasmid vector or a viral vector (preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector), encodes a fusion protein comprising an HBV Pol antigen having the amino acid sequence of SEQ ID NO: 4, and a truncated an HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14. Preferably, the vector contains a fusion protein coding sequence that contains a truncated HBV nuclear antigen coding sequence that is at least 90% identical to the sequence of SEQ ID NO : 1 or SEQ ID NO: 15, for example, by at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97 ,5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 15, preferably 98%, 99% or 100% identical to SEQ ID NO: 1

- 17 045279 or SEQ ID NO: 15, more preferably SEQ ID NO: 15, operably linked to an HBV Pol antigen coding sequence that is at least 90% identical to the sequence of SEQ ID NO: 3 or SEQ ID NO: 16, for example , by at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5 %, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 16, preferably 98%, 99% or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 16, more preferably SEQ ID NO: 16. Preferably the sequence encoding a truncated HBV nuclear antigen is operably linked to a HBV Pol antigen coding sequence through a linker coding sequence that is at least 90% identical to SEQ ID NO: 22, e.g., at least 90%, 91%, 92%, 93% , 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99 .3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 22 preferably 98, 99 or 100% identical is identical to SEQ ID NO: 22. In particular embodiments of the invention, the vector contains a sequence encoding a fusion protein having SEQ ID NO: 15, operably linked to SEQ ID NO: 22, which is further operably linked to SEQ ID NO: 16.

Polynucleotides and expression vectors encoding the HBV antigens of the invention can be prepared by any method known in the art in light of the present disclosure. For example, a polynucleotide encoding an HBV antigen can be introduced or cloned into an expression vector using standard molecular biology techniques, eg, polymerase chain reaction (PCR), etc., which are well known to those skilled in the art.

Cells, polypeptides and antibodies.

The invention also relates to cells, preferably isolated cells, containing any of the polynucleotides and vectors disclosed herein. The cells may, for example, be used to produce recombinant protein or to produce viral particles.

Thus, embodiments of the invention also relate to a method for producing the HBV antigen of the invention. The method comprises transfecting a host cell with an expression vector containing a polynucleotide encoding an HBV antigen of the invention operably linked to a promoter, growing the transfected cell under conditions suitable for expression of the HBV antigen, and optionally purifying or isolating the HBV antigen expressed in the cell. HBV antigen can be isolated or collected from a cell by any method known in the art, including affinity chromatography, size exclusion chromatography, etc. The methods used to express the recombinant protein are well known to one of ordinary skill in the art in light of the present disclosure. Expressed HBV antigens can also be studied without purification or isolation of the expressed protein, for example, by analyzing the supernatant of cells that have been transfected with an expression vector encoding the HBV antigen and that have been grown under conditions suitable for expression of the HBV antigen.

Thus, also provided are non-naturally occurring or recombinant polypeptides containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 14 or SEQ ID NO: 4. As described above and below , isolated nucleic acid molecules encoding these sequences, vectors containing these sequences operably linked to a promoter, and compositions containing a polypeptide, polynucleotide or vector are also presented in the description.

In one embodiment, the recombinant polypeptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, for example, 90%, 91%, 92%, 93%, 94%, 95%, 95.5 %, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99 .5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 2. Preferably, the non-naturally occurring or recombinant polypeptide consists of SEQ ID NO: 2.

In another embodiment, the non-naturally occurring or recombinant polypeptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4, for example 90%, 91%, 92%, 93%, 94%, 95% , 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99, 4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 4. Preferably, the non-naturally occurring or recombinant polypeptide contains SEQ ID NO: 4.

In another embodiment, the non-naturally occurring or recombinant polypeptide contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14, for example 90%, 91%, 92%, 93%, 94%, 95% , 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99, 4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 14. Preferably, the non-naturally occurring or recombinant polypeptide consists of SEQ ID NO: 14.

Also provided are antibodies or antigen-binding fragments thereof that specifically bind to a non-naturally occurring polypeptide of the invention. In one embodiment, an antibody specific for a non-naturally occurring HBV antigen of the invention does not bind specifically to another HBV antigen. For example, an antibody of the invention that specifically binds to an HBV Pol antigen having the amino acid sequence of SEQ ID NO: 4 will not specifically bind to an HBV Pol antigen not having the amino acid sequence of SEQ ID NO: 4.

As used herein, the term antibody includes polyclonal, monoclonal, chimeric, humanized, Fv, Fab and F(ab')2; bifunctional hybrid (eg, Lanzavecchia et al., Eur. J. Immunol. 17:105, 1987), single chain antibody (Huston et al., Proc. Natl. Acad. Sci. USA 85:5879, 1988; Bird et al. , Science 242: 423, 1988); and antibodies with modified constant regions (eg, US patent No. 5624821).

As used herein, the term antibody specifically binds to an antigen refers to an antibody that binds to an antigen with a KD of 1x10' ⁷ M or less. Preferably, an antibody that specifically binds to an antigen binds to an antigen with a KD of 1x10'8 M or less, more preferably 5x10'9 M or less, 1x10'9 M or less, 5x10' ¹⁰ M or less, or 1x10' ¹⁰ M or less . The term KD refers to the dissociation constant, which is derived from the ratio of Kd to Ka (ie Kd/Ka) and expressed as molar concentration (M). KD values for antibodies can be determined using methods known in the art in light of the present disclosure. For example, the KD of antibodies can be determined using surface plasmon resonance, such as a biosensor system such as the Biacore® system, or biolayer interferometry technology, such as the Octet RED96 system.

The lower the KD value of an antibody, the higher the binding affinity of the antibody to the target antigen.

Compositions, immunogenic combinations and vaccines.

The invention also relates to compositions, immunogenic combinations, more particularly to kits and vaccines containing one or more HBV antigens, polynucleotides and/or vectors encoding one or more HBV antigens of the invention. Any of the HBV antigens, polynucleotides (including RNA and DNA) and/or vectors of the invention disclosed herein can be used in the compositions, immunogenic combinations or kits and vaccines of the invention.

The invention relates to a composition containing an isolated or non-naturally occurring nucleic acid molecule (DNA or RNA) encoding a truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 2 or SEQ ID NO : 14, or an HBV polymerase antigen containing an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 4, a vector containing an isolated or unnatural nucleic acid molecule, and/or an isolated or unnatural polypeptide , encoded by an isolated or non-naturally occurring nucleic acid molecule.

In an embodiment of the invention, the composition comprises an isolated or unnatural nucleic acid molecule (DNA or RNA) encoding a truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 2 or SEQ ID NO : 14, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14.

In an embodiment of the invention, the composition comprises an isolated or unnatural nucleic acid molecule (DNA or RNA) encoding an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 4, preferably 100% identical SEQ ID NO: 4.

In one embodiment, the composition comprises an isolated or non-naturally occurring nucleic acid molecule (DNA or RNA) comprising a polynucleotide sequence encoding a truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14; and an isolated or unnatural nucleic acid molecule (DNA or RNA) containing a polynucleotide sequence encoding an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 4. The HBV truncated nuclear antigen and HBV Pol antigen coding sequences may be present in one isolated or unnatural nucleic acid molecule (DNA or RNA) or in two different isolated or unnatural nucleic acid molecules (DNA or RNA ).

In an embodiment of the invention, the composition contains a vector, preferably a DNA plasmid or viral vector (such as an adenoviral vector), containing a polynucleotide encoding a truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14.

In an embodiment of the invention, the composition contains a vector, preferably a DNA plasmid or viral vector (such as an adenoviral vector), containing a polynucleotide encoding an HBV Pol antigen containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 4.

In one embodiment, the composition comprises a vector, preferably a DNA plasmid or viral vector (such as an adenoviral vector), containing a polynucleotide encoding a truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14; and a vector, preferably a DNA plasmid or viral vector (such as an adenoviral vector), containing a polynucleotide encoding an HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 4. The vector containing the sequence encoding the truncated HBV nuclear antigen and the vector containing the sequence encoding the HBV Pol antigen may be the same vector or two different vectors.

In one embodiment, the composition contains a vector, preferably a DNA plasmid or viral vector (such as an adenoviral vector), containing a polynucleotide encoding a fusion protein containing a truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO : 2 or SEQ ID NO: 14, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14, operably linked to an HBV Pol antigen containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 4 , preferably 100% identical to SEQ ID NO: 4, or vice versa. Preferably, the fusion protein further comprises a linker that operably links the truncated HBV core antigen to the HBV Pol antigen or vice versa. Preferably, the linker has the amino acid sequence (AlaGly) _n , where n is an integer from 2 to 5.

In one embodiment, the composition comprises an isolated or non-naturally occurring truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14.

In an embodiment of the invention, the composition comprises an isolated or non-naturally occurring HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 4.

In one embodiment, the composition comprises an isolated or non-naturally occurring truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 14, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14; and an isolated or non-naturally occurring HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 4.

In one embodiment, the composition comprises an isolated or non-naturally occurring fusion protein comprising a truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 2 or SEQ ID NO: 14, preferably to 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14, operably linked to an HBV Pol antigen containing an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 4, or vice versa. Preferably, the fusion protein further comprises a linker that operably links the truncated HBV core antigen to the HBV Pol antigen or vice versa. Preferably, the linker has the amino acid sequence (AlaGly)n, where n is an integer from 2 to 5.

The invention also relates to an immunogenic combination or kit containing polynucleotides expressing truncated HBV nuclear antigen and HBV pol antigen in accordance with embodiments of the invention. Any polynucleotides and/or vectors encoding the HBV core and pol antigens of the invention disclosed herein can be used in the immunogenic combinations or kits of the invention.

According to embodiments of the invention, the vaccine combination or kit contains:

(a) a first unnatural nucleic acid molecule comprising a first polynucleotide encoding an HBV polymerase antigen having an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity, and RNase H activity;

(b) a second unnatural nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14; and (c) a pharmaceutically acceptable carrier, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in the same non-naturally occurring nucleic acid molecule or in two different non-naturally occurring nucleic acid molecules. 20 045279 leic acid.

In accordance with embodiments of the invention, the polynucleotides in a vaccine combination or kit may be linked or separated, and the HBV antigens expressed from such polynucleotides are fused together or produced as separate proteins, whether expressed from the same or different polynucleotides. In one embodiment, the first and second polynucleotides are present in separate vectors, such as DNA plasmids or viral vectors, used in combination, either in the same or in separate compositions, such that the expressed proteins are also separate proteins, but are used in combinations. In another embodiment, the HBV antigens encoded by the first and second polynucleotides can be expressed from the same vector to form a core-pol fusion HBV antigen. Optionally, the core and pol antigens can be connected or fused together by a short linker. Alternatively, the HBV antigens encoded by the first and second polynucleotides can be expressed independently from a single vector using a ribosome slip site (also known as a cis-hydrolase site) located between the sequences encoding the core and pol antigens. This strategy results in a bicistronic expression vector in which separate core and pol antigens are generated from a single mRNA transcript. The core and pol antigens produced by such a bicistronic expression vector may have additional N- or C-terminal residues depending on the ordering of the coding sequences on the mRNA transcript. Examples of ribosome slip sites that can be used for this purpose include, but are not limited to, the FA2 slip site from foot-and-mouth disease virus (FMDV). Another possibility is that the HBV antigens encoded by the first and second polynucleotides could be expressed independently from two separate vectors, one encoding the HBV core antigen and the other the HBV pol antigen.

In a preferred embodiment, the first and second polynucleotides are present in separate vectors, such as DNA plasmids or viral vectors. Preferably, the individual vectors are present in the same composition.

In accordance with preferred embodiments of the invention, the immunogenic combination or kit comprises a first polynucleotide present in a first vector and a second polynucleotide present in a second vector. The first and second vectors may be the same or different. Preferably, the vectors are DNA plasmids.

In a specific embodiment of the invention, the immunogenic combination or kit comprises: a first vector comprising a polynucleotide encoding a truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 2, preferably 100% identical to SEQ ID NO: 2; and a second vector comprising a polynucleotide encoding an HBV polymerase antigen comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 4.

In a specific embodiment of the invention, the first vector is a first DNA plasmid, and the second vector is a second DNA plasmid. Each of the first and second DNA plasmids contains an origin of replication, preferably pUC ORI SEQ ID NO: 10, and an antibiotic resistance cassette, preferably containing a codon optimized Kanr gene having a polynucleotide sequence that is at least 90% identical to the sequence SEQ ID NO: 12, preferably under the control of a Bla promoter, for example the Bla promoter shown in SEQ ID NO: 9. Each of the first and second DNA plasmids independently further comprises at least one of a promoter sequence, an enhancer sequence and a polynucleotide sequence, encoding a signal peptide sequence operably linked to the first polynucleotide sequence or the second polynucleotide sequence. Preferably, each of the first and second DNA plasmids contains an upstream sequence operably linked to the first polynucleotide or the second polynucleotide, the upstream sequence comprising, in a 5' to 3' direction, a promoter sequence of SEQ ID NO: 7 , an enhancer sequence of SEQ ID NO: 8, and a polynucleotide sequence encoding a signal peptide sequence having the amino acid sequence of SEQ ID NO: 6. Each of the first and second DNA plasmids may also contain a polyadenylation signal located downstream of the HBV antigen coding sequence, such as a signal bGH polyadenylation SEQ ID NO: 11.

In one particular embodiment, the first vector is a first viral vector and the second vector is a second viral vector. Preferably, each of the first and second viral vectors is an adenoviral vector, more preferably an Ad26 or Ad35 vector, containing an expression cassette comprising a polynucleotide encoding an HBV pol antigen or a truncated HBV core antigen of the invention; an upstream sequence operably linked to a polynucleotide encoding an HBV antigen, containing, in a 5' to 3' direction, a promoter sequence, preferably

- 21 045279 a CMV promoter sequence of SEQ ID NO: 17, an enhancer sequence, preferably an ApoAI gene fragment sequence of SEQ ID NO: 23, and a polynucleotide sequence encoding a signal peptide sequence, preferably an immunoglobulin secretion signal, having the amino acid sequence of SEQ ID NO: 19 ; and a downstream sequence operably linked to a polynucleotide encoding an HBV antigen containing a polyadenylation signal, preferably the SV40 polyadenylation signal of SEQ ID NO: 24.

In another preferred embodiment, the first and second polynucleotides are present in the same vector, such as a DNA plasmid or viral vector. Preferably, the unit vector is an adenoviral vector, more preferably an Ad26 vector, containing an expression cassette containing a polynucleotide encoding an HBV pol antigen and a truncated HBV core antigen of the invention, preferably encoding an HBV pol antigen and a truncated HBV core antigen of the invention as a fusion protein; an upstream sequence operably linked to the polynucleotide encoding the pol and truncated core antigens of HBV, containing, in a 5' to 3' direction, a promoter sequence, preferably the CMV promoter sequence of SEQ ID NO: 17, an enhancer sequence, preferably an ApoAI gene fragment sequence of SEQ ID NO: 23 and a polynucleotide sequence encoding a signal peptide sequence, preferably an immunoglobulin secretion signal, having the amino acid sequence of SEQ ID NO: 19; and a downstream sequence operably linked to a polynucleotide encoding an HBV antigen containing a polyadenylation signal, preferably the SV40 polyadenylation signal of SEQ ID NO: 24.

When the immunogenic combination of the invention includes a first vector, such as a DNA plasmid or a viral vector, and a second vector, such as a DNA plasmid or a viral vector, the amount of each of the first and second vectors is not particularly limited. For example, the first DNA plasmid and the second DNA plasmid may be present in a ratio of 10:1 to 1:10 by weight, such as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1 , 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1 :10 by weight. Preferably, the first and second DNA of the plasmid are present in a 1:1 ratio by weight.

The compositions and immunogenic combinations of the invention may contain additional polynucleotides or vectors encoding additional HBV antigens and/or additional HBV antigens or immunogenic fragments thereof. However, in specific embodiments, the compositions and immunogenic combinations of the invention do not contain certain antigens.

In a specific embodiment, the composition or immunogenic combination or kit of the invention does not contain HBsAg or a polynucleotide sequence encoding HBsAg.

In another specific embodiment, the composition or immunogenic combination or kit of the invention does not contain the HBV L protein or a polynucleotide sequence encoding the HBV L protein.

In yet another specific embodiment of the invention, the composition or immunogenic combination of the invention does not contain an HBV envelope protein or a polynucleotide sequence encoding an HBV envelope protein.

The compositions and immunogenic combinations of the invention may also contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is non-toxic and should not interfere with the effectiveness of the active ingredient. Pharmaceutically acceptable carriers may include one or more auxiliary agents such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavoring agents, sweeteners, preservatives, coloring agents, solubilizers, and coatings. Pharmaceutically acceptable carriers may include carriers such as lipid (nano)particles. The exact nature of the carrier or other material may depend on the route of administration, for example, intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous, intramuscular (eg, intestinal), intranasal, or intraperitoneal. For liquid injectable preparations, such as suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like. For solid oral preparations, such as powders, capsules, caplets, gel capsules and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrants and the like. For nasal sprays/inhalation mixtures, the aqueous solution/suspension may contain water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, fragrances, flavoring agents, and the like. as suitable carriers and additives.

The compositions and immunogenic combinations of the invention may be formulated with any substance suitable for administration to a subject to facilitate administration and enhance efficacy, including, without limitation, oral (enteral) administration and parenteral injections. Parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection. The compositions of the invention may also be formulated for other routes of administration, including transmucosal, ocular, rectal, chronic implantation, sublingual sublingual, transmucosal without portal circulation, inhalation or intranasal administration. .

In a preferred embodiment, the compositions and immunogenic combinations of the invention are formulated for parenteral injection, preferably subcutaneous injection, intradermal injection or intramuscular injection, more preferably intramuscular injection.

In accordance with embodiments of the invention, the compositions and immunogenic combinations of the invention typically comprise a buffered solution in a pharmaceutically acceptable carrier, eg, an aqueous vehicle such as buffered saline and the like, eg, phosphate buffered saline (PBS). The compositions and immunogenic combinations may also contain pharmaceutically acceptable substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents. For example, a composition or immunogenic combination of the invention comprising plasmid DNA may contain phosphate buffered saline (PBS) as a pharmaceutically acceptable carrier. The plasmid DNA may be present at a concentration of, for example, 0.5 mg/ml to 5 mg/ml, such as 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml , or 5 mg/ml, preferably 1 mg/ml.

The compositions and immunogenic combinations of the invention can be formulated as a vaccine (also referred to as an immunogenic composition) by methods well known in the art. Such compositions may include adjuvants to enhance immune responses. The optimal ratios of each component in the composition can be determined by methods well known to those skilled in the art in light of the present disclosure.

In a specific embodiment of the invention, the composition or immunogenic combination is a DNA vaccine. DNA vaccines typically contain bacterial plasmids containing a polynucleotide encoding the antigen of interest under the control of a strong eukaryotic promoter. Once the plasmids are delivered into the cytoplasm of the host cell, the encoded antigen is produced and processed endogenously. The resulting antigen typically elicits both humoral and cellular immune responses. DNA vaccines are advantageous to say the least because they provide improved safety, are heat stable, are easily adaptable to express antigenic variants, and are easy to manufacture. Any of the DNA plasmids of the invention can be used to prepare such a DNA vaccine.

In other specific embodiments of the invention, the composition or immunogenic combination is an RNA vaccine. RNA vaccines typically contain at least one single-stranded RNA molecule encoding an antigen of interest, such as an HBV antigen. Once the RNA is delivered into the cytoplasm of the host cell, the encoded antigen is produced and processed endogenously, inducing both humoral and cellular immune responses, similar to a DNA vaccine. The RNA sequence can be codon optimized to improve translation efficiency. The RNA molecule can be modified by any method known in the art in light of the present disclosure to improve stability and/or translation, for example, by adding a polyA tail, for example, at least 30 adenosine residues; and/or 5-terminal capping with a modified ribonucleotide, for example a 7-methylguanosine cap, which may be incorporated during RNA synthesis or enzymatically engineered after RNA transcription. The RNA vaccine may also be a self-replicating RNA vaccine developed from an alphavirus expression vector. Self-replicating RNA vaccines contain a replicase RNA molecule derived from a virus belonging to the alpha virus family with a subgenomic promoter that controls the replication of the HBV RNA antigen, followed by an artificial poly-tail located downstream of the replicase.

In some embodiments, an adjuvant is included in the composition or immunogenic combination of the invention, or an adjuvant is administered together with the composition or immunogenic combination of the invention. The use of an adjuvant is optional and may further enhance immune responses when the composition is used for vaccination purposes. Adjuvants suitable for co-administration or inclusion in the compositions of the invention should preferably be potentially safe, well tolerated and effective in humans. The adjuvant may be a small molecule or antibody, including, but not limited to, immune checkpoint inhibitors (e.g., αntu-PD1, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/or TLR8), RIG-1 agonists, IL-15 superagonists (Altor Bioscience), IRF3 and IRF7 mutant genetic adjuvant, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant and IL-7-hyFc. Adjuvants may also be selected, for example, from the following anti-HBV agents: HBV DNA polymerase inhibitors; immunomodulators; toll-like receptor 7 modulators; toll-like receptor modulators 8; toll-like receptor 3 modulators; interferon alpha receptor ligands; hyaluronidase inhibitors; IL-10 modulators; HBsAg inhibitors; toll-like receptor modulators 9; cyclophilin inhibitors; preventative vaccines

- 23 045279 against hepatitis B; HBV therapeutic vaccines; hepatitis B virus entry inhibitors; antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; small interfering RNAs (siRNAs), in particular anti-HBV siRNAs; endonuclease modulators; ribonucleotide reductase inhibitors; hepatitis B virus antigen inhibitors; anti-antibodies targeting hepatitis B virus surface antigens; anti-HBV antibodies; CCR2 chemokine antagonists; thymosin agonists; cytokines such as IL12; capsid assembly modulators, nucleoprotein inhibitors (HBV core or capsid protein inhibitors); nucleic acid polymers (NAP); stimulators of retinoic acid-inducible gene 1; NOD2 stimulants; recombinant thymosin alpha-1; hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors and CTLA-4 inhibitors; costimulatory receptor agonists that are expressed on immune cells (more specifically T cells) such as CD27, CD28, etc.; BTK inhibitors; other medicinal substances for the treatment of HBV; IDO inhibitors; arginase inhibitors; and KDM5 inhibitors.

The invention also relates to methods for preparing the compositions and immunogenic combinations according to the invention. A method of preparing a composition or immunogenic combination involves mixing an isolated polynucleotide encoding an HBV antigen, a vector and/or a polypeptide of the invention with one or more pharmaceutically acceptable carriers. One skilled in the art will be familiar with the traditional methods used to prepare such compositions.

Methods for inducing an immune response.

The invention also provides methods for inducing an immune response to hepatitis B virus (HBV) in a subject in need thereof, comprising administering to the subject an immunogenically effective amount of a composition or immunogenic composition of the invention. Any of the compositions and immunogenic combinations of the invention disclosed herein may be used in the methods of the invention.

As used herein, the term infection refers to the entry of a disease-causing agent into a host. A disease agent is considered infectious when it is capable of entering a host and replicating or multiplying within the host. Examples of infectious agents include viruses, such as HBV and some adenoviruses, prions, bacteria, fungi, protozoa, and the like. HBV infection specifically refers to the entry of HBV into a host, such as host cells and tissues.

The phrase induction of an immune response, when used with reference to the methods disclosed herein, includes initiating a desired immune response or effect in a subject in need thereof to an infection, such as an HBV infection. Inducing an immune response also includes providing therapeutic immunity for treatment directed against a pathogenic agent, such as HBV. As used herein, the term therapeutic immunity or therapeutic immune response means that a vaccinated subject is able to control an infection caused by a pathogenic agent against which vaccination has been given, for example, immunity against HBV infection provided by vaccination with an HBV vaccine. In an embodiment, inducing an immune response means generating immunity in a subject in need thereof, for example, to provide a therapeutic effect against a disease such as HBV infection. In some embodiments, induction of an immune response refers to the establishment or improvement of cellular immunity, such as a T cell response to HBV infection. In some embodiments, induction of an immune response refers to the establishment or improvement of a humoral immune response to HBV infection. In some embodiments, induction of an immune response refers to the initiation or improvement of cellular and humoral immune responses to HBV infection.

As used herein, the term protective immunity or protective immune response means that a vaccinated subject is able to control an infection caused by a pathogenic agent against which he has been vaccinated. Typically, a subject who has developed a protective immune response will develop only mild to moderate clinical symptoms or no symptoms at all. Typically, a subject who has a protective immune response or protective immunity to a particular agent does not die as a result of an infection caused by the specified agent.

In general, the purpose of administering the compositions and immunogenic combinations of the invention is to generate an immune response to HBV following HBV infection or the development of symptoms characteristic of HBV infection, for example, therapeutic vaccination.

As used herein, the term immunogenically effective amount or immunologically effective amount means an amount of a composition, polynucleotide, vector or antigen sufficient to induce the desired immune effect or immune response in a subject in need thereof. An immunogenically effective amount may be an amount sufficient to induce an immune response in a subject in need thereof. An immunogenically effective amount may be an amount sufficient to produce immunity in

- 24 045279 awaiting the subject, for example to provide a therapeutic effect against a disease such as HBV infection. The immunogenically effective amount may vary depending on several factors, such as the subject's physical condition, age, weight, medical condition, etc.; specific use, such as providing protective immunity or therapeutic immunity; and a specific disease, such as a viral infection, against which immunity is desired. An immunogenically effective amount can be readily determined by one skilled in the art in light of the present disclosure.

In specific embodiments, an immunogenically effective amount refers to an amount of a composition or immunogenic combination that is sufficient to achieve one, two, three, four or more of the following effects: (i) reducing or alleviating the severity of HBV infection or associated symptoms; (ii) reducing the duration of HBV infection or associated symptoms; (iii) preventing progression of HBV infection or associated symptoms; (iv) relief of HBV infection or associated symptoms; (v) preventing the development or occurrence of HBV infection or associated symptoms; (vi) preventing recurrence of HBV infection or associated symptoms; (vii) reducing the incidence of subject hospitalization with HBV infection; (viii) reducing the length of hospitalization of a subject infected with HBV; (ix) increasing survival of a subject with HBV infection; (x) eliminating the HBV infection in the subject; (xi) inhibiting or reducing HBV replication in the subject; and/or (xii) enhancing or improving the prophylactic or therapeutic effect(s) of another therapy.

An immunogenically effective amount may also be an amount sufficient to reduce HBsAg levels in accordance with the development of clinical manifestations of seroconversion; achieving sustained HBsAg clearance associated with a decrease in the number of infected hepatocytes provided by the subject's immune system; inducing the formation of HBV antigen-specific activated T cell populations; and/or achieving permanent HBsAg loss over 12 months. Examples of target index include a lower HBsAg value, below the 500 international unit (IU) HBsAg copy threshold, and/or a higher CD8 count.

As a general guide, an immunogenically effective amount when used in the context of a DNA plasmid can be from about 0.1 mg/ml to 10 mg/ml of the total amount of DNA plasmid, for example 0.1 mg/ml, 0.25 mg/ml, 0 .5 mg/ml, 0.75 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml or 10 mg/ml. Preferably, the immunogenically effective amount of DNA plasmid is less than 8 mg/ml, more preferably less than 6 mg/ml, even more preferably 3-4 mg/ml. An immunogenically effective amount can be obtained from a single vector or plasmid or multiple vectors or plasmids. The immunogenically effective amount may be administered in a single composition or in multiple compositions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compositions (e.g., as a tablet, capsule, or injectable, or as any a composition adapted for intradermal delivery (for example, for intradermal delivery using an intradermal delivery patch), wherein administration of multiple Capsules or injections together provides an immunogenically effective amount to the subject. For example, when two DNA plasmids are used, the immunogenically effective amount may be 3- 4 mg/ml, 1.5-2 mg/ml provided by each plasmid. It is also possible to administer an immunogenically effective amount to a subject and then administer another dose of an immunogenically effective amount to the same subject in a so-called priming regimen. This general concept of a stimulation regimen is good known to one skilled in the art of vaccines.If necessary, additional regimens can be added to the booster administrations.

An immunogenic combination containing two DNA plasmids, for example a first DNA plasmid encoding the HBV core antigen and a second DNA plasmid encoding the HBV pol antigen, can be administered to a subject by mixing both plasmids and delivering the mixture to a single anatomical site. Alternatively, two separate immunizations can be performed, each delivering one expression plasmid. In such embodiments, whether both plasmids are administered in one immunization as a mixture or in two separate immunizations, the first DNA plasmid and the second DNA plasmid can be administered in a ratio of 10:1 to 1:10 by weight, for example , 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1 :4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 by weight. Preferably, the first and second DNA of the plasmid are introduced in a ratio of 1:1 by weight.

Preferably, the subject to be treated according to the methods of the invention is a subject infected with HBV, in particular a subject with chronic HBV infection. Acute HBV infection is characterized by effective activation of the innate immune system, which is complemented by a subsequent broad adaptive response (eg, HBV-specific T cell neutralizing antibodies) that usually results in successful suppression of replication or elimination of infected hepatocytes. Otherwise, such responses are impaired or reduced due to high viral and antigenic load, for example, HBV envelope proteins are produced in abundance and can be released in subviral particles in 1000-fold excess to infection

- 25 045279 to this virus.

Chronic HBV infection is described in phases characterized by viral load, liver enzyme levels (necroinflammatory activity), HBeAg or HBsAg load, or the presence of antibodies to these antigens. Levels of cccDNA remain relatively constant at approximately 10 to 50 copies per cell, although the level of viremia can vary significantly. Persistence of cccDNA species leads to the development of a chronic condition. More specifically, the phases of chronic HBV infection include: (i) an immune-tolerant phase, characterized by a high viral load and normal or minimally elevated liver enzyme levels; (ii) an HBeAg-positive immune activation phase in which lower or declining levels of viral replication are observed with significantly elevated liver enzyme levels; (iii) the inactive carrier phase of HBsAg, which is a low-replication state with low viral loads and normal serum liver enzyme levels that may follow HBeAg seroconversion; and (iv) an HBeAg-negative phase, in which viral replication occurs periodically (reactivation) with accompanying fluctuations in liver enzyme levels, mutations in the pre-core and/or basal core promoter are common, such that HBeAg is not produced by the infected cell.

As used herein, the term chronic HBV infection refers to a subject in whom the detectable presence of HBV has been observed for more than 6 months. A subject having chronic HBV infection may be in any phase of chronic HBV infection. Chronic HBV infection should be understood in accordance with its usual meaning in the field. Chronic HBV infection may, for example, be characterized by persistence of HBsAg for 6 months or more after acute HBV infection. For example, chronic HBV infection referred to herein is consistent with the definition published by the Centers for Disease Control and Prevention (CDC), according to which chronic HBV infection can be characterized by laboratory criteria such as: (i) negative IgM antibodies to hepatitis B core antigen (IgM anti-HBc) and a positive hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) or hepatitis B virus DNA nucleic acid test, or (ii) a positive HBsAg or test nucleic acid test for HBV DNA, or positive for HBeAg obtained twice at least 6 months apart.

Preferably, an immunogenically effective amount refers to an amount of a composition or immunogenic combination of the invention that is sufficient to treat chronic HBV infection.

In some embodiments, a subject having chronic HBV infection is treated with a nucleoside analogue (NUC) and is NUC-suppressed. As used herein, the term NUC-suppressive refers to a subject having undetectable HBV levels and stable alanine aminotransferase (ALT) levels for at least six months. Examples of nucleoside/nucleotide analogue treatments include HBV polymerase inhibitors such as entacavir and tenofovir. Preferably, the subject having chronic HBV infection does not have advanced fibrosis or cirrhosis of the liver. Such a subject will typically have a METAVIR score of less than 3 for fibrosis and a fibroscan result of less than 9 kPa. The METAVIR system is a scoring system commonly used to assess the degree of inflammation and fibrosis, based on the histopathological evaluation of liver biopsies from patients with hepatitis B. The scoring system is based on two standardized numbers: one reflects the degree of inflammation and the other reflects the degree of fibrosis.

It is believed that eliminating or reducing chronic HBV will intercept early-onset severe liver disease, including viral cirrhosis and hepatocellular carcinoma. Thus, the methods of application can also be used as a therapy for the treatment of diseases caused by HBV. Examples of diseases caused by HBV include, but are not limited to, cirrhosis of the liver, cancer (eg, hepatocellular carcinoma) and fibrosis, particularly advanced fibrosis, characterized by a METAVIR score of 3 or higher for fibrosis. In such embodiments, an immunogenically effective amount is an amount sufficient to achieve sustained loss of HBsAg over 12 months and significantly reduce the severity of the clinical disease (eg, cirrhosis, hepatocellular carcinoma, etc.).

Methods according to embodiments of the invention further comprise administering to the subject in need thereof another immunogenic agent (such as another HBV antigen or another antigen) or another anti-HBV agent (such as a nucleoside analog or other anti-HBV agent) in combination with a composition of the invention. For example, another anti-HBV agent or immunogenic agent may be a small molecule or antibody, including, but not limited to, immune checkpoint inhibitors (eg, anti-PD1, anti-TIM-3, etc.), toll-like agonists receptors (eg, TLR7 agonists and/or TLR8 agonists), RIG-1 agonists, IL-15 superagonists (Altor Bioscience), IRF3 and IRF7 mutant genetic adjuvant, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant, IL -7-hyFc; CAR-Ts that bind to HBV env (S-CAR cells); capsid assembly modulators; cccDNA inhibitors, HBV polymerase inhibitors (eg

- 26 045279 entecavir and tenofovir). The one or more other active anti-HBV agents may be, for example, a small molecule, an antibody or an antigen-binding fragment thereof, a polypeptide, a protein, or a nucleic acid. One or more other anti-HBV agents may be selected, for example, from inhibitors of HBV DNA polymerase; immunomodulators; toll-like receptor 7 modulators; toll-like receptor modulators 8; toll-like receptor 3 modulators; interferon alpha receptor ligands; hyaluronidase inhibitors; IL-10 modulators; HBsAg inhibitors; toll-like receptor modulators 9; cyclophilin inhibitors; preventive vaccines against hepatitis B; HBV therapeutic vaccines; hepatitis B virus entry inhibitors; antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; small interfering RNAs (siRNAs), in particular anti-HBV siRNAs; endonuclease modulators; ribonucleotide reductase inhibitors; hepatitis B virus antigen inhibitors; anti-antibodies targeting hepatitis B virus surface antigens; anti-HBV antibodies; CCR2 chemokine antagonists; thymosin agonists; cytokines such as IL12; capsid assembly modulators, nucleoprotein inhibitors (HBV core or capsid protein inhibitors); nucleic acid polymers (NAP); stimulators of retinoic acid-inducible gene 1; NOD2 stimulants; recombinant thymosin alpha-1; hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors and CTLA-4 inhibitors; costimulatory receptor agonists that are expressed on immune cells (more specifically T cells) such as CD27, CD28, etc.; BTK inhibitors; other medicinal substances for the treatment of HBV; IIDO inhibitors; arginase inhibitors; and KDM5 inhibitors.

Delivery methods.

The compositions and immunogenic combinations of the invention may be administered to a subject by any method known in the art in light of the present invention, including, without limitation, parenteral administration (e.g., intramuscular, subcutaneous, intravenous, or intradermal injection), oral administration, transdermal administration, and nasal administration. introduction. Preferably, the compositions and immunogenic combinations are administered parenterally (eg, by intramuscular injection or intradermal injection) or transdermally.

In some embodiments, in which the composition or immunogenic combination contains one or more DNA plasmids, administration may be by transdermal injection, such as intramuscular or intradermal injection, preferably intramuscular injection. Intramuscular injection can be combined with electroporation, i.e. application of an electric field to facilitate the delivery of DNA plasmids to cells. As used herein, the term electroporation refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a biomembrane. During in vivo electroporation, electric fields of appropriate magnitude and duration are applied to cells, inducing a transient state of increased cell membrane permeability, allowing the uptake of molecules by the cell that are unable to pass through cell membranes on their own. The creation of such pores by electroporation facilitates the passage of biomolecules such as plasmids, oligonucleotides, siRNAs, drugs, etc. from one side of the cell membrane to the other. In vivo electroporation for the delivery of DNA vaccines has been shown to significantly increase plasmid uptake by host cells and also result in mild to moderate inflammation at the injection site. As a result, transfection efficiency and immune response are significantly improved (e.g., up to 1000-fold and 100-fold, respectively) with intradermal or intramuscular electroporation compared with conventional injection.

In a typical embodiment, electroporation is combined with intramuscular injection. However, electroporation can also be combined with other forms of parenteral administration, such as intradermal injection, subcutaneous injection, etc.

Administration of the composition, immunogenic combination or vaccine of the invention by electroporation can be accomplished using electroporation devices that are configured to apply a pulse of energy to the desired mammalian tissue effective to form reversible pores in cell membranes. The electroporation device may include an electroporation component, an electrode assembly, or a processing unit. The electroporation component may include one or more of the following electroporation device components: a controller, a current signal generator, an impedance tester, a signal recorder, an input element, a status message element, a communication port, a memory component, a power supply, and a power switch. Electroporation can be carried out using an in vivo electroporation device. Examples of electroporation devices and electroporation methods that can facilitate delivery of compositions and immunogenic combinations of the invention, particularly those containing DNA plasmids, include CELLECTRA® (Inovio Pharmaceuticals, Blue Bell, PA), Elgen Electroporator (Inovio Pharmaceuticals, Inc.), System delivery of Tri-Grid™ (Ichor Medical Systems, Inc., San Diego, CA 92121) and are described in US Patent No. 7,646,445, pa

- 27 045279 US Patent No. 8,209,006, US Patent No. 9,452,285, US Patent No. 5,273,525, US Patent No. 6,110,161, US Patent No. 6,261,281, US Patent No. 6,958,060 and US Patent No. 6,939,862, US Patent No. 7,328,064, US Patent No. 6,041,252, US Patent No. 5,873,849, U.S. Patent No. 6,278,895, U.S. Patent No. 6,319,901, U.S. Patent No. 6,912,417, U.S. Patent No. 8,187,249, U.S. Patent No. 9,364,664, U.S. Patent No. 9,802,035, U.S. Patent No. 6,117,660 and International Patent Application Publication WO2017172838, all of which are included in this disclosure is made in its entirety by reference. Other examples of in vivo electroporation devices are described in the international patent application entitled Method and apparatus for delivering vaccines against the hepatitis B virus, filed on the same date as the present application under patent attorney number 688097-405WO1, the contents of which are incorporated herein into in their entirety by reference. The application for delivery of the compositions and immunogenic combinations of the invention also provides for the use of a pulsed electric field, for example, as described, for example, in US Pat. No. 6,697,669, which is incorporated herein by reference in its entirety.

In other embodiments of the invention, in which the composition or immunogenic combination contains one or more DNA plasmids, the route of administration is transdermal. Transdermal administration can be combined with damage to the skin epidermis to facilitate delivery of DNA plasmids to cells. For example, a dermatological patch can be used to damage the skin epidermis. After removal of the dermatological patch, the composition or immunogenic combination can be applied to the damaged skin.

The delivery methods are not limited to the embodiments described above, and any means for intracellular delivery may be used. Other intracellular delivery methods contemplated by the methods of use include, but are not limited to, liposome encapsulation, nanoparticles, etc.

Adjuvants.

In some embodiments, the method of inducing an immune response to HBV further comprises administering an adjuvant. The terms adjuvant and immunostimulant are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance the immune response to HBV antigens and HBV antigenic polypeptides specified in the application.

In accordance with embodiments of the invention, the adjuvant may be present in the immunogenic combination or composition of the invention or administered in a separate composition. The adjuvant may be, for example, a small molecule or an antibody. Examples of adjuvants suitable for use in the invention include, but are not limited to, immune checkpoint inhibitors (e.g., anti-PD1, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists, and/or TLR8), RIG-1 agonists, IL-15 superagonists (Altor Bioscience), IRF3 and IRF7 mutant genetic adjuvant, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant and IL-7-hyFc. Examples of adjuvants may be selected, for example, from the following anti-HBV agents: HBV DNA polymerase inhibitors; immunomodulators; toll-like receptor 7 modulators; toll-like receptor modulators 8; toll-like receptor 3 modulators; interferon alpha receptor ligands; hyaluronidase inhibitors; IL-10 modulators; HBsAg inhibitors; toll-like receptor modulators 9; cyclophilin inhibitors; preventive vaccines against hepatitis B; HBV therapeutic vaccines; hepatitis B virus entry inhibitors; antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; small interfering RNAs (siRNAs), in particular anti-HBV siRNAs; endonuclease modulators; ribonucleotide reductase inhibitors; hepatitis B virus antigen inhibitors; anti-antibodies targeting hepatitis B virus surface antigens; anti-HBV antibodies; CCR2 chemokine antagonists; thymosin agonists; cytokines such as IL12; capsid assembly modulators, nucleoprotein inhibitors (HBV core or capsid protein inhibitors); nucleic acid polymers (NAP); stimulators of retinoic acid-inducible gene 1; NOD2 stimulants; recombinant thymosin alpha-1; hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors and CTLA-4 inhibitors; costimulatory receptor agonists that are expressed on immune cells (more specifically T cells) such as CD27, CD28, etc.; BTK inhibitors; other medicinal substances for the treatment of HBV; IDO inhibitors; arginase inhibitors; and KDM5 inhibitors.

The compositions and immunogenic combinations of the invention can also be administered in combination with at least one other anti-HBV agent. Examples of anti-HBV agents suitable for use in the invention include, but are not limited to, small molecules, antibodies and/or CAR-T therapies that bind HBV env (S-CAR cells), capsid assembly modulators, TLR agonists (e.g. TLR7 and/or TLR8), cccDNA inhibitors, HBV polymerase inhibitors (eg entecavir and tenofovir) and/or immune checkpoint inhibitors, etc. The at least one anti-HBV agent may be selected, for example, from inhibitors of HBV DNA polymerase; immunomodulators; mo

- 28 045279 toll-like receptor 7 duulators; toll-like receptor modulators 8; toll-like receptor 3 modulators; interferon alpha receptor ligands; hyaluronidase inhibitors; IL-10 modulators; HBsAg inhibitors; toll-like receptor modulators 9; cyclophilin inhibitors; preventive vaccines against hepatitis B; HBV therapeutic vaccines; hepatitis B virus entry inhibitors; antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; small interfering RNAs (siRNAs), in particular anti-HBV siRNAs; endonuclease modulators; ribonucleotide reductase inhibitors; hepatitis B virus antigen inhibitors; anti-antibodies targeting hepatitis B virus surface antigens; anti-HBV antibodies; CCR2 chemokine antagonists; thymosin agonists; cytokines such as IL12; capsid assembly modulators, nucleoprotein inhibitors (HBV core or capsid protein inhibitors); nucleic acid polymers (NAP); stimulators of retinoic acid-inducible gene 1; NOD2 stimulants; recombinant thymosin alpha-1; hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors and CTLA-4 inhibitors; costimulatory receptor agonists that are expressed on immune cells (more specifically T cells) such as CD27, CD28, etc.; BTK inhibitors; other medicinal substances for the treatment of HBV; IDO inhibitors; arginase inhibitors; and KDM5 inhibitors. Such anti-HBV agents can be administered together with the compositions and immunogenic combinations of the invention simultaneously or sequentially.

Methods of primary/booster immunization.

Embodiments of the invention also provide for administering an immunogenically effective amount of the composition or immunogenic combination to a subject and then administering another dose of an immunogenically effective amount of the composition or immunogenic combination to the same subject in a so-called stimulation mode. Thus, in one embodiment, the composition or immunogenic combination of the invention is a primer vaccine used to stimulate an immune response. In another embodiment, the composition or immunogenic combination of the invention is a booster vaccine used to enhance the immune response. The primer and booster vaccines of the invention can be used in the methods of the invention disclosed herein. This general concept of stimulation regimen is well known to one skilled in the art of vaccines. Any of the compositions and immunogenic combinations of the invention disclosed herein can be used as priming and/or booster vaccines to prime and/or initiate an immune response to HBV.

In some embodiments, the composition or immunogenic combination of the invention may be administered for primary immunization. The composition or immunogenic combination can be re-administered to enhance immunization. If necessary, the administration regimen may optionally be supplemented with additional booster administrations of the composition or vaccine combination. An adjuvant may be present in a composition of the invention used to enhance immunization, or may be present in a separate composition administered with a composition or immunogenic combination of the invention to enhance immunization, or may be administered alone as a booster immunization. In those embodiments in which an adjuvant is included in the regimen, the adjuvant is preferably used to enhance immunization.

An illustrative and non-limiting example of a prime-boost regimen includes administering to a subject a single dose of an immunogenically effective amount of a composition or immunogenic combination of the invention to stimulate an immune response; and then administering another dose of an immunogenically effective amount of the composition or immunogenic combination of the invention to enhance the immune response, with the first booster immunization being administered approximately two-six weeks, preferably four weeks after the initial primer immunization. Optionally, approximately 10-14 weeks, preferably 12 weeks, after the first primer immunization, an additional booster immunization is performed with a composition or immunogenic combination or other adjuvant.

Sets.

The present invention also relates to a kit containing the immunogenic combination of the invention. The kit may contain a first polynucleotide and a second polynucleotide in separate compositions, or the kit may contain a first polynucleotide and a second polynucleotide in a single composition. The kit may further contain one or more adjuvants or immunostimulants and/or other anti-HBV agents.

The ability to induce or stimulate an immune response to HBV when introduced into an animal or human body can be assessed either in vitro or in vivo using a variety of assays that are standard in the art. For a general description of the methods available to assess the initiation and activation of the immune response, see, for example, Coligan et al. (1992 and 1994, Current Protocols in Immunology; ed. J Wiley & Sons Inc, National Institute of Health). Measurement of cellular immunity can be performed by measuring the profiles of cytokines secreted by activated effec.

- 29 045279 tory cells, including those derived from CD4+ and CD8+ T cells (e.g., quantification of IL-10 or IFN-producing gamma cells using ELISPOT), by determining the activation status of immune effector cells (e.g., T proliferation assay -cells using a classical [ ³ H]thymidine assay or a flow cytometry-based assay), using an antigen-specific T-lymphocyte assay in a sensitized subject (eg, peptide-specific lysis in a cytotoxicity assay, etc.).

The ability to stimulate a cellular and/or humoral response can be determined by antibody binding and/or competition for binding (see, eg, Harlow, 1989, Antibodies, Cold Spring Harbor Press). For example, titers of antibodies produced in response to administration of a composition providing an immunogen can be measured using an enzyme-linked immunosorbent assay (ELISA). Immune responses can also be measured using a neutralizing antibody assay, where virus neutralization is defined as the loss of infectivity resulting from interaction/inhibition/neutralization of the virus by a specific antibody. The immune response can be further measured using an antibody-dependent cellular phagocytosis (ADCP) assay.

Embodiments

Embodiments. Section 1.

Embodiment 1 comprises an isolated or unnatural nucleic acid molecule comprising a polynucleotide sequence encoding a truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 2 or SEQ ID NO: 14, preferably 100% identical to SEQ ID NO: 2 or SEQ ID NO: 14; wherein preferably the truncated HBV nuclear antigen is capable of inducing an immune response in a mammal to at least two HBV genotypes; more preferably, the truncated HBV nuclear antigen is capable of inducing a T cell response in a mammal to at least HBV genotypes B, C and D; even more preferably, the truncated HBV nuclear antigen is capable of inducing a CD8 T cell response in a human subject to at least HBV genotypes A, B, C and D; and most preferably, the truncated HBV nuclear antigen coding sequence comprises a polynucleotide that is at least 90%, for example 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96 %, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5% , 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 15.

Embodiment 2 comprises the non-naturally occurring nucleic acid molecule of Embodiment 1, wherein the truncated HBV nuclear antigen consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14.

Embodiment 3 comprises the non-naturally occurring nucleic acid molecule of Embodiment 1 or Embodiment 2, further comprising a polynucleotide sequence encoding a signal sequence operably linked to an HBV polymerase antigen.

Embodiment 4 comprises the non-naturally occurring nucleic acid molecule of Embodiment 3, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, wherein preferably the signal sequence is encoded by the polynucleotide sequence of SEQ ID NO: 5 or SEQ ID NO : 18.

Embodiment 5 comprises the non-naturally occurring nucleic acid molecule of any one of Embodiments 1-4, wherein the polynucleotide sequence encoding the truncated HBV nuclear antigen is at least 90% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

Embodiment 6 comprises the non-naturally occurring nucleic acid molecule of Embodiment 5, wherein the polynucleotide sequence encoding a truncated HBV nuclear antigen comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

Embodiment 7 comprises a non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding an HBV polymerase antigen comprising an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity.

Embodiment 8 comprises the non-naturally occurring nucleic acid molecule of Embodiment 7, wherein the HBV polymerase antigen is capable of inducing an immune response in a mammal to at least two HBV genotypes, preferably the HBV polymerase antigen is capable of inducing a T cell response in a mammal to at least genotypes B, C and D of HBV, and more preferably, the HBV polymerase antigen is capable of inducing a CD8 T cell response in humans to at least genotypes A, B, C and D of HBV.

Embodiment 9 comprises the non-naturally occurring nucleic acid molecule of Embodiment 7, wherein the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 4.

- 30 045279

Embodiment 10 comprises the non-naturally occurring nucleic acid molecule of any one of embodiments 7-9, further comprising a polynucleotide sequence encoding a signal sequence operably linked to an HBV polymerase antigen.

Embodiment 11 comprises the non-naturally occurring nucleic acid molecule of Embodiment 10, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, wherein the signal sequence is preferably encoded by the polynucleotide sequence of SEQ ID NO: 5 or SEQ ID NO : 18.

Embodiment 12 comprises a non-naturally occurring nucleic acid molecule according to any of embodiments 7-11, wherein the first polynucleotide sequence is at least 90% identical to the sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

Embodiment 13 comprises the non-naturally occurring nucleic acid molecule of Embodiment 12, wherein the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

Embodiment 14 comprises the non-naturally occurring nucleic acid molecule of any one of embodiments 7-13, further comprising a second polynucleotide sequence encoding a truncated HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14.

Embodiment 15 comprises the non-naturally occurring nucleic acid molecule of Embodiment 14, wherein the second polynucleotide sequence is at least 90% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

Embodiment 16 comprises the non-naturally occurring nucleic acid molecule of embodiment 15, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

Embodiment 17 comprises the unnatural nucleic acid molecule of any one of embodiments 14-16 encoding a fusion protein comprising a truncated HBV nuclear antigen operably linked to an HBV polymerase antigen.

Embodiment 18 comprises the non-naturally occurring nucleic acid molecule of Embodiment 17, wherein the fusion protein comprises a truncated HBV nuclear antigen operably linked to an HBV polymerase antigen via a linker.

Embodiment 19 comprises the non-naturally occurring nucleic acid molecule of Embodiment 18, wherein the linker comprises the amino acid sequence (AlaGly) _n and n is an integer from 2 to 5, wherein the linker is preferably encoded by the polynucleotide sequence comprising SEQ ID NO: 22 .

Embodiment 20 comprises the non-naturally occurring nucleic acid molecule of Embodiment 19, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 20.

Embodiment 21 comprises the non-naturally occurring nucleic acid molecule of any one of embodiments 17-20, wherein the fusion protein further comprises a signal sequence, wherein the signal sequence preferably comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, more preferably the signal sequence the sequence is encoded by the polynucleotide sequence SEQ ID NO: 5 or SEQ ID NO: 18.

Embodiment 22 comprises the non-naturally occurring nucleic acid molecule of any of embodiments 7-21, further comprising a promoter sequence, optionally one or more additional regulatory sequences, wherein the promoter sequence preferably comprises the polynucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 17, and the additional regulatory sequence is selected from the group consisting of the enhancer sequences SEQ ID NO: 8 or SEQ ID NO: 23 and the polyadenylation signal sequence SEQ ID NO: 11 or SEQ ID NO: 24.

Embodiment 23 comprises the non-naturally occurring nucleic acid molecule of any one of embodiments 7-22, wherein the non-naturally occurring nucleic acid molecule does not encode an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), an HBV envelope antigen (Env) and HBV L protein antigen.

Embodiment 24 comprises a vector containing a non-naturally occurring nucleic acid molecule according to any of embodiments 1-24.

Embodiment 25 comprises the vector of Embodiment 24, wherein the non-naturally occurring nucleic acid molecule contains, in a 5' to 3' direction, a promoter sequence, an enhancer sequence, a signal peptide coding sequence, a first polynucleotide sequence, and a polyadenylation signal sequence, wherein the optionally non-naturally occurring nucleic acid molecule further comprises a second polynucleotide sequence.

Embodiment 26 contains the vector of Embodiment 24 or 25, which is a DNA plasmid vector, and the DNA plasmid vector further contains an origin of replication and an antibiotic resistance gene.

Embodiment 27 comprises the vector of Embodiment 26, wherein the DNA plasmid vector contains an origin of replication containing the polynucleotide sequence of SEQ ID NO: 10, an antibiotic resistance gene containing the polynucleotide sequence of SEQ ID NO: 12, a promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7, an enhancer sequence containing the polynucleotide sequence of SEQ ID NO: 8, a signal peptide coding sequence containing the polynucleotide sequence of SEQ ID NO: 5, a first polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 3, and a polyadenylation signal sequence, comprising the polynucleotide sequence of SEQ ID NO: 11, and optionally a second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 1.

Embodiment 28 contains the vector of embodiment 24 or 25, which is an adenoviral vector, preferably an Ad26 or Ad35 vector.

Embodiment 29 comprises the vector of Embodiment 28, which is an Ad26 vector containing a non-naturally occurring nucleic acid encoding a truncated HBV nuclear antigen of any of Embodiments 1-6.

Embodiment 30 comprises the vector of embodiment 28, which is an Ad26 vector containing a non-naturally occurring nucleic acid encoding an HBV polymerase antigen of any one of embodiments 7-13.

Embodiment 31 contains the vector of embodiment 28, which is an Ad26 vector containing a non-naturally occurring nucleic acid encoding the fusion protein of any of embodiments 17-21.

Embodiment 31a comprises the vector of any one of embodiments 28-31, wherein the adenoviral vector comprises a promoter sequence comprising the polynucleotide sequence of SEQ ID NO: 17, a regulatory sequence containing the polynucleotide sequence of SEQ ID NO: 23, a signal peptide encoding sequence containing the polynucleotide a sequence SEQ ID NO: 18, a second polynucleotide sequence comprising the polynucleotide sequence SEQ ID NO: 15, a linker encoding sequence comprising the polynucleotide sequence SEQ ID NO: 22, a first polynucleotide sequence comprising the polynucleotide sequence SEQ ID NO: 16, and a signal sequence polyadenylation containing the polynucleotide sequence SEQ ID NO: 24.

Embodiment 32 comprises a non-naturally occurring polypeptide encoded by the non-naturally occurring nucleic acid molecule of any one of embodiments 1-32.

Embodiment 33 comprises a host cell containing a non-naturally occurring nucleic acid molecule of any of embodiments 1-23 or a vector of any of embodiments 24-32.

Embodiment 34 comprises a composition comprising a non-naturally occurring nucleic acid molecule as in any of embodiments 1-23, a vector as in any of embodiments 24-32, or a non-naturally occurring polypeptide as in embodiment 33, and a pharmaceutically acceptable carrier.

Embodiment 35 comprises the composition of Embodiment 34, comprising the first polynucleotide of any of Embodiments 7-13, the second polynucleotide of any of Embodiments 14-16, and a pharmaceutically acceptable carrier, wherein the first and second polynucleotides are not contained in the same molecule nucleic acid or in the same nucleic acid vector.

Embodiment 36 comprises the composition of embodiment 35, wherein the first and second polynucleotides are contained in two separate vectors, preferably adenoviral vectors, more preferably Ad26 vectors.

Embodiment 37 comprises a kit containing:

(a) a first unnatural nucleic acid molecule comprising a first polynucleotide encoding an HBV polymerase antigen having an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity, and RNase H activity;

(b) a second unnatural nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14; and (c) a pharmaceutically acceptable carrier, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in the same non-naturally occurring nucleic acid molecule or in two different non-naturally occurring nucleic acid molecules. 32 045279 leic acid.

Embodiment 38 comprises the kit of embodiment 37, wherein the HBV polymerase antigen is capable of inducing an immune response in a mammal to at least two HBV genotypes, preferably the HBV polymerase antigen is capable of inducing a T cell response in a mammal to at least genotypes B, C, and HBV D, and more preferably, HBV polymerase antigen is capable of inducing a CD8 T cell response in humans to at least genotypes A, B, C and D of HBV.

Embodiment 39 comprises the kit of embodiment 37, wherein the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 4.

Embodiment 40 comprises the kit of any one of embodiments 37-39, wherein at least one of the first unnatural nucleic acid molecule and the second unnatural nucleic acid molecule further comprises a polynucleotide sequence encoding a signal sequence operably linked to at least one of HBV polymerase antigen and HBV truncated nuclear antigen.

Embodiment 41 comprises the kit of embodiment 40, wherein the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, preferably the signal sequence is independently encoded by the polynucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 18.

Embodiment 42 comprises the kit of any one of embodiments 37-41, wherein the first polynucleotide sequence is at least 90% identical to the sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

Embodiment 43 comprises the kit of embodiment 42, wherein the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

Embodiment 44 comprises the kit of any one of embodiments 37-43, wherein the second polynucleotide sequence is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 15.

Embodiment 45 comprises the kit of embodiment 44, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

Embodiment 46 comprises the kit of any one of embodiments 37-45, wherein at least one of the first unnatural nucleic acid molecule and the second unnatural nucleic acid molecule further comprises a promoter sequence, optionally an enhancer sequence, and, further, optionally a polyadenylation signal sequence, wherein the promoter sequence preferably has the polynucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 17, the enhancer sequence independently has the polynucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 23, and the polyadenylation signal sequence independently has the polynucleotide sequence SEQ ID NO: 11 or SEQ ID NO: 24.

Embodiment 47 comprises the kit of any one of embodiments 37-46, which does not contain a nucleic acid molecule encoding an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope antigen (Env), and HBV L protein antigen and an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope antigen (Env), and HBV L protein antigen.

Embodiment 48 comprises the kit as in any one of embodiments 37-47, wherein the first unnatural nucleic acid molecule and the second unnatural nucleic acid molecule are present in the same vector.

Embodiment 49 comprises a set of embodiment 48 in which the vector encodes HBV polymerase antigen and HBV truncated nuclear antigen as two separate proteins.

Embodiment 50 includes the kit of embodiment 49, wherein the vector encodes a fusion protein containing a truncated HBV nuclear antigen operably linked to an HBV polymerase antigen.

Embodiment 51 comprises the kit of Embodiment 50, wherein the fusion protein comprises a truncated HBV nuclear antigen operably linked to an HBV polymerase antigen via a linker.

Embodiment 52 comprises the kit of Embodiment 51, wherein the linker comprises the amino acid sequence (AlaGly) _n and n is an integer from 2 to 5, preferably the linker is encoded by the polynucleotide sequence comprising SEQ ID NO: 22.

Embodiment 53 comprises the kit of Embodiment 52, wherein the fusion protein contains the amino acid sequence of SEQ ID NO: 20.

Embodiment 54 comprises the kit of Embodiment 53, wherein the fusion protein further comprises a signal sequence, preferably the signal sequence having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, more preferably the signal sequence is encoded by a polynucleotide sequence

SEQ ID NO: 5 or SEQ ID NO: 18.

Embodiment 55 comprises the kit of Embodiment 54, wherein the fusion protein contains the amino acid sequence of SEQ ID NO: 21.

Embodiment 56 comprises the kit of any one of embodiments 48-55, wherein the vector contains, in a 5' to 3' direction, a promoter sequence, an enhancer sequence, a signal peptide coding sequence, a second polynucleotide sequence, a sequence a coding linker, a first polynucleotide sequence and a polyadenylation signal sequence.

Embodiment 57 contains the kit of embodiment 56, wherein the vector is an adenoviral vector, preferably an Ad26 or Ad35 vector.

Embodiment 58 comprises the kit of Embodiment 57, wherein the adenoviral vector comprises a promoter sequence comprising the polynucleotide sequence of SEQ ID NO: 17, a regulatory sequence comprising the polynucleotide sequence of SEQ ID NO: 23, a signal peptide encoding sequence comprising the polynucleotide sequence of SEQ ID NO: 18, a second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 15, a linker encoding sequence comprising the polynucleotide sequence of SEQ ID NO: 22, a first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 16, and a polyadenylation signal sequence containing polynucleotide sequence SEQ ID NO: 24.

Embodiment 59 comprises the kit of Embodiment 58, lacking a nucleic acid molecule encoding an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope antigen (Env), and HBV L protein antigen, or HBV antigen, selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope antigen (Env) and HBV L protein antigen.

Embodiment 60 comprises the kit of any one of embodiments 37-59, wherein the first unnatural nucleic acid molecule and the second unnatural nucleic acid molecule are present in two different vectors.

Embodiment 61 comprises the kit of embodiment 60, wherein the first unnatural nucleic acid molecule is present in the first DNA plasmid vector and the second unnatural nucleic acid molecule is present in the second DNA plasmid vector.

Embodiment 62 comprises the kit of Embodiment 61, wherein each of the first and second DNA plasmid vectors comprises an origin of replication, an antibiotic resistance gene, and, in a 5' to 3' direction, a promoter sequence, a regulatory sequence , a sequence encoding a signal peptide, a first polynucleotide sequence or a second polynucleotide sequence, and a polyadenylation signal sequence.

Embodiment 63 comprises the kit of embodiment 62, wherein the antibiotic resistance gene is a kanamycin resistance gene having a polynucleotide sequence at least 90% identical to SEQ ID NO: 12, preferably 100% identical to SEQ ID NO: 12.

Embodiment 64 includes the kit of Embodiment 63 containing:

(a) a first DNA plasmid vector containing, in the direction from the 3' end to the 5' end, a promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7, a regulatory sequence containing the polynucleotide sequence of SEQ ID NO: 8, a sequence, encoding a signal peptide containing the polynucleotide sequence of SEQ ID NO: 5, a first polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 3, and a polyadenylation signal sequence containing the polynucleotide sequence of SEQ ID NO: 11;

(b) a second DNA plasmid vector containing, in the direction from the 3' end to the 5' end, a promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7, a regulatory sequence containing the polynucleotide sequence of SEQ ID NO: 8, a sequence, encoding a signal peptide containing the polynucleotide sequence of SEQ ID NO: 5, a second polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 1, and a polyadenylation signal sequence containing the polynucleotide sequence of SEQ ID NO: 11; and (c) a pharmaceutically acceptable carrier, wherein each of the first DNA plasmid vector and the second DNA plasmid vector further comprises a kanamycin resistance gene having the polynucleotide sequence

- 34 045279

SEQ ID NO: 12, and an origin of replication having the polynucleotide sequence of SEQ ID

NO: 10, and wherein the first DNA plasmid vector and the second DNA plasmid vector are present in the same composition or in two different compositions.

Embodiment 65 comprises the kit of embodiment 60, wherein the first unnatural nucleic acid molecule is present in the first adenoviral vector, preferably the Ad26 vector, and the second unnatural nucleic acid molecule is present in the second adenoviral vector, preferably the Ad26 vector.

Embodiment 66 comprises the kit of embodiment 65, wherein each of the first and second adenoviral vectors comprises, in a 5' to 3' direction, a promoter sequence, a regulatory sequence, a signal peptide coding sequence, a first polynucleotide sequence, or a second polynucleotide sequence and a polyadenylation signal sequence.

Embodiment 67 includes the kit of Embodiment 66 containing:

(b) a first Ad26 vector containing, in the direction from the 3' end to the 5' end, a promoter sequence containing the polynucleotide sequence SEQ ID NO: 17, a regulatory sequence containing the polynucleotide sequence SEQ ID NO: 23, a signal encoding sequence a peptide comprising the polynucleotide sequence of SEQ ID NO: 17, a first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 15, and a polyadenylation signal sequence comprising the polynucleotide sequence of SEQ ID NO: 24;

(b) a second Ad26 vector containing, in the direction from the 3' end to the 5' end, a promoter sequence containing the polynucleotide sequence SEQ ID NO: 17, a regulatory sequence containing the polynucleotide sequence SEQ ID NO: 23, a signal encoding sequence a peptide containing the polynucleotide sequence of SEQ ID NO: 17, a second polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 16, and a polyadenylation signal sequence containing the polynucleotide sequence of SEQ ID NO: 24; and (c) a pharmaceutically acceptable carrier, wherein each of the first Ad26 vector and the second Ad26 vector is present in the same composition or in two different compositions.

Embodiment 68 comprises the kit of any one of embodiments 64-67 that does not contain a nucleic acid molecule encoding an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope antigen (Env), and HBV L protein antigen , and an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope antigen (Env), and HBV L protein antigen.

Embodiment 69 comprises the composition of any of embodiments 34-36 or the kit of any of embodiments 37-68 for use in inducing an immune response to hepatitis B virus (HBV) in a subject in need thereof, preferably the subject suffering from chronic HBV infection.

Embodiment 70 comprises the combination of another immunogenic agent, preferably another HBV antigen, with the composition of any of embodiments 34-36 or the kit of any of embodiments 37-68 for use in inducing in a subject in need thereof an immune response to the hepatitis B virus ( HBV), preferably the subject suffering from chronic HBV infection.

Embodiment 71 comprises the composition of any of embodiments 34-36 or the kit of any of embodiments 37-68 for use in treating hepatitis B virus (HBV)-induced disease in a subject in need thereof, preferably the subject suffering from chronic HBV infection and wherein the hepatitis B virus (HBV)-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).

Embodiment 72 comprises the combination of another immunogenic agent, preferably another HBV antigen, with the composition of any of embodiments 34-36 or the kit of any of embodiments 37-68 for use in treating hepatitis B virus (HBV)-induced disease in a patient in need wherein the subject, wherein preferably the subject suffers from chronic HBV infection, and wherein the hepatitis B virus (HBV)-induced disease is selected from the group consisting of progressive fibrosis, cirrhosis and hepatocellular carcinoma (HCC).

Embodiments. Section 2.

Embodiment 1 comprises a non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding an HBV polymerase antigen comprising an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 4, wherein the HBV polymerase antigen is not has reverse transcriptase activity and RNase H activity.

Embodiment 2 comprises the non-naturally occurring nucleic acid molecule of Embodiment 1, wherein the HBV polymerase antigen is capable of inducing an immune response in a mammal to at least two HBV genotypes, wherein preferably the HBV polymerase antigen is capable of inducing a T cell response in a mammal to at least at least genotypes B, C and D of HBV, and more preferably, the HBV polymerase antigen is capable of inducing a CD8 T cell response in a person to at least genotypes A, B, C and D of HBV.

Embodiment 3 comprises the non-naturally occurring nucleic acid molecule of Embodiment 1, wherein the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 4.

Embodiment 4 comprises the non-naturally occurring nucleic acid molecule of any one of embodiments 1-3, further comprising a polynucleotide sequence encoding a signal sequence operably linked to an HBV polymerase antigen.

Embodiment 5 comprises the non-naturally occurring nucleic acid molecule of Embodiment 4, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, preferably the signal sequence is encoded by the polynucleotide sequence SEQ ID NO: 5 or SEQ ID NO: 18.

Embodiment 6 comprises the non-naturally occurring nucleic acid molecule of any one of Embodiments 1-5, wherein the first polynucleotide sequence is at least 90% identical to the sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

Embodiment 7 comprises the non-naturally occurring nucleic acid molecule of Embodiment 6, wherein the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

Embodiment 8 comprises the non-naturally occurring nucleic acid molecule of any one of embodiments 1-7, further comprising a second polynucleotide sequence encoding a truncated HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14.

Embodiment 9 comprises the non-naturally occurring nucleic acid molecule of Embodiment 8, wherein the second polynucleotide sequence is at least 90% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

Embodiment 10 comprises the non-naturally occurring nucleic acid molecule of Embodiment 9, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

Embodiment 11 comprises the unnatural nucleic acid molecule of any one of embodiments 8-10 encoding a fusion protein comprising a truncated HBV nuclear antigen operably linked to an HBV polymerase antigen.

Embodiment 12 comprises the non-naturally occurring nucleic acid molecule of Embodiment 11, wherein the fusion protein comprises a truncated HBV nuclear antigen operably linked to an HBV polymerase antigen via a linker.

Embodiment 13 comprises the non-naturally occurring nucleic acid molecule of Embodiment 12, wherein the linker comprises the amino acid sequence (AlaGly) _n and n is an integer from 2 to 5, wherein preferably the linker is encoded by a polynucleotide sequence comprising SEQ ID NO: 22 .

Embodiment 14 comprises the non-naturally occurring nucleic acid molecule of Embodiment 13, wherein the fusion protein contains the amino acid sequence of SEQ ID NO: 20.

Embodiment 15 comprises the non-naturally occurring nucleic acid molecule of any one of Embodiments 11-14, wherein the fusion protein further comprises a signal sequence, wherein preferably the signal sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, more preferably the signal sequence the sequence is encoded by the polynucleotide sequence SEQ ID NO: 5 or SEQ ID NO: 18.

Embodiment 16 comprises the non-naturally occurring nucleic acid molecule of any one of embodiments 1-15, further comprising a promoter sequence, optionally one or more additional regulatory sequences, wherein preferably the promoter sequence comprises a polynucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 17, and the additional regulatory sequence is selected from the group consisting of SEQ ID NO: 8 or SEQ ID NO: 23, and the polyadenylation signal sequence SEQ ID NO: 11 or SEQ ID NO: 24.

Embodiment 17 comprises the non-naturally occurring nucleic acid molecule of any one of embodiments 1-16, wherein the non-naturally occurring nucleic acid molecule

- 36 045279 acid does not encode an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope antigen (Env) and HBV L protein antigen.

Embodiment 18 comprises a vector containing a non-naturally occurring nucleic acid molecule according to any of embodiments 1-17.

Embodiment 19 comprises the vector of Embodiment 18, wherein the non-naturally occurring nucleic acid molecule comprises, in a 5' to 3' direction, a promoter sequence, an enhancer sequence, a signal peptide coding sequence, a first polynucleotide sequence, and a polyadenylation signal sequence, wherein the optionally non-naturally occurring nucleic acid molecule further comprises a second polynucleotide sequence.

Embodiment 20 comprises the vector of embodiment 18 or 19, wherein the vector is a DNA plasmid vector, the DNA plasmid vector further comprising an origin of replication and an antibiotic resistance gene.

Embodiment 21 comprises the vector of Embodiment 20, wherein the DNA plasmid vector contains an origin of replication containing the polynucleotide sequence of SEQ ID NO: 10, an antibiotic resistance gene containing the polynucleotide sequence of SEQ ID NO: 12, a promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7, an enhancer sequence containing the polynucleotide sequence of SEQ ID NO: 8, a signal peptide coding sequence containing the polynucleotide sequence of SEQ ID NO: 5, a first polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 3, and a polyadenylation signal sequence, comprising the polynucleotide sequence of SEQ ID NO: 11, and optionally a second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 1.

Embodiment 22 contains the vector of embodiment 18 or 19, where the vector is an adenoviral vector, preferably an Ad26 or Ad35 vector.

Embodiment 23 comprises the vector of Embodiment 22, wherein the adenoviral vector comprises a promoter sequence comprising the polynucleotide sequence of SEQ ID NO: 17, a regulatory sequence comprising the polynucleotide sequence of SEQ ID NO: 23, a signal peptide encoding sequence comprising the polynucleotide sequence of SEQ ID NO: : 18, a second polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 15, a linker encoding sequence containing the polynucleotide sequence of SEQ ID NO: 22, a first polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 16, and a polyadenylation signal sequence containing the polynucleotide sequence SEQ ID NO: 24.

Embodiment 24 comprises a non-naturally occurring polypeptide encoded by the non-naturally occurring nucleic acid molecule of any one of embodiments 1-17.

Embodiment 25 comprises a host cell containing a non-naturally occurring nucleic acid molecule of any of embodiments 1-17 or a vector of any of embodiments 18-23.

Embodiment 26 comprises a composition comprising a non-naturally occurring nucleic acid molecule as in any of embodiments 1-17, a vector as in any of embodiments 18-23, or a non-naturally occurring polypeptide as in embodiment 24, and a pharmaceutically acceptable carrier.

Embodiment 27 comprises the composition of Embodiment 26, comprising the first polynucleotide of any of Embodiments 1-7, the second polynucleotide of any of Embodiments 8-10, and a pharmaceutically acceptable carrier, wherein the first and second polynucleotides are not contained in the same molecule nucleic acid or in the same nucleic acid vector.

Embodiment 28 contains a kit containing:

(a) a first unnatural nucleic acid molecule comprising a first polynucleotide encoding an HBV polymerase antigen having an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity, and RNase H activity;

(b) a second unnatural nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14; and (c) a pharmaceutically acceptable carrier, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in the same non-naturally occurring nucleic acid molecule or in two different non-naturally occurring nucleic acid molecules. 37 045279 leic acid.

Embodiment 29 comprises the kit of Embodiment 28, wherein the HBV polymerase antigen is capable of inducing an immune response in a mammal to at least two HBV genotypes, wherein preferably the HBV polymerase antigen is capable of inducing a T cell response in a mammal to at least genotypes B, C and D HBV, and more preferably, HBV polymerase antigen is capable of inducing a CD8 T cell response in humans to at least genotypes A, B, C and D of HBV.

Embodiment 30 comprises the kit of embodiment 29, wherein the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 4.

Embodiment 31 comprises the kit of any one of embodiments 28-30, wherein at least one of the first unnatural nucleic acid molecule and the second unnatural nucleic acid molecule further comprises a polynucleotide sequence encoding a signal sequence operably linked to at least one of HBV polymerase antigen and HBV truncated nuclear antigen.

Embodiment 32 comprises the kit of embodiment 31, wherein the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, wherein preferably the signal sequence is independently encoded by the polynucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 18.

Embodiment 33 comprises the kit of any one of embodiments 28-32, wherein the first polynucleotide sequence is at least 90% identical to the sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

Embodiment 34 comprises the kit of embodiment 33, wherein the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 16.

Embodiment 35 comprises the kit of any one of embodiments 28-34, wherein the second polynucleotide sequence is at least 90% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

Embodiment 36 comprises the kit of embodiment 35, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

Embodiment 37 comprises the kit as in any one of embodiments 28-36, wherein at least one of the first unnatural nucleic acid molecule and the second unnatural nucleic acid molecule further comprises a promoter sequence, optionally an enhancer sequence, and, further, optionally a polyadenylation signal sequence, preferably the promoter sequence having the polynucleotide sequence SEQ ID NO: 7 or SEQ ID NO: 17, the enhancer sequence independently having the polynucleotide sequence SEQ ID NO: 8 or SEQ ID NO: 23 and the polyadenylation signal sequence independently having the polynucleotide sequence SEQ ID NO: 11 or SEQ ID NO: 24.

Embodiment 38 comprises the kit of any one of embodiments 28-37, wherein the kit does not contain a nucleic acid molecule encoding an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope antigen (Env), and protein antigen HBV L, and an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope antigen (Env), and HBV L protein antigen.

Embodiment 39 comprises the kit as in any one of embodiments 28-38, wherein the first unnatural nucleic acid molecule and the second unnatural nucleic acid molecule are present in the same vector.

Embodiment 40 comprises the kit of embodiment 39, wherein the vector encodes the HBV polymerase antigen and the truncated HBV nuclear antigen as two separate proteins.

Embodiment 41 comprises the kit of embodiment 39, wherein the vector encodes a fusion protein containing a truncated HBV nuclear antigen operably linked to an HBV polymerase antigen.

Embodiment 42 comprises the kit of Embodiment 41, wherein the fusion protein comprises a truncated HBV nuclear antigen operably linked to an HBV polymerase antigen via a linker.

Embodiment 43 comprises the kit of Embodiment 42, wherein the linker comprises the amino acid sequence (AlaGly) _n and n is an integer from 2 to 5, wherein preferably the linker is encoded by the polynucleotide sequence comprising SEQ ID NO: 22.

Embodiment 44 contains the kit of embodiment 43, wherein the fusion protein contains the amino acid sequence of SEQ ID NO: 20.

Embodiment 45 comprises the kit of Embodiment 44, wherein the fusion protein

- 38 045279 further comprises a signal sequence, wherein preferably the signal sequence has the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19, more preferably the signal sequence is encoded by the polynucleotide sequence SEQ ID NO: 5 or SEQ ID NO: 18.

Embodiment 46 comprises the kit of embodiment 45, wherein the fusion protein contains the amino acid sequence of SEQ ID NO: 21.

Embodiment 47 comprises the kit of any one of embodiments 41-46, wherein the vector contains, in a 5' to 3' direction, a promoter sequence, an enhancer sequence, a signal peptide coding sequence, a second polynucleotide sequence, a sequence a coding linker, a first polynucleotide sequence and a polyadenylation signal sequence.

Embodiment 48 contains the kit of embodiment 47, wherein the vector is an adenoviral vector, preferably an Ad26 or Ad35 vector.

Embodiment 49 comprises the kit of Embodiment 48, wherein the adenoviral vector comprises a promoter sequence comprising the polynucleotide sequence of SEQ ID NO: 17, a regulatory sequence comprising the polynucleotide sequence of SEQ ID NO: 23, a signal peptide encoding sequence comprising the polynucleotide sequence of SEQ ID NO: 18, a second polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 15, a linker encoding sequence comprising the polynucleotide sequence of SEQ ID NO: 22, a first polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 16, and a polyadenylation signal sequence containing polynucleotide sequence SEQ ID NO: 24.

Embodiment 50 comprises the kit of Embodiment 49 which does not contain a nucleic acid molecule encoding an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen, and HBV L protein antigen, or HBV antigen , selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope antigen (Env), and HBV L protein antigen.

Embodiment 51 comprises the kit of any one of embodiments 28-38, wherein the first unnatural nucleic acid molecule and the second unnatural nucleic acid molecule are present in two different vectors.

Embodiment 52 comprises the kit of Embodiment 51, wherein the first unnatural nucleic acid molecule is present in the first DNA plasmid vector, and the second unnatural nucleic acid molecule is present in the second DNA plasmid vector.

Embodiment 53 comprises the kit of Embodiment 52, wherein each of the first and second DNA plasmid vectors comprises an origin of replication, an antibiotic resistance gene, and, in a 5' to 3' direction, a promoter sequence, a regulatory sequence , a sequence encoding a signal peptide, a first polynucleotide sequence or a second polynucleotide sequence, and a polyadenylation signal sequence.

Embodiment 54 comprises the kit of embodiment 53, wherein the antibiotic resistance gene is a kanamycin resistance gene having a polynucleotide sequence at least 90% identical to SEQ ID NO: 12, preferably 100% identical to SEQ ID NO: 12.

Embodiment 55 includes the kit of Embodiment 54 containing:

(a) a first DNA plasmid vector containing, in the direction from the 3' end to the 5' end, a promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7, a regulatory sequence containing the polynucleotide sequence of SEQ ID NO: 8, a sequence, encoding a signal peptide containing the polynucleotide sequence of SEQ ID NO: 5, a first polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 3, and a polyadenylation signal sequence containing the polynucleotide sequence of SEQ ID NO: 11;

(b) a second DNA plasmid vector containing, in the direction from the 3' end to the 5' end, a promoter sequence containing the polynucleotide sequence of SEQ ID NO: 7, a regulatory sequence containing the polynucleotide sequence of SEQ ID NO: 8, a sequence, encoding a signal peptide containing the polynucleotide sequence of SEQ ID NO: 5, a second polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 1, and a polyadenylation signal sequence containing the polynucleotide sequence of SEQ ID NO: 11; and (c) a pharmaceutically acceptable carrier, each of the first DNA plasmid vector and the second DNA plasmid vector further comprising a kanamycin resistance gene having the polynucleotide sequence

SEQ ID NO: 12, and an origin of replication having the polynucleotide sequence of SEQ ID

NO: 10, and wherein the first DNA plasmid vector and the second DNA plasmid vector are present in the same composition or in two different compositions.

Embodiment 56 comprises the kit of embodiment 55 which does not contain a nucleic acid molecule encoding an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope (Env) antigen and HBV L protein antigen, or HBV antigen , selected from the group consisting of hepatitis B surface antigen (HBsAg), HBV envelope antigen (Env), and HBV L protein antigen.

Embodiment 57 comprises the composition of Embodiment 26 or the kit of any of Embodiments 27-55 for use in inducing an immune response to hepatitis B virus (HBV) in a subject in need thereof, preferably the subject suffering from chronic HBV infection.

Embodiment 58 comprises a combination of another immunogenic agent, preferably another HBV antigen, with the composition of embodiment 26 or the kit of any of embodiments 27-55 for use in inducing an immune response to hepatitis B virus (HBV) in a subject in need thereof, wherein preferably the subject suffers from chronic HBV infection.

Embodiment 59 comprises the composition of Embodiment 26 or 27 or the kit of any of Embodiments 28-56 for use in treating hepatitis B virus (HBV)-induced disease in a subject in need thereof, preferably the subject suffering from chronic HBV infection, and wherein the hepatitis B virus (HBV) induced disease is selected from the group consisting of progressive fibrosis, cirrhosis and hepatocellular carcinoma (HCC).

Embodiment 60 comprises the combination of another immunogenic agent, preferably another HBV antigen, with the composition of Embodiment 26 or 27 or the kit of any of Embodiments 28-56 for use in treating hepatitis B virus (HBV)-induced disease in a patient in need thereof. the subject, wherein preferably the subject suffers from chronic HBV infection, and wherein the hepatitis B virus (HBV)-induced disease is selected from the group consisting of progressive fibrosis, cirrhosis and hepatocellular carcinoma (HCC).

Examples

The following examples of the invention are intended to further illustrate the nature of the invention. It should be understood that the following examples are not intended to limit the invention or its scope as defined by the appended claims.

Example 1: Generation of HBV Core and Pol antigen sequences and plasmid optimization.

It is proposed that the T cell epitope on the hepatitis B virus Core protein is important for clearance of hepatitis B infection, and that hepatitis B virus proteins such as polymerase may serve to increase the breadth of the response. Thus, the nuclear and polymerase proteins of the hepatitis B virus were selected for the development of a therapeutic vaccine against hepatitis B virus (HBV).

Obtaining consensus sequences of HBV nuclear and polymerase antigens.

Consensus sequences of HBV pol and core antigens were obtained based on HBV genotypes B, C, and D. Different sequences were obtained from different HBV sources and aligned separately for the nuclear protein and the polymerase protein. Initial sequence alignments for all subtypes (A to H) were subsequently restricted to HBV genotypes B, C, and D. Consensus sequences were determined for each protein alignment in each subtype separately and collectively for all BCD sequences. At varying alignment positions in the consensus sequence, the most frequently occurring amino acid was used.

Optimization of HBV nuclear antigen.

The consensus sequence of the HBV nuclear antigen was optimized by deletion of the native viral protein. In particular, a deletion of thirty-four amino acids was introduced, corresponding to the C-terminal segment with a high positive charge necessary for pre-genomic encapsulation of RNA.

Optimization of HBV antigen Pol.

The HBV antigen pol consensus sequence was optimized by replacing four residues to remove reverse transcriptase enzymatic activity and RNase H activity. Specifically, in the YXDD motif of the reverse transcriptase domain, aspartate residues (D) were replaced with aspartic residues (N) to completely eliminate the coordination function and thus , nucleotide/metal ion binding. In addition, in the DEDD motif of the RNase H domain, the first aspartate residue (D) was replaced by an asparagine residue (N), and the first glutamate residue (E) was replaced by a glutamine residue (A) to eliminate Mg ²⁺ coordination bonds. In addition, the HBV pol antigen sequence was codon optimized to scramble the internal open reading frames of the envelope proteins,

- 40 045279 including protein S and protein S variants with N-terminal extensions pre-S1 and pre-S2. As a result, the open reading frames for the envelope proteins (pre-S1, pre-S2 and S protein) and the X protein were removed. Optimization of strategies for the expression of HBV core and pol antigens To obtain maximum and equal levels of expression of both core and pol antigens from plasmid vectors, three different strategies were tested: (1) fusion of the core and pol antigens of HBV in frame through a short sequence AGAG inserted between the coding sequences to produce a single Core-Pol fusion protein (Fig. 2A); (2) expression of core and pol antigens from a single plasmid using a ribosomal slip site, specifically the foot-and-mouth disease FA2 slip site (FMDV), to form a bicistronic expression vector expressing separate core and pol proteins from a single mRNA (Fig. 2B); and (3) two separate plasmids encoding HBV core and pol antigens, respectively (Fig. 2C).

In vitro expression analysis.

The coding sequences of the consensus HBV core and pol antigens according to each of the above three expression strategies were cloned into the commercially available expression plasmid pcDNA3.1. HEK-293T cells were transfected with the vectors, and protein expression was assessed by Western blot analysis using HBV core-specific antigen.

Optimization of post-transcriptional regulatory elements.

Four different post-transcriptional regulatory elements were assessed for enhancing protein expression by stabilizing the primary transcript, facilitating its nuclear export, and/or improving transcription-translational coupling: (1) Woodchuck HBV post-transcriptional regulatory element (WPRE) (GenBank: J04514.1 ); (2) an intron/exon sequence derived from the human apolipoprotein A1 precursor (GenBank: X01038.1); (3) human leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) R-U5 untranslated domain (GenBank: KM023768.1); and (4) a composite sequence consisting of the HTLV-1 LTR, a synthetic rabbit β-globin intron (GenBank: V00882.1), and a splicing enhancer (triple composite sequence). Enhancer sequences were introduced into plasmids between the CMV promoter and the HBV antigen coding sequences. The results of Western blot analysis showed no significant difference in core antigen expression levels when expressed from a plasmid in the presence and absence of the WPRE element (Fig. 2D). However, when assessed by Western blot analysis of core antigen expression in HEK293T cells transfected with plasmids having the other three posttranscriptional regulatory sequences, it was found that the triple enhancer sequence resulted in the strongest core antigen expression (Fig. 2E).

Selection of signal peptide for efficient protein secretion.

Three different signal peptides framed at the N-terminus of the HBV core antigen were evaluated: (1) Ig heavy chain signal peptide SPIgG (BAA75024.1); (2) IgI epsilon heavy chain signal peptide SPIgE (AAB59424.1); and (3) cystatin S precursor signal peptide SPCS (NP_0018901.1). Signal peptide cleavage sites were optimized in silico for core fusion using the Signal P prediction program. Secretion efficiency was determined by analyzing core protein levels in the supernatant. Western blot analysis of core antigen secretion using three different signal peptides fused at the N terminus demonstrated that the cystatin S signal peptide resulted in the most efficient protein secretion (Fig. 2F).

Optimization of DNA vaccine vector.

Optimized expression cassettes containing a triple composite enhancer sequence upstream of the HBV antigen coding sequence with an N-terminal cystatin S signal peptide sequence were cloned into the DNA vaccine vector pVax-1 (Life Technologies, Thermo Fisher Scientific, Waltham, MA). The expression cassette in pVax-1 contains the human CMV-IE promoter followed by a polyadenylation (pA) sequence derived from bovine growth hormone (BGH). Bacterial reproduction is driven by the pUC ori replicon and the kanamycin resistance gene (Kan R) driven by a small eukaryotic promoter. The replication of pUC ori, the KanR expression cassette and the expression cassette driven by the CMVIE promoter are in the same orientation within the plasmid backbone. However, there was a marked reduction in core antigen expression in the pVax-1 vector compared to the expression level observed in the pcDNA3.1 vector.

Several strategies have been used to improve protein expression: (1) changing the orientation of the entire pUCori-KanR cassette in a counterclockwise direction (pVD core); and (2) changing the codon usage of the KanR gene together with replacing the Kan promoter with the Amp promoter from the pcDNA3.1 vector (pDKcore). Both strategies restored core antigen expression, but core antigen expression was highest in the pDK vector, which contained the codon-matched Kan R gene, the AmpR promoter (instead of the KanR promoter), and the pUCori-KanR cassette in reverse orientation.

Shown in FIG. 2G, four different optimized HBV core/pol antigen expression cassettes were introduced into the pDK plasmid backbone to test each of the three expression strategies shown in FIG. 2A-2C. Plasmids were tested in vitro for the expression of core and pol antigens by Western blot analysis using antibodies to core and pol. Most Compatible Expression Profile

- 41 045279 cellular and secreted core and pol antigens were obtained when the core and pol antigens were encoded as separate vectors, namely the separate DNA vectors pDK-core and pDK-pol (Fig. 2H). A schematic representation of the pDK-pol and pDK-core vectors is given in FIG. 3A and 3B, respectively.

Example 2. Creation of adenoviral vectors expressing a shortened HBV core antigen fused with the HBV Pol antigen.

The adenoviral vector was constructed as a fusion protein expressed from a single open reading frame. Additional configurations for expressing two proteins have also been considered, for example, using two separate expression cassettes or using a 2A-like sequence to separate the two sequences.

Construction of expression cassettes for adenoviral vectors.

The expression cassettes (see FIGS. 8A and 8B) consist of the CMV promoter (SEQ ID NO: 17), intron (SEQ ID NO: 23) (a fragment derived from the human ApoAI gene - pair 295-523 under GenBank accession number X01038 , carrying the second intron of ApoAI), followed by an optimized coding sequence - either a single core or core protein fused to a polymerase, preceded by a coding sequence for the human immunoglobulin secretion signal (SEQ ID NO: 18), followed by the SV40 polyadenylation signal (SEQ ID NO: 24).

The secretion signal was included because, based on past experience, it has improved the manufacturability of some adenoviral vectors containing secreted transgenes without affecting the elicited T cell response (mice experiments).

The last two residues of the core protein (VV) and the first two residues of the polymerase protein (MP), when fused, yield a junction sequence (VVMP) that is present in the human dopamine receptor protein (isoform D3), along with flanking homologs.

Introducing an AGAG linker between the core and polymerase sequences eliminates this homology, and Blast does not show any new hits in the human proteome.

Example 3: In vivo immunogenicity study of a DNA vaccine in mice.

An immunotherapeutic DNA vaccine containing DNA plasmids encoding HBV nuclear antigen or HBV polymerase antigen was tested in mice. The aim of the study was to detect vaccine-induced T cell responses following intramuscular delivery to BALB/c mice via electroporation. Initial immunogenicity studies were aimed at determining the cellular immune responses that might be induced by administered HBV antigens.

Specifically, the plasmids tested included plasmid pDK-Pol and plasmid pDK-Core, as shown in FIG. 3A and 3B, respectively, and as described above in Example 1. Plasmid pDK-Pol encoded a polymerase antigen having the amino acid sequence of SEQ ID NO: 4, and plasmid pDK-Core encoded a Core antigen having the amino acid sequence of SEQ ID NO: 2. First tested T cell responses induced by each plasmid separately. The DNA plasmid (pDNA) vaccine was delivered intramuscularly to Balb/c mice via electroporation using a commercially available TriGrid intramuscular delivery system (TDS-IM) adapted for use in the cranialis tibialis mouse model. Additional description of methods and apparatus for intramuscular delivery of DNA to mice by electroporation can be found in International Patent Application Publication WO2017172838 and US Patent Application entitled Method and Apparatus for Delivery of Hepatitis B Virus Vaccines, filed on the same date as the present application. with patent attorney authorization number 688097405U1, the disclosures of which are incorporated herein by reference in their entirety. Specifically, the TDS-IM array of the TDS-IM v1.0 device, having an electrode array with an interelectrode distance of 2.5 mm and an electrode diameter of 0.030 inch, was inserted percutaneously into the selected muscle with a guidewire length of 3.2 mm and an effective penetration depth of 3. 2 mm, and the main axis of the diamond-shaped electrode configuration was oriented parallel to the muscle fibers. After insertion of the electrode, an injection was started to introduce DNA (eg, 0.020 ml) into the muscle. After completion of the IM injection, an electric field of 250 V/cm was applied locally (applied voltage 59.4–65.6 V with applied current limit less than 4 A, 0.16 A/s) for a total duration of approximately 400 ms at 10% duty cycle ( i.e. the active voltage effect is a total of approximately 40 ms out of a total duration of approximately 400 ms) with 6 total pulses. After completion of the electroporation procedure, the TriGrid matrix was removed and the animals were allowed time to recover. High doses (20 μg) were administered to BALB/c mice as shown in Table. 1. Six mice were injected with plasmid DNA encoding HBV core antigen (pDK-core; group 1), six mice were injected with plasmid DNA encoding HBV pol antigen (pDK-pol; group 2), and two mice received an empty vector as a negative control. . Animals received two DNA immunizations two weeks apart, and splenocytes were collected one week after the last immunization.

- 42 045279

Table 1

Scheme of an experimental study on immunization of mice

Group N pDNA Section of one side, introduction (alternating sides) Dose Volume Days of input End point (splenocyte collection) day 1 6 Core ST+ER 20 mcg 20 µl 0.14 21 2 6 Pol ST+ER 20 mcg 20 µl 0.14 21 3 2 Empty vector (negative control) ST+ER 20 mcg 20 µl 0.14 21

CT, cranialis tibialis muscle; EP, electroporation.

Antigen-specific responses were analyzed and quantified by IFN-γ enzyme-linked immunosorbent spot assay (ELISPOT). In this assay, isolated splenocytes from immunized animals were incubated overnight with peptide pools covering the Core protein, Pol protein, or small peptide leader sequence and compound (2 μg/ml of each peptide). These pools consisted of 15-mer peptides that overlap 11 residues corresponding to the BCD consensus sequence of the Core and Pol vaccine vector genotypes. The large 94 kDa HBV Pol protein was split down the middle into two peptide pools. Antigen-specific T cells were stimulated with homologous peptide pools, and IFN-γ-positive T cells were assessed by ELISPOT assay. The release of IFN-γ by a single antigen-specific T cell was visualized using appropriate antibodies, followed by detection using a chromogenic substrate as a stained spot on a microplate called a spot-forming cell (SFC).

Significant T-cell responses to HBV Core were obtained in mice immunized with the pDK-Core DNA plasmid vaccine (Group 1) yielding 1000 SFC per 10 cells (Fig. 4). Pol T-cell responses to the Pol 1 peptide pool were strong (~1000 SFC per 10 ⁶ cells). The weak anti-Pol cellular responses targeting Pol 2 were likely caused by limited MHC diversity in mice, a phenomenon called T cell immunodominance, defined as unequal recognition of different epitopes from the same antigen. To confirm the results obtained in this study, a confirmatory study was performed (data not shown).

The above results demonstrate that vaccination with a DNA plasmid vaccine encoding HBV antigens induces cellular immune responses to the administered HBV antigens.

Example 4: Dose determination study of combined pDK-Core/pDK-Pol plasmids for mice.

The purpose of this combination plasmid dosing study was to evaluate immune responses in mice to a mixture of plasmid DNA (pDNA) vectors encoding HBV core and pol antigens administered at the same site using different doses of DNA. In this study, an immunotherapeutic DNA vaccine containing a 1:1 (w/v) mixture of two plasmids, plasmids pDK-pol and pDK-core, described in Example 1, was tested in mice. The DNA vaccine was delivered to Balb/c mice at one anatomical site intramuscularly via electroporation as described above in Example 3. Vaccination of combined plasmids expressing Core and Pol at doses of 10 μg, 1 μg and 0.1 μg of each plasmid DNA per site was performed , as shown in table. 2. Eight mice were tested in each group, and two mice were injected with the empty vector as a negative control. Animals received two DNA immunizations three weeks apart, and splenocytes were collected one week after the last immunization.

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table 2

Experimental scheme for immunization of mice in an experiment to determine the dose of combined plasmids

Group N pDNA Section of one side, introduction (alternating sides) Dose of each pDNA per site Dose of total pDNA per site Days of input End point (splenocyte collection) day 1 8 Core and Pol ST+ER 10 mcg 20 mcg 0.21 28 2 8 Core and Pol ST+ER 1 mcg 2 mcg 0.21 28 3 8 Core and Pol ST+ER 0.1 mcg 0.2 mcg 0.21 28 4 2 Empty vector (negative control) ST+ER 20 mcg 20 mcg 0.21 28

pDNA, plasmid DNA; CT, cranialis tibialis muscle; EP, electroporation.

Antigen-specific responses were analyzed and quantified using an IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay as described in Example 1. Significant T cell responses to Core and Pol were obtained in BALB/c mice immunized with the combination DNA vaccine, consisting of plasmids pDK-Core and pDK-Pol (Fig. 5). There were no statistical differences in the magnitude of immune responses between group 1, immunized with 10 μg of each plasmid, and group 2, immunized with only 1 μg of each plasmid. This result suggests that T cell responses reached maximal levels with approximately 1 μg of plasmids expressing Core and Pol antigens. However, with a 10-fold reduction in DNA exposure, i.e. at 0.1 μg of each plasmid, a significant decrease in the amount of SFC was observed. Pol T cell responses to the Pol 1 peptide pool were dominant. The weak anti-Pol cellular responses targeting Pol 2 were likely due to limited MHC diversity in inbred mice, a phenomenon called T cell immunodominance, defined as unequal recognition of different epitopes from the same antigen.

The above results showed that mice immunized with a combination of DNA plasmids expressing HBV Core and Pol antigens exhibited significant T cell responses at doses of 1 μg of each plasmid, while some level of immune response was still observed at a dose of 0.1 μg of each plasmid .

Example 5. Study of immune interference in mice.

For practical reasons, it would be desirable to develop a combination DNA vaccine containing the HBV Core and Pol antigens in the form of a combined (mixed) vector formulation. However, in multivalent vaccines, immunodominance may occur and immune responses to subdominant antigens may be suppressed. Therefore, immune interference was assessed, i.e. decreased Core- and/or Pol-specific cellular responses when administered with a combination of two antigen-expressing plasmids mixed together, compared with immunization of either vector at different anatomical sites.

Balb/c mice were vaccinated with DNA plasmids pDK-core and/or pDK-pol intramuscularly via electroporation as described in Example 3. DNA plasmids (pDNA) were administered at a dose of 5 μg per site, either individually or combined (mixed) in one area or combining into different areas, as shown in table. 3. Animals received two DNA immunizations three weeks apart, and splenocytes were collected one week after the last immunization.

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Table 3

Mice immunization scheme for experimental study of immune interference

Group N pDNA Section of one side, introduction (alternating sides) Dose of each pDNA per site Dose of total pDNA per site Days of input Ultimate dot (collection of splenocytes) day 1 6 Core Double-sided ST 5 mcg 10 mcg 0.21 28 2 6 Pol Double-sided ST 5 mcg 10 mcg 0.21 28 3 6 Blend Soge and Pol Double-sided ST 10 mcg 20 mcg 0.21 28 4 6 Core and Pol on a separate tee Core to left ST Pol to right ST 10 mcg 20 mcg 0.21 28 5 2 Empty vector (negative control) Bilateral ST 10 mcg 20 mcg 0.21 28

CT, cranial tibialis muscle.

Antigen-specific responses were analyzed and quantified using an IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay as described in Example 1. This experiment confirmed strong Core and Pol-specific responses in BALB/c mice (Fig. 6). No significant immune interference was observed according to virtually identical T cell responses obtained for group 3, in which both plasmids were mixed and introduced into the same site, and for group 4, in which pDNAs expressing Core and Pol antigens were electroporated separately in two different areas. One animal in group 1 showed a low abnormal response targeting the Pol 2 pool. The same experiment was repeated in C57/B16 mice with comparable results.

The above results demonstrate that when two plasmids expressing HBV antigens, pDK-Core and pDK-Pol, are combined, there is virtually no immune interference.

Example 6: Evaluation of DNA Vaccine Efficacy in Non-Human Primates.

The purpose of this study was to evaluate the efficacy of a therapeutic DNA vaccine against HBV delivered intramuscularly via electroporation and the induction and measurement of HBV-specific T cell response/cell activation in Cynomolgus monkeys (Macaca flavicularis).

Vaccine.

The vaccine used in this study was a combination of two separate DNA plasmids encoding HBV nuclear antigen and HBV polymerase antigen, respectively. Specifically, the DNA plasmids were plasmid pDK-Pol (encoding HBV polymerase antigen polymerase having the amino acid sequence of SEQ ID NO: 4) and plasmid pDK-Core (encoding HBV core antigen having the amino acid sequence SEQ ID NO: 2), as shown in fig. 3A and 3B, respectively, and described in example 1.

DNA plasmids were introduced into a 1:1 (wt/wt) mixture of both plasmids delivered to the same anatomical site. Non-human primates (NHP) were electroporated using the TriGrid intramuscular delivery system (TDS-IM) adapted for use in the NHP model. Additional description of methods and apparatus for intramuscular delivery of NHP DNA by electroporation can be found in International Patent Application Publication WO2017172838 and US Patent Application entitled Method and Apparatus for Delivery of Hepatitis B Virus Vaccines, filed on the same date as the present application. with patent attorney authorization number 688097-405U1, the disclosures of which are incorporated herein by reference in their entirety. Specifically, the TDS-IM array of a TDS-IM v1.0 or TDS-IM v2.0 device having an electrode array with an interelectrode distance of 6.0 mm and an electrode diameter of 0.021 or 0.023 inches was injected percutaneously into the

- 45 045279 brane muscle with a conductor length of 3.2 mm and an effective penetration depth of 3.2 mm, and the main axis of the diamond-shaped electrode configuration was oriented parallel to the muscle fibers. The guidewire length was 5.0 mm for TDS-IM v1.0 or TDS-IM v2.0, whereas the effective penetration depth was 15.5 mm for TDS-IM v1.0 and 9.0 mm for TDS-IM v2. 0. After insertion of the electrode, an injection was started to introduce DNA (eg, 1.0 ml) into the muscle. After completion of the IM injection, an electric field of 250 V/cm was applied locally (applied voltage 142.4-157.6 V with applied current limits less than 0.6-4 A, 0.16 A/s) for a total duration of approximately 400 ms for 10 % duty cycle (i.e. active voltage exposure is a total of approximately 40 ms out of a total duration of approximately 400 ms) with 6 total pulses. After completion of the electroporation procedure, the TriGrid matrix was removed and the animals were allowed time to recover. The initial immunogenicity study was aimed at determining the cellular immune responses that could be caused by the administered HBV antigens.

Non-human primates.

Cynomolgus macaques (n=30) were obtained from China (Covance Research Products Inc. USA) aged 2.5 to 5 years and weighing 3.0 to 5.0 kg at the start of the study. They were housed in an enriched environment in accordance with the recommendations of veterinary authorities and the National Research Council, Guide for the Care and Use of Laboratory Animals, ^8th Edition, Washington DC: National Academies Press (2011). Animals were given 2 weeks of acclimatization time before the start of the study. Before each electroporation plasmid administration, monkeys were anesthetized with ketamine. Blood was collected 2 weeks after each immunization into vials containing sodium heparin. PBMCs were isolated using a Ficoll gradient and stored in liquid nitrogen tanks until analysis.

Intramuscular/electroporation administration to non-human primates.

Plasmid administration was carried out three times (group 1) on days 0, 36 and 62, as shown in table. 4. pDKCore (1.0 mg) and pDK-Pol (1.0 mg) were administered via electroporation with a delivery system set to inject 19 mm (slight) into the quadriceps (vastus lateralis) muscle. Each injection was administered alternately into different muscles. The syringe containing the DNA plasmid was equipped with an injection depth stop suitable for insertion into the NHP quadriceps muscle, allowing injection into the muscle to a target depth of approximately 10 mm, with the major axis of the diamond configuration oriented parallel to the muscle fibers. Immediately after completion of the intramuscular injection, the electroporation apparatus was activated to electrically stimulate the muscle with an amplitude of up to 250 V per cm of electrode distance for a total of up to 40 ms over a 400 ms interval. Samples were collected on days 0, 14, 50, and 76 and analyzed by ELISPOT and intracellular cytokine staining.

Table 4

Experimental NHP Vaccination Scheme

Group N pDNA The plot is one-sided. introductions (alternating sides) Dose of each pDNA per site Dose of total pDNA per site Days of the Vedas Sample collection days 1 5 pDK-Core ST+ER 1.0 mg 2.0 mg 0, 36 and 62 0, 14, 50 and & pDK-Pol 76

ELISPOT Analysis.

Antigen-specific responses were analyzed and quantified by IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay using the Primate IFN-γ ELISpot kit (R&D Systems, USA, catalog number EL961). In this assay, isolated PBMCs from immunized animals were incubated in triplicate wells overnight with pools of peptides (2 μg/ml) covering the Core protein and the Pol protein. These pools consist of 15-mer peptides that overlap 11 residues corresponding to the ABCD consensus sequence of the Core and Pol vaccine vectors. Peptides were synthesized with 90% purity (JPT, Germany). The large 94 kDa HBV Pol protein was split down the middle into two peptide pools. The release of IFN-γ by a single antigen-specific T cell was visualized using appropriate antibodies followed by detection using a chromogenic substrate as a stained spot on a microplate called a spot-forming cell (SFC). The results are shown in Fig. 7A.

Intracellular cytokine staining (ICS).

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Intracellular cytokine staining (ICS) was used to study vaccine-induced T cell responses. Frozen PBMCs were thawed and left overnight in RPMI with 10% FBS before stimulation with a pool of Core, Pol-1, or Pol-2 peptides in the appropriate vaccine (2 μg/μl), DMSO, or leukocyte activation cocktail for 6 hours on medium. RPMI with 10% FBS containing Golgiplug protein transport inhibitor (1 μg/μl). Stimulated cells were stained with eFluor 780 viable cell fixation dye (65-0865-14, eBioscience) and treated for 20 min with fixation/permeabilization solution (554714, BD Biosciences) before staining for 30 min with intracellular staining mixture as indicated. in table 5 below. Stained cells were obtained using a Fortessa flow cytometer with appropriate single-color compensation controls. Response magnitudes were presented as the percentage of CD4+ or CD8+ T cells expressing IFN-γ, TNF-α, or IL-2 after stimulation. The results are shown in Fig. 7B.

Table 5

Panel of antibodies used to analyze intracellular cytokine staining

557705 CD3 Alexa fluor 488 SP34 563823 CD8 BUV786 RPA-T8 564107 CD4 BUV395 L200 554701 IFNg P.E. B27 554514 TNF APC MAbll 564164 IL-2 BV421 MQ1-17H12

Results.

ELISPOT data (Fig. 7A) showed strong Core and Pol-2 responses after two immunizations. The third immunization significantly increased the amount of IFN-β. The Po1-1 peptide pool induced an intermediate response, which also improved after the third immunization, although not as significantly as with Core and Pol-2. Data for day 76 only included results from four NHPs because a blood sample could not be obtained from the fifth monkey. The high variation within each group is due to the fact that NHPs are derived from different outbred populations, and genetic diversity may account for differing immune responses.

Data from the ICS assay (Fig. 7B) showed that the cytokine response induced by HBV peptide stimulation was driven by CD8 and was specific to the Core and Po1-2 peptide pools, as previously observed in ELISPOT. Responses in the ICS assay and in the ELISPOT assay were observed in the same individuals. Although some individual ICS responses were not positive as seen in the ELISPOT data, this may be due to the higher sensitivity of the ELISPOT assay.

Conclusion.

The above results demonstrate that NHPs immunized with the combination of pDK-Core and pDK-Pol vaccine by intramuscular injection via electroporation exhibit significant T cell responses at doses of 1.0 mg of each plasmid, with peptide-specific responses detected after two immunizations and even greater responses after three immunizations. At day 76, ELISPOT assay results showed that Core, Pol-1, and Pol-2 peptide pools induced positive IFN-T cell responses in every NHP tested (4/5 NHPs). ICS analysis of PBMCs from immunized NHPs showed that the specific response to HBV peptide was driven by CD8, with the highest responses observed for the Core and Pol-2 pools.

Example 7: Evaluating the effectiveness of a DNA vaccine in humans.

The efficacy of a therapeutic DNA HBV vaccine delivered intramuscularly via electroporation was evaluated in humans.

Human Subjects.

Subjects were adult patients with chronic HBV infection who were HBsAg positive. Subjects were treated with an HBV polymerase inhibitor (entecavir or tenofovir).

Vaccine.

Subjects were administered a combination of two separate DNA plasmids encoding HBV nuclear antigen and HBV polymerase antigen, respectively. Specifically, the DNA plasmids were plasmid pDKPol (encoding HBV polymerase antigen having the amino acid sequence of SEQ ID NO: 4) and plasmid pDK-Core (encoding HBV core antigen having the amino acid sequence of SEQ ID NO: 2), as shown in FIG. 3A and 3B, respectively, and described in Example 1. DNA plasmids were administered in a 1:1 mixture of both plasmids at various doses, in particular doses of 0.25 mg, 1 mg and 6 mg of total plasmid in accordance with a randomized, placebo-controlled study with dose escalation.

Intramuscular/electroporation in humans DNA plasmids were administered to humans by electroporation in 2-3 intramuscular immunizations using the TriGrid intramuscular system.

- 47 045279 muscle delivery (TDS-IM), adapted for use in humans. Some patients were given placebo (ie, plasmids lacking the coding sequences for HBV antigens) as a control. The TriGrid™ intramuscular delivery system (TDS-IM), adapted for human use, is used to deliver plasmid DNA by electroporation. Additional description of methods and apparatus for intramuscular delivery of DNA to humans via electroporation can be found in International Patent Application Publication WO2017172838 and US Patent Application entitled Method and Apparatus for Delivering Hepatitis B Virus Vaccines, filed on the same date as the present application. with patent attorney authorization number 688097405U1, the disclosures of which are incorporated herein by reference in their entirety. For example, the TDS-IM array of the TDS-IM v2.0 device, having an electrode array with an interelectrode spacing of 6.0 mm and an electrode diameter of 0.023 inches, was injected percutaneously into the selected muscle with the major axis of the diamond-shaped electrode configuration oriented parallel to the muscle fibers. The guidewire length was 5.0 mm, whereas the effective penetration depth was 19 mm. After insertion of the electrode, an injection was started to introduce DNA (eg, 1.0 ml) into the muscle. After completion of the IM injection, an electric field of 250 V/cm was applied locally (applied voltage 142.4-157.6 V with applied current limits less than 0.6-4 A, 0.16 A/s) for a total duration of approximately 400 ms for 10 % duty cycle (i.e. active voltage exposure is a total of approximately 40 ms out of a total duration of approximately 400 ms) with 6 total pulses. After completion of the electroporation procedure, the TriGrid™ matrix was removed and the subject was allowed time to recover.

Blood samples were collected from patients at various time points after immunization. The evolution of HBsAg levels after immunization, particularly levels consistent with evolution to clinical seroconversion, was assessed in patients 3 to 6 months after immunization. Persistent loss of HBsAg and reduction in clinical disease (eg, liver cirrhosis, hepatocellular carcinoma) were assessed in patients 6 to 12 months after immunization.

Example 8. In vivo study of the immunogenicity of adenoviral vectors in mice.

An immunotherapeutic vaccine containing adenoviral vectors encoding HBV nuclear antigen or HBV polymerase antigen was tested in mice. The aim of the study was to identify T cell responses induced by the vaccine following intramuscular delivery to F1 (C57BL/6xBa1b/C) mice. Initial immunogenicity studies were aimed at determining the cellular immune responses that could be induced by administered HBV antigens. In particular, the adenovectors tested contained the expression cassettes shown in FIG. 8A and 8B.

In vivo immunogenicity study.

To evaluate the immunogenicity of the adenovirus vaccine in vivo, HBV adenoviral vectors were administered intramuscularly to F1 mice. These immunogenicity studies were aimed at determining the cellular immune responses elicited by HBV nuclear and polymerase antigens. Administration to F1 mice was carried out as shown in Table 6. Animals received one immunization with the HBV adenoviral vector. Splenocytes were collected after nine weeks.

Table 6

Experimental scheme for immunization of mice with adenoviral vectors

Group N Priming Day 0 R dose (vp) End point day 1 4 Core Pol fused+Core I.M. ^10s 63 2 4 Core Pol fused+Core I.M. ^10y 63 3 4 Core Pol fused+Coge I.M. ^10w 63 7 4 Core Pol merged I.M. ^10s 63 8 4 Core Pol merged I.M. ^10y 63 9 4 Core Pol merged I.M. ^10w 63

IM: intramuscular; vp: viral particles.

Assessment of the immunogenicity of HBV adenoviral vectors.

Antigen-specific responses were analyzed and quantified by IFN-γ enzyme-linked immunosorbent spot assay (ELISPOT). In this assay, isolated splenocytes from immunized animals were incubated with peptide pools covering Core and Pol proteins (2 μg/ml of each peptide). The pools consisted of 15-mer peptides that overlap 11 residues corresponding to the ABCD consensus sequences of the Core and Pol genotypes of the adenoviral vectors. The large 94 kDa HBV Pol protein was split down the middle into two peptide pools. In ELISPOT, the release of IFN-γ by a single antigen-specific T cell was visualized using appropriate antibodies and subsequent detection using a chromogenic substrate as a stained spot on a microplate called a spot-forming cell (SFC).

The results are shown in Fig. 9. From the results it is clear that HBV adenoviral vectors, especially the combination of a Core and Pol fused adenovector with a Core adenovector, caused Core- and Pol-specific

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Claims (21)

T cell responses. These data indicate that adenoviral vectors encoding HBV Core and Pol antigens induce robust T cell responses to core and pol in F1 mice. It is understood that the examples and embodiments presented herein are for illustrative purposes only, and that changes may be made to the embodiments described above without deviating from the spirit of the invention. Accordingly, the present invention is not intended to be limited to the specific embodiments disclosed, but is intended to cover modifications falling within the spirit and scope of the present invention as defined by the appended claims. Literature. 1. Cohen et al. Is chronic hepatitis being undertreated in the United States? J. Viral Hepat. (2011) 18(6), 377-83. 2. Obeng-Adjei et al. DNA vaccine cocktail expressing genotype A and C HBV surface and consensus core antigens generates robust cytotoxic and antibody responses and mice and Rhesus macaques Cancer Gene Therapy (2013) 20, 652-662. 3. World Health Organization, Hepatitis B: Fact sheet no. 204 [Internet] 2015 March. Available from http://www.who.nt/mediacentre/factsheets/fs204/en/. 4. Belloni et al. IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome J. Clin. Invest. (2012) 122(2), 529537. 5. Michel et al. Therapeutic vaccines and immune-based therapies for the treatment of chronic hepatitis B: perspectives and challenges. J. Hepatol. (2011) 54(6), 1286-1296. CLAIM 1. Immunogenic composition containing: (a) a first unnatural nucleic acid molecule comprising a first polynucleotide encoding an HBV polymerase antigen having an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 4, wherein the HBV polymerase antigen does not have reverse transcriptase activity, and RNase H activity; (b) a second unnatural nucleic acid molecule comprising a second polynucleotide encoding a truncated HBV nuclear antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 14; and (c) a pharmaceutically acceptable carrier, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in the same non-naturally occurring nucleic acid molecule or in two different non-naturally occurring nucleic acid molecules and wherein the HBV polymerase antigen is capable of inducing a T cell response in a mammal against at least the B, C and D genotypes of HBV. 2. Immunogenic composition according to claim 1, in which: a) HBV polymerase antigen is capable of inducing a CD8 T cell response in humans against at least HBV genotypes A, B, C and D; b) the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are part of a non-viral vector selected from the group consisting of deoxyribonucleic acid (DNA) plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, ribonucleic acid replicons acids (RNA), messenger RNA (mRNA) replicons, modified mRNA replicons or self-amplifying mRNAs and closed linear deoxyribonucleic acids; c) the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are part of a viral vector selected from the group consisting of adenoviral vectors, adeno-associated virus vectors, smallpox virus vectors, enteric virus vectors, Venezuelan equine virus vectors encephalitis, Semliki forest virus vectors, tobacco mosaic virus vectors, lentiviral vectors, arenavirus viral vectors, replication-deficient arenavirus viral vectors or replication-competent arenavirus viral vectors, two-segmented or three-segmented arenaviruses, infectious arenavirus viral vectors, nucleic acids that contain an arenavirus genomic segment , wherein one open reading frame of the genomic segment is deleted or functionally inactivated and replaced by a nucleic acid encoding an antigen of HBV, lymphocytic choriomeningitis virus (LCMV) and Junin virus; and/or d) the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in the same non-naturally occurring nucleic acid, and the same non-naturally occurring nucleic acid is - 49 045279 is a bicistronic expression vector in which an individual HBV core and HBV polymerase antigens are expressed from a single mRNA transcript, wherein said bicistronic expression vector further comprises a ribosome slip site or a sequence encoding a cis-hydrolase site. 3. The immunogenic composition of any one of the preceding claims, wherein the first polynucleotide encodes an HBV polymerase antigen comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 4, and the second polynucleotide encodes an HBV nuclear antigen consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14. 4. The immunogenic composition according to claim 3, wherein the first polynucleotide encodes an HBV polymerase antigen containing the amino acid sequence of SEQ ID NO: 4. 5. The immunogenic composition of any one of the preceding claims, wherein at least one of the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule further comprises a polynucleotide sequence encoding a signal peptide sequence operably linked to at least one of HBV polymerase antigen and HBV truncated nuclear antigen. 6. The immunogenic composition of claim 5, wherein the signal peptide sequence independently comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 19. 7. An immunogenic composition according to any of the previous claims, wherein the first polynucleotide sequence is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 16. 8. The immunogenic composition according to claim 7, wherein the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 16. 9. The immunogenic composition according to any of the previous claims, wherein the second polynucleotide sequence is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 15. 10. The immunogenic composition of claim 9, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15. 11. The immunogenic composition according to any of the preceding claims, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in the same vector. 12. The immunogenic composition according to claim 11, wherein the vector encodes a fusion protein containing a truncated HBV nuclear antigen operably linked to an HBV polymerase antigen. 13. The immunogenic composition according to claim 12, wherein the fusion protein contains the amino acid sequence of SEQ ID NO: 20. 14. The immunogenic composition according to claim 12 or claim 13, in which the vector contains, in the direction from the 5' end to the 3' end, a promoter sequence, an enhancer sequence, a sequence encoding a signal peptide, a second polynucleotide sequence, a sequence encoding a linker , the first polynucleotide sequence and the polyadenylation signal sequence. 15. The immunogenic composition according to claim 14, wherein the vector is an adenoviral vector. 16. The immunogenic composition according to claim 15, in which the adenoviral vector contains a promoter sequence containing the polynucleotide sequence SEQ ID NO: 17, a regulatory sequence containing the polynucleotide sequence SEQ ID NO: 23, a sequence encoding a signal peptide containing the polynucleotide sequence SEQ ID NO : 18, a second polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 15, a linker encoding sequence containing the polynucleotide sequence of SEQ ID NO: 22, a first polynucleotide sequence containing the polynucleotide sequence of SEQ ID NO: 16, and a polyadenylation signal sequence containing the polynucleotide sequence SEQ ID NO: 24. 17. The immunogenic composition according to any one of claims 1 to 10, wherein the first non-naturally occurring nucleic acid molecule and the second non-naturally occurring nucleic acid molecule are present in two different vectors. 18. The immunogenic composition of claim 17, wherein the first unnatural nucleic acid molecule is present in the first DNA plasmid vector and the second unnatural nucleic acid molecule is present in the second DNA plasmid vector. 19. The immunogenic composition according to claim 18, wherein each of the first and second DNA plasmid vectors contains an origin of replication, an antibiotic resistance gene and, in the direction from the 5' end to the 3' end, a promoter sequence, a regulatory sequence, a , encoding a signal peptide, a first polynucleotide sequence or a second polynucleotide sequence, and a polyadenylation signal sequence. 20. The immunogenic composition according to claim 19, wherein the antibiotic resistance gene is a kanamycin resistance gene having a polynucleotide sequence at least 90% identical to SEQ ID NO: 12. 21. Immunogenic composition according to claim 20, containing: -