WO2020255010A1

WO2020255010A1 - Combination of recombinant interleukin 12 construct and hepatitis b virus (hbv) vaccines

Info

Publication number: WO2020255010A1
Application number: PCT/IB2020/055700
Authority: WO
Inventors: Helen Horton; An Martine M DE CREUS; Ellen Rosalie A VAN GULCK; Daniel BODEN; Dorien DE POOTER; Antony Chien-Hung CHEN
Original assignee: Janssen Sciences Ireland Unlimited Company
Priority date: 2019-06-18
Filing date: 2020-06-18
Publication date: 2020-12-24

Abstract

Immunogenic combinations of IL12 fusion proteins, or polynucleotides encoding such fusion proteins, and polynucleotides encoding hepatitis B virus (HBV) core antigen and/or polymerase antigen are described. The polynucleotides can be present in vectors, such as DNA plasmids or viral vectors. Methods of inducing an immune response against HBV or treating a HBV-induced disease, particularly in individuals having chronic HBV infection, using the disclosed immunogenic combinations are also described.

Description

TITLE OF THE INVENTION

Combination of Recombinant Interleukin 12 Construct and Hepatitis B Virus (HBV)

Vaccines

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to European Application No. EP19180980.5 filed on June 18, 2019, the disclosure of which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name“Sequence Listing” and a creation date of June 10, 2020 and having a size of 82.2 kb. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Interleukin 12 (IL12) is an interleukin that is naturally produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells in response to antigenic stimulation. IL12 is a proinflammatory cytokine that promotes differentiation of naive CD4 T cells into TH1 helper cells, induces proliferation, induces interferon gamma (IFNg) production by T cells and enhances cytotoxicity of natural killer (NK) and cytotoxic T cells (Trinchieri et al., Nat Rev Immunol. (2003) 3(2): 133-46). IL12 is a heterodimeric protein encoded by two separate genes, IL12A (p35) and IL12B (p40). When the subunits combine together, they form the functional protein, IL12 p70

(Kobayashi et al., J Exp Med. (1989)170(3):827-45).

Several studies have reported that IL12 as a protein has a critical role in inducing antiviral and antitumor effects in vivo. Direct administration of IL12 protein or cDNA expressing IL12 by gene gun can affect tumor progression and metastases in animal models (Dias et al, Int J Cancer. (1998) 75(1): 151-7; Rakhmilevich et al., Proc Natl Acad Sci U S A. (1996) 93(13):6291-6; Yu et al, J Leukoc Biol. (1997) 62(4):450-7). Similarly, therapeutic treatments with IL12 protein can result in protective responses to some infectious viral agents (Bi et al, J Immunol. (1995) 155(12):5684-9; Orange and Biron. J Immunol. (1996)156(12):4746-56). Hepatitis B virus (HBV) is a small 3.2-kb hepatotropic DNA virus that encodes four open reading frames and seven proteins. Approximately 240 million people have chronic hepatitis B infection (chronic HBV), characterized by persistent virus and subvirus particles in the blood for more than 6 months (Cohen et al. J. Viral Hepat.

(2011) 18(6), 377-83). Persistent HBV infection leads to T-cell exhaustion in circulating and intrahepatic HBV-specific CD4+ and CD8+ T-cells through chronic stimulation of HBV-specific T-cell receptors with viral peptides and circulating antigens. As a result, T- cell polyfunctionality is decreased (i.e., decreased levels of IL-2, tumor necrosis factor (TNF)-a, IFN-g, and lack of proliferation).

A safe and effective prophylactic vaccine against HBV infection has been available since the 1980s and is the mainstay of hepatitis B prevention (World Health Organization, Hepatitis B: Fact sheet No. 204 [Internet] 2015 March.). The World Health Organization recommends vaccination of all infants, and, in countries where there is low or intermediate hepatitis B endemicity, vaccination of all children and adolescents (<18 years of age), and of people of certain at risk population categories. Due to vaccination, worldwide infection rates have dropped dramatically. However, prophylactic vaccines do not cure established HBV infection.

Chronic HBV is currently treated with IFN-a and nucleoside or nucleotide analogs, but there is no ultimate cure due to the persistence in infected hepatocytes of an intracellular viral replication intermediate called covalently closed circular DNA

(cccDNA), which plays a fundamental role as a template for viral RNAs, and thus new virions. It is thought that induced virus-specific T-cell and B-cell responses can effectively eliminate cccDNA-carrying hepatocytes. Current therapies targeting the HBV polymerase suppress viremia, but offer limited effect on cccDNA that resides in the nucleus and related production of circulating antigen. The most rigorous form of a cure may be elimination of HBV cccDNA from the organism, which has neither been observed as a naturally occurring outcome nor as a result of any therapeutic intervention. However, loss of HBV surface antigens (HBsAg) is a clinically credible equivalent of a cure, since disease relapse can occur only in cases of severe immunosuppression, which can then be prevented by prophylactic treatment. Thus, at least from a clinical standpoint, loss of HBsAg is associated with the most stringent form of immune reconstitution against HBV.

For example, immune modulation with pegylated interferon (peglFN)-a has proven better in comparison to nucleoside or nucleotide therapy in terms of sustained off- treatment response with a finite treatment course. Besides a direct antiviral effect, IFN-a is reported to exert epigenetic suppression of cccDNA in cell culture and humanized mice, which leads to reduction of virion productivity and transcripts (Belloni et al. J.

Clin. Invest. (2012) 122(2), 529-537). However, this therapy is still fraught with side- effects and overall responses are rather low, in part because IFN-a has only poor modulatory influences on HBV-specific T-cells. In particular, cure rates are low (< 10%) and toxicity is high. Likewise, direct acting HBV antivirals, namely the HBV

polymerase inhibitors entecavir and tenofovir, are effective as monotherapy in inducing viral suppression with a high genetic barrier to emergence of drug resistant mutants and consecutive prevention of liver disease progression. However, cure of chronic hepatitis B, defined by HBsAg loss or seroconversion, is rarely achieved with such HBV polymerase inhibitors. Therefore, these antivirals in theory need to be administered indefinitely to prevent reoccurrence of liver disease, similar to antiretroviral therapy for human immunodeficiency virus (HIV).

Therapeutic vaccination has the potential to eliminate HBV from chronically infected patients (Michel et al. J. Hepatol. (2011) 54(6), 1286-1296). Many strategies have been explored, but to date therapeutic vaccination has not proven successful.

BRIEF SUMMARY OF THE INVENTION

Accordingly, there is an unmet medical need in the treatment of hepatitis B virus (HBV), particularly chronic HBV, for a finite well-tolerated treatment with a higher cure rate. The invention satisfies this need by providing immunogenic

compositions/combinations and methods for inducing an immune response against hepatitis B viruses (HBV) infection. The immunogenic compositions/combinations and methods of the invention can be used to provide therapeutic immunity to a subject, such as a subject having chronic HBV infection.

In a general aspect, the application relates to immunogenic combinations or compositions comprising IL12 fusion proteins, or polynucleotides encoding IL12 fusion proteins, and HBV antigens, or polynucleotides encoding HBV antigens.

In an embodiment, an immunogenic combination comprises

i) a fusion protein comprising:

a) an IL12 p40 subunit;

b) a linker consisting of the amino acid sequence of SEQ ID NO: 3; and c) an IL12 p35 subunit;

wherein the fusion protein is arranged from N-terminus to C-terminus in the order (a)- (b)-(c), and the C-terminus of the IL12 p40 subunit is fused to the N-terminus of the IL12 p35 subunit through the linker, or a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding the fusion protein; and

ii) at least one of:

a) an HBV polymerase antigen having an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity,

b) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding the HBV polymerase antigen;

c) a truncated HBV core antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 31, and

d) a third non-naturally occurring nucleic acid molecule comprising a third polynucleotide sequence encoding the truncated HBV core antigen.

In an embodiment, the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. In certain embodiments the immunogenic combination comprises the fusion protein. In another embodiment, the immunogenic composition comprises the first non-naturally occurring nucleic acid molecule comprising the first polynucleotide sequence encoding the fusion protein.

In an embodiment, the first polynucleotide sequence comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 27. Preferably, the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 27.

In an embodiment, the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 33 and the truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 31.

In an embodiment, the immunogenic combination comprises at least one of the HBV polymerase antigen and the truncated HBV core antigen. In certain embodiments, the immunogenic combination comprises the HBV polymerase antigen and the truncated HBV core antigen.

In an embodiment, the immunogenic combination comprises at least one of the second non-naturally occurring nucleic acid molecule comprising the second

polynucleotide sequence encoding the HBV polymerase antigen, and the third non- naturally occurring nucleic acid molecule comprising the third polynucleotide sequence encoding the truncated HBV core antigen. In certain embodiments, the HBV polymerase antigen and the truncated HBV core antigen each further comprises a signal sequence operably linked to the N-terminus of the antigen, preferably, the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 44, preferably the signal sequence is encoded by the polynucleotide sequence of SEQ ID NO: 34 or SEQ ID NO: 43.

In an embodiment, the HBV polymerase antigen and the truncated HBV core antigen each further comprises a signal sequence operably linked to the N-terminus of the antigen, preferably, the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 44, preferably the signal sequence is encoded by the polynucleotide sequence of SEQ ID NO: 34 or SEQ ID NO: 43. Preferably, the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 32 or SEQ ID NO: 39.

In an embodiment, the second polynucleotide sequence comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 32 or 39. Preferably, the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO:

32 or 39.

In an embodiment, the third polynucleotide sequence comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 38. Preferably, the third polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 38.

In an embodiment, each of the first, second and third non-naturally occurring nucleic acid molecules is a DNA molecule, preferably the DNA molecule is present on a plasmid or a viral vector.

In an embodiment, an immunogenic combination comprises

i) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding a fusion protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24;

ii) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding an HBV polymerase antigen having an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity; and

iii) a third non-naturally occurring nucleic acid molecule comprising a third polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 31.

Preferably, the immunogenic combination comprises a) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding a fusion protein having the amino acid sequence of SEQ ID NO: 24; b) a second non- naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding an HBV polymerase antigen consisting the amino acid sequence of SEQ ID NO: 33; and c) a third non-naturally occurring nucleic acid molecule comprising a third polynucleotide sequence encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 31.

More preferably, the immunogenic combination comprises a) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence of SEQ ID NO: 27; b) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence of SEQ ID NO: 32 or 39; and c) a third non-naturally occurring nucleic acid molecule comprising a third polynucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 38.

In another embodiment, each of the first, second and third non-naturally occurring nucleic acid molecules is an RNA molecule, preferably an mRNA or a self-replicating RNA molecule.

In some embodiments, each of the first, second and third non-naturally occurring nucleic acid molecules is independently formulated with a lipid nanoparticle (LNP).

In another general aspect, the application relates to a kit comprising an immunogenic combination of the application.

The application also relates to an immunogenic combination or kit of the application for use in inducing an immune response against hepatitis B virus (HBV); and use of an immunogenic combination, composition or kit of the application in the manufacture of a medicament for inducing an immune response against hepatitis B virus (HBV). The use can further comprise a combination with another immunogenic agent, preferably another HBV antigen. Preferably, the subject has chronic HBV infection.

The application further relates to an immunogenic combination or kit of the application for use in treating a HBV-induced disease in a subject in need thereof; and use of immunogenic combination, composition or kit of the application in the manufacture of a medicament for treating a HBV-induced disease in a subject in need thereof. The use can further comprise a combination with another therapeutic agent, preferably another anti -HBV antigen. Preferably, the subject has chronic HBV infection, and the HBV- induced disease 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 appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings.

FIG. 1 shows a DNA plasmid encoding a human IL12 fusion protein according to an embodiment of the application the IL12 fusion protein is expressed under control of a CMV promoter with a signal sequence located between the CMV promoter and the polynucleotide sequence encoding the fusion protein; transcriptional regulatory elements of the plasmid include a bGH polyadenylation sequence located downstream of the polynucleotide sequence encoding the fusion protein; a second expression cassette is included in the plasmid including an fl origin, a neomycin resistance gene under the control of an SV40 early promoter, and an SV40 polyadenylation sequence; a third expression cassette is included in the plasmid in reverse orientation including a ampicillin resistance gene under control of an Amp^r(bla) promoter; an origin of replication (pUC) is also included in reverse orientation.

FIG. 2 shows a DNA plasmid encoding a human IL12 fusion protein according to an embodiment of the application the IL12 fusion protein is expressed under control of a CMV promoter with a signal sequence located between the CMV promoter and the polynucleotide sequence encoding the fusion protein; transcriptional regulatory elements of the plasmid include an enhancer sequence located between the CMV promoter and the polynucleotide sequence encoding the fusion protein and a bGH polyadenylation sequence located downstream of the polynucleotide sequence encoding the fusion protein; a second expression cassette is included in the plasmid in reverse orientation including a kanamycin resistance gene under control of an Amp^r(bla) promoter; an origin of replication (pUC) is also included in reverse orientation.

FIG. 3 shows ELISA measurements of IL12 p70 concentrations from the media supernatant of HEK293T cells transfected with pcDNA-p35 and pcDNA-p40 expressing plasmids or pcDNA plasmids expressing fusion proteins with KE, FA, IR, and TC linkers located between the p40 and p35 subunits according to embodiments of the application; the IL12 p70 concentration is indicated on the x-axis expressed as pg/ml. FIG. 4 shows is a Western blot analysis showing a comparison of p40 expression in HEK293T cells transfected with either p40 and p35 expressing plasmids or p40-KE-p35 fusion protein expressing plasmid; lane 1 : pcDNA-p40-KE-p35 fusion construct cell lysate; lane 2: pcDNA-p40 and pcDNA-p35 cell lysate; lane 3: empty; lane 4: pcDNA- p40-KE-p35 media supernatant; lane 5: pcDNA-p40 and pcDNA-p35 media supernatant.

FIG. 5 shows interferon gamma (IFNy) protein expression in the media supernatant of two human CD3 T cell samples (DN921 and DN922) after stimulation with increasing concentrations of recombinant IL12 p70 or the supernatant of HEK293T cells transfected with either p40 and p35 expressing plasmids or p40-KE-p35 fusion protein expressing plasmid; IFNy concentration is indicated on the y-axis expressed as pg/ml; the IL12 p70 concentration is indicated on the x-axis expressed as pg/ml.

FIG. 6A and FIG. 6B show schematic representations of DNA plasmids according to embodiments of the application; FIG. 6A shows a DNA plasmid encoding an HBV core antigen according to an embodiment of the application; FIG. 6B shows a DNA plasmid encoding an HBV polymerase (pol) antigen according to an embodiment of the application; the HBV core and pol antigens are expressed under control of a CMV promoter with an N-terminal cystatin S signal peptide that is cleaved from the expressed antigen upon secretion from the cell; 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; a second expression cassette is included in the plasmid in reverse orientation including a kanamycin resistance gene under control of an Ampr (bla) promoter; an origin of replication (pUC) is also included in reverse orientation.

FIG. 7A and FIG. 7B. show the schematic representations of the expression cassettes in adenoviral vectors according to embodiments of the application; FIG. 7A shows the expression cassette for a truncated HBV core antigen, which contains a CMV promoter, an intron (a fragment derived from the human ApoAI gene - GenBank accession X01038 base pairs 295 - 523, harboring the ApoAI second intron), a human immunoglobulin secretion signal, followed by a coding sequence for a truncated HBV core antigen and a SV40 polyadenylation signal; FIG. 7B shows the expression cassette for a fusion protein of a truncated HBV core antigen operably linked to a HBV

polymerase antigen, which is otherwise identical to the expression cassette for the truncated HBV core antigen except the HBV antigen.

FIG. 8 shows ELISPOT responses of Balb/c mice immunized with different DNA plasmids expressing HBV core antigen or HBV pol antigen, as described in Example 4; peptide pools used to stimulate splenocytes isolated from the various vaccinated animal groups are indicated in gray scale; the number of responsive T-cells are indicated on the y- axis expressed as spot forming cells (SFC) per 10⁶ splenocytes;

FIG. 9 shows a DNA plasmid encoding a mouse IL12 fusion protein according to an embodiment of the application; the IL12 fusion protein is expressed under control of a CMV promoter with a signal sequence located between the CMV promoter and the polynucleotide sequence encoding the fusion protein and a SV40 polyadenylation sequence located downstream of the polynucleotide sequence encoding the fusion protein; a second expression cassette is included in the plasmid in reverse orientation including an ampicillin resistance gene under control of an Amp^r (bla) promoter; an origin of replication (pUC) is also included in reverse orientation.

FIG. 10 shows ELISPOT responses of Balb/C mice immunized with a

combination of DNA plasmids expressing an IL12 fusion protein and HBV antigens according the study described in Example 4; Group 1 , single Core and Pol pDNA; Group 2, Core and Pol pDNA and 0.1 ug mIL12 fusion protein pDNA; Group 3, Core and Pol pDNA and 0.5 ug mIL12 fusion protein pDNA; Group 4, Core and Pol pDNA and 2 ug mIL12 fusion protein pDNA; Group 5, Core and Pol pDNA and 0.1 ug pUMVC3 mIL12- IRES (Ichor) pDNA; Group 6, Core and Pol pDNA and 0.5 ug pUMVC3 mILl 2-IRES (Ichor) pDNA; Group 7, Core and Pol pDNA and 2 ug pUMVC3 mIL 12-IRES (Ichor) pDNA; Group 8, Empty pDK vector; peptide pools used to stimulate splenocytes isolated from the various vaccinated animal groups are indicated in gray scale; the number of responsive T-cells are indicated on the y-axis expressed as spot forming cells (SFC) per 10⁶ splenocytes.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein. It must be noted that as used herein and in the appended claims, the singular forms“a,”“an,” and“the” include plural reference unless the context clearly dictates otherwise.

Unless otherwise indicated, the term“at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word“comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term“comprising” can be substituted with the term “containing” or“including” or sometimes when used herein with the term“having”.

When used herein“consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein,“consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned terms of“comprising”,“containing”, “including”, and“having”, whenever used herein in the context of an aspect or embodiment of the application can be replaced with the term“consisting of’ or “consisting essentially of’ to vary scopes of the disclosure.

As used herein, the conjunctive term“and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by“and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term“and/or” as used herein.

Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term“and/or.”

Unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term“about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1 mg/mL to 10 mg/mL includes 0.9 mg/mL to 11 mg/mL. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

The phrases“percent (%) sequence identity” or“% identity” or“% identical to” when used with reference to an amino acid sequence describe the number of matches (“hits”) of identical amino acids of two or more aligned amino acid sequences as compared to the number of amino acid residues making up the overall length of the amino acid sequences. In other terms, using an alignment, for two or more sequences the percentage of amino acid residues that are the same (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity over the full-length of the amino acid sequences) may be determined, when the sequences are compared and aligned for maximum correspondence as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected. The sequences which are compared to determine sequence identity may thus differ by substitution(s), addition(s) or deletion(s) of amino acids. Suitable programs for aligning protein sequences are known to the skilled person. The percentage sequence identity of protein sequences can, for example, be determined with programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, e.g. 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“administered together with” in the context of the administration of two or more therapies or components to a subject refers to simultaneous administration or subsequent administration of two or more therapies or components, such as two vectors, e.g., DNA plasmids, peptides, or an immunogenic combination and an adjuvant.

“Simultaneous administration” can be administration of the two or more therapies or components at least within the same day. When two components are“administered together with” or“administered in combination with,” they can be administered in separate compositions sequentially within a short time period, such as 24, 20, 16, 12, 8 or 4 hours, or within 1 hour, or they can be administered in a single composition at the same time. “Subsequent administration” can be administration of the two or more therapies or components in the same day or on separate days. The use of the term“in combination with” does not restrict the order in which therapies or components are administered to a subject. For example, a first therapy or component (e.g. first DNA plasmid encoding an HBV antigen) can be administered prior to (e.g., 5 minutes to one hour before), concomitantly with or simultaneously with, or subsequent to (e.g., 5 minutes to one hour after) the administration of a second therapy or component (e.g., second DNA plasmid encoding an HBV antigen), and/or a third therapy or component (e.g., a IL12 fusion protein or vector encoding a IL12 fusion protein). In some embodiments, a first therapy or component (e.g. first DNA plasmid encoding an HBV antigen), a second therapy or component (e.g., second DNA plasmid encoding an HBV antigen), and a third therapy or component (e.g., a IL12 fusion protein or vector encoding a IL12 fusion protein) are administered in the same composition. In other embodiments, a first therapy or component (e.g. first DNA plasmid encoding an HBV antigen), a second therapy or component (e.g., second DNA plasmid encoding an HBV antigen), and a third therapy or component (e.g., an IL12 fusion protein or vector encoding a IL12 fusion protein) are administered in separate compositions, such as two or three separate compositions.

As used herein, a“non-naturally occurring” nucleic acid or polypeptide, refers to a nucleic acid or polypeptide that does not occur in nature. A“non-naturally occurring” nucleic acid or polypeptide can be synthesized, treated, fabricated, and/or otherwise manipulated in a laboratory and/or manufacturing setting. In some cases, a non-naturally occurring nucleic acid or polypeptide can comprise a naturally-occurring nucleic acid or polypeptide that is treated, processed, or manipulated to exhibit properties that were not present in the naturally-occurring nucleic acid or polypeptide, prior to treatment. 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 discovered, and it lacks covalent bonds to sequences with which it was associated in the natural source.

A“non-naturally occurring” nucleic acid or polypeptide can be made recombinantly or via other methods, such as chemical synthesis.

As used herein,“subject” means any animal, preferably a mammal, most preferably a human, to whom will be or has been treated by a method according to an embodiment of the application. The term“mammal” as used herein, encompasses 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 a human.

As used herein, the term“operably linked” refers to a linkage or a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence operably linked to a nucleic acid sequence of interest is capable of directing the 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 over a membrane.

In an attempt to help the reader of the application, the description has been separated in various paragraphs or sections, or is directed to various embodiments of the application. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiments. To the contrary, one skilled in the art will understand that the description has broad application and encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples. For example, while embodiments of HBV vectors of the application (e.g., plasmid DNA or viral vectors) described herein may contain particular components, including, but not limited to, certain promoter sequences, enhancer or regulatory sequences, signal peptides, coding sequence of an HBV antigen, polyadenylation signal sequences, etc. arranged in a particular order, those having ordinary skill in the art will appreciate that the concepts disclosed herein may equally apply to other components arranged in other orders that can be used in HBV vectors of the application. The application contemplates use of any of the applicable components in any combination having any sequence that can be used in HBV vectors of the application, whether or not a particular combination is expressly described. The invention generally relates to an immunogenic composition comprising an IL12 fusion protein and one or more HBV antigens.

I L 12 fusion construct

As used herein, the term“fusion protein” refers to a protein having two or more portions covalently linked together, where each of the portions is derived from different proteins.

As used herein, the terms“IL12” and“IL12 p70” and“NK cell stimulatory factor (NKSF)” are used interchangeably and refer to the interleukin 12 protein. IL12 p70 is a heterodimeric protein encoded by two separate genes, IL12A (p35) and IL12B (p40). As used herein, the terms“IL12A,”“IL12 p35,” and“p35” are used interchangeably and refer to IL12 subunit alpha protein. As used herein, the terms“FL12B,”“IL12 p40,” and “p40”are used interchangeably and refer to IL12 subunit beta protein.

Any suitable linker can be used in fusion proteins according to embodiments of the invention. As used herein, the term“linker” refers to a linking moiety comprising a peptide linker. Preferably, the linker helps insure correct folding, minimizes steric hindrance and does not interfere significantly with the structure of each functional component within the fusion protein.

In a general aspect, the invention relates to a fusion protein comprising a) an IL12 p40 subunit; b) a linker consisting of the amino acid sequence of SEQ ID NO: 3; and c) an IL12 p35 subunit; wherein the fusion protein is arranged from N-terminus to C-terminus in the order (a)-(b)-(c), and the C-terminus of the IL12 p40 subunit is fused to the N- terminus of the IL12 p35 subunit through the linker. In another aspect, the invention relates to a fusion protein comprising a) an IL12 p40 subunit; b) a linker consisting of the amino acid sequence of SEQ ID NO: 3; and c) an IL12 p35 subunit; wherein the fusion protein is arranged from N-terminus to C-terminus in the order (c)-(b)-(a), and the C- terminus of the IL12 p35 subunit is fused to the N-terminus of the IL12 p40 subunit through the linker. The IL12 subunits can be from any mammal, such as a human or another suitable mammal, such as a mouse, rabbit, rat, pig, dog, or a primate. In certain embodiments, the p40 subunit comprises an amino acid sequence having at least 90%

(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 1, 7, or 9. In certain embodiments, the p35 subunit comprises an amino acid sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence identity to SEQ ID NO: 2, 8, or 10.

In certain embodiments, the fusion protein comprises an amino acid sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the fusion protein comprises an amino acid sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the ammo acid sequence of SEQ ID NO: 25. In certain embodiments, the fusion protein comprises an amino acid sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 26.

In certain embodiments, the fusion protein further comprises a signal sequence operably linked to the N-terminus of the p40 subunit. As used herein, the term“signal sequence” refers to a sequence encoding a signal peptide that targets proteins for secretion and direct transport across the endoplasmic reticulum (ER) membrane. Any signal sequence known to those skilled in the art in view of the present disclosure can be used in the fusion protein of the invention. In preferred embodiments, the signal sequence is selected from the group consisting of SEQ ID NOs: 11, 12, and 13.

In a general aspect, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of the invention. In certain embodiments, the isolated nucleic acid molecule has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NOs: 27, 28, or 29.

Additional disclosure on IL12 fusion constructs that can be used in the present invention are described in European Patent Application No. 19180939.1 entitled “Recombinant Interleukin 12 Construct and Uses Thereof,” filed on the same day as this application with the attorney docket number ΉR1026, the contents of which are hereby incorporated by reference in their entireties.

Hepatitis B Virus (HBV)

As used herein“hepatitis B virus” or“HBV” refers to a virus of the

hepadnaviridae family. HBV is a small (e.g., 3.2 kb) hepatotropic DNA virus that encodes four open reading frames and seven proteins. The seven proteins encoded by HBV include small (S), medium (M), and large (L) surface antigen (HBsAg) or envelope (Env) proteins, pre-Core protein, core protein, viral polymerase (Pol), and HBx protein. HBV expresses three surface antigens, or envelope proteins, L, M, and S, with S being the smallest and L being the largest. The extra domains in the M and L proteins are named Pre-S2 and Pre-Si, respectively. Core protein is the subunit of the viral nucleocapsid. Pol is needed for synthesis of viral DNA (reverse transcriptase, RNaseH, and primer), which takes place in nucleocapsids localized to the cytoplasm of infected hepatocytes. PreCore is the core protein with an N-terminal signal peptide and is proteolytically processed at its N and C termini before secretion from infected cells, as the so-called hepatitis B e-antigen (HBeAg). HBx protein is required for efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a viral structural protein. All viral proteins of HBV have their own mRNA except for core and polymerase, which share an mRNA. With the exception of the protein pre-Core, none of the HBV viral proteins are subject to post-translational proteolytic processing.

The HBV virion contains a viral envelope, nucleocapsid, and single copy of the partially double-stranded DNA genome. The nucleocapsid comprises 120 dimers of core protein and is covered by a capsid membrane embedded with the S, M, and L viral envelope or surface antigen proteins. After entry into the cell, the virus is uncoated and the capsid-containing relaxed circular DNA (rcDNA) with covalently bound viral polymerase migrates to the nucleus. During that process, phosphorylation of the core protein induces structural changes, exposing a nuclear localization signal enabling interaction of the capsid with so-called importins. These importins mediate binding of the core protein to nuclear pore complexes upon which the capsid disassembles and polymerase/rcDNA complex is released into the nucleus. Within the nucleus the rcDNA becomes deproteinized (removal of polymerase) and is converted by host DNA repair machinery to a covalently closed circular DNA (cccDNA) genome from which overlapping transcripts encode for HBeAg, HBsAg, Core protein, viral polymerase and HBx protein. Core protein, viral polymerase, and pre-genomic RNA (pgRNA) associate in the cytoplasm and self-assemble into immature pgRNA-containing capsid particles, which further convert into mature rcDNA-capsids and function as a common intermediate that is either enveloped and secreted as infectious virus particles or transported back to the nucleus to replenish and maintain a stable cccDNA pool.

To date, 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) based on the sequence of the viral genome. The HBV genotypes are distributed over different geographic regions. For example, the most prevalent genotypes in Asia are genotypes B and C. Genotype D is dominant in Africa, the Middle East, and India, whereas genotype A is widespread in Northern Europe, sub-Saharan Africa, and West Africa.

HBV Antigens

As used herein, the terms“HBV antigen,”“antigenic polypeptide of HBV,”

“HBV antigenic polypeptide,”“HBV antigenic protein,”“HBV immunogenic polypeptide,” and“HBV immunogen” all refer to a polypeptide capable of inducing an immune response, e.g., a humoral and/or cellular mediated response, against an HBV in a subject. The HBV antigen can be a polypeptide of HBV, a fragment or epitope thereof, or a combination of multiple HBV polypeptides, portions or derivatives thereof. An HBV antigen is capable of raising in a host a protective immune response, e.g., inducing an immune response against a viral disease or infection, and/or producing an immunity (i.e., vaccinates) in a subject against a viral disease or infection, that protects the subject against the viral disease or infection. For example, an HBV antigen can comprise a polypeptide or immunogenic fragment(s) thereof from any HBV protein, such as HBeAg, pre-core protein, HBsAg (S, M, or L proteins), core protein, viral polymerase, or HBx protein derived from any HBV genotype, e.g., genotype A, B, C, D, E, F, G, and/or H, or combination thereof.

(Tj HBV Core Antigen

As used herein, each of the terms“HBV core antigen,”“HBc” and“core antigen” refers to an HBV antigen capable of inducing an immune response, e.g., a humoral and/or cellular mediated response, against an HBV core protein in a subject. Each of the terms “core,”“core polypeptide,” and“core protein” refers to the HBV viral core protein. Full- length core 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 pre-genomic RNA encapsidation. This domain also functions as a nuclear import signal. It comprises 17 arginine residues and is highly basic, consistent with its function. HBV core protein is dimeric in solution, with the dimers self-assembling into icosahedral capsids. Each dimer of core protein has four a-helix bundles flanked by an a-helix domain on either side. Truncated HBV core proteins lacking the nucleic acid binding domain are also capable of forming capsids.

In an embodiment of the application, an HBV antigen is a truncated HBV core antigen. As used herein, a“truncated HBV core antigen,” refers to an HBV antigen that does not contain the entire length of an HBV core protein, but is capable of inducing an immune response against the HBV core protein in a subject. For example, an HBV core antigen can be modified to delete one or more amino acids of the highly positively charged (arginine rich) C-terminal nucleic acid binding domain of the core antigen, which typically contains seventeen arginine (R) residues. A truncated HBV core antigen of the application is preferably a C-terminally truncated HBV core protein which does not comprise the HBV core nuclear import signal and/or a truncated HBV core protein from which the C-terminal HBV core nuclear import signal has been deleted. In an embodiment, a truncated HBV core antigen comprises a deletion in the C-terminal nucleic acid binding domain, such as a deletion of 1 to 34 amino acid residues of the C- terminal nucleic acid binding domain, e.g., 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 ammo acid residues, preferably a deletion of all 34 amino acid residues. In a preferred embodiment, a truncated HBV core antigen comprises a deletion in the C-terminal nucleic acid binding domain, preferably a deletion of all 34 amino acid residues.

An HBV core antigen of the application can be a consensus sequence derived from multiple HBV genotypes (e.g., genotypes A, B, C, D, E, F, G, and H). As used herein,“consensus sequence” means an artificial sequence of amino acids based on an alignment of amino acid sequences of homologous proteins, e.g., as determined by an alignment (e.g., using Clustal Omega) of amino acid sequences of homologous proteins.

It can be the calculated order of most frequent amino acid residues, found at each position in a sequence alignment, based upon sequences of HBV antigens (e.g., core, pol, etc.) from at least 100 natural HBV isolates. A consensus sequence can be non- naturally occurring and different from the native viral sequences. Consensus sequences can be designed by aligning multiple HBV antigen sequences from different sources using a multiple sequence alignment tool, and at variable alignment positions, selecting the most frequent amino acid. Preferably, a consensus sequence of an HBV antigen 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 exemplary truncated HBV core antigen according to the application lacks the nucleic acid binding function, and is capable of inducing an immune response in a mammal against at least two HBV genotypes. Preferably a truncated HBV core antigen is capable of inducing a T cell response in a mammal against at least HBV genotypes B,

C and D. More preferably, a truncated HBV core antigen is capable of inducing a CD8 T cell response in a human subject against at least HBV genotypes A, B, C and D.

Preferably, an HBV core antigen of the application is a consensus antigen, preferably a consensus antigen derived from HBV genotypes B, C, and D, more preferably a truncated consensus antigen derived from HBV genotypes B, C, and D. An exemplary truncated HBV core consensus antigen according to the application consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or SEQ ID NO: 37, such as 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: 31 or SEQ ID NO: 37. SEQ ID NO: 31 and SEQ ID NO: 37 are core consensus antigens derived from HBV genotypes B, C, and D. SEQ ID NO: 31 and SEQ ID NO: 37 contain a 34-amino acid C-terminal deletion of the highly positively charged (arginine rich) nucleic acid binding domain of the native core antigen.

In a particular embodiment of the application, an HBV core antigen is a truncated HBV antigen consisting of the amino acid sequence of SEQ ID NO: 34. In another particular embodiment, an HBV core antigen is a truncated HBV antigen consisting of the amino acid sequence of SEQ ID NO: 37.

(21 HBV Polymerase Antigen

As used herein, the term“HBV polymerase antigen,”“HBV Pol antigen” or “HBV pol antigen” refers to an HBV antigen capable of inducing an immune response, e.g., a humoral and/or cellular mediated response, against an HBV polymerase in a subject. Each of the terms“polymerase,”“polymerase polypeptide,”“Pol” and“pol” refers to the HBV viral DNA polymerase. The HBV viral DNA polymerase has four domains, including, from the N terminus to the C terminus, a terminal protein (TP) domain, which acts as a primer for minus-strand DNA synthesis; a spacer that is nonessential for the polymerase functions; a reverse transcriptase (RT) domain for transcription; and a RNase H domain.

In an embodiment of the application, an HBV antigen comprises an HBV Pol antigen, or any immunogenic fragment or combination thereof. An HBV Pol antigen can contain further modifications to improve immunogenicity of the antigen, such as by introducing mutations into the active sites of the polymerase and/or RNase domains to decrease or substantially eliminate certain enzymatic activities.

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

Thus, in some embodiments, an HBV Pol antigen is an inactivated Pol antigen. In an embodiment, an inactivated HBV Pol antigen comprises one or more amino acid mutations in the active site of the polymerase domain. In another embodiment, an inactivated HBV Pol antigen comprises one or more amino acid mutations in the active site of the RNaseH domain. In a preferred embodiment, an inactivated HBV pol antigen comprises one or more amino acid mutations in the active site of both the polymerase domain and the RNaseH domain. For example, the“YXDD” motif in the polymerase domain of an HBV pol antigen that can be required for nucleotide/metal ion binding can be mutated, e.g., by replacing one or more of the aspartate residues (D) with asparagine residues (N), eliminating or reducing metal coordination function, thereby decreasing or substantially eliminating reverse transcriptase function. Alternatively, or in addition to mutation of the“YXDD” motif, the“DEDD” motif in the RNaseH domain of an HBV pol antigen required for Mg2+ coordination can be mutated, e.g., by replacing one or more aspartate residues (D) with asparagine residues (N) and/or replacing the glutamate residue (E) with glutamine (Q), thereby decreasing or substantially eliminating RNaseH function. In a particular embodiment, an HBV pol antigen is modified by (1) mutating the aspartate residues (D) to asparagine residues (N) in the“YXDD” motif of the polymerase domain; and (2) mutating 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 decreasing or substantially eliminating both the reverse transcriptase and RNaseH functions of the pol antigen.

In a preferred embodiment of the application, an 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.

An exemplary HBV pol consensus antigen according to the application comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, such as 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: 33, preferably at least 98% identical to SEQ ID NO: 33, such as 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: 33. SEQ ID NO: 33 is a pol consensus antigen derived from HBV genotypes B, C, and D comprising four mutations located in the active sites of the polymerase and RNaseH domains. In particular, the four mutations include mutation of the aspartic acid residues (D) to asparagine residues (N) 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 RNaseH domain.

In a particular embodiment of the application, an HBV pol antigen comprises the amino acid sequence of SEQ ID NO: 33. In other embodiments of the application, an HBV pol antigen consists of the amino acid sequence of SEQ ID NO: 33.

(31 Fusion of HBV Core Antigen and HBV Polymerase Antigen

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

For example, in a fusion protein containing a first polypeptide and a second heterologous polypeptide, a linker serves primarily as a spacer between the first and second polypeptides. In an embodiment, a linker is made up of amino acids linked together by peptide bonds, preferably from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. In an embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Exemplary linkers are polyglycines, particularly (Gly)5, (Gly)8; poly(Gly-Ala), and polyalanines. One exemplary suitable linker as shown in the Examples below is (AlaGly)n, wherein n is an integer of 2 to 5.

Preferably, a fusion protein of the application is capable of inducing an immune response in a mammal against HBV core and HBV Pol of at least two HBV genotypes. Preferably, a fusion protein is capable of inducing a T cell response in a mammal against at least HBV genotypes B, C and D. More preferably, the fusion protein is capable of inducing a CD8 T cell response in a human subject against at least HBV genotypes A, B,

C and D.

In an embodiment of the application, a fusion protein comprises a truncated HBV core antigen having an amino acid sequence at least 90%, such as 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:

31 or SEQ ID NO: 37, a linker, and a HBV Pol antigen having an amino acid sequence at least 90%, such as 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: 33. In a preferred embodiment of the application, a fusion protein comprises a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 37, a linker comprising (AlaGly)n, wherein n is an integer of 2 to 5, and a HBV Pol antigen having the amino acid sequence of SEQ ID NO: 33. More preferably, a fusion protein according to an embodiment of the application comprises the amino acid sequence of SEQ ID NO: 45.

In an embodiment of the application, a fusion protein further comprises a signal sequence. Preferably, the signal sequence has the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 44. More preferably, a fusion protein comprises the amino acid sequence of SEQ ID NO: 46.

Additional disclosure on HBV vaccines that can be used for the present invention are described in U.S. Patent Application No: 16/223,251, filed December 18, 2018, the contents of the application, more preferably the examples of the application, are hereby incorporated by reference in their entireties.

Polynucleotides and Vectors

In another general aspect, the application provides a first non-naturally occurring nucleic acid molecule encoding an IL12 fusion protein according to the application and a second and/or third non-naturally occurring nucleic acid molecule encoding an HBV antigen according to the application, and vectors comprising the non-naturally occurring nucleic acids. A first non-naturally occurring nucleic acid molecule can comprise any polynucleotide sequence encoding an IL12 fusion protein of the application, which can be made using methods known in the art in view of the present disclosure. A second or third non-naturally occurring nucleic acid molecule can comprise any polynucleotide sequence encoding an HBV antigen of the application, which can be made using methods known in the art in view of the present disclosure. Preferably, a second polynucleotide encodes at least one of a truncated HBV core antigen and an HBV polymerase antigen of the application. A polynucleotide can be in the form of RNA or in the form of DNA obtained by recombinant techniques (e.g., cloning) or produced synthetically (e.g., chemical synthesis). The DNA can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. The DNA can, for example, comprise genomic DNA, cDNA, or combinations thereof. The polynucleotide can also be a DNA/RNA hybrid. The polynucleotides and vectors of the application can be used for recombinant protein production, expression of the protein in host cell, or the production of viral particles. Preferably, a polynucleotide is DNA.

In an embodiment of the application, a first non-naturally occurring nucleic acid molecule comprises a first polynucleotide sequence encoding a fusion protein consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 24, SEQ ID NO:25 or SEQ ID NO:26, such as 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: 24, SEQ ID NO:25 or SEQ ID NO:26, preferably 98%, 99% or 100% identical to SEQ ID NO: 24, SEQ ID NO:25 or SEQ ID NO:26. In a particular embodiment of the application, a first non- naturally occurring nucleic acid molecule encodes a fusion protein consisting the amino acid sequence of SEQ ID NO: 24, SEQ ID NO:25 or SEQ ID NO:26.

Examples of polynucleotide sequences of the application encoding an IL12 fusion protein comprising the amino acid sequence of SEQ ID NO: 24, SEQ ID NO:25 or SEQ ID NO:26 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 27, SEQ ID NO:28 or SEQ ID NO:29, such as 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:

27, SEQ ID NO:28 or SEQ ID NO:29, preferably 98%, 99% or 100% identical to SEQ ID NO: 27, SEQ ID NO:28 or SEQ ID NO:29. Exemplary non-naturally occurring nucleic acid molecules encoding an IL12 fusion protein have the polynucleotide sequence of SEQ ID NO: 27, SEQ ID NO:28 or SEQ ID NO:29.

In an embodiment of the application, second a non-naturally occurring nucleic acid molecule comprises a second polynucleotide sequence encoding a HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, such as 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: 33, preferably 100% identical to SEQ ID NO: 33. In a particular embodiment of the application, a second non-naturally occurring nucleic acid molecule comprises a second polynucleotide sequence encoding a HBV polymerase antigen consisting of the amino acid sequence of SEQ ID NO: 33.

Examples of polynucleotide sequences of the application encoding a HBV Pol antigen comprising the amino acid sequence of SEQ ID NO: 33 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 32 or SEQ ID NO: 39, such as 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: 32 or SEQ ID NO: 39, preferably 98%, 99% or 100% identical to SEQ ID NO: 32 or SEQ ID NO: 39. Exemplary non-naturally occurring nucleic acid molecules encoding a HBV pol antigen have the polynucleotide sequence of SEQ ID NOs: 32 or 39. In an embodiment of the application, a third non-naturally occurring nucleic acid molecule comprises a third polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or SEQ ID NO: 37, such as 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: 31, preferably 98%, 99% or 100% identical to SEQ ID NO: 31 or SEQ ID NO: 37. In a particular embodiment of the application, a third non-naturally occurring nucleic acid molecule comprises a third polynucleotide sequence encoding a truncated HBV core antigen consisting the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 37.

Examples of polynucleotide sequences of the application encoding a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 37 include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 30 or SEQ ID NO: 38, such as 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: 30 or SEQ ID NO: 38, preferably 98%, 99% or 100% identical to SEQ ID NO: 30 or SEQ ID NO: 38. Exemplary non-naturally occurring nucleic acid molecules encoding a truncated HBV core antigen have the polynucleotide sequence of SEQ ID NOs: 30 or 38.

In another embodiment of the application, a non-naturally occurring nucleic acid molecule encodes an HBV antigen fusion protein comprising a truncated HBV core antigen operably linked to a HBV Pol antigen, or a HBV Pol antigen operably linked to a truncated HBV core antigen. In a particular embodiment, a non-naturally occurring nucleic acid molecule of the application encodes a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or SEQ ID NO: 37, such as 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: 31 or SEQ ID NO: 37, preferably 100% identical to SEQ ID NO: 31 or SEQ ID NO: 37, more preferably 100% identical to SEQ ID NO: 37; a linker; and a HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, such as 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: 33, preferably 98%, 99% or 100% identical to SEQ ID NO: 33. In a particular embodiment of the application, a non-naturally occurring nucleic acid molecule encodes a fusion protein comprising a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 37, a linker comprising (AlaGly)n, wherein n is an integer of 2 to 5; and a HBV Pol antigen comprising the amino acid sequence of SEQ ID NO: 33. In a particular embodiment of the application, a non-naturally occurring nucleic acid molecule encodes an HBV antigen fusion protein comprising the amino acid sequence of SEQ ID NO: 45.

Examples of polynucleotide sequences of the application encoding an HBV antigen fusion protein include, but are not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO: 30 or SEQ ID NO: 38, such as 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:

30 or SEQ ID NO: 38, preferably 98%, 99% or 100% identical to SEQ ID NO: 30 or SEQ ID NO: 38, operably linked to a linker coding sequence at least 90% identical to SEQ ID NO: 40, such as 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: 40, preferably 98%, 99% or 100% identical to SEQ ID NO: 40, which is further operably linked a polynucleotide sequence at least 90% identical to SEQ ID NO: 32 or SEQ ID NO: 39, such as 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: 32 or SEQ ID NO: 39, preferably 98%, 99% or 100% identical to SEQ ID NO: 32 or SEQ ID NO: 39. In particular embodiments of the application, a non-naturally occurring nucleic acid molecule encoding an HBV antigen fusion protein comprises SEQ ID NO:

30 or SEQ ID NO: 38, operably linked to SEQ ID NO: 40, which is further operably linked to SEQ ID NO: 32 or SEQ ID NO: 39.

The application also relates to a vector comprising the first, second, and/or third non-naturally occurring nucleic acid molecules. As used herein, a“vector” is a nucleic acid molecule used to carry genetic material into another cell, where it can be replicated and/or expressed. Any vector known to those skilled in the art in view of the present disclosure can be used. Examples of vectors include, but are not limited to, plasmids, viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs). Preferably, a vector is a DNA plasmid. A vector can be a DNA vector or an RNA vector. One of ordinary skill in the art can construct a vector of the application through standard recombinant techniques in view of the present disclosure.

A vector of the application can be an expression vector. As used herein, the term “expression vector” refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed. Expression vectors include, but are not limited to, vectors for recombinant protein expression, such as a DNA plasmid or a viral vector, and vectors for delivery of nucleic acid into a subject for expression in a tissue of the subject, such as a DNA plasmid or a viral vector. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.

Vectors of the application can contain a variety of regulatory sequences. As used herein, the term“regulatory sequence” refers to any sequence that allows, contributes or modulates the functional regulation of the nucleic acid molecule, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (e.g., mRNA) into the host cell or organism. In the context of the disclosure, this term encompasses promoters, enhancers and other expression control elements (e.g., polyadenylation signals and elements that affect mRNA stability).

In some embodiments of the application, a 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, RNA replicon, mRNA replicon, modified mRNA replicon or self-amplifying mRNA, closed linear deoxyribonucleic acid, e.g. a linear covalently closed DNA such as linear covalently closed double stranded DNA molecule. Preferably, a non-viral vector is a DNA plasmid. A“DNA plasmid”, which is used interchangeably with“DNA plasmid vector,”“plasmid DNA” or“plasmid DNA vector,” refers to a double-stranded and generally circular DNA sequence that is capable of autonomous replication in a suitable host cell. DNA plasmids used for expression of an encoded polynucleotide typically comprise an origin of replication, a multiple cloning site, and a selectable marker, which for example, can be an antibiotic resistance gene. Examples of DNA plasmids suitable 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 production and/or expression of protein in

Escherichia coli; pYES2 (Invitrogen, Thermo Fisher Scientific), which can be used for production and/or expression in Saccharomyces cerevisiae strains of yeast; MAXBAC® complete baculovirus expression system (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 for high level constitutive protein expression in mammalian cells; and pVAX or pVAX-1 (Life Technologies, Thermo Fisher Scientific), which can be used for high-level transient expression of a protein of interest in most mammalian cells. The backbone of any commercially available DNA plasmid can be modified to optimize protein expression in the host cell, such as to reverse the orientation of certain elements (e.g., origin of replication and/or antibiotic resistance cassette), replace a promoter endogenous to the plasmid (e.g., the promoter in the antibiotic resistance cassette), and/or replace the polynucleotide sequence encoding transcribed proteins (e.g., the coding sequence of the antibiotic resistance gene), by using routine techniques and readily available starting materials. (See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)).

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

A vector of the application can also be a viral vector. In general, viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that has been rendered non-infectious, but still contains viral promoters and transgenes, thus allowing for translation of the transgene through a viral promoter. Because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Examples of viral vectors that can be used include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors, etc. Examples of viral vectors that can be used include, but are not limited to, arenavirus viral vectors, replication- deficient arenavirus viral vectors or replication-competent arenavirus viral vectors, bi- segmented or tri-segmented arenavirus, infectious arenavirus viral vectors, nucleic acids which comprise 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 a HBV antigen as described herein), arenavirus such as lymphocytic choriomeningitidis virus (LCMV), e.g., clone 13 strain or MP strain, and arenavirus such as Junin virus e.g., Candid #1 strain. The vector can also be a non-viral vector.

Preferably, a viral vector is an adenovirus vector, e.g., a recombinant adenovirus vector. A recombinant adenovirus vector can for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV) or rhesus adenovirus (rhAd). Preferably, an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc. In other embodiments, an adenovirus vector is a rhAd vector, e.g.

rhAd51, rhAd52 or rhAd53.

The vector can also be a linear covalently closed double-stranded DNA vector.

As used herein, a“linear covalently closed double-stranded DNA vector” refers to a closed linear deoxyribonucleic acid (DNA) that is structurally distinct from a plasmid DNA. It has many of the advantages of plasmid DNA as well as a minimal cassette size similar to RNA strategies. For example, it can be a vector cassette generally comprising an encoded antigenic sequence, a promoter, a polyadenylation sequence, and telomeric ends. The plasmid-free construct can be synthesized through an enzymatic process without the need for bacterial sequences. Examples of suitable linear covalently closed DNA vectors include, but are not limited to, commercially available expression vectors such as‘Doggybone™ closed linear DNA’ (dbDNA™) (Touchlight Genetics Ltd.;

London, England). See, e.g., Scott et al, Hum Vaccin Immunother. 2015 Aug; 11(8): 1972-1982, the entire content of which is incorporated herein by reference. Some examples of linear covalently closed double-stranded DNA vectors, compositions and methods to create and use such vectors for delivering DNA molecules, such as active molecules of this invention, are described in US2012/0282283, US2013/0216562, and US2018/0037943, the relevant content of each of which is hereby incorporated by reference in its entirety.

A recombinant vector useful for the application can be prepared using methods known in the art in view of the present disclosure. For example, in view of the degeneracy of the genetic code, several nucleic acid sequences can be designed that encode the same polypeptide. A polynucleotide encoding an HBV antigen of the application can optionally be codon-optimized to ensure proper expression in the host cell (e.g., bacterial or mammalian cells). Codon-optimization is a technology widely applied in the art, and methods for obtaining codon- optimized polynucleotides will be well known to those skilled in the art in view of the present disclosure. A vector of the application, e.g., a DNA plasmid, a viral vector (particularly an adenoviral vector), an RNA vector (such as a self-replicating RNA replicon), or a linear covalently closed double-stranded DNA vector, can comprise any regulatory elements to establish conventional function(s) of the vector, including but not limited to replication and expression of the HBV antigen(s) encoded by the polynucleotide sequence of the vector. Regulatory elements include, but are not limited to, a promoter, an enhancer, a polyadenylation signal, translation stop codon, a ribosome binding element, a

transcription terminator, selection markers, origin of replication, etc. A vector can comprise one or more expression cassettes. An“expression cassette” is part of a vector that directs the cellular machinery to make RNA and protein. An expression cassette typically comprises three components: a promoter sequence, an open reading frame, and a 3’-untranslated region (UTR) optionally comprising a polyadenylation signal. An open reading frame (ORF) is a reading frame that contains a coding sequence of a protein of interest (e.g., HBV antigen) from a start codon to a stop codon. Regulatory elements of the expression cassette can be operably linked to a polynucleotide sequence encoding an HBV antigen of interest. As used herein, the term“operably linked” is to be taken in its broadest reasonable context, and refers to a linkage of polynucleotide elements in a functional relationship. A polynucleotide is“operably linked” when it is placed into a functional relationship with another polynucleotide. For instance, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. Any components suitable for use in an expression cassette described herein can be used in any combination and in any order to prepare vectors of the application.

A vector can comprise a promoter sequence, preferably within an expression cassette, to control expression of an IL12 fusion protein or an HBV antigen of interest. The term“promoter” is used in its conventional sense, and refers to a nucleotide sequence that initiates the transcription of an operably linked nucleotide sequence. A promoter is located on the same strand near the nucleotide sequence it transcribes.

Promoters can be a constitutive, inducible, or repressible. Promoters can be naturally occurring or synthetic. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source). For example, if the vector to be employed is a DNA plasmid, the promoter can be endogenous to the plasmid

(homologous) or derived from other sources (heterologous). Preferably, the promoter is located upstream of the polynucleotide encoding an HBV antigen within an expression cassette. Examples of promoters that can be used include, but are not limited to, a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter (CMV-IE), Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. A promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. A promoter can also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic.

Preferably, a promoter is a strong eukaryotic promoter, preferably a

cytomegalovirus immediate early (CMV-IE) promoter. A nucleotide sequence of an exemplary CMV-IE promoter is shown in SEQ ID NO: 18 and SEQ ID NO: 47.

A vector can comprise additional polynucleotide sequences that stabilize the expressed transcript, enhance nuclear export of the RNA transcript, and/or improve transcriptional-translational coupling. Examples of such sequences include

polyadenylation signals and enhancer sequences. A polyadenylation signal is typically located downstream of the coding sequence for a protein of interest (e.g., an IL12 fusion protein or an HBV antigen) within an expression cassette of the vector. Enhancer sequences are regulatory DNA sequences that, when bound by transcription factors, enhance the transcription of an associated gene. An enhancer sequence is preferably located upstream of the polynucleotide sequence encoding an IL12 fusion protein or HBV antigen, but downstream of a promoter sequence within an expression cassette of the vector.

Any polyadenylation signal known to those skilled in the art in view of the present disclosure can be used. For example, the polyadenylation signal can be a SV40 polyadenylation signal (e.g., SEQ ID NO: 42), LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human b-globin polyadenylation signal. Preferably, a polyadenylation signal is a bovine growth hormone (bGH) polyadenylation signal or a SV40 polyadenylation signal. A nucleotide sequence of an exemplary bGH

polyadenylation signal is shown in SEQ ID NO: 19. A nucleotide sequence of an exemplary SV40 polyadenylation signal is shown in SEQ ID NO: 42.

Any enhancer sequence known to those skilled in the art in view of the present disclosure can be used. For example, an enhancer sequence can be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as one from CMV, HA, RSV, or EBV. Examples of particular enhancers include, but are not limited to, Woodchuck HBV Post-transcriptional regulatory element (WPRE), intron/exon sequence derived from human apolipoprotein A1 precursor (ApoAI), untranslated R-U5 domain of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR), a splicing enhancer, a synthetic rabbit b-globin intron, or any combination thereof. Preferably, an enhancer sequence is a composite sequence of three consecutive elements of the untranslated R-U5 domain of HTLV-1 LTR, rabbit b-globin intron, and a splicing enhancer, which is referred to herein as“a triple enhancer sequence.” A nucleotide sequence of an exemplary triple enhancer sequence is shown in SEQ ID NO: 36. Another exemplary enhancer sequence is an ApoAI gene fragment shown in SEQ ID NO: 41.

A vector can comprise a polynucleotide sequence encoding a signal peptide sequence. Preferably, the polynucleotide sequence encoding the signal peptide sequence is located upstream of the polynucleotide sequence encoding an HBV antigen. Signal peptides typically direct localization of a protein, facilitate secretion of the protein from the cell in which it is produced, and/or improve antigen expression and cross-presentation to antigen-presenting cells. A signal peptide can be present at the N-terminus of an HBV antigen when expressed from the vector, but is cleaved off by signal peptidase, e.g., upon secretion from the cell. An expressed protein in which a signal peptide has been cleaved is often referred to as the“mature protein.” Any signal peptide known in the art in view of the present disclosure can be used. For example, a signal peptide can be a cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as the Ig heavy chain gamma signal peptide SPIgG or the Ig heavy chain epsilon signal peptide SPIgE.

Preferably, a 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: 34 and 35, respectively. Exemplary nucleic acid and amino acid sequences of an immunoglobulin secretion signal are shown in SEQ ID NOs: 43 and 44, respectively.

A vector, such as a DNA plasmid, can also include a bacterial origin of replication and an antibiotic resistance expression cassette for selection and maintenance of the plasmid in bacterial cells, e.g., E. coli. Bacterial origins of replication and antibiotic resistance cassettes can be located in a vector in the same orientation as the expression cassette encoding an IL12 fusion protein or an HBV antigen, or in the opposite (reverse) orientation. An origin of replication (ORI) is a sequence at which replication is initiated, enabling a plasmid to reproduce and survive within cells. Examples of ORIs suitable for use in the application include, but are not limited to ColEl, pMBl, pUC, pSClOl, R6K, and 15 A, preferably pUC. An exemplary nucleotide sequence of a pUC ORI is shown in SEQ ID NO: 21.

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 an antibiotic resistance gene differs from the promoter sequence operably linked to a polynucleotide sequence encoding a protein of interest, e.g., HBV antigen. The antibiotic resistance gene can be codon optimized, and the sequence composition of the antibiotic resistance gene is normally adjusted to bacterial, e.g., E. coli, codon usage. Any antibiotic resistance gene known to those skilled in the art in view of the present disclosure can be used, including, but not limited to, kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr), and tetracycline resistance gene (Tetr), as well as genes conferring resistance to chloramphenicol, bleomycin, spectinomycin, carbenicillin, etc.

Preferably, an antibiotic resistance gene in the antibiotic expression cassette of a vector is a kanamycin resistance gene (Kanr). The sequence of Kanr gene is shown in SEQ ID NO: 22. Preferably, the Kanr gene is codon optimized. An exemplary nucleic acid sequence of a codon optimized Kanr gene is shown in SEQ ID NO: 23. The Kanr can be operably linked to its native promoter, or the Kanr gene can be linked to a heterologous promoter. In a particular embodiment, the Kanr gene is operably linked to the ampicillin resistance gene (Ampr) promoter, known as the bla promoter. An exemplary nucleotide sequence of a bla promoter is shown in SEQ ID NO: 20.

In a particular embodiment of the application, a vector is a DNA plasmid comprising an expression cassette including a polynucleotide encoding an IL12 fusion protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 24; an upstream sequence operably linked to the polynucleotide encoding the IL12 fusion protein comprising, from 5’ end to 3’ end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID NO: 18, and an enhancer sequence; and a downstream sequence operably linked to the polynucleotide encoding the IL12 fusion protein comprising a polyadenylation signal, preferably a bGH polyadenylation signal of SEQ ID NO: 19.

Such vector further comprises an antibiotic resistance expression cassette including a polynucleotide encoding an antibiotic resistance gene, preferably a Kanr gene, more preferably a codon optimized Kanr gene that is at least 90% identical to SEQ ID NO: 22, such as 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 100% identical to SEQ ID NO: 22, operably linked to an Ampr (bla) promoter of SEQ ID NO: 20, upstream of and operably linked to the polynucleotide encoding the antibiotic resistance gene; and an origin of replication, preferably a pUC ori of SEQ ID NO: 21. Preferably, the antibiotic resistance cassette and the origin of replication are present in the plasmid in the reverse orientation relative to the IL12 fusion protein expression cassette. Exemplary DNA plasmids comprising the above mentioned features are shown in FIG. 1 and FIG. 2.

In another embodiment, a vector is a DNA plasmid comprising an expression cassette including a polynucleotide encoding an IL12 fusion protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 26; an upstream sequence operably linked to the polynucleotide encoding the IL12 fusion protein comprising, from 5’ end to 3’ end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID NO: 18, and an enhancer sequence; and a downstream sequence operably linked to the

polynucleotide encoding the IL12 fusion protein comprising a polyadenylation signal, preferably a bGH polyadenylation signal of SEQ ID NO: 19. Such vector further comprises an antibiotic resistance expression cassette including a polynucleotide encoding an antibiotic resistance gene, preferably a Kanr gene, more preferably a codon optimized Kanr gene that is at least 90% identical to SEQ ID NO: 23, such as 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: 23, preferably 100% identical to SEQ ID NO: 23, operably linked to an Ampr (bla) promoter of SEQ ID NO: 20, upstream of and operably linked to the polynucleotide encoding the antibiotic resistance gene; and an origin of replication, preferably a pUC ori of SEQ ID NO: 21. Preferably, the antibiotic resistance cassette and the origin of replication are present in the plasmid in the reverse orientation relative to the IL12 fusion protein expression cassette.

In a particular embodiment of the application, a vector is a DNA plasmid comprising an expression cassette including a polynucleotide encoding at least one of an HBV antigen selected from the group consisting of an HBV pol antigen comprising an amino acid sequence at least 98% identical to SEQ ID NO: 33, such as 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: 33, and a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 31; an upstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising, from 5’ end to 3’ end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID NO: 18, an enhancer sequence, preferably a triple enhancer sequence of SEQ ID NO: 36, and a polynucleotide sequence encoding a signal peptide sequence, preferably a cystatin S signal peptide having the amino acid sequence of SEQ ID NO: 35; and a downstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising a polyadenylation signal, preferably a bGH polyadenylation signal of SEQ ID NO: 19. Such vector further comprises an antibiotic resistance expression cassette including a polynucleotide encoding an antibiotic resistance gene, preferably a Kanr gene, more preferably a codon optimized Kanr gene of at least 90% identical to SEQ ID NO: 23, such as 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: 23, preferably 100% identical to SEQ ID NO: 23, operably linked to an Ampr (bla) promoter of SEQ ID NO: 20, upstream of and operably linked to the polynucleotide encoding the antibiotic resistance gene; and an origin of replication, preferably a pUC ori of SEQ ID NO: 21. Preferably, the antibiotic resistance cassette and the origin of replication are present in the plasmid in the reverse orientation relative to the HBV antigen expression cassette.

In another particular embodiment of the application, a vector is a viral vector, preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector, comprising an expression cassette including a polynucleotide encoding at least one of an HBV antigen selected from the group consisting of an HBV pol antigen comprising an amino acid sequence at least 98% identical to SEQ ID NO: 33, such as 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: 33, and a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 37; an upstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising, from 5’ end to 3’ end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID NO: 47, an enhancer sequence, preferably an ApoAI gene fragment sequence of SEQ ID NO: 41, and a polynucleotide sequence encoding a signal peptide sequence, preferably an immunoglobulin secretion signal having the amino acid sequence of SEQ ID NO: 44; and a downstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising a polyadenylation signal, preferably a SV40 polyadenylation signal of SEQ ID NO: 42.

In an embodiment of the application, a vector, such as a plasmid DNA 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: 33.

Preferably, the vector comprises a coding sequence for the HBV Pol antigen that is at least 90% identical to the polynucleotide sequence of SEQ ID NO: 32, such as 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: 32, preferably 100% identical to SEQ ID NO: 32. In an embodiment of the application, a vector, such as a plasmid DNA vector or a viral vector (preferably an adenoviral vector, more preferably an Ad26 or Ad35 vector), encodes a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 37. Preferably, the vector comprises a coding sequence for the truncated HBV core antigen that is at least 90% identical to the polynucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 38, such as 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: 30 or SEQ ID NO: 38, preferably 100% identical to SEQ ID NO: 30 or SEQ ID NO: 38.

In yet another embodiment of the application, a vector, such as a plasmid DNA 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: 33 and a truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 37. Preferably, the vector comprises a coding sequence for the fusion, which contains a coding sequence for the truncated HBV core antigen at least 90% identical to SEQ ID NO: 30 or SEQ ID NO: 38, such as 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: 30 or SEQ ID NO: 38, preferably 98%, 99% or 100% identical to SEQ ID NO: 30 or SEQ ID NO: 38, more preferably SEQ ID NO: 38, operably linked to a coding sequence for the HBV Pol antigen at least 90% identical to SEQ ID NO: 32 or SEQ ID NO: 39, such as 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: 32 or SEQ ID NO: 39, preferably 98%, 99% or 100% identical to SEQ ID NO: 32 or SEQ ID NO: 39, more preferably SEQ ID NO: 39. Preferably, the coding sequence for the truncated HBV core antigen is operably linked to the coding sequence for the HBV Pol antigen via a coding sequence for a linker at least 90% identical to SEQ ID NO: 40, such as 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: 40, preferably 98%, 99% or 100% identical to SEQ ID NO: 40. In particular embodiments of the application, a vector comprises a coding sequence for the fusion having SEQ ID NO: 38 operably linked to SEQ ID NO: 40, which is further operably linked to SEQ ID NO: 39.

The polynucleotides and expression vectors encoding the HBV antigens of the application can be made by any method known in the art in view 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, e.g., polymerase chain reaction (PCR), etc., which are well known to those skilled in the art.

Cells. Polypeptides and Antibodies

The application also provides cells, preferably isolated cells, comprising any of the polynucleotides and vectors described herein. The cells can, for instance, be used for recombinant protein production, or for the production of viral particles.

Embodiments of the application thus also relate to a method of making an HBV antigen of the application. The method comprises transfecting a host cell with an expression vector comprising a polynucleotide encoding an HBV antigen of the application 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. The HBV antigen can be isolated or collected from the cell by any method known in the art including affinity chromatography, size exclusion chromatography, etc. Techniques used for recombinant protein expression will be well known to one of ordinary skill in the art in view of the present disclosure. The expressed HBV antigens can also be studied without purifying or isolating the expressed protein, e.g., by analyzing the supernatant of cells transfected with an expression vector encoding the HBV antigen and grown under conditions suitable for expression of the HBV antigen.

Thus, also provided are non-naturally occurring or recombinant polypeptides comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 37, or SEQ ID NO: 33. As described above and below, isolated nucleic acid molecules encoding these sequences, vectors comprising these sequences operably linked to a promoter, and compositions comprising the polypeptide, polynucleotide, or vector are also contemplated by the application.

In an embodiment of the application, a recombinant polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 31, such as 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: 31. Preferably, a non-naturally occurring or recombinant polypeptide consists of SEQ ID NO: 31.

In another embodiment of the application, a non-naturally occurring or recombinant polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 33, such as 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: 33. Preferably, a non-naturally occurring or recombinant polypeptide comprises SEQ ID NO: 33.

In another embodiment of the application, a non-naturally occurring or recombinant polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 37, such as 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: 37. Preferably, a non-naturally occurring or recombinant polypeptide consists of SEQ ID NO: 37.

Also provided are antibodies or antigen binding fragments thereof that specifically bind to a non-naturally occurring polypeptide of the application. In an embodiment of the application, an antibody specific to a non-naturally HBV antigen of the application does not bind specifically to another HBV antigen. For example, an antibody of the application that binds specifically to an HBV Pol antigen having the amino acid sequence of SEQ ID NO: 33 will not bind specifically to an HBV Pol antigen not having the amino acid sequence of SEQ ID NO: 33.

As used herein, the term“antibody” includes polyclonal, monoclonal, chimeric, humanized, Fv, Fab and F(ab')2; bifunctional hybrid (e.g., Lanzavecchia et al, Eur. J. Immunol. 17: 105, 1987), single-chain (Huston et al, Proc. Natl. Acad. Sci. USA

85:5879, 1988; Bird et al., Science 242:423, 1988); and antibodies with altered constant regions (e.g., U.S. Pat. No. 5,624,821).

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

The smaller the value of the KD of an antibody, the higher affinity that the antibody binds to a target antigen.

Compositions, Immunogenic Combinations, and Vaccines

The application also relates to compositions, immunogenic combinations, more particularly kits, and vaccines comprising a fusion protein comprising a) an IL12 p40 subunit b) a linker consisting of the amino acid sequence of SEQ ID NO: 3; and c) an IL12 p35 subunit; and one or more HBV antigens, polynucleotides, and/or vectors encoding one or more HBV antigens according to the application. Any of the fusion proteins, HBV antigens, polynucleotides (including RNA and DNA), and/or vectors of the application described herein can be used in the compositions, immunogenic combinations or kits, and vaccines of the application.

The application provides a composition comprising an isolated or non-naturally occurring nucleic acid molecule comprising a polynucleotide sequence encoding the fusion protein consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 24, SEQ ID NO:25 or SEQ ID NO:26, a vector comprising the isolated or non- naturally occurring nucleic acid molecule, and/or an isolated or non-naturally occurring polypeptide encoded by the isolated or non-naturally occurring nucleic acid molecule.

In an embodiment of the application, a composition comprises an isolated or non- naturally occurring nucleic acid molecule (DNA or RNA) comprising polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or SEQ ID NO: 37, or a HBV polymerase antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, a vector comprising the isolated or non-naturally occurring nucleic acid molecule, and/or an isolated or non-naturally occurring polypeptide encoded by the isolated or non- naturally occurring nucleic acid molecule.

In an embodiment of the application, a composition comprises an isolated or non- naturally occurring nucleic acid molecule (DNA or RNA) comprising a polynucleotide sequence encoding a HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, preferably 100% identical to SEQ ID NO: 33.

In an embodiment of the application, a composition comprises an isolated or non- naturally occurring nucleic acid molecule (DNA or RNA) encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or SEQ ID NO: 37, preferably 100% identical to SEQ ID NO: 31 or SEQ ID NO: 37.

In an embodiment of the application, a composition comprises an isolated or non- naturally occurring nucleic acid molecule (DNA or RNA) comprising a polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or SEQ ID NO: 37, preferably 100% identical to SEQ ID NO: 31 or SEQ ID NO: 37; and an isolated or non-naturally occurring nucleic acid molecule (DNA or RNA) comprising a polynucleotide sequence encoding a HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, preferably 100% identical to SEQ ID NO: 33. The coding sequences for the truncated HBV core antigen and the HBV Pol antigen can be present in the same isolated or non-naturally occurring nucleic acid molecule (DNA or RNA), or in two different isolated or non-naturally occurring nucleic acid molecules (DNA or RNA).

In an embodiment of the application, a composition comprises a vector, preferably a DNA plasmid or a viral vector comprising a polynucleotide encoding IL12 fusion protein consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 24, SEQ ID NO:25 or SEQ ID NO:26, preferably 100% identical to SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

In an embodiment of the application, a composition comprises a vector, preferably a DNA plasmid or a viral vector (such as an adenoviral vector) comprising a

polynucleotide encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or SEQ ID NO: 37, preferably 100% identical to SEQ ID NO: 31 or SEQ ID NO: 37.

In an embodiment of the application, a composition comprises a vector, preferably a DNA plasmid or a viral vector (such as an adenoviral vector), comprising a

polynucleotide encoding a HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, preferably 100% identical to SEQ ID NO: 33.

polynucleotide encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or SEQ ID NO: 37, preferably 100% identical to SEQ ID NO: 31 or SEQ ID NO: 37; and a vector, preferably a DNA plasmid or a viral vector (such as an adenoviral vector), comprising a polynucleotide encoding a HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, preferably 100% identical to SEQ ID NO: 33. The vector comprising the coding sequence for the truncated HBV core antigen and the vector comprising the coding sequence for the HBV Pol antigen can be the same vector, or two different vectors.

polynucleotide encoding a fusion protein comprising a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or SEQ ID NO: 37, preferably 100% identical to SEQ ID NO: 31 or SEQ ID NO: 37, operably linked to a HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, preferably 100% identical to SEQ ID NO: 33, 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 of (AlaGly)n, wherein n is an integer of 2 to 5.

In an embodiment of the application, a composition comprises an isolated or non- naturally occurring truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or SEQ ID NO: 37, preferably 100% identical to SEQ ID NO: 31 or SEQ ID NO: 37.

In an embodiment of the application, a 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: 33, preferably 100% identical to SEQ ID NO: 33.

In an embodiment of the application, a composition comprises an isolated or non- naturally occurring truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or SEQ ID NO: 37, preferably 100% identical to SEQ ID NO: 31 or SEQ ID NO: 37; 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: 33, preferably 100% identical to SEQ ID NO: 33.

In an embodiment of the application, a composition comprises an isolated or non- naturally occurring fusion protein comprising a truncated HBV core antigen consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 31 or SEQ ID NO:

14, preferably 100% identical to SEQ ID NO: 31 or SEQ ID NO: 37, operably linked to a HBV Pol antigen comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, preferably 100% identical to SEQ ID NO: 33, 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 of (AlaGly)n, wherein n is an integer of 2 to 5.

The application also relates to an immunogenic combination or a kit comprising polynucleotides expressing a truncated HBV core antigen and an HBV pol antigen according to embodiments of the application. Any polynucleotides and/or vectors encoding HBV core and pol antigens of the application described herein can be used in the immunogenic combinations or kits of the application.

According to embodiments of the application, an immunogenic combination or kit comprises:

i) a fusion protein comprising:

a) an IL12 p40 subunit;

b) a linker consisting of the amino acid sequence of SEQ ID NO: 3; and c) an IL12 p35 subunit; wherein the fusion protein is arranged from N-terminus to C-terminus in the order (a)-(b)- (c), and the C-terminus of the IL12 p40 subunit is fused to the N-terminus of the IL12 p35 subunit through the linker, or

a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding the fusion protein; and

ii) at least one of:

In a particular embodiment of the application, an immunogenic combination or kit comprises: i) a first non-naturally occurring nucleic acid molecule comprising a first polynucleotide sequence encoding a fusion protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24; ii) a second non-naturally occurring nucleic acid molecule comprising a second polynucleotide sequence encoding an HBV polymerase antigen having an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity; and iii) a third non-naturally occurring nucleic acid molecule comprising a third polynucleotide sequence encoding a truncated HBV core antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 31.

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

In a preferred embodiment, the first, second, and third polynucleotides are present in separate vectors, e.g., DNA plasmids or viral vectors. Preferably, the separate vectors are present in the same composition.

According to preferred embodiments of the application, an immunogenic combination or kit comprises a first polynucleotide present in a first vector, a second polynucleotide present in a second vector, and a third polynucleotide present in a third vector. The first, second, and third vectors can be the same or different. Preferably the vectors are DNA plasmids.

In a particular embodiment of the application, the first vector is a first DNA plasmid, the second vector is a second DNA plasmid, and the third is a third DNA plasmid. Each of the first, second, and third DNA plasmids comprises an origin of replication, preferably pUC ORI of SEQ ID NO: 21, and an antibiotic resistance cassette, preferably comprising a codon optimized Kanr gene having a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 23, preferably under control of a bla promoter, for instance the bla promoter shown in SEQ ID NO: 20. Each of the first, second and third DNA plasmids independently further comprises at least one of a promoter sequence, enhancer sequence, and a polynucleotide sequence encoding a signal peptide sequence operably linked to the first polynucleotide sequence, the second polynucleotide sequence, or the third polynucleotide sequence. Preferably, each of the second and third DNA plasmids comprises an upstream sequence operably linked to the first polynucleotide or the second polynucleotide, wherein the upstream sequence comprises, from 5’ end to 3’ end, a promoter sequence of SEQ ID NO: 18, an enhancer sequence, and a polynucleotide sequence encoding a signal peptide sequence having the amino acid sequence of SEQ ID NO: 35. Each of the first and second DNA plasmids can also comprise a polyadenylation signal located downstream of the coding sequence of the HBV antigen, such as the bGH polyadenylation signal of SEQ ID NO: 19.

In one particular embodiment of the application, the second vector is a viral vector and the third vector is a viral vector. Preferably, each of the viral vectors is an adenoviral vector, more preferably an Ad26 or Ad35 vector, comprising an expression cassette including the polynucleotide encoding an HBV pol antigen or an truncated HBV core antigen of the application; an upstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising, from 5’ end to 3’ end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID NO: 47, an enhancer sequence, preferably an ApoAI gene fragment sequence of SEQ ID NO: 41, and a polynucleotide sequence encoding a signal peptide sequence, preferably an immunoglobulin secretion signal having the amino acid sequence of SEQ ID NO: 44; and a downstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising a

polyadenylation signal, preferably a SV40 polyadenylation signal of SEQ ID NO: 42.

In another preferred embodiment, the second and third polynucleotides are present in a single vector, e.g., DNA plasmid or viral vector. Preferably, the single vector is an adenoviral vector, more preferably an Ad26 vector, comprising an expression cassette including a polynucleotide encoding an HBV pol antigen and a truncated HBV core antigen of the application, preferably encoding an HBV pol antigen and a truncated HBV core antigen of the application as a fusion protein; an upstream sequence operably linked to the polynucleotide encoding the HBV pol and truncated core antigens comprising, from 5’ end to 3’ end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID NO: 47, an enhancer sequence, preferably an ApoAI gene fragment sequence of SEQ ID NO: 41, and a polynucleotide sequence encoding a signal peptide sequence, preferably an immunoglobulin secretion signal having the amino acid sequence of SEQ ID NO: 44; and a downstream sequence operably linked to the polynucleotide encoding the HBV antigen comprising a polyadenylation signal, preferably a SV40 polyadenylation signal of SEQ ID NO: 42.

When an immunogenic combination of the application comprises a first vector, such as a DNA plasmid or viral vector, and a second vector, such as a DNA plasmid or 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 can 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 plasmids are present in a ratio of 1 : 1, by weight. The immunogenic combination of the application can further comprise a third vector. Compositions and immunogenic combinations of the application can comprise additional polynucleotides or vectors encoding additional HBV antigens and/or additional HBV antigens or immunogenic fragments thereof. However, in particular embodiments, the compositions and immunogenic combinations of the application do not comprise certain antigens.

In a particular embodiment, a composition or immunogenic combination or kit of the application does not comprise a HBsAg or a polynucleotide sequence encoding the HBsAg.

In another particular embodiment, a composition or immunogenic combination or kit of the application does not comprise a HBV L protein or a polynucleotide sequence encoding the HBV L protein.

In yet another particular embodiment of the application, a composition or immunogenic combination of the application does not comprise a HBV envelope protein or a polynucleotide sequence encoding the HBV envelope protein.

Compositions and immunogenic combinations of the application can also comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is non-toxic and should not interfere with the efficacy of the active ingredient.

Pharmaceutically acceptable carriers can include one or more excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings.

Pharmaceutically acceptable carriers can include vehicles, such as lipid nanoparticles (LNPs). The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes. For liquid injectable preparations, for example, suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like. For solid oral preparations, for example, powders, capsules, caplets, gel caps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. For nasal sprays/inhalant mixtures, the aqueous solution/suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives.

Compositions and immunogenic combinations of the application can be formulated in any matter suitable for administration to a subject to facilitate

administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections. The parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection. Compositions of the application can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal.

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

According to embodiments of the application, compositions and immunogenic combinations for administration will typically comprise a buffered solution in a pharmaceutically acceptable carrier, e.g., an aqueous carrier such as buffered saline and the like, e.g., phosphate buffered saline (PBS). The compositions and immunogenic combinations can also contain pharmaceutically acceptable substances as required to approximate physiological conditions such as pH adjusting and buffering agents. For example, a composition or immunogenic combination of the application comprising plasmid DNA can contain phosphate buffered saline (PBS) as the pharmaceutically acceptable carrier. The plasmid DNA can be present in a concentration of, e.g., 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 at 1 mg/mL.

Compositions and immunogenic combinations of the application can be formulated as a vaccine (also referred to as an“immunogenic composition”) according to methods well known in the art. Such compositions can include adjuvants to enhance immune responses. The optimal ratios of each component in the formulation can be determined by techniques well known to those skilled in the art in view of the present disclosure.

In a particular embodiment of the application, a composition or immunogenic combination is a DNA vaccine. DNA vaccines typically comprise bacterial plasmids containing a polynucleotide encoding an antigen of interest under control of a strong eukaryotic promoter. Once the plasmids are delivered to the cell cytoplasm of the host, the encoded antigen is produced and processed endogenously. The resulting antigen typically induces both humoral and cell-medicated immune responses. DNA vaccines are advantageous at least because they offer improved safety, are temperature stable, can be easily adapted to express antigenic variants, and are simple to produce. Any of the DNA plasmids of the application can be used to prepare such a DNA vaccine. In other particular embodiments of the application, a composition or immunogenic combination is an RNA vaccine. RNA vaccines typically comprise at least one single- stranded RNA molecule encoding an antigen of interest, e.g., a fusion protein or HBV antigen according to the application. Once the RNA is delivered to the cell cytoplasm of the host, the encoded antigen is produced and processed endogenously, inducing both humoral and cell-mediated 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 view of the present disclosure to enhance stability and/or translation, such by adding a polyA tail, e.g., of at least 30 adenosine residues; and/or capping the 5-end with a modified ribonucleotide, e.g., 7- methylguanosine cap, which can be incorporated during RNA synthesis or enzymatically engineered after RNA transcription. An RNA vaccine can also be self-replicating RNA vaccine developed from an alphavirus expression vector. Self-replicating RNA vaccines comprise a replicase RNA molecule derived from a virus belonging to the alphavirus family with a subgenomic promoter that controls replication of the fusion protein or HBV antigen RNA followed by an artificial poly A tail located downstream of the replicase.

While not wishing to be bound by theories, it is believed that an IL 12 fusion construct according to embodiment of the invention services as an adjuvant and enhances the immune response stimulated by a HBV vaccine.

In certain embodiments, a further adjuvant or active agent can be included in a composition or immunogenic combination of the application, or co-administered with a composition or immunogenic combination of the application. Use of another adjuvant or active agent is optional, and can further enhance immune responses when the composition is used for vaccination purposes. Other adjuvants or active agents suitable for co-administration or inclusion in compositions in accordance with the application should preferably be ones that are potentially safe, well tolerated and effective in humans. An adjuvant or active agent can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PDl, anti-HM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/or TLR8 agonists), RIG-1 agonists, IL-15 superagonists (Altor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, and IL-7-hyFc. For example, adjuvants or active agents can e.g., be chosen from among the following anti -HBV agents: HBV DNA polymerase inhibitors; Immunomodulators; Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll-like receptor 3 modulators; Interferon alpha receptor ligands; Hyaluronidase inhibitors; Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators; Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of ribonucleotide reductase; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; 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 (NAPs); Stimulators of retinoic acid-inducible gene 1 ; Stimulators of NOD2; 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, HGIT inhibitors, Lag3 inhibitors, CTLA-4 inhibitors; Agonists of co- stimulatory receptors that are expressed on immune cells (more particularly T cells), such as CD27 and CD28; BTK inhibitors; Other drugs for treating HBV; IDO inhibitors; Arginase inhibitors; and KDM5 inhibitors.

In certain embodiments, the combination further comprises a surface antigen of the hepatitis B virus or a nucleic acid encoding the surface antigen. Examples of a surface antigen of the hepatitis B virus or a nucleic acid encoding the surface antigen useful for the invention are described in European Patent Application Ser. No. 19180926.8 entitled “Hepatitis B Virus (HBV) Vaccines and Uses Thereof,” filed on the same day as this application with the Attorney Docket Number TIP 1023, the content of which is hereby incorporated by reference in its entirety.

In certain embodiments, each of the first, second and third non-naturally occurring nucleic acid molecules is independently formulated with a lipid nanoparticle (LNP).

The application also provides methods of making compositions and immunogenic combinations of the application. A method of producing a composition or immunogenic combination comprises mixing an isolated polynucleotide encoding an HBV antigen, vector, and/or polypeptide of the application with one or more pharmaceutically acceptable carriers. One of ordinary skill in the art will be familiar with conventional techniques used to prepare such compositions.

Methods of Inducing an Immune Response

The application also provides methods of inducing an immune response against 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 application. Any of the compositions and immunogenic combinations of the application described herein can be used in the methods of the application. As used herein, the term“infection” refers to the invasion of a host by a disease causing agent. A disease causing agent is considered to be“infectious” when it is capable of invading a host, and replicating or propagating within the host. Examples of infectious agents include viruses, e.g., HBV and certain species of adenovirus, prions, bacteria, fungi, protozoa and the like. “HBV infection” specifically refers to invasion of a host organism, such as cells and tissues of the host organism, by HBV.

The phrase“inducing an immune response” when used with reference to the methods described herein encompasses causing a desired immune response or effect in a subject in need thereof against an infection, e.g., an HBV infection. “Inducing an immune response” also encompasses providing a therapeutic immunity for treating against a pathogenic agent, e.g., HBV. As used herein, the term“therapeutic immunity” or “therapeutic immune response” means that the vaccinated subject is able to control an infection with the pathogenic agent against which the vaccination was done, for instance immunity against HBV infection conferred by vaccination with HBV vaccine. In an embodiment,“inducing an immune response” means producing an immunity in a subject in need thereof, e.g., to provide a therapeutic effect against a disease, such as HBV infection. In certain embodiments,“inducing an immune response” refers to causing or improving cellular immunity, e.g., T cell response, against HBV infection. In certain embodiments,“inducing an immune response” refers to causing or improving a humoral immune response against HBV infection. In certain embodiments,“inducing an immune response” refers to causing or improving a cellular and a humoral immune response against HBV infection.

As used herein, the term“protective immunity” or“protective immune response” means that the vaccinated subject is able to control an infection with the pathogenic agent against which the vaccination was done. Usually, the subject having developed a “protective immune response” develops only mild to moderate clinical symptoms or no symptoms at all. Usually, a subject having a“protective immune response” or“protective immunity” against a certain agent will not die as a result of the infection with said agent.

Typically, the administration of compositions and immunogenic combinations of the application will have a therapeutic aim to generate an immune response against HBV after HBV infection or development of symptoms characteristic of HBV infection, e.g., for therapeutic vaccination.

As used herein,“an immunogenically effective amount” or“immunologically effective amount” means an amount of a composition, polynucleotide, vector, or antigen sufficient to induce a desired immune effect or immune response in a subject in need thereof. An immunogenically effective amount can be an amount sufficient to induce an immune response in a subject in need thereof. An immunogenically effective amount can be an amount sufficient to produce immunity in a subject in need thereof e.g., provide a therapeutic effect against a disease such as HBV infection. An immunogenically effective amount can vary depending upon a variety of factors, such as the physical condition of the subject, age, weight, health, etc.; the particular application, e.g., providing protective immunity or therapeutic immunity; and the particular disease, e.g., viral infection, for which immunity is desired. An immunogenically effective amount can readily be determined by one of ordinary skill in the art in view of the present disclosure.

In particular embodiments of the application, an immunogenically effective amount refers to the amount of a composition or immunogenic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an HBV infection or a symptom associated therewith; (ii) reduce the duration of an HBV infection or symptom associated therewith; (iii) prevent the progression of an HBV infection or symptom associated therewith; (iv) cause regression of an HBV infection or symptom associated therewith; (v) prevent the development or onset of an HBV infection, or symptom associated therewith; (vi) prevent the recurrence of an HBV infection or symptom associated therewith; (vii) reduce hospitalization of a subject having an HBV infection; (viii) reduce hospitalization length of a subject having an HBV infection; (ix) increase the survival of a subject with an HBV infection; (x) eliminate an HBV infection in a subject; (xi) inhibit or reduce HBV replication in a subject; and/or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

An immunogenically effective amount can also be an amount sufficient to reduce HBsAg levels consistent with evolution to clinical seroconversion; achieve sustained HBsAg clearance associated with reduction of infected hepatocytes by a subject’s immune system; induce HBV-antigen specific activated T-cell populations; and/or achieve persistent loss of HBsAg within 12 months. Examples of a target index include lower HBsAg below a threshold of 500 copies of HBsAg international units (IU) and/or higher CD8 counts.

As general guidance, an immunogenically effective amount when used with reference to a DNA plasmid can range from about 0.1 mg/mL to 10 mg/mL of DNA plasmid total, such as 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, an 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 from one vector or plasmid, or from multiple vectors or plasmids. As further general guidance, an immunogenically effective amount when used with reference to a peptide can range from about 10 pg to 1 mg per administration, such as 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 9000, or 1000 pg per administration. An immunogenically effective amount can 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., tablets, capsules or injectables, or any composition adapted to intradermal delivery, e.g., to intradermal delivery using an intradermal delivery patch), wherein the administration of the multiple capsules or injections collectively provides a subject with an immunogenically effective amount. For example, when two DNA plasmids are used, an immunogenically effective amount can be 3-4 mg/mL, with 1.5-2 mg/mL of each plasmid. It is also possible to administer an immunogenically effective amount to a subject, and subsequently administer another dose of an immunogenically effective amount to the same subject, in a so-called prime-boost regimen. This general concept of a prime-boost regimen is well known to the skilled person in the vaccine field. Further booster administrations can optionally be added to the regimen, as needed.

An immunogenic combination comprising two DNA plasmids, e.g., a first DNA plasmid encoding an HBV core antigen and second DNA plasmid encoding an HBV pol antigen can be administered to a subject by mixing both plasmids and delivering the mixture to a single anatomic site. Alternatively, two separate immunizations each delivering a single expression plasmid can be performed. In such embodiments, whether both plasmids are administered in a single immunization as a mixture of 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, 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 plasmids are administered in a ratio of 1 : 1 , by weight.

Preferably, a subject to be treated according to the methods of the application is an HBV-infected subject, particular a subject having chronic HBV infection. Acute HBV infection is characterized by an efficient activation of the innate immune system complemented with a subsequent broad adaptive response (e.g., HBV-specific T-cells, neutralizing antibodies), which usually results in successful suppression of replication or removal of infected hepatocytes. In contrast, such responses are impaired or diminished due to high viral and antigen load, e.g., HBV envelope proteins are produced in abundance and can be released in sub-viral particles in 1,000-fold excess to infectious virus.

Chronic HBV infection is described in phases characterized by viral load, liver enzyme levels (necroinflammatory activity), HBeAg, or HBsAg load or presence of antibodies to these antigens. cccDNA levels stay relatively constant at approximately 10 to 50 copies per cell, even though viremia can vary considerably. The persistence of the cccDNA species leads to chronicity. More specifically, the phases of chronic HBV infection include: (i) the immune-tolerant phase characterized by high viral load and normal or minimally elevated liver enzymes; (ii) the immune activation HBeAg-positive phase in which lower or declining levels of viral replication with significantly elevated liver enzymes are observed; (iii) the inactive HBsAg carrier phase, which is a low replicative state with low viral loads and normal liver enzyme levels in the serum that may follow HBeAg seroconversion; and (iv) the HBeAg-negative phase in which viral replication occurs periodically (reactivation) with concomitant 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,“chronic HBV infection” refers to a subject having the detectable presence of HBV for more than 6 months. A subject having a chronic HBV infection can be in any phase of chronic HBV infection. Chronic HBV infection is understood in accordance with its ordinary meaning in the field. Chronic HBV infection can for example be characterized by the persistence of HBsAg for 6 months or more after acute HBV infection. For example, a chronic HBV infection referred to herein follows the definition published by the Centers for Disease Control and Prevention (CDC), according to which a chronic HBV infection can be characterized by laboratory criteria such as: (i) negative for IgM antibodies to hepatitis B core antigen (IgM anti-HBc) and positive for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), or nucleic acid test for hepatitis B virus DNA, or (ii) positive for HBsAg or nucleic acid test for HBV DNA, or positive for HBeAg two times at least 6 months apart.

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

In some embodiments, a subject having chronic HBV infection is undergoing nucleoside analog (NUC) treatment, and is NUC-suppressed. As used herein,“NUC- suppressed” refers to a subject having an undetectable viral level of HBV and stable alanine aminotransferase (ALT) levels for at least six months. Examples of

nucleoside/nucleotide analog treatment include HBV polymerase inhibitors, such as entacavir and tenofovir. Preferably, a subject having chronic HBV infection does not have advanced hepatic fibrosis or cirrhosis. Such subject would typically have a METAVTR score of less than 3 for fibrosis and a fibroscan result of less than 9 kPa. The METAVIR score is a scoring system that is commonly used to assess the extent of inflammation and fibrosis by histopathological evaluation in a liver biopsy of patients with hepatitis B. The scoring system assigns two standardized numbers: one reflecting the degree of inflammation and one reflecting the degree of fibrosis.

It is believed that elimination or reduction of chronic HBV may allow early disease interception of severe liver disease, including virus-induced cirrhosis and hepatocellular carcinoma. Thus, the methods of the application can also be used as therapy to treat HBV-induced diseases. Examples of HBV-induced diseases include, but are not limited to cirrhosis, cancer (e.g., 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 persistent loss of HBsAg within 12 months and significant decrease in clinical disease (e.g., cirrhosis, hepatocellular carcinoma, etc.).

Methods according to embodiments of the application further comprises administering to the subject in need thereof another immunogenic agent (such as another HBV antigen or other antigen) or another anti-HBV agent (such as a nucleoside analog or other anti-HBV agent) in combination with a composition of the application. For example, another anti-HBV agent or immunogenic agent can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PDl, anti- ΉM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/oror TLR8 agonists), RIG-1 agonists, IL-15 superagonists (Altor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL12 genetic adjuvant, IL- 7-hyFc; CAR-T which bind HBV env (S-CAR cells); capsid assembly modulators;

cccDNA inhibitors, HBV polymerase inhibitors (e.g., entecavir and tenofovir). The one or other anti-HBV active agents can be, for example, a small molecule, an antibody or antigen binding fragment thereof, a polypeptide, protein, or nucleic acid. The one or other anti-HBV agents can e.g., be chosen from among HBV DNA polymerase inhibitors; Immunomodulators; Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll-like receptor 3 modulators; Interferon alpha receptor ligands; Hyaluronidase inhibitors; Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators;

Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of ribonucleotide reductase;

Hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; 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 (NAPs); Stimulators of retinoic acid- inducible gene 1; Stimulators of NOD2; Recombinant thymosin alpha- 1; Hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, ΉM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, and CTLA-4 inhibitors; Agonists of co-stimulatory receptors that are expressed on immune cells (more particularly T cells), such as CD27, CD28; BTK inhibitors; Other drugs for treating HBV; IDO inhibitors; Arginase inhibitors; and KDM5 inhibitors.

In certain embodiments, the method further comprises administering to the subject a surface antigen of the hepatitis B virus or a nucleic acid encoding the surface antigen. Examples of a surface antigen of the hepatitis B virus or a nucleic acid encoding the surface antigen useful for the invention are described in European Patent Application Ser. No. 19180926.8 entitled“Hepatitis B Virus (HBV) Vaccines and Uses Thereof,” filed on the same day as this application with the Attorney Docket Number TIPI 023, the content of which is hereby incorporated by reference in its entirety.

Methods of Delivery

Compositions and immunogenic combinations of the application can be administered to a subject by any method known in the art in view of the present disclosure, including, but not limited to, parenteral administration (e.g., intramuscular, subcutaneous, intravenous, or intradermal injection), oral administration, transdermal administration, and nasal administration. Preferably, compositions and immunogenic combinations are administered parenterally (e.g., by intramuscular injection or intradermal injection) or transdermally.

In some embodiments of the application in which a composition or immunogenic combination comprises one or more DNA plasmids, administration can be by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection. Intramuscular injection can be combined with electroporation, i.e., application of an electric field to facilitate delivery of the 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 bio-membrane. During in vivo electroporation, electrical fields of appropriate magnitude and duration are applied to cells, inducing a transient state of enhanced cell membrane permeability, thus enabling the cellular uptake of molecules unable to cross cell membranes on their own. Creation of such pores by electroporation facilitates passage of biomolecules, such as plasmids, oligonucleotides, siRNAs, drugs, etc., from one side of a cellular membrane to the other. In vivo electroporation for the delivery of DNA vaccines has been shown to significantly increase plasmid uptake by host cells, while also leading to mild-to-moderate inflammation at the injection site. As a result, transfection efficiency and immune response are significantly improved (e.g., up to 1,000 fold and 100 fold respectively) with intradermal or intramuscular electroporation, in comparison to conventional injection.

In a typical embodiment, electroporation is combined with intramuscular injection. However, it is also possible to combine electroporation with other forms of parenteral administration, e.g., intradermal injection, subcutaneous injection, etc.

Administration of a composition, immunogenic combination or vaccine of the application via electroporation can be accomplished using electroporation devices that can be configured to deliver to a desired tissue of a mammal a pulse of energy effective to cause reversible pores to form in cell membranes. The electroporation device can include an electroporation component and an electrode assembly or handle assembly.

The electroporation component can include one or more of the following components of electroporation devices: controller, current waveform generator, impedance tester, waveform logger, input element, status reporting element, communication port, memory component, power source, and power switch. Electroporation can be accomplished using an in vivo electroporation device. Examples of electroporation devices and

electroporation methods that can facilitate delivery of compositions and immunogenic combinations of the application, particularly those comprising DNA plasmids, include CELLECTRA® (Inovio Pharmaceuticals, Blue Bell, PA), Eigen electroporator (Inovio Pharmaceuticals, Inc.) Tri-GridTM delivery system (Ichor Medical Systems, Inc., San Diego, CA 92121) and those described in U.S. Patent No. 7,664,545, U.S. Patent No. 8,209,006, U.S. Patent No. 9,452,285, U.S. Patent No. 5,273,525, U.S. Patent No.

6,110,161, U.S. Patent No. 6,261,281, U.S. Patent No. 6,958,060, and U.S. Patent No. 6,939,862, U.S. Patent No. 7,328,064, U.S. Patent No. 6,041,252, U.S. 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, the relevant content of each of which is herein incorporated by reference in their entireties. Other examples of in vivo electroporation devices are described in International Patent Application entitled“Method and Apparatus for the Delivery of Hepatitis B Virus (HBV) Vaccines,” filed on the same day as this application with the Attorney Docket Number 688097-405WO, the contents of which are hereby incorporated by reference in their entireties. Also contemplated by the application for delivery of the compositions and immunogenic combinations of the application are use of a pulsed electric field, for instance as described in, e.g., U.S. Patent No. 6,697,669, which is herein incorporated by reference in its entirety. In other embodiments of the application in which a composition or immunogenic combination comprises one or more DNA plasmids, the method of administration is transdermal. Transdermal administration can be combined with epidermal skin abrasion to facilitate delivery of the DNA plasmids to cells. For example, a dermatological patch can be used for epidermal skin abrasion. Upon removal of the dermatological patch, the composition or immunogenic combination can be deposited on the abraised skin.

Methods of delivery are not limited to the above described embodiments, and any means for intracellular delivery can be used. Other methods of intracellular delivery contemplated by the methods of the application include, but are not limited to, liposome encapsulation, lipid nanoparticles (LNPs), etc.

In certain embodiments of the application, the method of administration is a lipid composition, such as a lipid nanoparticle (LNP). Lipid compositions, preferably lipid nanoparticles, that can be used to deliver a therapeutic product (such as one or more nucleic acid molecules of the invention), include, but are not limited to, liposomes or lipid vesicles, wherein an aqueous volume is encapsulated by amphipathic lipid bilayers, or wherein the lipids coat an interior that comprises a therapeutic product; or lipid aggregates or micelles, wherein the lipid-encapsulated therapeutic product is contained within a relatively disordered lipid mixture.

In particular embodiments, the LNPs comprise a cationic lipid to encapsulate and/or enhance the delivery of a nucleic acid molecule, such as a DNA or RNA molecule of the invention, into the target cell. The cationic lipid can be any lipid species that carries a net positive charge at a selected pH, such as physiological pH. The lipid nanoparticles can be prepared by including multi- component lipid mixtures of varying ratios employing one or more cationic lipids, non-cationic lipids and polyethylene glycol (PEG) - modified lipids. Several cationic lipids have been described in the literature, many of which are commercially available. For example, suitable cationic lipids for use in the compositions and methods of the invention include l,2-dioleoyl-3 -trimethyl- ammonium-propane (DOTAP).

The LNP formulations can include anionic lipids. The anionic lipids can be any lipid species that carries a net negative charge at a selected pH, such as physiological pH. The anionic lipids, when combined with cationic lipids, are used to reduce the overall surface charge of LNPs and to introduce pH-dependent disruption of the LNP bilayer structure, facilitating nucleotide release. Several anionic lipids have been described in the literature, many of which are commercially available. For example, suitable anionic lipids for use in the compositions and methods of the invention include 1 ,2-dioleoyl-.v«-glycero- 3-phosphoethanolamine (DOPE). LNPs can be prepared using methods well known in the art in view of the present disclosure. For example, the LNPs can be prepared using ethanol injection or dilution, thin film hydration, freeze-thaw, French press or membrane extrusion, diafiltration, sonication, detergent dialysis, ether infusion, and reverse phase evaporation.

Some examples of lipids, lipid compositions, and methods to create lipid carriers for delivering active nucleic acid molecules, such as those of this invention, are described in: US2017/0190661, US2006/0008910, US2015/0064242, US2005/0064595,

WO/2019/036030, US2019/0022247, WO/2019/036028, WO/2019/036008,

WO/2019/036000, US2016/0376224, US2017/0119904, WO/2018/200943,

WO/2018/191657, US2014/0255472, and US2013/0195968, the relevant content of each of which is hereby incorporated by reference in its entirety.

Adjuvants

In some embodiments of the application, a method of inducing an immune response against HBV further comprises administering an adjuvant. The terms “adjuvant” and "immune stimulant" 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 an immune response to HBV antigens and antigenic HBV polypeptides of the application.

According to embodiments of the application, an adjuvant can be present in an immunogenic combination or composition of the application, or administered in a separate composition. An adjuvant can be, e.g., a small molecule or an antibody.

Examples of adjuvants suitable for use in the application include, but are not limited to, immune checkpoint inhibitors (e.g., anti-PDl, anti-HM-3, etc.), toll-like receptor agonists (e.g., TLR7 and/or TLR8 agonists), RIG-1 agonists, IL-15 superagonists (Altor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL12 genetic adjuvant, and IL-7-hyFc. Examples of adjuvants can e.g., be chosen from among the following anti-HBV agents: HBV DNA polymerase inhibitors; Immunomodulators; Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll-like receptor 3 modulators; Interferon alpha receptor ligands;

Hyaluronidase inhibitors; Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators; Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of

ribonucleotide reductase; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; 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 (NAPs); Stimulators of retinoic acid-inducible gene 1; Stimulators of NOD2;

Recombinant thymosin alpha- 1; Hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, ΉM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, and CTLA-4 inhibitors; Agonists of co-stimulatory receptors that are expressed on immune cells (more particularly T cells), such as CD27, CD28; BTK inhibitors; Other drugs for treating HBV; IDO inhibitors; Arginase inhibitors; and KDM5 inhibitors.

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

The at least one anti-HBV agent can e.g., be chosen from among HBV DNA polymerase inhibitors; Immunomodulators; Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll-like receptor 3 modulators; Interferon alpha receptor ligands; Hyaluronidase inhibitors; Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators; Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of

Recombinant thymosin alpha- 1; Hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, ΉM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, and CTLA-4 inhibitors; Agonists of co- stimulatory receptors that are expressed on immune cells (more particularly T cells), such as CD27, CD28; BTK inhibitors; Other drugs for treating HBV; IDO inhibitors; Arginase inhibitors; and KDM5 inhibitors. Such anti-HBV agents can be administered with the compositions and immunogenic combinations of the application simultaneously or sequentially.

Methods of Prime/Boost Immunization

Embodiments of the application also contemplate administering an

immunogenically effective amount of a composition or immunogenic combination to a subject, and subsequently administering another dose of an immunogenically effective amount of a composition or immunogenic combination to the same subject, in a so-called prime-boost regimen Thus, in an embodiment, a composition or immunogenic combination of the application is a primer vaccine used for priming an immune response. In another embodiment, a composition or immunogenic combination of the application is a booster vaccine used for boosting an immune response. The priming and boosting vaccines of the application can be used in the methods of the application described herein. This general concept of a prime-boost regimen is well known to the skilled person in the vaccine field. Any of the compositions and immunogenic combinations of the application described herein can be used as priming and/or boosting vaccines for priming and/or boosting an immune response against HBV.

In some embodiments of the application, a composition or immunogenic combination of the application can be administered for priming immunization. The composition or immunogenic combination can be re-administered for boosting immunization. Further booster administrations of the composition or vaccine

combination can optionally be added to the regimen, as needed. An adjuvant can be present in a composition of the application used for boosting immunization, present in a separate composition to be administered together with the composition or immunogenic combination of the application for the boosting immunization, or administered on its own as the boosting immunization. In those embodiments in which an adjuvant is included in the regimen, the adjuvant is preferably used for boosting immunization.

An illustrative and non-limiting example of a prime-boost regimen includes administering a single dose of an immunogenically effective amount of a composition or immunogenic combination of the application to a subject to prime the immune response; and subsequently administering another dose of an immunogenically effective amount of a composition or immunogenic combination of the application to boost the immune response, wherein the boosting immunization is first administered about two to six weeks, preferably four weeks after the priming immunization is initially administered. Optionally, about 10 to 14 weeks, preferably 12 weeks, after the priming immunization is initially administered, a further boosting immunization of the composition or

immunogenic combination, or other adjuvant, is administered.

Kits

Also provided herein is a kit comprising an immunogenic combination of the application. A kit can comprise the first polynucleotide, the second polynucleotide, and the third polynucleotide in separate compositions, or a kit can comprise the first polynucleotide, the second polynucleotide, and the third polynucleotide in a single composition. A kit can further comprise one or more adjuvants or immune stimulants, and/or other anti-HBV agents.

The ability to induce or stimulate an anti-HBV immune response upon administration in an animal or human organism can be evaluated either in vitro or in vivo using a variety of assays which are standard in the art. For a general description of techniques available to evaluate the onset and activation of an 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 measurement of cytokine profiles secreted by activated effector cells including those derived from CD4+ and CD8+ T-cells (e.g. quantification of IL-10 or IFN gamma-producing cells by ELISPOT), by determination of the activation status of immune effector cells (e.g. T cell proliferation assays by a classical [3H] thymidine uptake or flow cytometry-based assays), by assaying for antigen-specific T lymphocytes in a sensitized subject (e.g. peptide-specific lysis in a cytotoxicity assay, etc.).

The ability to stimulate a cellular and/or a humoral response can be determined by antibody binding and/or competition in binding (see for example 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 by enzyme-linked immunosorbent assay (ELISA). The immune responses can also be measured by neutralizing antibody assay, where a neutralization of a virus is defined as the loss of infectivity through reaction/inhibition/neutralization of the virus with specific antibody. The immune response can further be measured by Antibody-Dependent Cellular Phagocytosis (ADCP) Assay.

EMBODIMENTS

The invention provides also the following non-limiting embodiments.

Embodiment 1 is an immunogenic combination comprising:

i) a fusion protein comprising:

a) an IL12 p40 subunit; b) a linker consisting of the amino acid sequence of SEQ ID NO: 3; and c) an IL12 p35 subunit;

wherein the fusion protein is arranged from N-terminus to C-terminus in the order (a)-(b)-(c), and the C-terminus of the IL12 p40 subunit is fused to the N-terminus of the IL12 p35 subunit through the linker, or

ii) at least one of:

Embodiment 2 is the immunogenic combination of embodiment 1 , wherein the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

Embodiment 3 is he immunogenic combination of embodiment 1 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 24, SEQ ID NO:25 or SEQ ID NO:26.

Embodiment 4 is the immunogenic combination of any one of embodiments 1-3, comprising the fusion protein.

Embodiment 5 is the immunogenic combination of any one of embodiments 1-3, comprising the first non-naturally occurring nucleic acid molecule comprising the first polynucleotide sequence encoding the fusion protein.

Embodiment 6 is the immunogenic combination of embodiment 5, wherein the fusion protein further comprises a signal sequence operably linked to the N-terminus of the p40 subunit, preferably, the signal sequence is selected from the group consisting of SEQ ID NOs: 11, 12, and 13.

Embodiment 7 is the immunogenic combination of embodiment 5 or 6, wherein the first polynucleotide sequence comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 27. Embodiment 8 is the immunogenic combination of embodiment 7, wherein the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 27.

Embodiment 9 is the immunogenic combination of any one of embodiments 1 to 8, wherein the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 33.

Embodiment 9a is the immunogenic combination of any one of embodiments 1 to 8, wherein and the truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 31.

Embodiment 10 is the immunogenic combination of any one of embodiments 1 to 9a, comprising at least one of the HBV polymerase antigen and the truncated HBV core antigen.

Embodiment 11 is the immunogenic combination of embodiment 10, comprising the HBV polymerase antigen and the truncated HBV core antigen.

Embodiment 12 is the immunogenic combination of any one of embodiment 1 to 9a, comprising at least one of the second non-naturally occurring nucleic acid molecule comprising the second polynucleotide sequence encoding the HBV polymerase antigen, and the third non-naturally occurring nucleic acid molecule comprising the third polynucleotide sequence encoding the truncated HBV core antigen.

Embodiment 13 is the immunogenic combination of embodiment 12, wherein the HBV polymerase antigen and the truncated HBV core antigen each further comprises a signal sequence operably linked to the N-terminus of the antigen, preferably, the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 44, preferably the signal sequence is encoded by the polynucleotide sequence of SEQ ID NO: 34 or SEQ ID NO: 43.

Embodiment 14 is the immunogenic combination of embodiment 12 or 13, comprising the second non-naturally occurring nucleic acid molecule and the third non- naturally occurring nucleic acid molecule in the same non-naturally occurring nucleic acid molecule.

Embodiment 15 is the immunogenic combination of embodiment 12 or 13, comprising the second non-naturally occurring nucleic acid molecule and the third non- naturally occurring nucleic acid molecule in two different non-naturally occurring nucleic acid molecules.

Embodiment 16 the immunogenic combination of any one of embodiment 12 to 15, wherein the second polynucleotide sequence comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 32 or SEQ ID NO: 39. Embodiment 17 is the immunogenic combination of embodiment 16, wherein the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 32 or SEQ ID NO: 39.

Embodiment 18 the immunogenic combination of any one of embodiment 12 to 17, wherein the third polynucleotide sequence comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 38.

Embodiment 19 is the immunogenic combination of embodiment 18, wherein the third polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 38.

Embodiment 20 is an immunogenic combination, comprising

Embodiment 21 is the immunogenic combination of embodiment 20, wherein a. the fusion protein comprises the amino acid sequence of SEQ ID NO: 24; b. the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 33; and

c. the truncated HBV core antigen consists of the amino acid sequence of SEQ ID NO: 31.

Embodiment 22 is the immunogenic combination of any one of embodiments 1 - 21, wherein each of the first, second and third non-naturally occurring nucleic acid molecules is a DNA molecule, preferably the DNA molecule is present on a DNA vector.

Embodiment 22a is the immunogenic combination of embodiment 22, wherein the DNA vector is selected from the group consisting of DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, and closed linear deoxyribonucleic acid.

Embodiment 22b is the immunogenic combination of any one of embodiments 1 - 21, wherein each of the first, second and third non-naturally occurring nucleic acid molecules is present on a viral vector. Embodiment 22c is the immunogenic combination of embodiment 22b, wherein the viral vector is selected from the group consisting of bacteriophages, animal viruses, and plant viruses.

Embodiment 22d is the vector of embodiment 22, wherein the vector is a linear covalently closed double-stranded DNA vector that is structurally distinct from plasmid DNA.

Embodiment 22e is the vector of embodiment 22d, wherein the vector is “Doggybone™ closed linear DNA” (dbDNA™) (Touchlight Genetics Ltd.; London, England).

Embodiment 23 is the immunogenic combination of any one of embodiments 22 to 22e, wherein the first non-naturally occurring nucleic acid molecule is present on a first plasmid, a first viral vector or a first linear covalently closed double-stranded DNA vector.

Embodiment 24 is the immunogenic combination of embodiment 23, wherein the second and third non-naturally occurring nucleic acid molecules are also present on the first plasmid, the first viral vector or the first linear covalently closed double-stranded DNA vector.

Embodiment 25 is the immunogenic combination of embodiment 23, wherein the second and third non-naturally occurring nucleic acid molecules are present on a second plasmid, a second viral vector or a second linear covalently closed double-stranded DNA vector.

Embodiment 26 is the immunogenic combination of embodiment 23, wherein the second non-naturally occurring nucleic acid molecule is present on a second plasmid, a second viral vector, or a second linear covalently closed double-stranded DNA vector, and the third non-naturally occurring nucleic acid molecule is present on a third plasmid, a third viral vector or a third linear covalently closed double-stranded DNA vector.

Embodiment 27 is the immunogenic combination of any one of embodiments 1 - 21, wherein each of the first, second and third non-naturally occurring nucleic acid molecules is an RNA molecule.

Embodiment 27a is the immunogenic combination of embodiment 27, wherein the RNA molecule is an RNA replicon, preferably a self-replicating RNA replicon, an mRNA replicon, a modified mRNA replicon, or self-amplifying mRNA.

Embodiment 28 is the immunogenic combination of embodiment 27 or 27a, wherein the first non-naturally occurring nucleic acid molecule is present on a first RNA molecule, preferably a first mRNA or a first self-replicating RNA molecule. Embodiment 29 is the immunogenic combination of embodiment 28, wherein the second and third non-naturally occurring nucleic acid molecules are also present on the first RNA molecule, preferably the first mRNA or the first self-replicating RNA molecule.

Embodiment 30 is the immunogenic combination of embodiment 28, wherein the second and third non-naturally occurring nucleic acid molecules are present on a second RNA molecule, preferably a second mRNA or a second self-replicating RNA molecule.

Embodiment 31 is the immunogenic combination of embodiment 28, wherein the second non-naturally occurring nucleic acid molecule is present on a second RNA molecule, preferably a second mRNA or a second self-replicating RNA molecule, and the third non-naturally occurring nucleic acid molecule is present on a third RNA molecule, preferably a third mRNA or a third self-replicating RNA molecule.

Embodiment 32 is the immunogenic combination of any one of embodiments 1 to 31, wherein each of the first, second and third non-naturally occurring nucleic acid molecules is independently formulated with a lipid composition, preferably a lipid nanoparticle (LNP).

Embodiment 32a is the immunogenic combination of any one of embodiments 1 to 32, further comprising one or more other anti-HBV agents.

Embodiment 32b is the immunogenic combination of embodiment 32a, wherein the anti-HBV agents are HBV DNA polymerase inhibitors; Immunomodulators; Toll-like receptor 7 modulators; Toll-like receptor 8 modulators; Toll-like receptor 3 modulators; Interferon alpha receptor ligands; Hyaluronidase inhibitors; Modulators of IL-10; HBsAg inhibitors; Toll like receptor 9 modulators; Cyclophilin inhibitors; HBV Prophylactic vaccines; HBV Therapeutic vaccines; HBV viral entry inhibitors; Antisense

oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense

oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; Endonuclease modulators; Inhibitors of ribonucleotide reductase; Hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; 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 (NAPs); Stimulators of retinoic acid-inducible gene 1; Stimulators of NOD2; 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, CTLA-4 inhibitors; Agonists of co- stimulatory receptors that are expressed on immune cells (more particularly T cells), such as CD27 and CD28; BTK inhibitors; Other drugs for treating HBV; IDO inhibitors; Arginase inhibitors; or KDM5 inhibitors.

Embodiment 32c is the immunogenic combination of embodiment 32a, wherein the other anti-HBV agents comprise one or more surface antigens of the hepatitis B virus, preferably one or more surface antigens of the hepatitis B virus described in European Patent Application Ser. No. 19180926.8 entitled“Hepatitis B Virus (HBV) Vaccines and Uses Thereof,” filed on the same day as this application with the Attorney Docket Number TIPI 023, the content of which is hereby incorporated by reference in its entirety.

Embodiment 33 is a kit comprising the immunogenic combination of any one of embodiments 1 to 32c, and instructions for use.

Embodiment 34 is the immunogenic combination of any one of embodiments 1 to 32 or the kit of embodiment 33 for use in inducing an immune response against a hepatitis B virus in a subject in need thereof, preferably the subject has chronic HBV infection.

Embodiment 34a are products containing the immunogenic combination of any one of embodiments 1 to 32c or the kit of embodiment 33 as a combined preparation for simultaneous, separate or sequential use in inducing an immune response against a hepatitis B virus in a subject in need thereof, preferably the subject has chronic HBV infection.

Embodiment 35 is the immunogenic combination of any one of embodiments 1 to 32c or the kit of embodiment 33 for use in treating a hepatitis B virus (HBV)-induced disease in a subject in need thereof, preferably the subject has chronic HBV infection, and the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis and hepatocellular carcinoma (HCC).

Embodiment 35a are products containing the immunogenic combination of any one of embodiments 1 to 32c or the kit of embodiment 33 as a combined preparation for simultaneous, separate or sequential use in treating a hepatitis B virus (HBV)-induced disease in a subject in need thereof, preferably the subject has chronic HBV infection, and the HBV-induced disease is selected from the group consisting of advanced fibrosis, cirrhosis and hepatocellular carcinoma (HCC).

Embodiment 36 is the immunogenic combination for use of embodiment 34 or 35, wherein each of the first, second and third non-naturally occurring nucleic acid molecules is a DNA molecule, and the DNA molecule is administered to the subject via

electroporation or a lipid nanoparticle (LNP).

Embodiment 36a are the products of embodiment 34a or 35a, wherein each of the first, second and third non-naturally occurring nucleic acid molecules is a DNA molecule, and the DNA molecule is administered to the subject via electroporation or a lipid nanoparticle (LNP).

Embodiment 37 is the immunogenic combination for use of embodiment 34 or 35, wherein each of the first, second and third non-naturally occurring nucleic acid molecules is an RNA molecule, and the RNA molecule is administered to the subject via a lipid nanoparticle (LNP).

Embodiment 37a is the products of embodiment 34a or 35a, wherein each of the first, second and third non-naturally occurring nucleic acid molecules is an RNA molecule, and the RNA molecule is administered to the subject via a lipid nanoparticle (LNP).

Embodiment 38 is a method of inducing an immune response against HBV infection in a subject in need thereof, comprising administering to the subject an immunogenic combination of any one of embodiments 1 to 32c.

Embodiment 39 is the method of embodiments 38, wherein the immunogenic combination of any one of embodiments 1 to 32c is administered by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection.

Embodiment 40 is the method of embodiment 39, wherein the immunogenic combination comprises the first, second and third non-naturally occurring nucleic acid molecules.

Embodiment 41 is the method of embodiment 40, wherein the non-naturally occurring nucleic acid molecules are administered to the subject by intramuscular injection in combination with electroporation.

Embodiment 42 is the method of embodiment 40, wherein the non-naturally occurring nucleic acid molecules are administered to the subject by a lipid composition, preferably by a lipid nanoparticle.

Embodiment 43 is the method of any one of embodiments 38-42, wherein the IL12 fusion protein enhances the immune response induced by the one or more HBV antigens.

EXAMPLES

It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description. Example 1. Generation of pcDNA-P40-KE-P35 construct

Preparation of IL12 p35 and p40 subunit plasmids

A pcDNA-P40-KE-P35 construct was generated by first preparing two separate DNA plasmids, containing the human IL12 p40 subunit (SEQ ID NO: 1) and human IL12 p35 subunit (SEQ ID NO: 2), respectively, using standard molecular cloning procedures. Briefly, gBlocks® Gene Fragments were ordered from Integrated DNA Technologies (IDT; Coralville, I A) and used in the following PCR reaction mixtures to amplify the subunits:

PCR mixture 1 :

PCR mastermix (dNTP, MgCl2, Taq polymerase)

Nhe-p40 primer (SEQ ID NO: 14)

Xho-p40 primer (SEQ ID NO: 15)

gBlock DNA

H₂0

PCR mixture 2:

PCR mastermix (dNTP, MgCF, Taq polymerase)

Nhe-P35 primer (SEQ ID NO: 16)

Xho-P35 primer (SEQ ID NO: 17)

gBlock DNA

H₂0

The PCR products were double digested with Nhe and Xho enzymes (Fermentas; Waltham, MA) by adding 1 mΐ of each enzyme and 3.4 mΐ of 10X FastDigest buffer (Catalog #B64; Thermo Scientific; Waltham, MA) to the PCR product and incubating at 37° C for 30 minutes.

Next, the pcDNA3.1 backbone was double digested with Nhe and Xho enzymes in 10X FastDigest buffer and H₂0. The reaction was incubated at 37°C for 60 minutes. Then 1 mΐ FastAp Thermosensitive Alkaline Phosphatase (Catalog #EF0564; Thermo Scientific) was added to the reaction and incubated for 10 minutes at 37°C. The enzymes were then inactivated by incubating the reaction mixture for 10 minutes at 80°C. The digested vectors were then run on an agarose gel at 100V for a minimum 30 minutes with the total time depending on the size of fragment/vectors. The digested DNA was then cut from the gel and purified according to standard techniques.

The p35 and p40 DNA fragments were each ligated into a digested pcDNA3.1 backbone using Quick Ligation™ kit (Catalog #M2200L; NEB) to generate pcDNA-p35 and pcDNA-p40 plasmids. Fusion of p35 and p40 plasmids

To generate the p40-KE-p35 fusion plasmid, an intermediate plasmid with a TC linker was first generated. The plasmid was generated using standard molecular cloning techniques as described above for the generation of the pcDNA-p40 and pcDNA-p35 plasmids. Briefly, PCR reactions were carried out on a p40-TC-p35 gBlock DNA fragment using Apa-p40 and Xho-EcoRV-p35 primers. Next, the Apa-p40-TC PCR product and the pcDNA-p40 plasmid were double digested with Apa and Xho enzymes. After DNA purification, the Apa-p40-TC fragment was ligated into the cut pcDNA-p40 plasmid to generate pcDNA-P40-TC plasmid. Next, EcoRV-p35-Pme fragments were generated by PCR on the pcDNA-p35 plasmid using EcoRV-p35 and Pme-p35 primers. The EcoRV-p35-Pme PCR product and pcDNA-p40-TC plasmid were then double digested with EcoRV and Pme. After DNA purification, the EcoRV-p35-Pme fragment was ligated into the pcDNA-p40-TC plasmid to generate pcDNA-p40-TC-p35 fusion plasmid.

Next, PCR was carried out on a p40-KE-p35 gBlock DNA fragment using Apa- p40 and Pme-Xho-EcoRV-p35 primers. The Apa-p40-KE-p35-EcoRV PCR product and pcDNA-p40-TC-p35 plasmid were then double digested with Apa and EcoRV enzymes. After DNA purification, the p40-KE-p35 fragment was ligated into the cut pcDNA-p40- TC-p35 plasmid to generate the pcDNA-p40-KE-p35 fusion plasmid (FIG. 1).

Similar molecular cloning methods to those described above were used to generate pDK-p40-KE-p35 fusion construct. This construct contains a kanamycin resistance cassette which replaces the ampicillin resistance cassette of the pcDNA plasmid, making it suitable for use in vivo (FIG. 2).

In addition to the KE linker (SEQ ID NO: 3), plasmids were generated using linkers such as RNA-binding protein 3 IRES (IR; SEQ ID NO: 4) and FMDV (Foot Mouth Disease Virus) 2A peptide (FA; SEQ D NO: 5) and an overlapping stop and start codon, termed translational coupling spacer (TC; SEQ ID NO: 6) in a similar manner as described above.

Example 2. IL12 protein production and secretion

To confirm that recombinant IL12 p70 protein was expressed from the pcDNA- P40-KE-P35 construct, the pcDNA-KE-P35 construct was transfected into human embryonic kidney (HEK) 293T (ATCC 11268) cells. Prior to transfection, cells were grown as adherent cultures. The transfections were performed according to standard procedures using PEI transfection reagent (Polyplus-transfection; Illkirch-Graffenstaden, France). The second day after transfection, the cell media was collected and centrifuged for 5 minutes at 1500 rpm to remove cells/cell debris. The supernatant was stored at - 20°C or used immediately for a IL12 p70 ELISA. Next, the Human IL12 p70 Quantikine ELISA Kit (R&D Systems Catalog #D1200; Minneapolis, MN) protocol was performed according to the kit’s manual. The concentration of the IL12 p70 secreted protein from cells transfected with pcDNA-P40-FA-P35, pcDNA-p40-KE-p35, pcDNA-p40-IR-p35, pcDNA-p40-TC-p35, or pcDNA-p35 and pcDNA-p40 was determined by comparison to a standard curve of known IL12 p70 concentrations. FIG. 3 shows that transfection of cells with pcDNA-p40-KE-p35 fusion plasmid resulted in the highest IL12 p70 compared to fusion plasmids with other linkers.

Western Blot analysis was used to assess whether the secreted recombinant IL12 p70 protein was maintained as a fused IL12 p70 protein or separated into IL12 p40 and IL12 p35 protein subunits. Cell lysates and supernatant were collected from cells transfected with the pDK-p40-KE-p35 construct and cells transfected with both the pcDNA-p35 and the pcDNA-p40 constructs. IL12 p40 expression was assessed in cell lysates and supernatant using an anti-IL12 p40 antibody (Thermo Scientific Catalog #701233). FIG. 4 shows that for pDK-p40-KE-p35, the IL12 protein only results in a p40 subunit that is maintained in the IL12 p70 heterodimeric protein.

Example 3. Biological Activity of secreted IL12p70 protein

IL12 is a proinflammatory cytokine that induces Interferon gamma (IFNg) production by T cells. To assess whether the p40-KE-p35 fusion protein expressed from the plasmid functioned as a IL12 p70 protein, the fusion protein’s ability to stimulate CD3 T cells to produce IFNg was tested. CD3 T cells were isolated from two human donor samples (DN921 and DN922) using CD3 MicroBeads (MiltenyiBiotec; Bergisch Gladbach, Germany) according to manufacturer’s instructions. CD3 T cells were maintained in IMDM Iscove’s Modified Dulbecco’s medium with 20% FBS. CD3 T cells were stimulated with anti-CD3 antibody (BD Biosciences Catalog #555336; Franklin Lakes, New Jersey), anti-CD28 antibody (Sanquin Catalog #M1650; Amsterdam, The Netherlands), and supernatant containing IL12 p70. Supernatants from cells transfected with the pcDNA-p40-KE-p35 construct and cells transfected with both the pcDNA-P35 and the pcDNA-P40 constructs were tested for biological activity. Recombinant human IL12 p70 (Peprotech; Rocky Hill, NJ) was used as a positive control. Prior to stimulation, concentrations of IL12 p70 in the supernatants were first measured by ELISA as described above in order to ensure equal concentrations of IL12 p70 from the supernatant and the recombinant IL12 p70 were used to stimulate the CD3 T cells. After incubating the CD3 T cells in stimulation media for 3 days at 37°C, the culture plates were spun down and supernatant was collected. Supernatant was either frozen at -20°C or immediately tested for IFNg concentration.

IFNg concentrations were measured using the V-PLEX NHP IFN-g Kit (Meso Scale Discovery Catalog #K156QOD; Rockville, Maryland). IL12 p70 expressed from the pcDNA-p40-KE-p35 construct induced CD3 T cells to produce IFNg comparable to the recombinant IL12p70 positive control. Increasing the concentration of IL12p70 led to a corresponding increase in IFNg production (FIG. 5). These results confirmed that the p40-KE-p35 fusion protein had the functional activity of an IL12 p70 protein.

Example 4. Generation of pDF-core and pDF-pol

A schematic representation of the pDK-pol and pDK-core vectors is shown in Fig. 6A and 6B, respectively. An HBV core or pol antigen optimized expression cassette containing a CMV promoter (SEQ ID NO: 18), a splicing enhancer (triple composite sequence) (SEQ ID NO: 36), Cystatin S precursor signal peptide SPCS (NP OOl 8901.1) (SEQ ID NO: 34), and pol (SEQ ID NO: 32) or core (SEQ ID NO: 30) gene was introduced into a pDK plasmid backbone, using standard molecular biology techniques.

The plasmids were tested in vitro for core and pol antigen expression by Western blot analysis using core and pol specific antibodies, and were shown to provide consistent expression profile for cellular and secreted core and pol antigens (data not shown).

Example 5. Generation of Adenoviral Vectors Expressing a Fusion of Truncated HBV Core Antigen with HBV Pol Antigen

The creation of an adenovirus vector has been designed as a fusion protein expressed from a single open reading frame. Additional configurations for the expression of the two proteins, e.g. using two separate expression cassettes, or using a 2A-like sequence to separate the two sequences, can also be envisaged.

Design of expression cassettes for adenoviral vectors

The expression cassettes (diagrammed in FIG. 7A and FIG. 7B) are comprised of the CMV promoter (SEQ ID NO: 47), an intron (SEQ ID NO:41) (a fragment derived from the human ApoAI gene - GenBank accession X01038 base pairs 295 - 523, harboring the ApoAI second intron), followed by the optimized coding sequence - either core alone or the core and polymerase fusion protein preceded by a human

immunoglobulin secretion signal coding sequence (SEQ ID NO: 43), and followed by the SV40 polyadenylation signal (SEQ ID NO: 42). A secretion signal was included because of past experience showing improvement in the manufacturability of some adenoviral vectors harboring secreted transgenes, without influencing the elicited T-cell response (mouse experiments).

The last two residues of the Core protein (W) and the first two residues of the Polymerase protein (MP) if fused results in a junction sequence (VVMP) that is present on the human dopamine receptor protein (D3 isoform), along with flanking homologies.

The interjection of an AGAG linker between the core and the polymerase sequences eliminates this homology and returned no further hits in a Blast of the human proteome.

Example 6. In Vivo Immunogenicity Study of DNA Vaccine in Mice

An immunotherapeutic DNA vaccine containing DNA plasmids encoding an HBV core antigen or HBV polymerase antigen was tested in mice. The purpose of the study was designed to detect T-cell responses induced by the vaccine after intramuscular delivery via electroporation into B ALB/c mice. Initial immunogenicity studies focused on determining the cellular immune responses that would be elicited by the introduced HBV antigens.

In particular, the plasmids tested included a pDK-Pol plasmid and pDK-Core plasmid, as shown in FIGS. 6A and 6B, respectively, and as described above in Example 4. The pDK-Pol plasmid encoded a polymerase antigen having the amino acid sequence of SEQ ID NO: 33, and the pDK-Core plasmid encoding a Core antigen having the amino acid sequence of SEQ ID NO: 31. First, T-cell responses induced by each plasmid individually were tested. The DNA plasmid (pDNA) vaccine was intramuscularly delivered via electroporation to Balb/c mice using a commercially available TriGrid™ delivery system-intramuscular (TDS-IM) adapted for application in the mouse model in cranialis tibialis. See International Patent Application Publication WO2017172838, and U.S. Patent Application No. 62/607,430, entitled“Method and Apparatus for the Delivery of Hepatitis B Virus (HBV) Vaccines,” filed on December 19, 2017 for additional description on methods and devices for intramuscular delivery of DNA to mice by electroporation, the disclosures of which are hereby incorporated by reference in their entireties. In particular, the TDS-IM array of a TDS-IM vl.O device having an electrode array with a 2.5 mm spacing between the electrodes and an electrode diameter of 0.030 inch was inserted percutaneously into the selected muscle, with a conductive length of 3.2 mm and an effective penetration depth of 3.2 mm, and with the major axis of the diamond configuration of the electrodes oriented in parallel with the muscle fibers.

Following electrode insertion, the injection was initiated to distribute DNA (e.g., 0.020 ml) in the muscle. Following completion of the IM injection, a 250 V/cm electrical field (applied voltage of 59.4 -65.6 V, applied current limits of less than 4 A, 0.16 A/sec) was locally applied for a total duration of about 400 ms at a 10% duty cycle (i.e., voltage is actively applied for a total of about 40 ms of the about 400 ms duration) with 6 total pulses. Once the electroporation procedure was completed, the TriGridTM array was removed and the animals were recovered. High-dose (20 pg) administration to BALB/c mice was performed as summarized in Table 1. Six mice were administered plasmid DNA encoding the HBV core antigen (pDK-core; Group 1), six mice were administered plasmid DNA encoding the HBV pol antigen (pDK-pol; Group 2), and two mice received empty vector as the negative control. Animals received two DNA immunizations two weeks apart and splenocytes were collected one week after the last immunization.

Table 1: Mouse immunization experimental design of the pilot study.

Group N pDNA Unilateral Dose Vol Admin Endpoint

Admin Site Days (spleen (alternate harvest) sides) Day

1 6 Core CT + EP 20 pg 20 0, 14 21 pL

2 6 Pol CT + EP 20 pg 20 0, 14 21 pL

3 2 Empty CT + EP 20 pg 20 0, 14 21

Vector pL

(neg

control)

CT, cranialis tibialis muscle; EP, electroporation. Antigen-specific responses were analyzed and quantified by IFN-g enzyme-linked immunospot (ELISPOT). In this assay, isolated splenocytes of immunized animals were incubated overnight with peptide pools covering the Core protein, the Pol protein, or the small peptide leader and junction sequence (2pg/ml of each peptide). These pools consisted of 15 mer peptides that overlap by 11 residues matching the Genotypes BCD consensus sequence of the Core and Pol vaccine vectors. The large 94 kDa HBV Pol protein was split in the middle into two peptide pools. Antigen-specific T cells were stimulated with the homologous peptide pools and IFN-y-positive T cells were assessed using the ELISPOT assay. IFN-g release by a single antigen-specific T cell was visualized by appropriate antibodies and subsequent chromogenic detection as a colored spot on the microplate referred to as spot-forming cell (SFC).

Substantial T-cell responses against HBV Core were achieved in mice immunized with the DNA vaccine plasmid pDK-Core (Group 1) reaching 1,000 SFCs per 10⁶ cells (FIG. 8). Pol T-cell responses towards the Pol 1 peptide pool were strong (-1,000 SFCs per 10⁶ cells). The weak Pol-2-directed anti -Pol cellular responses were likely due to the limited MHC diversity in mice, a phenomenon called T-cell immunodominance defined as unequal recognition of different epitopes from one antigen. A confirmatory study was performed confirming the results obtained in this study (data not shown).

The above results demonstrate that vaccination with a DNA plasmid vaccine encoding HBV antigens induces cellular immune responses against the administered HBV antigens in mice. Similar results were also obtained with non-human primates (data not shown).

Example 7. In vivo immune stimulation with p40-KE-p35 construct

This example describes experiments testing whether a plasmid encoding the p40- KE-p35 fusion protein can enhance T cell responses to HBV core and Pol antigens in mice. For these experiments, a mouse p40-KE-p35 fusion plasmid was first constructed. Plasmids containing mouse IL12 p40 (SEQ ID NO: 7) and p35 (SEQ ID NO: 8) subunits were generated as described in Example 1 using gBlock® MP40KEP35 (IDT) with Eco and Xho restriction sites. The mouse p40-KE-p35 fusion construct was then cloned into a pDF vector using standard molecular cloning techniques as described in Example 1 (FIG. 9). The pDF vector contains an ampicillin resistance cassette in place of the kanamycin resistance cassette.

Immunization

The following plasmids were used in the experiment: pDF-HBV core, pDF-HBV pol, pDF-p40-KE-p35, pUMVC3 mIL12-IRES, pDK empty vector. The pUMVC3 mIL12-IRES plasmid was provided by Ichor Medical Systems (San Diego, CA). The pUMVC3 mIL 12-IRES construct is a bicistronic construct whereby p35 and p40 are linked together on the nucleotide level with an EMCV IRES sequence, thus the plasmid makes the p35 and p40 proteins separately.

The DNA plasmid (pDNA) vaccine along with various amounts of the IL12- expressing plasmids was intramuscularly delivered via electroporation to Balb/c mice using a commercially available TriGridTM delivery system-intramuscular (TDS-IM) (Ichor Medical Systems) adapted for application in the mouse model in cranialis tibialis. Forty-four female BALB/c mice, 8-9 weeks old, were injected with a combination of plasmids as outlined in Table 2. Six mice were administered plasmid DNA encoding the HBV core antigen and HBV pol antigen (pDF-core + pDF-pol; Group 1), groups of six mice were administered plasmid DNA encoding the HBV core antigen and HBV pol antigen with 0.1 pg, 0.5 pg, 2.0 pg of plasmid DNA encoding p40-KE-p35 fusion,

5 respectively (pDF-core + pDF-pol + pDF-p40-KE-p35; Groups 2, 3, 4); groups of six mice were administered plasmid DNA encoding the HBV core antigen and HBV pol antigen with 0.1 pg, 0.5 pg, 2.0 pg of plasmid DNA encoding Ichor’s mIL12, respectively (pDF-core + pDF-pol + pUMVC3 mIL12-IRES; Group 5, 6, and 7), and two mice received empty vector as the negative control (pDK-empty; Group 8). Animals 10 received two DNA immunizations three weeks apart and splenocytes were collected one week after the last immunization.

Table 2 Mouse immunization experimental design

N, number of mice per group; pDNA, plasmid DNA; CT, cranial tibialis; EP electroporation; D, days

T cell activity assay

5 Antigen-specific responses were analyzed and quantified by an IFNg ELISPOT assay. In this assay, isolated splenocytes of immunized animals were incubated overnight with peptide pools covering the HBV Core protein and the HBV Pol protein. A pool of 35 peptides was used for the HBV Core protein. A pool of 103 peptides was used for the HBV Poll. A pool of 105 peptides was used for HBV Pol2. Dimethyl sulfoxide (DMSO) 10 was used as a negative control, and Concanavalin A (ConA) was used as a positive

control.

Antigen-specific T cells were stimulated with the homologous peptide pools and IFNg-positive T cells were assessed using the ELISPOT assay. IFN-g release by a single antigen-specific T cell was visualized by appropriate antibodies and subsequent

15 chromogenic detection as a colored spot on the microplate referred to as spot-forming cell (SFC). A spot is formed for every T cell that secretes IFNg which is a marker for T cell activity.

Administration of the pDF-p40-KE-p35 adjuvant in combination with HBV core and pol plasmids resulted in a significantly (p<0.05) higher amount of T cells producing 0 IFNg compared to the Core and Pol plasmids administered without adjuvant (FIG. 10).

There was no difference in activity with increasing concentrations of pDF-p40-KE-p35 (0.1 pg/0.5pg/2pg). Administration of the pUMVC3 mIL12-IRES plasmid in

combination HBV core and pol plasmids did not lead to a significant increase in T cell activity compared to the HBV Core and Pol plasmids administered alone. These results demonstrate the IL12 p40-KE-p35 fusion protein acts as an adjuvant to enhance immune responses to vaccines in vivo.

It is understood that the examples and embodiments described herein are for illustrative purposes only, and that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the invention as defined by the appended claims.

Claims

CLAIMS claimed:

1. An immunogenic combination comprising:

i) a fusion protein comprising:

a) an IL12 p40 subunit;

ii) at least one of:

a) an HBV polymerase antigen having an amino acid sequence that is at least 90% identical to SEQ ID NO: 33, wherein the HBV polymerase antigen does not have reverse transcriptase activity and RNase H activity, b) a second non-naturally occurring nucleic acid molecule comprising a

second polynucleotide sequence encoding the HBV polymerase antigen; c) a truncated HBV core antigen consisting of an amino acid sequence that is at least 95% identical to SEQ ID NO: 31, and

2. The immunogenic combination of claim 1 , wherein the fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

3. The immunogenic combination of claim 1, wherein the fusion protein comprises the ammo acid sequence of SEQ ID NO: 24, SEQ ID NO:25 or SEQ ID NO:26.

4. The immunogenic combination of any one of claims 1-3, comprising the first non- naturally occurring nucleic acid molecule comprising the first polynucleotide sequence encoding the fusion protein.

5. The immunogenic combination of claim 4, wherein the fusion protein further comprises a signal sequence operably linked to the N-terminus of the p40 subunit, preferably, the signal sequence is selected from the group consisting of SEQ ID NOs: 11, 12, and 13.

6. The immunogenic combination of claim 5, wherein the first polynucleotide

sequence comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 27, preferably, the first polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 27.

7. The immunogenic combination of any one of claims 1 to 6, wherein the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 33 and the truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO: 31.

8. The immunogenic combination of any one of claims 1 to 7, comprising at least one of the HBV polymerase antigen and the truncated HBV core antigen.

9. The immunogenic combination of claim 8, comprising the HBV polymerase antigen and the truncated HBV core antigen.

10. The immunogenic combination of any one of claims 1 to 7, comprising at least one of the second non-naturally occurring nucleic acid molecule comprising the second polynucleotide sequence encoding the HBV polymerase antigen, and the third non-naturally occurring nucleic acid molecule comprising the third polynucleotide sequence encoding the truncated HBV core antigen.

11. The immunogenic combination of claim 10, wherein the HBV polymerase antigen and the truncated HBV core antigen each further comprises a signal sequence operably linked to the N-terminus of the antigen, preferably, the signal sequence independently comprises the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 44, preferably the signal sequence is encoded by the polynucleotide sequence of SEQ ID NO: 34 or SEQ ID NO: 43.

12. The immunogenic combination of claim 10 or 11, comprising the second non- naturally occurring nucleic acid molecule and the third non-naturally occurring nucleic acid molecule in the same non-naturally occurring nucleic acid molecule, or the second non-naturally occurring nucleic acid molecule and the third non- naturally occurring nucleic acid molecule are in two different non-naturally occurring nucleic acid molecules.

13. The immunogenic combination of any one of claims 10 to 12, wherein the second polynucleotide sequence comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 32 or SEQ ID NO: 39; preferably, the second polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 32 or SEQ ID NO: 39.

14. The immunogenic combination of any one of claims 10 to 13, wherein the third polynucleotide sequence comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 38, preferably, the third polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 38.

15. An immunogenic combination, comprising:

16. The immunogenic combination of claim 15, wherein

a) the fusion protein comprises the amino acid sequence of SEQ ID NO: 24; b) the HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 33; and

c) the truncated HBV core antigen consists of the amino acid sequence of SEQ ID NO: 31.

17. The immunogenic combination of any one of claims 1-16, wherein each of the first, second and third non-naturally occurring nucleic acid molecules is present on a DNA plasmid, a viral vector or a linear covalently closed double-stranded DNA vector.

18. The immunogenic combination of claim 17, wherein the first non-naturally

occurring nucleic acid molecule is present on a first plasmid, a first viral vector or a first linear covalently closed double-stranded DNA vector; optionally, wherein the second and third non-naturally occurring nucleic acid molecules are also present on the first plasmid, the first viral vector or the first linear covalently closed double-stranded DNA vector.

19. The immunogenic combination of claim 18, wherein the second and third non- naturally occurring nucleic acid molecules are present on a second plasmid, a second viral vector or a second linear covalently closed double-stranded DNA vector, or the second non-naturally occurring nucleic acid molecule is present on a second plasmid, a second viral vector or a second linear covalently closed double-stranded DNA vector, and the third non-naturally occurring nucleic acid molecule is present on a third plasmid, a third viral vector or a third linear covalently closed double-stranded DNA vector.

20. The immunogenic combination of any one of claims 1 to 19 for use in inducing an immune response against a hepatitis B virus in a subject in need thereof, preferably in treating a hepatitis B virus (HBV)-induced disease in a subject in need thereof, preferably the subject has chronic HBV infection and the HBV- induced disease is selected from the group consisting of advanced fibrosis, cirrhosis and hepatocellular carcinoma (HCC).

21. Products containing the immunogenic combination of any one of claims 1 to 19 as a combined preparation for simultaneous, separate or sequential use in inducing an immune response against a hepatitis B virus in a subject in need thereof, preferably in treating a hepatitis B virus (HBV)-induced disease in a subject in need thereof, preferably the subject has chronic HBV infection and the HBV- induced disease is selected from the group consisting of advanced fibrosis, cirrhosis and hepatocellular carcinoma (HCC).