Methods and compositions for mrna- and DNA-based treatment of hepatitis b infection

Abstract

Disclosed herein are compositions and methods for the treatment of hepatitis B infection, including chronic hepatitis B (CHB).

Description

Atty. Dkt. No.117586-0146 METHODS AND COMPOSITIONS FOR mRNA- AND DNA-BASED TREATMENT OF HEPATITIS B INFECTION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No.63 / 650,222, filed May 21, 2024, the contents of which are incorporated herein by reference in their entireties. FIELD OF TECHNOLOGY

[0002] The present technology relates to compositions and methods for the treatment of hepatitis B infection, including chronic hepatitis B (CHB). BACKGROUND

[0003] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0004] Hepatitis B is the most common viral hepatitis, potentially life threatening, with long term complications and is one of the major public health challenges worldwide. Currently, vaccination is the most effective tool against hepatitis B infection. While the availability of a prophylactic vaccine has reduced the number of new hepatitis B virus (HBV) infections, it does not benefit the 296 million people already chronically infected by the virus with an estimated 1.5 million new infections and 820,000 deaths each year (WHO Fact Sheet 18 July 2023). It is estimated that a cumulative 63 million new cases of chronic HBV infection and 17 million HBV-related deaths to occur between 2015 and 2030 (Nayagam et al. Lancet Infectious Dis 201616:1399-1408).

[0005] Chronic infection with hepatitis B virus (HBV) leads to the clinical outcome of liver disease, including cirrhosis and hepatocellular carcinoma (HCC). Despite the introduction of a prophylactic hepatitis B vaccine more than 30 years ago, chronic hepatitis B (CHB) infection remains a global health issue (Lozano R, et al. Lancet 2012; 380(9859):2095-128), contributing to more than 50% of the world’s liver cancer burden.Atty. Dkt. No.117586-0146 Approximately one-third of individuals with chronic hepatitis B (CHB) will die from serious liver diseases, such as cirrhosis, hepatocellular carcinoma (HCC), and liver failure, if left untreated. By numbers, there are estimated to be 260-350 million people living with the virus worldwide and more than 780,000 people dying each year from HBV-related liver disease including cirrhosis and liver cancer (Lozano R, et al. Lancet.2012; 380(9859):2095- 128). Current nucleos(t)ide analogue (NA) therapies for CHB effectively target viral DNA suppression (HBV DNA undetectable), but not the clearance of the HBV surface antigen (HBsAg), continued expression of which is associated with ongoing risk for developing HCC (Fattovich G, et al. Am J Gastroenterol.1998;93(6):896-900; Yuen MF et al. Gastroenterology 2008; 135(4):1192-1199). Hepatitis B complete cure is defined by the eradication of the virus and all its replicative intermediates (Revill P, et al. Nature Reviews Gastroenterology and Hepatology, 2016, 13(4):239-248), which is presently considered an unrealistic outcome. The clinical endpoint of HBsAg loss and / or seroconversion to anti- HBs is the current goal for CHB therapy, but it is rarely achieved. The basis for this clearance is presumably the selective pressure of an effective host antiviral (antibody driven) response. However, the innate and adaptive immune responses in patients with CHB have been shown to be compromised, characterized by suboptimal antigen presentation, exhaustion of antigen-specific T-cells and insufficient antibody production (Wang L, et al. World J Hepatol.2015, 7(30):2980-91). Accordingly, there is a need to develop additional therapeutic approaches for CHB.

[0006] At present, the preferred first-line treatment choices are pegylated-interferon alpha-2a (pegIFN-α), entecavir, and tenofovir, based on their superior antiviral efficacy and / or high resistance barrier. However, even with the first-line treatment options, pegIFN- α is effective in achieving sustained virological response in only 30% of HBeAg-positive and 40% of HBeAg-negative cases and is usually associated with severe side-effects. On the other hand, the nucleos(t)ide analogs are well tolerated and potently suppress HBV replication in the vast majority of treated patients. However, even the most potent nucleos(t)ide analogs rarely induce HBV surface antigen (HBsAg) seroconversion, the hallmark of a successful immunologic response to HBV with complete and durable control of infection, or a “functional cure.” Hence, long-term, and possibly life-long, NA treatment is required to continuously suppress HBV replication, which may be associated with significant cost burden and limited by drug-associated toxicity. It is, therefore, a pressing need for the introduction of therapeutic regimens that are safer and effective in achieving aAtty. Dkt. No.117586-0146 functional cure.

[0007] A significant issue in HBV vaccine development is the diminished capacity of an aged immune system and immunosenescence being associated with a decreased vaccine efficacy in the elderly (Derhovanessian E and Pawelec G. Microbial Biotechnology 2012, 5:226-232; Pera A et al. Maturitas 2015, 82: 50-55). Accordingly, there is a need to develop an effective vaccine for the treatment of CHB. SUMMARY

[0008] In one aspect, the present disclosure provides an isolated mRNA molecule comprising a 5’ UTR sequence and a coding sequence, wherein the 5’ UTR sequence is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, and 6; and wherein the coding sequence encodes a protein comprising one or more HBsAg epitopes. In some embodiments, the one or more HBsAg epitopes comprises (i) a first epitope and a second epitope comprising CKTCTTPAQGNSMFPS (SEQ ID NO: 40); or (ii) a first epitope, a second epitope, and a third epitope comprising CTKP(T / S)DGNC (SEQ ID NO: 36). In some embodiments, the coding sequence is selected from the group consisting of SEQ ID NOs: 10, 11, 64, and 65. In some embodiments, the mRNA molecule further comprises a 5’ cap. In some embodiments, the 5’ cap is added enzymatically or chemically using anti- reverse cap analogues (ARCA). In some embodiments, the mRNA further comprises a 3’ UTR, wherein the 3’ UTR sequence is selected from the group consisting of SEQ ID NOs: 5 and 7. In some embodiments, the 5’ UTR comprises SEQ ID NO: 1. In some embodiments, the 5’ UTR comprises SEQ ID NO: 3. In some embodiments, the 5’ UTR comprises SEQ ID NO: 6. In some embodiments, the 3’ UTR comprises SEQ ID NO: 5. In some embodiments, the 3’ UTR comprises SEQ ID NO: 7. In some embodiments, the coding sequence comprises SEQ ID NO: 10 or SEQ ID NO: 64. In some embodiments, the coding sequence comprises SEQ ID NO: 11 or SEQ ID NO: 65. In some embodiments, the coding sequence comprises SEQ ID NOs: 10 and 64 or SEQ ID NOs: 11 and 65. In some embodiments, the mRNA molecule comprises SEQ ID NOs: 12-21.

[0009] In one aspect, the present disclosure provides an immunogenic composition comprising a first mRNA molecule comprising the mRNA molecule of any one of the preceding embodiments and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a second mRNA molecule of any one of the preceding embodiments. In some embodiments, the first mRNA molecule comprises: a 5’ UTRAtty. Dkt. No.117586-0146 sequence comprising SEQ ID NO: 1; and a coding sequence selected from the group consisting of: SEQ ID NOs: 10, 11, 64, and 65; and the second mRNA sequence comprises: a 5’ UTR sequence comprising SEQ ID NO: 3; and a coding sequence selected from the group consisting of: SEQ ID NOs: 10, 11, 64, and 65. In some embodiments, the first mRNA sequence comprises a 5’ UTR sequence comprising SEQ ID NO: 1 and a coding sequence comprising SEQ ID NOs: 64 or 65. In some embodiments, the second mRNA sequence comprises a 5’ UTR sequence comprising SEQ ID NO: 3 and a coding sequence comprising SEQ ID NOs: 64 or 65. In some embodiments, the immunogenic composition is formulated as a vaccine. In some embodiments, the mRNA molecule is formulated in a carrier. In some embodiments, the carrier is a lipid nanoparticle.

[0010] In one aspect, the present disclosure provides a method of treating or preventing hepatitis B infection in a subject in need thereof comprising administering to the subject the immunogenic compositions of any one of the preceding embodiments. In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from the group consisting of an siRNA, an anti-sense oligonucleotide, an antibody, a serum composition comprising antibodies, a protein vaccine, an mRNA vaccine, an antiviral, an immunomodulator, an adjuvant, and combinations thereof. In some embodiments, the additional therapeutic and the immunogenic composition are administered, separately, sequentially, or simultaneously to the subject. In some embodiments, the method is effective to reduce circulating hepatitis B surface antigen (HBsAg) levels, to reduce serum HBV DNA levels, and / or to improve survival in the subject as compared to a control subject. In some embodiments, the method is effective to reduce circulating HBsAg levels to undetectable levels in the subject. In some embodiments, the amount of mRNA in the immunogenic compositions ranges from about 1 µg to about 200 µg. In some embodiments, the immunogenic composition is administered to the subject at least once. In some embodiments, the immunogenic composition is administered to the subject at an interval. In some embodiments, the immunogenic composition is administered to the subject at least once weekly. In some embodiments, the immunogenic composition is administered orally, intravenously, intraperitoneally, subcutaneously, intrabuccally, intradermally, intranasally, intrahepatically, or intramuscularly to the subject. In some embodiments, the subject is human. In some embodiments, the method comprises inducing a T-cell response.Atty. Dkt. No.117586-0146

[0011] In one aspect, the present disclosure provides an expression vector comprising the mRNA molecule of any one of the preceding embodiments.

[0012] In one aspect, the present disclosure provides a cell comprising the mRNA molecule of any one of the preceding embodiments or the expression vector of any one of the preceding embodiments. In some embodiments, the cell is selected from the group consisting of monocytic cells, dermal fibroblasts, lung cancer cells, skeletal muscle cells.

[0013] In one aspect, the present disclosure provides a lipid nanoparticle comprising the mRNA molecule of any one of the preceding embodiments.

[0014] In one aspect, the present disclosure provides a composition comprising one or more lipid nanoparticles of any of the preceding embodiments. In some embodiments, the one or more lipid nanoparticles are lyophilized, in a suspension, or emulsified. In some embodiments, the lipid nanoparticle composition further comprises a pharmaceutically acceptable carrier.

[0015] In one aspect, the present disclosure provides an isolated DNA molecule comprising a first amino acid coding sequence operably linked to a promoter and a second amino acid coding sequence operably linked to a promoter; wherein the first amino acid coding sequence encodes SEQ ID NO: 8; and the second amino acid coding sequence encodes SEQ ID NO: 9. In some embodiments, the first amino acid coding sequence is operably linked to a CAG promoter comprising SEQ ID NO: 62 and / or wherein the second amino acid coding sequence is operably linked to a CMV promoter comprising SEQ ID NO: 63. In some embodiments, the DNA molecule comprises SEQ ID NO: 24.

[0016] In one aspect, the present disclosure provides an adenovirus construct comprising a genome comprising the isolated DNA molecule of any one of the preceding embodiments.

[0017] In one aspect, the present disclosure provides a method of treating or preventing hepatitis B infection in a subject in need thereof comprising administering to the subject a DNA vaccine comprising the isolated DNA molecule of any one of the preceding embodiments or the adenovirus construct of any one of the preceding embodiments, and an anti-hepatitis B siRNA. In some embodiments, the anti-hepatitis B siRNA and the DNA vaccine are administered, separately, sequentially, or simultaneously to the subject. In some embodiments, the method is effective to reduce circulating hepatitis B surface antigenAtty. Dkt. No.117586-0146 (HBsAg) levels, to reduce serum HBV DNA levels, and / or to improve survival in the subject as compared to a control subject. In some embodiments, the method is effective to reduce circulating HBsAg levels to undetectable levels in the subject. In some embodiments, the amount of DNA in the DNA vaccine ranges from about 1 µg to about 500 µg. In some embodiments, the DNA vaccine is administered to the subject at least once. In some embodiments, the DNA vaccine is administered to the subject at an interval. In some embodiments, the DNA vaccine is administered to the subject at least once weekly. In some embodiments, the DNA vaccine is administered orally, intravenously, intraperitoneally, subcutaneously, intradermal, intranasally, intrabuccally, or intramuscularly to the subject. In some embodiments, the subject is human. In some embodiments, the method comprises administering the adenovirus construct of any one of the preceding embodiments in combination with an anti-hepatitis B siRNA. In some embodiments, the anti-HBV siRNA comprises AD66810, wherein the AD66810 sense strand comprises SEQ ID NO: 22 and wherein the AD66810 anti-sense strand comprises SEQ ID NO: 23. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG.1 is a schematic showing the general design for mRNA-based therapeutics of the present technology.

[0019] FIGs.2A-2H show the results of RNA secondary structure modelling for the 1- 40X (FIG.2A), 2-40X (FIG.2B), 3-40X (FIG.2C), 4-40X (FIG.2D), 1-50X (FIG.2E), 2-50X (FIG.2F), 3-50X (FIG.2G), and 4-50X (FIG.2H) constructs, including the structures predicted using the Minimum Free Energy (MFE) model and the Centroid model and the entropy and height of those predicted models at each position in the RNA sequence.

[0020] FIGs.3A-3D show the expression of protein products from A549 cells transfected with 1-40X, 2-40X, 3-40X, 4-40X, 1-50X, 2-50X, 3-50X, and 4-50X, with the 40X and 50X products generating bands indicated with arrows indicating a larger glycosylated 40X product and a smaller unglycosylated 40X product, and a larger glycosylated 50X product and a lower unglycosylated 50X product. FIG.3A is a western blot showing the expression of 40X protein isolated from the supernatant of A549 cells transfected with either a mock transfection or mRNA candidates 1-40X, 2-40X, 3-40X, or 4-40X, with a 40X protein positive control (CLB-405). FIG.3B is a quantification of the 40X product and CLB-405 band intensity in FIG.3A. FIG.3C is a western blot showing the expression of 50XAtty. Dkt. No.117586-0146 protein isolated from the supernatant of A549 cells transfected with either a mock transfection or mRNA candidates 1-50X, 2-50X, 3-50X, or 4-50X, with a 50X protein positive control (CLB-505). FIG.3D is a quantification of the 50X product and CLB-505 band intensity in FIG.3C.

[0021] FIGs.4A-4B show the expression of protein products from U937 cells transfected with 1-40X, 3-40X, 1-50X, or 3-50X. FIG.4A is a western blot showing expression of 1- 40X or 3-40X protein and a mock transfection control, with samples loaded into lanes A1- A15 and B1-B15 as indicated in Table 7. FIG.4B is a western blot showing expression of 1-50X or 3-50X protein and a mock transfection control, with samples loaded into lanes C1- C11 and D1-D11 as indicated in Table 7. Arrows indicate where 40X product was identified in FIG.4A and where 50X product was identified in FIG.4B.

[0022] FIGs.5A-5G show the results of two experiments where the mRNA candidates 1- 40X, 3-40X, 1-50X, and 3-50X (“mRNA”) or a protein vaccine (CLB-405 and CLB-505) were used to treat mice in an HDI model of persistent HBV infection, with placebo controls. FIGs.5A-5D show the individual mouse data from the first experiment for the placebo treatment group (FIG.5A; placebo 1), the 5 µg mRNA construct treatment group (FIG. 5B), the 20 µg mRNA construct treatment group (FIG.5C), and the 3 µg protein vaccine (CLB-405 and CLB-505) treatment group (FIG.5D). FIGs.5E-5G show the individual mouse data from the second experiment for the placebo treatment group (FIG.5E; placebo 2), the 10 µg mRNA construct treatment group (FIG.5F), and the 12 µg protein vaccine (CLB-405 and CLB-505) treatment group (FIG.5G).

[0023] FIGs.6A-6I show that each pairwise combination of the mRNA candidates successfully reduced HBsAg levels in an in vivo HDI mouse model of persistent HBV infection. Individual mouse data is shown for the placebo (FIG.6B), anti-HBV siRNA (FIG.6C), 1-40X+3-40X +1-50X+3-50X (FIG.6D), 1-40X+1-50X (FIG.6E), 1-40X+3- 50X (FIG.6F), 3-40X+1-50X (FIG.6G), and 3-40X+3-50X (FIG.6H) treatment groups, and FIG.6A shows the average HBsAg levels for each treatment group. FIG.6I shows the results of an HBcAg immunohistochemistry analysis of liver cells from mice from each treatment group.

[0024] FIGs.7A-7D show that the mRNA candidates 1-40X, 3-40X, 1-50X, and 3-50X (“mRNA”) displayed synergistic therapeutic effects when combined with siRNA treatmentAtty. Dkt. No.117586-0146 in an AAV-HBV in vivo model. FIG.7A shows the average HBsAg levels for AAV-HBV mice receiving a placebo, an anti-HBV siRNA, anti-HBV siRNA and 1 µg 1-40X, 3-40X, 1-50X, and 3-50X; siRNA and 5 µg 1-40X, 3-40X, 1-50X, and 3-50X; siRNA and 20 µg 1- 40X, 3-40X, 1-50X, and 3-50X; or 5 µg 1-40X, 3-40X, 1-50X, and 3-50X. FIG.7B shows the average HBsAg levels for AAV-HBV mice receiving a placebo, 30 µg of a protein vaccine (15 µg CLB-405 and 15 µg CLB-505), 5 µg 1-40X, 3-40X, 1-50X, and 3-50X (mRNA vaccine), an anti-HBV siRNA AD-66810, anti-HBV siRNA AD-66810 and 30 µg of a protein vaccine (15 µg CLB-405 and 15 µg CLB-505), or siRNA and 5 µg 1-40X, 3- 40X, 1-50X, and 3-50X. FIG.7C shows the number of IFN-^^secreting splenocytes from each treatment group. FIG.7D shows the number of mice with greater than a 1.5 log10decrease in HBsAg levels over time. FIG.7E is two graphs showing the anti-CLB-405 (top) and anti-CLB-505 (bottom) antibody titers over time in uninfected mice administered a placebo, 5µg mRNA (1-40X + 3-40X + 1-50X+ 3-50X), or 10µg mRNA (1-40X + 3-40X + 1-50X+ 3-50X).

[0025] FIGs.8A-8C are a series of western blots that show that the mRNA candidates 5- 40X and 5-50X were successfully expressed from multiple different cell types. FIG.8A is a western blot showing that 5-40X and 5-50X were expressed in fibroblast cells (HDF) 48 hours post transfection, with the arrow and box indicating where positive bands are expected. FIG.8B is a western blot showing that 5-40X and 5-50X were expressed in skeletal muscle cells (SkMc) 48 hours post transfection, with the arrow and box indicating where positive bands are expected. FIG.8C is a western blot showing that 5-40X and 5- 50X were expressed in monocytic cells (U937) 48 hours post transfection, with the box and arrow indicating where positive bands are expected. UT = untransfected control; 40X = 5- 40X; 50X = 5-50X; Marker = Ladder.

[0026] FIGs.9A-9B show that the DNA candidate Ad5-BNP4-BNP5 is successfully expressed in transfected HEK293A cells and that Ad5-BNP4-BNP5 displays synergistic benefits when combined with siRNA therapy in vivo. FIG.9A is a western blot showing that Ad5-BNP4-BNP5 was successfully expressed in HEK293A cells. FIG.9B shows the reduction in HBsAg levels over time in an AAV / HBV mouse model for CHB for mice treated with a placebo, the anti-HBV siRNA AD-66810 (siRNA), a DNA vector control (GFP DNA), the DNA vaccine candidate Ad5-BNP4-BNP5 and the anti-HBV siRNA AD- 66810, or Ad5-BNP4-BNP5.Atty. Dkt. No.117586-0146 DETAILED DESCRIPTION

[0027] It is to be appreciated that certain aspects, modes, embodiments, variations, and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology. The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present technology belongs. I. General

[0028] Functional hepatitis B cure is defined as HbsAg loss with or without seroconversion to anti-HbsAg antibodies (“anti-HBs”) whilst maintaining serum HBV DNA undetectability (Revill P, et al. Nature Reviews Gastroenterology and Hepatology.2016, 13(4):239-248)). Described herein are RNA and DNA vaccines which encode HbsAg bio- nanoparticles (BNPs) with enhanced antigenic display of clearance profile (CP)-associated epitopes to mimic the selective pressure of a CP and promote efficient and specific B cell anti-HBs immune responses for HBsAg clearance. The immunogenicity of the vaccines and the capacity to drive HBsAg clearance and seroconversion were determined in both the HDI mouse model of persistent HBV infection (See, Chen et al, PNAS 2006, vol.103 no.47, 17862–17867) and the AAV / HBV mouse model of CHB (See, Yang et al, Cellular & Molecular Immunology (2014) 11, 71–78). In some embodiments, the aim of a RNA or DNA therapeutic vaccine, when delivered to patients with CHB, is to accelerate and drive functional hepatitis B cure. In some embodiments, the RNA or DNA therapeutic vaccine comprises novel 5’ and 3’ untranslated region (UTR) combinations that enhance therapeutic efficacy (e.g., increased BNP expression or increased vaccine half-life.). In some embodiments, the therapeutic function of RNA or DNA vaccines can be further enhanced by modification of the encoded BNP delivery backbone to minimize neutralization of the BNPs by pre-existing circulating anti-HBs antibodies in CHB patients. II. Definitions

[0029] The following terms are used herein, the definitions of which are provided for guidance.

[0030] As used in the subject specification, the singular forms “a,” “an,” and “the” includeAtty. Dkt. No.117586-0146 plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to “a profile” includes a single profile, as well as two or more profiles; reference to “an epitope” includes a single epitope, as well as two or more epitopes, “an antibody” includes a single antibody, as well as two or more antibodies; reference to “the disclosure” includes a single and multiple aspects taught by the disclosure; and so forth.

[0031] The term “about” and the use of ranges in general, whether or not qualified by the term about, means that the number comprehended is not limited to the exact number set forth herein, and is intended to refer to ranges substantially within the quoted range while not departing from the scope of the technology. As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

[0032] The term “antigen” is used herein in its broadest sense to refer to a substance that is capable of reacting in and / or inducing an immune response. Reference to an “antigen” includes an antigenic determinant or epitope.

[0033] By “antigenic determinant” or “epitope” is meant that part of an antigenic molecule against which a particular immune response is directed and includes a hapten. Typically, in an animal, antigens present several or even many antigenic determinants simultaneously. A “hapten” is a substance that can combine specificity with an antibody but cannot or only poorly induces an immune response unless bound to a carrier. A hapten typically comprises a single antigenic determinant or epitope.

[0034] As used herein, the term “bio-nanoparticle” or “BNP” refers to a virus-like particle (VLP) that has been modified to include or display one or more target insert epitopes. For the purposes of this disclosure the terms “bio-nanoparticle” or “BNP” also refer to “recombinant virus-like particle antigen” or “VLP-Ag.”

[0035] As used herein, the terms “effective amount” or “therapeutically effective amount” or “pharmaceutically effective amount” refer to a quantity sufficient to achieve a desired biological, therapeutic, and / or prophylactic effect, e.g., an amount which results in the prevention of a disease, condition and / or symptom(s) thereof. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject willAtty. Dkt. No.117586-0146 depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to the composition drugs. It will also depend on the degree, severity and type of disease or condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. In some embodiments, multiple doses are administered. Additionally or alternatively, in some embodiments, multiple therapeutic compositions or compounds (e.g., immunogenic compositions, such as vaccines) are administered.

[0036] As used herein, the term “functional cure” refers to a functional cure of CHB and is defined by loss of detectable HBsAg with or without presence of naturally occurring or introduced anti-HBsAg antibodies (also referred to herein as “anti-HBs antibodies” or “anti- HBs”), which include a population of antibodies that selectively bind to select epitopes on HBsAg, which when occupied by anti-HBs antibodies, results in clearance of HBsAg and ultimately HBV. HBV is non-detectable in a fully cured subject.

[0037] The term “immunogenic composition” is used herein to refer to a composition that will elicit an immune response in a mammal that has been exposed to the composition. In some embodiments, an immunogenic composition includes at least one or more nucleic acid sequences, such as mRNA and / or DNA sequences, encoding one or more of the eight DNA sequences for CP-BNPs (e.g., BNP 1, 2, 3, 4, 5, 6, 7, 8), and / or one or more of RNA 40X and 50X sequences, and / or one or more amino acid sequences (e.g., CLB-405, CLB-505).

[0038] In some embodiments, the immunogenic compositions described herein may be formulated for administration in a number of forms. For example, in some embodiments, the immunogenic compositions are prepared for intravenous, intramuscular, subcutaneous, intradermal, parenteral, oral, nasal, or topical administration. Compositions may also be formulated for specific dosage forms. For example, in some embodiments, the immunogenic composition may be formulated as a lipid nanoparticle liquid, gel, aerosol, ointment, cream, lyophilized formulation, powder, cake, tablet, or capsule. In other embodiments, the immunogenic composition is formulated as a controlled release formulation, delayed release formulation, extended release formulation, pulsatile release formulation, and mixed immediate release formulation. In some embodiments, the immunogenic composition is provided as a liquid. In other embodiments, the immunogenic composition is provided in lyophilized form. In some embodiments, the immunogenic composition is formulated with an excipient. In some embodiments, the immunogenicAtty. Dkt. No.117586-0146 composition is formulated without an excipient. In some embodiments, the excipient is selected from one or more of solvents, salts, sugars, or cryoprotectants. In some embodiments, the excipient is sucrose.

[0039] As used herein, the term “infected” refers to harboring a disease or pathogen, such as a virus. An infection can be intentional, such as by administration of a virus or pathogen (e.g., by vaccination), or unintentional, such as by natural transfer of the pathogen from one organism to another, or from a contaminated surface to the organism. In some embodiments, infection is induced in a model organism (e.g., murine model) by in vivo transfection of replication-competent DNA using a hydrodynamic injection approach.

[0040] As used herein “subject” and “patient” are used interchangeably and refer to an animal, for example, a member of any vertebrate species. In some embodiments, the subject is a human.

[0041] As used herein, the terms “treating” or “treatment” or “alleviation” refers to therapeutic treatment, wherein the object is to reduce, alleviate, or slow down the progression or advancement of, and / or reverse the progression of the targeted pathological condition or disorder. For example, a subject is successfully “treated” for an existing and / or persisting hepatitis B infection, including chronic hepatitis B (CHB) infection if, after receiving a therapeutic amount of the compositions of the present technology, according to the methods described herein, the subject shows observable and / or measurable induction of antibodies that clear HBsAg, and / or loss of detectable HBsAg, and / or reduced levels of HBsAg and / or HBV DNA.

[0042] The term “vaccine” is used herein to refer to a composition that is administered to a subject to produce or increase immunity to a particular disease. In some embodiments, vaccines include a pharmaceutically acceptable adjuvant and / or a pharmaceutically acceptable carrier. In some embodiments, a vaccine comprises nucleic acid sequences, such as RNA and / or DNA sequences, and / or amino acid sequences (e.g., CLB-405, CLB-505). In some embodiments, the RNA sequence is a linear sequence. In some embodiments, the DNA sequence is a circular plasmid sequence. In some embodiments, a vaccine is incorporated into a carrier such as a lipid nanoparticle.

[0043] In some embodiments, components of the vaccine comprise non-naturally occurring nucleotides or amino acids. In some embodiments, components of the vaccineAtty. Dkt. No.117586-0146 comprise chemical modifications. One of ordinary skill in the art is aware of the numerous, well-characterized chemical modifications and non-naturally occurring nucleotides and amino acids that can be incorporated into vaccines to improve various characteristics, such as stability or expression.

[0044] As used herein, “BNP 1” or “CP-BNP 1” refers to SEQ ID NO: 46, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO 46, such as the nucleotide sequence set forth in SEQ ID NO: 54, virus-like particles (VLPs) or bio- nanoparticles (BNPs) comprising SEQ ID NO: 46, immunogenic compositions comprising SEQ ID NO: 46, or a vaccine comprising SEQ ID NO: 46.

[0045] As used herein, “BNP 2” or “CP-BNP 2” refers to SEQ ID NO: 47, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 47, such as the nucleotide sequence set forth in SEQ ID NO: 55, VLPs or BNPs comprising SEQ ID NO: 47, immunogenic compositions comprising SEQ ID NO: 47, or a vaccine comprising SEQ ID NO: 47.

[0046] As used herein, “BNP 3” or “CP-BNP 3” refers to SEQ ID NO: 48, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 48, such as the nucleotide sequence set forth in SEQ ID NO: 56, VLPs or BNPs comprising SEQ ID NO: 48, immunogenic compositions comprising SEQ ID NO: 48, or a vaccine comprising SEQ ID NO: 48.

[0047] As used herein, “BNP 4” or “CP-BNP 4” refers to SEQ ID NO: 49, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 49, such as the nucleotide sequence set forth in SEQ ID NO: 57, VLPs or BNPs comprising SEQ ID NO: 49, immunogenic compositions comprising SEQ ID NO: 49, or a vaccine comprising SEQ ID NO: 49.

[0048] As used herein, “BNP 5” or “CP-BNP 5” refers to SEQ ID NO: 50, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 50, such as the nucleotide sequence set forth in SEQ ID NO: 58, VLPs or BNPs comprising SEQ ID NO: 50, immunogenic compositions comprising SEQ ID NO: 50, or a vaccine comprising SEQ ID NO: 50.

[0049] As used herein, “BNP 6” or “CP-BNP 6” refers to SEQ ID NO: 51, nucleotideAtty. Dkt. No.117586-0146 sequences encoding the amino acid sequence set forth in SEQ ID NO: 51, such as the nucleotide sequence set forth in SEQ ID NO: 59, VLPs or BNPs comprising SEQ ID NO: 51, immunogenic compositions comprising SEQ ID NO: 51, or a vaccine comprising SEQ ID NO: 51.

[0050] As used herein, “BNP 7” or “CP-BNP 7” refers to SEQ ID NO: 52, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 52, such as the nucleotide sequence set forth in SEQ ID NO: 60, VLPs or BNPs comprising SEQ ID NO: 52, immunogenic compositions comprising SEQ ID NO: 52, or a vaccine comprising SEQ ID NO: 52.

[0051] As used herein, “BNP 8” or “CP-BNP 8” refers to SEQ ID NO: 53, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 53, such as the nucleotide sequence set forth in SEQ ID NO: 61, VLPs or BNPs comprising SEQ ID NO: 53, immunogenic compositions comprising SEQ ID NO: 53, or a vaccine comprising SEQ ID NO: 53.

[0052] As used herein, “40X” refers to SEQ ID NOs: 10 or 64, mRNA constructs comprising SEQ ID NOs: 10 or 64, and lipid nanoparticles comprising mRNA constructs comprising SEQ ID NOs: 10 or 64. Sequences comprising 40X may further comprise one or more regions or features that are not translated, including, but not limited to, 3’ UTRs, 5’ UTRs, promoters, 5’ caps, and / or poly-A tails.

[0053] As used herein, “50X” refers to SEQ ID NOs: 11 or 65, mRNA constructs comprising SEQ ID NOs: 11 or 65, and lipid nanoparticles comprising mRNA constructs comprising SEQ ID NOs: 11 or 65. Sequences comprising 50X may further comprise a one or more regions or features that are not translated, including, but not limited to, 3’ UTRs, 5’ UTRs, promoters, 5’ caps, and / or poly-A tails.

[0054] As used herein, CLB-405 refers to SEQ ID NO: 8 and protein constructs comprising SEQ ID NO: 8. In some embodiments, CLB-405, alone or in combination with CLB-505, acts as a protein vaccine against HBV.

[0055] As used herein, CLB-505 refers to SEQ ID NO: 9 and protein constructs comprising SEQ ID NO: 9. In some embodiments, CLB-505, alone or in combination with CLB-405, acts as a protein vaccine against HBV.Atty. Dkt. No.117586-0146

[0056] As used herein, “AD66810” refers to the anti-HBV siRNA AD66810, which targets the X gene expression in the HBV genome. See WO2018 / 195165 for a discussion of AD66810. The sense sequence of AD66810 is gsusguGfcAfCfUfucgcuucacaL96 (SEQ ID NO: 22) and the anti-sense sequence is usGfsugaAfgCfGfaaguGfcAfcacsusu (SEQ ID NO: 23), wherein g stands for 2’-O-methylguanosine-3’-phosphate, s stands for phosphorothioate linkage, u stands for 2’-O-methyluridine-3’-phosphate, gs stands for 2’-O- methylguanosine-3’-phosphorothioate, us stands for 2’-O-methyluridine-3’- phosphorothioate, Gf stands for 2’-fluoroguanosine-3’-phosphate, c stands for 2’-O- methylcytidine-3’-phosphate, Af stands for 2’-fluoroadenosine-3’-phosphate, Cf stands for 2’-fluorocytidine-3’-phosphate, Uf stands for 2’-fluorouridine-3’-phosphate, a stands for 2’- O-methyladenosine-3’-phosphate, Gfs stands for 2’-fluoroguanosine-3’-phosphorothioate, and L96 is an abbreviation for N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4- hydroxyprolinolHyp-(GalNAc-alkyl)3..

[0057] As used herein, the antibodies within an anti-HBs response which result in functional cure are referred to as “clearance antibodies.” These antibodies define a “clearance profile” of antibodies which target the specific HBsAg epitopes and which ultimately result in clearance of HBsAg and functional cure. The epitopes are referred to as “clearance epitopes” meaning once occupied by antibodies in the host subject, the subject will result or likely result in a functional cure. Hence, a “clearance profile” can refer to the fingerprint of non-available epitopes on HbsAg or the suite or population of an individual’s antibodies which occupy these epitopes and when they do occupy the epitopes are predictive of a functional cure being achieved. The clearance profile of antibodies or epitopes on HbsAg represent biomarkers of the potential or likelihood that a subject on treatment will achieve a functional cure. Reference to a “likelihood” of a functional cure generally means that the likelihood is 100%, that is, once a clearance profile of antibodies is detected, the subject will reach a state of functional cure or, in the absence of circulating HbsAg, has achieved a functional cure. However, as in any biological system, variability can occur. Hence, for the purposes of the present technology, reference to a “likelihood” of a functional cure means at least 80% probability that a subject with a clearance profile of antibodies will achieve a functional cure. By “at least 80% means 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

[0058] The “clearance profile of epitopes” or “CP of epitopes” on HBsAg is the presenceAtty. Dkt. No.117586-0146 of available or non-available epitopes on HBsAg which have the potential to be occupied by the clearance antibodies. When the clearance profile of epitopes on HBsAg comprise no epitopes available for binding, then the epitopes are deemed “non-available” and this means that the epitopes are occupied by antibodies and a functional cure will likely occur. Alternatively, if no HBsAg is detected nor any circulating HBV DNA, then a functional cure has been achieved and only the clearance profile of antibodies is present (or can be induced in an immune response following exposure to HBV).

[0059] The term “standardized” also encompasses “normalization” of level of HBsAg or HBsAg-Ab complex and represents optimal level of HBsAg or HBsAg-Ab complex for the assay. A “blood-derived sample” includes serum. III. Chronic Hepatitis B (CHB) Functional Cure

[0060] The clearance of serum HBsAg and / or seroconversion to anti-HBs, with undetectable serum HBV DNA, is considered to be a functional cure for CHB, as it allows not only for the cessation of therapy, but is also associated with significant reduction in the rates of liver cirrhosis and the development of hepatocellular carcinoma (HCC), and an overall increased survival rate (Fattovich G, et al. Am J Gastroenterol.1998; 93(6):896- 900; Yuen MF, et al. Gastroenterology 2008, 135(4):1192-1199; Simonetti J, et al. Hepatology 2010, 51: 1531-1537). During spontaneous resolution of acute hepatitis B infection, a coordinated interaction of HBV-specific T cells and B cells eliminate infected hepatocytes, suppress viral replication via non-cytolytic pathways, and neutralize virions through the production of virus-specific antibodies (anti-HBs). Chronic infection represents failure of the host immune response to control HBV replication, with spontaneous clearance of HbsAg being uncommon, and found to occur in only 1-2% of patients. This rate is not enhanced with current antiviral therapies, but a key factor associated with this spontaneous loss is a low serum level of HBsAg of <100 IU / ml (Tseng TC, et al. Gastroenterology 2011, 141(2):517-525, 25 e1-2), and strategies designed to achieve such a level will form part of the treatment armamentarium. The mechanism(s) associated with HBsAg loss and / or seroconversion are unknown, but an effective B-cell response has been shown to be essential to maintain a functional hepatitis B cure, with B-cell depleting therapies (e.g., rituximab, anti-CD 20) being associated with HBV reactivation leading to liver failure and death (Shouval D. Semin Liver Dis.2013; 33(2):167-177). Clinical and diagnostic observations suggest that B-cell clones encoding anti-HBs with low affinity to theAtty. Dkt. No.117586-0146 homologous HBsAg exist in patients with CHB (Gerlich WH. Clin Infect Dis.2007; 44(9):1170-1172). Concurrently expressed anti-HBs in HBsAg-positive patients with CHB further suggests that the anti-HBs response is quantitatively and / or qualitatively insufficient or inadequate (weak) to overcome chronic infection, but does indicate that specific antibody production is not completely inhibited (Zhang JM, et al. Clin Infect Dis.2007; 44(9):1161- 1169). IV. Chronic Hepatitis B (CHB) Disease

[0061] Chronic hepatitis B (CHB) is an infectious disease that covers five recognizable and separate phases: Phase 1 is the immune tolerant (IT) phase covering the time from infection and establishment of chronic infection to the first signs of active disease. It is typically asymptomatic (no significant liver disease) and these individuals acquired infection at birth or shortly thereafter, and they are HBeAg-positive. Phase 2 or the immune clearance (IC) phase is a disease active phase with significant progression in the person’s liver disease. These patients are also HBeAg-positive. In phase 3 or the non-replicative phase (NR) there is little or no evidence of active liver disease; viruses can be identified at low level, and the person is HBeAg-negative. Phase 4 is a relapse or recrudescence in liver disease activity and viral replication and this phase is also known as HbeAg-negative disease. This phase tends to eventually burn itself out and then patients often have cirrhosis. The final phase, phase 5, is identified as HBsAg loss and anti-HBs seroconversion. This is also recognized as the functional cure (FC) phase.

[0062] CHB comprises at least 4 different diseases based on HBV genotypes. Thus, CHB can be considered under Asian CHB (genotypes B and C), European CHB (genotypes A-2 and D), African CHB (genotypes A-1 and E) and Latin American CHB (genotypes F and H) and each of these 4 groups have different ethnicities (Asian versus European / Caucasian versus African versus Latino), age of acquisition (perinatal versus early childhood versus early adult hood) and mode of transmission (mother-to-baby, child-to-child, cultural scarification, iatrogenic / parenteral and sexual), respectively. Recombination between different genotypes (e.g., A and D, or B and C) is not uncommon. CHB can be further stratified on the basis of HBeAg and disease status; either HBeAg-positive or HBeAg- negative, with or without liver disease.

[0063] A FC-P is associated with FC and is demonstrated by loss of epitope recognition atAtty. Dkt. No.117586-0146 loop 1 and loop 2 within the external loop region, which includes the “a” determinant of HbsAg. Anti-HBs responses following a cure outcome “induce” an FC-P against reference HbsAg samples. In some embodiments described herein, vaccines comprising a nucleic acid sequence (e.g., RNA or DNA) encoding FC-P associated epitopes (e.g., 40X, 50X, BNP5, or BNP5) generate an anti-HBs response associated with an FC-P enhancing the chance for cure when administered to a subject in need thereof. V. HBsAg, anti-HBs, and CP / F-CP

[0064] Measurement of the epitope profile of the envelope protein (HBsAg) of the HBV circulating in a patient’s blood is a direct measure of the virus; it comprises the detection of a foreign antigen found in the patient’s blood. The logic or argument developed in the CP is that an infected person will mount an immune response against the virus and the effects of that immune response selection pressure on the virus can be measured. The effect can be demonstrated as HBsAg epitope changes on the envelope of the virus in that person. The CP is measured across a multiplex reporting panel. The FC-P is reported against reactivity to a 4plex panel applied to a natural history cohort run at St. Vincent’s Hospital Melbourne, within which the patients were HBeAg negative and had minimal or no disease activity, were in phase 4 and transitioning to phase 5, with the infecting genotype unrestricted (i.e., genotypes A, B, C, or D). In contrast, the measure of an antibody made by the patient is a direct measure of the host’s response to the virus; it is also found circulating in the patient’s blood. The logic or argument developed in the FC-P is that a person who has cured their CHB has successfully mounted an antibody response, whose effect can be measured. The effect can be demonstrated by the ability of that antibody to induce epitope changes onto exogenously supplied HBsAg of panels of HBV; that is, the patients’ antibody “induces” an epitope profile response on supplied, exogenous antigen. An “induced” FC-P applies to the Natural History cohort of phase 4 / 5, HBeAg negative, genotype unrestricted, CHB patients, which is based on a 4plex reporting panel. VI. HBsAg, VLPs, and BNPs

[0065] HBV encodes three HBsAg proteins that form the viral envelope; small (S), middle (M) and large (L) (Carman WF, et al. J Hepatol.1999; 31(2):195-201; Seeger C, et al. Hepadnaviruses.2013). All share a common 226-residue HBsAg-S domain (Seeger C, et al. Hepadnaviruses. In “Fields Virology”, 2013, 6th edition. pp.2180-2221), whilst N-Atty. Dkt. No.117586-0146 terminal extensions of M and L encode the PreS2 and PreS1 domains respectively. The HBsAg envelope proteins are key viral antigens; during infection, S, M, and L are expressed and co-packaged through the ER membrane of the host cell, for the formation of the envelope of infectious HBV particles. S proteins are the sole antigenic components of the hepatitis B vaccine able to induce protective antibodies mainly directed to the major antigenic ‘a’ determinant of the external loop region (residues 99-169). S proteins readily self-assemble into lipid-associated VLPs, and form the basis of the current recombinant vaccines, which are made in yeast and are generally not glycosylated. The S protein is considered a conformationally dynamic protein containing numerous cysteine and proline residues, predicted to form discrete loop domains (loop 1:aa 107-135; loop 2:aa 139-149) within the immunodominant ‘a’ determinant (Stirk HJ, et al. Intervirology.1992, 33(3):148- 158). The cDNA sequence encoding the S protein is set forth in SEQ ID NO: 25. The cDNA sequence encoding the S protein with an AgeI restriction site and FLAG tag is set forth in SEQ ID NO: 26. Alterations (variants and antibody occupancy) within the ‘a’ determinant can modify HBsAg topology and directly influence the HBV neutralization phenotype (Carman WF, et al. J Hepatol.1999; 31(2):195-201).

[0066] The cDNA sequence encoding the HBsAg-S protein having the amino acid sequence of SEQ ID NO: 27 is set forth in SEQ ID NO: 25. The cDNA sequence encoding the S protein having the amino acid sequence of SEQ ID NO: 28 is set forth in SEQ ID NO: 26. The HBsAg-S cDNA and amino acid sequences are provided below in Table A. Table A. HBsAg-S cDNA and Amino Acid SequencesAtty. Dkt. No.117586-0146

[0067] HBsAg VLPs are highly compact due to the large number of intra- and intermolecular disulfide bonds within and between the individual subunits (Seeger C, et al. Hepadnaviruses. In “Fields Virology”, 2013, 6th edition. pp.2180-2221; Mangold CM, et al. Arch Virol.1997, 142(11):2257-2267), and clinical trials have established that they can be successfully modified to display inserted foreign antigenic and medically-relevant sequences as bio-nanoparticle (BNP) delivery platforms (Beaumont E, et al. Vaccine.2015, 33(8):973-976; Buonaguro L, et al. Expert Rev Vaccines.2011, 10(11):1569-1583; Cheong WS, et al. Antiviral Res.2009, 81(2):113-122; Moffat JM et al. Vaccine.2013,Atty. Dkt. No.117586-0146 31(18):2310-2316; Netter HJ, et al. J Virology 2001;75(5):2130-2141; Phogat S et al. Virology.2008, 373(1):72-84). VII. Clearance Epitopes of HBsAg Loop 1 and Loop 2

[0068] The clearance epitopes on HBsAg are those that when occupied by antibodies in a subject, the likely result is a functional cure. The epitopes are located on each of Loop 1 and Loop 2 of HBsAg-S. In relation to the assay, monoclonal antibodies (mAbs) are selected for use in a multiplex assay which target a range of epitopes on HBsAg-S. One set of mAbs designated mAb5 and 6 targets the Loop 1 epitopes. Two Loop 1 epitopes are screened defined by the consensus amino acid sequence: CX1TCX2X3X4X5QGX6SMX7PC (SEQ ID NO: 29), wherein: X1 is sK122 (Genotypes A1, A2, A6, B1, B2, B3, B6, C, F, G, H, I) or sR122 (Genotypes A3, A4, A5, B3, B4, B5, B7, B8, B9, C2, C4, D, E); X2is sT125 (Genotype A, B, C, D, E, F, G, H, I) or sM125 (genotype D3, D5); X3 is sT126 (Genotypes A, B, C, D, E, F, G, H, I) or sI126 (Genotype C); X4is sP127 (Genotypes A, B, E, C, D, G, I) or sT127 (genotype C4, D2, D5) or sL127 (genotype E, F, H); X5 is sA128 (A, B, C, D, E, F, G, H, I) or sV128 (D2); X6is sN131 (Genotypes A, G, I) or sT131 (Genotypes B, C, D, E, F, H); X7is sF134 (Genotype A, B, C, E, F, H) or sY134 (Genotype D, G, I); and the consensus amino acid sequence to which mAb 10 binds PCX8TCX9X10X11 (SEQ ID NO: 30), wherein: X8is sK122 (Genotypes A1, A2, A6, B1, B2, B3, B6, C, F, G, H, I) or sR122 (Genotypes A3, A4, A5, B3, B4, B5, B7, B8, B9, C2, C4, D, E); X9 is sT125 (Genotype A, B, C, D, E, F, G, H, I) or sM125 (genotype D3, D5);Atty. Dkt. No.117586-0146 X10 is sT126 (Genotypes A, B, C, D, E, F, G, H, I) or sI126 (Genotype C) or sS126 or sA126 (common variants); X11is sP127 (Genotypes A, B, E, C, D, G, I) or sT127 (genotype C4, D2, D5) or sL127 (genotype E, F, H);

[0069] Another set of mAbs (designated mAb, 7, 8, 11, 12, 16, and 17) targets an epitope on Loop 2 defined by the consensus amino acid sequence: CCCTKPX12DGNCX13(SEQ ID NO: 31) wherein: X12is sT140 (Genotype A, B, C, D, G, H, I) or sS140 (Genotype E or F) ; and X13is sT143 (Genotype A, B) or sS143 (Genotype C, D, E, F, G, H, I).

[0070] Without wishing to be bound by theory, it is proposed herein that when either epitope at Loop 1 is occupied, when the epitope at Loop 2 is occupied, or when either epitope at Loop 1 is occupied together with the epitope at Loop 2, then a clearance profile of antibodies has been achieved resulting in functional clearance.

[0071] In an embodiment, the epitopes occupied are CKTCTTPAQGNSMFPSC (SEQ ID NO: 32); and / or PCKTCTTP (SEQ ID NO: 33); and CCCTKPTDGNCT (SEQ ID NO: 34).

[0072] In an embodiment, the epitopes occupied are CKTCTIPAQGTSMFPSC (SEQ ID NO: 35); and / or PCKTCTTP (SEQ ID NO: 33); and CCTKPSDGNCT (SEQ ID NO: 36).

[0073] In an embodiment the epitopes occupied are CRTCTTPAQGTSMFPSC (SEQ ID NO: 37); and / or PCKTCTTP (SEQ ID NO: 33); and CCTKPSDGNCT (SEQ ID NO: 36). VIII. Clearance Profile-Bio Nanoparticle (CP-BNP) Therapeutic Vaccine Formulations

[0074] Eight CP-BNPs (SEQ ID NOs: 46-53) encompassing the pan-genotypic / serotypic loop 1 and loop 2 HBsAg target epitopes, alongside a control wild-type (WT) BNP, are referenced in various aspects of the present disclosure. The BNP backbone consists of HBsAg-S envelope protein non-infectious subviral particles (virus-like particles, or VLPs, that do not contain all the components of the complete virus). In some embodiments, theAtty. Dkt. No.117586-0146 HBV genotype is genotype A, A1, A2, A3, A4, A5, A6, B, B1, B2, B3, B4, B5, B6, B7, B8, B9, C, C1, C2, C4, D, D2, D3, D5, E, F, G, H, or I. In some embodiments, the HBV genotype is genotype D. In some embodiments, the serotype is ayw or adw. In some embodiments, the serotype is ayw. The modification of VLPs as bio-nanoparticles (BNPs) to deliver target insert epitopes represents an active approach, which stimulates the immune system to induce a response able to treat or cure (clear) a persistent hepatitis B virus infection. The CP epitope target (insert) sequences are listed in Table 1 below. BNPs are produced and purified (endo-free), and assessed for immunogenicity individually and in formulations that consider combinations of loop 1 and loop 2 target epitopes. The formulations analysed are listed in Table 2 below.Atty. Dkt. No.117586-0146 Table 2. CP-BNP Formulations

[0075] The amino acid sequences of the 8 CP-BNP constructs are provided in Table 3, Construct 1 (BNP 1) (SEQ ID NO: 46), Construct 2 (BNP 2) (SEQ ID NO: 47), Construct 3 (BNP 3) (SEQ ID NO: 48), Construct 4 (BNP 4) (SEQ ID NO: 49), Construct 5 (BNP 5) (SEQ ID NO: 50), Construct 6 (BNP 6) (SEQ ID NO: 51), Construct 7 (BNP 7) (SEQ ID NO: 52), and Construct 8 (BNP 8) (SEQ ID NO: 53). Table 3. CP-BNP Construct Amino Acid SequencesAtty. Dkt. No.117586-0146Atty. Dkt. No.117586-0146

[0076] Exemplary nucleotide sequences encoding the 8 CP-BNP amino acid constructs are provided in Table 4.Atty. Dkt. No.117586-0146 Table 4. CP-BNP Construct Exemplary Nucleotide SequencesAtty. Dkt. No.117586-0146Atty. Dkt. No.117586-0146Atty. Dkt. No.117586-0146IX. RNA Immunogenic Compositions (e.g., Vaccines)

[0077] In some embodiments, the present disclosure relates to the use of RNA, including mRNA, constructs to elicit an immune response in existing and / or persistent hepatitis B, including chronic hepatitis B (CHB). In some embodiments, the RNA encodes an antigenic epitope repeat region from antigenic epitopes expressed in the loop 1 and loop 2 regions of the HBsAg-S domain. In some embodiments, RNA vaccines of the present technology comprise a nucleic acid sequence that encodes one or more HBsAg epitopes selected from the group consisting of SEQ ID NOs: 33 and 38-45, or sequences with about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% homology thereto. In some embodiments, RNA vaccines of the present technology comprise a nucleic acid sequence that encodes one or more HBsAg epitopes selected from the group consisting of SEQ ID NOs: 40 and 41, or sequences with about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% homology thereto. In some embodiments, RNA vaccines of the present technology comprise a nucleic acid sequence that encodes two HBsAg epitopes comprising SEQ ID NO: 40. In some embodiments, RNA vaccines of the present technology comprise a nucleic acid sequence that encodes three HBsAg epitopes comprising SEQ ID NO: 41. In some embodiments, RNA vaccines of the present technology comprise a nucleic acid sequence that encodes the amino acid sequences of SEQ ID NO: 8 and / or SEQ ID NO: 9, or sequences with or sequences with about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% homology thereto. In some embodiments, RNA vaccines of the present technology encode the nucleotide sequences of SEQ ID NO: 10, and / or SEQ ID NO: 11, or sequences with about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% homology thereto, and / or SEQ ID NO: 64, and / or SEQ ID NO: 65, or sequences with about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% homology thereto. In some embodiments, RNA vaccines of the present technology comprise one of SEQ ID NOs: 12-21, or sequences with about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% homology thereto. In some embodiments, RNA vaccines of the present technology are formulated in a carrier. In some embodiments, RNA vaccines of the present technology are formulated in lipid nanoparticles (LNPs). In some embodiments, the RNA vaccines of the present technologyAtty. Dkt. No.117586-0146 comprise pseudouridine residues. In some embodiments, the RNA vaccines of the present technology are codon optimized to improve translation, or other properties, in a specific cell type (e.g., mammalian), while still encoding the desired protein. Accordingly, in some embodiments the RNA vaccines of the present technology comprise sequences having about 80% similarity, about 85% similarity, about 90% similarity, about 95% similarity, about 96% similarity, about 97% similarity, about 98% similarity, or about 99% similarity to any of SEQ ID NOs: 12-21.

[0078] In some embodiments, RNA constructs of the present technology comprise regions or features that are not translated and one or more open reading frames of one or more proteins. In some embodiments, the one or more open reading frames encode 40X and / or 50X. In some embodiments, the one or more open reading frames encode nucleic acid sequences encoding CLB-405 and / or CLB-505. In some embodiments, the one or more open reading frames are operably linked to a promoter, such as a T7 promoter, a CMV promoter, or a CAG promoter. In some embodiments, the promoter is tissue-specific, inducible, or constitutive. In some embodiments, the RNA constructs of the present technology comprise two open reading frames, each of which is operably linked to a promoter. Regions or features that are not translated can include, for example, one or more ribozyme binding sites, a promoter, a 5’ cap, a 3’ poly-A tail, a 5’ UTR, and a 3’ UTR. Regions or features that are not translated impact multiple RNA properties, including, but not limited to, RNA processing, stability, transport, splicing, and translation initiation and efficiency. In some embodiments, RNA constructs of the present technology comprise a 5’ RE. In some embodiments, RNA constructs of the present technology comprise a 3’ RE. In some embodiments, RNA constructs of the present technology comprise a 5’ cap. In some embodiments, mRNA constructs of the present technology comprise a 3’ poly-A tail. In some embodiments, RNA constructs of the present technology comprise a 5’ cap and a 3’ poly-A tail. In some embodiments, RNA constructs of the present technology contain a 5’ UTR. In some embodiments, RNA constructs of the present technology contain a 3’ UTR. In some embodiments, RNA constructs of the present technology contain a 5’ UTR and a 3’ UTR. In some embodiments, RNA constructs of the present technology contain a 5’ cap, a 3’ poly-A tail, a 5’ UTR, and a 3’ UTR. In some embodiments, the 3’ poly-A tail is encoded by DNA. In some embodiments, the 3’ poly-A tail is added to the RNA construct enzymatically. In some embodiments, the 5’ cap is added enzymatically. In some embodiments the 3’ UTR of the RNA construct enhances and improves one or more ofAtty. Dkt. No.117586-0146 RNA processing, stability, transport, and splicing and translation initiation and efficiency as compared to sequences with a different 3’ UTR. In some embodiments the 5’ UTR of the RNA construct enhances and improves one or more of RNA processing, stability, transport, and splicing and translation initiation and efficiency as compared to sequences with a different 5’ UTR. In some embodiments the 5’ UTR and the 3’ UTR of the RNA construct enhances and improves one or more of mRNA processing, stability, transport, and splicing and translation initiation and efficiency as compared to sequences with a different 5’ and / or 3’ UTRs.

[0079] In some embodiments, the RNA vaccines of the present technology encode 40X (SEQ ID NOs: 10 or 64) or 50X (SEQ ID NOs: 11 or 65). In some embodiments, the RNA vaccines of the present technology comprise 1-40X (SEQ ID NO: 12), 2-40X (SEQ ID NO: 13), 3-40X (SEQ ID NO: 14), 4-40X (SEQ ID NO: 15), 5-40X (SEQ ID NO: 16), 1-50X (SEQ ID NO: 17), 2-50X (SEQ ID NO: 18), 3-50X (SEQ ID NO: 19), 4-50X (SEQ ID NO: 20), 5-50X (SEQ ID NO: 21), or any combination thereof. X. DNA Immunogenic Compositions (e.g., Vaccines)

[0080] In some embodiments, the present disclosure relates to the use of DNA constructs to elicit an immune response in existing and / or persistent hepatitis B, including chronic hepatitis B (CHB). In some embodiments, the DNA encodes for an antigenic epitope repeat region from antigenic epitopes expressed in the loop 1 and loop 2 regions of the HBsAg-S domain. In some embodiments, DNA vaccines of the present technology comprise a nucleic acid sequence that encodes one or more HBsAg epitopes selected from the group consisting of SEQ ID NOs: 33 and 38-45, or sequences with about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% homology thereto. In some embodiments, DNA vaccines of the present technology comprise a nucleic acid sequence that encodes one or more HBsAg epitopes selected from the group consisting of SEQ ID NOs: 40 and 41, or sequences with about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% homology thereto. In some embodiments, DNA vaccines of the present technology comprise a nucleic acid sequence that encodes two HBsAg epitopes comprising SEQ ID NO: 40. In some embodiments, DNA vaccines of the present technology comprise a nucleic acid sequence that encodes three HBsAg epitopes comprising SEQ ID NO: 41. In some embodiments, DNA vaccines of the present technology comprise a nucleic acid sequence that encodes the amino acid sequences of SEQAtty. Dkt. No.117586-0146 ID NO: 8 and / or SEQ ID NO: 9, or sequences with about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% homology thereto. In some embodiments, DNA vaccines of the present technology encode the nucleotide sequences of SEQ ID NO: 24, or sequences with about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% homology thereto. In some embodiments, DNA vaccines of the present technology are formulated in a carrier. In some embodiments, DNA vaccines of the present technology are formulated in lipid nanoparticles (LNPs). In some embodiments, the DNA vaccines of the present technology are codon optimized to improve transcription, or other properties, in a specific cell type (e.g., mammalian), while still encoding the desired protein. Accordingly, in some embodiments the DNA vaccines of the present technology comprise sequences having about 80% similarity, about 85% similarity, about 90% similarity, about 95% similarity, about 96% similarity, about 97% similarity, about 98% similarity, or about 99% similarity to SEQ ID NO: 24.

[0081] In some embodiments, DNA constructs of the present technology comprise one or more open reading frames of one or more proteins and one or more regions or features that are not transcribed and one or more regions or features that are not translated. In some embodiments, the one or more open reading frames comprise nucleic acid sequences encoding CLB-405 and CLB-505. In some embodiments, the one or more open reading frames are operably linked to a promoter, such as a T7 promoter, a CMV promoter, or a CAG promoter. In some embodiments, the promoter is an inducible, tissue-specific, or constitutive promoter. In some embodiments, the DNA constructs of the present technology comprise two open reading frames, each of which is operably linked to a promoter. Regions or features that are not transcribed effect transcription initiation, regulation, and efficiency. Regions or features that are not transcribed can include, for example, regulatory sequences, methylation sites, replication origin sites, promoters and enhancer, repressor, or insulator binding sites. In some embodiments, DNA constructs of the present technology encode one or more regulatory sequences, methylation sites, replication origin sites, promoters and enhancer, repressor, or insulator binding sites. Regions or features that are not translated can include, for example, one or more introns, a poly-A tail, a 5’ UTR, and a 3’ UTR. Regions or features that are not translated multiple RNA properties, including, but not limited to, RNA processing, stability, transport, and splicing and translation initiation and efficiency. In some embodiments, DNA constructs of the present technology encode a 3’ poly-A tail. In some embodiments, DNA constructs of the present technology encode a 5’Atty. Dkt. No.117586-0146 UTR. In some embodiments, DNA constructs of the present technology encode a 3’ UTR. In some embodiments, DNA constructs of the present technology encode a 3’ UTR and a 5’ UTR. In some embodiments, DNA constructs of the present technology encode a 3’ poly-A tail, a 5’ UTR, and a 3’ UTR. In some embodiments the 3’ UTR, the 5’UTR, or a combination thereof enhances and improves the transcribed RNA processing, stability, transport, and splicing and translation initiation and efficiency. In some embodiments the 3’ UTR of the DNA construct enhances and improves one or more of transcribed RNA processing, stability, transport, and splicing and translation initiation and efficiency as compared to sequences with a different 3’ UTR. In some embodiments the 5’ UTR of the DNA construct enhances and improves one or more of transcribed RNA processing, stability, transport, and splicing and translation initiation and efficiency as compared to sequences with a different 5’ UTR. In some embodiments the 5’ UTR and the 3’ UTR of the DNA construct enhances and improves one or more of transcribed RNA processing, stability, transport, and splicing and translation initiation and efficiency as compared to sequences with a different 5’ and / or 3’ UTRs. In some embodiments, the DNA vaccines of the present technology comprise SEQ ID NO: 24.

[0082] In some embodiments, the DNA vaccine is delivered via a viral vector. In some embodiments, the viral vector is an adenovirus vector. Adenovirus vectors can be of any serotype suitable for vaccine administration, including, for example, serotype 5. XI. Modes of Administration and Effective Dosages

[0083] An immunogenic composition (e.g., vaccine) as disclosed herein may be administered by any of the routes conventionally used or recommended for vaccines (e.g., parenteral route), and may be in various forms (e.g., injectable liquid). Vaccines may be administered by means of a syringe or by means of a needle-free injector for intramuscular, subcutaneous, intravenous, or intradermal injection. In some embodiments, vaccines are administered topically by means of a patch, gel, or ointment. In some embodiments, vaccines are administered orally or buccally.

[0084] According to the present technology, an “effective amount” of an immunogenic composition is one that is sufficient to achieve a desired biological effect. It is understood that, in some embodiments, the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, andAtty. Dkt. No.117586-0146 the nature of the effect wanted. The ranges of effective doses provided below are not intended to be limiting and represent exemplary dose ranges. Thus, in some embodiments, the dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art. The dosage of a protein, RNA, or DNA vaccine for a mammalian (e.g., human) adult can be from 0.01 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 0.10 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 1 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 2 µg to 10, 000 µg, or any range or value therein. In some embodiments, the dosage can be from 3 µg to 10, 000 µg, or any range or value therein. In some embodiments, the dosage can be from 4 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 5 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 10 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 15 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 20 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 25 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 50 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 100 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 500 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 1,000 µg to 10,000 µg, or any range or value therein. In some embodiments, the dosage can be from 5,000 µg to 10,000 µg, or any range or value therein. XII. Therapeutic Methods

[0085] The following discussion is presented by way of example only, and is not intended to be limiting.

[0086] One aspect of the present technology includes methods of treating existing and / or persistent hepatitis B, including chronic hepatitis B (CHB) in a subject diagnosed as having or suspected as having a hepatitis B infection. In therapeutic applications, compositions or medicaments comprising the protein, DNA, or mRNA constructs of the present technology are administered to a subject suspected of, or already suffering from, a hepatitis B infection in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease. In additional therapeutic applications, compositions or medicaments comprising the protein,Atty. Dkt. No.117586-0146 DNA, or mRNA constructs of the present technology are administered along with a co- therapeutic to a subject suspected of, or already suffering from, a hepatitis B infection in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease. Appropriate co-therapeutics will be recognized by those with skill in the art, and can include vaccines, antibodies, antivirals (such as capsid inhibitors, nucleos(t)ide analogues, or other therapeutics), siRNAs, and / or immunomodulators (such as anti-PD1, anti-PD-L1, or interferon alpha). In some embodiments, the co-therapeutic is the anti-HBV siRNA AD66810 (SEQ ID NOs: 22-23).

[0087] Another aspect of the present technology includes a method for treating a subject in need thereof by first measuring HBV antigen in a sample, and then, if HBV antigen is identified, using the protein, DNA, or mRNA constructs of the present technology to treat said subject. Additionally, a co-therapeutic may be administered. Appropriate co- therapeutics will be recognized by those with skill in the art, and can include vaccines, antibodies, antivirals (such as capsid inhibitors, nucleos(t)ide analogues, or other therapeutics), siRNAs, anti-sense oligonucleotides, and / or immunomodulators (such as anti- PD1, anti-PD-L1, or interferon alpha). In some embodiments, the co-therapeutic is the anti- HBV siRNA AD66810 (SEQ ID NOs: 22-23).

[0088] Another aspect of the present technology includes heterologous and homologous prime boost methods of vaccination using the RNA and / or DNA vaccines of the present technology. Prime-boost vaccinations comprise an administration of one or more types of vaccine composition to a subject via multiple administrations to generate an enhanced immune response. The skilled artisan will appreciate that the dosage and nature of the vaccine(s) will necessitate adjustments to the prime boost methodology in terms of administration timing and frequency and total dosage. Prime boost administrations can be given, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14, days, 21 days, 28 days, 35 days, or 42 days apart. In some embodiments, prime boost administrations can be given as a booster, for example, 3 months, 4 months, 5 months, 6 months, or more than 12 months apart. Prime boost administrations can be given at regular or irregular intervals. Prime-boost methods of vaccination can include as many administrations as is necessary to achieve a satisfactory immune response, including, for example, 2 administrations, 3 administrations, 4 administrations, or 5 administrations. In some embodiments, a prime boost vaccination schedule for a subject in need thereof comprises the following: a first dose with one of anAtty. Dkt. No.117586-0146 anti-HBV siRNA, a protein HBV vaccine, an mRNA HBV vaccine, or a DNA HBV vaccine, followed by a second dose with one of an anti-HBV siRNA, a protein HBV vaccine, an mRNA HBV vaccine, or a DNA HBV vaccine between 7-14 days after the first dose, followed by a third dose with one of an anti-HBV siRNA, a protein HBV vaccine, an mRNA HBV vaccine, or a DNA HBV vaccine between 7-14 days after the second dose. In some embodiments, a prime boost vaccination schedule for a subject in need thereof comprises the following: a first dose with an mRNA HBV vaccine, followed by a second dose with a protein HBV vaccine between 7-14 days after the first dose, followed by a third dose with a protein HBV vaccine between 7-14 days after the second dose. In some embodiments, a prime boost vaccination schedule for a subject in need thereof comprises the following: a first dose with a DNA HBV vaccine, followed by a second dose with a protein HBV vaccine between 7-14 days after the first dose, followed by a third dose with a protein HBV vaccine between 7-14 days after the second dose. In some embodiments, a prime boost vaccination schedule for a subject in need thereof comprises the following: a first dose with an anti-HBV siRNA, followed by a second dose with an mRNA HBV vaccine between 7-14 days after the first dose, followed by a third dose with a protein HBV vaccine between 7-14 days after the second dose, followed by a fourth dose with a protein HBV vaccine between 7-14 days after the third dose. In some embodiments, a prime boost vaccination schedule for a subject in need thereof comprises the following: a first dose with an anti-HBV siRNA, followed by a second dose with a DNA HBV vaccine between 7-14 days after the first dose, followed by a third dose with a protein HBV vaccine between 7-14 days after the second dose, followed by a fourth dose with a protein HBV vaccine between 7-14 days after the third dose.

[0089] Another aspect of the present technology includes kits for treating a subject in need thereof, wherein the kit includes the protein, DNA, or RNA constructs of the present technology. Such a kit can optionally include other components, such as a co-therapeutic. Appropriate co-therapeutics will be recognized by those with skill in the art, and can include vaccines, antibodies. antivirals (such as capsid inhibitors, nucleos(t)ide analogues, or other therapeutics), siRNAs, and / or immunomodulators (such as anti-PD1, anti-PD-L1, or interferon alpha). In some embodiments, the co-therapeutic is the anti-HBV siRNA AD66810 (SEQ ID NOs: 22-23).Atty. Dkt. No.117586-0146 XIII. Determination of the Biological Effect of the DNA or mRNAs of the Present Technology

[0090] Where a functional cure has been achieved or will be achieved based on the results of the HBsAg immunoassay described herein, a clinician is then able to decide to cease treatment. Hence, the HBsAg immunoassay described herein can monitor treatment to determine if an individual will achieve a functional cure, determine if an individual has achieved a functional cure, and determine if an individual has achieved a cure through natural defense mechanisms.

[0091] The protocol may be varied without departing from the essence of the present technology. The critical endpoint is the determination of the fingerprint of epitopes on HBsAg which have been occupied by an individual's antibody response. Where the fingerprint of epitopes at Loop 1 and Loop 2 has been occupied, then a functional cure can be expected. Where the antibodies which have the capacity to bind to this fingerprint are present or are indicative upon exposure to HBV but HBsAg is not detectable, then a functional cure has been achieved. At that point, treatment can cease. EXAMPLES

[0092] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. Example 1: Development of RNA Candidates and in silico Modelling

[0093] RNA candidates as described in Table 5 were synthesized using the method as described in Tiwari et al. Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection. Nat Commun 9, 3999 (2018). Briefly, the 5’ UTR, codon optimized CDS, and 3’ UTR were inserted into a pMA-7 vector (Thermo Fisher Scientific, GeneArt), modified to include a T7 promoter, via Gibson assembly at the T7 promoter site to generate DNA plasmid templates for the RNA vaccine constructs. Plasmids were transformed into E. coli, and ampicillin plates were used to select for positive transformants, with sequence fidelity being confirmed using Sanger sequencing. Plasmids were amplifiedAtty. Dkt. No.117586-0146 and purified from successful transformants, and then linearized via overnight restriction enzyme digestion before being purified using a PCR clean-up kit (Qiagen) per the manufacturer’s protocol. Linearized templates were then in vitro transcribed using a T7 mScript kit (Cellscript) to generate capped RNAs with poly-A tails, either encoded or added enzymatically per the T7 mScript kit protocol. A depiction of the general construct design for RNA-based therapeutics of the present technology is shown in FIG.1 and a description of the mRNA candidates is provided in Table 5. Table 5. RNA vaccine candidates

[0094] The 5’ UTR sequences in Table 5 are as follows: 5’-ARNV1 (SEQ ID NO: 1): AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC 5’-ARNV2 (SEQ ID NO: 2): UCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCU AUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUC ACCAUUUACGAACGAUAGC 5’-ARNV3 (SEQ ID NO: 3): AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCCAACAAUCUCAAAtty. Dkt. No.117586-0146 CACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUACUUCUAUUGCA GCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUGAAAAUUUUCACCAUU UACGAACGAUAGC 5’-ARNV4 (SEQ ID NO: 4): CACUCGCGCUGCCAUCACUCUUCCGCCGUCUUCGCCGCCAUCCUCGGCGCGACUCGCU UCUUUCGGUUCUACCAGGUAGAGUCCGCCGCCAUCCUCCACCCAACAACUUGUCUCG CUCCGGGGAACGCUCGGAAACUCCCGGCCGCCGCCACCCGCGUCUGUUCUGUUACAC AAGGGAAGAAAAGCCGCUGCCGCACUCCGAGUGU

[0095] The 3’ UTR sequence in Table 5 is as follows: 3’-ARNV1 (SEQ ID NO: 5): GCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAAC UACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAA AAACAUUUAUUUUCAUUGC

[0096] mRNA GC content and secondary structure and stability thereof are known to impact mRNA half-life and translation levels. The secondary structure of mRNA vaccine candidates 1-40X, 2-40X, 3-40X, 4-40X, 1-50X, 2-50X, 3-50X, and 4-50X was modelled to predict the stability and product expression. Briefly, RNAfold was used per the website’s instructions to generate anticipated secondary structures for each construct and to assess the stability thereof. See Gruber et al 2008, Nucleic Acids Research, 36 (2) W70-W74 for a detailed discussion of the RNAfold methodology. FIGs.2A-2H show the predicted secondary structures of 1-40X (FIG.2A), 2-40X (FIG.2B), 3-40X (FIG.2C), 4-40X (FIG.2D), 1-50X (FIG.2E), 2-50X (FIG.2F), 3-50X (FIG.2G), and 4-50X (FIG.2H) using a minimum free energy (MFE) model and a Centroid model, and the predicted entropy levels and height at each position in the RNA sequence for each of those models. The mountain plots in FIGs.2A-2H represent a secondary structure in a plot of height versus position, where the height m(k) is given by the number of base pairs enclosing the base at position k. Loops correspond to plateaus (hairpin loops are peaks), helices to slope. The results of these models are further shown in Table 6 below, which lists the minimum free energy (MFE) for the predicted MFE model secondary structure (kcal / mol), the average MFE for the ensemble of predicted RNA secondary structures, the ensemble diversity, which is the expected distance between the MFE secondary structure and all the other secondary structures, the MFE for the predicted Centroid model secondary structure (kcal / mol), and the GC percent of the sequence. Structures from either the MFE or centroid model with more negative MFE values are predicted to have more stability, which promotes RNA half-life and translation. Structures with higher GC content (%GC) are similarly predicted to have enhanced half-life and translation capacity. Per the results of thisAtty. Dkt. No.117586-0146 modelling, the 4-40X and 4-50X constructs were predicted to be the most translated in vivo based on their relatively high GC content and lower MFE values. Table 6. Secondary Structure Modeling for mRNA candidatesExample 2: mRNA Candidate in vivo Expression Analysis

[0097] The mRNA vaccine candidates in Table 5 were tested in vivo to determine the translation efficiency of each construct. 1-40X, 2-40X, 3-40X, 4-40X, 1-50X, 2-50X, 3- 50X, and 4-50X were transfected into A549 cells, a human lung cancer cell line. Briefly, A549 cells were transfected with mRNA Lipofectamine™ MessengerMAX™ Transfection Reagent. Cell lysates were obtained using RIPA lysis buffer and protein concentration was determined by BCA. The respective protein samples prepared from the transfected cell lysates and the culture supernatants were analyzed via Western blot as follows. Samples were mixed with 5X sample buffer (final concentration 1X) and boiled for 5 min at 100˚C. Samples loaded into a SDS polyacrylamide gel (4–12%) and electrophoresis was performed. Samples were then transferred to a nitrocellulose membrane (Cat #1620112; Bio-Rad) and blocked with 5% skim milk in TBS-T. The expressed HBsAg was detected by anti-HBsAg primary antibody (Novus, NB100-62652) followed by HRP-labeled goat polyclonal anti-rabbit immunoglobulins (LI-Cor, 926-32211)) as a secondary antibody 5 % milk solution. FIG.3A is a western blot showing successful translation of the 1-40X, 2- 40X, 3-40X, and 4-40X constructs in transfected A549 cells. FIG.3B is the quantified 40X expression levels from FIG.3A, and indicates that the 1-40X and 3-40X constructs were substantially more translated compared to the 2-40X and 4-40X constructs. FIG.3C is aAtty. Dkt. No.117586-0146 western blot showing that the 1-50X, 2-50X, 3-50X, and 4-50X constructs were successfully translated in transfected A549 cells. FIG.3D is the quantified expression levels from FIG.3C, and indicates that the 1-50X and 3-50X were substantially more translated than the 2-50X and 4-50X constructs. The substantial differences in translation were unexpected, given that all the vaccine candidates share the same coding sequence and promoter, and primarily differ in the 5’ and 3’ UTR combinations. Moreover, the in vivo results surprisingly diverged from what was predicted by the in silico models as reflected in Table 6. Despite having the highest GC content and predicted stability, the 4-40X / 50X constructs were among the least productive in vitro, whereas the 1-40X / 50X and 3-40X / 50X constructs were the most productive despite having comparably lower GC content and predicted stability.

[0098] To further assess expression and determine product localization, candidates 1-40X, 3-40X, 1-50X, and 3-50X were transfected into U937 cells, a human monocytic cell line, along with a mock transfection control. Media and cell pellet protein samples were taken at 12-, 24-, 48-, and 72-hours post-transfection and assayed via western blotting. Briefly, U937 cells were transfected with mRNA Lipofectamine™ MessengerMAX™ Transfection Reagent. Cell lysates were obtained using RIPA lysis buffer and protein concentration was determined by BCA. Protein samples were mixed with 3x Urea-Cholate buffer and heated at 95°C for 15 minutes, before being run on 4-12 % Bis-Tris protein gels at 200 V for 20 minutes. Samples were loaded onto the Bis-Tris protein gels as indicated in Table 7 below. The protein gels were transferred onto nitrocellulose membranes at 125 mA for 25 minutes. Membranes were blocked in 5 % milk for 1 hour, then incubated with Novus polyclonal antibody (Novus Catalog #NB100-62652) at a dilution of 1:2500. Membranes were washed in TBST three times and then incubated with Goat anti-Rabbit secondary antibody diluted 1:10,000 for 30 minutes. As shown in FIGs.4A-4B, 1-40X, 3-40X, 1-50X, and 3-50X product was detected in pellet samples at 12-hours post-transfection, as indicated by the arrows in lanes A7, B7, C7, and D7. These results show that the 1-40X, 3-40X, 1-50X, and 3-50X constructs are translated in vitro, and that the protein products are cellularly localized and are not secreted. Table 7. Sample loading for FIGs.4A-4B western blotsAtty. Dkt. No.117586-0146 Table 7. Sample loading for FIGs.4A-4B western blotsAtty. Dkt. No.117586-0146 Table 7. Sample loading for FIGs.4A-4B western blots

[0099] Accordingly, these results demonstrate that the mRNA immunogenic compositions (e.g., vaccines) of the present disclosure are effective for expressing protein products in human cells, and are useful in compositions and methods for the treatment and prevention of chronic hepatitis B infection. Example 3: Combination Treatment with mRNA Candidates using in vivo Chronic Hepatitis B Infection Model

[0100] To evaluate the therapeutic efficacy of the mRNA candidates in vivo a mouse model of chronic hepatitis B infection was established. The hydrodynamic infection (HDI) model is a well-established murine CHB model. See Chou H-H et al., 2015. PNAS 112(7) pp2175. CBA / CaJ mice were injected with plasmids expressing the hepatitis B A2 adw genome (pHBV-DNA) through the tail vein at high pressure, which permeabilized hepatocytes, allowing for uptake of the HBV plasmid and stable HBV infection, with approximately 3 log HBsAg (log IU / ml). CHB mice were then used for two experiments. In the first experiment, four treatment groups were established: placebo (placebo 1); 5 µg mRNA vaccine treatment; 20 µg mRNA vaccine treatment; and 3 µg protein vaccine (CLB- 405 and CLB-505) treatment. In the second experiment, three treatment groups were established: placebo (placebo 2); 10 µg mRNA vaccine treatment; and 12 µg protein vaccine (CLB-405 and CLB-505) treatment. For both the first and second experiments, placebo treated mice received the adjuvant Alum alone, mRNA vaccine treated mice received an equal parts mixture of lipid-encapsulated 1-40X, 3-40X, 1-50X, and 3-50X at the indicated dosage, and protein vaccine treated mice received an equal parts mixture of CLB-405 and CLB-505 at the indicated dosage. Lipid encapsulation was achieved according to the method of Yanez Arteta et al. PNAS, 115 (15) E3351-E3360 (2018). Briefly, stocks of lipids were dissolved in ethanol and mixed in the appropriate molar ratios to obtain a lipid concentration of 12.5 mM (1.85 mg / mL). mRNA was diluted in RNase free 50 mM citrate buffer pH 3.0 to obtain a mRNA:lipid weight ratio of 10:1 (CIL:nucleotide 3:1 molar ratio). The aqueous and ethanol solutions were mixed in a 3:1 volume ratio using a microfluidic apparatus NanoAssemblr (Precision NanoSystems Inc.) atAtty. Dkt. No.117586-0146 a mixing rate of 12 mL / min. LNPs were dialyzed overnight against 500× sample volume using Slide-A-Lyzer G2 dialysis cassettes (Thermo Scientific) with a molecular weight cutoff of 10 K. For all groups in both experiments, treatment was given subcutaneously at 8-, 10-, and 12-weeks post injection with pHBV-DNA, and HBsAg was measured at 8-, 10-, 12-, 14-, 16-, and 18-weeks post injection with pHBV-DNA. At 18-weeks post injection with pHBV-DNA mice were classified based on whether HBsAg was undetectable and whether they responded to treatment, as indicated by a 0.5 log decrease to HBsAg levels. The averages for each group in the two experiments are summarized in Tables 8A and 8B below and the results for individual mice in each group are shown in FIGs.5A-5G as follows: placebo 1 (FIG.5A), 5 µg mRNA vaccine treatment (FIG.5B), 20 µg mRNA vaccine treatment (FIG.5C), 3 µg protein vaccine treatment (FIG.5D), placebo 2 (FIG. 5E), 10 µg mRNA vaccine treatment (FIG.5F), and 12 µg protein vaccine treatment (FIG. 5G). Table 8A. mRNA Vaccination Results for First ExperimentTable 8B. mRNA Vaccination Results for Second Experiment

[0101] Tables 8A-8B and FIGs.5A-5G show that the mRNA candidates of the present disclosure are not only effective at reducing circulating HBsAg levels in a dose-dependent manner, but they can also render circulating HBsAg undetectable in subjects. Table 8A and FIG.5B show that treatment with 5 µg of lipid-encapsulated 1-40X, 3-40X, 1-50X, and 3- 50X reduced circulating HBsAg substantially. Moreover, when the dosage was increased to 20 µg of lipid-encapsulated 1-40X, 3-40X, 1-50X, and 3-50X, HBsAg was undetectable inAtty. Dkt. No.117586-0146 all mice by week 14, and remained undetectable until the end of the experiment, which represents an improved outcome over the protein 3µg treatment group. See Table 8A and FIG.5C. Treatment with 3 µg of the protein vaccine (CLB-405 and CLB-505) resulted in broadly similar trends to the RNA vaccine treatment groups. See Table 8A and FIG.5D. Table 8B and FIG.5F show that treatment with 10 µg of 1-40X, 3-40X, 1-50X, and 3-50X reduced circulating HBsAg substantially. Taken together, the results shown in Tables 8A and 8B and FIGs.5B, 5C, and 5F demonstrate a dose dependent response to the mRNA vaccines of the present technology. Treatment with 12 µg of the protein vaccine (CLB-405 and CLB-505) resulted in broadly similar trends to the mRNA vaccine treatment groups. See Table 8B and FIG.5G.

[0102] Accordingly, these results demonstrate that the mRNA immunogenic compositions (e.g., vaccines) of the present disclosure are effective for expressing protein products in human cells, and are useful in compositions and methods for the treatment and prevention of chronic hepatitis B infection. Example 4: mRNA Candidates Pairwise Treatment using in vivo Chronic Hepatitis B Infection Model

[0103] The mRNA candidates were then evaluated in pairwise combinations for their efficacy in treating CHB. CBA / CaJ mice were injected with plasmids expressing the hepatitis B A2 adw genome (pHBV-DNA) through the tail vein at high pressure, which permeabilized hepatocytes, allowing for uptake of the HBV plasmid and stable HBV infection, with approximately 3 log HBsAg (log IU / ml). Mice were then given one of 7 treatments: placebo, anti-HBV siRNA, an equal parts mixture of 1-40X+3-40X+1-50X+3- 50X, an equal parts mixture of 1-40X+1-50X, an equal parts mixture of 1-40X+3-50X, an equal parts mixture of 3-40X+1-50X, or an equal parts mixture of 3-40X +3-50X. Treatment groups, doses, dosing day, sample collection day, and study termination date are summarized in Table 9 below (SC = sub-cutaneous; TA = test article; IM = intramuscular). siRNA treatment was performed subcutaneously (SC) which served as a positive control and lipid-encapsulated mRNA treatment was given intramuscularly (IM), with lipid encapsulation achieved as described in Example 3. Mice were terminated at day 66.Atty. Dkt. No.117586-0146 Table 9. mRNA candidate pairwise comparison experimental conditions

[0104] FIGs.6A-6H and Table 10 show that each pairwise combination of the lipid- encapsulated mRNA candidates successfully reduced HBsAg levels in vivo. Individual mouse data is shown for the placebo (FIG.6B), siRNA (FIG.6C), 1-40X+3-40X+1- 50X+3-50X (FIG.6D), 1-40X+1-50X (FIG.6E), 1-40X+3-50X (FIG.6F), 3-40X+1-50X (FIG.6G), and 3-40X +3-50X (FIG.6H) treatment groups. FIG.6A shows the average HBsAg levels for each treatment group. FIGs.6B-6C show that average HBsAg levels did not decrease in placebo treated mice, while siRNA treatment reduced average HBsAg, with a partial rebound in HBsAg levels by the end of the study. FIG.6D shows that mice receiving the mixture of 1-40X+3-40X+1-50X+3-50X exhibited a rapid and sustained decrease in HBsAg levels. Treatment with 1-40X+1-50X (FIG.6E), 1-40X+3-50X (FIG. 6F), 3-40X+1-50X (FIG.6G), and 3-40X +3-50X (FIG.6H) also caused a rapid and sustained decrease in HBsAg levels, with Table 10 showing that the treatment effect was sustained even as late as the termination of the study. Liver cells from treated mice were further analyzed for HBcAg positivity. Tissues from formalin fixed and paraffin embedded blocks of liver collected from experimental mice were sectioned at 4 µm and placed onto glass slides. Sections were stained to detect the presence of HBcAg. Specific expression of HBcAg was observed from all HDI / HBV liver samples, and mostly situated in nucleus. As shown in FIG.6I, HBcAg levels were substantially reduced in liver samples from mice treated with 1-40X+3-40X+1-50X+3-50X, 1-40X+1-50X, 1-40X+3-50X, 3-40X+1-50X, and 3-40X +3-50X compared to the placebo treated control. Taken together, these findings indicate that any combination of the 40X and 50X mRNA candidates of the present technology is effective in methods of treating CHB in and in vivo model. Table 10. HBsAg response to mRNA treatmentsAtty. Dkt. No.117586-0146

[0105] Accordingly, these results demonstrate that the mRNA immunogenic compositions (e.g., vaccines) of the present disclosure are effective for expressing protein products in human cells, and are useful in compositions and methods for the treatment and prevention of chronic hepatitis B infection. Example 5: mRNA Candidates and siRNA Treatment of CHB

[0106] The mRNA candidates were then evaluated for their efficacy in treating CHB when paired with an additional siRNA therapeutic. For this experiment, the AAV / HBV model was used. See Yang et al, Cellular & Molecular Immunology (2014) 11, 71–78. Briefly, male C57BL / 6 mice were infected with the AAV8 virus carrying 1.3 copies of the HBV genome (genotype D, serotype ayw) at 5×1010viral genome equivalents to establish a stable infection of ~4 log IU / mL HbsAg in hepatocytes.

[0107] As explained in Table 11, 8 treatment groups (n = 7) were given the following treatments at days 14, 28, and 42: a placebo, an anti-HBV siRNA AD66810, anti-HBV siRNA AD66810 on day 0 followed by 1 µg 1-40X, 3-40X, 1-50X, and 3-50X on days 14, 28 and 42, anti-HBV siRNA AD66810 on day 0 followed by 5 µg 1-40X, 3-40X, 1-50X, and 3-50X on days 14, 28 and 42, anti-HBV siRNA AD66810 on day 0 followed by 20 µg 1-40X, 3-40X, 1-50X, and 3-50X on days 14, 28 and 42, 5 µg 1-40X, 3-40X, 1-50X, and 3- 50X on days 14, 28 and 42, 30 µg protein vaccine (15 µg CLB-405 and 15 µg CLB-505) on day 14, 28 and 42, or siRNA AD66810 on day 0 and 30 µg protein vaccine (15 µg CLB-405 and 15 µg CLB-505) on day 14, 28 and 42. For all treatment groups the siRNA was given subcutaneously at 3 mg / kg and the lipid-encapsulated mRNA candidates were given intramuscularly with lipid encapsulation achieved as described in Example 3. Table 11: Study design to evaluate efficacy of mRNA candidates in combination with siRNAAtty. Dkt. No.117586-0146

[0108] As shown in FIG.7A, the combination therapy with siRNA and the mRNA candidates 1-40X, 3-40X, 1-50X, and 3-50X had a synergistic effect and reduced HBsAg levels to a greater degree than either therapy alone. The mice treated with siRNA followed by 1, 5 or 20 µg dose levels of the mRNA candidates (1-40X, 3-40X, 1-50X, and 3-50X) displayed 1.4, 2.1 and 1.6 log10IU / ml HBsAg reduction respectively from baseline one week after the last dose of mRNA was administered compared to 1.0 log10 IU / ml HBsAg reduction seen in siRNA monotherapy group. A statistically significant difference in efficacy was seen at peak response (Day 42) in the group dosed with 5 µg mRNA in combination with siRNA compared to the group dosed with siRNA alone. The mice in the placebo group resulted in no significant change in plasma HBsAg levels over the study duration. All mice showed a rebound in plasma HBsAg, with the group dosed with 5 µg mRNA in combination with siRNA showing the most delayed rebound with 7 / 7 mice demonstrating greater than 1.5 log10 decrease in HBsAg from baseline on day 42 (peak response day) compared to 0 / 7 in the group dosed with siRNA alone (FIG.7D). As shown in FIG.7B, the combination therapy with siRNA and 5 µg of the mRNA candidates 1-40X, 3-40X, 1-50X, and 3-50X had a synergistic effect and reduced HBsAg levels to a greater degree than either therapy alone, with levels remaining depressed throughout the course of the study. Combination therapy with siRNA and 5 µg of the mRNA candidates 1-40X, 3- 40X, 1-50X, and 3-50X had a similar effect to combination therapy with siRNA and a protein sub-unit vaccine (FIG.7B). FIG.7C shows the results of an ELISPOT analysis of splenocytes on termination (day 70). The splenocytes were harvested and frozen in 10% DMSO + 90% FBS for further analysis. The cells were thawed and resuspended with culture medium at 2e6 cells / mL. Splenocytes were stimulated with HBsAg peptide pool, negative control and positive control (ConA) for 40 hours (36-48 hours). The assay was performed according to the protocol of Mouse IFN-γ ELISpot kit. As shown by FIG.7C,Atty. Dkt. No.117586-0146 splenocytes from mice receiving siRNA and mRNA vaccine combination treatment had the highest rate of IFN-γ secretion, followed by splenocytes from mice receiving siRNA and protein vaccine (CLB-405 / CLB-505) combination treatment and splenocytes from mice receiving siRNA monotreatment respectively.

[0109] Separately, the mRNA vaccines were tested for immunogenicity in naïve mice. C57BL / 6 mice were administered three injections of mRNA candidates, 1-40X, 3-40X, 1- 50X and 3-50X at a total dose of 5 µg or 10 µg (candidates given at a 1:1:1:1 mass ratio). mRNA candidates were given intramuscularly on Days 0, 14 and 28 (n=6 / group). A placebo group was also included in the study as a negative control and dosed with phosphate buffered saline. During the course of the study, body weights were recorded every 2 weeks, and cage side observations were performed daily. Blood samples were collected on days 0, 14, 28, 42 and the terminal collection day which was Day 56. The plasma samples were analyzed for anti-CLB-405 and anti-CLB-505 end-point titers using a semi-quantitative titration method. The plasma samples were serially diluted 5-fold with blocking buffer for a total of 8 test points, with an initial dilution of 1:10. The data were reported as end point titers (EPT) which is the plasma dilution the cut point of 3 folds of blank average. Table 12 shows the study design. Table 12: Immunogenicity analysis study design.

[0110] FIG.7E shows the anti-CLB-405 and anti-CLB-505 end-point titer over time on days 0, 14, 28 and 42, with both dosages of 1-40X + 3-40X + 1-50X+ 3-50X generating a robust immunogenic response that increased over time.

[0111] Taken together, FIGs.7A-7E show that the mRNA candidates of the present technology are effective in another in vivo CHB model, induce immunogenic responses in naïve mice, and the mRNA candidates display therapeutic synergy when paired with an anti-CHB siRNA treatment. Accordingly, these results demonstrate that the mRNAAtty. Dkt. No.117586-0146 immunogenic compositions (e.g., vaccines) of the present disclosure are effective for expressing protein products in human cells, and are useful in compositions and methods for the treatment and prevention of chronic hepatitis B infection. Example 6: Development of Additional mRNA Candidates and Expression Analysis

[0112] To further assess mRNA-based therapeutics for CHB, two additional mRNA candidates were synthesized. mRNAs encoding the modified HBsAg sequence of 40X (SEQ ID NO: 10) and 50X (SEQ ID NO: 11) were synthesized by T7 polymerase-mediated in vitro transcription (IVT) based on a linearized DNA template (pIVT-A) containing codon-optimized 40X- and 50X-encoding DNA flanked by 5’ and 3’ untranslated regions (UTRs) and an encoded 45 residue poly-A tail. During IVT N1-Methyl pseudouridine was incorporated into the mRNAs and co-transcriptional capping was carried out using ARCA (Anti-Reverse Cap Analog). IVT products were then purified by RP-FPLC followed by buffer exchange against Nuclease free water using 10 kDa MWCO Amicon Ultra (Regenerated Cellulose) membrane. A depiction of the general construct design for mRNA- based therapeutics of the present technology is shown in FIG.1 and a description of the additional mRNA candidate is provided in Table 13. Table 13. RNA vaccine candidates

[0113] The 5’ UTR sequence in Table 13 is as follows 5’-AUR1 (SEQ ID NO: 6): UUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAG AAGAGUAAGAAGAAAUAUAAGA

[0114] The 3’ UTR sequence in Table 13 is as follows: 3’-AUR1 (SEQ ID NO: 7): GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUG UACCUCUUGGUCUUUGAAUAAAGCCUGAGUA

[0115] The 5’ UTR for 5-40X and 5-50X was derived from the Modern mRNA1273 expression vector, complete sequence (GenBank: OR134578.1) while the 3’ UTR from 5- 40X and 5-50X was derived from Mus musculus isolate LaPaz_047A alpha-globin (Hbat1)Atty. Dkt. No.117586-0146 gene, complete coding sequence (GenBank: EF605436.1). This combination of a 5’ UTR from a human mRNA vaccine candidate sequence with a 3’ UTR from a highly but selectively transcribed murine gene represents a novel combination of regulatory, untranslated regions. The 5-40X and 5-50X candidates were then assessed for in vivo expression. Briefly, both mRNAs were transfected into three different cell lines: a human monocytic cell line (U937), a human dermal fibroblast line (HDF), and a skeleton muscle line (SkMC). HDF and SKMC cells were seeded in a 6-well plate at a density of 0.15 x 106cells / well and were transfected using the Lipofectamine 3000 (Thermo Fischer cat. No. L3000001) transfection reagent according to the manufacturer’s protocol. U937 cells were transfected by electroporation and were then seeded into a 6-well plate at 0.15 x 106cells / well. 48 hours post-transfection, cell lysates were prepared in a reducing loading dye, heated at 95°C for 10 min and loaded on NuPAGE 4-12% gels for electrophoresis. Post- electrophoresis the protein was transferred to Nitrocellulose membranes using iBlot2 (Thermo Fischer cat. No. IB23001) according to the manufacturers protocol. The membranes were blocked in 5% Milk in TBS-T followed by overnight primary antibody incubation (Novus, NB100-62652) at 4°C followed by HRP-labeled goat polyclonal anti- rabbit immunoglobulins (LI-Cor, 926-32211)) as a secondary antibody in 5 % milk solution. CLB-405 and CLB-505 protein served as positive controls, and untransfected cells served as a negative control. The results for each cell type are shown in FIGs.8A-8C. Both the 5- 40X and 5-50X constructs were successfully expressed at 48 hours post transfection in HDF cells (FIG.8A), SkMc cells (FIG.8B), and U937 cells (FIG.8C).

[0116] Taken together, these results demonstrate that the 5-40X and 5-50X mRNA constructs of the present technology are successfully expressed in multiple human cell lines of differing type and origin. Accordingly, these results demonstrate that the mRNA constructs of the present technology are useful vectors for expressing HBV antigenic epitopes in subjects in need thereof. Example 7: 5-40X and 5-50X mRNA Candidates in vivo Analysis (prophetic)

[0117] The 5-40X and 5-50X mRNA candidates of the present disclosure will be compared to ascertain therapeutic efficacy in a murine model. Briefly, 5-40X and 5-50X will be formulated to prepare mRNA-encapsulated LNPs. LNP formation will be achieved using standard methodologies, for example by using Precision Nanosystems GenVoy-ILM commercial reagent on the NanoAssemblr system according to the manufacturer’sAtty. Dkt. No.117586-0146 protocols. The formulated candidates will then be evaluated for efficacy at a 1:1 ratio in the HDI and AAV-HBV models as described herein. Example 8: Comparison of mRNA Candidates of the Present Disclosure (prophetic)

[0118] The two sets of mRNA candidates of the present disclosure, 1-40X, 3-40X, 1-50X, and 3-50X and 5-40X and 5-50X, will be compared to ascertain therapeutic efficacy in a murine model. Briefly, the 5-40X and 5-50X encapsulated LNPs of Example 7 will be evaluated for in vivo efficacy in the HDI and AAV HBV models, as described herein, in comparison with various combinations of 1-40X or 3-40X with 1-50X or 3-50X encapsulated in LNPs. Example 9: Development of DNA Candidates

[0119] DNA vaccines were next examined for their potential therapeutic efficacy. Briefly, a serotype 5 adenovirus was genetically engineered to contain the following heterologous construct: CAG promoter-DNA encoding CLB-405-CMV promoter-DNA encoding CLB-505. The CAG promoter (SEQ ID NO: 62) was operably linked to drive expression of CLB-405, and the CMV promoter (SEQ ID NO: 63) was operably linked to drive expression of CLB-505. The sequence of the entire construct, identified as Ad5- BNP4-BNP5, is (SEQ ID NO: 24).

[0120] The expression of Ad5-BNP4-BNP5 in target cells was confirmed using HEK293A cells and western blotting. HEK293A cells were transduced with Ad5-BNP4- BNP5 as follows: Briefly, HEK293A cells are seeded at a suitable concentration incubated overnight at 37℃, 5% CO2. The recombinant adenovirus carrying plasmid pAd-BNP4- BNP5 is added to the cells and incubated at 37℃, 5% CO2 for 2 days. The cells are then collected and centrifuged and media is discarded followed by two wash steps with PBS. The cells are then lysed with RIPA lysis buffer and centrifuged to collect the supernatant, the pellet is discarded. The concentration of protein is determined using Bradford protein concentration determination kit. 10µg protein samples from transduced cells were analyzed using standard western blotting techniques with a primary antibody against CLB-405 (A-I 0828 at a 1:1000 dilution) or a primary antibody against CLB-505 (6F3A1 / A9 at a 1:1000 dilution) and a goat anti-mouse HRP IgG secondary antibody at a 1:10000 dilution. CLB- 405 (100ng) and CLB-505 (100ng) protein samples served as positive controls, non- transduced HEK293A cells (293A cell only) served as a negative control, and TubulinAtty. Dkt. No.117586-0146 served as a loading control, with Marker (Ladder) used to determine band size. FIG.9A shows that HEK293A cells transduced with Ad5-BNP4-BNP5 successfully expressed BNP4 and BNP5 as indicated by the 30 kDa and 27 kDa bands.

[0121] To assess the therapeutic efficacy of Ad5-BNP4-BNP5, the DNA vaccine construct was tested in an in vivo CHB model. For this experiment, the AAV / HBV model was used. See Yang et al, Cellular & Molecular Immunology (2014) 11, 71–78). Briefly, male C57BL / 6 mice were infected with the AAV8 virus carrying 1.3 copies of the HBV genome (genotype D, serotype ayw) at 5×1010viral genome equivalents to establish a stable infection of ~4 log IU / mL HBsAg in hepatocytes. Once infection was established, mice received one of five treatments: a placebo, the anti-HBV siRNA AD-66810 (siRNA) on day 0, a DNA vector control (GFP DNA) on days 49, 63, and 77, the DNA vaccine candidate Ad5-BNP4-BNP5 (2X109viral particles) on days 49, 63, and 77 and the AD-66810 siRNA on day 0, or Ad5-BNP4-BNP5 on days 49, 63, and 77. FIG.9B shows that treatment with siRNA alone resulted in a substantial early drop in HBsAg levels, although they rebounded over the course of the experiment. Surprisingly, FIG.9B also shows that combination treatment with AD66810 and Ad5-BNP4-BNP5 resulted in a later decrease in HBsAg levels which was sustained throughout the course of the experiment without any rebound.

[0122] Taken together, these results show that the Ad5-BNP4-BNP5 construct is expressed in human cells and that in an in vivo model for CHB infection Ad5-BNP4-BNP5 displayed an unexpected and substantial synergistic therapeutic effect when combined with anti-HBV siRNA treatment. Accordingly, these results demonstrate that the DNA immunogenic compositions (e.g., vaccines) of the present disclosure are effective for expressing protein products in human cells, and are useful in compositions and methods for the treatment and prevention of chronic hepatitis B infection.Atty. Dkt. No.117586-0146 EQUIVALENTS

[0123] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0124] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0125] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.Atty. Dkt. No.117586-0146

[0126] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0127] Other embodiments are set forth within the following claims.Atty. Dkt. No.117586-0146 SEQUENCE LISTING RNA Sequence of 5’ UTR ARNV1: AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 1) RNA Sequence of 5’ UTR ARNV2: UCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUA CUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCU GAAAAUUUUCACCAUUUACGAACGAUAGC (SEQ ID NO: 2) RNA Sequence of 5’ UTR ARNV3: AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCCAACAAU CUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCUAC UUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUUCUG AAAAUUUUCACCAUUUACGAACGAUAGC (SEQ ID NO: 3) RNA Sequence of 5’ UTR ARNV4: CACUCGCGCUGCCAUCACUCUUCCGCCGUCUUCGCCGCCAUCCUCGGCGCGAC UCGCUUCUUUCGGUUCUACCAGGUAGAGUCCGCCGCCAUCCUCCACCCAACA ACUUGUCUCGCUCCGGGGAACGCUCGGAAACUCCCGGCCGCCGCCACCCGCGU CUGUUCUGUUACACAAGGGAAGAAAAGCCGCUGCCGCACUCCGAGUGU (SEQ ID NO: 4) RNA Sequence of 3’ UTR ARNV1: GCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGU CCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUG CCUAAUAAAAAACAUUUAUUUUCAUUGC (SEQ ID NO: 5) RNA Sequence of 5’ UTR AUR1: UUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGA AAAGAAGAGUAAGAAGAAAUAUAAGA (SEQ ID NO: 6) RNA Sequence of 3’ UTR AUR1: GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGC ACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUA (SEQ ID NO: 7) Amino Acid Sequence of CLB-405 (HBV epitope underlined and linker bolded): MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTS NHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTST GPCRTCMTTGSGSCKTCTTPAQGNSMFPSGSGSCKTCTTPAQGNSMFPSGSGSTGQ GTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVG LSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI (SEQ ID NO: 8) Amino Acid Sequence of CLB-505 (HBV epitope underlined and linker bolded): MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTS NHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTST GPCRTCMTTGQGTSMYPSCCCTKPSDGNCGSGSCTKPTDGNCGSGSCTKPTDGNCAtty. Dkt. No.117586-0146 TCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYW GPSLYSILSPFLPLLPIFFCLWVYI (SEQ ID NO: 9) RNA sequence of 40X (used in 5-40X): AUGGAGAACAUCACCUCUGGCUUUCUGGGCCCUCUGCUGGUGCUGCAGGCUG GCUUCUUUCUGCUGACCCGCAUCCUGACAAUCCCUCAGAGCCUGGAUAGCUG GUGGACCAGCCUGAAUUUUCUCGGCGGCACAACAGUGUGCCUGGGCCAGAAU AGCCAGUCUCCUACCAGCAAUCACAGCCCCACCAGCUGUCCUCCAACCUGUCC UGGCUACAGAUGGAUGUGCCUGCGGCGGUUCAUCAUCUUUCUGUUCAUCCUG CUGCUGUGCCUGAUCUUCCUGCUCGUGCUGCUGGAUUACCAGGGAAUGCUGC CUGUGUGUCCUCUGAUCCCUGGCAGCAGCACAACAAGCACAGGCCCUUGCAG AACCUGUAUGACAACAGGCUCUGGCUCCUGCAAGACCUGCACAACACCAGCU CAGGGCAACAGCAUGUUUCCUAGCGGCAGCGGCAGCUGCAAGACAUGUACUA CCCCUGCACAGGGCAACUCUAUGUUCCCAUCUGGCAGCGGCUCUACCGGCCA GGGCACAUCUAUGUACCCUAGCUGCUGUUGCACCAAGCCUAGCGACGGCAAC UGCACAUGCAUCCCCAUUCCUAGCAGCUGGGCCUUCGGCAAGUUUCUGUGGG AAUGGGCCAGCGCCAGAUUCAGCUGGCUGAGCCUGCUGGUUCCUUUCGUGCA GUGGUUCGUGGGCCUGUCUCCUACAGUGUGGCUGAGCGUGAUCUGGAUGAUG UGGUAUUGGGGCCCUAGCCUGUACAGCAUUCUGAGCCCUUUUCUGCCCCUGC UGCCUAUCUUCUUCUGCCUGUGGGUGUACAUCUGAUGA (SEQ ID NO: 10) RNA sequence of 50X (used in 5-50X): AUGGAGAACAUCACCUCUGGCUUUCUGGGCCCUCUGCUGGUGCUGCAGGCUG GCUUCUUUCUGCUGACCCGCAUCCUGACAAUCCCUCAGAGCCUGGAUAGCUG GUGGACCAGCCUGAAUUUUCUCGGCGGCACAACAGUGUGCCUGGGCCAGAAU AGCCAGUCUCCUACCAGCAAUCACAGCCCCACCAGCUGUCCUCCAACCUGUCC UGGCUACAGAUGGAUGUGCCUGCGGCGGUUCAUCAUCUUUCUGUUCAUCCUG CUGCUGUGCCUGAUCUUCCUGCUCGUGCUGCUGGAUUACCAGGGAAUGCUGC CUGUGUGUCCUCUGAUCCCUGGCAGCAGCACAACAAGCACAGGCCCUUGCAG AACCUGUAUGACAACCGGCCAGGGCACCAGCAUGUACCCUAGCUGUUGUUGC ACCAAGCCUAGCGACGGCAAUUGUGGCAGCGGCAGCUGUACAAAGCCCACCG AUGGAAAUUGCGGCUCCGGCUCUUGUACCAAGCCAACAGAUGGCAACUGCAC AUGCAUCCCCAUUCCUAGCAGCUGGGCCUUCGGCAAGUUUCUGUGGGAAUGG GCCAGCGCCAGAUUCAGCUGGCUGAGCCUGCUGGUUCCUUUCGUGCAGUGGU UCGUGGGCCUGUCUCCUACAGUGUGGCUGAGCGUGAUCUGGAUGAUGUGGUA UUGGGGCCCUAGCCUGUACAGCAUUCUGAGCCCUUUUCUGCCCCUGCUGCCU AUCUUCUUCUGCCUGUGGGUGUACAUCUGAUGA (SEQ ID NO: 11) RNA sequence of 1-40X (5’ and 3’ UTR bolded): AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAG AACAUCACCAGCGGCUUCCUGGGCCCCCUGCUGGUGCUGCAGGCCGGCUUCU UCCUGCUGACCAGGAUCCUGACCAUCCCCCAGAGCCUGGACAGCUGGUGGAC CAGCCUGAACUUCCUGGGCGGCACCACCGUGUGCCUGGGCCAGAACAGCCAG AGCCCCACCAGCAACCACAGCCCCACCAGCUGCCCCCCCACCUGCCCCGGCUA CAGGUGGAUGUGCCUGAGGAGGUUCAUCAUCUUCCUGUUCAUCCUGCUGCUG UGCCUGAUCUUCCUGCUGGUGCUGCUGGACUACCAGGGCAUGCUGCCCGUGU GCCCCCUGAUCCCCGGCAGCAGCACCACCAGCACCGGCCCCUGCAGGACCUGC AUGACCACCGGCAGCGGCAGCUGCAAGACCUGCACCACCCCCGCCCAGGGCAA CAGCAUGUUCCCCAGCGGCAGCGGCAGCUGCAAGACCUGCACCACCCCCGCCC AGGGCAACAGCAUGUUCCCCAGCGGCAGCGGCAGCACCGGCCAGGGCACCAGAtty. Dkt. No.117586-0146 CAUGUACCCCAGCUGCUGCUGCACCAAGCCCAGCGACGGCAACUGCACCUGC AUCCCCAUCCCCAGCAGCUGGGCCUUCGGCAAGUUCCUGUGGGAGUGGGCCA GCGCCAGGUUCAGCUGGCUGAGCCUGCUGGUGCCCUUCGUGCAGUGGUUCGU GGGCCUGAGCCCCACCGUGUGGCUGAGCGUGAUCUGGAUGAUGUGGUACUGG GGCCCCAGCCUGUACAGCAUCCUGAGCCCCUUCCUGCCCCUGCUGCCCAUCUU CUUCUGCCUGUGGGUGUACAUCUAGUAAUGAGCUCGCUUUCUUGCUGUCCA AUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGG GAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUU UAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 12) RNA sequence of 2-40X (5’ and 3’ UTR bolded): UCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCU ACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUU CUGAAAAUUUUCACCAUUUACGAACGAUAGCAUGGAGAACAUCACCAGCGG CUUCCUGGGCCCCCUGCUGGUGCUGCAGGCCGGCUUCUUCCUGCUGACCAGG AUCCUGACCAUCCCCCAGAGCCUGGACAGCUGGUGGACCAGCCUGAACUUCC UGGGCGGCACCACCGUGUGCCUGGGCCAGAACAGCCAGAGCCCCACCAGCAA CCACAGCCCCACCAGCUGCCCCCCCACCUGCCCCGGCUACAGGUGGAUGUGCC UGAGGAGGUUCAUCAUCUUCCUGUUCAUCCUGCUGCUGUGCCUGAUCUUCCU GCUGGUGCUGCUGGACUACCAGGGCAUGCUGCCCGUGUGCCCCCUGAUCCCC GGCAGCAGCACCACCAGCACCGGCCCCUGCAGGACCUGCAUGACCACCGGCAG CGGCAGCUGCAAGACCUGCACCACCCCCGCCCAGGGCAACAGCAUGUUCCCCA GCGGCAGCGGCAGCUGCAAGACCUGCACCACCCCCGCCCAGGGCAACAGCAU GUUCCCCAGCGGCAGCGGCAGCACCGGCCAGGGCACCAGCAUGUACCCCAGC UGCUGCUGCACCAAGCCCAGCGACGGCAACUGCACCUGCAUCCCCAUCCCCAG CAGCUGGGCCUUCGGCAAGUUCCUGUGGGAGUGGGCCAGCGCCAGGUUCAGC UGGCUGAGCCUGCUGGUGCCCUUCGUGCAGUGGUUCGUGGGCCUGAGCCCCA CCGUGUGGCUGAGCGUGAUCUGGAUGAUGUGGUACUGGGGCCCCAGCCUGUA CAGCAUCCUGAGCCCCUUCCUGCCCCUGCUGCCCAUCUUCUUCUGCCUGUGGG UGUACAUCUAGUAAUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAG GUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGG GCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAA (SEQ ID NO: 13) RNA sequence of 3-40X (5’ and 3’ UTR bolded): AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCCAACAA UCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCU ACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUU CUGAAAAUUUUCACCAUUUACGAACGAUAGCAUGGAGAACAUCACCAGCGG CUUCCUGGGCCCCCUGCUGGUGCUGCAGGCCGGCUUCUUCCUGCUGACCAGG AUCCUGACCAUCCCCCAGAGCCUGGACAGCUGGUGGACCAGCCUGAACUUCC UGGGCGGCACCACCGUGUGCCUGGGCCAGAACAGCCAGAGCCCCACCAGCAA CCACAGCCCCACCAGCUGCCCCCCCACCUGCCCCGGCUACAGGUGGAUGUGCC UGAGGAGGUUCAUCAUCUUCCUGUUCAUCCUGCUGCUGUGCCUGAUCUUCCU GCUGGUGCUGCUGGACUACCAGGGCAUGCUGCCCGUGUGCCCCCUGAUCCCC GGCAGCAGCACCACCAGCACCGGCCCCUGCAGGACCUGCAUGACCACCGGCAGAtty. Dkt. No.117586-0146 CGGCAGCUGCAAGACCUGCACCACCCCCGCCCAGGGCAACAGCAUGUUCCCCA GCGGCAGCGGCAGCUGCAAGACCUGCACCACCCCCGCCCAGGGCAACAGCAU GUUCCCCAGCGGCAGCGGCAGCACCGGCCAGGGCACCAGCAUGUACCCCAGC UGCUGCUGCACCAAGCCCAGCGACGGCAACUGCACCUGCAUCCCCAUCCCCAG CAGCUGGGCCUUCGGCAAGUUCCUGUGGGAGUGGGCCAGCGCCAGGUUCAGC UGGCUGAGCCUGCUGGUGCCCUUCGUGCAGUGGUUCGUGGGCCUGAGCCCCA CCGUGUGGCUGAGCGUGAUCUGGAUGAUGUGGUACUGGGGCCCCAGCCUGUA CAGCAUCCUGAGCCCCUUCCUGCCCCUGCUGCCCAUCUUCUUCUGCCUGUGGG UGUACAUCUAGUAAUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAG GUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGG GCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAA (SEQ ID NO: 14) RNA sequence of 4-40X (5’ and 3’ UTR bolded): CACUCGCGCUGCCAUCACUCUUCCGCCGUCUUCGCCGCCAUCCUCGGCGC GACUCGCUUCUUUCGGUUCUACCAGGUAGAGUCCGCCGCCAUCCUCCACC CAACAACUUGUCUCGCUCCGGGGAACGCUCGGAAACUCCCGGCCGCCGCC ACCCGCGUCUGUUCUGUUACACAAGGGAAGAAAAGCCGCUGCCGCACUCC GAGUGUAUGGAGAACAUCACCAGCGGCUUCCUGGGCCCCCUGCUGGUGCUGC AGGCCGGCUUCUUCCUGCUGACCAGGAUCCUGACCAUCCCCCAGAGCCUGGA CAGCUGGUGGACCAGCCUGAACUUCCUGGGCGGCACCACCGUGUGCCUGGGC CAGAACAGCCAGAGCCCCACCAGCAACCACAGCCCCACCAGCUGCCCCCCCAC CUGCCCCGGCUACAGGUGGAUGUGCCUGAGGAGGUUCAUCAUCUUCCUGUUC AUCCUGCUGCUGUGCCUGAUCUUCCUGCUGGUGCUGCUGGACUACCAGGGCA UGCUGCCCGUGUGCCCCCUGAUCCCCGGCAGCAGCACCACCAGCACCGGCCCC UGCAGGACCUGCAUGACCACCGGCAGCGGCAGCUGCAAGACCUGCACCACCC CCGCCCAGGGCAACAGCAUGUUCCCCAGCGGCAGCGGCAGCUGCAAGACCUG CACCACCCCCGCCCAGGGCAACAGCAUGUUCCCCAGCGGCAGCGGCAGCACCG GCCAGGGCACCAGCAUGUACCCCAGCUGCUGCUGCACCAAGCCCAGCGACGG CAACUGCACCUGCAUCCCCAUCCCCAGCAGCUGGGCCUUCGGCAAGUUCCUG UGGGAGUGGGCCAGCGCCAGGUUCAGCUGGCUGAGCCUGCUGGUGCCCUUCG UGCAGUGGUUCGUGGGCCUGAGCCCCACCGUGUGGCUGAGCGUGAUCUGGAU GAUGUGGUACUGGGGCCCCAGCCUGUACAGCAUCCUGAGCCCCUUCCUGCCC CUGCUGCCCAUCUUCUUCUGCCUGUGGGUGUACAUCUAGUAAUGAGCUCGCU UUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAAC UACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCU AAUAAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 15) RNA sequence of 5-40X (5’ and 3’ UTR bolded): UUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGA GAAAAGAAGAGUAAGAAGAAAUAUAAGAAUGGAGAACAUCACCUCUGGCUU UCUGGGCCCUCUGCUGGUGCUGCAGGCUGGCUUCUUUCUGCUGACCCGCAUC CUGACAAUCCCUCAGAGCCUGGAUAGCUGGUGGACCAGCCUGAAUUUUCUCG GCGGCACAACAGUGUGCCUGGGCCAGAAUAGCCAGUCUCCUACCAGCAAUCA CAGCCCCACCAGCUGUCCUCCAACCUGUCCUGGCUACAGAUGGAUGUGCCUGAtty. Dkt. No.117586-0146 CGGCGGUUCAUCAUCUUUCUGUUCAUCCUGCUGCUGUGCCUGAUCUUCCUGC UCGUGCUGCUGGAUUACCAGGGAAUGCUGCCUGUGUGUCCUCUGAUCCCUGG CAGCAGCACAACAAGCACAGGCCCUUGCAGAACCUGUAUGACAACAGGCUCU GGCUCCUGCAAGACCUGCACAACACCAGCUCAGGGCAACAGCAUGUUUCCUA GCGGCAGCGGCAGCUGCAAGACAUGUACUACCCCUGCACAGGGCAACUCUAU GUUCCCAUCUGGCAGCGGCUCUACCGGCCAGGGCACAUCUAUGUACCCUAGC UGCUGUUGCACCAAGCCUAGCGACGGCAACUGCACAUGCAUCCCCAUUCCUA GCAGCUGGGCCUUCGGCAAGUUUCUGUGGGAAUGGGCCAGCGCCAGAUUCAG CUGGCUGAGCCUGCUGGUUCCUUUCGUGCAGUGGUUCGUGGGCCUGUCUCCU ACAGUGUGGCUGAGCGUGAUCUGGAUGAUGUGGUAUUGGGGCCCUAGCCUG UACAGCAUUCUGAGCCCUUUUCUGCCCCUGCUGCCUAUCUUCUUCUGCCUGU GGGUGUACAUCUGAUGAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUG CCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCU GAGUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 16) RNA sequence of 1-50X (5’ and 3’ UTR bolded): AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAG AACAUCACCAGCGGCUUCCUGGGCCCCCUGCUGGUGCUGCAGGCCGGCUUCU UCCUGCUGACCAGGAUCCUGACCAUCCCCCAGAGCCUGGACAGCUGGUGGAC CAGCCUGAACUUCCUGGGCGGCACCACCGUGUGCCUGGGCCAGAACAGCCAG AGCCCCACCAGCAACCACAGCCCCACCAGCUGCCCCCCCACCUGCCCCGGCUA CAGGUGGAUGUGCCUGAGGAGGUUCAUCAUCUUCCUGUUCAUCCUGCUGCUG UGCCUGAUCUUCCUGCUGGUGCUGCUGGACUACCAGGGCAUGCUGCCCGUGU GCCCCCUGAUCCCCGGCAGCAGCACCACCAGCACCGGCCCCUGCAGGACCUGC AUGACCACCGGCCAGGGCACCAGCAUGUACCCCAGCUGCUGCUGCACCAAGC CCAGCGACGGCAACUGCGGCAGCGGCAGCUGCACCAAGCCCACCGACGGCAA CUGCGGCAGCGGCAGCUGCACCAAGCCCACCGACGGCAACUGCACCUGCAUCC CCAUCCCCAGCAGCUGGGCCUUCGGCAAGUUCCUGUGGGAGUGGGCCAGCGC CAGGUUCAGCUGGCUGAGCCUGCUGGUGCCCUUCGUGCAGUGGUUCGUGGGC CUGAGCCCCACCGUGUGGCUGAGCGUGAUCUGGAUGAUGUGGUACUGGGGCC CCAGCCUGUACAGCAUCCUGAGCCCCUUCCUGCCCCUGCUGCCCAUCUUCUUC UGCCUGUGGGUGUACAUCUAGUAAUGAGCUCGCUUUCUUGCUGUCCAAUUU CUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUA UUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUU UUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 17) RNA sequence of 2-50X (5’ and 3’ UTR bolded): UCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCU ACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUU CUGAAAAUUUUCACCAUUUACGAACGAUAGCAUGGAGAACAUCACCAGCGG CUUCCUGGGCCCCCUGCUGGUGCUGCAGGCCGGCUUCUUCCUGCUGACCAGG AUCCUGACCAUCCCCCAGAGCCUGGACAGCUGGUGGACCAGCCUGAACUUCC UGGGCGGCACCACCGUGUGCCUGGGCCAGAACAGCCAGAGCCCCACCAGCAA CCACAGCCCCACCAGCUGCCCCCCCACCUGCCCCGGCUACAGGUGGAUGUGCC UGAGGAGGUUCAUCAUCUUCCUGUUCAUCCUGCUGCUGUGCCUGAUCUUCCU GCUGGUGCUGCUGGACUACCAGGGCAUGCUGCCCGUGUGCCCCCUGAUCCCC GGCAGCAGCACCACCAGCACCGGCCCCUGCAGGACCUGCAUGACCACCGGCCAAtty. Dkt. No.117586-0146 GGGCACCAGCAUGUACCCCAGCUGCUGCUGCACCAAGCCCAGCGACGGCAAC UGCGGCAGCGGCAGCUGCACCAAGCCCACCGACGGCAACUGCGGCAGCGGCA GCUGCACCAAGCCCACCGACGGCAACUGCACCUGCAUCCCCAUCCCCAGCAGC UGGGCCUUCGGCAAGUUCCUGUGGGAGUGGGCCAGCGCCAGGUUCAGCUGGC UGAGCCUGCUGGUGCCCUUCGUGCAGUGGUUCGUGGGCCUGAGCCCCACCGU GUGGCUGAGCGUGAUCUGGAUGAUGUGGUACUGGGGCCCCAGCCUGUACAGC AUCCUGAGCCCCUUCCUGCCCCUGCUGCCCAUCUUCUUCUGCCUGUGGGUGU ACAUCUAGUAAUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUU CCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCC UUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAA (SEQ ID NO: 18) RNA sequence of 3-50X (5’ and 3’ UTR bolded): AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCCAACAA UCUCAACACAACAUAUACAAAACAAACGAAUCUCAAGCAAUCAAGCAUUCU ACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAAUUUU CUGAAAAUUUUCACCAUUUACGAACGAUAGCAUGGAGAACAUCACCAGCGG CUUCCUGGGCCCCCUGCUGGUGCUGCAGGCCGGCUUCUUCCUGCUGACCAGG AUCCUGACCAUCCCCCAGAGCCUGGACAGCUGGUGGACCAGCCUGAACUUCC UGGGCGGCACCACCGUGUGCCUGGGCCAGAACAGCCAGAGCCCCACCAGCAA CCACAGCCCCACCAGCUGCCCCCCCACCUGCCCCGGCUACAGGUGGAUGUGCC UGAGGAGGUUCAUCAUCUUCCUGUUCAUCCUGCUGCUGUGCCUGAUCUUCCU GCUGGUGCUGCUGGACUACCAGGGCAUGCUGCCCGUGUGCCCCCUGAUCCCC GGCAGCAGCACCACCAGCACCGGCCCCUGCAGGACCUGCAUGACCACCGGCCA GGGCACCAGCAUGUACCCCAGCUGCUGCUGCACCAAGCCCAGCGACGGCAAC UGCGGCAGCGGCAGCUGCACCAAGCCCACCGACGGCAACUGCGGCAGCGGCA GCUGCACCAAGCCCACCGACGGCAACUGCACCUGCAUCCCCAUCCCCAGCAGC UGGGCCUUCGGCAAGUUCCUGUGGGAGUGGGCCAGCGCCAGGUUCAGCUGGC UGAGCCUGCUGGUGCCCUUCGUGCAGUGGUUCGUGGGCCUGAGCCCCACCGU GUGGCUGAGCGUGAUCUGGAUGAUGUGGUACUGGGGCCCCAGCCUGUACAGC AUCCUGAGCCCCUUCCUGCCCCUGCUGCCCAUCUUCUUCUGCCUGUGGGUGU ACAUCUAGUAAUGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUU CCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCC UUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAA (SEQ ID NO: 19) RNA sequence of 4-50X (5’ and 3’ UTR bolded): CACUCGCGCUGCCAUCACUCUUCCGCCGUCUUCGCCGCCAUCCUCGGCGC GACUCGCUUCUUUCGGUUCUACCAGGUAGAGUCCGCCGCCAUCCUCCACC CAACAACUUGUCUCGCUCCGGGGAACGCUCGGAAACUCCCGGCCGCCGCC ACCCGCGUCUGUUCUGUUACACAAGGGAAGAAAAGCCGCUGCCGCACUCC GAGUGUAUGGAGAACAUCACCAGCGGCUUCCUGGGCCCCCUGCUGGUGCUGC AGGCCGGCUUCUUCCUGCUGACCAGGAUCCUGACCAUCCCCCAGAGCCUGGA CAGCUGGUGGACCAGCCUGAACUUCCUGGGCGGCACCACCGUGUGCCUGGGC CAGAACAGCCAGAGCCCCACCAGCAACCACAGCCCCACCAGCUGCCCCCCCAC CUGCCCCGGCUACAGGUGGAUGUGCCUGAGGAGGUUCAUCAUCUUCCUGUUCAtty. Dkt. No.117586-0146 AUCCUGCUGCUGUGCCUGAUCUUCCUGCUGGUGCUGCUGGACUACCAGGGCA UGCUGCCCGUGUGCCCCCUGAUCCCCGGCAGCAGCACCACCAGCACCGGCCCC UGCAGGACCUGCAUGACCACCGGCCAGGGCACCAGCAUGUACCCCAGCUGCU GCUGCACCAAGCCCAGCGACGGCAACUGCGGCAGCGGCAGCUGCACCAAGCC CACCGACGGCAACUGCGGCAGCGGCAGCUGCACCAAGCCCACCGACGGCAAC UGCACCUGCAUCCCCAUCCCCAGCAGCUGGGCCUUCGGCAAGUUCCUGUGGG AGUGGGCCAGCGCCAGGUUCAGCUGGCUGAGCCUGCUGGUGCCCUUCGUGCA GUGGUUCGUGGGCCUGAGCCCCACCGUGUGGCUGAGCGUGAUCUGGAUGAUG UGGUACUGGGGCCCCAGCCUGUACAGCAUCCUGAGCCCCUUCCUGCCCCUGC UGCCCAUCUUCUUCUGCCUGUGGGUGUACAUCUAGUAAUGAGCUCGCUUUC UUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUAC UAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAU AAAAAACAUUUAUUUUCAUUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 20) RNA sequence of 5-50X (5’ and 3’ UTR bolded): UUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGA GAAAAGAAGAGUAAGAAGAAAUAUAAGAAUGGAGAACAUCACCUCUGGCUU UCUGGGCCCUCUGCUGGUGCUGCAGGCUGGCUUCUUUCUGCUGACCCGCAUC CUGACAAUCCCUCAGAGCCUGGAUAGCUGGUGGACCAGCCUGAAUUUUCUCG GCGGCACAACAGUGUGCCUGGGCCAGAAUAGCCAGUCUCCUACCAGCAAUCA CAGCCCCACCAGCUGUCCUCCAACCUGUCCUGGCUACAGAUGGAUGUGCCUG CGGCGGUUCAUCAUCUUUCUGUUCAUCCUGCUGCUGUGCCUGAUCUUCCUGC UCGUGCUGCUGGAUUACCAGGGAAUGCUGCCUGUGUGUCCUCUGAUCCCUGG CAGCAGCACAACAAGCACAGGCCCUUGCAGAACCUGUAUGACAACCGGCCAG GGCACCAGCAUGUACCCUAGCUGUUGUUGCACCAAGCCUAGCGACGGCAAUU GUGGCAGCGGCAGCUGUACAAAGCCCACCGAUGGAAAUUGCGGCUCCGGCUC UUGUACCAAGCCAACAGAUGGCAACUGCACAUGCAUCCCCAUUCCUAGCAGC UGGGCCUUCGGCAAGUUUCUGUGGGAAUGGGCCAGCGCCAGAUUCAGCUGGC UGAGCCUGCUGGUUCCUUUCGUGCAGUGGUUCGUGGGCCUGUCUCCUACAGU GUGGCUGAGCGUGAUCUGGAUGAUGUGGUAUUGGGGCCCUAGCCUGUACAGC AUUCUGAGCCCUUUUCUGCCCCUGCUGCCUAUCUUCUUCUGCCUGUGGGUGU ACAUCUGAUGAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUC UUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 21) Sequence of AD66810 siRNA sense strand: gsusguGfcAfCfUfucgcuucacaL96 (SEQ ID NO: 22) Sequence of AD66810 siRNA anti-sense strand: usGfsugaAfgCfGfaaguGfcAfcacsusu (SEQ ID NO: 23) Sequence of Ad5-BNP4-BNP5(First bolded section is the CAG promoter, second bolded sequence is DNA encoding CLB-405, third bolded sequence is the CMV promoter, and fourth bolded sequence is DNA encoding CLB505): GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGT TCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCAtty. Dkt. No.117586-0146 GCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTA TGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGA CTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA TGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCA CTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTT TTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCA GGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGT GCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGC GAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG GGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCG CCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGC GGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGC TCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCC CTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGT GGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCG GGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCG GCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGG CTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCG GCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGC ACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCT CGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCG GGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCG GAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTAT GGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGGCGG AGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGA AGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGC GTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTCGGGGCTGCCGCA GGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTC TGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTC TTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTaTTGTGCTGTCTCATCATT TTGGCAAAGAATTCAGATCTGCCGCCACCATGGAGAACATCACCTCCGGCTT CCTGGGACCACTGCTGGTGCTGCAGGCTGGCTTCTTTCTGCTGACCAGAAT CCTGACAATCCCCCAGTCCCTGGACTCTTGGTGGACCTCTCTGAATTTTCT GGGAGGAACCACCGTGTGCCTGGGACAGAACAGCCAGTCCCCTACAAGCA ATCACTCCCCAACCTCTTGTCCCCCTACATGCCCAGGATACAGGTGGATGT GCCTGCGGAGGTTCATCATCTTCCTGTTTATCCTGCTGCTGTGCCTGATCT TTCTGCTGGTGCTGCTGGATTATCAGGGCATGCTGCCCGTGTGCCCACTGA TCCCAGGCAGCTCCACCACAAGCACCGGACCTTGTCGGACATGCATGACC ACAGGCTCTGGCAGCTGTAAGACCTGCACCACACCAGCCCAGGGCAACAG CATGTTCCCCTCCGGCTCTGGCAGCTGCAAAACATGCACCACACCTGCTCA GGGCAATTCCATGTTTCCATCCGGCTCTGGCAGCACCGGACAGGGCACAT CTATGTACCCCAGCTGCTGTTGCACCAAGCCTTCTGACGGCAACTGTACAT GCATCCCAATCCCCTCTAGCTGGGCTTTCGGCAAGTTTCTGTGGGAGTGGG CCAGCGCCAGATTCAGCTGGCTGTCCCTGCTGGTGCCCTTCGTGCAGTGGT TTGTGGGCCTGTCCCCTACCGTGTGGCTGTCTGTGATCTGGATGATGTGGT ACTGGGGCCCTTCTCTGTATAGCATCCTGTCCCCTTTTCTGCCACTGCTGC CCATCTTCTTTTGTCTGTGGGTGTATATCTGACTCGAGtctagagggcccgtttaaacccgc tgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccAtty. Dkt. No.117586-0146 actgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacag caagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggtcgacGACATTGATTA TTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCAT ATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGAC CGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAG TAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGT AAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCC CTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACA TGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCG CTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGC GGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGA GTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACT CCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTAT ATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGA AATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAG CTTGGTACCGAGCTCGGATCCGCCGCCACCATGGAAAATATCACAAGTGGGTT TCTGGGTCCTCTGCTGGTGCTGCAGGCTGGGTTCTTTCTGCTGACTCGCAT TCTGACTATCCCCCAGTCCCTGGATTCTTGGTGGACCTCTCTGAATTTCCT GGGAGGAACCACCGTGTGCCTGGGACAGAACTCCCAGTCTCCTACAAGCA ATCACTCCCCAACCTCTTGTCCCCCTACATGCCCCGGCTACAGATGGATGT GCCTGCGGAGGTTCATCATCTTCCTGTTTATCCTGCTGCTGTGCCTGATCT TTCTGCTGGTGCTGCTGGACTATCAGGGCATGCTGCCCGTGTGCCCACTGA TCCCAGGCAGCTCCACCACAAGCACCGGACCTTGTCGCACATGCATGACCA CAGGCCAGGGCACCTCTATGTACCCCAGCTGCTGTTGCACAAAGCCTTCCG ACGGCAACTGTGGCAGCGGCTCCTGCACCAAGCCTACAGATGGCAATTGT GGCTCTGGCAGCTGCACCAAGCCAACAGACGGCAACTGTACCTGCATCCC AATCCCCTCTAGCTGGGCCTTCGGCAAGTTTCTGTGGGAGTGGGCTTCCGC TCGGTTCAGCTGGCTGTCCCTGCTGGTGCCATTCGTGCAGTGGTTTGTGGG CCTGTCCCCCACCGTGTGGCTGTCTGTGATCTGGATGATGTGGTACTGGGG CCCCTCTCTGTATAGCATCCTGTCCCCTTTTCTGCCACTGCTGCCCATCTTC TTTTGTCTGTGGGTCTACATTTAACTCGAGTCTAGAGGGCCCGTTTAAACCCG CTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCC CCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGC TGGGGATGCGGTGGGCTCTATGG (SEQ ID NO: 24) cDNA encoding the small version of the hepatitis B surface antigen ATGGAGAACATCACATCAGGATTCCTAGGACCCCTTCTCGTGTTACAGGCGGGGTTT TTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCT CTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACC TCCAATCACTCACCAACCTCTTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGT CTGCGGCGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTG GTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCCTCA ACAACCAGCACGGGACCATGCCGGACCTGCATGACTACCGGTCAAGGAACCTCTATG TATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGAAATTGCACCTGTATTCCCATC CCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCC TGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTT TGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCTTGAtty. Dkt. No.117586-0146 AGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAA (SEQ ID NO: 25) cDNA encoding the small version of the hepatitis B surface antigen, with the introduced AgeI restriction site, and a cDNA sequence encoding the tag “FLAG” Start ATG and stop codon TAA in bold FLAG-specific DNA sequence (in bold and italic) The introduced AgeI restriction site in italic The sequence encoding the external loop region (amino acids 100-160) is underlined ATGGACTATAAAGACGACGATGACAAAGAGAACATCACATCAGGATTCCTAGGACCC CTTCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAG AGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTT GGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAACT TGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCCTG CTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTT TGTCCTCTAATTCCAGGATCCTCAACAACCAGCACGGGACCATGCCGGACCTGCATG ACTACCGGTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGAC GGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGG GAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGG TTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGG GGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTT TGTCTTTGGGTATACATTTAA (SEQ ID NO: 26) Hepatitis B surface antigen S (HBsAg-S) protein External loop region between 100 and 160 amino acid position is underlined MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPT SNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSS TTSTGPCRTCMTTGQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFS WLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI* (SEQ ID NO: 27) Hepatitis B surface antigen S (HBsAg-S) protein and a FLAG tag FLAG sequence (in bold and italic) External loop region between 100 and 160 amino acid position is underlined MDYKDDDDKENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCL GQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPV CPLIPGSSTTSTGPCRTCMTTGQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLW EWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFF CLWVYI* (SEQ ID NO: 28) Loop 1 first epitope consensus amino acid sequence: CX1TCX2X3X4X5QGX6SMX7PC (SEQ ID NO: 29) Loop 1 second epitope consensus amino acid sequence: PCX8TCX9X10X11 (SEQ ID NO: 30)Atty. Dkt. No.117586-0146 Loop 2 first epitope consensus amino acid sequence: CCCTKPX12DGNCX13 (SEQ ID NO: 31) HBsAg epitope: CKTCTTPAQGNSMFPSC (SEQ ID NO: 32) HBsAg epitope: PCKTCTTP (SEQ ID NO: 33) HBsAg epitope: CCCTKPTDGNCT (SEQ ID NO: 34). HBsAg epitope: CKTCTIPAQGTSMFPSC (SEQ ID NO: 35) HBsAg epitope: CCTKPSDGNCT (SEQ ID NO: 36). HBsAg epitope: CRTCTTPAQGTSMFPSC (SEQ ID NO: 37) HBsAg epitope (residues 120-127): PCRTCTTP (SEQ ID NO: 38) HBsAg epitope (residues 139-147): CTKPTDGNC (SEQ ID NO: 39) HBsAg epitope (residues 121-136): CKTCTTPAQGNSMFPS (SEQ ID NO: 40) HBsAg epitope (residues 139-147): CTKP(T / S)DGNC (SEQ ID NO: 41) HBsAg epitope (residues 120-127): PC(K / R)TC(T / M)TP (SEQ ID NO: 42) HBsAg epitope (residues 121-136): C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS (SEQ ID NO: 43) HBsAg epitope (residues 120-147): PCRTCMTTAQGTSMYPSCCCTKPSDGNC (SEQ ID NO: 44) HBsAg epitope (residues 120-147): PCKTCTTPAQGNSMFPSCCCTKPTDGNC (SEQ ID NO: 45)Atty. Dkt. No.117586-0146 BNP 1 amino acid sequence Insert via AgeI restriction site Insertion: Miniloop 1120-PCKTCTTP-127 (trimer): genotype A-specific insert, adw, (in bold) linker: GSGS (underlined and italics) FLAG-tag (underlined) MDYKDDDDKENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNS QSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSS TTSTGPCRTCMTTGSGSPCKTCTTPGSGSPCKTCTTPGSGSPCKTCTTPGSGSTGQGTSMY PSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVI WMMWYWGPSLYSILSPFLPLLPIFFCLWVYI* (SEQ ID NO: 46) BNP 2 amino acid sequence Insert via AgeI restriction site Insertion: Miniloop 1 PCRTCTTP (trimer): genotypes B / C-specific insert, ayw (in bold) linker: GSGS (underlined and italics) FLAG-tag (underlined) MDYKDDDDKENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNS QSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSS TTSTGPCRTCMTTGSGSPCRTCTTPGSGSPCRTCTTPGSGSPCRTCTTPGSGSTGQGTSMY PSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVI WMMWYWGPSLYSILSPFLPLLPIFFCLWVYI* (SEQ ID NO: 47) BNP 3 amino acid sequence Insert via AgeI restriction site Insertion: Loop 2139-CTKPTDGNC-147 (trimer) (in bold) linker: GSGS (underlined and italics) FLAG-tag (underlined) MDYKDDDDKENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNS QSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSS TTSTGPCRTCMTTGSGSCTKPTDGNCGSGSCTKPTDGNCGSGSCTKPTDGNCGSGSTGQGT SMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWL SVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI* (SEQ ID NO: 48) BNP 4 amino acid sequence Insert via AgeI restriction site Insertion: Loop 1121-CKTCTTPAQGNSMFPS-136 (dimer), adw (in bold) Start ATG and stop codon: enlarged font linker: GSGS (underlined and italics) FLAG-tag (underlined) MDYKDDDDKENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNS QSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSS TTSTGPCRTCMTTGSGSCKTCTTPAQGNSMFPSGSGSCKTCTTPAQGNSMFPSGSGSTGQG TSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVW LSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI* (SEQ ID NO: 49) BNP 5 amino acid sequence Loop 2 original sequence encoding 139-CTKPSDGNC-147 (ayw), unchanged (in bold), then twoAtty. Dkt. No.117586-0146 additional loop 2 sequences (adw) 139-CTKPTDGNC-147 (dimer) (in bold and italic) Linker: GSGS (underlined and italic) FLAG-tag (underlined) MDYKDDDDKENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNS QSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSS TTSTGPCRTCMTTGQGTSMYPSCCCTKPSDGNCGSGSCTKPTDGNCGSGSCTKPTDGNCTC IPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILS PFLPLLPIFFCLWVYI* (SEQ ID NO: 50) BNP 6 amino acid sequence Loop 1 and loop 2 repeats (in total trimer); in addition to the original ayw loop 1 and ayw loop2 sequences, two adw loop 1 (dimer) and loop 2 (dimer) sequences added. Loop 1 region (original ayw sequence in bold, and two additional adw loop 1 sequences, in bold and italic). Miniloop 1120-PCKTCTTP-127 (dimer, serotype adw) followed by original 120- PCRTCMTT-127 (serotype ayw) Loop 2 region (enlarged font): Loop 2 original sequence 139-CTKPSDGNC-147 (ayw) (in bold) followed by adw dimer 139- CTKPTDGNC-147 (in bold and italic) Linker: GSGS (underlined and italic) FLAG-tag (underlined) MDYKDDDDKENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNS QSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSS TTSTGPCKTCTTPGSGSPCKTCTTPGSGSPCRTCMTTAQGTSMYPSCCCTKPSDGNCGSGS CTKPTDGNCGSGSCTKPTDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGL SPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI* (SEQ ID NO: 51) BNP 7 amino acid sequence Loop 1, adw, dimer.121-CKTCTTPAQGNSMFPS-136 (in bold and italic) Loop 1 ayw, monomer.121-PCRTCMTTAQGTSMYPS-136 (in bold) Start ATG and stop codon: enlarged font Linker: GSGS (underlined and italics) FLAG-tag (underlined) MDYKDDDDKENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNS QSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSS TTSTGPCKTCTTPAQGNSMFPSCGSGSCKTCTTPAQGNSMFPSCGSGSPCRTCMTTAQGTS MYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLS VIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI* (SEQ ID NO: 52) BNP 8 amino acid sequence Original ayw loop in bold Added adw derived loop sequence, in bold and italic Linker: GSGS (underlined and italic) FLAG-tag (underlined) MDYKDDDDKENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNS QSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSS TTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCGSGSPCKTCTTPAQGNSMFPSCCCTKPT DGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPS LYSILSPFLPLLPIFFCLWVYI* (SEQ ID NO: 53)Atty. Dkt. No.117586-0146 BNP 1 DNA sequence Insert via AgeI restriction site (ACCGGT, bold and italic, note overlap with GSGS at first restriction site) Insertion: Miniloop 1120-PCKTCTTP-127 (trimer): genotype A-specific insert, adw (in bold) Start ATG and stop codon: enlarged font linker: GSGS (underlined and italic) FLAG-tag (underlined) GAGCTCGCCACCATGGACTATAAAGACGACGATGACAAAGAGAACATCACATCAGGATTCCTAGGACCCCTTC TCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACT TCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACC AACCTCTTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCC TGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCA GGATCCTCAACAACCAGCACGGGACCATGCCGGACCTGCATGACTACCGGTTCCGGTTCACCCTGCAAAACCTG TACTACCCCAGGTTCCGGATCACCCTGCAAAACCTGTACTACCCCAGGTTCCGGATCACCCTGCAAAACCTGTA CTACCCCAGGTTCCGGATCAACCGGTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGAC GGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCG TTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAG TTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTACCAATT TTCTTTTGTCTTTGGGTATACATTTAACTCGAG (SEQ ID NO: 54) BNP 2 DNA sequence Insert via AgeI restriction site (ACCGGT, bold and italic, note overlap with GSGS at first restriction site) Insertion: Miniloop 1 PCRTCTTP (trimer): genotypes B / C-specific insert, ayw (in bold) Start ATG and stop codon: enlarged font linker: GSGS (underlined and italic) FLAG-tag (underlined) GAGCTCGCCACCATGGACTATAAAGACGACGATGACAAAGAGAACATCACATCAGGATTCCTAGGACCCCTTC TCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACT TCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACC AACCTCTTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCC TGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCA GGATCCTCAACAACCAGCACGGGACCATGCCGGACCTGCATGACTACCGGTTCCGGTTCACCCTGCCGGACCTG TACTACCCCAGGTTCCGGATCACCCTGCCGGACCTGTACTACCCCAGGTTCCGGATCACCCTGCCGGACCTGTA CTACCCCAGGTTCCGGATCAACCGGTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGAC GGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCG TTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAG TTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTACCAATT TTCTTTTGTCTTTGGGTATACATTTAACTCGAG (SEQ ID NO: 55) BNP 3 DNA sequence Insert via AgeI restriction site (ACCGGT, bold and italic, note overlap with GSGS at first restriction site) Insertion: Loop 2139-CTKPTDGNC-147 (trimer) Start ATG and stop codon: enlarged font linker: GSGS (underlined and italic) FLAG-tag (underlined) GAGCTCGCCACCATGGACTATAAAGACGACGATGACAAAGAGAACATCACATCAGGATTCCTAGGACCCCTTC TCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACT TCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACC AACCTCTTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCC TGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCA GGATCCTCAACAACCAGCACGGGACCATGCCGGACCTGCATGACTACCGGTTCCGGTTCATGTACTAAACCAACAtty. Dkt. No.117586-0146 CGACGGAAATTGCGGTTCCGGTTCATGTACTAAACCAACCGACGGAAATTGCGGTTCCGGTTCATGTACTAAAC CAACCGACGGAAATTGCGGTTCCGGTTCAACCGGTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAA CCTTCGGACGGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGC CTCAGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTT GGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCTG TTACCAATTTTCTTTTGTCTTTGGGTATACATTTAACTCGAG (SEQ ID NO: 56) BNP 4 DNA sequence Insert via AgeI restriction site (ACCGGT, bold and italic, note overlap with GSGS at first restriction site) Insertion: Loop 1121-CKTCTTPAQGNSMFPS-136 (dimer), adw (in bold) Start ATG and stop codon: enlarged font Linker: GSGS (underlined and italic) FLAG-tag (underlined) GAGCTCGCCACCATGGACTATAAAGACGACGATGACAAAGAGAACATCACATCAGGATTCCTAGGACCCCTTC TCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACT TCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACC AACCTCTTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCC TGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCA GGATCCTCAACAACCAGCACGGGACCATGCCGGACCTGCATGACTACCGGTTCCGGTTCATGTAAAACTTGCAC TACCCCAGCTCAAGGAAATTCTATGTTCCCTTCCGGTTCCGGATCATGTAAAACTTGCACTACCCCAGCTCAAG GAAATTCTATGTTCCCTTCCGGTTCCGGATCAACCGGTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACC AAACCTTCGGACGGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTG GGCCTCAGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTG TTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCG CTGTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAACTCGAG (SEQ ID NO: 57) BNP 5 DNA sequence Three repeats in loop 2 region - Original sequence (in bold) and two additional inserts (in bold and italics) Loop 2 original sequence encoding 139-CTKPSDGNC-147 (ayw), unchanged (in bold), then two additional loop 2 sequences (adw) 139-CTKPTDGNC-147 (dimer) (bold and italic) Linker: GSGS (underlined and italic) FLAG-tag (underlined) GAGCTCGCCACCATGGACTATAAAGACGACGATGACAAAGAGAACATCACATCAGGATTCCTAGGACCCCTTC TCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACT TCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACC AACCTCTTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCC TGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCA GGATCCTCAACAACCAGCACGGGACCATGCCGGACCTGCATGACTACCGGTCAAGGAACCTCTATGTATCCCTC CTGTTGCTGTACCAAACCTTCGGACGGAAATTGCGGTTCCGGTTCATGTACTAAACCAACCGACGGAAATTGCG GTTCCGGTTCATGTACTAAACCAACCGACGGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGA AAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGT AGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCT TGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAACTCGAG (SEQ ID NO: 58) BNP 6 DNA sequence Loop 1 region (original ayw sequence in bold, and two additional adw loop 1 sequences, in bold and italic). Miniloop 1120-PCKTCTTP-127 (dimer, serotype adw) followed by original 120- PCRTCMTT-127 (serotype ayw) Loop 2 region (enlarged font): Loop 2 original sequence 139-CTKPSDGNC-147 (ayw) (in bold) followed by adw dimer 139-Atty. Dkt. No.117586-0146 CTKPTDGNC-147 (in bold and italic) Start ATG and stop codon: enlarged font Linker: GSGS (underlined and italic) FLAG-tag (underlined) GAGCTCGCCACCATGGACTATAAAGACGACGATGACAAAGAGAACATCACATCAGGATTCCTAGGACCCCTTC TCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACT TCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACC AACCTCTTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCC TGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCA GGATCCTCAACAACCAGCACGGGACCATGCAAAACCTGTACTACCCCAGGTTCCGGATCACCATGCAAAACCTG TACTACCCCAGGTTCCGGATCACCATGCCGGACCTGCATGACTACTGCTCAAGGAACCTCTATGTATCCCTCCT GTTGCTGTACCAAACCTTCGGACGGAAATTGCGGTTCCGGTTCATGTACTAAACCAACCGACGGAAA TTGCGGTTCCGGTTCATGTACTAAACCAACCGACGGAAATTGCACCTGTATTCCCATCCCATCATCCTG GGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTC AGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTG TACAGCATCTTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAACTCGAG (SEQ ID NO: 59) BNP 7 DNA sequence Loop 1, adw, dimer.121-CKTCTTPAQGNSMFPS-136 (in bold and italic) Loop 1 original ayw, monomer.121-PCRTCMTTAQGTSMYPS-136 (in bold) Start ATG and stop codon: enlarged font Linker: GSGS (underlined and italic) FLAG-tag (underlined) GAGCTCGCCACCATGGACTATAAAGACGACGATGACAAAGAGAACATCACATCAGGATTCCTAGGACCCCTTC TCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACT TCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACC AACCTCTTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCC TGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCA GGATCCTCAACAACCAGCACGGGACCATGCAAAACCTGCACCACTCCAGCGCAAGGAAATTCTATGTTTCCCTC CTGTGGTTCCGGTTCATGCAAAACCTGCACCACTCCAGCGCAAGGAAATTCTATGTTTCCCTCCTGTGGTTCCG GTTCACCATGCCGGACCTGCATGACTACTGCTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCT TCGGACGGAAATTGCACCTGTATTCCCATCCCATCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTC AGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGC TTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTA CCAATTTTCTTTTGTCTTTGGGTATACATTTAACTCGAG (SEQ ID NO: 60) BNP 8 DNA sequence Original ayw loop in bold Added adw derived loop sequence, in bold and italic Linker: GSGS (underlined and italic) FLAG-tag (underlined) GAGCTCGCCACCATGGACTATAAAGACGACGATGACAAAGAGAACATCACATCAGGATTCCTAGGACCCCTTC TCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACT TCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACC AACCTCTTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTCATCC TGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCA GGATCCTCAACAACCAGCACGGGACCATGCCGGACCTGCATGACTACTGCTCAAGGAACCTCTATGTATCCCTC CTGTTGCTGTACCAAACCTTCGGACGGAAATTGCGGTTCCGGTTCACCATGCAAAACCTGTACTACCCCAGCGC AAGGAAATTCTATGTTTCCCTCCTGCTGTTGCACTAAACCAACCGACGGAAATTGCACCTGTATTCCCATCCCA TCATCCTGGGCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCC ATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCAtty. Dkt. No.117586-0146 CAAGTCTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGTATACATTTA ACTCGAG (SEQ ID NO: 61) CAG promoter GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTC ATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTG GCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCA TAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGT AAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCT TATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT GGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCC ACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCG GGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGC GGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAA AGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGC GCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCC GCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGC GGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGG CTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCT TTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGA GCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCG CGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTG CCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGT GCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCC CCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCT CCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGG TGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGA GGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAG CCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCA AATCTGGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCG CGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTC GTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGCCTCGGGGCTGCCGCA GGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGC GTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTT CCTACAG (SEQ ID NO: 62) CMV promoter GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTC ATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTG GCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCA TAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGT AAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCT TATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT GGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACG GGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCA AAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAAT GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT (SEQ ID NO: 63)Atty. Dkt. No.117586-0146 RNA sequence of 40X as used in 1-40X, 2-40X, 3-40X, and 4-40X: AUGGAGAACAUCACCAGCGGCUUCCUGGGCCCCCUGCUGGUGCUGCAGGCCG GCUUCUUCCUGCUGACCAGGAUCCUGACCAUCCCCCAGAGCCUGGACAGCUG GUGGACCAGCCUGAACUUCCUGGGCGGCACCACCGUGUGCCUGGGCCAGAAC AGCCAGAGCCCCACCAGCAACCACAGCCCCACCAGCUGCCCCCCCACCUGCCC CGGCUACAGGUGGAUGUGCCUGAGGAGGUUCAUCAUCUUCCUGUUCAUCCUG CUGCUGUGCCUGAUCUUCCUGCUGGUGCUGCUGGACUACCAGGGCAUGCUGC CCGUGUGCCCCCUGAUCCCCGGCAGCAGCACCACCAGCACCGGCCCCUGCAGG ACCUGCAUGACCACCGGCAGCGGCAGCUGCAAGACCUGCACCACCCCCGCCCA GGGCAACAGCAUGUUCCCCAGCGGCAGCGGCAGCUGCAAGACCUGCACCACC CCCGCCCAGGGCAACAGCAUGUUCCCCAGCGGCAGCGGCAGCACCGGCCAGG GCACCAGCAUGUACCCCAGCUGCUGCUGCACCAAGCCCAGCGACGGCAACUG CACCUGCAUCCCCAUCCCCAGCAGCUGGGCCUUCGGCAAGUUCCUGUGGGAG UGGGCCAGCGCCAGGUUCAGCUGGCUGAGCCUGCUGGUGCCCUUCGUGCAGU GGUUCGUGGGCCUGAGCCCCACCGUGUGGCUGAGCGUGAUCUGGAUGAUGUG GUACUGGGGCCCCAGCCUGUACAGCAUCCUGAGCCCCUUCCUGCCCCUGCUGC CCAUCUUCUUCUGCCUGUGGGUGUACAUCUAGUAAUGA (SEQ ID NO: 64) RNA sequence of 50X as used in 1-50X, 2-50X, 3-50X, and 4-50X: AUGGAGAACAUCACCAGCGGCUUCCUGGGCCCCCUGCUGGUGCUGCAGGCCG GCUUCUUCCUGCUGACCAGGAUCCUGACCAUCCCCCAGAGCCUGGACAGCUG GUGGACCAGCCUGAACUUCCUGGGCGGCACCACCGUGUGCCUGGGCCAGAAC AGCCAGAGCCCCACCAGCAACCACAGCCCCACCAGCUGCCCCCCCACCUGCCC CGGCUACAGGUGGAUGUGCCUGAGGAGGUUCAUCAUCUUCCUGUUCAUCCUG CUGCUGUGCCUGAUCUUCCUGCUGGUGCUGCUGGACUACCAGGGCAUGCUGC CCGUGUGCCCCCUGAUCCCCGGCAGCAGCACCACCAGCACCGGCCCCUGCAGG ACCUGCAUGACCACCGGCCAGGGCACCAGCAUGUACCCCAGCUGCUGCUGCA CCAAGCCCAGCGACGGCAACUGCGGCAGCGGCAGCUGCACCAAGCCCACCGAC GGCAACUGCGGCAGCGGCAGCUGCACCAAGCCCACCGACGGCAACUGCACCU GCAUCCCCAUCCCCAGCAGCUGGGCCUUCGGCAAGUUCCUGUGGGAGUGGGC CAGCGCCAGGUUCAGCUGGCUGAGCCUGCUGGUGCCCUUCGUGCAGUGGUUC GUGGGCCUGAGCCCCACCGUGUGGCUGAGCGUGAUCUGGAUGAUGUGGUACU GGGGCCCCAGCCUGUACAGCAUCCUGAGCCCCUUCCUGCCCCUGCUGCCCAUC UUCUUCUGCCUGUGGGUGUACAUCUAGUAAUGA (SEQ ID NO: 65) DNA sequence of 40X as used in 1-40X, 2-40X, 3-40X, and 4-40X: ATGGAGAACATCACCAGCGGCTTCCTGGGCCCCCTGCTGGTGCTGCAGGCCGGC TTCTTCCTGCTGACCAGGATCCTGACCATCCCCCAGAGCCTGGACAGCTGGTGG ACCAGCCTGAACTTCCTGGGCGGCACCACCGTGTGCCTGGGCCAGAACAGCCA GAGCCCCACCAGCAACCACAGCCCCACCAGCTGCCCCCCCACCTGCCCCGGCTA CAGGTGGATGTGCCTGAGGAGGTTCATCATCTTCCTGTTCATCCTGCTGCTGTGC CTGATCTTCCTGCTGGTGCTGCTGGACTACCAGGGCATGCTGCCCGTGTGCCCC CTGATCCCCGGCAGCAGCACCACCAGCACCGGCCCCTGCAGGACCTGCATGAC CACCGGCAGCGGCAGCTGCAAGACCTGCACCACCCCCGCCCAGGGCAACAGCA TGTTCCCCAGCGGCAGCGGCAGCTGCAAGACCTGCACCACCCCCGCCCAGGGC AACAGCATGTTCCCCAGCGGCAGCGGCAGCACCGGCCAGGGCACCAGCATGTA CCCCAGCTGCTGCTGCACCAAGCCCAGCGACGGCAACTGCACCTGCATCCCCAT CCCCAGCAGCTGGGCCTTCGGCAAGTTCCTGTGGGAGTGGGCCAGCGCCAGGTT CAGCTGGCTGAGCCTGCTGGTGCCCTTCGTGCAGTGGTTCGTGGGCCTGAGCCCAtty. Dkt. No.117586-0146 CACCGTGTGGCTGAGCGTGATCTGGATGATGTGGTACTGGGGCCCCAGCCTGTA CAGCATCCTGAGCCCCTTCCTGCCCCTGCTGCCCATCTTCTTCTGCCTGTGGGTG TACATCTAGTAATGA (SEQ ID NO: 66) DNA sequence of 40X as used in 5-40X: ATGGAGAACATCACCTCTGGCTTTCTGGGCCCTCTGCTGGTGCTGCAGGCTGGC TTCTTTCTGCTGACCCGCATCCTGACAATCCCTCAGAGCCTGGATAGCTGGTGG ACCAGCCTGAATTTTCTCGGCGGCACAACAGTGTGCCTGGGCCAGAATAGCCAG TCTCCTACCAGCAATCACAGCCCCACCAGCTGTCCTCCAACCTGTCCTGGCTAC AGATGGATGTGCCTGCGGCGGTTCATCATCTTTCTGTTCATCCTGCTGCTGTGCC TGATCTTCCTGCTCGTGCTGCTGGATTACCAGGGAATGCTGCCTGTGTGTCCTCT GATCCCTGGCAGCAGCACAACAAGCACAGGCCCTTGCAGAACCTGTATGACAA CAGGCTCTGGCTCCTGCAAGACCTGCACAACACCAGCTCAGGGCAACAGCATG TTTCCTAGCGGCAGCGGCAGCTGCAAGACATGTACTACCCCTGCACAGGGCAAC TCTATGTTCCCATCTGGCAGCGGCTCTACCGGCCAGGGCACATCTATGTACCCT AGCTGCTGTTGCACCAAGCCTAGCGACGGCAACTGCACATGCATCCCCATTCCT AGCAGCTGGGCCTTCGGCAAGTTTCTGTGGGAATGGGCCAGCGCCAGATTCAGC TGGCTGAGCCTGCTGGTTCCTTTCGTGCAGTGGTTCGTGGGCCTGTCTCCTACAG TGTGGCTGAGCGTGATCTGGATGATGTGGTATTGGGGCCCTAGCCTGTACAGCA TTCTGAGCCCTTTTCTGCCCCTGCTGCCTATCTTCTTCTGCCTGTGGGTGTACATC TGATGA (SEQ ID NO: 67) DNA sequence of 50X as used in 1-50X, 2-50X, 3-50X, and 4-50X: ATGGAGAACATCACCAGCGGCTTCCTGGGCCCCCTGCTGGTGCTGCAGGCCGGC TTCTTCCTGCTGACCAGGATCCTGACCATCCCCCAGAGCCTGGACAGCTGGTGG ACCAGCCTGAACTTCCTGGGCGGCACCACCGTGTGCCTGGGCCAGAACAGCCA GAGCCCCACCAGCAACCACAGCCCCACCAGCTGCCCCCCCACCTGCCCCGGCTA CAGGTGGATGTGCCTGAGGAGGTTCATCATCTTCCTGTTCATCCTGCTGCTGTGC CTGATCTTCCTGCTGGTGCTGCTGGACTACCAGGGCATGCTGCCCGTGTGCCCC CTGATCCCCGGCAGCAGCACCACCAGCACCGGCCCCTGCAGGACCTGCATGAC CACCGGCCAGGGCACCAGCATGTACCCCAGCTGCTGCTGCACCAAGCCCAGCG ACGGCAACTGCGGCAGCGGCAGCTGCACCAAGCCCACCGACGGCAACTGCGGC AGCGGCAGCTGCACCAAGCCCACCGACGGCAACTGCACCTGCATCCCCATCCCC AGCAGCTGGGCCTTCGGCAAGTTCCTGTGGGAGTGGGCCAGCGCCAGGTTCAG CTGGCTGAGCCTGCTGGTGCCCTTCGTGCAGTGGTTCGTGGGCCTGAGCCCCAC CGTGTGGCTGAGCGTGATCTGGATGATGTGGTACTGGGGCCCCAGCCTGTACAG CATCCTGAGCCCCTTCCTGCCCCTGCTGCCCATCTTCTTCTGCCTGTGGGTGTAC ATCTAGTAATGA (SEQ ID NO: 68) DNA sequence of 50X as used in 5-50X: ATGGAGAACATCACCTCTGGCTTTCTGGGCCCTCTGCTGGTGCTGCAGGCTGGC TTCTTTCTGCTGACCCGCATCCTGACAATCCCTCAGAGCCTGGATAGCTGGTGG ACCAGCCTGAATTTTCTCGGCGGCACAACAGTGTGCCTGGGCCAGAATAGCCAG TCTCCTACCAGCAATCACAGCCCCACCAGCTGTCCTCCAACCTGTCCTGGCTAC AGATGGATGTGCCTGCGGCGGTTCATCATCTTTCTGTTCATCCTGCTGCTGTGCC TGATCTTCCTGCTCGTGCTGCTGGATTACCAGGGAATGCTGCCTGTGTGTCCTCT GATCCCTGGCAGCAGCACAACAAGCACAGGCCCTTGCAGAACCTGTATGACAA CCGGCCAGGGCACCAGCATGTACCCTAGCTGTTGTTGCACCAAGCCTAGCGACG GCAATTGTGGCAGCGGCAGCTGTACAAAGCCCACCGATGGAAATTGCGGCTCC GGCTCTTGTACCAAGCCAACAGATGGCAACTGCACATGCATCCCCATTCCTAGCAtty. Dkt. No.117586-0146 AGCTGGGCCTTCGGCAAGTTTCTGTGGGAATGGGCCAGCGCCAGATTCAGCTGG CTGAGCCTGCTGGTTCCTTTCGTGCAGTGGTTCGTGGGCCTGTCTCCTACAGTGT GGCTGAGCGTGATCTGGATGATGTGGTATTGGGGCCCTAGCCTGTACAGCATTC TGAGCCCTTTTCTGCCCCTGCTGCCTATCTTCTTCTGCCTGTGGGTGTACATCTG ATGA (SEQ ID NO: 69) DNA sequence of 1-40X (5’ and 3’ UTR bolded): AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGAAC ATCACCAGCGGCTTCCTGGGCCCCCTGCTGGTGCTGCAGGCCGGCTTCTTCCTGCTG ACCAGGATCCTGACCATCCCCCAGAGCCTGGACAGCTGGTGGACCAGCCTGAACTT CCTGGGCGGCACCACCGTGTGCCTGGGCCAGAACAGCCAGAGCCCCACCAGCAAC CACAGCCCCACCAGCTGCCCCCCCACCTGCCCCGGCTACAGGTGGATGTGCCTGAG GAGGTTCATCATCTTCCTGTTCATCCTGCTGCTGTGCCTGATCTTCCTGCTGGTGCTG CTGGACTACCAGGGCATGCTGCCCGTGTGCCCCCTGATCCCCGGCAGCAGCACCAC CAGCACCGGCCCCTGCAGGACCTGCATGACCACCGGCAGCGGCAGCTGCAAGACCT GCACCACCCCCGCCCAGGGCAACAGCATGTTCCCCAGCGGCAGCGGCAGCTGCAA GACCTGCACCACCCCCGCCCAGGGCAACAGCATGTTCCCCAGCGGCAGCGGCAGCA CCGGCCAGGGCACCAGCATGTACCCCAGCTGCTGCTGCACCAAGCCCAGCGACGGC AACTGCACCTGCATCCCCATCCCCAGCAGCTGGGCCTTCGGCAAGTTCCTGTGGGA GTGGGCCAGCGCCAGGTTCAGCTGGCTGAGCCTGCTGGTGCCCTTCGTGCAGTGGT TCGTGGGCCTGAGCCCCACCGTGTGGCTGAGCGTGATCTGGATGATGTGGTACTGG GGCCCCAGCCTGTACAGCATCCTGAGCCCCTTCCTGCCCCTGCTGCCCATCTTCTTC TGCCTGTGGGTGTACATCTAGTAATGAGCTCGCTTTCTTGCTGTCCAATTTCTAT TAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAA GGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAA (SEQ ID NO: 70) DNA sequence of 2-40X (5’ and 3’ UTR bolded): TCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACT TCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAA TTTTCACCATTTACGAACGATAGCATGGAGAACATCACCAGCGGCTTCCTGGGCC CCCTGCTGGTGCTGCAGGCCGGCTTCTTCCTGCTGACCAGGATCCTGACCATCCCCC AGAGCCTGGACAGCTGGTGGACCAGCCTGAACTTCCTGGGCGGCACCACCGTGTGC CTGGGCCAGAACAGCCAGAGCCCCACCAGCAACCACAGCCCCACCAGCTGCCCCCC CACCTGCCCCGGCTACAGGTGGATGTGCCTGAGGAGGTTCATCATCTTCCTGTTCAT CCTGCTGCTGTGCCTGATCTTCCTGCTGGTGCTGCTGGACTACCAGGGCATGCTGCC CGTGTGCCCCCTGATCCCCGGCAGCAGCACCACCAGCACCGGCCCCTGCAGGACCT GCATGACCACCGGCAGCGGCAGCTGCAAGACCTGCACCACCCCCGCCCAGGGCAA CAGCATGTTCCCCAGCGGCAGCGGCAGCTGCAAGACCTGCACCACCCCCGCCCAGG GCAACAGCATGTTCCCCAGCGGCAGCGGCAGCACCGGCCAGGGCACCAGCATGTA CCCCAGCTGCTGCTGCACCAAGCCCAGCGACGGCAACTGCACCTGCATCCCCATCC CCAGCAGCTGGGCCTTCGGCAAGTTCCTGTGGGAGTGGGCCAGCGCCAGGTTCAGC TGGCTGAGCCTGCTGGTGCCCTTCGTGCAGTGGTTCGTGGGCCTGAGCCCCACCGTG TGGCTGAGCGTGATCTGGATGATGTGGTACTGGGGCCCCAGCCTGTACAGCATCCT GAGCCCCTTCCTGCCCCTGCTGCCCATCTTCTTCTGCCTGTGGGTGTACATCTAGTA ATGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAA GTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCT GCCTAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAtty. Dkt. No.117586-0146 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 71) DNA sequence of 3-40X (5’ and 3’ UTR bolded): AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCCAACAATCT CAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCT ATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTT TCACCATTTACGAACGATAGCATGGAGAACATCACCAGCGGCTTCCTGGGCCCCC TGCTGGTGCTGCAGGCCGGCTTCTTCCTGCTGACCAGGATCCTGACCATCCCCCAGA GCCTGGACAGCTGGTGGACCAGCCTGAACTTCCTGGGCGGCACCACCGTGTGCCTG GGCCAGAACAGCCAGAGCCCCACCAGCAACCACAGCCCCACCAGCTGCCCCCCCA CCTGCCCCGGCTACAGGTGGATGTGCCTGAGGAGGTTCATCATCTTCCTGTTCATCC TGCTGCTGTGCCTGATCTTCCTGCTGGTGCTGCTGGACTACCAGGGCATGCTGCCCG TGTGCCCCCTGATCCCCGGCAGCAGCACCACCAGCACCGGCCCCTGCAGGACCTGC ATGACCACCGGCAGCGGCAGCTGCAAGACCTGCACCACCCCCGCCCAGGGCAACA GCATGTTCCCCAGCGGCAGCGGCAGCTGCAAGACCTGCACCACCCCCGCCCAGGGC AACAGCATGTTCCCCAGCGGCAGCGGCAGCACCGGCCAGGGCACCAGCATGTACC CCAGCTGCTGCTGCACCAAGCCCAGCGACGGCAACTGCACCTGCATCCCCATCCCC AGCAGCTGGGCCTTCGGCAAGTTCCTGTGGGAGTGGGCCAGCGCCAGGTTCAGCTG GCTGAGCCTGCTGGTGCCCTTCGTGCAGTGGTTCGTGGGCCTGAGCCCCACCGTGTG GCTGAGCGTGATCTGGATGATGTGGTACTGGGGCCCCAGCCTGTACAGCATCCTGA GCCCCTTCCTGCCCCTGCTGCCCATCTTCTTCTGCCTGTGGGTGTACATCTAGTAATG AGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTC CAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCC TAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 72) DNA sequence of 4-40X (5’ and 3’ UTR bolded): CACTCGCGCTGCCATCACTCTTCCGCCGTCTTCGCCGCCATCCTCGGCGCGAC TCGCTTCTTTCGGTTCTACCAGGTAGAGTCCGCCGCCATCCTCCACCCAACAA CTTGTCTCGCTCCGGGGAACGCTCGGAAACTCCCGGCCGCCGCCACCCGCGT CTGTTCTGTTACACAAGGGAAGAAAAGCCGCTGCCGCACTCCGAGTGTATGGA GAACATCACCAGCGGCTTCCTGGGCCCCCTGCTGGTGCTGCAGGCCGGCTTCTTCCT GCTGACCAGGATCCTGACCATCCCCCAGAGCCTGGACAGCTGGTGGACCAGCCTGA ACTTCCTGGGCGGCACCACCGTGTGCCTGGGCCAGAACAGCCAGAGCCCCACCAGC AACCACAGCCCCACCAGCTGCCCCCCCACCTGCCCCGGCTACAGGTGGATGTGCCT GAGGAGGTTCATCATCTTCCTGTTCATCCTGCTGCTGTGCCTGATCTTCCTGCTGGT GCTGCTGGACTACCAGGGCATGCTGCCCGTGTGCCCCCTGATCCCCGGCAGCAGCA CCACCAGCACCGGCCCCTGCAGGACCTGCATGACCACCGGCAGCGGCAGCTGCAA GACCTGCACCACCCCCGCCCAGGGCAACAGCATGTTCCCCAGCGGCAGCGGCAGCT GCAAGACCTGCACCACCCCCGCCCAGGGCAACAGCATGTTCCCCAGCGGCAGCGGC AGCACCGGCCAGGGCACCAGCATGTACCCCAGCTGCTGCTGCACCAAGCCCAGCGA CGGCAACTGCACCTGCATCCCCATCCCCAGCAGCTGGGCCTTCGGCAAGTTCCTGT GGGAGTGGGCCAGCGCCAGGTTCAGCTGGCTGAGCCTGCTGGTGCCCTTCGTGCAG TGGTTCGTGGGCCTGAGCCCCACCGTGTGGCTGAGCGTGATCTGGATGATGTGGTA CTGGGGCCCCAGCCTGTACAGCATCCTGAGCCCCTTCCTGCCCCTGCTGCCCATCTT CTTCTGCCTGTGGGTGTACATCTAGTAATGAGCTCGCTTTCTTGCTGTCCAATTTC TATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATG AAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAtty. Dkt. No.117586-0146 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAA (SEQ ID NO: 73) DNA sequence of 5-40X (5’ and 3’ UTR bolded): TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAG AAAAGAAGAGTAAGAAGAAATATAAGAATGGAGAACATCACCTCTGGCTTTC TGGGCCCTCTGCTGGTGCTGCAGGCTGGCTTCTTTCTGCTGACCCGCATCCTGAC AATCCCTCAGAGCCTGGATAGCTGGTGGACCAGCCTGAATTTTCTCGGCGGCAC AACAGTGTGCCTGGGCCAGAATAGCCAGTCTCCTACCAGCAATCACAGCCCCAC CAGCTGTCCTCCAACCTGTCCTGGCTACAGATGGATGTGCCTGCGGCGGTTCAT CATCTTTCTGTTCATCCTGCTGCTGTGCCTGATCTTCCTGCTCGTGCTGCTGGATT ACCAGGGAATGCTGCCTGTGTGTCCTCTGATCCCTGGCAGCAGCACAACAAGCA CAGGCCCTTGCAGAACCTGTATGACAACAGGCTCTGGCTCCTGCAAGACCTGCA CAACACCAGCTCAGGGCAACAGCATGTTTCCTAGCGGCAGCGGCAGCTGCAAG ACATGTACTACCCCTGCACAGGGCAACTCTATGTTCCCATCTGGCAGCGGCTCT ACCGGCCAGGGCACATCTATGTACCCTAGCTGCTGTTGCACCAAGCCTAGCGAC GGCAACTGCACATGCATCCCCATTCCTAGCAGCTGGGCCTTCGGCAAGTTTCTG TGGGAATGGGCCAGCGCCAGATTCAGCTGGCTGAGCCTGCTGGTTCCTTTCGTG CAGTGGTTCGTGGGCCTGTCTCCTACAGTGTGGCTGAGCGTGATCTGGATGATG TGGTATTGGGGCCCTAGCCTGTACAGCATTCTGAGCCCTTTTCTGCCCCTGCTGC CTATCTTCTTCTGCCTGTGGGTGTACATCTGATGAGCTGCCTTCTGCGGGGCT TGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCT TTGAATAAAGCCTGAGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAA (SEQ ID NO: 74) DNA sequence of 1-50X (5’ and 3’ UTR bolded): AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGAAC ATCACCAGCGGCTTCCTGGGCCCCCTGCTGGTGCTGCAGGCCGGCTTCTTCCTGCTG ACCAGGATCCTGACCATCCCCCAGAGCCTGGACAGCTGGTGGACCAGCCTGAACTT CCTGGGCGGCACCACCGTGTGCCTGGGCCAGAACAGCCAGAGCCCCACCAGCAAC CACAGCCCCACCAGCTGCCCCCCCACCTGCCCCGGCTACAGGTGGATGTGCCTGAG GAGGTTCATCATCTTCCTGTTCATCCTGCTGCTGTGCCTGATCTTCCTGCTGGTGCTG CTGGACTACCAGGGCATGCTGCCCGTGTGCCCCCTGATCCCCGGCAGCAGCACCAC CAGCACCGGCCCCTGCAGGACCTGCATGACCACCGGCCAGGGCACCAGCATGTACC CCAGCTGCTGCTGCACCAAGCCCAGCGACGGCAACTGCGGCAGCGGCAGCTGCACC AAGCCCACCGACGGCAACTGCGGCAGCGGCAGCTGCACCAAGCCCACCGACGGCA ACTGCACCTGCATCCCCATCCCCAGCAGCTGGGCCTTCGGCAAGTTCCTGTGGGAG TGGGCCAGCGCCAGGTTCAGCTGGCTGAGCCTGCTGGTGCCCTTCGTGCAGTGGTT CGTGGGCCTGAGCCCCACCGTGTGGCTGAGCGTGATCTGGATGATGTGGTACTGGG GCCCCAGCCTGTACAGCATCCTGAGCCCCTTCCTGCCCCTGCTGCCCATCTTCTTCT GCCTGTGGGTGTACATCTAGTAATGAGCTCGCTTTCTTGCTGTCCAATTTCTATT AAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAG GGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAA (SEQ ID NO: 75) DNA sequence of 2-50X (5’ and 3’ UTR bolded): TCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACT TCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAA TTTTCACCATTTACGAACGATAGCATGGAGAACATCACCAGCGGCTTCCTGGGCCAtty. Dkt. No.117586-0146 CCCTGCTGGTGCTGCAGGCCGGCTTCTTCCTGCTGACCAGGATCCTGACCATCCCCC AGAGCCTGGACAGCTGGTGGACCAGCCTGAACTTCCTGGGCGGCACCACCGTGTGC CTGGGCCAGAACAGCCAGAGCCCCACCAGCAACCACAGCCCCACCAGCTGCCCCCC CACCTGCCCCGGCTACAGGTGGATGTGCCTGAGGAGGTTCATCATCTTCCTGTTCAT CCTGCTGCTGTGCCTGATCTTCCTGCTGGTGCTGCTGGACTACCAGGGCATGCTGCC CGTGTGCCCCCTGATCCCCGGCAGCAGCACCACCAGCACCGGCCCCTGCAGGACCT GCATGACCACCGGCCAGGGCACCAGCATGTACCCCAGCTGCTGCTGCACCAAGCCC AGCGACGGCAACTGCGGCAGCGGCAGCTGCACCAAGCCCACCGACGGCAACTGCG GCAGCGGCAGCTGCACCAAGCCCACCGACGGCAACTGCACCTGCATCCCCATCCCC AGCAGCTGGGCCTTCGGCAAGTTCCTGTGGGAGTGGGCCAGCGCCAGGTTCAGCTG GCTGAGCCTGCTGGTGCCCTTCGTGCAGTGGTTCGTGGGCCTGAGCCCCACCGTGTG GCTGAGCGTGATCTGGATGATGTGGTACTGGGGCCCCAGCCTGTACAGCATCCTGA GCCCCTTCCTGCCCCTGCTGCCCATCTTCTTCTGCCTGTGGGTGTACATCTAGTAATG AGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTC CAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCC TAATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 76) DNA sequence of 3-50X (5’ and 3’ UTR bolded): AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCCAACAATCT CAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCT ATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTT TCACCATTTACGAACGATAGCATGGAGAACATCACCAGCGGCTTCCTGGGCCCCC TGCTGGTGCTGCAGGCCGGCTTCTTCCTGCTGACCAGGATCCTGACCATCCCCCAGA GCCTGGACAGCTGGTGGACCAGCCTGAACTTCCTGGGCGGCACCACCGTGTGCCTG GGCCAGAACAGCCAGAGCCCCACCAGCAACCACAGCCCCACCAGCTGCCCCCCCA CCTGCCCCGGCTACAGGTGGATGTGCCTGAGGAGGTTCATCATCTTCCTGTTCATCC TGCTGCTGTGCCTGATCTTCCTGCTGGTGCTGCTGGACTACCAGGGCATGCTGCCCG TGTGCCCCCTGATCCCCGGCAGCAGCACCACCAGCACCGGCCCCTGCAGGACCTGC ATGACCACCGGCCAGGGCACCAGCATGTACCCCAGCTGCTGCTGCACCAAGCCCAG CGACGGCAACTGCGGCAGCGGCAGCTGCACCAAGCCCACCGACGGCAACTGCGGC AGCGGCAGCTGCACCAAGCCCACCGACGGCAACTGCACCTGCATCCCCATCCCCAG CAGCTGGGCCTTCGGCAAGTTCCTGTGGGAGTGGGCCAGCGCCAGGTTCAGCTGGC TGAGCCTGCTGGTGCCCTTCGTGCAGTGGTTCGTGGGCCTGAGCCCCACCGTGTGGC TGAGCGTGATCTGGATGATGTGGTACTGGGGCCCCAGCCTGTACAGCATCCTGAGC CCCTTCCTGCCCCTGCTGCCCATCTTCTTCTGCCTGTGGGTGTACATCTAGTAATGA GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCC AACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCT AATAAAAAACATTTATTTTCATTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 77) DNA sequence of 4-50X (5’ and 3’ UTR bolded): CACTCGCGCTGCCATCACTCTTCCGCCGTCTTCGCCGCCATCCTCGGCGCGAC TCGCTTCTTTCGGTTCTACCAGGTAGAGTCCGCCGCCATCCTCCACCCAACAA CTTGTCTCGCTCCGGGGAACGCTCGGAAACTCCCGGCCGCCGCCACCCGCGT CTGTTCTGTTACACAAGGGAAGAAAAGCCGCTGCCGCACTCCGAGTGTATGGA GAACATCACCAGCGGCTTCCTGGGCCCCCTGCTGGTGCTGCAGGCCGGCTTCTTCCT GCTGACCAGGATCCTGACCATCCCCCAGAGCCTGGACAGCTGGTGGACCAGCCTGA ACTTCCTGGGCGGCACCACCGTGTGCCTGGGCCAGAACAGCCAGAGCCCCACCAGC AACCACAGCCCCACCAGCTGCCCCCCCACCTGCCCCGGCTACAGGTGGATGTGCCTAtty. Dkt. No.117586-0146 GAGGAGGTTCATCATCTTCCTGTTCATCCTGCTGCTGTGCCTGATCTTCCTGCTGGT GCTGCTGGACTACCAGGGCATGCTGCCCGTGTGCCCCCTGATCCCCGGCAGCAGCA CCACCAGCACCGGCCCCTGCAGGACCTGCATGACCACCGGCCAGGGCACCAGCATG TACCCCAGCTGCTGCTGCACCAAGCCCAGCGACGGCAACTGCGGCAGCGGCAGCTG CACCAAGCCCACCGACGGCAACTGCGGCAGCGGCAGCTGCACCAAGCCCACCGAC GGCAACTGCACCTGCATCCCCATCCCCAGCAGCTGGGCCTTCGGCAAGTTCCTGTG GGAGTGGGCCAGCGCCAGGTTCAGCTGGCTGAGCCTGCTGGTGCCCTTCGTGCAGT GGTTCGTGGGCCTGAGCCCCACCGTGTGGCTGAGCGTGATCTGGATGATGTGGTAC TGGGGCCCCAGCCTGTACAGCATCCTGAGCCCCTTCCTGCCCCTGCTGCCCATCTTC TTCTGCCTGTGGGTGTACATCTAGTAATGAGCTCGCTTTCTTGCTGTCCAATTTCT ATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGA AGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA (SEQ ID NO: 78) DNA sequence of 5-50X (5’ and 3’ UTR bolded): TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAG AAAAGAAGAGTAAGAAGAAATATAAGAATGGAGAACATCACCTCTGGCTTTC TGGGCCCTCTGCTGGTGCTGCAGGCTGGCTTCTTTCTGCTGACCCGCATCCTGAC AATCCCTCAGAGCCTGGATAGCTGGTGGACCAGCCTGAATTTTCTCGGCGGCAC AACAGTGTGCCTGGGCCAGAATAGCCAGTCTCCTACCAGCAATCACAGCCCCAC CAGCTGTCCTCCAACCTGTCCTGGCTACAGATGGATGTGCCTGCGGCGGTTCAT CATCTTTCTGTTCATCCTGCTGCTGTGCCTGATCTTCCTGCTCGTGCTGCTGGATT ACCAGGGAATGCTGCCTGTGTGTCCTCTGATCCCTGGCAGCAGCACAACAAGCA CAGGCCCTTGCAGAACCTGTATGACAACCGGCCAGGGCACCAGCATGTACCCT AGCTGTTGTTGCACCAAGCCTAGCGACGGCAATTGTGGCAGCGGCAGCTGTACA AAGCCCACCGATGGAAATTGCGGCTCCGGCTCTTGTACCAAGCCAACAGATGG CAACTGCACATGCATCCCCATTCCTAGCAGCTGGGCCTTCGGCAAGTTTCTGTG GGAATGGGCCAGCGCCAGATTCAGCTGGCTGAGCCTGCTGGTTCCTTTCGTGCA GTGGTTCGTGGGCCTGTCTCCTACAGTGTGGCTGAGCGTGATCTGGATGATGTG GTATTGGGGCCCTAGCCTGTACAGCATTCTGAGCCCTTTTCTGCCCCTGCTGCCT ATCTTCTTCTGCCTGTGGGTGTACATCTGATGAGCTGCCTTCTGCGGGGCTTG CCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTT GAATAAAGCCTGAGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAA (SEQ ID NO: 79) DNA Sequence of 5’ UTR ARNV1: AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC (SEQ ID NO: 80) DNA Sequence of 5’ UTR ARNV2: TCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACT TCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAAT TTTCACCATTTACGAACGATAGC (SEQ ID NO: 81) DNA Sequence of 5’ UTR ARNV3: AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCCAACAATC TCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCAtty. Dkt. No.117586-0146 TATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTT TCACCATTTACGAACGATAGC (SEQ ID NO: 82) DNA Sequence of 5’ UTR ARNV4: CACTCGCGCTGCCATCACTCTTCCGCCGTCTTCGCCGCCATCCTCGGCGCGACTC GCTTCTTTCGGTTCTACCAGGTAGAGTCCGCCGCCATCCTCCACCCAACAACTTG TCTCGCTCCGGGGAACGCTCGGAAACTCCCGGCCGCCGCCACCCGCGTCTGTTC TGTTACACAAGGGAAGAAAAGCCGCTGCCGCACTCCGAGTGT (SEQ ID NO: 83) DNA Sequence of 3’ UTR ARNV1: GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCA ACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAA TAAAAAACATTTATTTTCATTGC (SEQ ID NO: 84) DNA Sequence of 5’ UTR AUR1: TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAA AAGAAGAGTAAGAAGAAATATAAGA (SEQ ID NO: 85) DNA Sequence of 3’ UTR AUR1: GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACC TGTACCTCTTGGTCTTTGAATAAAGCCTGAGTA (SEQ ID NO: 86)

Claims

Atty. Dkt. No.117586-0146 CLAIMS What is claimed is:

1. An isolated mRNA molecule comprising a 5’ UTR sequence and a coding sequence, wherein the 5’ UTR sequence is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, and 6; and wherein the coding sequence encodes a protein comprising one or more HBsAg epitopes.

2. The isolated mRNA molecule of claim 1, wherein the one or more HBsAg epitopes comprises (i) a first epitope and a second epitope comprising CKTCTTPAQGNSMFPS (SEQ ID NO: 40); or (ii) a first epitope, a second epitope, and a third epitope comprising CTKP(T / S)DGNC (SEQ ID NO: 36).

3. The isolated mRNA of claim 2 or claim 3, wherein the coding sequence is selected from the group consisting of SEQ ID NOs: 10, 11, 64, and 65.

4. The mRNA molecule of any one of claims 1-3, further comprising a 5’ cap.

5. The mRNA molecule of claim 4, wherein the 5’ cap is added enzymatically or chemically using anti-reverse cap analogues (ARCA).

6. The mRNA molecule of any one of claims 1-5, further comprising a 3’ UTR, wherein the 3’ UTR sequence is selected from the group consisting of SEQ ID NOs: 5 and 7.

7. The mRNA molecule of any one of claims 1-6, wherein the 5’ UTR comprises SEQ ID NO:

1.

8. The mRNA molecule of any one of claims 1-6, wherein the 5’ UTR comprises SEQ ID NO:

3.

9. The mRNA molecule of any one of claims 1-6, wherein the 5’ UTR comprises SEQ ID NO: 6.Atty. Dkt. No.117586-0146 10. The mRNA molecule of any one of claims 1-9, wherein the 3’ UTR comprises SEQ ID NO:

5.

11. The mRNA molecule of any one of claims 1-9, wherein the 3’ UTR comprises SEQ ID NO:

7.

12. The mRNA molecule of any one of claims 1-11, wherein the coding sequence comprises SEQ ID NO: 10 or SEQ ID NO:

64.

13. The mRNA molecule of any one of claims 1-11, wherein the coding sequence comprises SEQ ID NO: 11 or SEQ ID NO:

65.

14. The mRNA molecule of any one of claims 1-11, wherein the coding sequence comprises SEQ ID NOs: 10 and 64 or SEQ ID NOs: 11 and 65.

15. The mRNA molecule of any one of claims 1-14, wherein the mRNA molecule comprises SEQ ID NOs: 12-21.

16. An immunogenic composition comprising a first mRNA molecule comprising the mRNA molecule of any one of claims 1-15 and a pharmaceutically acceptable carrier.

17. The immunogenic composition of claim 16, further comprising a second mRNA molecule of any one of claims 1-15.

18. The immunogenic composition of claim 17, wherein the first mRNA molecule comprises: a 5’ UTR sequence comprising SEQ ID NO: 1; and a coding sequence selected from the group consisting of: SEQ ID NOs: 10, 11, 64, and 65; and wherein the second mRNA sequence comprises: a 5’ UTR sequence comprising SEQ ID NO: 3; and a coding sequence selected from the group consisting of: SEQ ID NOs: 10, 11, 64, and 65.Atty. Dkt. No.117586-0146 19. The immunogenic composition of claim 18, wherein the first mRNA sequence comprises a 5’ UTR sequence comprising SEQ ID NO: 1 and a coding sequence comprising SEQ ID NOs: 64 or 65.

20. The immunogenic composition of claim 18, wherein the second mRNA sequence comprises a 5’ UTR sequence comprising SEQ ID NO: 3 and a coding sequence comprising SEQ ID NOs: 64 or 65.

21. The immunogenic composition of any one of claims 16-20, wherein the immunogenic composition is formulated as a vaccine.

22. The immunogenic composition of any one of claims 16-21, wherein the mRNA molecule is formulated in a carrier.

23. The immunogenic composition of claim 22, wherein the carrier is a lipid nanoparticle.

24. A method of treating or preventing hepatitis B infection in a subject in need thereof comprising administering to the subject the immunogenic compositions of any one of claims 16-23.

25. The method of claim 24, further comprising administering an additional therapeutic agent.

26. The method of claim 25, wherein the additional therapeutic agent is selected from the group consisting of an siRNA, an anti-sense oligonucleotide, an antibody, a serum composition comprising antibodies, a protein vaccine, an mRNA vaccine, an antiviral, an immunomodulator, an adjuvant, and combinations thereof.

27. The method of claim 25 or 26, wherein the additional therapeutic and the immunogenic composition are administered, separately, sequentially, or simultaneously to the subject.

28. The method of any one of claims 24-27, wherein the method is effective to reduce circulating hepatitis B surface antigen (HBsAg) levels, to reduce serum HBV DNA levels, and / or to improve survival in the subject as compared to a control subject.Atty. Dkt. No.117586-0146 29. The method of claim 28, wherein the method is effective to reduce circulating HBsAg levels to undetectable levels in the subject.

30. The method of any one of claims 24-29, wherein the amount of mRNA in the immunogenic compositions ranges from about 1 µg to about 200 µg.

31. The method of any one of claims 24-30, wherein the immunogenic composition is administered to the subject at least once.

32. The method of any one of claims 24-30, wherein the immunogenic composition is administered to the subject at an interval.

33. The method of any one of claims 24-30, wherein the immunogenic composition is administered to the subject at least once weekly.

34. The method of any one of claims 24-33, wherein the immunogenic composition is administered orally, intravenously, intraperitoneally, subcutaneously, intrabuccally, intradermally, intranasally, intrahepatically, or intramuscularly to the subject.

35. The method of any one of claims 24-34, wherein the subject is human.

36. The method of any one of claims 24-35, wherein the method comprises inducing a T-cell response.

37. An expression vector comprising the mRNA molecule of any one of claims 1-15.

38. A cell comprising the mRNA molecule of any one of claims 1-15 or the expression vector of claim 36.

39. The cell of claim 38, wherein the cell is selected from the group consisting of monocytic cells, dermal fibroblasts, lung cancer cells, skeletal muscle cells.

40. A lipid nanoparticle comprising the mRNA molecule of any one of claims 1-15.

41. A composition comprising one or more lipid nanoparticles of claim 40.

42. The composition of claim 41, wherein the one or more lipid nanoparticles are lyophilized, in a suspension, or emulsified.Atty. Dkt. No.117586-0146 43. The lipid nanoparticle composition of claim 41 or claim 42, further comprising a pharmaceutically acceptable carrier.

44. An isolated DNA molecule comprising a first amino acid coding sequence operably linked to a promoter and a second amino acid coding sequence operably linked to a promoter; wherein the first amino acid coding sequence encodes SEQ ID NO: 8; and the second amino acid coding sequence encodes SEQ ID NO:

9.

45. The isolated DNA molecule of claim 42, wherein the first amino acid coding sequence is operably linked to a CAG promoter comprising SEQ ID NO: 62 and / or wherein the second amino acid coding sequence is operably linked to a CMV promoter comprising SEQ ID NO:

63.

46. The isolated DNA molecule of claims 42 or 43, wherein the DNA molecule comprises SEQ ID NO:

24.

47. An adenovirus construct comprising a genome comprising the isolated DNA molecule of claims 43 or 44.

48. A method of treating or preventing hepatitis B infection in a subject in need thereof comprising administering to the subject a DNA vaccine comprising the isolated DNA molecule of claims 43 or 44 or the adenovirus construct of claim 42, and an anti-hepatitis B siRNA.

49. The method of claim 48, wherein the anti-hepatitis B siRNA and the DNA vaccine are administered, separately, sequentially, or simultaneously to the subject.

50. The method of any one of claims 48-49, wherein the method is effective to reduce circulating hepatitis B surface antigen (HBsAg) levels, to reduce serum HBV DNA levels, and / or to improve survival in the subject as compared to a control subject.

51. The method of claim 50, wherein the method is effective to reduce circulating HBsAg levels to undetectable levels in the subject.

52. The method of any one of claims 48-51, wherein the amount of DNA in the DNA vaccine ranges from about 1 µg to about 500 µg.Atty. Dkt. No.117586-0146 53. The method of any one of claims 48-52, wherein the DNA vaccine is administered to the subject at least once.

54. The method of any one of claims 48-52, wherein the DNA vaccine is administered to the subject at an interval.

55. The method of any one of claims 48-52, wherein the DNA vaccine is administered to the subject at least once weekly.

56. The method of any one of claims 48-55, wherein the DNA vaccine is administered orally, intravenously, intraperitoneally, subcutaneously, intradermal, intranasally, intrabuccally, or intramuscularly to the subject.

57. The method of any one of claims 48-56, wherein the subject is human.

58. The method of claim 48, wherein the method comprises administering the adenovirus construct of claim 47 in combination with an anti-hepatitis B siRNA.

59. The methods of any one of claims 48-58, wherein the anti-HBV siRNA comprises AD66810, wherein the AD66810 sense strand comprises SEQ ID NO: 22 and wherein the AD66810 anti-sense strand comprises SEQ ID NO: 23.

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