Hepatitis b virus vaccine and methods of use

Abstract

Provided is a hepatitis B vaccine comprising at least one mRNA molecule encoding a hepatitis B antigen and methods of use thereof to treat or prevent hepatitis B.

Description

Attorney Docket No.206664-0001-00WOHEPATITIS B VIRUS VACCINE AND METHODS OF USECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Application No. 63 / 719,510, filed November 12, 2024, which is hereby incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under AI146101 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an XML Document formatted sequence listing with a file name “206664-0001-00WO_SequenceListing.xml,” having a creation date of November 10, 2025, and having a size of 23,075 bytes. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0004] Hepatitis B virus (HBV) is a small-enveloped DNA virus, which selectively infects human hepatocytes (Kim et al., 2022, Clin Mol Hepatol 28, 17-30). Upon infection, the circular viral genome (relaxed circular DNA) is transported to the nucleus of the infected cell where host enzymes convert it into an episomal transcriptional template (covalently closed circular DNA, cccDNA) leading to chronic infection. Even though an effective prophylactic antihepatitis B vaccine exists, there are an estimated 300 million people worldwide who suffer from chronic hepatitis B (CHB) infection and 1.5 million new infections occur every year. The principal route of HBV transmission in high-endemic countries is from mother to child; the most relevant transmission routes in low-endemic countries are sexual and parenteral. Recently, mother-to-child transmission has been reduced significantly by administering a prophylactic vaccine during the first 24 hours of life (Terrault et al., 2018, Hepatology 67, 1560-1599). In many cases, however, the induction of protective immunity, represented by anti-hepatitis BAttorney Docket No.206664-0001-00WOsurface antigen (HBsAg) levels of 10-100 IU / L for low-responders and anti-HBsAg levels > 100 IU / L for high-responders, is not sufficient, as evidenced by a breakthrough infection rate of 5-10%. On the other hand, unvaccinated children infected with HBV exhibit a 90% probability of developing the chronic infection and a 25% chance of eventually suffering liver damage (Pooter et al., 2021, Vaccines (Basel) 9). Pediatric CHB patients often fail to exhibit symptoms during childhood but carry a high risk of developing hepatocellular carcinoma (HCC) as adults (Defresne et al., 2017, J Gastroenterol Hepatol 32, 368-371). In fact, CHB is a major cause of HCC (Kim et al., 2022, Clin Mol Hepatol 28, 17-30).

[0005] Ineffective innate and adaptive immune responses characterize CHB infection. HBV has the ability to modulate innate immunity, which leads to the inability to mount an adaptive immune response in CHB infection, eventually resulting in viral persistence. In this regard, excessive quantities of subviral HBV particles negatively impact innate immune pathways by inhibiting the secretion of inflammatory cytokines and suppressing interferon-stimulated genes (Shi et al., 2012, PLoS One 7; Jiang et al., 2014, J Viral Hepat 21, 860-872). Regarding adaptive immunity, HBV-specific CD4+ and CD8+ T cells suffer from a state of constant exhaustion (Yuen et al., 2018, Nat Rev Dis Primers 4, 18035). In summary, CHB infection is characterized by T cell exhaustion with reduced cytotoxic activity, impaired cytokine production, and sustained expression of cell-surface inhibitory receptors (Bertoletti et al., 2018, Gut and Liver 12, 497-507). High levels of HBsAg found in most CHB patients impair B and T cell responses. It has been suggested that treatment options that clear systemic HBsAg levels could reverse immune dysfunction (lannacone et al., 2022, Nat Rev Immunol 22, 19-32). The concept of “functional cure” is used in this context to describe the loss of detectable serum HBsAg with or without seroconversion to anti-HBsAg antibody production (Terrault et al., 2018, Hepatology 67, 1560-1599). Unfortunately, functional cure is not achieved with current treatment options that include the use of pegylated IFN-a protein and nucleos(t)ide analogs.

[0006] The coordinated activation of anti-HBV-specific humoral and cell-mediated immunity in patients who completely recover from HBV infections indicates that immune activation and functional cure are achievable (Zhou et al., 2019, Lancet Gastroenterol Hepatol 4, 227-238; Bertoletti et al., 2012, Gut 61, 1754-1764). B cells capable of secreting anti-HBV antibodies and robust helper and cytotoxic T cell responses observed in patients who recovered indicate that therapeutic approaches that elicit anti-viral immune mechanisms could induce aAttorney Docket No.206664-0001-00WOfunctional cure.

[0007] Prophylactic vaccines tested for their therapeutic effects in CHB patients achieved little success, i.e., the induction of specific antibody production, but no cytotoxic T-cell response (Vandepapeliere et al., 2007, Vaccine 25, 8585-8597; Mancini-Bourgine et al., 2004, Hepatology 40, 874-882). New vaccine formulations showed only limited success when used curatively (Lok et al., 2016, J Hepatol 65, 509-516; Godon et al., 2014, Molecular Therapy 22, 675-684; Xu et al., 2013, J Hepatol 59, 450-456). A therapeutic vaccine is needed that could induce both adaptative immunity and a robust innate immune response, optimized by incorporating the appropriate adjuvants into the vaccine formulation (Fanning et al., 2019, Nat Rev Drug Discov 18, 827-844).

[0008] Due to the absence of a highly effective treatment for CHB, there remains a need in the art for improved novel immunotherapies aiming to restore life-long patient immunity. The present invention addresses this need.SUMMARY OF THE INVENTION

[0009] The invention relates to compositions and methods for inducing an immune response against hepatitis B in a subject. In some embodiments, the invention provides a composition comprising at least one optimized RNA molecule encoding a hepatitis B antigen. In some embodiments, the invention provides a combination of at least one optimized RNA molecule encoding a hepatitis B antigen lacking a signal peptide and at least one optimized RNA molecule encoding a hepatitis B antigen linked to a signal peptide to induce both humoral and cellular immune responses.

[0010] In some embodiments, the invention relates to a composition comprising at least one optimized RNA molecule encoding at least one hepatitis B antigen, wherein the optimized RNA molecule is transcribed from a sequence having at least 80% identity to SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 14, wherein the optimized RNA molecule encodes SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. In some embodiments, the composition comprises at least one RNA molecule transcribed from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 12.

[0011] In some embodiments, the hepatitis B antigen is operably linked to a signalAttorney Docket No.206664-0001-00WOpeptide.

[0012] In some embodiments, the composition comprises a combination of at least two optimized RNA molecules encoding the hepatitis B antigen, wherein the combination comprises at least two of:a) a RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 2, wherein the optimized RNA molecule encodes SEQ ID NO: 1;b) a RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 4, wherein the optimized RNA molecule encodes SEQ ID NO: 3;a RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 10 or SEQ ID NO: 11, wherein the RNA molecule encodes SEQ ID NO: 9; andd) a RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 13 or SEQ ID NO: 14, wherein the RNA molecule encodes SEQ ID NO: 12.

[0013] In some embodiments, the composition comprises at least one lipid nanoparticle (LNP) encapsulating the optimized RNA molecule encoding the hepatitis B antigen.

[0014] In some embodiments, the RNA molecule is an mRNA molecule, aself-amplifying RNA (saRNA) molecule or a circular RNA (circRNA) molecule.

[0015] In some embodiments, the optimized RNA molecule comprises at least one modified nucleoside.

[0016] In some embodiments, the composition induces a cellular immune response against hepatitis B. In some embodiments, the composition comprises an optimized RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 2, wherein the optimized RNA molecule encodes SEQ ID NO: 1.

[0017] In some embodiments, the composition induces a humoral immune response against hepatitis B. In some embodiments, the composition comprises an optimized RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 4, wherein the optimized RNA molecule encodes SEQ ID NO: 3.

[0018] In some embodiments, the composition induces both a cellular and humoral immune response against hepatitis B.

[0019] In some embodiments, the composition comprises a combination of:a) an optimized RNA molecule transcribed from SEQ ID NO: 2, wherein the optimized RNA molecule encodes SEQ ID NO: 1; andAttorney Docket No. 206664-0001-00WOb) an optimized RNA molecule transcribed from SEQ ID NO: 4, wherein the optimized RNA molecule encodes SEQ ID NO: 3.

[0020] In some embodiments, the composition comprises a combination of at least one RNA encoding a hepatitis B surface antigen (HBsAg) and at least one RNA encoding a hepatitis B core antigen (HBcAg). In some embodiments, the ratio of RNA encoding the HBsAg to RNA encoding the HBcAg is about 2:1. In some embodiments, the ratio of RNA encoding the HBsAg to RNA encoding the HBcAg is about 1: 1. In some embodiments, the ratio of RNA encoding the HBsAg to RNA encoding the HBcAg is about 1:2.

[0021] In some embodiments, the invention relates to a method of inducing an immune response against hepatitis B in a subject comprising administering to the subject an effective amount of a composition comprising at least one optimized RNA molecule encoding at least one hepatitis B antigen, wherein the optimized RNA molecule is transcribed from a sequence having at least 80% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 11 or SEQ ID NO: 14, wherein the optimized RNA molecule encodes SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO: 12. In some embodiments, the composition comprises at least one RNA molecule transcribed from SEQ ID NO:2, SEQ IDNO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO: 11 or SEQ ID NO: 14.

[0022] In some embodiments, the method comprises inducing a cellular immune response in a subject in need thereof, the method comprising administering a composition comprising an optimized RNA molecule transcribed from SEQ ID NO: 2, wherein the optimized RNA molecule encodes SEQ ID NO: 1 to the subject.

[0023] In some embodiments, the method comprises inducing a humoral immune response in a subject in need thereof, the method comprising administering a composition comprising an optimized RNA molecule transcribed from SEQ ID NO: 4, wherein the optimized RNA molecule encodes SEQ ID NO: 3 to the subject. In some embodiments, the method comprises administering the composition to a healthy subject.

[0024] In some embodiments, the method comprises inducing a combination of a cellular and humoral immune response in a subject in need thereof, the method comprising administering a composition comprising a combination of at least two of:a) an optimized RNA molecule transcribed from SEQ ID NO: 2, wherein the optimized RNA molecule encodes SEQ ID NO: 1;Attorney Docket No. 206664-0001-00WOb) an optimized RNA molecule transcribed from SEQ ID NO: 4, wherein the optimized RNA molecule encodes SEQ ID NO: 3 to the subject. In some embodiments, the subject has chronic hepatitis B;c) a RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 10 or SEQ ID NO:11, wherein the RNA molecule encodes SEQ ID NO: 9; and d) a RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 13 or SEQ ID NO: 14, wherein the RNA molecule encodes SEQ ID NO: 12 to the subject.

[0025] In some embodiments, the method comprises administering a priming, a first boosting and a second boosting vaccine composition comprising a combination of:a) an RNA molecule transcribed from SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1; andb) an RNA molecule transcribed from SEQ ID NO: 10 or SEQ ID NO: 11, wherein the RNA molecule encodes SEQ ID NO: 9

[0026] In some embodiments, the method comprises administering:i) a priming vaccine composition comprising an optimized RNA molecule transcribed from SEQ ID NO: 2, wherein the optimized RNA molecule encodes SEQ ID NO: 1;ii) a first boosting vaccine composition comprising a combination of:a) an optimized RNA molecule transcribed from SEQ ID NO: 2, wherein the optimized RNA molecule encodes SEQ ID NO: 1; andb) an optimized RNA molecule transcribed from SEQ ID NO: 4, wherein the optimized RNA molecule encodes SEQ ID NO: 3; andiii) a second boosting vaccine composition comprising an optimized RNA molecule transcribed from SEQ ID NO: 4, wherein the optimized RNA molecule encodes SEQ ID NO: 3.

[0027] In some embodiments, the method comprises administering:i) a priming vaccine composition comprising a combination of an RNA molecule transcribed from SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1 and an RNA molecule transcribed from SEQ ID NO: 10 or SEQ ID NO: 11, wherein the RNA molecule encodes SEQ ID NO:9;Attorney Docket No.206664-0001-00WOii) a first boosting vaccine composition comprising a combination of:a) an RNA molecule transcribed from SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1; andb) an RNA molecule transcribed from SEQ ID NO: 4, wherein the RNA molecule encodes SEQ ID NO: 3;c) an RNA molecule transcribed from SEQ ID NO: 10 or SEQ ID NO: 11, wherein the RNA molecule encodes SEQ ID NO:9; andd) an RNA molecule transcribed from SEQ ID NO: 13 or SEQ ID NO: 14, wherein the RNA molecule encodes SEQ ID NO: 12;andiii) a second boosting vaccine composition comprising a combination of an RNA molecule transcribed from SEQ ID NO: 4, wherein the RNA molecule encodes SEQ ID NO: 3 and an RNA molecule transcribed from SEQ ID NO: 13 or SEQ ID NO: 14, wherein the RNA molecule encodes SEQ ID NO: 12.

[0028] In some embodiments, the composition further comprises an adjuvant.

[0029] In some embodiments, the composition is administered by a delivery route selected from the group consisting of intravenous, intradermal, subcutaneous, inhalation, intranasal, and intramuscular.

[0030] In some embodiments, the method comprises administration of a combination of two or more separate LNP, wherein each LNP encapsulates a single species of RNA molecule and administering the combination of LNPs to the subject at two or more separate injection sites.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0032] Figure 1, comprising Figure 1A through Figure IF, depicts data demonstrating that mRNA encoding HBsAg leads to high antigen expression and immune activation in primary human cells in vitro. Figure 1 A and Figure IB depict intracellular and Figure 1C and Figure IDAttorney Docket No.206664-0001-00WOdepict extracellular staining of HBsAg in transfected hMoDCs was evaluated by flow cytometry. As control HBsAg protein was incubated directly with hMoDCs. Figure IE depicts up-regulation of activation markers (HLA-DR, CD40, CD80 & CD86) of hMoDCs transfected with HBsAg mRNA was studied by flow cytometry. Ova mRNA was used as an irrelevant mRNA control. ENGERIX®-B, HBsAg protein and Lipofectamine Messenger Max were also used as controls for comparison. Mean fluorescence intensity (MFI) of the treatments was compared with the untreated control. Figure IF depicts data demonstrating that cell culture supernatants were collected for cytokine profile evaluation and fold control of the cytokine concentration was calculated in comparison with the untreated control. Results are the means ± SEM obtained in 4 independent experiments. Significance tested compared to the control groups: *p < 0.0332; ***p < 0.0002, ****p < 0.0001 (two-tailed Student’s t-test and two-way ANOVA, Tukey’s multiple comparisons test).

[0033] Figure 2, comprising Figure 2A through Figure 2D, depicts data demonstrating that HBsAg mRNA formulated in LNPs induces early activation of DCs and T cells in secondary lymphoid organs. Figure 2A depicts a schematic representation of the preparation process of mRNA-based vaccines. HBsAg mRNA was formulated into lipid nanoparticles (LNPs) by mixing the lipid-containing ethanolic phase (GenVoy-ILM™) with the RNA-aqueous phase using a microfluidic device. After synthesis, mRNA-LNPs were characterized. Figure 2B depicts an experimental schedule in which mice were administered with 5 pg of HBsAg mRNA-LNPs by intramuscular (i.m.) injection. Spleen and inguinal lymph nodes were collected for evaluation 24 hours after treatment. Figure 2C depicts data demonstrating that early activation markers CD69 in CD4+ and CD8+ T cells, and CD80 and CD86 in DCs, as depicted in Figure 2D, isolated from the spleen and lymph nodes were studied and compared against PBS control group. Data are the means ± SEM for animal cohorts (4 mice / group). Significantly different from the control group: *p < 0.0332; **p < 0.0021, ****p < 0.0001 (two-way ANOVA, Sidak’s multiple comparisons test).

[0034] Figure 3, comprising Figure 3A through Figure 3G, depicts data demonstrating that mice immunized with HBsAg mRNA-LNPs generate HBsAg-specific immunity. Figure 3A depicts an experimental schedule in which mice were immunized with HBsAg mRNA-LNPs on day 0. Two weeks after priming, the second dose (boost) was injected (dl 4). ENGERIX®-B was used as a control treatment, and PBS was used as a negative control. The experiment wasAttorney Docket No. 206664-0001-00WOfinalized on day 28, and blood and spleen were collected for further analyses. Blood withdrawal was also executed on day -1 (pre-bleeding) and on day 14. Figure 3B depicts IgG2c / IgGl antibody ratio measured on day 28. Figure 3C depicts anti-HBsAg antibody titers on day 14 (post-prime) and on day 28 (post-boost). Figure 3D depicts Ki-67+ spleen cells were analyzed for the assessment of the proliferation index. Figure 3E depicts the frequencies of HBsAg-specific IFN-y+, IL-2+, and TNF-a+ CD4+ T cells. Figure 3F depicts the frequencies of HBsAg-specific IFN- y+, IL-2+, and TNF- a+ CD8+ T cells. Figure 3G depicts cytokine profiles from supernatants collected from peptide-stimulated spleen cell cultures for 72 hours. Data are the means ± SEM for animal cohorts (6 mice / group). Significance tested compared to the other groups: no significant differences (ns); *p < 0.0332; **p < 0.0021; ***p < 0.0002; ****p < 0.0001 (two-tailed Student’s t-test, two-way ANOVA and one-way ANOVA, Tukey’s multiple comparisons test).

[0035] Figure 4, comprising Figure 4A through Figure 4H, depicts data demonstrating antigen optimization by incorporating signal peptides into mRNA constructs leads to high anti-HBsAg antibody titers and to an improvement in memory T cell response. Figure 4A depicts data demonstrating that 2nd generation mRNA constructs were sequence optimized. An MHC class I signal peptide (SP) was included in the 5’ region of the HBsAg mRNA construct (SP HBsAg mRNA). Another construct (SP HBsAg MITD mRNA) was modified by adding the transmembrane and cytosolic trafficking domain of MHC class I (MITD) into the 3’ region of the HBsAg sequence. Luciferase mRNA (Luc mRNA) was prepared and use as irrelevant mRNA control. Figure 4B depicts an experimental schedule in which mice were immunized with 2nd gen mRNA vaccines on day 0 (Prime). Four weeks after priming, the second dose (boost) was injected (d28). ENGERIX®-B was used as a control treatment, Luc mRNA-LNPs were used as irrelevant mRNA-LNPs control, and PBS was used as negative control. The experiment was finalized on day 42, and blood and spleen were collected for further analyses. Blood withdrawal was also executed on day -1 (pre-bleeding) and day 28. Figure 4C depicts the total anti-HBsAg IgG titers at termination (d42). Figure 4D depicts the IgG2c / IgGl ratio at termination (d42). Figure 4E depicts the proportion of CD4+ effector cells. Figure 4G depicts the proportion of CD8+ effector cells. Figure 4F and Figure 4H respectively present pie charts depicting the percentages of CD4+ and CD8+ memory T cells including T central memory cells (TCM), Naive T cells, T effector cells (TE), and T effector memory cells (TEM) for all immunized groups.Attorney Docket No.206664-0001-00WOData are the means ± SEM for animal cohorts (8 mice / group). Significance tested compared to the other groups: *p < 0.0332; **p < 0.0021; ***p < 0.0002; ****p < 0.0001 (one-way ANOVA, Tukey’s multiple comparisons test).

[0036] Figure 5, comprising Figure 5A through Figure 5F, depicts data demonstrating that sequence optimized mRNA-LNP vaccines induce robust CD4+ and CD8+ immune responses. Figure 5A depicts the percentages of IFN-y+, IL-2+ and TNF-a+ -producing CD4+ and CD8+ T cells, as depicted in Figure 5B, after peptide stimulation of cell isolates from spleen. Figure 5C depicts secreted cytokine levels from spleen cells were analyzed 72 hours after stimulation. Heatmap cytokine values represent the fold control of the cytokine concentration in comparison with the unstimulated cells. Figure 5D depicts secreted IL-2 levels after peptide-stimulation overnight. Values are represented as the fold control with the unstimulated cells. Figure 5E depicts Ki67+ spleen cells were analyzed for the assessment of the proliferation index. Figure 5F depicts a western blot analysis of HepG2 cell lysates after transfection with nonoptimized (lane 5: HBsAg mRNA) and optimized mRNA constructs (lane 2: HBsAg mRNA, lane 3: SP HBsAg mRNA, lane 4: SP HBsAg MITD mRNA). Non-transfected HepG2 cells were used as negative control (lane 1). HBsAg protein is expressed as glycosylated (gp27) and nonglycosylated (p24) isomers. Data are the means ± SEM for animal cohorts (8 mice / group).Significance tested compared to the other groups: *p < 0.0332; **p < 0.0021; ***p < 0.0002; ****p < 0.0001 (one-way ANOVA, Tukey’s multiple comparisons test).

[0037] Figure 6, comprising Figure 6A through Figure 6F, depicts data demonstrating that a heterologous immunization schedule enhances immune responses compared with proteinbased anti -hepatitis B commercial vaccine. Figure 6A depicts percentages of IFN-y+, IL-2+ and TNF-a+-producing CD4+ and CD8+ T cells, as depicted in Figure 6B, after peptide stimulation of cell isolates from spleen. Figure 6C depicts the proportion of CD4+ and CD8+ T effector cells. Figure 6D depicts the total anti-HBsAg IgG titers at termination of the immunization schedule (d28). Figure 6E depicts secreted cytokine levels from spleen cells analyzed 72 hours after stimulation. Heatmap cytokine values represent the fold control of the cytokine concentration in comparison with the unstimulated cells. Figure 6F depicts secreted IL-2 levels after peptide-stimulation overnight. Values are represented as the fold control compared to the respective unstimulated cells. Data are the means ± SEM for animal cohorts (8 mice / group). Significance tested compared to the other groups: no significant differences (ns); *p < 0.0332;Attorney Docket No.206664-0001-00WO**p < 0.0021; ***p < 0.0002; ****p < 0.0001 (one-way ANOVA, Tukey’s multiple comparisons test).

[0038] Figure 7, comprising Figure 7A through Figure 7F, depicts data demonstrating that sequence-optimized mRNA-LNP vaccines prevent HBV replication in an HBV challenge setting. Figure 7A depicts an experimental schedule in which mice were immunized with sequence-optimized mRNA-LNP vaccines on day 0 (Prime). Four weeks after priming, the second dose (Boost) was injected (d28). PBS was used as negative control. On day 56, animals were challenged with AAV / HBV viral genome and the experiment was finalized on day 70, and blood and spleen were collected for further analyses. Blood withdrawal was also executed on day 63. Figure 7B depicts the total anti-HBsAg IgG titers at termination of the immunization schedule (d70). Figure 7C depicts the measurement of plasma HBsAg and (Figure 7d) HBeAg on d7 and dl4 post-challenge. Figure 7E and Figure 7F depict the percentages of IFN-γ+, IL-2+ and TNF-α+-producing (Figure 7E) CD4+ and (Figure 7F) CD8+ T cells after peptide stimulation of cell isolates from spleen overnight. Data are the means ± SEM for animal cohorts (8 mice / group). Significance tested compared to the other groups: *p < 0.0332; **p < 0.0021; ***p < 0.0002; ****p < 0.0001 (one-way ANOVA, Dunn's and Tukey 's multiple comparisons test; two-way ANOVA, Tukey’s multiple comparisons test). LLOQ: lower limit of quantification.

[0039] Figure 8, comprising Figure 8A through Figure 8F, depicts data demonstrating HBsAg mRNA-LNP vaccination reduces serum HBsAg and induces cellular immunity in CHB mice. Figure 8A depicts an experimental design in which mice were injected i.v. with AAV / HBV viral genome on day -28 before prime (CHB mice). CHB mice were vaccinated i.m. with 10 pg HBsAg mRNA-LNP on days 0 (Prime), 14 (Boost 1) and 28 (Boost 2). PBS was used as negative control. The experiment was terminated on day 70, and the blood and spleens were collected for further analyses. Blood was also collected on day -1, and days 7, 14, 21, 28, 35, 42, 49, 56 and 63 post-prime. Figure 8B depicts the total anti-HBsAg IgG concentration at days -1, 14, 28, 49 and 70 post-prime. Figure 8C depicts the measurement of plasma HBsAg and Figure 8D depicts the adverse correlation of serum anti-HBsAg and plasma HBsAg concentrations over time. Figure 8E depicts the percentages of IFN-y+, IL-2+ and TNF-a+-producing CD4+ and Figure 8F depicts the CD8+ T splenocytes from their respective previous CD4+ and CD8+ T cell population quantified after overnight stimulation with peptides. Data are the means ± SEM forAttorney Docket No.206664-0001-00WOanimal cohorts (6 mice / group). Significantly different from other groups: *p <0.05; **p <0.01; ****p <o 0001 (two-tailed Student’s t-test). LLOQ: lower limit of quantitation.

[0040] Figure 9, comprising Figure 9A and Figure 9B, depicts data demonstrating an analysis of HBsAg protein production in hmoDCs transfected with different amounts of HBsAg mRNA. Figure 9A depicts a western blot analysis of lysates of 24-hour transfected hmoDCs with 250 ng (lane 2), 500 ng (lane 3), 750 ng (lane 4) and 1000 ng (lane 5) of HBsAg mRNA mixed with 1 pL Lipofectamine MessengerMax. Lane 1 corresponds to the negative control. Figure 9B depicts the quantification of HBsAg protein bands was calculated using an HBsAg standard curve in ImageJ software. Data represent the means ± SEM (n= 2).

[0041] Figure 10 depicts data demonstrating an analysis of OVA protein production in ova mRNA-transfected hmoDCs. Western blot analysis of lysates (lanes 1 and 2) and supernates (lanes 3 and 4) of 24-hour transfected hmoDCs with ova mRNA mixed with 1 pL Lipofectamine MessengerMax. Lanes 1 and 3 correspond to the negative controls.

[0042] Figure 11 depicts data demonstrating a cytometry bead array from cell culture supernatants collected from 48-hour stimulated splenocytes isolated from mice immunized with the non-optimized HBsAg mRNA-LNPs. Values are represented as the fold control compared to the respective unstimulated cells. Data represent the means ± SEM for animal cohorts (6 mice / group).

[0043] Figure 12, comprising Figure 12A and Figure 12B, depicts data demonstrating memory T cell subpopulations in 72-hour ex vivo peptide-stimulated splenocytes isolated from mice immunized with the heterologous immunization schedule. Figure 12A depicts pie charts depicting the percentages of CD4+ and Figure 12B depicts CD8+ memory T cells including T central memory cells (TCM), Naive T cells, T effector cells (TE), and T effector memory cells (TEM) for all immunized groups. Data represent the means for animal cohorts (8 mice / group).

[0044] Figure 13 depicts data demonstrating a cytometry bead array from cell culture supernatants collected from 24-h stimulated splenocytes isolated from mice immunized with the sequence-optimized HBsAg mRNA-LNP formulations. Values are represented as the fold control compared to the respective unstimulated cells. Data represent the means ± SEM for animal cohorts (8 mice / group).

[0045] Figure 14 depicts data demonstrating a cytometry bead array from cell culture supernatants collected from 24-h stimulated splenocytes isolated from mice immunized with theAttorney Docket No.206664-0001-00WOheterologous immunization schedule. Values are represented as the fold control compared to the respective unstimulated cells. Data represent the means ± SEM for animal cohorts (8 mice / group).

[0046] Figure 15 depicts data demonstrating a proliferation index of 72-h stimulated splenocytes isolated from mice immunized with the heterologous immunization schedule. Ki67+ spleen cells were analyzed for the assessment of the proliferation index. Data are the means ± SEM for animal cohorts (8 mice / group). Significance tested compared to the other groups: no significant differences (ns); *p < 0.0332; **p < 0.0021 (one-way ANOVA, Tukey’s multiple comparisons test).

[0047] Figure 16 depicts data demonstrating anti-HBsAg IgG subtypes antibodies from mice immunized with the heterologous immunization schedule. IgG2c / IgGl ratio at termination (d42). Data are the means ± SEM for animal cohorts (8 mice / group). Significance tested compared to the other groups: no significant differences (ns) (one-way ANOVA, Tukey’s multiple comparisons test).

[0048] Figure 17, comprising Figure 17A and Figure 17B, depicts data demonstrating HBsAg-specific cellular response in HBV-carrier mice in a proof of concept immunization with HBsAg mRNA-LNP vaccine candidates. Percentage of specific IFN-y+, IL-2+ and TNF-a+ cells in CD4+ (Figure 17A) and CD8+ (Figure 17B) T cells. Data is represented as violin plot with the corresponding median (n=6). Significance tested compared to the other groups: *p < 0.0332; **p < 0.0021; ***p < 0.0002; ****p < 0.0001 (one-way ANOVA, Tukey’s multiple comparisons test).

[0049] Figure 18 depicts a diagram of the experimental design for bivalent vaccine immunogenicity studies. C57BL / 6 mice were vaccinated with HBsAg and HBcAg nucleoside-modified mRNA-LNP vaccines on day 0 (prime). Two weeks and four weeks after priming, the second dose (boost 1) and third dose (boost 2) were administered (days 14 and 28, respectively). PBS was used as a negative control. The experiment was terminated on day 42, and blood and spleens were collected for immunological analyses.

[0050] Figure 19, comprising Figure 19A through Figure 19D, depicts data demonstrating that monovalent mRNA-LNP vaccines induce robust humoral and cellular immune responses. Figure 19A and Figure 19B depict the total anti-HBsAg and anti-HBcAg IgG titers measured at day 42 post-immunization. Data represent means ± SEM (n=5 mice / group).Attorney Docket No.206664-0001-00WO****p <• o 0001 vs. PBS control (one-way ANOVA with Tukey's multiple comparisons test). Figure 19C and Figure 19D depict the frequencies of IFN-γ+, IL-2+, and TNF-α+ antigen-specific CD8+ and CD4+ T cells following peptide pool restimulation of splenocytes.

[0051] Figure 20, comprising Figure 20A through Figure 20E, depicts data demonstrating that bivalent mRNA-LNP vaccines encoding HBsAg and HBcAg elicit immunity against both antigens. Mice were immunized with bivalent vaccines at HBs: HBc ratios of 2: 1, 1: 1, or 1:2. Figure 20A and Figure 20B depict the anti-HBsAg and anti-HBcAg IgG titers at day 42. Figure 20C through Figure 20E depict the Frequencies of IFN-y+, IL-2+, and TNF-a+ CD8+ and CD4+ T cells, and perforin / granzyme B expression in CD8+ T cells following antigen-specific peptide pool stimulation. Data represent means ± SEM (n=5 mice / group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Tukey's multiple comparisons test).

[0052] Figure 21, comprising Figure 21 A through Figure 21C, depicts data demonstrating that incorporation of signal peptide into bivalent vaccine configuration enhances humoral immune responses. Figure 21 A depicts an experimental design in which mice were immunized with mRNA-LNP vaccines encoding HBsAg and HBcAg antigens with or without incorporation of a SP sequence into the N-terminal domain of each construct. Vaccines were tested as individual antigen configurations using Luc mRNA as an irrelevant construct to normalize the total mRNA dose to 6 pg (HBs: Luc; Luc: HBc; SP-HBs: Luc; Luc: SP-HBc).Bivalent mRNA-LNP vaccines were administered as HBs: HBc and SP-HBs: SP-HBc at 2:1 ratio. Figure 21B and Figure 21C depict the total anti-HBsAg and anti-HBcAg IgG titers at day 42. Data represent means ± SEM (n=5 mice / group). *p < 0.05 (one-way ANOVA with Tukey's multiple comparisons test).

[0053] Figure 22, comprising Figure 22A through Figure 22F, depicts data demonstrating that incorporation of signal peptide into bivalent vaccine configuration enhances cellular immune responses. Mice were vaccinated with bivalent vaccines at 2: 1 ratio, and corresponding individual antigen controls. At the experimental endpoint (day 42), splenocytes were isolated and analyzed. Figure 22A, 22C, 22D and 22F demonstrate the frequencies of IFN-y+, IL-2+, and TNF-a+ CD8+ and CD4+ T cells. Figure 22B and Figure 22E depict perforin / granzyme B expression in CD8+ T cells following antigen-specific peptide pool stimulation. Data represent means ± SEM (n=5 mice / group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Tukey's multiple comparisons test).Attorney Docket No. 206664-0001-00WO

[0054] Figure 23, comprising Figure 23A through Figure 23F, depicts data demonstrating that combinatorial bivalent vaccine strategy employing sequential SP-modified and nonmodified constructs elicits robust Thl-biased immune responses. Figure 23A depicts an experimental design in which mice were immunized with mRNA-LNP vaccines encoding HBsAg and HBcAg antigens with or without SP modification in different bivalent configurations: HBs: HBc vaccine (without SP); SP-HBs: SP-HBc (SP in both antigen constructs); a combinatorial approach where the HBs: HBc vaccine without SP was used for prime immunization, followed by a combination of constructs with and without SP modification (HBsAg; SP-HBsAg; HBcAg; SP-HBcAg) for boost 1, and SP-HBs: SP-HBc (constructs with SP only) for boost 2. Figure 23B and Figure 23C depict the total anti-HBsAg and anti-HBcAg IgG titers at day 42. Figure 23D and Figure 23F depict the frequencies of IFN-y+, IL-2+, and TNF-a+ CD8+ and CD4+ T cells. Figure 23E depicts the granzyme B expression in CD8+ T cells following antigen-specific peptide pool stimulation. Data represent means ± SEM (n=5 mice / group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Tukey's multiple comparisons test).

[0055] Figure 24, comprising Figure 24A through Figure 23F, depicts data demonstrating that combinatorial bivalent mRNA vaccine breaks immune tolerance and reduces viral markers in a chronic hepatitis B mouse model. Figure 24A depicts an experimental design in which mice were injected intravenously with AAV8-HBV1.3 viral genome 28 days before prime immunization to establish chronic HBV infection (CHB mice). CHB mice were vaccinated intramuscularly with: (i) 10 pg HBsAg mRNA-LNP; (ii) 10 pg HBs mRNA-LNP + 5 pg HBc mRNA-LNP; or (iii) a combinatorial approach comprising 10 pg HBs mRNA-LNP + 5 pg HBc mRNA-LNP for prime (day 0), 5 pg HBs mRNA-LNP + 5 pg SP-HBs mRNA-LNP + 2.5 pg HBc mRNA-LNP + 2.5 pg SP-HBc mRNA-LNP for boost 1 (day 14), and 10 pg SP-HBs mRNA-LNP + 5 pg SP-HBc mRNA-LNP for boost 2 (day 28). PBS was used as a negative control. The experiment was terminated on day 56, and blood was collected for analyses. Figure 24B depicts the total anti-HBsAg IgG concentration determined at the experimental endpoint (day 56). Figure 24C depicts the total anti-HBeAg IgG concentration determined at the experimental endpoint (day 56). Figure 24D and Figure 24E depict plasma HBsAg and HBeAg levels. Figure 24F depicts the quantification of HBV DNA in plasma by Cobas method. Data represent means ± SEM (n=6 mice / group). *p < 0.05, **p < 0.01, ****p < 0.0001 (one-wayAttorney Docket No.206664-0001-00WOANOVA with Tukey's multiple comparisons test). LLOQ, lower limit of quantitation.DETAILED DESCRIPTION

[0056] The invention relates to compositions and methods for inducing an immune response against hepatitis B in a subject. In some embodiments, the composition comprises at least one optimized mRNA molecule encoding a hepatitis B antigen. In one embodiment, the composition comprises a lipid nanoparticle (LNP) comprising an optimized mRNA molecule encoding a hepatitis B antigen.

[0057] In some embodiments, the invention a composition comprising an optimized nucleoside modified mRNA molecule encoding a HBsAg, HBeAg, HBcAg or polymerase antigen. In one embodiment, the composition comprises a LNP comprising an optimized RNA molecule encoding a HBsAg, HBeAg, HBcAg or polymerase antigen operably linked to a signal peptide. In some embodiments, the signal peptide comprises SEQ ID NO: 15. In some embodiments, the RNA molecule is an mRNA molecule, a self-amplifying RNA (saRNA) molecule or a circular RNA (circRNA) molecule.

[0058] In some embodiments, the vaccine induces a cellular immune response, seroconversion, or a combination thereof in the subject to hepatitis B. Therefore, the invention also provides methods of treating chronic hepatitis B or preventing hepatitis B by administering the vaccine to a subject. In one embodiment, the invention provides a method of protecting a healthy subject from hepatitis B by administering the vaccine to a subject. In one embodiment, the invention provides a method of promoting clearance of hepatitis B from a subject having chronic hepatitis B by administering the vaccine to the subject.Definitions

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0060] As used herein, each of the following terms has the meaning associated with it in this section.

[0061] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” meansAttorney Docket No.206664-0001-00WOone element or more than one element.

[0062] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0063] The term “antibody,” as used herein, refers to an immunoglobulin molecule, which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0064] The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0065] An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0066] An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. K and X light chains refer to the two major antibody light chain isotypes.

[0067] By the term “synthetic antibody” as used herein, is meant an antibody, which is generated using recombinant DNA technology. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art. The term should also be construed to mean an antibody, which has been generated by the synthesis of anAttorney Docket No.206664-0001-00WORNA molecule encoding the antibody. The RNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the RNA has been obtained by transcribing DNA (synthetic or cloned), synthesizing the RNA, or other technology, which is available and well known in the art.

[0068] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such crossspecies reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0069] The term “immunogen” as used herein, is intended to denote a substance of matter, which is capable of inducing an adaptive immune response in an individual, where said adaptive immune response is capable of inducing an immune response, which significantly engages pathogenic agents, which share immunological features with the immunogen.“Immunogen” refers to any substance introduced into the body in order to generate an immune response. That substance can a physical molecule, such as a protein, or can be encoded by a vector, such as DNA, mRNA, or a virus.

[0070] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an adaptive immune response. This immune response may involve either antibody production, or the activation of specific immunogenically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. AAttorney Docket No.206664-0001-00WOskilled artisan will understand that any DNA or RNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an adaptive immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0071] “Immune response,” as the term is used herein, means a process involving the activation and / or induction of an effector function in, by way of non-limiting examples, a T cell, B cell, natural killer (NK) cell, and / or an antigen-presenting cell (APC). Thus, an immune response, as would be understood by the skilled artisan, includes, but is not limited to, any detectable antigen-specific activation and / or induction of a helper T cell or cytotoxic T cell activity or response, production of antibodies, antigen presenting cell activity or infiltration, macrophage activity or infiltration, neutrophil activity or infiltration, and the like.

[0072] As used herein, an “immunogenic composition” may comprise an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding an antigen, a cell expressing or presenting an antigen or cellular component, a virus expressing or presenting an antigen or cellular component, or a combination thereof. In particular embodiments, the composition comprises or encodes all or part of any peptide antigen described herein, or an immunogenically functional equivalent thereof. In other embodiments, the composition is in a mixture that comprises an additional immunostimulatory agent or nucleic acids encoding such an agent. Immunostimulatory agents include but are not limited to an additional antigen, an immunomodulator, an antigen presenting cell, lipid nanoparticle, or an adjuvant. In other embodiments, one or more of the additional agent(s) is covalently bonded to the antigen or an immunostimulatory agent, in any combination.

[0073] As used herein, the term “vaccine” refers to a composition that induces an immune response upon inoculation into a subject. In some embodiments, the induced immune response provides protective immunity.Attorney Docket No.206664-0001-00WO

[0074] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0075] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0076] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0077] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function ofAttorney Docket No.206664-0001-00WOthe number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0078] As used herein, a nucleotide sequence is “substantially homologous” to any of the nucleotide sequences described herein when its nucleotide sequence has a degree of identity with respect to the original nucleotide sequence at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

[0079] As used herein, an amino acid sequence is “substantially homologous” to any of the amino acid sequences described herein when its amino acid sequence has a degree of identity with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%. The identity between two amino acid sequences can be determined by using the BLASTN algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)).

[0080] The term “variant” as used herein with respect to a nucleic acid refers (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto. A variant may be a nucleic acid sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof.

[0081] The term “variant” as used with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, orAttorney Docket No.206664-0001-00WOconservative substitution of amino acids, but retain at least one biological activity. Variant may also refer to a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., 1982, J. Mol. Biol. 157:105-132). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U. S. Patent No. 4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0082] As used herein, the terms “fragment” or “functional fragment” refer to a fragment of a hepatitis B antigen or a nucleic acid sequence encoding a hepatitis B antigen that, when administered to a subject, provides an increased immune response. Fragments are generally 10 or more amino acids or nucleic acids in length. “Fragment” may mean a polypeptide fragment of anAttorney Docket No. 206664-0001-00WOantigen that is capable of eliciting an immune response in a subject. A fragment of an antigen may be 100% identical to the full length except missing at least one amino acid from the N and / or C terminal, in each case with or without signal peptides and / or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length antigen, excluding any heterologous signal peptide added. The fragment may comprise a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antigen and additionally comprise an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity.

[0083] A fragment of a nucleic acid sequence that encodes an antigen may be 100% identical to the full length except missing at least one nucleotide from the 5’ and / or 3’ end, in each case with or without sequences encoding signal peptides and / or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length coding sequence, excluding any heterologous signal peptide added. The fragment may comprise a fragment that encode a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antigen and additionally optionally comprise sequence encoding an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity.

[0084] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living subject is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0085] In the context of the invention, the following abbreviations for the commonly occurring nucleosides (nucleobase bound to ribose or deoxyribose sugar via N-glycosidicAttorney Docket No.206664-0001-00WOlinkage) are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0086] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translated by translational machinery in a cell. Exemplary modified nucleosides are described elsewhere herein. For example, an mRNA where some or all of the uridines have been replaced with pseudouridine, 1 -methyl pseudouridine, 5-methyl-uridine or another modified nucleoside, such as those described elsewhere herein. In some embodiments, the nucleotide sequence may contain a sequence where some or all cytidines are replaced with methylated cytidine, or another modified nucleoside, such as those described elsewhere herein.

[0087] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0088] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.Attorney Docket No.206664-0001-00WO

[0089] In some instances, the polynucleotide or nucleic acid of the invention is a “nucleoside-modified nucleic acid,” which refers to a nucleic acid comprising at least one modified nucleoside. A “modified nucleoside” refers to a nucleoside with a modification. For example, over one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196- 197).

[0090] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0091] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. By way of one non-limiting example, a promoter that is recognized by bacteriophage RNA polymerase and is used to generate the mRNA by in vitro transcription.

[0092] The term “adjuvant” as used herein is defined as any molecule to enhance an antigen-specific adaptive immune response.

[0093] In some embodiments, “pseudouridine” refers to m1acp3vP (l-methyl-3-(3-amino-3 -carboxypropyl) pseudouridine). In another embodiment, the term refers to mlvP (1-methylpseudouridine). In another embodiment, the term refers to m (2’-O-methylpseudouridine. In another embodiment, the term refers to m5D (5-methyldihydrouridine). In another embodiment, the term refers to m3(3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In anotherAttorney Docket No.206664-0001-00WOembodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the invention.

[0094] The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm), which includes one or more lipids.

[0095] The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0096] As used herein, the term “cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In some embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

[0097] The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

[0098] The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH.

[0099] The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid.

[0100] The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and includel-(monom ethoxy-poly ethyl eneglycol)-2, 3 -dimyristoylglycerol (PEG-s- DMG) and the like.

[0101] “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueousAttorney Docket No.206664-0001-00WOsolution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0102] The terms “subject,” “patient,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In some non-limiting embodiments, the patient, subject or individual is a mammal, bird, poultry, cattle, pig, horse, sheep, ferret, primate, dog, cat, guinea pig, rabbit, bat, or human.

[0103] A “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate.

[0104] In contrast, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health.

[0105] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, such as a human.

[0106] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0107] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0108] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, prevention, or eradication of at least one sign or symptom of a disease or disorder.Attorney Docket No.206664-0001-00WO

[0109] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0110] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0111] The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0112] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington’s Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0113] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on theAttorney Docket No.206664-0001-00WOscope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.Description

[0114] In various embodiments, the invention relates to compositions and methods for inducing an immune response against hepatitis B. In some embodiments, the invention is a composition comprising an optimized RNA molecule encoding a hepatitis B antigen. In some embodiments, the invention is a composition comprising LNP encapsulating an optimized RNA molecule encoding a hepatitis B antigen.

[0115] In some embodiments, the composition comprises an optimized mRNA molecule encoding a HBsAg, HBeAg, HBcAg or polymerase antigen. In some embodiments, the composition comprises an optimized RNA molecule encoding a HBsAg, HBeAg, HBcAg or polymerase antigen operably linked to a signal peptide. In one embodiment, the signal peptide comprises SEQ ID NO: 15. In some embodiments, the optimized RNA molecule comprises at least one modified nucleoside.

[0116] For example, in one embodiment, the composition is a vaccine comprising an optimized RNA molecule corresponding to, or encoded by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 14 or a variant thereof, wherein the vaccine induces an immune response in the subject to at least one hepatitis B antigen. In some embodiments, the variant of SEQ ID NO: 2 comprises a sequence having at least 80% identity to SEQ ID NO: 2, wherein the variant encodes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant of SEQ ID NO: 4 comprises a sequence having at least 80% identity to SEQ ID NO: 4, wherein the variant encodes the amino acid sequence of SEQ ID NO: 3. In some embodiments, the variant of SEQ ID NO: 6 comprises a sequence having at least 80% identity to SEQ ID NO: 6, wherein the variant encodes the amino acid sequence of SEQ ID NO: 5. In some embodiments, the variant of SEQ ID NO: 8 comprises a sequence having at least 80% identity to SEQ ID NO: 8, wherein the variant encodes the aminoAttorney Docket No.206664-0001-00WOacid sequence of SEQ ID NO: 7. In some embodiments, the variant of SEQ ID NO: 10 comprises a sequence having at least 80% identity to SEQ ID NO: 10, wherein the variant encodes the amino acid sequence of SEQ ID NO: 9. In some embodiments, the variant of SEQ ID NO: 11 comprises a sequence having at least 80% identity to SEQ ID NO: 11, wherein the variant encodes the amino acid sequence of SEQ ID NO: 9. In some embodiments, the variant of SEQ ID NO: 13 comprises a sequence having at least 80% identity to SEQ ID NO: 13, wherein the variant encodes the amino acid sequence of SEQ ID NO: 12. In some embodiments, the variant of SEQ ID NO: 14 comprises a sequence having at least 80% identity to SEQ ID NO: 14, wherein the variant encodes the amino acid sequence of SEQ ID NO: 12.Vaccine

[0117] In one embodiment, the invention provides an immunogenic composition for inducing an immune response against hepatitis B in a subject. In some embodiments, the composition comprises an optimized mRNA molecule encoding aHBsAg, HBeAg, HBcAg or polymerase antigen. In some embodiments, composition comprises an optimized nucleoside modified RNA molecule encoding a HBsAg, HBeAg, HBcAg or polymerase antigen. In one embodiment, the composition comprises a LNP comprising an optimized mRNA molecule encoding a HBsAg, HBeAg, HBcAg or polymerase antigen operably linked to a signal peptide. In some embodiments, the signal peptide comprises a MHC 1 signal peptide. In some embodiments, the signal peptide comprises SEQ ID NO: 15.

[0118] In some embodiments, the composition induces a cellular immune response against hepatitis B in a cell, tissue or subject. In some embodiments, the composition induces a cellular immune response against hepatitis B in a cell, tissue or subject. In some embodiments, the vaccine induces a humoral immune response in the subject. In some embodiments, the vaccine induces both cellular and humoral immune responses in the subject.

[0119] In various embodiments, the immunogenic composition of the invention may vary in its composition of nucleic acid and / or cellular components. In one embodiment, the immunogenic composition comprises at least one lipid nanoparticle comprising an optimized RNA molecule encoding a hepatitis B antigen. In one embodiment, the immunogenic composition comprises at least one lipid nanoparticle comprising an optimized RNA molecule encoding a hepatitis B antigen operably linked to a MHC 1 signal peptide (SP). In oneAttorney Docket No. 206664-0001-00WOembodiment, the signal peptide comprises SEQ ID NO: 15. In some embodiments, the optimized RNA molecule is a nucleoside-modified mRNA molecule.

[0120] In various embodiments, the vaccine of the invention may vary in its composition of nucleic acid and / or cellular components.

[0121] In various embodiments, the immunogenic composition of the invention comprises at least one lipid nanoparticle comprising an RNA molecule corresponding to, or encoded by, a DNA sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 14, or a variant thereof. In various embodiments, the immunogenic composition of the invention comprises at least one lipid nanoparticle comprising a nucleoside-modified RNA molecule corresponding to, or encoded by, a DNA sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 14, or a variant thereof. In some embodiments, the variant of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 14 comprises a sequence having at least 80% identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 14, wherein the variant encodes the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 12.

[0122] In a non-limiting example, one or more nucleic acid molecules encoding an immunogen might also be formulated with an adjuvant. In another non-limiting example, the vaccine may comprise one or more adjuvants. In various embodiments, a vaccine of the invention, and its various components, may be prepared and / or administered by any method disclosed herein or as would be known to one of ordinary skill in the art, in light of the present disclosure.

[0123] In various embodiments, the induction of immunity by the expression of the hepatitis B antigen can be detected by observing in vivo or in vitro the response of all or any part of the immune system in the host against a native hepatitis B antigen.

[0124] For example, a method for detecting the induction of cytotoxic T lymphocytes is well known. A foreign substance that enters the living body is presented to T cells and B cells by the action of antigen presenting cells (APCs). Some T cells that respond to the antigen presented by APC in an antigen specific manner differentiate into cytotoxic T cells (also referred to asAttorney Docket No.206664-0001-00WOcytotoxic T lymphocytes or CTLs) due to stimulation by the antigen. These antigen-stimulated cells then proliferate. This process is referred to herein as “activation” of T cells. Therefore, CTL induction by an epitope of a polypeptide or peptide or combinations thereof can be evaluated by presenting an epitope of a polypeptide or peptide or combinations thereof to a T cell by APC, and detecting the induction of CTL. Furthermore, APCs have the effect of activating B cells, CD4+ T cells, CD8+ T cells, macrophages, eosinophils and NK cells.

[0125] A method for evaluating the inducing action of CTL using dendritic cells (DCs) as APC is well known in the art. DC is a representative APC having a robust CTL inducing action among APCs. In the methods of the invention, the epitope of a polypeptide or peptide or combinations thereof is initially expressed by the DC and then this DC is contacted with T cells. Detection of T cells having cytotoxic effects against the cells of interest after the contact with DC shows that the epitope of a polypeptide or peptide or combinations thereof has an activity of inducing the cytotoxic T cells. Furthermore, the induced immune response can also be examined by measuring IFN-gamma produced and released by CTL in the presence of antigen-presenting cells that carry immobilized peptide or a combination of peptides by visualizing using anti-IFN-gamma antibodies, such as an ELISPOT assay.

[0126] Apart from DC, peripheral blood mononuclear cells (PBMCs) may also be used as the APC. The induction of CTL is reported to be enhanced by culturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL has been shown to be induced by culturing PBMC in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

[0127] The antigens confirmed to possess CTL -inducing activity by these methods are antigens having DC activation effect and subsequent CTL-inducing activity. Furthermore, CTLs that have acquired cytotoxicity due to presentation of the antigen by APC can be also used as vaccines against antigen-associated disorders.

[0128] The induction of immunity by expression of the hepatitis B antigen can be further confirmed by observing the induction of antibody production against the hepatitis B antigen. For example, when antibodies against an antigen are induced in a laboratory subject immunized with the composition encoding the antigens, and when antigen-associated pathology is suppressed by those antibodies, the composition is determined to induce immunity.

[0129] The specificity of the antibody response induced in a subject can include binding to many regions of the delivered antigen, as well as, the induction of neutralization capableAttorney Docket No.206664-0001-00WOantibodies that that prevent infection or reduce disease severity.

[0130] The induction of immunity by expression of the hepatitis B antigen can be further confirmed by observing the induction of T cells, such as CD4+ T cells, CD8+ T cells, or a combination thereof. For example, CD4+ T cells can also lyse target cells, but mainly supply help in the induction of other types of immune responses, including CTL and antibody generation. The type of CD4+ T cell help can be characterized, as Thl, Th2, Th9, Th 17, Tregulatory (Treg), or T follicular helper (Tfh) cells. Each subtype of CD4+ T cell supplies help to certain types of immune responses. In one embodiment, the composition selectively induces T follicular helper cells, which drive potent antibody responses.

[0131] The therapeutic compounds or compositions of the invention may be administered prophylactically (i.e., to prevent a disease or disorder) or therapeutically (i.e., to treat a disease or disorder) to subjects suffering from, or at risk of (or susceptible to) developing a disease or disorder. Such subjects may be identified using standard clinical methods. In the context of the invention, prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or alternatively delayed in its progression. In the context of the field of medicine, the term “prevent” encompasses any activity, which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications.Antigen

[0132] In some embodiments, the invention a composition that induces an immune response in a subject.

[0133] In various embodiments, the immunogenic composition of the invention comprises at least one nucleic acid molecule encoding a hepatitis B antigen. In some embodiments, the hepatitis B antigen comprises an amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO: 12. In some embodiments, the hepatitis B antigen is operably linked to a signal peptide.Attorney Docket No.206664-0001-00WO

[0134] In one embodiment, the optimized nucleic acid molecule encoding the hepatitis B antigen comprises a sequence encoding a tag or signal peptide (SP). Other signal peptides that may be used include, but are not limited to, signal sequences derived from IL-2, tPA, mouse and human IgG, and synthetic optimized signal sequences. In some instances, the nucleic acid sequence comprises include additional sequences that encode linker or tag sequences that are linked to the antigen by a peptide bond.

[0135] In some embodiments, the optimized nucleotide sequence encoding the hepatitis B antigen comprises a nucleotide sequence that is substantially homologous, or substantially identical, to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 14, and retains the immunogenic function of the hepatitis B antigen. Therefore in some embodiments, the nucleotide sequence of the optimized nucleic acid molecule encoding the hepatitis B antigen has a degree of identity with respect to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 14 of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%. In some embodiments, the variant of SEQ ID NO: 2 comprises a sequence having at least 80% identity to SEQ ID NO: 2, wherein the variant encodes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant of SEQ ID NO: 4 comprises a sequence having at least 80% identity to SEQ ID NO: 4, wherein the variant encodes the amino acid sequence of SEQ ID NO: 3. In some embodiments, the variant of SEQ ID NO: 6 comprises a sequence having at least 80% identity to SEQ ID NO: 6, wherein the variant encodes the amino acid sequence of SEQ ID NO: 5. In some embodiments, the variant of SEQ ID NO: 8 comprises a sequence having at least 80% identity to SEQ ID NO: 8, wherein the variant encodes the amino acid sequence of SEQ ID NO: 7. In some embodiments, the variant of SEQ ID NO: 10 comprises a sequence having at least 80% identity to SEQ ID NO: 10, wherein the variant encodes the amino acid sequence of SEQ ID NO: 9. In some embodiments, the variant of SEQ ID NO: 11 comprises a sequence having at least 80% identity to SEQ ID NO: 11, wherein the variant encodes the amino acid sequence of SEQ ID NO: 9. In some embodiments, the variant of SEQ ID NO: 13 comprises a sequence having at least 80% identity to SEQ ID NO: 13, wherein the variant encodes the amino acid sequence of SEQ ID NO: 12. In some embodiments, the variantAttorney Docket No. 206664-0001-00WOof SEQ ID NO: 14 comprises a sequence having at least 80% identity to SEQ ID NO: 14, wherein the variant encodes the amino acid sequence of SEQ ID NO: 12.

[0136] In some embodiments the vaccine comprises a combination of SEQ ID NO: 2 and SEQ ID NO: 4, or variants thereof. In some embodiments, the variant of SEQ ID NO: 2 comprises at least 80% identity to SEQ ID NO: 2. In some embodiments, the variant of SEQ ID NO: 2 encodes SEQ ID NO: 1. In some embodiments, the variant of SEQ ID NO: 4 comprises at least 80% identity to SEQ ID NO: 4. In some embodiments, the variant of SEQ ID NO: 4 encodes SEQ ID NO: 3.

[0137] In some embodiments the vaccine comprises a combination of at least two of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 11 and SEQ ID NO: 14 or variants thereof. In some embodiments, the variant of SEQ ID NO: 2 comprises at least 80% identity to SEQ ID NO: 2. In some embodiments, the variant of SEQ ID NO: 2 encodes SEQ ID NO: 1. In some embodiments, the variant of SEQ ID NO: 4 comprises at least 80% identity to SEQ ID NO: 4. In some embodiments, the variant of SEQ ID NO: 4 encodes SEQ ID NO: 3. In some embodiments, the variant of SEQ ID NO: 11 comprises at least 80% identity to SEQ ID NO: 11. In some embodiments, the variant of SEQ ID NO: 11 encodes SEQ ID NO: 9. In some embodiments, the variant of SEQ ID NO: 14 comprises at least 80% identity to SEQ ID NO: 14. In some embodiments, the variant of SEQ ID NO: 14 encodes SEQ ID NO: 12.Adjuvant

[0138] In one embodiment, the composition comprises an adjuvant. In one embodiment, the composition comprises a nucleic acid molecule encoding an adjuvant. In one embodiment, the adjuvant-encoding nucleic acid molecule is IVT RNA. In one embodiment, the adjuvantencoding nucleic acid molecule is an optimized RNA molecule. In one embodiment, the adjuvant-encoding nucleic acid molecule is a nucleoside-modified mRNA molecule.

[0139] Exemplary adjuvants include, but are not limited to, Flt3-L, alpha-interferon, beta-interferon, gamma-interferon, platelet derived growth factor (PDGF), TN Fa, TNFp, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86. Other genes which may be useful adjuvants include those encoding: MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES, L-selectin, P-selectin, E-selectin,Attorney Docket No. 206664-0001-00WOCD34, GlyCAM-1, MadCAM-1, LFA-T, VLA-I, Mac-1, pl50.95, PECAM, TCAM-T, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-I, Ap-I, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP 1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig, anti-TIM3-Ig, and functional fragments thereof.

[0140] In some embodiments, the composition comprises an LNP, where the LNP acts as an adjuvant.

[0141] In some embodiments, the adjuvant comprises IFN-P (SEQ ID NO: 17) or a nucleic acid molecule encoding IFN-p.

[0142] In some embodiments, the adjuvant comprises Flt3-L or a nucleic acid molecule encoding Flt3-L.Nucleic Acids

[0143] In one embodiment, the invention includes an optimized nucleic acid molecule encoding a hepatitis B antigen. The nucleic acid molecule can be made using any methodology in the art, including, but not limited to, in vitro transcription, chemical synthesis, or the like.

[0144] In various embodiments, the optimized nucleotide sequences encoding the hepatitis B antigen can alternatively comprise sequence variations with respect to the original nucleotide sequences, for example, substitutions, insertions and / or deletions of one or more nucleotides, with the condition that the resulting polynucleotide encodes a polypeptide according to the invention. Therefore, the scope of the invention includes nucleotide sequences that are substantially homologous or substantially identical to the optimized nucleotide sequences recited herein and encode a hepatitis B antigen.

[0145] A nucleotide sequence that is substantially homologous to a nucleotide sequence encoding an antigen can typically be isolated from a producer organism of the antigen based onAttorney Docket No.206664-0001-00WOthe information contained in the nucleotide sequence by means of introducing conservative or non-conservative substitutions, for example. Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides in any of the ends of the sequence, or the deletion of one or more nucleotides in any end or inside the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.

[0146] Further, the scope of the invention includes nucleotide sequences that encode amino acid sequences that are substantially homologous to the amino acid sequences recited herein and preserve the immunogenic function of the original amino acid sequence.

[0147] In one embodiment, the invention relates to a construct comprising a nucleotide sequence encoding a hepatitis B antigen. In one embodiment, the construct comprises a plurality of nucleotide sequences encoding a plurality of hepatitis B antigens.

[0148] In one embodiment, the construct comprises a nucleotide sequence encoding a hepatitis surface (HBsAg) antigen. In one embodiment, the construct comprises an optimized nucleotide sequence encoding SEQ ID NO: 1. In one embodiment, the optimized nucleotide sequence encoding SEQ ID NO: 1 comprises SEQ ID NO: 2.

[0149] In one embodiment, the construct comprises a nucleotide sequence encoding a hepatitis HBsAg antigen operably linked to an MHC 1 signal peptide (SEQ ID NO: 15). In one embodiment, the construct comprises an optimized nucleotide sequence encoding SEQ ID NO: 3. In one embodiment, the optimized nucleotide sequence encoding SEQ ID NO: 3 comprises SEQ ID NO: 4.

[0150] In one embodiment, the construct comprises a nucleotide sequence encoding a hepatitis HBsAg antigen operably linked to a MITD. In one embodiment, the construct comprises an optimized nucleotide sequence encoding SEQ ID NO: 5. In one embodiment, the optimized nucleotide sequence encoding SEQ ID NO: 5 comprises SEQ ID NO: 6.

[0151] In one embodiment, the construct comprises a nucleotide sequence encoding a hepatitis HBsAg antigen operably linked to a MITD and a MHC 1 signal peptide (SEQ ID NO: 15). In one embodiment, the construct comprises an optimized nucleotide sequence encoding SEQ ID NO: 7. In one embodiment, the optimized nucleotide sequence encoding SEQ ID NO: 7 comprises SEQ ID NO: 8.

[0152] In one embodiment, the construct comprises a nucleotide sequence encoding aAttorney Docket No.206664-0001-00WOhepatitis HBcAg antigen. In one embodiment, the construct comprises an optimized nucleotide sequence encoding SEQ ID NO: 9. In one embodiment, the optimized nucleotide sequence encoding SEQ ID NO: 9 comprises SEQ ID NO: 11.

[0153] In one embodiment, the construct comprises a nucleotide sequence encoding a hepatitis HBcAg antigen operably linked to an MHC 1 signal peptide (SEQ ID NO: 15). In one embodiment, the construct comprises an optimized nucleotide sequence encoding SEQ ID NO: 12. In one embodiment, the optimized nucleotide sequence encoding SEQ ID NO: 12 comprises SEQ ID NO: 14.

[0154] In some embodiments, the construct is operatively bound to a translational control element. In some embodiments, the construct can incorporate an operatively bound regulatory sequence for the expression of the nucleotide sequence of the invention, thus forming an expression cassette.

[0155] In some embodiments, the nucleic acid molecule is an mRNA molecule, a self-amplifying RNA (saRNA) molecule or a circular RNA (circRNA) molecule.Vectors

[0156] The nucleic acid molecules can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, a PCR-generated linear DNA sequence, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors and vectors optimized for in vitro transcription.

[0157] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, carbohydrates, peptides, cationic polymers, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0158] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to aAttorney Docket No. 206664-0001-00WOliposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / RNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources.

[0159] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to a composition of the invention, in order to confirm the presence of the mRNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Northern blotting and RT-PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunogenic means (ELIS As and Western blots) or by assays described herein to identify agents falling within the scope of the invention.In vitro transcribed RNA

[0160] In one embodiment, the composition of the invention comprises an in vitro transcribed (IVT) RNA molecule encoding the hepatitis B antigen of the invention. In one embodiment, an IVT RNA can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a plasmid DNA template generated synthetically. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. In one embodiment, the desired template for in vitro transcription is an optimized DNA sequence encoding a hepatitis B antigen capable of inducing an adaptive immune response. In one embodiment, the desired template for in vitroAttorney Docket No.206664-0001-00WOtranscription is an adjuvant capable of enhancing an adaptive immune response.

[0161] In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the DNA is a full-length gene of interest of a portion of a gene. The gene can include some or all of the 5’ and / or 3’ untranslated regions (UTRs). The gene can include exons and introns. In one embodiment, the DNA to be used for PCR is a human gene. In another embodiment, the DNA to be used for PCR is a human gene including the 5’ and 3’ UTRs. In another embodiment, the DNA to be used for PCR is a gene from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi. In another embodiment, the DNA to be used for PCR is from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi, including the 5’ and 3’ UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

[0162] Genes that can be used as sources of DNA for PCR include genes that encode polypeptides that induce or enhance an adaptive immune response in an organism. In some instances, the genes are useful for a short term treatment. In some instances, the genes have limited safety concerns regarding dosage of the expressed gene.

[0163] In various embodiments, a plasmid is used to generate a template for in vitro transcription of mRNA, which is used for transfection.

[0164] Chemical structures with the ability to promote stability and / or translation efficiency may also be used. In some embodiments, the RNA has 5’ and 3’ UTRs. In one embodiment, the 5’ UTR is between zero and 3000 nucleotides in length. The length of 5’ and 3’ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0165] The 5’ and 3’ UTRs can be the naturally occurring, endogenous 5’ and 3’ UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primersAttorney Docket No.206664-0001-00WOor by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3’ UTR sequences can decrease the stability of mRNA. Therefore, 3’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[0166] In one embodiment, the 5’ UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5’ UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5’ UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments the 5’ UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.

[0167] To enable synthesis of RNA from a DNA template, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5’ end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one embodiment, the promoter is a T7 RNA polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0168] In one embodiment, the mRNA has both a cap on the 5’ end and a 3’ poly(A) tail which determine ribosome binding, initiation of translation and stability of mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatemeric product, which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3’ UTR results in normal sized mRNA, which is effective in eukaryotic transfection when it is polyadenylated after transcription.

[0169] On a linear DNA template, phage T7 RNA polymerase can extend the 3’ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).Attorney Docket No. 206664-0001-00WO

[0170] The conventional method of integration of polyA / T stretches into a DNA template is molecular cloning. However, polyA / T sequence integrated into plasmid DNA can cause plasmid instability, which can be ameliorated through the use of recombination incompetent bacterial cells for plasmid propagation.

[0171] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP) or yeast polyA polymerase. In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3’ end can increase mRNA stability. Such attachment can contain modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[0172] 5’ caps also provide stability to mRNA molecules. In one embodiment, RNAs produced by the methods to include a 5’ capl structure. Such capl structure can be generated using Vaccinia capping enzyme and 2’-O-methyltransferase enzymes (CellScript, Madison, WI). Alternatively, 5’ cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0173] RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)). In some embodiments RNA of the invention is introduced to a cell with a method comprising the use of TransIT®-mRNA transfection Kit (Minis, Madison WI), which, in some instances, provides high efficiency, low toxicity, transfection.Nucleoside-modified RNA

[0174] In one embodiment, the composition of the invention comprises a nucleoside-Attorney Docket No.206664-0001-00WOmodified nucleic acid encoding a hepatitis B antigen as described herein. In one embodiment, the composition of the invention comprises a nucleoside-modified nucleic acid encoding an adjuvant as described herein. In one embodiment, the composition of the invention comprises a nucleoside-modified nucleic acid encoding one or more hepatitis B antigens and one or more adjuvants. In some embodiments, the adjuvant comprises IFN-P (SEQ ID NO: 17).

[0175] For example, in one embodiment, the composition comprises a nucleoside-modified RNA. In one embodiment, the composition comprises a nucleoside-modified mRNA. Nucleoside-modified mRNA have particular advantages over non-modified mRNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside-modified mRNA useful in the invention is further described in U. S. Patent Nos. 8,278,036, 8,691,966, and 8,835,108, each of which is incorporated by reference herein in its entirety.

[0176] In some embodiments, nucleoside-modified mRNA does not activate any pathophysiologic pathways, translates very efficiently and almost immediately following delivery, and serve as templates for continuous protein production in vivo lasting for several days to weeks (Kariko et al., 2008, Mol Ther 16:1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). The amount of mRNA required to exert a physiological effect is small, making it applicable for human therapy. For example, in some embodiments, the nucleoside-modified mRNA encoding a hepatitis B antigen has the ability to induce antigen-specific antibody production. For example, in some instances administration of the optimized nucleoside-modified mRNA induces greater production of antigen-specific antibody production as compared to antigen encoded by non-modified mRNA.

[0177] In some instances, expressing a protein by delivering the encoding mRNA has many benefits over methods that use protein, plasmid DNA or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to cells, thus avoiding all the side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery. For example, high levels of circulating proteins have been measured within 15 to 30 minutes of in vivo injection of the encoding mRNA. In some embodiments, using mRNA rather than the protein also has many advantages. Half-lives of proteins in the circulation or inAttorney Docket No.206664-0001-00WOtissues are often short, thus protein treatment would need frequent dosing, while mRNA provides a template for continuous protein production for several days to weeks. Purification of proteins is problematic and they can contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0178] In some embodiments, the nucleoside-modified RNA comprises the naturally occurring modified-nucleoside pseudouridine. In some embodiments, inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Kariko et al., 2008, Mol Ther 16:1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:el42; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23:165-175).

[0179] It has been demonstrated that the presence of modified nucleosides, including pseudouridines in RNA suppress their innate immunogenicity (Kariko et al., 2005, Immunity 23:165-175). Further, protein-encoding, in vitro-transcribed RNA containing pseudouridine can be translated more efficiently than RNA containing no or other modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). Subsequently, it is shown that the presence of pseudouridine improves the stability of RNA (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and abates both activation of PKR and inhibition of translation (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892).

[0180] Similar effects as described for pseudouridine have also been observed for RNA containing 1 -methyl -pseudouridine.

[0181] In some embodiments, the nucleoside-modified nucleic acid molecule is a purified nucleoside-modified nucleic acid molecule. For example, in some embodiments, the composition is purified to remove double-stranded contaminants. In some instances, a preparative high-performance liquid chromatography (HPLC) purification procedure is used to obtain pseudouridine-containing RNA that has superior translational potential and no innate immunogenicity (Kariko et al., 2011, Nucleic Acids Research 39:el42). Administering HPLC-purified, pseudouridine-containing RNA coding for erythropoietin into mice and macaques resulted in a significant increase of serum EPO levels (Kariko et al., 2012, Mol Ther 20:948-953), thus confirming that pseudouridine-containing mRNA is suitable for in vivo protein therapy. In some embodiments, the nucleoside-modified nucleic acid molecule is purified using non-HPLC methods. In some instances, the nucleoside-modified nucleic acid molecule isAttorney Docket No.206664-0001-00WOpurified using chromatography methods, including but not limited to HPLC and fast protein liquid chromatography (FPLC). An exemplary FPLC -based purification procedure is described in Weissman et al., 2013, Methods Mol Biol, 969: 43-54. Exemplary purification procedures are also described in U. S. Patent Application Publication No. US2016 / 0032316, which is hereby incorporated by reference in its entirety.

[0182] The invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside. In some embodiments, the composition comprises an isolated nucleic acid encoding an antigen, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside. In some embodiments, the composition comprises a vector, comprising an isolated nucleic acid encoding an antigen, adjuvant, or combination thereof, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside.

[0183] In one embodiment, the nucleoside-modified RNA of the invention is IVT RNA, as described elsewhere herein. For example, in some embodiments, the nucleoside-modified RNA is synthesized by T7 phage RNA polymerase. In another embodiment, the nucleoside-modified mRNA is synthesized by SP6 phage RNA polymerase. In another embodiment, the nucleoside-modified RNA is synthesized by T3 phage RNA polymerase.

[0184] In one embodiment, the modified nucleoside is m1acp3'P (l-methyl-3-(3-amino-3-carboxypropyl) pseudouridine. In another embodiment, the modified nucleoside is m1(P (1-methylpseudouridine). In another embodiment, the modified nucleoside is m (2’-O-methylpseudouridine). In another embodiment, the modified nucleoside is m5D (5-methyldihydrouridine). In another embodiment, the modified nucleoside is m3,P (3-methylpseudouridine). In another embodiment, the modified nucleoside is a pseudouridine moiety that is not further modified. In another embodiment, the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0185] In another embodiment, the nucleoside that is modified in the nucleoside-modified RNA the invention is uridine (U). In another embodiment, the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenosine (A). In another embodiment, the modified nucleoside is guanosine (G).Attorney Docket No. 206664-0001-00WO

[0186] In another embodiment, the modified nucleoside of the invention is nfC (5-methylcytidine). In another embodiment, the modified nucleoside is m5U (5-methyluridine). In another embodiment, the modified nucleoside is m6A (N6-methyladenosine). In another embodiment, the modified nucleoside is s2U (2 -thiouridine). In another embodiment, the modified nucleoside is T (pseudouridine). In another embodiment, the modified nucleoside is Um (2’-O-methyluridine).

[0187] In other embodiments, the modified nucleoside is m'A (1 -methyladenosine); m2A (2-m ethyladenosine); Am (2’-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2’-O-ribosyladenosine (phosphate)); I (inosine); m1! (1 -methylinosine); nflrn (l,2’-O-dimethylinosine); m3C (3-methylcytidine); Cm (2’-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f’C (5-formylcytidine); nfCm (5,2’-O-dimethylcytidine); ac4Cm (N4-acetyl-2’-O-methylcytidine); k2C (lysidine); nfG (1 -methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2’-O-methylguanosine); m22G (N2, N2-dimethylguanosine); m2Gm (N2,2’-O-dimethylguanosine); m22Gm (N2, N2,2’-O-trimethylguanosine); Gr(p) (2’-O-ribosylguanosine (phosphate)); yW (wybutosine); O2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G+(archaeosine); D (dihydrouridine); m’Um (5,2’ -O-dimethyluri dine); s4U (4-thiouridine); m3s2U (5-methyl-2-thiouridine); s2Um (2-thio-2’-O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxy carbonylmethyluridine); mcm5Um (5-methoxycarbonylmethyl-2’-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-Attorney Docket No.206664-0001-00WOthiouridine); mnm5U (5-methylaminomethyluridine); mnnris2!! (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncnriUm (5-carbamoylmethyl-2’-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cmnnfUm (5-carboxymethylaminomethyl-2’-O-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m A (N6, N6-dimethyladenosine); Im (2’-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4, 2’-0-dimethylcytidine); hnfC (5-hydroxymethylcytidine); m3U (3 -methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,2’-O-dimethyladenosine); m Am (N6, N6, O-2’-trimethyladenosine); m27G (N2,7-dimethylguanosine); m2,2’7G (N2, N2,7-trimethylguanosine); m3Um (3,2’ -O-dimethyluri dine); m5D (5-methyldihydrouridine); fCm (5-formyl-2’-O-methylcytidine); m'Gm (l,2’-O-dimethylguanosine); m'Am (l,2’-O-dimethyladenosine); Tm?U (5-taurinomethyluridine); imVU (5-taurinomethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); or ac6A (N6-acetyladenosine).

[0188] In another embodiment, a nucleoside-modified RNA of the invention comprises a combination of 2 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of 3 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of more than 3 of the above modifications.

[0189] In various embodiments, between 0.1% and 100% of the residues in the nucleoside-modified RNA of the invention are modified (e.g., either by the presence of pseudouridine, 1-methyl-pseudouridine, 5-methyl-uridine or another modified nucleoside base). In one embodiment, the fraction of modified residues is 0.1%. In another embodiment, the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.7%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 0.9%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 7%. In another embodiment, the fraction is 8%. In anotherAttorney Docket No.206664-0001-00WOembodiment, the fraction is 9%. In another embodiment, the fraction is 10%. Tn another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 55%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 65%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 75%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 85%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 91%. In another embodiment, the fraction is 92%. In another embodiment, the fraction is 93%. In another embodiment, the fraction is 94%. In another embodiment, the fraction is 95%. In another embodiment, the fraction is 96%. In another embodiment, the fraction is 97%. In another embodiment, the fraction is 98%. In another embodiment, the fraction is 99%. In another embodiment, the fraction is 100%.

[0190] In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0191] In another embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In another embodiment, the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.7%. In another embodiment, theAttorney Docket No.206664-0001-00WOfraction is 0.8%. Tn another embodiment, the fraction is 0.9%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 7%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 9%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 55%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 65%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 75%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 85%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 91%. In another embodiment, the fraction is 92%. In another embodiment, the fraction is 93%. In another embodiment, the fraction is 94%. In another embodiment, the fraction is 95%. In another embodiment, the fraction is 96%. In another embodiment, the fraction is 97%. In another embodiment, the fraction is 98%. In another embodiment, the fraction is 99%. In another embodiment, the fraction is 100%. In another embodiment, the fraction of the given nucleotide that is modified is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.Attorney Docket No.206664-0001-00WO

[0192] In some embodiments, the composition comprises a purified preparation of single-stranded RNA. For example, in some embodiments, the purified preparation of singlestranded RNA is substantially free of double stranded RNA (dsRNA). In some embodiments, the purified preparation is at least 90%, or at least 91%, or at least 92%, or at least 93 % or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% single stranded RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0193] In another embodiment, a nucleoside-modified RNA of the invention is translated in the cell more efficiently than an unmodified RNA molecule with the same sequence. In another embodiment, the nucleoside-modified RNA exhibits enhanced ability to be translated by a target cell. In another embodiment, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In another embodiment, translation is enhanced by a 3-fold factor. In another embodiment, translation is enhanced by a 4-fold factor. In another embodiment, translation is enhanced by a 5-fold factor. In another embodiment, translation is enhanced by a 6-fold factor. In another embodiment, translation is enhanced by a 7-fold factor. In another embodiment, translation is enhanced by an 8-fold factor. In another embodiment, translation is enhanced by a 9-fold factor. In another embodiment, translation is enhanced by a 10-fold factor. In another embodiment, translation is enhanced by a 15-fold factor. In another embodiment, translation is enhanced by a 20-fold factor. In another embodiment, translation is enhanced by a 50-fold factor. In another embodiment, translation is enhanced by a 100-fold factor. In another embodiment, translation is enhanced by a 200-fold factor. In another embodiment, translation is enhanced by a 500-fold factor. In another embodiment, translation is enhanced by a 1000-fold factor. In another embodiment, translation is enhanced by a 2000-fold factor. In another embodiment, the factor is 10-1000-fold. In another embodiment, the factor is 10-100-fold. In another embodiment, the factor is 10-200-fold. In another embodiment, the factor is 10-300-fold. In another embodiment, the factor is 10-500-fold. In another embodiment, the factor is 20-1000-fold. In another embodiment, the factor is 30-1000-fold. In another embodiment, the factor is 50-1000-fold. In another embodiment, the factor is 100-1000-fold. In another embodiment, the factor is 200-1000-fold. In another embodiment, translation is enhanced by any other significant amount or range of amounts.

[0194] In another embodiment, the nucleoside-modified antigen-encoding RNA of theAttorney Docket No.206664-0001-00WOinvention induces a significantly more robust adaptive immune response as compared with an unmodified in vitro-synthesized RNA molecule of the same sequence. In another embodiment, the modified RNA molecule induces an adaptive immune response that is 2-fold greater than its unmodified counterpart. In another embodiment, the adaptive immune response is increased by a 3-fold factor. In another embodiment, the adaptive immune response is increased by a 4-fold factor. In another embodiment, the adaptive immune response is increased by a 5-fold factor. In another embodiment, the adaptive immune response is increased by a 6-fold factor. In another embodiment, the adaptive immune response is increased by a 7-fold factor. In another embodiment, the adaptive immune response is increased by an 8-fold factor. In another embodiment, the adaptive immune response is increased by a 9-fold factor. In another embodiment, the adaptive immune response is increased by a 10-fold factor. In another embodiment, the adaptive immune response is increased by a 15-fold factor. In another embodiment, the adaptive immune response is increased by a 20-fold factor. In another embodiment, the adaptive immune response is increased by a 50-fold factor. In another embodiment, the adaptive immune response is increased by a 100-fold factor. In another embodiment, the adaptive immune response is increased by a 200-fold factor. In another embodiment, the adaptive immune response is increased by a 500-fold factor. In another embodiment, the adaptive immune response is increased by a 1000-fold factor. In another embodiment, the adaptive immune response is increased by a 2000-fold factor. In another embodiment, the adaptive immune response is increased by another fold difference.

[0195] In another embodiment, “induces significantly more robust adaptive immune response” refers to a detectable increase in an adaptive immune response. In another embodiment, the term refers to a fold increase in the adaptive immune response (e.g., 1 of the fold increases enumerated above). In another embodiment, the term refers to an increase such that the nucleoside-modified RNA can be administered at a lower dose or frequency than an unmodified RNA molecule while still inducing a similarly effective adaptive immune response. In another embodiment, the increase is such that the nucleoside-modified RNA can be administered using a single dose to induce an effective adaptive immune response.

[0196] In another embodiment, the nucleoside-modified RNA of the invention exhibits significantly less innate immunogenicity than an unmodified in vitro-synthesized RNA molecule of the same sequence. In another embodiment, the modified RNA molecule exhibits an innateAttorney Docket No.206664-0001-00WOimmune response that is 2-fold less than its unmodified counterpart. In another embodiment, innate immunogenicity is reduced by a 3-fold factor. In another embodiment, innate immunogenicity is reduced by a 4-fold factor. In another embodiment, innate immunogenicity is reduced by a 5-fold factor. In another embodiment, innate immunogenicity is reduced by a 6-fold factor. In another embodiment, innate immunogenicity is reduced by a 7-fold factor. In another embodiment, innate immunogenicity is reduced by a 8-fold factor. In another embodiment, innate immunogenicity is reduced by a 9-fold factor. In another embodiment, innate immunogenicity is reduced by a 10-fold factor. In another embodiment, innate immunogenicity is reduced by a 15-fold factor. In another embodiment, innate immunogenicity is reduced by a 20-fold factor. In another embodiment, innate immunogenicity is reduced by a 50-fold factor. In another embodiment, innate immunogenicity is reduced by a 100-fold factor. In another embodiment, innate immunogenicity is reduced by a 200-fold factor. In another embodiment, innate immunogenicity is reduced by a 500-fold factor. In another embodiment, innate immunogenicity is reduced by a 1000-fold factor. In another embodiment, innate immunogenicity is reduced by a 2000-fold factor. In another embodiment, innate immunogenicity is reduced by another fold difference.

[0197] In another embodiment, “exhibits significantly less innate immunogenicity” refers to a detectable decrease in innate immunogenicity. In another embodiment, the term refers to a fold decrease in innate immunogenicity (e g., 1 of the fold decreases enumerated above). In another embodiment, the term refers to a decrease such that an effective amount of the RNA can be administered without triggering a detectable innate immune response. In another embodiment, the term refers to a decrease such that the RNA molecule can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the RNA molecule. In another embodiment, the decrease is such that the RNA molecule can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the RNA molecule.

[0198] In some embodiments, the RNA molecule is an mRNA molecule, aself-amplifying RNA (saRNA) molecule or a circular RNA (circRNA) molecule.Lipid Nanoparticle

[0199] In one embodiment, delivery of an optimized RNA molecule comprises anyAttorney Docket No.206664-0001-00WOsuitable delivery method, including exemplary RNA transfection methods described elsewhere herein. In some embodiments, delivery of an optimized RNA molecule to a subject comprises mixing the optimized RNA molecule with a transfection reagent prior to the step of contacting. In another embodiment, a method of invention further comprises administering an optimized RNA molecule together with the transfection reagent. In another embodiment, the transfection reagent is a cationic lipid reagent. In another embodiment, the transfection reagent is a cationic polymer reagent.

[0200] In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a carbohydrate-based transfection reagent. In another embodiment, the transfection reagent is a cationic lipid-based transfection reagent. In another embodiment, the transfection reagent is a cationic polymer-based transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0201] In another embodiment, the transfection reagent forms a liposome. Liposomes, in another embodiment, increase intracellular stability, increase uptake efficiency and improve biological activity. In another embodiment, liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids, which make up the cell membrane. They have, in another embodiment, an internal aqueous space for entrapping water-soluble compounds and range in size from 0.05 to several microns in diameter. In another embodiment, liposomes can deliver RNA to cells in a biologically active form.

[0202] In some embodiments, the lipid nanoparticle is a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm). In some embodiments, the lipid nanoparticle comprises one or more lipids. For example, in some embodiments, the lipid comprises a lipid of Formula (I), (II) or (III).

[0203] In some embodiments, lipid nanoparticles are included in a formulation comprising an RNA molecule as described herein. In some embodiments, such lipid nanoparticles comprise a cationic lipid (e.g., a lipid of Formula (I), (II) or (III)) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipidsAttorney Docket No.206664-0001-00WO(e g., a pegylated lipid such as a pegylated lipid of structure (IV). In some embodiments, the RNA molecule is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.

[0204] In some embodiments, the RNA molecule is a nucleoside-modified RNA as described herein. In some embodiments, the nucleoside-modified RNA is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.

[0205] In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In some embodiments, the RNA molecule is a nucleoside-modified RNA molecule which, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease.

[0206] The LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.

[0207] In one embodiment, the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

[0208] In one embodiment, the LNP comprises a cationic lipid. In some embodiments, the cationic lipid comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N, N-dioleyL N, N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA); N, N-distearyl-N, N-dimethylammonium bromideAttorney Docket No.206664-0001-00WO(DDAB); N-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP); 3-(N — (N ', N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N, N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxy spermine (DOGS), l,2-dioleoyl-3 -dimethylammonium propane (DODAP), N, N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(l,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N-hydroxy ethyl ammonium bromide (DMRIE).Additionally, a number of commercial preparations of cationic lipids are available which can be used in the invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and l,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N. Y ); LIPOFECT AMINE® (commercially available cationic liposomes comprising N-(l -(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N, N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, l,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), N, N-dimethyl-2,3-bi s(((9Z, 12Z, 15Z)-octadeca-9, 12,15 -trien- 1 -y l)oxy)propan- 1 -amine (DLenDMA).

[0209] In one embodiment, the cationic lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in WO 2012 / 016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2-dilinol ey oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoley oxy-3 -morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA. Cl), l,2-dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin-TAP. Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N, N-dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N, N-dioleylamino)-l,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2-N, N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA).

[0210] Suitable amino lipids include those having the formula:Attorney Docket No.206664-0001-00WOwherein Ri and R2 are either the same or different and independently optionally substituted C10-C24 alkyl, optionally substituted C10-C24 alkenyl, optionally substituted C10-C24 alkynyl, or optionally substituted Cio-C24acyl;R3 and R4 are either the same or different and independently optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl or R3 and R4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;Rs is either absent or present and when present is hydrogen or Ci-Ce alkyl; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0;q is 0, 1, 2, 3, or 4; andY and Z are either the same or different and independently O, S, or NH.

[0211] In one embodiment, Ri and R2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid.

[0212] A representative useful dilinoleyl amino lipid has the formula:DLiti-K-DMAwherein n is 0, 1, 2, 3, or 4.

[0213] In one embodiment, the cationic lipid is a DLin-K-DMA. In one embodiment, theAttorney Docket No. 206664-0001-00WOcationic lipid is DLin-KC2-DMA (DLin-K-DMA above, wherein n is 2).

[0214] In one embodiment, the cationic lipid component of the LNPs has the structure of Formula (I):R9(I)or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:L1and L2are each independently -O(C=O)-, -(C=O)O- or a carbon-carbon double bond;Rlaand Rlbare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) Rlais H or C1-C12 alkyl, and Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond;R2aand R2bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R2ais H or C1-C12 alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon-carbon double bond;R3aand R3bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R3ais H or C1-C12 alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R3band the carbon atom to which it is bound to form a carbon-carbon double bond;R4aand R4bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond;R5and R6are each independently methyl or cycloalkyl;R7is, at each occurrence, independently H or C1-C12 alkyl;Attorney Docket No. 206664-0001-00WOR8and R9are each independently C1-C12 alkyl; or R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom;a and d are each independently an integer from 0 to 24;b and c are each independently an integer from 1 to 24; ande is 1 or 2.

[0215] In some embodiments of Formula (I), at least one of Rla, R2a, R3aor R4ais C1-C12 alkyl, or at least one of L1or L2is -O(C=O)- or -(C=O)O-. In other embodiments, Rlaand Rlbare not isopropyl when a is 6 or n-butyl when a is 8.

[0216] In still further embodiments of Formula (I), at least one of Rla, R2a, R3aor R4ais C1-C12 alkyl, or at least one of L1or L2is -O(C=O)- or -(C=O)O-; andRlaand Rlbare not isopropyl when a is 6 or n-butyl when a is 8.

[0217] In other embodiments of Formula (I), R8and R9are each independently unsubstituted C1-C12 alkyl; or R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom.

[0218] In some embodiments of Formula (I), any one of L1or L2may be -O(C=O)- or a carbon-carbon double bond. L1and L2may each be -O(C=O)- or may each be a carbon-carbon double bond.

[0219] In some embodiments of Formula (I), one of L1or L2is -O(C=O)-. In other embodiments, both L1and L2are -O(C=O)-.

[0220] In some embodiments of Formula (I), one of L1or L2is -(C=O)O-. In otherembodiments, both L1and L2are -(C=O)O-

[0221] In some other embodiments of Formula (I), one of L1or L2is a carbon-carbon double bond. In other embodiments, both L1and L2are a carbon-carbon double bond.

[0222] In still other embodiments of Formula (I), one of L1or L2is -O(C=O)- and theother of L1or L2is -(C=O)O- In more embodiments, one of L1or L2is -O(C=O)- and the other of L1or L2is a carbon-carbon double bond. In yet more embodiments, one of L1or L2is -(C=O)O- and the other of L1or L2is a carbon-carbon double bond.Attorney Docket No. 206664-0001-00WO

[0223] It is understood that “carbon-carbon” double bond, as used throughout the specification, refers to one of the following structures:.. obRa, Rb / wherein Raand Rbare, at each occurrence, independently H or a substituent. For example, in some embodiments Raand Rbare, at each occurrence, independently H, C1-C12 alkyl or cycloalkyl, for example H or C1-C12 alkyl.

[0224] In other embodiments, the lipid compounds of Formula (I) have the following structure (la):R1 aR2aR3aR4a(la)

[0225] In other embodiments, the lipid compounds of Formula (I) have the following structure (lb):(lb)

[0226] In yet other embodiments, the lipid compounds of Formula (I) have the following structure (Ic):(Ic)

[0227] In some embodiments of the lipid compound of Formula (I), a, b, c and d are eachAttorney Docket No. 206664-0001-00WOindependently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[0228] In some other embodiments of Formula (I), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[0229] In some more embodiments of Formula (I), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[0230] In some other embodiments of Formula (I), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.

[0231] In some other various embodiments of Formula (I), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

[0232] The sum of a and b and the sum of c and d in Formula (I) are factors which may be varied to obtain a lipid of Formula (I) having the desired properties. In one embodiment, a andAttorney Docket No.206664-0001-00WOb are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such the sum of a and b and the sum of c and d is 12 or greater.

[0233] In some embodiments of Formula (I), e is 1. In other embodiments, e is 2.

[0234] The substituents at Rla, R2a, R3aand R4aof Formula (I) are not particularly limited. In some embodiments Rla, R2a, R3aand R4aare H at each occurrence. In some other embodiments at least one of Rla, R2a, R3aand R4ais C1-C12 alkyl. In some other embodiments at least one of Rla, R2a, R3aand R4ais Ci-Cs alkyl. In some other embodiments at least one of Rla, R2a, R3aand R4ais Ci-Ce alkyl. In some of the foregoing embodiments, the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0235] In some embodiments of Formula (I), Rla, Rlb, R4aand R4bare C1-C12 alkyl at each occurrence.

[0236] In further embodiments of Formula (I), at least one of Rlb, R211, R3band R411is H or Rlb, R2b, R3band R4bare H at each occurrence.

[0237] In some embodiments of Formula (I), Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0238] The substituents at R5and R6of Formula (I) are not particularly limited in the foregoing embodiments. In some embodiments one or both of R5or R6is methyl. In some other embodiments one or both of R5or R6is cycloalkyl for example cyclohexyl. In these embodiments the cycloalkyl may be substituted or not substituted. In some other embodiments the cycloalkyl is substituted with C1-C12 alkyl, for example tert-butyl.

[0239] The substituents at R7are not particularly limited in the foregoing embodiments of Formula (I). In some embodiments at least one R7is H. In some other embodiments, R7is H at each occurrence. In some other embodiments R7is C1-C12 alkyl.

[0240] In some other of the foregoing embodiments of Formula (I), one of R8or R9isAttorney Docket No.206664-0001-00WOmethyl. Tn other embodiments, both R8and R9are methyl.

[0241] In some different embodiments of Formula (I), R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R8and R9, together with the nitrogen atom to which they are \attached, form a 5-membe z —red heterocyclic ring, for example a pyrrolidinyl ring.

[0242] In various different embodiments, the lipid of Formula (I) has one of theZ 7—structures set forth in Table 1 below. TLo oTab e 1: of FormulaPrep. No. Structureo o Method1-1 B Z—'1 \ Z —o 1 1 '1-2 Ao 1 1 1 11-3 A o1 1 1 11-4 BAttorney Docket No.206664-0001-00WOAttorney Docket No.206664-0001-00WOPrep. No. Structure\z— Method \z—Z / —1-11 A z / —BB BBBo o o o1-12 A ° / \o=\o o° / \c 'o=—\ / 1 T o o1-13 A oz z — —\ \ 1 11-14 A o 1 11-15 A CK_O.i T1-16 A o1-17 AAttorney Docket No.206664-0001-00WOPrep. No. StructureMethod1-18 A0i T1-19 A o oo \ )°= —\ / \ ° o oo — '2 \°=1-20 / )o=AZ—> Z.—' - OZ — z —1-21 \ \ A1-22 A1-23 Ai T T1-24 Ao 1Attorney Docket No.206664-0001-00WOPrep. No. StructureMethod i T1-25 A o1, N. / s. N y _O.i 'x / 'k^-x^^ ° o o1-26 y < \O= — A O / \q / \ ° ' —2 \°=o o\ °1-27 / / °^ A jjo>"Z.—' ' 0z—>\ Z —\ 1 Z — 1^ l~- 1-28 A 01-29 A1-30 A1-31 CAttorney Docket No.206664-0001-00WOPrep. No. StructureMethod1-32 C1 Xx / XZ1-33 C \ O ' —\°=01 / )o=?1-34 z.—' B1 GL o ' l1-35 B \ / zX^^XX^0\ / XzX / XX0 1 11 1 11-36 C Ix^^x^Ox^x / x^^x,1-37 O^N^xJ CAttorney Docket No.206664-0001-00WO

[0243] In some embodiments, the LNPs comprise a lipid of Formula (I), an RNA molecule and one or more excipients selected from neutral lipids, steroids and pegylated lipids. In some embodiments the lipid of Formula (I) is compound 1-5. In some embodiments the lipid of Formula (I) is compound 1-6.Attorney Docket No.206664-0001-00WO

[0244] In some other embodiments, the cationic lipid component of the LNPs has the structure of Formula (II):(II)or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:L1and L2are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-,-S(O)X-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa, -OC(=O)NRa-, -NRaC(=O)O-, or a direct bond;G1is C1-C2 alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -NRaC(=O)- or a direct bond;G2is -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NRaor a direct bond;G3is Ci-Ce alkylene;Rais H or C1-C12 alkyl;Rlaand Rlbare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) Rlais H or C1-C12 alkyl, and Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond;R2aand R2bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R2ais H or C1-C12 alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon-carbon double bond;R3aand R3bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R3ais H or C1-C12 alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R3band the carbon atom to which it is bound to form a carbon-carbon double bond;Attorney Docket No.206664-0001-00WOR4aand R4bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond;R5and R6are each independently H or methyl;R7is C4-C20 alkyl;R8and R9are each independently C1-C12 alkyl; or R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring;a, b, c and d are each independently an integer from 1 to 24; andx is 0, 1 or 2.

[0245] In some embodiments of Formula (II), L1and L2are each independently -O(C=O)-, -(C=O)O- or a direct bond. In other embodiments, G1and G2are each independently -(C=O)- or a direct bond. In some different embodiments, L1and L2are each independently -O(C=O)-, -(C=O)O- or a direct bond; and G1and G2are each independently -(C=O)- or a direct bond.

[0246] In some different embodiments of Formula (II), L1and L2are each independently -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, -SC(=O)-, -NRa-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa, -OC(=O)NRa-, -NRaC(=O)O-, -NRaS(O)xNRa-, -NRaS(O)x- or -S(O)xNRa-.

[0247] In other of the foregoing embodiments of Formula (II), the lipid compound has one of the following structures (IIA) or (IIB):^1a R?a R3aR4a(IIA) (IIB)

[0248] In some embodiments of Formula (II), the lipid compound has structure (IIA). In other embodiments, the lipid compound has structure (IIB).

[0249] In any of the foregoing embodiments of Formula (II), one of L1or L2Attorney Docket No. 206664-0001-00WOis -0(C=0)-. For example, in some embodiments each of L1and L2are -O(C=O)-.

[0250] In some different embodiments of Formula (II), one of L1or L2is -(C=O)O-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0251] In different embodiments of Formula (II), one of L1or L2is a direct bond. As used herein, a “direct bond” means the group (e.g., L1or L2) is absent. For example, in some embodiments each of L1and L2is a direct bond.

[0252] In other different embodiments of Formula (II), for at least one occurrence of Rlaand Rlb, Rlais H or C1-C12 alkyl, and Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carboncarbon double bond.

[0253] In still other different embodiments of Formula (II), for at least one occurrence of R4aand R4b, R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carboncarbon double bond.

[0254] In more embodiments of Formula (II), for at least one occurrence of R2aand R2b, R2ais H or C1-C12 alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0255] In other different embodiments of Formula (II), for at least one occurrence of R3aand R3b, R3ais H or Ci-C 12 alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R3band the carbon atom to which it is bound to form a carboncarbon double bond.

[0256] In various other embodiments of Formula (II), the lipid compound has one of the following structures (IIC) or (IID):Attorney Docket No.206664-0001-00WOIR\ / G3N IR8(IID) wherein e, f, g and h are each independently an integer from 1 to 12.

[0257] In some embodiments of Formula (II), the lipid compound has structure (IIC). In other embodiments, the lipid compound has structure (IID).

[0258] In various embodiments of structures (IIC) or (IID), e, f, g and h are each independently an integer from 4 to 10.

[0259] In some embodiments of Formula (II), a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[0260] In some embodiments of Formula (II), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[0261] In some embodiments of Formula (II), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yetAttorney Docket No.206664-0001-00WOother embodiments, c is 12. Tn some embodiments, c is 13. Tn other embodiments, c is 14. Tn more embodiments, c is 15. In yet other embodiments, c is 16.

[0262] In some embodiments of Formula (IT), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.

[0263] In some embodiments of Formula (II), e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In yet other embodiments, e is 12.

[0264] In some embodiments of Formula (II), f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In yet other embodiments, fis 12.

[0265] In some embodiments of Formula (II), g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In yet other embodiments, g is 12.

[0266] In some embodiments of Formula (II), h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In yet other embodiments, h is 12.

[0267] In some other various embodiments of Formula (II), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments and a and d are the same and b and c are the same.Attorney Docket No.206664-0001-00WO

[0268] The sum of a and b and the sum of c and d of Formula (II) are factors which may be varied to obtain a lipid having the desired properties. In one embodiment, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such that the sum of a and b and the sum of c and d is 12 or greater.

[0269] The substituents at Rla, R2a, R3aand R4aof Formula (II) are not particularly limited. In some embodiments, at least one of Rla, R2a, R3aand R4ais H. In some embodiments Rla, R2a, R3aand R4aare H at each occurrence. In some other embodiments at least one of Rla, R2a, R3aand R4ais C1-C12 alkyl. In some other embodiments at least one of Rla, R2a, R3aand R4ais Ci-Cs alkyl. In some other embodiments at least one of Rla, R2a, R3aand R4ais Ci-Ce alkyl. In some of the foregoing embodiments, the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0270] In some embodiments of Formula (II), Rla, Rlb, R4aand R4bare C1-C12 alkyl at each occurrence.

[0271] In further embodiments of Formula (II), at least one of Rlb, R2b, R3band R4bis H or Rlb, R2b, R3band R4bare H at each occurrence.

[0272] In some embodiments of Formula (II), Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond.

[0273] The substituents at R5and R6of Formula (II) are not particularly limited in the foregoing embodiments. In some embodiments one of R5or R6is methyl. In other embodiments each of R5or R6is methyl.

[0274] The substituents at R7of Formula (II) are not particularly limited in the foregoing embodiments. In some embodiments R7is Ce-Ci6 alkyl. In some other embodiments, R7is C6-C9 alkyl. In some of these embodiments, R7is substituted with -(C=O)ORb, -O(C=O)Rb, -C(=O)Rb, -ORb, -S(O)xRb, -S-SRb, -C(=O)SRb,Attorney Docket No. 206664-0001-00WO-SC(=O)Rb, -NRaRb, -NRaC(=O)Rb, -C(=O)NRaRb, -NRaC(=O)NRaRb, -OC(=O)NRaRb, -NRaC(=O)ORb, -NRaS(O)xNRaRb, -NRaS(O)xRbor -S(O)xNRaRb, wherein: Rais H or C1-C12 alkyl; Rbis C1-C15 alkyl; and x is 0, 1 or 2. For example, in some embodiments R7is substituted with -(C=O)ORbor -O(C=O)Rb.

[0275] In various of the foregoing embodiments of Formula (II), Rbis branched C1-C15 alkyl. For example, in some embodiments Rbhas one of the following structures:

[0276] In some other of the foregoing embodiments of Formula (II), one of R8or R9is methyl. In other embodiments, both R8and R9are methyl.

[0277] In some different embodiments of Formula (II), R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R8and R9, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring. In some different embodiments of the foregoing, R8and R9, together with the nitrogen atom to which they are attached, form a 6-membered heterocyclic ring, for example a piperazinyl ring.

[0278] In still other embodiments of the foregoing lipids of Formula (II), G3is C2-C4 alkylene, for example C3 alkylene.

[0279] In various different embodiments, the lipid compound has one of the structures set forth in Table 2 below.Table 2: Representative Lipids of Formula (II).Attorney Docket No.206664-0001-00WOPrep. No. StructureMethod 1 / ^x^xx^xX^' z —II- 1 / NxX^XXND ° / / NZ— NII-2 D o \ — 'i11-3 / NxX~XXN'y^X^X^^^ Y \ ° D / °\ / \°x( o °==\o oII-4 E \ z—ozOx / X / X / xz —\Ni T11-5 X x / \xND Oxx^xX\X~\X~\ _ Xx _ x'x. X'x Xi TII-6 XNxx^xxNx^XX^^X^^^^ D Ox JxII-7N1 YX \ / \xNx^XX^XX^X^^ DoYx X\^\^x / 11-8!XNYx^xNx^Xx^Xx^ DII-9 DAttorney Docket No.206664-0001-00WOAttorney Docket No.206664-0001-00WOAttorney Docket No.206664-0001-00WOPrep. No. StructureMethod 0' z —i. N. J k / x 11-22 D pz\U HU: o o ■ / \ O( / / —o=—\ 2 / o / \ \ °11-23 ° / \ / \ / ' 220= — D o o\ z— \ z—o oz\\ 1 / Z — Z — o / N. X^. N. x^ / x x^ A. A- 4o. / x / 112 D11-25 E11-26 EAttorney Docket No.206664-0001-00WOPrep. No. StructureMethod \z —IT-27 E P p° / z—\o o^ \o°==11-28 E ° \ ° / / \ / / > / \ / \o=° — / / Q O o oo o==\\o o°^X / X o / IT-29 \ z— E I l I Ioz\0^ ^--- 11-30 E 1 0IT-31 E11-32 EAttorney Docket No.206664-0001-00WO

[0280] In some embodiments, the LNPs comprise a lipid of Formula (II), an RNA molecule and one or more excipient selected from neutral lipids, steroids and pegylated lipids. In some embodiments the lipid of Formula (II) is compound II-9. In some embodiments the lipid of Formula (II) is compound 11-10. In some embodiments the lipid of Formula (II) is compound II-11. In some embodiments the lipid of Formula (II) is compound 11-12. In some embodiments the lipid of Formula (II) is compound 11-32.

[0281] In some other embodiments, the cationic lipid component of the LNPs has the structure of Formula (III):Attorney Docket No.206664-0001-00WOR3^G3A1. ^L2R1<32R2(III)or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:one ofL1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-,-C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond;G1and G2are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3is C1-C24 alkylene, C1-C24 alkenylene, Ca-Cs cycloalkylene, Ca-Cs cycloalkenylene; Rais H or C1-C12 alkyl;R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl;R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or -NR5C(=O)R4;R4is C1-C12 alkyl;R5is H or Ci-Ce alkyl; andx is 0, 1 or 2.

[0282] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB):(IIIA) (IIIB) wherein:A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;R6is, at each occurrence, independently H, OH or C1-C24 alkyl;n is an integer ranging from 1 to 15.

[0283] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).Attorney Docket No.206664-0001-00WO

[0284] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID):(IIIC) (IIID)wherein y and z are each independently integers ranging from 1 to 12.

[0285] In any of the foregoing embodiments of Formula (III), one of L1or L2is -O(C=O)-. For example, in some embodiments each of L1and L2are -O(C=O)-. In some different embodiments of any of the foregoing, L1and L2are eachindependently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0286] In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF):(IIIE) (IIIF)

[0287] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):Attorney Docket No. 206664-0001-00WO

[0288] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0289] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0290] In some of the foregoing embodiments of Formula (III), R6is H. In other of the foregoing embodiments, R6is C1-C24 alkyl. In other embodiments, R6is OH.

[0291] In some embodiments of Formula (III), G3is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3is linear C1-C24 alkylene or linear C1-C24 alkenylene.

[0292] In some other foregoing embodiments of Formula (III), R1or R2, or both, is Ce-C24 alkenyl. For example, in some embodiments, R1and R2each, independently have the following structure:wherein:R7aand R7bare, at each occurrence, independently H or C1-C12 alkyl; anda is an integer from 2 to 12,wherein R7a, R7band a are each selected such that R1and R2each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0293] In some of the foregoing embodiments of Formula (III), at least one occurrence of R7a is H. For example, in some embodiments, R7ais H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R7bis Ci-Cs alkyl. For example, in some embodiments, Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tertbutyl, n-hexyl or n-octyl.

[0294] In different embodiments of Formula (III), R1or R2, or both, has one of the following structures:Attorney Docket No.206664-0001-00WO

[0295] In some of the foregoing embodiments of Formula (III), R3is OH,CN, -C(=O)OR4, -OC(=O)R4or -NHC(=O)R4. In some embodiments, R4is methyl or ethyl.

[0296] In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in Table 3 below.Table 3: Representative Compounds of Formula (III).Prep.No. StructureMethodL oIII-l FoI0III-2 F0l0III-3 F00III-4 10FAttorney Docket No.206664-0001-00WOPrep. No. StructureMethod 0I \X\X\III-5 o F 1 °z / —oHO-^^N> ° \\\ oIII-6 F 1 ( ° \0° — \ / I oIII-7 F oI0III-8 F 0OH I 0III-9 F 0III- 10 F HOX^N-S^^YOXXXX^ / III- 11 FoAttorney Docket No.206664-0001-00WOPrep. No. StructureMethodIll- 12 k / V / X / ^Ox / X / X F To0kx'\X'III- 13 F Ao o^, / X / X / y-xX / \ / \ / \kx^\ ° XXX / X / XIII- 14 \X>.^ / \XX^ F O VX / X / Xo^'^’xXX^X^'N^X^Xx^k^Qxx^xx^x^x^x^xx^xIII- 15 FHO XX XNz ^xX^ / x\^ xxX^ XX xX'xX XXX^X zxX'X z'xX^ / XIII- 16 k / X ° X^XX^xX' F k^-Oxxk^Xx^ / ^ / x^XO HOxx^N-xXx^ojC^k^x\ 0 xx^xx^III- 17 F k^O\xkx^X''xxx'x~X0III- 18 k ° X^xX^xX' F k<Oxxkxx^XxX\X0HO^X^. / x^x^O xX^CXxX / L oIII- 19 FoAttorney Docket No.206664-0001-00WOPrep. No. StructureMethodIII-20 F 0III-21 F 00III-22 F 10I0III-23 F 00III-24 1 ° F 0III-25 F 10I 0III-26 F0Attorney Docket No.206664-0001-00WOPrep. No. StructureMethod I0III-27 F 0H 00I 0III-28 F 0L0111-29 F l^OyOx / ^00 JLOH I O III-30 F 0 HO^N^^^OyCO^ / I0III-31 F 0HO^ JI III-320F0o L oIII-33 F0Attorney Docket No. 206664-0001-00WO

[0297] In some embodiments, the LNPs comprise a lipid of Formula (III), an RNA molecule and one or more excipient selected from neutral lipids, steroids and pegylated lipids. In some embodiments the lipid of Formula (III) is compound III-3. In some embodiments the lipid of Formula (III) is compound III-7.

[0298] In some embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In one embodiment, the LNP comprises only cationic lipids.

[0299] In some embodiments, the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation.

[0300] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0301] Exemplary anionic lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, N-Attorney Docket No. 206664-0001-00WOglutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0302] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoyl-phosphatidy ethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0303] In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, dimyristyl phosphatidylcholine (“DMPC”), DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to the neutral lipid ranges from about 2: 1 to about 8:1.

[0304] In various embodiments, the LNPs further comprise a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton:

[0305] In some embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to cholesterol ranges from about 2:1 to 1:1.

[0306] In some embodiments, the LNP comprises glycolipids (e.g., monosialoganglioside GM1). In some embodiments, the LNP comprises a sterol, such as cholesterol.

[0307] In some embodiments, the LNPs comprise a polymer conjugated lipid.

[0308] In some embodiments, the LNP comprises an additional, stabilizing -lipid whichAttorney Docket No.206664-0001-00WOis a polyethylene glycol-lipid (pegylated lipid). Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modifieddi acylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2ooo)carbamyl]-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as l-(m on om ethoxy-poly ethyl eneglycol)-2, 3 -dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-0-(2’,3’-di(tetradecanoyloxy)propyl-l-0-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100:1 to about 25:1.

[0309] In some embodiments, the LNPs comprise a pegylated lipid having the following structure (IV):O(IV)or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; andz has mean value ranging from 30 to 60.

[0310] In some of the foregoing embodiments of the pegylated lipid (IV), R10and R11are not both n-octadecyl when z is 42. In some other embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms. In someAttorney Docket No.206664-0001-00WOembodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. In still other embodiments, R10is a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms and R11is a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

[0311] In various embodiments, z spans a range that is selected such that the PEG portion of (II) has an average molecular weight of about 400 to about 6000 g / mol. In some embodiments, the average z is about 45.

[0312] In other embodiments, the pegylated lipid has one of the following structures:wherein n is an integer selected such that the average molecular weight of the pegylated lipid is about 2500 g / mol.

[0313] In some embodiments, the additional lipid is present in the LNP in an amount from about 1 to about 10 mole percent. In one embodiment, the additional lipid is present in the LNP in an amount from about 1 to about 5 mole percent. In one embodiment, the additional lipid is present in the LNP in about 1 mole percent or about 1.5 mole percent.

[0314] In some embodiments, the LNPs comprise a lipid of Formula (I), an RNA molecule, a neutral lipid, a steroid and a pegylated lipid. In some embodiments the lipid ofAttorney Docket No.206664-0001-00WOFormula (T)is compound 1-6. In different embodiments, the neutral lipid is DSPC. In other embodiments, the steroid is cholesterol. In still different embodiments, the pegylated lipid is compound IVa.

[0315] In some embodiments, the LNP comprises one or more targeting moieties, which are capable of targeting the LNP to a cell or cell population. For example, in one embodiment, the targeting moiety is a ligand, which directs the LNP to a receptor found on a cell surface.

[0316] In some embodiments, the LNP comprises one or more internalization domains. For example, in one embodiment, the LNP comprises one or more domains, which bind to a cell to induce the internalization of the LNP. For example, in one embodiment, the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP. In some embodiments, the LNP is capable of binding a biomolecule in vivo, where the LNP-bound biomolecule can then be recognized by a cell-surface receptor to induce internalization. For example, in one embodiment, the LNP binds systemic ApoE, which leads to the uptake of the LNP and associated cargo.

[0317] Other exemplary LNPs and their manufacture are described in the art, for example in U. S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2): 172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1: e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, el39; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which are incorporated by reference in their entirety.

[0318] The following Reaction Schemes illustrate methods to make lipids of Formula (I), (II) or (III).GENERAL REACTION SCHEME 1Attorney Docket No.206664-0001-00WO

[0319] Embodiments of the lipid of Formula (T) (e.g., compound A-5) can be prepared according to General Reaction Scheme 1 (“Method A”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 1, compounds of structure A-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of A-l, A-2 and DMAP is treated with DCC to give the bromide A-3. A mixture of the bromide A-3, a base (e.g., N, N-diisopropylethylamine) and the N, N-dimethyldiamine A-4 is heated at a temperature and time sufficient to produce A-5 after any necessarily workup and or purification step.GENERAL REACTION SCHEME 2

[0320] Other embodiments of the compound of Formula (I) (e.g., compound B-5) can be prepared according to General Reaction Scheme 2 (“Method B”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. As shown in General Reaction Scheme 2, compounds of structure B-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A solution of B-l (1 equivalent) is treated with acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine). The crude product is treated with an oxidizing agent (e g., pyridinum chlorochromate) and intermediate product B-3 is recovered. A solution of crude B-3, an acid (e.g., acetic acid), and N, N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain B-5 after any necessary work up and / or purification.Attorney Docket No.206664-0001-00WO

[0321] It should be noted that although starting materials A-l and B-l are depicted above as including only saturated methylene carbons, starting materials which include carbon-carbon double bonds may also be employed for preparation of compounds which include carbon-carbon double bonds.GENERAL REACTION SCHEME 3C-7c’9

[0322] Different embodiments of the lipid of Formula (I) (e.g., compound C-7 or C9) can be prepared according to General Reaction Scheme 3 (“Method C”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 3, compounds of structure C-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.GENERAL REACTION SCHEME 4Attorney Docket No. 206664-0001-00WOD-7

[0323] Embodiments of the compound of Formula (II) (e.g., compounds D-5 and D-7) can be prepared according to General Reaction Scheme 4 (“Method D”), wherein Rla, Rlb, R2a, R2b, R3a, R3b, R4a, R4b, R5, R6, R8, R9, L1, L2, G1, G2, G3, a, b, c and d are as defined herein, and R7represents R7or a C3-C19 alkyl. Referring to General Reaction Scheme 1, compounds of structure D-l and D-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A solution of D-l and D-2 is treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain D-3 after any necessary work up. A solution of D-3 and a base (e.g. trimethylamine, DMAP) is treated with acyl chloride D-4 (or carboxylic acid and DCC) to obtain D-5 after any necessary work up and / or purification. D-5 can be reduced with LiAlH4 D-6 to give D-7 after any necessary work up and / or purification.GENERAL REACTION SCHEME 5Attorney Docket No. 206664-0001-00WOXR7R®. G® E-2 N NH2N NHR7IR9X=CI, Br or I R9Y= Cl or OH

[0324] Embodiments of the lipid of Formula (II) (e.g., compound E-5) can be prepared according to General Reaction Scheme 5 (“Method E”), wherein Rla, Rlb, R2a, R2b, R3a, R3b, R4a, R4b, R5, R6, R7, R8, R9, L1, L2, G3, a, b, c and d are as defined herein. Referring to General Reaction Scheme 2, compounds of structure E-l and E-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of E-l (in excess), E-2 and a base (e.g., potassium carbonate) is heated to obtain E-3 after any necessary work up. A solution of E-3 and a base (e.g. trimethylamine, DMAP) is treated with acyl chloride E-4 (or carboxylic acid and DCC) to obtain E-5 after any necessary work up and / or purification.GENERAL REACTION SCHEME 69 HO— G1— OH 9R1A OH — - — - R1A O^k OH[0]»F'1F-303R1A X x Xx -HJNMR3- (III)F-4 3General Reaction Scheme 6 provides an exemplary method (Method F) for preparation ofAttorney Docket No.206664-0001-00WOLipids of Formula (III). G1, G3, R1and R3in General Reaction Scheme 6 are as defined herein for Formula (III), and GL refers to a one-carbon shorter homologue of Gl. Compounds of structure F-l are purchased or prepared according to methods known in the art. Reaction of F-l with diol F-2 under appropriate condensation conditions (e.g., DCC) yields ester / alcohol F-3, which can then be oxidized (e.g., PCC) to aldehyde F-4. Reaction of F-4 with amine F-5 under reductive amination conditions yields a lipid of Formula (III).

[0325] It should be noted that various alternative strategies for preparation of lipids of Formula (III) are available to those of ordinary skill in the art. For example, other lipids of Formula (III) wherein L1and L2are other than ester can be prepared according to analogous methods using the appropriate starting material. Further, General Reaction Scheme 6 depicts preparation of a lipids of Formula (III), wherein G1and G2are the same; however, this is not a required aspect of the invention and modifications to the above reaction scheme are possible to yield compounds wherein G1and G2are different.

[0326] It will be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or di arylalkyl silyl (for example, / -butyldimethylsilyl, / -butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include / -butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl or arylalkyl), / ;-mcthoxybcnzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T. W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.Pharmaceutical Compositions

[0327] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general,Attorney Docket No.206664-0001-00WOsuch preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0328] Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to subjects of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various subjects is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0329] Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.

[0330] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient, which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0331] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.Attorney Docket No.206664-0001-00WO

[0332] In some embodiments, in addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.

[0333] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[0334] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.

[0335] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0336] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient,Attorney Docket No.206664-0001-00WOadditional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0337] In various embodiments, a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers. In some embodiments, the formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent / powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. In some embodiments, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. In some embodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. In some embodiments, dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0338] Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0.1 to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (in some instances having a particle size ofAttorney Docket No.206664-0001-00WOthe same order as particles comprising the active ingredient).

[0339] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0340] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.Methods of Treatment or Prevention

[0341] In some embodiments, the invention is a method of inducing an adaptive immune response against hepatitis B in a subject comprising administering an effective amount of aAttorney Docket No.206664-0001-00WOcomposition comprising at least one optimized RNA molecule encoding a hepatitis B antigen. In some embodiments, the composition comprising at least one optimized nucleoside modified RNA molecule encoding a hepatitis B antigen.

[0342] In one embodiment, the method provides protection against hepatitis B. In one embodiment, the method provides treatment for chronic hepatitis B. In some embodiments, the invention thus provides a method of treating or preventing the infection, disease, or disorder associated with hepatitis B infection. In some embodiments, the invention a method of treating or preventing maternal transmission of hepatitis B. In some embodiments, the invention a method of clearance of hepatitis B.

[0343] In one embodiment, the composition is administered to a subject having an infection, disease, or disorder associated with hepatitis B infection. In one embodiment, the composition is administered to a subject at risk for developing the infection, disease, or disorder associated with hepatitis B infection. In one embodiment, the composition is administered to a subject at risk for developing hepatitis B. In one embodiment, the composition is administered to a subject at risk for transmitting hepatitis B. For example, the composition may be administered to a maternal subject who is at risk of vertical transmission of hepatitis B to an infant during childbirth. In one embodiment, the composition is administered to a subject who has knowingly been exposed to an infected person's blood, semen, or other bodily fluids through their occupation, or other contact.

[0344] In one embodiment, the method comprises administering a composition comprising one or more optimized nucleic acid molecules encoding a hepatitis B antigen.

[0345] In some embodiments, the method comprises administering to subject a plurality of nucleic acid molecules encoding a plurality of hepatitis B antigens, adjuvants, or a combination thereof. In some embodiments, the method comprises administering to subject a plurality of nucleoside-modified nucleic acid molecules encoding a plurality of hepatitis B antigens, adjuvants, or a combination thereof.

[0346] In some embodiments, the method comprises administering to subject a combination of at least one RNA encoding a hepatitis B surface antigen (HBsAg) and at least one RNA encoding a hepatitis B core antigen (HBcAg). In some embodiments, the ratio of RNA encoding the HBsAg to RNA encoding the HBcAg is about 2: 1. In some embodiments, the ratio of RNA encoding the HBsAg to RNA encoding the HBcAg is about 1:1. In some embodiments,Attorney Docket No.206664-0001-00WOthe ratio of RNA encoding the HBsAg to RNA encoding the HBcAg is about 1:2.

[0347] In some embodiments, the method of the invention allows for sustained expression of the hepatitis B antigen or adjuvant, described herein, for at least several days following administration. In some embodiments, the method of the invention allows for sustained expression of the hepatitis B antigen or adjuvant, described herein, for at least 2 weeks following administration. In some embodiments, the method of the invention allows for sustained expression of the hepatitis B antigen or adjuvant, described herein, for at least 1 month following administration. However, the method, in some embodiments, also provides for transient expression, as in some embodiments, the nucleic acid is not integrated into the subject genome.

[0348] In some embodiments, the method comprises administering an optimized RNA molecule which provides stable expression of the hepatitis B antigen or adjuvant described herein. In some embodiments, administration of the RNA molecule results in little to no innate immune response, while inducing an effective adaptive immune response.

[0349] In some embodiments, the method comprises administering nucleoside-modified RNA, which provides stable expression of the hepatitis B antigen or adjuvant described herein. In some embodiments, administration of nucleoside-modified RNA results in little to no innate immune response, while inducing an effective adaptive immune response.

[0350] In some embodiments, the method provides sustained protection against hepatitis B. For example, in some embodiments, the method provides sustained protection against a disease or disorder associated with hepatitis B for more than 2 weeks. In some embodiments, the method provides sustained protection against hepatitis B for 1 month or more. In some embodiments, the method provides sustained protection against hepatitis B for 2 months or more. In some embodiments, the method provides sustained protection against hepatitis B for 3 months or more. In some embodiments, the method provides sustained protection against hepatitis B for 4 months or more. In some embodiments, the method provides sustained protection against hepatitis B for 5 months or more. In some embodiments, the method provides sustained protection against hepatitis B for 6 months or more. In some embodiments, the method provides sustained protection against hepatitis B for 1 year or more.

[0351] In one embodiment, a single immunization of the composition induces a sustained immune response against hepatitis B for 1 month or more, 2 months or more, 3 months or more,Attorney Docket No.206664-0001-00WO4 months or more, 5 months or more, 6 months or more, or 1 year or more.

[0352] Administration of the compositions of the invention in a method of treatment can be achieved in a number of different ways, using methods known in the art. In one embodiment, the method of the invention comprises systemic administration of the subject, including for example enteral or parenteral administration. In some embodiments, the method comprises intradermal delivery of the composition. In another embodiment, the method comprises intravenous delivery of the composition. In some embodiments, the method comprises intramuscular delivery of the composition. In one embodiment, the method comprises subcutaneous delivery of the composition. In one embodiment, the method comprises inhalation of the composition. In one embodiment, the method comprises intranasal delivery of the composition.

[0353] It will be appreciated that the composition of the invention may be administered to a subject either alone, or in conjunction with another agent.

[0354] The therapeutic and prophylactic methods of the invention thus encompass the use of pharmaceutical compositions comprising the optimized nucleic acid molecule encoding the hepatitis B antigen, adjuvant, or a combination thereof, described herein to practice the methods of the invention. The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of from 1 ng / kg / day and 100 mg / kg / day. In one embodiment, the invention envisions administration of a dose, which results in a concentration of the compound of the invention from 10 nM and 10 pM in a mammal.

[0355] Typically, dosages which may be administered in a method of the invention to a mammal, such as a human, range in amount from 0.01 pg to about 50 mg per kilogram of body weight of the mammal, while the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of mammal and type of disease state being treated, the age of the mammal and the route of administration. In some embodiments, the dosage of the compound will vary from about 0.1 pg to about 10 mg per kilogram of body weight of the mammal. In some embodiments, the dosage will vary from about 1 pg to about 1 mg per kilogram of body weight of the mammal.

[0356] The composition may be administered to a mammal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months, several years,Attorney Docket No.206664-0001-00WOor even less frequently, such as every 10-20 years, 15-30 years, or even less frequently, such as every 50-100 years. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc.

[0357] In some embodiments, administration of an immunogenic composition or combination vaccine of the invention may be performed by single administration or performed using multiple administrations (e.g., in a prime boost regimen). In some embodiments, administration includes a prime administration and at least one boost administration. In some embodiments, the administration includes a prime administration and at least two boost administrations. In some embodiments, the prime and one or more boost administration comprise administration of the same composition. In some embodiments, the prime and one or more boost administration comprise administration of different compositions.

[0358] In some embodiments, the treatment regimen comprises priming with HBsAg mRNA without a signal peptide to induce a cellular immune response, followed by boosting with HBsAg mRNA linked to a signal peptide to induce a humoral immune response. In some embodiments, the treatment regimen comprises administering a priming, a first boosting and a second boosting vaccine composition comprising a combination of an RNA molecule transcribed from SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1; and an RNA molecule transcribed from SEQ ID NO: 10 or SEQ ID NO:11, wherein the RNA molecule encodes SEQ ID NO:9. In some embodiments, the treatment regimen comprises priming with HBsAg mRNA-LNP (SEQ ID NO:2); a 1st boost with the combination of HBsAg mRNA-LNP (SEQ ID NO:2) and SP-HBsAg mRNA-LNP (SEQ ID NO:4); and a 2nd boost with SP-HBsAg mRNA-LNP (SEQ ID NO:4). In some embodiments, the treatment regimen comprises priming with a combination of HBsAg mRNA-LNP (SEQ ID NO:2) and HBcAg mRNA-LNP (SEQ ID NO: 10 or SEQ ID NO: 11); a 1st boost with the combination of HBsAg mRNA-LNP (SEQ ID NO:2), SP-HBsAg mRNA-LNP (SEQ ID NO:4), HBcAg mRNA-LNP (SEQ ID NO: 10 or SEQ ID NO: 11) and SP -HBcAg mRNA-LNP (SEQ ID NO: 13 or SEQ ID NO: 14); and a 2nd boost with a combination of SP-HBsAg mRNA-LNP (SEQ ID NO:4) and SP-HBcAg mRNA-LNP (SEQ ID NO: 13 or SEQ ID NO: 14).Attorney Docket No.206664-0001-00WOEXPERIMENTAL EXAMPLES

[0359] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0360] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.EXAMPLE 1: AN MRNA-LNP VACCINE AGAINST HEPATITIS B INDUCES POTENT IMMUNE RESPONSES IN MICE

[0361] mRNA-based vaccine platforms possess a competitive advantage over other platforms since the production and purification processes are similar regardless of the antigenic sequence. In addition, these processes are cell-free and do not require delivery to the nucleus for expression as is the case for DNA-based platforms. The use of mRNA-based platforms leads to important immunological advantages as well. In principle, mRNA vaccines do not need adjuvants to stimulate innate immunity since both the mRNA molecule and LNP components are inherently able to induce innate immune responses (Verbeke et al., 2022, Immunity, 55, 1993-2005). Th1-polarized immune responses, which generally promote anti-viral immunity, often occur in response to mRNA-based vaccination (Cagigi et al., 2021, Vaccines (Basel) 9, 61). Furthermore, mRNA-encoded antigens lead to the production of the antigenic protein by host cells which may induce presentation in major histocompatibility complex (MHC) class I molecules consequently priming CD8+T cells. Lastly, the antigenic proteins encoded by mRNA vaccines are synthesized in a manner that conserves their conformation and glycosylation congruous with the actual viral protein that is synthesized during infection resulting in maximum antibody specificity (Espeseth et al., 2020, NPJ Vaccines 5).

[0362] HBsAg was selected as the antigenic protein because of its use in licensed antihepatitis B vaccines. Moreover, HBsAg-specific seroconversion and decreases in HBsAg serumAttorney Docket No.206664-0001-00WOlevels achieved in some CHB patients are often associated with the obtention of a functional cure. In the present study, a stabilized and nucleoside-modified mRNA construct that encodes the HBsAg protein sequence (i.e., HBsAg mRNA) was synthesized, and hmoDCs were transfected as an immunologically relevant approach to characterizing HBsAg mRNA expression. HBsAg was observed in DC lysates prepared just 24 hours after mRNA transfection. Transfection with a relatively small amount of mRNA proved to be sufficient to induce HBsAg protein production. Noteworthy, adaptive immunity is induced only if epitopes derived from the translated antigen are properly presented by class I and class II MHC molecules on the surface of professional antigen-presenting cells (APCs) such as DCs. In this regard, flow cytometric analysis demonstrated the presence of HBsAg both intracellularly and on the surface of HBsAg mRNA-transfected hmoDCs. Collectively, these findings demonstrate the ability of the HBsAg mRNA to transfect immunologically relevant cells and induce the production of HBsAg.

[0363] Optimal T cell priming only occurs when 1) the antigenic peptide sequences are presented in association with MHC molecules, 2) the expression of costimulatory molecules on the surface of APCs is augmented and 3) the secretion of inflammatory cytokines takes place. In the experiments reported here, HBsAg mRNA significantly up-regulated the cell-surface expression of CD80, CD86, HLA-DR, and CD40 by transfected hmoDCs. HBsAg mRNA transfection also induced IP-10 and TNF-a secretion by hmoDCs. Similarly, Liang et al. reported that the APCs in rhesus macaques immunized with influenza H10 hemagglutinin encoding mRNA formulated in LNPs were principally responsible for internalizing and translating the mRNA (Liang et al., 2017, Molecular Therapy 25, 2635-2647). mRNA translation was followed by DC maturation and the upregulation of costimulatory molecules (e.g., CD80, CD86, CD40, and HLA-DR) and type 1 IFN inducible genes such as CXCL10 that encodes IP- 10.

[0364] It remains a matter of conjecture whether the activation of innate immunity following immunization with mRNA-LNPs occurs as a function of the adjuvant properties associated with mRNA constructs or the vaccine’s LNP component (Verbeke et al., 2022, Immunity, 55, 1993-2005). In the current study, the enhanced expression of cell-surface costimulatory molecules and cytokine secretion occurred only after hmoDCs were transfected with HBsAg mRNA, not ova mRNA. This seems to indicate, in principle, that mRNA alone does not account for any specific adjuvant effect on cells in this in vitro setting. It is relevant to note, however, that HBsAg protein alone translated from the mRNA-transfected cells exerts a selfAttorney Docket No.206664-0001-00WOadjuvant effect on hmoDCs in vitro, up regulating the expression of CD80, CD83, CD86, and MHC class II (Jan et al., 2012, Microbiol Immunol 56, 719-727). Conceivably, the combined effects of HBsAg mRNA and the translated protein product stimulate the innate immune response of hmoDCs observed in this work.

[0365] The early effects of HBsAg mRNA formulated in LNPs and injected i.m. into mice were assessed 24 hours after administration. Either DCs or T cells in the spleen and inguinal lymph nodes were effectively activated indicating that immunization with HBsAg mRNA-LNPs generated a protein product (HBsAg) in a setting that was optimal for the induction of adaptive B and T cell responses.

[0366] Mice were immunized i.m. with two doses of HBsAg mRNA-LNP or the licensed anti-hepatitis B vaccine, ENGERIX®-B. HBsAg-specific antibody titers were significantly higher following a single first dose of HBsAg mRNA-LNPs compared to ENGERIX®-B, evidencing the improved ability of HBsAg mRNA-LNPs to stimulate antibody production.Remarkably, anti -HBsAg antibody titers increased 10-fold after the second dose of any vaccines administered. Importantly, HBsAg mRNA-LNPs proved superior in stimulating anti-HBsAg seroconversion. In this regard, other investigators report that mRNA-LNP vaccines draining into lymph nodes promote increases in T follicular helper cells, formation of germinal center, B cell activation, and high avidity antibody production (Cagigi et al., 2021, Vaccines (Basel) 9, 61; Lindgren et al., 2017, Front Immunol 8). Consequently, studies that compare vaccine preparations traditionally find that mRNA-LNP vaccines outperform adjuvanted protein vaccines (Espeseth et al., 2020, NPJ Vaccines 5; Pardi et al., 2018, Journal of Experimental Medicine 215, 1571-1588).

[0367] Seroconversion alone is not enough to overcome CHB infection. A Thl-, rather than a Th2-type immune response is preferred for generating effector T cell-mediated immunity. IgG2a / IgGl and IgG2c / IgGl ratios are reliable parameters for determining polarization towards Thl or Th2. In the current study, immunization with HBsAg mRNA-LNPs elicited a marked increase in either IgG2a / IgGl or IgG2c / IgGl ratios indicating polarization towards a Thl-type immune response. IgG2a activates effector functions such as antibody-dependent cell-mediated cytotoxicity and opsonophagocytosis by macrophages, functions that would be advantageous for treating CHB infection (Huber et al., 2006, Clinical and Vaccine Immunology 13, 981-990). Moreover, IgG2c antibodies possess a high capacity to bind to all activating Fcy-receptorAttorney Docket No.206664-0001-00WO(FcyRs) achieving thus a robust activation of innate immunity (Kao et al., 2017, Eur J Immunol 47, 2070-2079; Kao et al., 2015, Cell Rep 13, 2376-2385). In contrast, mice immunized with the protein-based vaccine failed to exhibit increments in the IgG2a / IgGl or IgG2c / IgGl ratios. This was evidenced by the predominant presence of IgGl-type antibodies in serum, whereas IgG2a and IgG2c titers were close to zero. ENGERIX®-B is adjuvanted with alum, which is widely known to elicit Th2 immune responses and the production of antigen-specific antibodies; it is incapable, however, of stimulating Thl or cytotoxic T cell responses (Ebensen et al., 2019, Front Cell Infect Microbiol 9).

[0368] HBV-specific CD4+and CD8+T cell responses are essential for virus clearance and control. Activated effector T cells can eliminate virus-infected cells as a function of their cytolytic activity. Additionally, secreted antiviral cytokines can interfere with cccDNA activity and induce its degradation (Kosinska et al., 2021, Clin Exp Immunol, 205, 106-118). Indeed, mRNA vaccines that target different diseases elicit CD4+and CD8+T cells that express strong, biased Thl-type responses (Espeseth et al., 2020, NPJ Vaccines 5; Arevalo et al., 2022, Science 378, 899-904; Monslow et al., 2020, Vaccine 38, 5793-5802; Wollner et al., 2021, J Virol 95; Awasthi et al., 2019, Sci Immunol, 4, eaaw7083; John et al., 2018, Vaccine 36, 1689-1699). Humans immunized with COVID-19 mRNA vaccines, for example, exhibit long-lasting antigenspecific CD4+T cells and IFN-y-producing CD8+T cells (Verbeke et al., 2022, Immunity, 55, 1993-2005). Similarly, vaccination of mice with HBsAg mRNA-LNPs in the current study induced HBsAg-specific IFN-y+CD4+, IFN-y+CD8+T cells, as well as TNF-oc+CD4+, TNF-oc+CD8+T cells. To a lesser extent, mRNA-base immunization induced IL-2+CD4+and IL-2+CD8+T cells. The elevated production of IFN-y, TNF-oc and IL -2 in response to HBsAg stimulation is consistent with the Thl-type responses normally seen following the administration of mRNA vaccines (Pardi et al., 2018, Journal of Experimental Medicine 215, 1571-1588). On the contrary, very high levels of IL-5 synthesized by cultured splenocytes derived from ENGERIX®-B vaccinated mice are consistent with a Th2-type response. Worth mentioning, that only splenocytes derived from HBsAg mRNA-LNPs vaccinated mice proliferated in response to HBsAg stimuli.

[0369] Aiming to improve even more the generation of HBsAg-specific immunity by mRNA-LNPs, 2ndgeneration mRNA constructs were designed and optimized. Additionally, trafficking sequences were incorporated into the mRNA construct to enhance antigenAttorney Docket No.206664-0001-00WOpresentation by optimizing routing through MHC class T and MHC class IT intracellular compartments. Many cellular compartments, such as endoplasmic reticulum (ER), early and late endosomes, and lysosomes, have been described as part of the essential machinery for antigen processing and presentation into MHC class I and class II molecules (Pishesha et al., 2022, Nat Rev Immunol 22, 751-764). Different studies have shown that engineering the antigen sequence by attaching endoplasmic reticulum translocation signal sequences to the N-terminus may improve antigen presentation and, therefore, optimize specific immune responses (Kang et al., 2006, Immunol Lett 106, 126-134; Kim et al., 2003, Gene Ther 10, 1268-1273; Langlade-Demoyen et al., 2003, 33, 720-728). Furthermore, introducing modifications into the antigen C-terminal has been also studied for the improvement of antigen trafficking. Signal motifs such as lysosome-associated membrane proteins (LAMPs) and the MHC class I trafficking domain (MITD) linked to C-terminal regions of the antigen, in combination with N-terminal signal peptides, have been linked with a strong enhancement of CTL responses (Zhang et al., 2021, Proc Natl Acad Sci U S A, 118, e2005191118; Wang et al., 2023, Biomedicines 11; Kreiter et al., 2008, J Immunol, 180, 309-318; Kang et al., 2006, Immunol Lett 106, 126-134; De Arruda et al., 2004, Immunology 112, 126-135). In this context, an MHC class I signal peptide (SP) and MITD were bound to the N-terminal and C-terminal regions of the HBsAg mRNA construct, respectively. After analyzing anti-HBsAg titers in immunized mice, a robust humoral response was observed for HBsAg and SP-HBsAg mRNA vaccines. On one side, this 2ndgeneration of optimized HBsAg mRNA vaccine generated an estimated 10-fold increase in total IgG levels when compared with the results obtained from our 1stgeneration 7 / A.sd - RNA vaccine candidate. Secondly, the incorporation of the N-terminal signal peptide did not alter antibody levels produced by the HBsAg mRNA vaccine, both outperforming by far the response obtained with protein-based adjuvanted vaccine. Conversely, the C-terminal modification in the SP-HBsAg-MITD candidate produced dramatically lower antibody titers. In the case IgG subtypes, while IgGl levels were high in every immunized cohort, the SP-HBsAg mRNA vaccine led to the production of large amounts of IgG2a and IgG2c antibodies. High IgG2a / IgGl and IgG2c / IgGl ratios expressed polarization towards Thl-type responses, as observed with the 1stgeneration of mRNA-based vaccines. Notably, immunization with all mRNA-based vaccines elicited remarkable cellular responses. Although the incorporation of MITD into the antigenic sequence has been used for stimulation and expansion of specific CD4 T cells (Zhang et al.,Attorney Docket No.206664-0001-00WO2021, Proc Natl Acad Sci U S A, 118, e2005191118; Wang et al., 2023, Biomedicines 11;Kreiter et al., 2008, J Immunol, 180, 309-318), in the particular case of HBsAg, more robust effects were observed without this modification. Interestingly, the N-terminal modification using the ER translocation signal from MHC class I led to the highest levels of CD8+and CD4+cytokine-producing T cells. Taking this into consideration together with the highest IgG2c / IgGl ratios, we considered the SP-HBsAg mRNA-LNP vaccine as the leading optimized candidate to mount the highest specific immune response against HBV. It is worth mentioning that the additional presence of MITD, on the other side, impaired the vaccine’s ability to create such immunity. A potential reason for these weak responses was the very low translation efficiency of SP-HBsAg-MITD mRNA. We speculate that the incorporation of the C-terminal peptide can be detrimental to HBsAg protein production and stability. Further studies would need to be performed to better understand and overcome this problem in MITD-modified mRNA translation efficiency.

[0370] Of additional interest was noticed that when a heterologous vaccination was applied, namely ENGERIX®-B priming followed by an mRNA-LNP booster, similar antibody levels were displayed in comparison with the mRNA prime-boost scheme. An interesting observation was that the heterologous vaccination elicited a similar robust cellular response either in CD4+or CD8+T cells when compared with homologous mRNA immunizations.Additionally, a mixed cytokine pattern was found after splenocyte stimulation. Consistent with previous studies, these results suggest that once priming immunization is done with a proteinbased vaccine, the mRNA vaccine boosting induces a robust CD8+T-cell response (Park et al., 2023, NPJ Vaccines 8).

[0371] A widely studied mouse model for hepatitis B virus infection is obtained by the injection of a hepatotropic AAV8 containing 1.3-fold HBV genome. After infection, mice establish persistent HBV replication and secretion of infectious HBV virions (Du et al., 2021, Front Immunol, 12:766534). After immunization with either HBsAg or SP-HBsAg mRNA vaccines, mice were challenged with rAAV8-1.3HBV. Both vaccine candidates mounted humoral and cellular immunity preventing increments in HBsAg levels. Detectable HBsAg levels in serum are widely recognized as markers of persistent HBV infection (Jindal et al., 2013, Liver Int, 33 Suppl 1:164-75).Attorney Docket No.206664-0001-00WO

[0372] Perinatal transmission of HBV is highly associated with unvaccinated pregnant women who are positive for Hepatitis B “e” Antigen (HBeAg), while transmission is significantly less frequent in HBeAg-negative mothers (Milich et al., 2019, Hum Vaccin Immunother 15, 2187-2191). Besides being a marker for HBV infection, HBeAg is the only antigenic protein capable of trespassing through the placenta. In this regard, HBeAg has been described as an immunomodulator and tolerogenic protein. Despite role the of HBeAg in chronicity induction is still not completely understood, probably its presence as tolerogen in the context of an immature immune system is contributing to the development of CHB in newborns (Milich et al., 2019, Hum Vaccin Immunother 15, 2187-2191; Kramvis et al., 2016, Rev Med Virol, 26(4):285-303).

[0373] In this context, high HBeAg levels were found as fast as 7 days after the challenge in the three groups. Notably, SP-HBsAg- but not HBsAg- mRNA vaccine successfully reduced HBeAg to baseline levels 2 weeks after HBV challenge.

[0374] In the context of chronic infection, HBV-carrier mice modeling a CHB infection were immunized with mRNA-based vaccines. Independently whether HBV-carrier mice were vaccinated with HBsAg-mRNA-LNP, SP-HBsAg mRNA-LNP, or a combination of both, a strong immune response was induced after the three immunizations over 4 weeks. Remarkably, seroconversion and the clearance of HBsAg in plasma were achieved and maintained at least for 70 days post-immunization. Reversing immune tolerance alone fails to restore antibody production. Virus-specific CD8+ and CD4+ T cell activation must accompany seroconversion (Su et al., 2023, J Hepatol 78, 717-730; Fumagalli et al., 2020, Journal of Experimental Medicine 217; Zhu et al., 2016, The Journal of Immunology 196, 3079-3087). The responses of antigen-specific CD8+ and CD4+ T cells after immunization resulted in the robust secretion of cytokines. Successful T-cell activation represents another contribution to reversing immune tolerance. Of particular interest was the cell-mediated immune response obtained in the combination group, where an improvement in CD8+ T cell responses was observed compared to the individual vaccination schemes. Further studies need to be performed to comprehend the immunological causes of this finding.

[0375] In summary, this work establishes an optimized mRNA-based platform used to develop an anti-hepatitis B vaccine for both prophylactic and therapeutic applications. In contrast to the licensed ENGERIX®-B vaccine, immunization with HBsAg mRNA-LNP induced robust TAttorney Docket No.206664-0001-00WOcell responses, as well as elevated HBsAg-specific antibody titers. Notably, these results were obtained in the absence of an added (external) adjuvant. The ability of HBsAg mRNA-LNP to induce both strong humoral and cell-mediated immune responses could ultimately lead to the eradication of HBV in chronically infected patients. Similarly, HBsAg mRNA-LNP vaccination could provide more protective immunity in newborns, eliminating breakthrough infections that occur consequent to mother-to-child transmissions. These data encourage the continued evaluation of mRNA-LNP vaccine constructs as therapeutic candidates to cure CHB.Methods

[0376] Cell line. HepG2 cells were purchased from The American Type Culture Collection (ATCC, Manassas, VA, USA), ATCC* Number: HB-8065™. HepG2 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (iFBS), 100 U / mL of penicillin, 100 pg / mL streptomycin, 1% L -glutamine and 5 mMHEPES.

[0377] Animals. Eight- to 10-week-old C57BL / 6Ncr mice were purchased from Charles River Laboratories and used in the experiments described. Five mice / cage were housed in a dedicated vivarium (18-23°C ambient temperature, 40-60% humidity, 14 h:10 h light cycle) with clean food, water and bedding. Experimental cohorts were composed of both males and females selected at random; randomized littermate controls were included in each experiment. All animal procedures were approved by the local authorities (Landesuntersuchungsamt Rhineland-Palatinate). Ethical approval was granted by the Landesuntersuchungsamt LUA, Koblenz, AK G 23-1-006.

[0378] For the challenge and the chronic hepatitis B experiments, five-week-old C57BL / 6 mice were obtained from SLAC ( Shanghai Laboratory Animal Center of Chinese Academy of Sciences) and the studies were approved by the WuXi IACUC (Institutional Animal Care and Use Commitee, IACUC protocol IDO 1-013-202 Iv 1.0) and carried out by WuXi AppTec (Shanghai) Co., Ltd.

[0379] mRNA-LNP vaccine production. An mRNA sequence (HBsAg mRNA) that encodes hepatitis B, sub-genotype A surface antigen was designed (GenBank: BAD91275.1). HBsAg mRNA was synthesized by TriLink Biotechnologies (San Diego, CA, USA) substituting uridine for pseudouridine, incorporating proprietary 5' UTR and 3' UTR sequences, and using CleanCap technology and polyadenylation. The mRNA was treated with DNase andAttorney Docket No.206664-0001-00WOphosphatase, followed by silica-membrane purification. Final mRNA solutions were adjusted to 1 g / L in 1 mM sodium citrate buffer (pH 6.4) and stored at -80°C until use. The mRNA was encapsulated in lipid nanoparticles (LNPs) using a NanoAssemblr Ignite™ machine for formulation (Precision Nanosystems, Vancouver, Canada) (Figure 2A). All procedures were conducted according to the manufacturer’s protocols. Briefly, Genvoy-ILM™ lipid mix (Precision Nanosystems, Vancouver, Canada), containing 50% ionizable lipid, 10% distearoylphosphatidylcholine, 37.5% cholesterol and 2.5% proprietary stabilizer, was mixed with mRNA in proprietary formulation buffer, pH 5.0, using an N / P ratio of 6. The final solution was filter sterilized and used within the next 24-hour period. mRNA-LNP particle size was determined by dynamic light scattering using a Zetasizer Pro (Malvern, UK). Encapsulation efficiency and mRNA concentration were determined using the RiboGreen™ RNA Assay Kit (ThermoFisher Scientific, Watham, MA, United States). To enhance HBsAg-specific immune responses, mRNA constructs were optimized (2ndgeneration mRNA vaccines) by incorporating an MHC class I signal peptide (SP) in the 5' region and a transmembrane and cytosolic trafficking domain of MHC class I (MITD) in the 3 ' region flanking the HBsAg mRNA sequence. As a result, three 2ndgen constructs were obtained: HBsAg mRNA, SP HBsAg mRNA and SP HBsAg MITD mRNA.

[0380] HmoDCs. Blood samples were obtained from healthy volunteers who donated at the blood bank (University Medical Center Mainz, Germany). All donations were collected upon informed consent. Peripheral blood mononuclear cells (PBMCs) were enriched and collected by density gradient centrifugation on Histopaque-1077 (Sigma-Aldrich, Steinheim am Albuch, Germany). The monocytes were isolated from PBMCs by positive selection using a CD14 Microbeads kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol, and seeded into six-well suspension culture plates at a density of 106cells / mL of serum-free X-VIVO 15 medium (Lonza, Walkersville, MD) supplemented with 1% penicillinstreptomycin, 25 ng / mL of recombinant human IL-4 (ImmunoTools GmbH, Friesoythe, Germany) and 100 ng / mL of recombinant human granulocyte macrophage colony stimulating factor (ImmunoTools). Monocytes were incubated 4 days at 37°C in a humidified environment containing 5% CO2; half the spent culture medium was replaced with fresh supplemented X-VIVO 15 medium on day 2.5.Attorney Docket No. 206664-0001-00WO

[0381] Transfection of immature hmoDCs and HepG2 cells. The immature hmoDCs that remained in suspension on day 4 were collected and seeded at 1.2 x 106cells / mL of supplemented X-VIVO 15 medium into 24-well tissue culture plates. After an additional 24 h incubation, the cells were transfected with 500 ng HBsAg mRNA mixed with 1 pL Lipofectamine MessengerMax (ThermoFisher) in accordance with the manufacturer's instructions. HmoDCs transfected with 500 ng ova mRNA mixed with 1 pL Lipofectamine MessengerMax served as a negative mRNA control. Recombinant HBsAg (Hytest, Turku, Finland) and anti-hepatitis B ENGERIX®-B vaccine (GlaxoSmithKline Biologicals s.a., GSK; Rixensart, Belgium) were added 1 pg / mL to the culture medium as HBsAg protein positive controls. Lipofectamine MessengerMax alone served as an addition negative control and the results were compared to unstimulated hmoDCs derived from the same donor. Cells and cell culture supernatants were analysed 24 h after transfection or stimulation. HepG2 cells were seeded at 5 x 105cells / mL in RPMI-1640 culture medium into 24-well tissue culture plates. After 24 h of incubation, the cells were transfected with 500 ng of the different 2ndgen constructs mixed with 1 pL Lipofectamine MessengerMax (ThermoFisher) according to the manufacturer's indications. Non-optimized HBsAg mRNA and non-transfected HepG2 cells were used as comparative control and negative control, respectively.

[0382] Intracellular (ICS) and extracellular (ECS) staining. HmoDCs transfected with HBsAg mRNA or incubated with HBsAg protein (positive control) were subjected to ICS and ECS to characterize the expression of HBsAg mRNA by the antigen-presenting cells. After 24 h of transfection, the cells were detached from the substratum by incubating with IX DPBS (2 mM EDTA and 0.5% BSA) for 30 min on ice and then washed with fluorescence-activated cell sorting (FACS) buffer (IX DPBS containing 2% iFBS). Subsequently, cells were incubated at 4°C for 15 min with 10% Privigen® Immunoglobulin solution (CSL Behring; Marburg, Germany), an Fc receptor blocker. For ECS, hmoDCs were incubated with biotin-conjugated anti-HBsAg antibody (Abeam) at 4°C for 30 min, washed, then incubated with the LEGENDplex SA-PE (BioLegend; San Diego, CA, USA) for 30 min at RT in the dark and washed twice. For ICS, the cells were fixed and permeabilized using Cytofix / Cytoperm fixation / permeabilization kit (BD Biosciences; Franklin, NJ, USA) according to the manufacturer's instructions. The cells were then incubated with biotin-conjugated anti-HBsAg antibody (abeam). After washing, cells were incubated with LEGENDplex SA-PE (BioLegend) then washed twice. Stained cells wereAttorney Docket No.206664-0001-00WOacquired with an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10.8 (Ashland, OR; Becton, Dickinson and Company).

[0383] Western blot analysis. HepG2 cells were transfected with the different 2ndgen-HBsAg mRNA constructs. After 24 h, the cells were detached from the substratum by incubating with Trypsin for 10 min at 37°C. The cells were collected and centrifuged, and the pellet was resuspended in 40 pL lysis buffer (Pierce™ IP lysis buffer, IX protease inhibitor cocktail and 1 mM DTT) and incubated for 30 min on ice. Total protein in lysates was quantified using Qubit™ Protein Assay Kit (ThermoFisher), and SDS-PAGE was performed by seeding equal protein concentrations per sample. Subsequently, the proteins were transferred to a nitrocellulose membrane which was blocked with IX blocking solution (IX TBS and 5% milk powder) at RT for 1 h. The membrane was then incubated with 1 / 2,000 biotin-conjugated rabbit anti-HBsAg antibody (ab68520; Abeam, Cambridge, United Kingdom) and 1 / 100,000 mouse anti-P-tubulin I antibody (T7816; Sigma-Aldrich, Saint Louis, MO, USA) as housekeeping control at 4°C overnight. The membrane was then washed and stained with 1 / 20,000 IRDye® 800CW goat antirabbit antibody and 1 / 20,000 IRDye® 680RD goat anti-mouse antibody (LI-COR Biosciences, Lincoln, NE, USA) and the signal was acquired using an Odyssey® DLx instrument (LI-COR). Images were analyzed using Empiria Studio® Software 2.3 (LI-COR).

[0384] Mouse immunization. C57BL / 6Ncr mice were immunized on days 0 and 14 i.m. in both anterior tibialis muscles (Figure 3A). Whole blood was collected by submandibular extraction (0.02 mL) on day 1 prior to the first injection and on day 14 prior to the second injection. On day 28, the experimental endpoint, blood was collected by intracardiac puncture under anesthesia (120 mg ketamine and 16 mg xylazine per Kg body weight). Three groups of mice (n= 6) were immunized with: (i) 5 pg per dose of HBsAg mRNA-LNPs; (ii) 1 pg per dose of ENGERIX®-B vaccine (GSK); and (ill) PBS control. Following the final blood collection, mice were euthanized by cervical dislocation under anesthesia and the spleens were dissected for functional analysis.

[0385] To analyze innate immune activation by DCs and early T cell activation, C57BL / 6Ncr mice (n= 4) were injected once i.m. with 5 pg of HBsAg mRNA-LNPs (Figure 2B). Control animals were injected with PBS. Twenty-four hours later, the mice were euthanized and the spleens and iLNs were collected for flow cytometric analyses.Attorney Docket No.206664-0001-00WO

[0386] To evaluate the immune response elucidated by the 2ndgen constructs, C57BL / 6Ncr mice were immunized on days 0 and 28 i.m. (Figure 4B). Whole blood was collected on day 1 prior the prime and on day 28 prior the boost. On day 42, the termination of the experiment, blood was collected and the spleens were dissected as explained before. Eight groups of mice (n= 8) were immunized with: (i) 5 pg per dose of 2ndgen HBsAg mRNA-LNPs; (ii) 5 pg per dose of 2ndgen SP HBsAg mRNA-LNPs; (iii) 5 pg per dose of 2ndgen SP HBsAg MITD mRNA-LNPs; (iv) PBS control; (v) 5 pg per dose of Luc mRNA-LNPs as inert mRNA control; (vi) 1 pg per dose of ENGERIX®-B vaccine (GSK) and the last two animal groups were immunized with an heterologous immunization schedule: (vii) 1 pg per dose of ENGERIX®-B vaccine (GSK) as prime and 5 pg per dose of 2ndgen HBsAg mRNA-LNPs as boost; and (viii) 1 pg per dose of ENGERIX®-B vaccine (GSK) as prime and 5 pg per dose of 2ndgen SP HBsAg mRNA-LNPs as boost. To evaluate the efficacy of the 2ndgen vaccines, C57BL / 6 mice were first immunized on days 0 and 28 i.m. (Figure 7A). On day 56 after priming, mice were challenged with 0.5 x IO10of rAAV8-1.3HBV viral genome / mL. Each animal was injected intravenously with 200 pL (1 x 109AAV / HBV) via tail vein and the experiment termination was set on day 70. Whole blood was withdrawal on days 63 and 70; and spleens were dissected at termination for further functional analysis. Three groups of mice (n= 8) were immunized with: (i) 5 pg per dose of 2ndgen HBsAg mRNA-LNPs; (ii) 5 pg per dose of 2ndgen SP HBsAg mRNA-LNPs; and (iii) PBS control.

[0387] To assess the immunological protection exerted by the 2ndgen vaccines, HBV-carrier mice were generated by injecting C57BL / 6 mice with 0.5 x 1010of rAAV8-1.3HBV viral genome / mL as indicated for the challenge experiment on day -28 before prime. Animals were then immunized on days 0 (prime), 14 (boost 1) and 28 (boost 2) i.m. (Figure 8A). Experiment termination was set on day 70 and whole blood was withdrawal on days -1, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70. Spleens were dissected at termination for further functional analysis. Two groups of mice (n= 6) were immunized with: (i) 10 pg per dose of 2ndgen HBsAg mRNA-LNPs; and (ii) PBS control.

[0388] Secondary lymphoid organ cell isolation and stimulation. The spleens and iLNs were dissected from euthanized mice and disaggregated by passing through a nylon cells 70 pm strainer. Splenocyte suspensions were washed and the red blood cells were lysed by treating with RBC lysis buffer (ThermoFisher) for 5 min at RT. The RBC-free spleen cells were washedAttorney Docket No.206664-0001-00WOand counted. To evaluate the HBsAg-specific T cell responses elicited by immunization, singlecell splenocyte suspensions were seeded into 96-well U-bottom culture plates at a density of 5 x 105cells / 100 pL / well in RPMI-1640 medium (Life Technologies Ltd.; Scotland, United Kingdom) supplemented with 10% iFBS, 1% penicillin-streptomycin and 2% L-glutamine. Subsequently, the cells were either unstimulated or stimulated by the addition of 2 pg / mL PepMix™ HBV (JPT Peptide Technologies GmbH, Berlin, Germany) per well and incubated at 37 °C overnight in 95% air and 5% CO2 for intracellular cytokine staining and for IL-2 levels measurements in supemates. For proliferation assay, supemate cytokine measurements and T cell memory, splenocytes were incubated for 72 h. Concanavalin A (2.5 pg / mL) was used as the positive control (data not shown).

[0389] To assess DC and T cell activation single-cell splenocyte and iLN suspensions were counted and stained for flow cytometric analysis as described below.

[0390] Flow cytometric analyses. The 24-h transfected / stimulated hmoDCs were detached and collected as detailed above, and washed with FACS buffer. After blocking Fc receptors with Privigen® Immunoglobulin solution (described above), the cells were stained at 4°C for 30 min with fluorochrome-conjugated anti-human monoclonal antibodies specific for the following determinants and analyzed by flow cytometry: HLA-DR (APC), CD86 (BD Horizon™ V450), CD80 (PE) and CD40 (PE-Cy™7) (BD Biosciences). Stained cells were washed with FACS buffer and stained with 7-AAD (BD Biosciences) viability dye at RT for 5 min in the dark before acquisition.

[0391] For in vivo DC and T cell activation experiment, 106freshly-isolated splenocytes and iLN cells were washed with FACS buffer and the FC receptors were blocked by incubation with anti-CD16 / CD32 monoclonal antibody (eBioscience, San Diego, CA, USA). The fluorochrome-conjugated anti-mouse monoclonal antibodies were used to stain the following determinants at 4°C for 30 min in the dark: CD8a (Super Bright™ 436), CD3 (eFluor™ 506) and CD80 (PerCP-eFluor™ 710) purchased from eBioscience; CD4 (FITC) and CD69 (PE-Cy™7) purchased from BD Biosciences; and CDllc (PE) and CD86 (APC) purchased from BioLegend. Splenocytes stimulated overnight were evaluated by intracellular cytokine staining to detect the HBsAg-specific T cell responses. The protein transport inhibitor Brefeldin A was added 15 h before cell analysis to enhance the signals detected of the intracellular cytokines. The cells were then washed with FACS buffer and the Fc receptors were blocked by treatment with antiAttorney Docket No. 206664-0001-00WOCD16 / CD32 monoclonal antibody (eBioscience). Subsequently, cells were stained with fluorochrome-conjugated anti-mouse monoclonal antibodies specific for the following extracellular determinants at 4°C for 30 min: CD8a (Super Bright™ 436) and CD3 (eFluor™ 506) purchased from eBioscience, and CD4 (FITC) and CD45R / B220 (PerCP) purchased from BD Biosciences. After fixation and permeabilization with the BD Cytofix / Cy toperm™ Fixation / Permeabilization Kit (BD Biosciences), cells were intracellularly stained with the following fluorochrome-conjugated anti-mouse monoclonal antibodies at 4°C for 30 min: IL-2 (PE) and IFN-y (APC) purchased from BD Biosciences and TNF-a (PE-Cy™7) purchased from eBioscience. All stained cells were acquired with an LSR II flow cytometer (BD Biosciences) and analyzed with Flowlo software version 10.8 (Becton, Dickinson and Company).

[0392] To evaluate the T cell memory subpopulations, splenocytes stimulated 72 h were assessed by flow cytometry. The cells were harvested, washed with FACS buffer and the Fc receptors were blocked as explained before. After, cells were first stained with anti-mouse CCR7 monoclonal antibody conjugated with APC (eBioscience) at 37°C for 30 min, then washed and stained with the following fluorochrome-conjugated anti-mouse monoclonal antibodies at 4°C for 30 min: CD8a (Super Bright™ 436), CD3 (eFluor™ 506) and CD44 (PE-Cy7) purchased from eBioscience and CD4 (FITC) purchased from BD Biosciences. Stained cells were acquired with an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10.8 (Becton, Dickinson and Company).

[0393] Cytokine and chemokine quantitation. The supernatants fromtransfected / stimulated hmoDCs cultures were collected after 24 h, and the concentrations of cytokines and chemokines were quantified using the LEGENDplex Human Essential Immune Response Panel (BioLegend) following the manufacturer's instructions. The panel included the following human cytokines / chemokines: IL-2, CXCL10 (IP-10), IL-ip, TNF-a, CCL2 (MCP-1), IL-17A, IL-6, IL-10, IFN-g, IL-12p70, IL-8 and free active TGF-pi. Similarly, the supernates were collected from cultures of mouse splenocytes following 72-hour stimulation and the concentration of the following cytokines was determined using the LEGENDplex mouse Th Cytokine Panel (BioLegend): IFN-g, IL-2, TNF-a, IL-6, IL-4, IL-5, IL-10, IL-9, IL-17A, IL-17F, IL-22 and IL-13. The level of IL-2 in supernates was also measured after overnight stimulation. Samples derived from both human and mouse cell cultures were acquired with an LSR II flowAttorney Docket No. 206664-0001-00WOcytometer (BD Biosciences) and data were analyzed using the Legendplex Data Analysis Software Suite (BioLegend).

[0394] Proliferation assay. To assess splenocyte proliferation, Ki-67 nuclear protein was quantified (Di Rosa et al., 2021, Front Immunol 12). Briefly, the splenocytes were harvested after 72-h stimulation and washed with FACS buffer. The cells were then fixed and permeabilized using the Foxp3 / Transcription Factor Buffer Set (ThermoFisher) following the one-step protocol for intracellular proteins according to the manufacturer. Fixed and permeabilized cells were incubated with PE-conjugated anti-human / mouse Ki-67 antibody REAfinity (Miltenyi Biotec). The cells were washed, acquired with an LSR II flow cytometer (BD Biosciences) and data were analyzed with FlowJo software version 10.8 (Becton, Dickinson and Company). The Ki-67 proliferation index was calculated as the percentage of peptide-stimulated PE-positive cells / percentage of unstimulated PE-positive cells, i.e., basal stimulation.

[0395] Serological analyses. The sera were collected from whole blood and anti-HBsAg antibodies were quantified by enzyme linked immunosorbent assay. Anti-mouse IgG H& L (HRP) (ab6789; Abeam) was used to titer total IgG in serial dilutions of the sera using regression analysis. Anti-mouse IgGl (HRP) (ab97240; Abeam) and anti-mouse IgG2c (HRP) (ab97255) were used to calculate the IgG2c / IgGl ratio with the optical density obtained from the assay at the same fixed dilutions of the sera. Plasma HBsAg was quantified using the HBsAg ELISA kit (Autobio, CL 0310). Mouse plasma samples were either 20-fold or 600-fold diluted and HBsAg measurement was carried out according to the manufacturer's instructions.

[0396] Statistical analyses. All graphs were performed, and data were analyzed using GraphPad Prism version 9.4.1 (GraphPad Software Inc., San Diego, CA, USA). A one-way ANOVA test followed by Dunnett post hoc multiple comparison test was used to compare two groups; a two-way ANOVA test followed by Sidak post hoc multiple comparison test was used to compare three or more groups. All values are expressed as mean ± SEM; statistically significant / ^-values were: *,p < 0.0332; **,p < 0.0021; ***, p < 0.0002; ****, p < 0.0001.The Experimental Results are Now DescribedAttorney Docket No.206664-0001-00WOHuman monocyte-derived dendritic cells (hmoDCs) transfected in vitro with HBsAg mRNA produce HBsAg protein.

[0397] Human moDCs transfected with HBsAg mRNA produced HBsAg protein detected by Western blot analysis of cell lysates prepared 24 hours post transfection (Figure 1 A and lb). Next, the presence of intracellular and extracellular HBsAg protein in hmoDCs transfected with HBsAg mRNA or incubated with HBsAg protein was assessed and compared in flow cytometry studies. In both cases, there was a marked increase in the frequency of intracellular HBsAg-positive hmoDCs (Figure 1C). However, a significantly higher signal was observed in the HBsAg mRNA-transfected cells. Moreover, only HBsAg mRNA-transfected hmoDCs exhibited a significant signal for HBsAg protein stained extracellularly (Figure ID). Further, HBsAg mRNA-transfected hmoDCs exhibited elevated expression of cell surface activation markers such as CD80, CD86, HLA-DR and CD40 activation as measured by flow cytometry (Figure IE). Transfection with ovalbumin-encoding mRNA (ova mRNA), an irrelevant mRNA control, showed no effect. Similarly, incubation with neither HBsAg protein nor ENGERIX®-B, a licensed anti -hepatitis B vaccine, exerted a significant effect on hmoDC activation marker expression. Cytokines secreted into the cell culture supernatants were also analyzed. HmoDCs transfected with HBsAg mRNA generated relatively large amounts of CXCL10 (IP- 10) and TNF-a, but no presence of the other cytokines or chemokines included in the panel was detected (Figure IF). Ova mRNA transfection, or incubation with HBsAg protein or ENGERIX®-B failed to stimulate cytokine / chemokine secretion by hmoDCs.Vaccination with HBsAg mRNA formulated in LNPs stimulates DCs and T cells

[0398] A vaccine composed of HBsAg mRNA formulated with a lipid mix (GenVoy-ILM™) was synthesized using a Nanoassemblr Ignite™ microfluidic system (Figure 2A). The resultant formulated LNPs displayed an average 83.9 nm (±3.15) size with a poly dispersity index of 0.148 (±0.028). mRNA encapsulation exhibited an average efficiency of 98.75% (±0.19).HBsAg mRNA-LNP formulations were prepared within 24 hours before use to guarantee mRNA and LNP stability. Mice were injected intramuscularly (i.m.) with 5 pg HBsAg mRNA-LNPs and the early immune-related effects were evaluated 24 hours after administration (Figure 2B).

[0399] T cell and DC activation were evaluated in cells isolated from spleens and inguinal lymph nodes (iLNs). Significantly upregulated expression of the early activationAttorney Docket No.206664-0001-00WOmarker, CD69, by CD4+and CD8+T cells was observed in mice vaccinated with HBsAg mRNA-LNPs (Figure 2C). Similarly, the DC populations in the spleens and draining lymph nodes of vaccinated animals exhibited significant increases in the expression of activation markers: CD80 and CD86 (Figure 2D).HBsAg mRNA-LNPs elicit robust anti-HBsAg antibody production and a polarized Thl / Th2 response.

[0400] Experiments were undertaken to characterize the HBsAg-specific humoral and cellular responses of wild-type mice vaccinated twice (days 0 and 14) with HBsAg mRNA-LNPs (Figure 3 A). Mice injected with PBS or immunized with the ENGERIX®-B vaccine served as negative and positive controls, respectively. HBsAg-specific antibody titers assessed 14 days following the priming dose demonstrated a significant humoral response to mRNA-based vaccination (Figure 3B). Vaccination with ENGERIX®-B, on the other hand, did not elicit significant antibody titers post-priming, measured on day 14. Specific antibody titers evaluated at the end of the experiment (day 28), i.e., 14 days following vaccine boosts, were approximately 10-fold higher than those determined after a single dose (Figure 3B). The HBsAg mRNA-LNPs vaccinated group exhibited the highest antibody titers compared with those found in either the PBS control group or the ENGERIX®-B vaccinated group.

[0401] The ratio IgG2c / IgGl in sera reflect the nature of the immune response. Ratio values of less than 1 is indicative of a Th2-type humoral response while increments on IgG2c that results in ratios above 1 indicate a Thl-type cellular immune response. Elevated IgG2c / IgGl ratio in sera collected at the experimental endpoint from mice vaccinated with HBsAg mRNA-LNPs indicated the preferential stimulated cellular immunity (Figure 3C). In contrast, despite showing reasonable total IgG values, the ENGERIX®-B group did not exhibit increments in the levels of IgG2c.HBsAg mRNA-LNP vaccination elicits strong anti-HBsAg T-cell responses.

[0402] The HBsAg-specific proliferation of splenocytes derived from vaccinated animals was assessed and compared by staining the intranuclear protein, Ki -67, which is only expressed during the active cell cycle. A significant increase in the Ki-67 proliferation index was onlyAttorney Docket No.206664-0001-00WOobserved in the HBsAg-specific response of splenocytes derived from mice vaccinated with HBsAg mRNA-LNPs (Figure 3D).

[0403] The HBsAg-specific CD4+and CD8+T cell responses were determined from the splenocytes obtained from mice on day 28. HBsAg mRNA-LNPs induced potent CD8+and CD4+T cell responses as demonstrated by IFN- y, IL-2 and TNF-oc production of antigen-specific cells (Figure 3E and 3F). No significant differences were observed for CD4 responses between mRNA-based and protein-based (ENGERIX®-B) vaccinations. Regarding CD8-type responses, similar CD8+IL-2+frequencies were obtained for mRNA and protein-based approaches. While significantly high HBsAg-specific CD8+IFN-y+and CD8+TNF-oc+T cell levels were observed after mRNA-LNP vaccination, the group immunized with the ENGERIX®-B scheme failed to elicit significant responses (Figures 3F).

[0404] The supernatants were collected from cultures of HBsAg-stimulated splenocytes and cytokine production was quantified. Cells derived from mice vaccinated with HBsAg mRNA-LNPs secreted a large amount of IFN-D, and moderate amounts of IL-2, IL-17A, and IL-22 (Figure 3G). In contrast, very low concentrations of IFN-y+and IL-2, elevated IL-5 levels, and a minor presence of IL- 13 were determined in culture supernatants derived from ENGERIX®-B-vaccinated mice.

[0405] These findings are consistent with previous results indicating that the mRNA-based vaccine platform elicits significant cellular immune responses.Optimization of HBsAg-mRNA constructs improves humoral antigen-specific immune responses and generates a shift towards Th-1 type response.

[0406] To enhance HBsAg-specific immune responses, mRNA constructs were engineered by incorporating trafficking sequences for optimized routing through MHC class I and MHC class II intracellular compartments (Figure 4A). An MHC class I signal peptide (SP) was included in the 5’ region of the mRNA construct, immediately to the left of the HBsAg sequence. Moreover, the transmembrane and cytosolic trafficking domain of MHC class I (MITD) was incorporated into the 3’ region immediately to the HBsAg sequence. In parallel, a construct with the 5’ SP sequence but without the 3’ MITD was generated and tested. Both modified mRNA constructs (SP-HBsAg & SP-HBsAg-MITD) were LNP formulated forAttorney Docket No.206664-0001-00WOimmunization studies in wild-type mice, as well as the HB gmRNA without trafficking signals. Luc mRNA-LNPs were used as a control group, as well as the ENGERIX®-B (proteinbased licensed vaccine) (Figure 4B). Two weeks after the boost, anti-HBsAg titers were analyzed. While protein-based vaccination generated similar antibody levels as with the 1stgeneration mRNA vaccine (Figure 3B), either HBsAg or SP-HBsAg mRNA-LNPs showed a strong increase in antigen-specific IgG levels (Figure 4C). In contrast, the cohort treated with SP-HBsAg-MITD mRNA-LNPs failed to elicit such high antibody titers generating levels only comparable with protein-based immunization. Next, IgG subtypes (IgG2c, and IgGl) were analyzed to assess the balance between Thl- and Th2-biased immune responses. In this context, only SP-HBsAg mRNA-LNPs induced a shift towards Thl -type response exhibiting IgG2c / IgGl ratios higher than 1 (Figure 4D).HBsAg mRNA-LNPs vaccination promotes CD8+and CD4+T cells functional differentiation towards effector sub-types.

[0407] Splenocytes isolated at the termination of the immunization schemes (d42) were studied with and without peptide stimulation for the assessment of effector and memory T cell subtypes. All three mRNA constructs were responsive generating robust CD8+and CD4+T effector cell populations (CCR7 ) after peptide stimulation (Figures 4E & 4G). Although proteinbased vaccination elicited CD8+and CD4+T cell effector responses, mRNA-based vaccines showed significantly higher CD8+and CD4+T effector cell populations. Furthermore, complete T cell memory profiles including T effector (TE), T effector memory (TEM), and T central memory (TCM) cells were analyzed after peptide stimulation in mRNA-LNP vaccinated cohorts, as shown in the plots of Figure 4F and 4H. Interestingly, the T cell memory profiles showed a similar pattern to the ones observed in Engerix®-B vaccinated animals.Optimization of HBsAg mRNA constructs improves T cell immunity.

[0408] T cell immune responses in mice immunized with the 2ndgeneration mRNA constructs were evaluated. Two weeks after the second immunization, as described in Figure 4B, splenocytes were isolated and analyzed by intracellular cytokine staining (ICS). As shown in Figures 5A and 5B, mRNA-LNPs elicited HBsAg-specific CD4+and CD8+T cell immuneAttorney Docket No.206664-0001-00WOresponses expressing high intracellular levels of IFN-y, TNF-a and IL-2. Either in the case of CD4+or CD8+T cells, mRNA-based approaches outperform immune responses generated by protein-based immunization. Differences between the mRNA constructs were found especially after the incorporation of both SP and MITD into the HBsAg mRNA sequence. In the context of CD8+T cells, SP-HBsAg-MITD mRNA-LNPs showed significantly lower frequencies of IFN-y, TNF-a, and IL-2 positive cells in comparison with HBsAg or SP-HBsAg mRNAs (Figure 5B). Interestingly, immunizations with SP-HBsAg mRNA significantly increased IFN-y+, IL-2+and TNF-a+cells for both CD4+or CD8+T cell populations. To further analyze the generated cellular responses, cytokine levels in supernatants were measured after peptide stimulation of splenocytes. Regardless of the presence of the trafficking signals, high levels of IFN-y, TNF-a, IL-2, IL-6, and IL-22 were detected in all groups immunized with mRNA-based vaccines (Figure 5C). In correlation with the ICS studies, however, there were significant differences between the groups immunized with HBsAg or SP-HBsAg mRNAs and those vaccinated with SP-HBsAg-MITD mRNA, being the latter the lowest responders. Noteworthy, the N-terminal incorporation of SP into HBsAg mRNA showed a reduction in IL-2 levels while IFN-y and TNF-a levels were higher than those treated with unmodified HBsAg mRNA after 72 hours of stimulation.Importantly, IL-2 levels measured 16 hours after splenocyte culture were significantly higher in HBsAg or SP-HBsAg mRNA groups, with the latter reaching the highest IL-2 levels (Figure 5D). These results also correlate with those observed at intracellular levels. All vaccinated animals showed no significant differences in Ki67-proliferation indexes (Figure 5E). Furthermore, the translation efficiency was evaluated for the different mRNA constructs by in vitro transfection and subsequent western blot analysis. Results showed that, while HBsAg and SP-HBsAg mRNAs produced an important amount of protein, the presence of MITD in the 3’ region of the construct dramatically impacted protein synthesis (Figure 5F). In addition, consistent differences in protein size were detected in the presence of the signal peptides. For instance, the presence of SP generated a slight increase in HBsAg protein size while the incorporation of both SP and MITD produced the highest size evidenced by the slower gel migration rate.HBsAg mRNA-LNPs used as a boost in a heterologous vaccination context led to a significant enhancement in immunogenicity.Attorney Docket No.206664-0001-00WO

[0409] Next to priming with a protein-based vaccine, HBsAg and SP-HBsAg mRNA-LNPs were used as a boost immunization (Figure 6). Notably, cellular immune responses were significantly boosted when mRNA vaccines were used as a second immunization. While this improvement was observed for both HBsAg and SP-HBsAg mRNA-LNP vaccines, the latter provoked a more robust stimulation of immunity achieving changes of 2-3-fold in cytokinesecreting CD4 and CD8 T cells (Figure 6A & 6B). Furthermore, mRNA-treated groups presented improved T-effector cell responses (Figure 6C) and total anti -HBsAg IgGs were significantly higher in comparison to the control cohort (Figure 6D). In this context, cytokine levels were evaluated after stimulating splenocytes. In correlation with the previous descriptions of ICS, those spleen cells from mRNA-boosted mice secreted high levels of IFN-y, TNF-a and IL -2.Predominantly, the group immunized with SP-HBsAg mRNA-LNP showed the highest values on IFN-y and TNF-a (Figure 6E). IL-2 levels were measured 16 hours post peptide stimulation resulting in significantly higher values for SP-HBsAg mRNA-LNP (Figure 6F).mRNA-based vaccines mount immunity and protect mice against HBV challenge.

[0410] Mice were immunized either with HBsAg or SP-HBsAg mRNA-LNPs followed by a challenge infection using rAAV8-1.3HBV, as depicted in the scheme in Figure 7A. Anti-HBsAg antibodies measured in serum at termination showed that the control group did not generate antibodies whereas high IgG titers were detected for those immunized mice (Figure 7B). No significant differences were observed in serum HBsAg or anti-HBsAg levels between groups immunized with HBsAg and SP-HBsAg mRNA-LNPs. One and two weeks after the challenge infection, high levels of HBsAg were detected in the plasma of the control cohort. In contrast, HBsAg levels remained undetectable in mice pre-immunized with either mRNA vaccine (Figure 7C). Also, one and two weeks after the challenge, HBeAg was measured in blood as another descriptor of HBV infection (Figure 7D). During the first week after the challenge infection, an increment in HBeAg levels was detected in all cohorts without observing significant differences between the groups. After the second week post-challenge, HBeAg levels kept increasing in the control group but remained unaltered in HBsAg mRNA-LNP vaccinated mice. Interestingly, SP-HBsAg mRNA-LNPs led to a decrease in HBeAg to baseline levels.Following the workflow used before, at the termination of the experiment the cellular responses were studied. In correlation with previous results, immunizations with mRNA vaccinesAttorney Docket No. 206664-0001-00WOsignificantly increased IFN-y, IL-2+and TNF-of CD4+or CD8+T cell populations. Either at CD4 or CD8 level, the strongest responses were observed in the group immunized with SP-HBsAg mRNA-LNPs.HBsAg mRNA-LNPs led to seroconversion and robust cellular immune responses suppressing viral load and overcoming immune tolerance in HBV-carrier mice.

[0411] Among the mouse models used to represent CHB, AAV-mediated transfection mouse model is based on viral transduction of the HBV genome, initiating HBV replication and secretion of infectious HBV virions (Du et al., 2021, Front Immunol 12). The viral construct used in this work (rAAV8-1.3HBV) was injected i.v. to C57BL / 6 mice to generate the CHB mouse model (HBV-carrier mice). HBV-carrier mice are characterized by persistent viral replication with long-lasting HBsAg levels in serum. Moreover, these mice were reported to have T-cell immune tolerance and to be completely resistant to immunization with a conventional aluminum-adjuvanted vaccine (Yang et al., 2014, Cell Mol Immunol 11, 71-78).

[0412] To overcome this immune tolerance associated with CHB, HBV-carrier mice were vaccinated and then boosted twice with mRNA-LNP vaccines. As depicted in Figure 8A, experimental cohorts were organized in four groups: (i) control, injected with PBS, (ii) 10 pg HBsAg mRNA-LNP, (iii) 10 pg SP-HBsAg mRNA-LNP, and (iv) a combinatorial approach priming with 10 pg HBsAg mRNA-LNP, followed by first boost with a mix of 5 pg HBsAg mRNA-LNP and 5 pg SP-HBsAg mRNA-LNP; and finally a second boost with 10 pg SP-HBsAg mRNA-LNP. Seroconversion was observed in all groups following the prime. Those HBV-carrier mice immunized only with the HBsAg mRNA-LNP vaccine showed the highest anti-HBsAg antibody levels. A plateau in antibody concentrations was reached after the second boost (Figure 8B). Conversely, plasma HBsAg fell to almost undetectable levels after the second boost in all vaccinated groups (Figure 8C). Noteworthy, HBsAg levels in mice immunized only with SP-HBsAg mRNA-LNP vaccine remained low but above the LLOQ during the 73-day study (Figure 8C). Interestingly, the group using a combination of HBsAg mRNA-LNP and SP-HBsAg mRNA-LNP exhibited an improvement in HBsAg plasma levels when comparing it with the SP-HBsAg group. Figures 8E and 8F show that mRNA-LNP vaccines elicited strong CD4+ and CD8+ T cell responses by expressing anti-viral cytokines such as IFN-y, IL-2, and TNF-a inAttorney Docket No.206664-0001-00WOresponse to HBsAg peptide pool stimulation. Notably, the combinatorial immunization strategy generated the highest levels of cytokine-secreting CD8+ T cells outperforming the cellular responses observed in those groups that received the individual candidate vaccines. In summary, the data demonstrates that both vaccine candidates were effective against CHB in a mouse model. Seroconversion was successful, independently of the incorporation of the signal peptide into the antigenic sequence. An improvement in the cell-mediated immunity by the presence of the signal peptide SP was only observed in the combinatorial approach, indicating a greater restitution of immune control.EXAMPLE 2: LNP -FORMULATED MRNA ENCODING FOR IFN-B

[0413] Therapeutic vaccine candidates could be improved by combination with adjuvants to induce a more robust innate and adaptative immune response. In this regard, an LNP-formulated mRNA encoding for IFN-P is used in combination with the different mRNA-based vaccine candidates. The clinical implementation of IFN-P intends to activate innate immune responses resembling anti-viral immune mechanisms. In fact, IFN-P has been used as a therapy to reduce viral load in CHB patients. More recently, the ability of IFN-P to boost the SARS-CoV-2 specific humoral response after vaccination was observed. In summary, these data provide evidence regarding the potential use of IFN-P as an additional tool in an mRNA-based vaccine setting to boost host innate immunity and generate a strong therapeutic approach against CHB infections.IFN beta with modified Nu construct: CAA23795.1 (GenBank)(SEQ ID NO: 17) MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMN FDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHL KTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFIN RLTGYLRNEXAMPLE 3: BIVALENT mRNA-LNP VACCINE ENCODING HEPATITIS B SURFACE ANTIGEN (HBsAg) AND CORE ANTIGEN (HBcAg)

[0414] To enhance the breadth and robustness of immune responses necessary for breaking immune tolerance in chronic hepatitis B (CHB) infection, a bivalent mRNA-LNP vaccine formulation was developed. This formulation comprises mRNA constructs encodingAttorney Docket No.206664-0001-00WOboth Hepatitis B surface antigen (HBsAg) and Hepatitis B core antigen (HBcAg).

[0415] The inclusion of HBcAg is strategically significant, as HBcAg shares substantial sequence homology with Hepatitis B e antigen (HBeAg), particularly within T and B cell epitope regions. Both antigens are translated from the pre-core / core gene and differ primarily at the N-terminus. This sequence conservation enables HBcAg-induced immune responses to cross-react with HBeAg, thereby targeting multiple viral antigens simultaneously and contributing to control of viral persistence and immune tolerance mechanisms characteristic of CHB.

[0416] The general experimental setting utilices monovalent vaccines (HBs mRNA-LNP and HBc mRNA-LNP) to evaluate the specific immune responses exerted in a context without the presence of the other antigen. For bivalent formulations, HBsAg-encoding and HBcAg-encoding mRNA constructs were formulated separately in distinct LNP preparations and coadministered intramuscularly into opposite hind limbs to prevent potential immunodominance effects. The total mRNA dose was maintained at 6 pg per mouse across all bivalent groups, with relative amounts of each antigen varying according to the tested ratio. Plasma and splenocytes were collected on day 42 for immunological analyses. PBS-treated mice served as negative controls.Monovalent mRNA-LNP Vaccines Elicit Robust Humoral and Cellular Responses Against HBsAg and HBcAg

[0417] Monovalent vaccination with either HBsAg or HBcAg mRNA-LNPs (Figure 18) generated potent antigen-specific immune responses. Plasma samples collected at the experimental endpoint (day 42) demonstrated high total IgG antibody titers against the respective antigens (Figures 19A and 19B). Anti-HBsAg and anti-HBcAg IgG levels were significantly elevated compared to PBS controls (p < 0.0001), with comparable magnitudes between the two monovalent vaccines.

[0418] Cellular immune responses were assessed by intracellular cytokine staining of splenocytes following antigen-specific peptide pool restimulation. HBsAg mRNA-LNP vaccination induced high frequencies of CD8+ T cells producing IFN-y (approximately 40%), IL-2, and TNF-a (Figure 19C). HBcAg mRNA-LNP vaccination elicited even higher CD8+ IFN-y+ T cell responses, reaching approximately 60%. Similar patterns were observed for IL-2 and TNF-a production by CD8+ T cells. CD4+ T cells from both HBsAg- and HBcAg-vaccinatedAttorney Docket No.206664-0001-00WOmice also exhibited robust cytokine secretion profiles for TFN-y, TL-2, and TNF-a (Figure 19D). These data demonstrate that both HBsAg and HBcAg mRNA-LNP formulations independently induce strong antigen-specific humoral and cellular immunity in wild-type mice.Bivalent mRNA-LNP Vaccines Generate Broad Immunity Against Both HBsAg and HBcAg

[0419] To evaluate the immunogenicity of bivalent vaccine formulations, mice were coimmunized with HBsAg and HBcAg mRNA-LNPs at three different ratios (HBs: HBc = 2:1, 1:1, and 1:2) (Figure 18). The total mRNA dose remained fixed at 6 pg per injection, with the relative contributions of each antigen adjusted according to the ratio tested.Humoral Immune Responses:

[0420] All three bivalent vaccine ratios elicited high anti-HBsAg IgG titers, with no significant differences observed between ratios (Figure 20A). Anti-HBsAg antibody levels in bivalent-vaccinated mice were comparable to those achieved with monovalent HBsAg vaccination, indicating that co-administration with HBcAg did not diminish HBsAg-specific humoral immunity.

[0421] Anti -HBcAg IgG titers were also elevated across all bivalent groups (Figure 20B). However, the HBs: HBc 2:1 ratio generated slightly lower anti-HBcAg antibody levels compared to the 1: 1 and 1:2 ratios, suggesting a dose-dependent relationship between HBcAg mRNA content and anti-HBcAg antibody production.Cellular Immune Responses:

[0422] Bivalent vaccination induced robust CD8+ T cell responses against both antigens. Mice immunized with HBs: HBc ratios of 1: 1 and 2: 1 exhibited the highest frequencies of CD8+ IFN-y+ T cells following stimulation with either HBsAg or HBcAg peptide pools (Figure 20C). The 1:2 ratio resulted in significantly lower CD8+ IFN-y+ responses, suggesting that an excess of HBcAg mRNA relative to HBsAg may reduce overall CD8+ T cell activation. Similar trends were observed for IL-2 and TNF-a production by CD8+ T cells.

[0423] Cytotoxic effector function was further assessed by measuring perforin and granzyme B expression in CD8+ T cells. All three bivalent vaccine ratios significantly increased perforin and granzyme B expression, regardless of whether splenocytes were stimulated with HBsAg or HBcAg peptides (Figure 20D). This indicates that bivalent vaccination promotesAttorney Docket No.206664-0001-00WOfunctional cytotoxic T lymphocyte (CTL) differentiation capable of targeting both antigens. CD4+ T cell responses showed moderate increases in cytokine production across all bivalent groups. The HBs: HBc 2: 1 ratio exhibited particularly strong CD4+ responses upon HBsAg peptide stimulation (Figure 20E). While all ratios demonstrated significant increases in CD4+ cytokine production compared to controls, no substantial differences were observed between ratios for HBcAg-specific CD4+ responses.Incorporation of Signal Peptide into Bivalent Vaccine Configuration Enhances Humoral and Cellular Immune Responses

[0424] To determine whether incorporation of the signal peptide (SP) modification into HBcAg mRNA constructs enhances immune responses analogous to previously observed improvements with SP-modified HBsAg, both mRNA constructs were engineered with SP sequences fused to the N-terminal region and evaluated in vivo (Figure 21A).Humoral Immune Responses:

[0425] Incorporation of the second antigen into the vaccine formulation, whether HBsAg or HBcAg, did not negatively impact antibody production. As depicted in Figure 21 A, antigens were tested both in bivalent configurations (HBs: HBc) and as monovalent formulations combined with irrelevant luciferase-encoding mRNA (Luc) to maintain a total mRNA dose of 6 pg (HBs: Luc; Luc: HBc; SP-HBs: Luc; Luc: SP-HBc). No immunodominance effects were observed in total IgG production when HBsAg was combined with HBcAg (Figures 2 IB and 21C). Remarkably, incorporation of the SP sequence into the HBcAg mRNA construct led to a significant increase in anti -HBcAg antibody titers (Figure 21C).Cellular Immune Responses:

[0426] Results depicted in Figures 22A and 22B provide clear evidence of the absence of CD8+ T cell immunodominance or interference between HBsAg and HBcAg when administered at the 2: 1 ratio. Both antigens administered together or separately, using Luc mRNA to normalize total RNA dose, elicited highly similar CD8+ responses. An interesting observation emerged regarding cytokine-secreting CD4+ T cells. For HBsAg-stimulated immune responses, the frequencies of CD4+ T cells secreting IFN-y, IL-2, and TNF-a were significantly higher when animals were immunized with the bivalent vaccine (HBs: HBc) compared to monovalent controls (HBs: Luc; HBc: Luc) (Figure 22C). This finding provides evidence of a potentialAttorney Docket No.206664-0001-00WOsynergistic effect occurring in the bivalent vaccine configuration.

[0427] Notably, incorporation of the SP sequence into both antigen-encoding mRNA constructs (SP-HBs: SP-HBc) led to a substantial increment in HBsAg-specific CD8+ responses (Figures 22D and 22E). This effect was observed only when both SP-modified antigens were administered together in the bivalent vaccine setting and not when tested individually (SP-HBs: Luc or SP-HBc: Luc). Remarkably, the SP-modified bivalent vaccine elicited stronger CD4+ immune responses. Splenocytes stimulated with either HBsAg or HBcAg antigens showed significant increases in the frequencies of IFN-y- and TNF-a-secreting CD4+ T cells (Figure 22F). This finding further supports the conclusion that SP-modified HBsAg and HBcAg mRNA constructs, when administered together, generate a synergistic effect enhancing CD4+ responses against both antigens.Combinatorial Bivalent Vaccine Strategy Employing Sequential SP-Modified and Non-Modified Constructs Elicits Robust Thl-Biased Immune Responses

[0428] Utilizing a strategy analogous to that previously employed for the monovalent HBsAg vaccine, a combinatorial approach was designed to capitalize on the distinct immune response profiles elicited by each vaccine configuration (Figure 23 A). The HBs: HBc vaccine without SP modification, characterized by strong CD4+ T cell responses, was administered as the prime immunization. A combination of constructs with and without SP modification (HBsAg; SP-HBsAg; HBcAg; SP-HBcAg) was administered for boost 1 to achieve a more balanced immune response. Finally, for boost 2, animals were immunized with the combination SP-HBs: SP-HBc (constructs with SP only) with the intention of mounting a stronger CD8+ T cell response.Humoral Immune Responses:

[0429] Anti-HBsAg antibody levels were equally high across all tested bivalent vaccine configurations (Figure 23B). Consistent with previous observations, the presence of SP modification in the HBcAg mRNA construct led to improved anti-HBcAg antibody titers, even within the combinatorial vaccination strategy (Figure 23C).Cellular Immune Responses:

[0430] Results demonstrated that the bivalent vaccine configuration with SP incorporated into both HBsAg and HBcAg constructs elicited superior cytokine-secreting CD8+ T cellAttorney Docket No.206664-0001-00WOresponses, predominantly in terms of HBsAg-specific immunity (Figure 23D). No significant improvements were observed for HBcAg-specific CD8+ responses. Furthermore, the combinatorial approach exhibited a synergistic effect, evidenced by superior levels of CD8+ T cells secreting IFN-y, TNF-a, and IL-2 when stimulated with either HBsAg or HBcAg peptide pools (Figure 23D). Moreover, the combinatorial approach led to higher cytotoxic effector function, as demonstrated by increased granzyme B+ CD8+ T cells (Figure 23E). No improvement in CD4+ T cell responses was observed compared to the bivalent configuration without SP (Figure 23F).Combinatorial Bivalent mRNA Vaccine Breaks Immune Tolerance and Reduces Viral Markers in a Chronic Hepatitis B Mouse Model

[0431] A CHB mouse model was established via intravenous injection of AAV8-HBV1.3 to induce stable viral load in hepatocytes, recapitulating key characteristics of chronic HBV infection. The experiment was designed to evaluate the benefits of incorporating the second antigen, HBcAg, into the vaccine formulation. Additionally, the positive effects of SP incorporation into both antigens within the combinatorial vaccination strategy were assessed (Figure 24A). The vaccination schedule comprised a prime injection followed by two booster immunizations, each delivering 15 pg of mRNA-LNP with 14-day intervals between doses. Chronic HBV infection is characterized by depleted and functionally impaired HBV-specific CD4+ and CD8+ T cell responses resulting from high antigen-dose exhaustion and liver-tolerizing pathways. Thus, the presence of HBcAg in a therapeutic vaccine configuration is of critical importance for restoring HBV-specific CD8+ T cells capable of cytolytic viral control, activating CD4+ T cells to support humoral immunity, and generating memory T cell populations capable of clearing infected hepatocytes.

[0432] Among HBV-associated antigens, HBeAg serves as a critical orchestrator of immune evasion and viral persistence in CHB infections. By crossing the placental barrier during vertical transmission, HBeAg establishes early-life immune tolerance, effectively "educating" the developing immune system to ignore HBV-related antigens. This foundational tolerance allows the virus to persist unchecked, as HBeAg continues to skew immune responses toward a non-protective Th2-dominated profile while suppressing Th 1 -mediated antiviral defenses. The antigen actively undermines innate immunity by silencing crucial pathogen sensors such asAttorney Docket No.206664-0001-00WOTLR2 and sabotaging interferon signaling pathways through SOCS2 activation, creating a permissive environment for viral replication. As chronic infection progresses, HBeAg's immunomodulatory mechanisms become more sophisticated. It promotes T cell exhaustion by upregulating PD-L1 checkpoint molecules and recruits myeloid-derived suppressor cells that paralyze antiviral T cell responses. Persistent HBeAg expression accelerates fibrosis progression and increases cancer risk, while its clearance — whether spontaneous or therapy-induced — often improves long-term outcomes.

[0433] However, the lack of an endogenous initiation codon in the mature HBeAg sequence presents a major hurdle to designing an mRNA-based construct encoding HBeAg alone. In this context, it is notable that HBeAg shares a high degree of sequence homology with HBeAg, as both are translated from the pre-core / core gene, differing primarily at the N-terminus where HBeAg contains a short pre-core extension. This substantial sequence overlap means that many T and B cell epitopes are conserved between the two antigens, enabling the immune system to recognize shared regions following vaccination. Indeed, previous studies have demonstrated that immune responses elicited by HBcAg-based vaccines can cross-react with HBeAg, leading to reductions in circulating HBeAg levels and enhanced antiviral immunity in preclinical models. By leveraging this sequence similarity, HBcAg-encoded mRNA vaccines are anticipated to induce robust HBcAg-specific responses while also generating cross-reactive immunity against HBeAg, thereby contributing to the control of viral persistence and immune tolerance mechanisms that characterize chronic HBV infection.

[0434] Robust HBsAg- and HBcAg-specific humoral and cellular immune responses were established in multiple previous experiments in wild-type mice. In the current CHB mouse model experiment (Figure 24A), not only were antibodies against HBsAg generated, but anti-HBeAg antibodies were also produced in bivalent vaccines containing HBsAg and HBeAg (Figures 24B and 24C). These results confirm that the presence of HBeAg successfully induced cross-reactive immunity against HBeAg. Remarkably, the combinatorial approach, characterized by the incorporation of formulations containing the SP modification, elicited significantly higher levels of anti-HBeAg antibodies (Figure 24C).

[0435] Importantly, at the early timepoint of day 56 from the start of the experiment, seroconversion was accompanied by reductions in HBsAg and HBeAg levels (Figures 24D and 24E). HBV DNA was also measured as a complementary marker of viral activity. ResultsAttorney Docket No.206664-0001-00WOshowed that all tested vaccines significantly reduced HBV DNA levels. However, bivalent vaccines generated a decrease in HBV DNA levels below the lower limit of quantitation (LLOQ) (Figure 24F), demonstrating superior virological control.

[0436] The present data demonstrate that bivalent mRNA-LNP vaccines encoding HBsAg and HBcAg are capable of inducing broad, balanced, and functionally competent immune responses against both antigens. This multi-antigen approach addresses a critical unmet need in CHB immunotherapy by simultaneously targeting multiple viral epitopes, thereby reducing the likelihood of immune escape and enhancing the potential to break HBV-associated immune tolerance.Strategic Advantages of Bivalent Vaccination:

[0437] The incorporation of HBcAg into the vaccine formulation provides several strategic benefits. HBcAg shares extensive sequence homology with HBeAg, a key mediator of immune evasion in CHB. HBeAg establishes and maintains immune tolerance by promoting Th2-biased responses, suppressing Th 1 -mediated antiviral immunity, inducing T cell exhaustion, and recruiting immunosuppressive myeloid-derived suppressor cells. By targeting HBcAg, the vaccine generates cross-reactive immunity against HBeAg, as many T and B cell epitopes are conserved between the two antigens. Previous studies have shown that HBcAg-based vaccines can reduce circulating HBeAg levels and enhance antiviral immunity in preclinical models. Thus, the bivalent approach effectively targets three viral antigens (HBsAg, HBcAg, and HBeAg) with only two mRNA constructs, maximizing immunological coverage while maintaining manufacturing simplicity.Optimization of Antigen Ratios:

[0438] The evaluation of multiple HBs: HBc ratios (2:1, 1:1, and 1:2) was conducted to identify optimal dosing configurations and to assess potential immunodominance effects. Results indicate that both 2: 1 and 1: 1 ratios generate strong CD8+ T cell responses against both antigens, with the 2: 1 ratio showing a trend toward enhanced HBsAg-specific immunity and the 1: 1 ratio providing balanced responses. The 1:2 ratio, while still immunogenic, resulted in reduced CD8+ T cell activation, suggesting that excessive HBcAg mRNA relative to HBsAg may suboptimallyAttorney Docket No.206664-0001-00WOengage the immune system under the fixed total mRNA dose constraint.

[0439] Importantly, the strategy of formulating HBsAg and HBcAg mRNAs in separate LNP preparations and administering them into opposite limbs effectively mitigated immunodominance. Humoral and cellular responses against both antigens were successfully elicited without significant suppression of either antigen-specific immunity, demonstrating the feasibility of multi-antigen mRNA vaccination strategies.Signal Peptide Enhancement of Immunogenicity:

[0440] Incorporation of the SP modification into both HBsAg and HBcAg mRNA constructs significantly enhanced humoral and cellular immune responses. The SP-modified bivalent vaccine elicited superior antibody titers, particularly against HBcAg, and generated substantially increased frequencies of cytokine-secreting CD8+ and CD4+ T cells. Critically, synergistic effects were observed only when both SP-modified constructs were administered together, suggesting that combinatorial SP modification optimizes antigen presentation and T cell priming. These findings support the use of SP modifications as a platform technology for enhancing mRNA vaccine immunogenicity across multiple antigens.Combinatorial Prime-Boost Strategy:

[0441] The sequential administration of non-SP-modified and SP-modified formulations within a combinatorial prime-boost strategy successfully balanced CD4+ and CD8+ T cell responses. This approach capitalized on the distinct immunological profiles elicited by each formulation, generating robust Thl-biased immunity characterized by high frequencies of IFN-y-, TNF-a-, and IL-2-producing T cells and enhanced cytotoxic effector function. Such balanced, polyfunctional T cell responses are critical for achieving sustained viral control and functional cure in CHB.Implications for Functional Cure of CHB:

[0442] The induction of cytotoxic effector functions, as evidenced by granzyme B expression in CD8+ T cells, is critical for achieving viral control in CHB. The ability of bivalent vaccines to generate CTL responses capable of recognizing both HBsAg and HBcAg epitopes enhances the likelihood of targeting infected hepatocytes displaying diverse viral antigens.Attorney Docket No. 206664-0001-00WOFurthermore, the generation of robust CD4+ T helper responses supports sustained antibody production and CTL maintenance, both of which are essential for long-term immune control. The cross-reactive immunity against HBeAg conferred by HBcAg-specific responses is anticipated to contribute significantly to breaking immune tolerance, a major barrier to CHB cure. By simultaneously targeting tolerance-promoting (HBeAg) and structural (HBsAg) viral antigens, the bivalent vaccine addresses multiple mechanisms of viral persistence. Importantly, the demonstration of HBsAg and HBeAg antigen clearance and HBV DNA suppression below the LLOQ in a chronic HBV mouse model provides compelling preclinical evidence of therapeutic efficacy.

[0443] These results establish that bivalent mRNA-LNP vaccines encoding HBsAg and HBeAg elicit broad, balanced, and functionally competent immune responses in both wild-type and chronic HBV-infected mice. The data support the use of bivalent formulations, particularly those incorporating SP modifications within a combinatorial prime-boost strategy, as a superior therapeutic vaccination approach for CHB. The ability to co-deliver multiple mRNA constructs without immunodominance, combined with cross-reactive targeting of HBeAg and demonstrated virological control in a preclinical CHB model, positions this platform as a highly promising candidate for advancing functional cure strategies in chronic hepatitis B.EXAMPLE 4: SEQUENCES

[0444] mRNA constructs encoding for HBsAg were designed:HBsAg sequence: BAD91275.1 (GenBank)Construct 1: HBsAg antigen without signal peptide(SEQ ID NO: 1) MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHS PTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTC TTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWF VGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI(SEQ ID NO: 2) ATGGAGAACATCACCTCCGGCTTCCTGGGCCCCCTGCTGGTGCTGCAGGCCGGCTTC TTCCTGCTGACCCGCATCCTGACCATCCCCCAGTCCCTGGACTCCTGGTGGACCTCCC TGAACTTCCTGGGCGGCTCCCCCGTGTGCCTGGGCCAGAACTCCCAGTCCCCCACCTAttorney Docket No.206664-0001-00WOCCAACCACTCCCCCACCTCCTGCCCCCCCATCTGCCCCGGCTACCGCTGGATGTGCC TGCGCCGCTTCATCATCTTCCTGTTCATCCTGCTGCTGTGCCTGATCTTCCTGCTGGT GCTGCTGGACTACCAGGGCATGCTGCCCGTGTGCCCCCTGATCCCCGGCTCCACCAC CACCTCCACCGGCCCCTGCAAGACCTGCACCACCCCCGCCCAGGGCAACTCCATGTT CCCCTCCTGCTGCTGCACCAAGCCCACCGACGGCAACTGCACCTGCATCCCCATCCC CTCCTCCTGGGCCTTCGCCAAGTACCTGTGGGAGTGGGCCTCCGTGCGCTTCTCCTG GCTGTCCCTGCTGGTGCCCTTCGTGCAGTGGTTCGTGGGCCTGTCCCCCACCGTGTG GCTGTCCGCCATCTGGATGATGTGGTACTGGGGCCCCTCCCTGTACTCCATCGTGTC CCCCTTCATCCCCCTGCTGCCCATCTTCTTCTGCCTGTGGGTGTACATCConstruct 2: HBsAg antigen with MHC 1 signal peptide (SEQ ID NO: 3) MRVTAPRTLILLLSGALALTETWAGSMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSW WTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLL DYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFA KYLWEW AS VRF S WL SLLVPF VQWF VGL SPTVWL S AIWMMWYWGP SLYSIVSPFIPLLPI FFCLWVYI(SEQ ID NO: 4) ATGCGCGTGACCGCCCCCCGCACCCTGATCCTGCTGCTGTCCGGCGCCCTGGCCCTG ACCGAGACCTGGGCCGGCTCCATGGAGAACATCACCTCCGGCTTCCTGGGCCCCCTG CTGGTGCTGCAGGCCGGCTTCTTCCTGCTGACCCGCATCCTGACCATCCCCCAGTCC CTGGACTCCTGGTGGACCTCCCTGAACTTCCTGGGCGGCTCCCCCGTGTGCCTGGGC CAGAACTCCCAGTCCCCCACCTCCAACCACTCCCCCACCTCCTGCCCCCCCATCTGC CCCGGCTACCGCTGGATGTGCCTGCGCCGCTTCATCATCTTCCTGTTCATCCTGCTGC TGTGCCTGATCTTCCTGCTGGTGCTGCTGGACTACCAGGGCATGCTGCCCGTGTGCC CCCTGATCCCCGGCTCCACCACCACCTCCACCGGCCCCTGCAAGACCTGCACCACCC CCGCCCAGGGCAACTCCATGTTCCCCTCCTGCTGCTGCACCAAGCCCACCGACGGCA ACTGCACCTGCATCCCCATCCCCTCCTCCTGGGCCTTCGCCAAGTACCTGTGGGAGT GGGCCTCCGTGCGCTTCTCCTGGCTGTCCCTGCTGGTGCCCTTCGTGCAGTGGTTCGT GGGCCTGTCCCCCACCGTGTGGCTGTCCGCCATCTGGATGATGTGGTACTGGGGCCC CTCCCTGTACTCCATCGTGTCCCCCTTCATCCCCCTGCTGCCCATCTTCTTCTGCCTGT GGGTGTACATCConstruct 3: HBsAg antigen with MHC1 Trafficking Domain (MITD)(SEQ ID NO: 5) MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHS PTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTC TTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWF VGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYIIVGIVAGLAVLAVVVIG AVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTAAttorney Docket No. 206664-0001-00WO(SEQ ID NO: 6) ATGGAGAACATCACCTCCGGCTTCCTGGGCCCCCTGCTGGTGCTGCAGGCCGGCTTC TTCCTGCTGACCCGCATCCTGACCATCCCCCAGTCCCTGGACTCCTGGTGGACCTCCC TGAACTTCCTGGGCGGCTCCCCCGTGTGCCTGGGCCAGAACTCCCAGTCCCCCACCT CCAACCACTCCCCCACCTCCTGCCCCCCCATCTGCCCCGGCTACCGCTGGATGTGCC TGCGCCGCTTCATCATCTTCCTGTTCATCCTGCTGCTGTGCCTGATCTTCCTGCTGGT GCTGCTGGACTACCAGGGCATGCTGCCCGTGTGCCCCCTGATCCCCGGCTCCACCAC CACCTCCACCGGCCCCTGCAAGACCTGCACCACCCCCGCCCAGGGCAACTCCATGTT CCCCTCCTGCTGCTGCACCAAGCCCACCGACGGCAACTGCACCTGCATCCCCATCCC CTCCTCCTGGGCCTTCGCCAAGTACCTGTGGGAGTGGGCCTCCGTGCGCTTCTCCTG GCTGTCCCTGCTGGTGCCCTTCGTGCAGTGGTTCGTGGGCCTGTCCCCCACCGTGTG GCTGTCCGCCATCTGGATGATGTGGTACTGGGGCCCCTCCCTGTACTCCATCGTGTC CCCCTTCATCCCCCTGCTGCCCATCTTCTTCTGCCTGTGGGTGTACATCATCGTGGG CATCGTGGCCGGCCTGGCCGTGCTGGCCGTGGTGGTGATCGGCGCCGTGGTG GCCACCGTGATGTGCCGCCGCAAGTCCTCCGGCGGCAAGGGCGGCTCCTACTC CCAGGCCGCCTCCTCCGACTCCGCCCAGGGCTCCGACGTGTCCCTGACCGCCConstruct 4: HBsAg antigen with MHC 1 signal peptide and MHC1 Trafficking Domain (MITD)(SEQ ID NO: 7) MRVTAPRTLILLLSGALALTETWAGSMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSW WTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLL DYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFA KYLWEW AS VRF S WL SLLVPF VQWF VGL SPTVWL S AIWMMWYWGP SLYSIVSPFIPLLPI FFCLWVYIIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGS DVSLTA(SEQ ID NO: 8) ATGCGCGTGACCGCCCCCCGCACCCTGATCCTGCTGCTGTCCGGCGCCCTGGCCCTG ACCGAGACCTGGGCCGGCTCCATGGAGAACATCACCTCCGGCTTCCTGGGCCCCCTG CTGGTGCTGCAGGCCGGCTTCTTCCTGCTGACCCGCATCCTGACCATCCCCCAGTCC CTGGACTCCTGGTGGACCTCCCTGAACTTCCTGGGCGGCTCCCCCGTGTGCCTGGGC CAGAACTCCCAGTCCCCCACCTCCAACCACTCCCCCACCTCCTGCCCCCCCATCTGC CCCGGCTACCGCTGGATGTGCCTGCGCCGCTTCATCATCTTCCTGTTCATCCTGCTGC TGTGCCTGATCTTCCTGCTGGTGCTGCTGGACTACCAGGGCATGCTGCCCGTGTGCC CCCTGATCCCCGGCTCCACCACCACCTCCACCGGCCCCTGCAAGACCTGCACCACCC CCGCCCAGGGCAACTCCATGTTCCCCTCCTGCTGCTGCACCAAGCCCACCGACGGCA ACTGCACCTGCATCCCCATCCCCTCCTCCTGGGCCTTCGCCAAGTACCTGTGGGAGT GGGCCTCCGTGCGCTTCTCCTGGCTGTCCCTGCTGGTGCCCTTCGTGCAGTGGTTCGTAttorney Docket No. 206664-0001-00WOGGGCCTGTCCCCCACCGTGTGGCTGTCCGCCATCTGGATGATGTGGTACTGGGGCCC CTCCCTGTACTCCATCGTGTCCCCCTTCATCCCCCTGCTGCCCATCTTCTTCTGCCTGT GGGTGTACATCATCGTGGGCATCGTGGCCGGCCTGGCCGTGCTGGCCGTGGTG GTGATCGGCGCCGTGGTGGCCACCGTGATGTGCCGCCGCAAGTCCTCCGGCGG CAAGGGCGGCTCCTACTCCCAGGCCGCCTCCTCCGACTCCGCCCAGGGCTCCG ACGTGTCCCTGACCGCCConstruct: HBcAg antigen without signal peptide(SEQ ID NO:9) MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTAAALYRDALESPEHCSPHHTALRQAIL CWGDLITLSTWVGTNLEDPASRDLVVSYVNSNMGLKFRQLLWFHISCLTFGRETVLEYL VSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRES QCDNA sequence (SEQ ID NO: 10):ATGGATATTGATCCGTATAAAGAATTTGGCGCGACCGTGGAACTGCTGAGCTTTCTG CCGAGCGATTTTTTTCCGAGCGTGCGCGATCTGCTGGATACCGCGGCGGCGCTGTAT CGCGATGCGCTGGAAAGCCCGGAACATTGCAGCCCGCATCATACCGCGCTGCGCCA GGCGATTCTGTGCTGGGGCGATCTGATTACCCTGAGCACCTGGGTGGGCACCAACCT GGAAGATCCGGCGAGCCGCGATCTGGTGGTGAGCTATGTGAACAGCAACATGGGCC TGAAATTTCGCCAGCTGCTGTGGTTTCATATTAGCTGCCTGACCTTTGGCCGCGAAA CCGTGCTGGAATATCTGGTGAGCTTTGGCGTGTGGATTCGCACCCCGCCGGCGTATC GCCCGCCGAACGCGCCGATTCTGAGCACCCTGCCGGAAACCACCGTGGTGCGCCGC CGCGGCCGCAGCCCGCGCCGCCGCACCCCGAGCCCGCGCCGCCGCCGCAGCCAGAG CCCGCGCCGCCGCCGCAGCCAGAGCCGCGAAAGCCAGTGCCodon optimized DNA sequence (SEQ ID NO:11):ATGGACATCGACCCCTACAAGGAGTTCGGCGCCACCGTGGAGCTGCTGTCCTTCCTG CCCTCCGACTTCTTCCCCTCCGTGCGCGACCTGCTGGACACCGCCGCCGCCCTGTAC CGCGACGCCCTGGAGTCCCCCGAGCACTGCTCCCCCCACCACACCGCCCTGCGCCAG GCCATCCTGTGCTGGGGCGACCTGATCACCCTGTCCACCTGGGTGGGCACCAACCTG GAGGACCCCGCCTCCCGCGACCTGGTGGTGTCCTACGTGAACTCCAACATGGGCCTG AAGTTCCGCCAGCTGCTGTGGTTCCACATCTCCTGCCTGACCTTCGGCCGCGAGACC GTGCTGGAGTACCTGGTGTCCTTCGGCGTGTGGATCCGCACCCCCCCCGCCTACCGC CCCCCCAACGCCCCCATCCTGTCCACCCTGCCCGAGACCACCGTGGTGCGCCGCCGC GGCCGCTCCCCCCGCCGCCGCACCCCCTCCCCCCGCCGCCGCCGCTCCCAGTCCCCC CGCCGCCGCCGCTCCCAGTCCCGCGAGTCCCAGTGCHBcAg antigen with signal peptideAttorney Docket No.206664-0001-00WO(SEQ ID NO: 12) MRVTAPRTLILLLSGALALTETWAGSMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTA AALYRDALESPEHCSPHHTALRQAILCWGDLITLSTWVGTNLEDPASRDLVVSYVNSNM GLKFRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGR SPRRRTPSPRRRRSQSPRRRRSQSRESQCNative DNA sequence (SEQ ID NO: 13):ATGCGCGTGACCGCGCCGCGCACCCTGATTCTGCTGCTGAGCGGCGCGCTGGCGCTG ACCGAAACCTGGGCGGGCAGCATGGATATTGATCCGTATAAAGAATTTGGCGCGAC CGTGGAACTGCTGAGCTTTCTGCCGAGCGATTTTTTTCCGAGCGTGCGCGATCTGCT GGATACCGCGGCGGCGC...

Claims

Attorney Docket No. 206664-0001-00WOCLAIMSWhat is claimed is:

1. A composition comprising at least one RNA molecule encoding at least one hepatitis B antigen, wherein the mRNA molecule is transcribed from a sequence having at least 80% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO: 14 wherein the RNA molecule encodes SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO: 12.

2. The composition of claim 1, wherein the composition comprises at least one RNA molecule transcribed from SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 11 or SEQ ID NO: 14.

3. The composition of claim 1, wherein the hepatitis B antigen is operably linked to a signal peptide.

4. The composition of claim 1, comprising a combination of at least two mRNA molecules encoding the hepatitis B antigen, wherein the combination comprises at least two of:a) a RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1;b) a RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 4, wherein the RNA molecule encodes SEQ ID NO: 3c) a RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NOTO or SEQ ID NO:11, wherein the RNA molecule encodes SEQ ID NO: 9; and d) a RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 13 or SEQ ID NO: 14, wherein the RNA molecule encodes SEQ ID NO: 12.

5. The composition of any one of claims 1-4, wherein the composition comprises at least one lipid nanoparticle (LNP) encapsulating the RNA molecule encoding the hepatitis B antigen.Attorney Docket No.206664-0001-00WO6. The composition of any one of claims 1-5, wherein the RNA molecule comprises at least one modified nucleoside.

7. The composition of any one of claims 1-6, wherein the composition induces a cellular immune response against hepatitis B.

8. The composition of claim 7, wherein the composition comprises an RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1.

9. The composition of any one of claims 1-6, wherein the composition induces a humoral immune response against hepatitis B.

10. The composition of claim 9, wherein the composition comprises an RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 4, wherein the RNA molecule encodes SEQ ID NO: 3.

11. The composition of any one of claims 1-6, wherein the composition induces both a cellular and humoral immune response against hepatitis B.

12. The composition of claim 11, wherein the composition comprises a combination of:a) an RNA molecule transcribed from SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1; andb) an RNA molecule transcribed from SEQ ID NO: 4, wherein the RNA molecule encodes SEQ ID NO: 3.

13. The composition of claim 4, wherein the composition comprises a combination of at least one RNA encoding a hepatitis B surface antigen (HBsAg) and at least one RNA encoding a hepatitis B core antigen (HBcAg).Attorney Docket No. 206664-0001-00WO14. The composition of claim 13, wherein the ratio of RNA encoding the HBsAg to RNA encoding the HBcAg is about 2:1.

15. The composition of claim 13, wherein the ratio of RNA encoding the HBsAg to RNA encoding the HBcAg is about 1:1.

16. The composition of claim 13, wherein the ratio of RNA encoding the HBsAg to RNA encoding the HBcAg is about 1:2.

17. The composition of any one of claims 1-16, wherein the RNA molecule is selected from the group consisting of an mRNA molecule, a self-amplifying RNA (saRNA) molecule and a circular RNA (circRNA) molecule.

18. A method of inducing an immune response against hepatitis B in a subject comprising administering to the subject an effective amount of a composition as set forth in any one of claims 1 to 17.

19. The method of claim 18, wherein the method comprises inducing a cellular immune response in a subject in need thereof, the method comprising administering a composition comprising an RNA molecule transcribed from SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1 to the subject.

20. The method of claim 18, wherein the method comprises inducing a humoral immune response in a subject in need thereof, the method comprising administering a composition comprising an RNA molecule transcribed from SEQ ID NO: 4, wherein the RNA molecule encodes SEQ ID NO: 3 to the subject.

21. The method of claim 20, wherein the method comprises administering the composition to a healthy subject.Attorney Docket No.206664-0001-00WO22. The method of claim 18, wherein the method comprises inducing a combination of a cellular and humoral immune response in a subject in need thereof, the method comprising administering a composition comprising a combination of at least two of:a) an RNA molecule transcribed from SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1;b) an RNA molecule transcribed from SEQ ID NO: 4, wherein the RNA molecule encodes SEQ ID NO: 3;c) a RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NOTO or SEQ ID NO:11, wherein the RNA molecule encodes SEQ ID NO:9; and d) a RNA molecule transcribed from a sequence having at least 80% identity to SEQ ID NO: 13 or SEQ ID NO: 14, wherein the RNA molecule encodes SEQ ID NO: 12 to the subject.

23. The method of claim 22, wherein the subject has chronic hepatitis B.

24. The method of any one of claims 22-23, wherein the method comprises administering:i) a priming vaccine composition comprising an RNA molecule transcribed from SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1;ii) a first boosting vaccine composition comprising a combination of:a) an RNA molecule transcribed from SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1; andb) an RNA molecule transcribed from SEQ ID NO: 4, wherein the RNA molecule encodes SEQ ID NO: 3; andiii) a second boosting vaccine composition comprising an RNA molecule transcribed from SEQ ID NO: 4, wherein the RNA molecule encodes SEQ ID NO: 3.

25. The method of any one of claims 22-23, wherein the method comprises administering a priming, a first boosting and a second boosting vaccine composition comprising a combination of:a) an RNA molecule transcribed from SEQ ID NO: 2, wherein the RNAAttorney Docket No.206664-0001-00WOmolecule encodes SEQ ID NO: 1; andb) an RNA molecule transcribed from SEQ ID NO: 10 or SEQ ID NO: 11, wherein the RNA molecule encodes SEQ ID NO:9.

26. The method of any one of claims 22-23, wherein the method comprises administering:i) a priming vaccine composition comprising a combination of an RNA molecule transcribed from SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1 and an RNA molecule transcribed from SEQ ID NOTO or SEQ ID NOT 1, wherein the RNA molecule encodes SEQ ID NO:9;ii) a first boosting vaccine composition comprising a combination of:a) an RNA molecule transcribed from SEQ ID NO: 2, wherein the RNA molecule encodes SEQ ID NO: 1; andb) an RNA molecule transcribed from SEQ ID NO: 4, wherein the RNA molecule encodes SEQ ID NO: 3;c) an RNA molecule transcribed from SEQ ID NOTO or SEQ ID NO: 11, wherein the RNA molecule encodes SEQ ID NO:9; andd) an RNA molecule transcribed from SEQ ID NO: 13 or SEQ ID NO: 14, wherein the RNA molecule encodes SEQ ID NO: 12;andiii) a second boosting vaccine composition comprising a combination of an RNA molecule transcribed from SEQ ID NO: 4, wherein the RNA molecule encodes SEQ ID NO: 3 and an RNA molecule transcribed from SEQ ID NO: 13 or SEQ ID NO: 14, wherein the RNA molecule encodes SEQ ID NO: 12.

27. The method of any one of claims 18-26, wherein the composition further comprises an adjuvant.

28. The method of any one of claims 18-27, wherein the composition is administered by a delivery route selected from the group consisting of intravenous, intradermal, subcutaneous, inhalation, intranasal, and intramuscular.Attorney Docket No.206664-0001-00WO29. The method of any one of claims 18-28 wherein the method comprises administration of a combination of two or more separate LNP, wherein each LNP encapsulates a single species of RNA molecule and administering the combination of LNPs to the subject at two or more separate injection sites.

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