Immunogenic composition for treating hepatitis b

Abstract

To solve a problem of presence of need for improvement in treating a chronic hepatitis B patient, particularly treatment capable of extracting enhancement of cellular immune response in a subject despite long exploration of a vaccine to hepatitis B infection.SOLUTION: Provided is a composition and a method useful for inducing Th1 cell response in a subject affected with hepatitis B. As described in the specification, the composition contains HBsAg having S-protein, Pre-S1 protein and Pre-S2 protein, and aluminum phosphate adjuvant. In a preferable embodiment, an immunogenic composition contains at least 20 μg / mL of HBsAg antigen, and the amount of non-adsorptive antigen is at least 30%.SELECTED DRAWING: None

Description

[Technical field]

[0001]

[0002] The present invention is in the field of immunogenic compositions, particularly immunogenic compositions useful in the treatment of Hepatitis B infection. [Background technology]

[0002]

[0003] Hepatitis B is a viral infection that is the causative agent of acute and chronic liver disease. Hepatitis B is transmitted in humans by contact with the blood or other bodily fluids of an infected person. Hepatitis B infection is widespread and a major health problem causing chronic liver disease in over 250 million people worldwide and nearly 1 million deaths per year (Global Hepatitis Report 2017, World Health Organization). Upon infection, hepatitis B causes an acute phase of the disease in most people with no symptoms at all. Those who do show symptoms experience jaundice, fatigue, and abdominal pain, and a small percentage of affected individuals experience acute liver failure that can be fatal. After infection with hepatitis B, the patient's immune system may clear the virus and be cured, or the patient may develop chronic hepatitis B infection. The consequences of chronic hepatitis B are severe, as 30% of chronically infected adults eventually develop cirrhosis and / or liver cancer. The likelihood of developing chronic disease decreases with age; infants have a 90% chance of chronic infection, while healthy adults have only a 5%-10% chance.

[0003]

[0004] Although vaccines against hepatitis B infection have been explored, there is a need for improved treatments for patients with chronic hepatitis B, particularly treatments that can elicit an enhanced cell-mediated immune response in subjects. Summary of the Invention

[0004]

[0005] Various embodiments are provided relating to an immunogenic composition comprising an HBsAg antigen comprising the S, Pre-S1 and Pre-S2 domains and an aluminum phosphate adjuvant, uses of the immunogenic compositions of the disclosure, methods of inducing a Th1 cell response by administering the immunogenic compositions of the disclosure, and methods of treating Hepatitis B infection by administering the immunogenic compositions of the disclosure.

[0005]

[0006] In some embodiments, the present disclosure provides an immunogenic composition comprising an HBsAg envelope antigen comprising the S, Pre-S1 and Pre-S2 domains (e.g., the S, Pre-S1 and Pre-S2 proteins described herein) and an aluminum phosphate adjuvant. In a preferred embodiment, the present disclosure provides an immunogenic composition comprising an HBsAg envelope antigen comprising the S, Pre-S1 and Pre-S2 domains and an aluminum phosphate adjuvant at least about 20 μg / mL (e.g., at least about 30 μg / mL, at least about 40 μg / mL, at least about 50 μg / mL or at least about 60 μg / mL), wherein the amount of unbound antigen is at least 30% (e.g., at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more). In particularly preferred embodiments, the present disclosure provides a vaccine formulation comprising an HBsAg antigen comprising the S, Pre-S1 and Pre-S2 domains at about 20 μg / mL to 60 μg / mL (e.g., about 20 μg / mL, about 30 μg / mL, about 40 μg / mL, about 50 μg / mL, or about 60 μg / mL) and an aluminum phosphate adjuvant, wherein the amount of unbound antigen is at least about 30% (e.g., at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more).

[0006]

[0007] In some embodiments, the immunogenic composition of the present disclosure comprises between 62.5 μg / mL and 500 μg / mL of aluminum present as an aluminum phosphate adjuvant. In a preferred embodiment, the immunogenic composition of the present disclosure comprises 500 μg / mL of aluminum as an aluminum phosphate adjuvant. In a particularly preferred embodiment, the immunogenic composition of the present disclosure comprises 500 μg / mL of aluminum as an aluminum phosphate adjuvant and 20 μg / mL of HBsAg antigen having all three of the S, Pre-S1 and Pre-S2 proteins.

[0007]

[0008] In another embodiment, the present disclosure provides an immunogenic composition for eliciting a Th1 response in a subject, the composition comprising an HBsAg envelope antigen comprising S, Pre-S1 and Pre-S2 proteins and an aluminum phosphate adjuvant. In a preferred embodiment, the present disclosure provides an immunogenic composition for eliciting a Th1 response in a mammal, the composition comprising at least 20 μg / mL of an HBsAg envelope antigen comprising S, Pre-S1 and Pre-S2 proteins and an aluminum phosphate adjuvant, and the amount of unbound antigen is at least 30%. In a particularly preferred embodiment, the present disclosure provides an immunogenic composition for eliciting a Th1 response in a mammal, the composition comprising 20 μg / mL to 60 μg / mL of an HBsAg antigen comprising S, Pre-S1 and Pre-S2 proteins and an aluminum phosphate adjuvant, and the amount of unbound antigen is at least 30%. In some embodiments, the immunogenic composition for eliciting a Th1 response in a mammal comprises 62.5 μg / mL to 500 μg / mL of aluminum present as an aluminum phosphate adjuvant. In a preferred embodiment, the immunogenic composition for eliciting a Th1 response in a mammal comprises 500 μg / mL of aluminum as an aluminum phosphate adjuvant and 20 μg / mL to 60 μg / mL of an HBsAg antigen comprising S, Pre-S1 and Pre-S2 proteins. In a particularly preferred embodiment, the immunogenic composition for eliciting a Th1 response in a mammal comprises 500 μg / mL of aluminum as an aluminum phosphate adjuvant and 20 μg / mL of an HBsAg antigen comprising S, Pre-S1 and Pre-S2 proteins.

[0008]

[0009] In another embodiment, the present disclosure provides an immunogenic composition for treating hepatitis B in a human subject in need thereof, the composition comprising an HBsAg envelope antigen comprising S, Pre-S1 and Pre-S2 proteins and an aluminum phosphate adjuvant. In a preferred embodiment, the present disclosure provides an immunogenic composition for treating hepatitis B in a human subject in need thereof, the composition comprising at least 20 μg / mL of an HBsAg envelope antigen comprising S, Pre-S1 and Pre-S2 proteins and an aluminum phosphate adjuvant, and the amount of unbound antigen is at least 30%. In a particularly preferred embodiment, the present disclosure provides an immunogenic composition for treating hepatitis B in a human subject in need thereof, the composition comprising 20 μg / mL to 60 μg / mL of an HBsAg antigen comprising S, Pre-S1 and Pre-S2 proteins and an aluminum phosphate adjuvant, and the amount of unbound antigen is at least 30%. In some embodiments, an immunogenic composition for treating hepatitis B in a human subject in need thereof comprises 62.5 μg / mL to 500 μg / mL of aluminum present as an aluminum phosphate adjuvant. In a preferred embodiment, an immunogenic composition for treating hepatitis B in a human subject in need thereof comprises 500 μg / mL of aluminum as an aluminum phosphate adjuvant and 20 μg / mL to 60 μg / mL of an HBsAg antigen comprising S, Pre-S1 and Pre-S2 proteins. In a particularly preferred embodiment, an immunogenic composition for treating hepatitis B in a human subject in need thereof comprises 500 μg / mL of aluminum as an aluminum phosphate adjuvant and 20 μg / mL to 60 μg / mL of an HBsAg antigen comprising S, Pre-S1 and Pre-S2 proteins.

[0009]

[0010] The present disclosure also encompasses the use of at least one of the immunogenic compositions of the present disclosure in the preparation of a pharmaceutical composition for treating Hepatitis B infection.

[0010]

[0011] The present disclosure further provides a pharmaceutical composition comprising the immunological composition of the present disclosure for administration to a subject in need thereof.

[0011]

[0012] The present disclosure also provides a method for inducing a Th1 response in a mammal comprising administering a therapeutically effective amount of a composition of the present disclosure.

[0012]

[0013] The present disclosure also provides a method for treating hepatitis B infection, particularly chronic hepatitis B infection, comprising administering to a subject in need thereof a therapeutically effective amount of a composition of the present disclosure.

[0013]

[0014] In any of the aspects or embodiments described herein, the composition comprises an S protein (e.g., an S protein comprising or consisting of an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1); a Pre-S2 protein (e.g., an S protein comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:2); 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 3); and Pre-S1 protein (e.g., Pre-S1 protein comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 3). Embodiments described herein may include about 75-90% by weight S protein, about 2-8% by weight Pre-S1 protein, and about 5-15% by weight Pre-S2 protein (e.g., 83±3.3% S protein, 6±3% Pre-S1 protein, and 11±3% Pre-S2 protein).

[0014]

[0015] Other features, objects, and advantages of the present disclosure will become apparent in the detailed description that follows. It should be understood, however, that the detailed description, while illustrating embodiments of the present disclosure, is given by way of illustration and not by way of limitation. Various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art from the detailed description.

[0015] [List of arrays]

[0016] The following is a list of sequences referenced herein:

[0017] [Table 1]

[0018] [Table 2]

[0019] SEQ ID NO:3 is the large protein of HBsAg amino acid sequence: MGLSWTVPLE WGKNQSTSNP LGFFPDHQLD PAFGANSNNPDWDLNSNKDH WPQANQVGVG AFGPGFTPPH GGLLGWSSQA QGTLHTVPAVPPPASTNRQT KRQPTPISPP LRDSHPQAMQ WNSTAFHQAL QHPRVRGLYFPAGGSSSGTV NPAQNIASHI SSISSRTGDP APNMENITSG FLGPLLVLQAGFFLLTRILT IPQSLDSWWT SLNFLGGSPV CLGQNSQSPT SNHSPTSCPPICPGYRWMCL RRFIIFLFIL LLCLIFLLVL LDYQGMLPVC PLIPGSTTTSTGPCKTCTTP AQGNSMFPSC CCTKPTDGNC TCIPIPSSWA FAKYLWEWGSVRFSWLSLLV PFVQWFVGLS PTVWLSVIWM MWYWGPNLYN ILSPFIPLLPIFFCLWVYI DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016]

[0020] The inventors of the present application have generated an improved therapeutic Hepatitis B vaccine that is effective in inducing a Th1 cellular immune response.

[0017]

[0021] Hepatitis B virus is a member of the Orthohepadnavirus genus of the Hepadnaviridae family of viruses. Hepatitis B virus is a small, enveloped DNA virus that contains a nucleocapsid and an outer coat of the Hepatitis B surface antigen (HBsAg). The HBsAg antigen is composed of three related envelope proteins that are all encoded by the same open reading frame ("S ORF") on the viral DNA. These three proteins have the same C-terminus but differ at the N-terminus due to the presence of three in-frame ATG start codons that divide the S ORF into three regions. The S or "small" envelope protein is the most abundant and the smallest at 226 amino acids. The M or "medium" surface protein contains the S protein and has an additional protein domain of 55 amino acids known as pre-S2. The protein known as the L or "large" protein consists of S, Pre-S2, and a third protein domain known as pre-S1 with 118 amino acids. The Hepatitis B viral genome is prone to replication errors, resulting in many genotypes and genetic variants. At least 10 different genotypes of the virus have been identified, with many mutations occurring in the S and Pre-S domains. Thus, many sequence changes exist in each of the three protein domains.

[0018]

[0022] In some embodiments, the S protein described herein comprises or consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the S protein described herein comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the Pre-S2 protein described herein comprises or consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the Pre-S2 protein described herein comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the Pre-S1 protein described herein comprises or consists of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the Pre-S1 protein described herein comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:3.

[0019]

[0023] No effective treatment for hepatitis B infection has been found. Acute infection is treated with bed rest. Chronic infection is usually treated with antiviral drugs, specifically nucleoside antivirals such as tenofovir, lamivudine or entecavir, or pegylated interferon alpha (PEG-IFNα). Antiviral drugs can slow the progression of cirrhosis and reduce the incidence of liver cancer, but have proven unable to achieve elimination of the virus from patients. Furthermore, antiviral drugs are costly and must be continued for the patient's lifetime to remain effective. PEG-IFNα is associated with side effects and produces a sustained antiviral response in only about 30% of patients. Thus, drug treatment for chronic hepatitis B has generally only been effective in slowing the progression of the disease and has not been successful in providing a cure. To eliminate the hepatitis B virus and thereby avoid chronic symptoms, patients must mount a strong and diverse immune response. In particular, strong and specific T cell responses are essential to achieve viral clearance of hepatitis B [Bertoletti and Rivino (2014) Curr Opin Infect. Dis 27:528-534]. This type of response is almost exclusively seen in adult patients with a mature immune system [Bertoletti and Gehring (2013) PLOS Path. 9:1-4].

[0020]

[0024] Given the severity of the health impact of hepatitis B infection, much attention has focused on preventing the disease. Prophylactic vaccines against hepatitis B have been available for over 30 years, and in many countries, widespread vaccination against hepatitis B is performed on children. The first prophylactic vaccine against hepatitis B was developed in the late 1970s using plasma from hepatitis B patients. Improved hepatitis B vaccines became available in the 1980s and were produced in yeast cells using recombinant DNA technology to express the S-domain of HBsAg. These more modern vaccines quickly supplanted the first vaccines and are still the major commercial vaccines on the market today. A more advanced hepatitis B vaccine was developed in the 1990s, produced in mammalian cells, and contains all three domains of HBsAg, specifically S, Pre-S1, and Pre-S2. The presence of the Pre-S1 and Pre-S2 domains was associated with enhanced immunogenic responses and inhibition of viral binding and infectivity (U.S. Pat. No. 5,242,812). The biological functions of the various envelope proteins and the nature of the immune response to each of the components (S, pre-S2 and pre-S1) are only partially understood. However, it is known that in acute hepatitis B infection, Pre-S1 and pre-S2 antigens and antibodies appear and disappear early after acute infection, whereas antibodies to the S domain appear several weeks later. The above findings and studies using either Pre-S1 or Pre-S2 domain antigens suggested that pre-S1 and pre-S2 antigens may be involved in the induction of helper T cells to produce antibodies to the S domain [Milich et al. (1985) PNAS 82 8170-8172; Milich et al. (1986) J Immunol 137 315-322].

[0021]

[0025] Commercially available prophylactic hepatitis B vaccines have been shown to successfully induce strong antibody responses against the S protein domain of HBsAG in healthy subjects, particularly those who are young and have an unimpaired immune system. However, attempts to use these vaccines as therapeutic treatments in patients with chronic hepatitis B have proven unsuccessful. Clinical trials conducted to measure the therapeutic efficacy of hepatitis B vaccines containing the HBsAG S domain alone as well as the Pre-S2 and S domains together have shown that the vaccines cannot achieve elimination of the hepatitis B virus [Pol et al., (2001) J. Hepatol 34:917-921]. The failure of immunogenic therapy to treat chronic hepatitis B infection is generally attributed to the phenomenon of "immune tolerance", whereby the immune system of hepatitis B patients becomes unresponsive to further exposure to hepatitis B antigens, particularly HBsAg. Immune tolerance in patients with chronic hepatitis B is associated with the viral mass production of hepatitis B antigens, which causes the exhaustion of T cells, specifically hepatitis B-specific CD8+ T cells [Boni, C. et al., (2007) J. Virol. 81:4215-4225]. Exhausted hepatitis B-specific T cells overexpress programmed cell death protein 1 (PD-1), which promotes the death of antigen-specific T cells. In patients with high hepatitis B viral loads, the destruction of antigen-specific T cells may result in the complete disappearance of these cells from the liver. As a result, the T cell response to stimulation with hepatitis B antigens is attenuated.

[0022]

[0026] In addition to the challenges presented by immune tolerance, hepatitis B vaccines containing only HBsAg S protein may not be able to clear the virus from chronic patients due to their weak ability to stimulate cellular immunity by T cell responses in most subjects. In an attempt to stimulate T cell responses more effectively, therapeutic vaccination studies have been carried out using hepatitis B vaccines containing all three of the Pre-S1, Pre-S2 and S protein domains in chronic hepatitis B patients. These studies showed hepatitis B-specific T cell responses in some patients. However, the effect was transient and did not result in elimination of the disease, probably because the vaccine stimulated Th2 responses but not CD8+ T lymphocyte responses. [Jung et al. (2002) Vaccine 20:3598-3612; Kosinka et al. (2015) Med. Microbiol. Immunol 204]. Th2 cells, characterized by secretion of IL-4, IL-5 and IL-13 cytokines, promote antibody production, are commonly associated with allergic responses, and are less effective against viral infections than Th1 cells, characterized by secretion of IFN-γ. Further attempts to induce good immunological responses using DNA vaccines also failed to achieve sustained responses [Bertoletti and Gehring (2009) Exp. Rev. Gastro. Hep. 3:561-569].

[0023]

[0027] Many researchers have concluded that a cure for hepatitis B is many years away, and that by using a combination of treatments, chronic patients may be able to achieve a so-called "functional cure" that achieves a sustained reduction in hepatitis B virus and other markers of the disease as well as a reduction in the incidence of liver cancer after treatment is stopped [Lok et al., (2017) J. Hep. 67:847-861]. Due to the failure of antiviral treatment alone and prophylactic vaccines alone to achieve a functional cure, some researchers have concluded that it may be necessary to combine a prophylactic vaccine with a hepatitis B treatment that reduces the viral load. A reduction in viral load may reduce the level of immune tolerance, thereby improving the ability of vaccine treatment to induce an immune response that can control the infection [Zoulim, F. et al. (2018) Cold Spring Harb. Perspect. Med. (2015):5:a21501]. For example, a combination of a prophylactic vaccine with a nucleoside inhibitor has been attempted. However, to date, combination therapy using commercially available hepatitis B vaccines has not been successful in achieving viral clearance from patients with chronic hepatitis B [Vandepapeliere et al. (2007) Vaccine 51:8585-8597]. In a Vietnamese study, an attempt was made to improve responsiveness by combining a newer vaccine, Genhevac B (Pre-S1, Pre-S2 and S protein vaccine with aluminum hydroxide adjuvant), with a nucleoside inhibitor (lamivudine). This combination was superior to each individual therapy alone in reducing the levels of viral DNA in patients. However, the effect was not sustained after the vaccine treatment was discontinued. Thus, this combination treatment was unable to restore the patient's immune system to effectively eliminate the virus [Hoa, (2009) Antimicrob. Agents and Chemo. 53:5134-5140].

[0024]

[0028] A more complex triple combination treatment has been proposed, including antibodies blocking PD-1 activity in addition to nucleoside inhibitors and a prophylactic hepatitis B vaccine. Studies of this triple combination in a woodchuck model using a virus similar to hepatitis B were effective in only one-third of the animals [Kosinka et al., (2015) supra: pp. 103-114; Liu et al., Virologica, (2014) 29: pp. 10-16]. Furthermore, the efficacy of this triple combination has not been demonstrated in humans, and the triple combination would require the use of expensive treatments, possibly indefinitely. Thus, the use of conventional prophylactic vaccines for therapeutic purposes, even in combination with other drugs, has not proven successful as a functional cure for hepatitis B in chronic patients. Therefore, to achieve a sustained response in chronic hepatitis B patients, improved means must be found to enhance the ability of these patients to mount a T cell response against the virus.

[0025]

[0029] Newer strategies have emerged that utilize gene silencing techniques to treat chronic hepatitis B. Examples of these therapies include the use of small interfering RNA (SiRNA) to interfere with viral gene expression, particularly HBsAg. Like nucleoside inhibitors, these therapies have the potential to reduce viral load and levels of secreted HBsAg [Woodell et al. (2015) Mol. Ther. 21:973-985], and it has been suggested that the treatments may be useful in combination with treatments that induce immune responses [Ren et al. (2013) PLOSone 8(3):e57525]. However, the efficacy of this combination has not been established, and given the failure of earlier studies with antiviral drugs, its efficacy may depend on combinations that include immunological agents capable of stimulating a robust Th1 cell response.

[0026]

[0030] Previous attempts have been made to enhance T cell responses to hepatitis B vaccines by modifying the vaccine formulation. Most of the commercially available hepatitis B vaccines, including the widely used hepatitis B vaccine, Engerix B®, and the hepatitis B vaccine containing S / Pre-S1 and Pre-S2, Sci-B-Vac®, are formulated with aluminum hydroxide adjuvants. Aluminum hydroxide adjuvants are one of a family of aluminum-containing adjuvants sometimes informally referred to as "alum" (although the word "alum" is more correctly used to describe hydrated potassium aluminum sulfate [KAl(SO4)2·12H2O], which was used as an early adjuvant for diphtheria toxoid but was abandoned due to difficulties in manufacture). Aluminum-based adjuvants have been used in human vaccines since 1932 and have a long record of safety, although their mechanism of action is not fully understood. Aluminum-based adjuvants are commonly believed to enhance immune responses through activation of dendritic cells.

[0027]

[0031] Research has been conducted to better understand aluminum-based adjuvants and to enhance their effectiveness. The two most commonly used aluminum-based adjuvants are called "aluminum phosphate" and "aluminum hydroxide", with aluminum hydroxide adjuvant being the most widely used commercially. Aluminum hydroxide adjuvant is not Al(OH)3 but crystalline aluminum oxyhydroxide (AlOOH), which has a larger surface area than crystalline aluminum hydroxide. Aluminum phosphate adjuvant is actually amorphous aluminum hydroxyphosphate [Al(OH)3], in which some of the hydroxyl groups of aluminum hydroxide adjuvant have been replaced by phosphate groups. x (PO4) y

[0001] . The surface of aluminum phosphate adjuvant is composed of Al-OH and Al-OPO3 groups. Although they are chemically similar, the chemical properties of the two adjuvants are different. Aluminum hydroxide adjuvant has a crystalline structure, a large surface area, and a positive charge (+30 mVolts) at neutral pH. Aluminum phosphate adjuvant is amorphous and has a negative charge (approximately -20 mVolts) at neutral pH. Aluminum phosphate adjuvant has also been shown to dissolve more readily after injection.

[0028]

[0032] By "adsorbing" an antigen to an aluminum-based adjuvant, we mean that the antigen attaches to the adjuvant and forms a layer on the surface. Antigens adsorb to the surface of aluminum-based adjuvants through electrostatic interactions, van der Waals forces, and ligand (mainly phosphate) exchange. Ligand exchange is the strongest force of adsorption and can occur even in the presence of electrostatic repulsion. HBsAg is composed primarily of phospholipids containing phosphate groups that adsorb strongly to the hydroxylated mineral surface of the adjuvant through ligand exchange between phosphate groups in the antigen and surface hydroxyls in the aluminum-based adjuvant. Electrostatic attraction is not the dominant adsorption force for HBsAg [Iver et al., (2004) Vaccine, 22:1475-1479].

[0029]

[0033] Traditionally, antigen adsorption to aluminum-based adjuvants was purposely maximized, since adsorption was generally considered important for immunostimulatory effects, as it retains the antigen at the injection site and creates a "depot" effect that releases the antigen to the immune system in a sustained manner. Furthermore, complete adsorption of the antigen results in higher long-term stability of the vaccine formulation during long-term storage. Engelix B® contains 10 μg of HBsAg per 250 μg of aluminum from the aluminum hydroxide adjuvant. This ratio, 0.04 μg of HBsAg / μg of aluminum, ensures complete adsorption. However, more recently, the relationship between antibody and T-cell immunity, efficacy, and adsorption has become unclear. Studies on the effect of the tightness of binding of HBsAg to aluminum hydroxide on antibody production in immunized mice have shown that vaccine formulations containing antigens that adsorb most tightly to the adjuvant produced the lowest antibody responses. [Clapp et al. (2011) J. Pharm Sci 100(2) pp. 388-401]. One study using the HBsAg S antigen domain showed that aluminum-based adjuvants with higher phosphate content showed reduced adsorption, weaker antigen binding, and greater antibody responses [Egan et al. (2009) Vaccine 27:3175-3180]. Similarly, a single antigen hepatitis B vaccine containing an aluminum phosphate adjuvant elicited a greater antibody response than a similar vaccine formulated with an aluminum hydroxide adjuvant, and was effective at lower adjuvant concentrations [Fazeli et al. (2008) DARU 3:143-148]. At least one study has shown that adsorption of antigens with aluminum-based adjuvants is not necessary for the adjuvant to enhance antibody responses, leading to the conclusion that inflammation, not adsorption, is associated with immunogenicity [Noe et al. (2010) Vaccine 28:3588-3594]. Little is known about the extent to which modified aluminum compositions and amounts affect Th2 versus Th1 T cell immunity, but those of skill in the art would assume that studies reporting higher antibody responses promoted enhancement of Th2 but not Th1 responses.

[0030]

[0034] It is well known that aluminum-based adjuvants improve immunogenicity, but it is also well known that aluminum-based adjuvants act primarily to enhance antibody production, and are therefore most effective in targeting pathogens that are killed or inhibited by antibody interaction.It is generally believed that aluminum-based adjuvants are ineffective in eliciting Th1 responses, but rather induce inflammatory, Th2 responses by improving antigen attraction and uptake by antigen-presenting cells (APCs).Therefore, aluminum-based adjuvants have not been utilized in the search for better stimulators of Th1 cell immunity.

[0031]

[0035] More modern adjuvants have been added to Hepatitis B vaccines to improve the cellular or "natural" immune response. Such modern adjuvants are endotoxin, monophosphoryl lipid A (MPL), and bacterial liposaccharides. WO 93 / 19780 discloses the stimulation of T cell responses by HBsAg vaccines with the combination of adjuvants. No T cell response (measured by IL-2, IFN-γ, and IL-4) was observed after immunization with HBsAg absorbed to aluminum hydroxide in Balb / c mice. However, when 3-O-deacylated monophosphoryl lipid A (3D-MPL) was added to the formulation, a Th1 cell response was observed (measured by IL-2 and IFN-γ). A later patent filed by the same research group, U.S. Pat. No. 5,972,346, disclosed improved humoral immunogenicity when aluminum phosphate was used instead of aluminum hydroxide in the HBsAg / 3D MPL vaccine formulation. This discovery led to the development of a commercial vaccine sold under the brand name Fendrix®. Subsequent studies have shown that Fendrix elicits a Th1 cellular immune response. However, Fendrix is ​​an expensive product and has only been approved for use in patients over 15 years of age with renal deficiency in Europe. Furthermore, other studies have shown that the effects of the 3D-MPL adjuvant are temporary and mostly limited to the injection site and local lymph nodes (De Pasquale et al., Vaccine 2015).

[0032]

[0036] More recently, US Patent Publication No. 20160136264 discloses a hepatitis B vaccine consisting of a hepatitis B antigen fragment and an adjuvant, the adjuvant consisting of an immunomodulatory DNA sequence containing a CpG motif capable of mediating a Th1 response in mice. However, safety concerns were raised about the use of CpG-based adjuvants in hepatitis B vaccines, and marketing approval was not obtained in all jurisdictions.

[0033]

[0037] The inventors of the present application have discovered that immunogenic compositions comprising all three HBsAg proteins (S, Pre-S1 and Pre S-2) and an aluminum phosphate adjuvant are effective in stimulating Th1 cell responses in mammals when they have significant amounts of unbound antigen. Physicochemical analysis of the compositions of the present disclosure indicates that the adjuvant binds weakly to the HBsAg antigen. As further demonstrated in the Examples herein, this binding is significantly weaker than that seen in Hepatitis B vaccines formulated with an aluminum hydroxide adjuvant. This binding is also significantly weaker than that seen in formulations containing HBsAg antigen and an aluminum phosphate adjuvant that have a lower ratio of HBsAg antigen to aluminum phosphate adjuvant.

[0034]

[0038] Surprisingly, the compositions of the present disclosure induced enhanced Th1 cell responses in mammals in the absence of other, more modern adjuvants known to stimulate cellular immunity, such as MPL or CpG. These Th1 cell responses were experimentally demonstrated using different immunological markers, specifically antigen-specific IFN-γ response and IgG2a / IgG1 ratio. This is surprising considering previous studies that showed no Th1 response to conventional Hepatitis B vaccines formulated with aluminum-based adjuvants alone.

[0035]

[0039] Even more surprisingly, the immunogenic compositions of the present disclosure are effective in enhancing Th1 cell responses compared to commercially available prophylactic vaccines when a significantly reduced concentration of aluminum phosphate adjuvant is used relative to the amount of antigen in the composition, In particular, the compositions of the present disclosure are effective in enhancing Th1 responses at a 50% lower aluminum phosphate adjuvant to antigen ratio compared to the most widely used commercially available prophylactic vaccines.

[0036]

[0040] The vaccine of the present disclosure includes HBsAg that includes all three of the S, Pre-S1 and Pre-S2 proteins. The HBsAg may be derived from any genotype, strain or isolate of Hepatitis B. Furthermore, the HBsAg may be derived from natural or modified HBsAg. The HBsAg antigen may be isolated from a natural source of Hepatitis B virus, such as a biological sample (e.g., blood, plasma, serum, semen, saliva, tissue section, biopsy specimen, etc.) collected from an infected subject, cultured cells or tissue culture. The HBsAg may be produced using recombinant techniques in cells. In some embodiments, the HBsAg is expressed in a mammalian cell line. In some embodiments, the HBsAg is expressed in a Chinese hamster ovary cell line. The HBsAg antigen that includes the S, Pre-S1 and Pre-S2 domains may be produced using the methods disclosed in U.S. Pat. No. 5,242,812. Nucleotide sequences encoding different HBsAg can be found in data banks such as Genbank and in published literature (e.g. Fukimori et al. (1990) 18 Nuc. Acid Res 4587; Vaudin et al. (1988) 69 J. Gen Virol. 1383-1389). The amino acid sequence of the large protein of HBsAg is shown in SEQ ID NO:1. In a preferred embodiment, the immunogenic composition of the present disclosure comprises HBsAg antigen having 20 μg / mL or more of all three of the S, Pre-S1 and Pre-S2 proteins. In another preferred embodiment, the immunogenic composition of the present disclosure comprises HBsAg antigen having 20 μg / mL to 60 μg / mL of all three of the S, Pre-S1 and Pre-S2 proteins.

[0037]

[0041] The vaccine formulation of the present disclosure further comprises an aluminum phosphate adjuvant. One example of an aluminum phosphate adjuvant suitable for use in the present disclosure is Adju-Phos®, an aluminum phosphate wet gel suspension manufactured by Brenntag.

[0038]

[0042] As shown in Example 2, HBsAg vaccine formulations using aluminum hydroxide adjuvant (including the commercial prophylactic formulation of the vaccine containing all three of the S, Pre-S1 and Pre-S2 antigens sold under the trademark Sci-B-Vac) contain less than 5% non-adsorbed antigen. As shown in Table 3, the amount of non-adsorbed antigen in a formulation containing the same amount of antigen used in the prophylactic vaccine, 10 μg / mL of HBsAg antigen with all three of the S, Pre-S1 and Pre-2 domains, and aluminum phosphate adjuvant rather than aluminum hydroxide, was not increased, and in fact, there was no non-adsorbed antigen in this formulation, as shown in Table 3. This was surprising, since aluminum phosphate adjuvant is known to bind HBsAg antigen weaker than aluminum hydroxide adjuvant. Nevertheless, at an antigen concentration of 10 μg / mL of HBsAg, similar levels of adsorption were seen when using both aluminum phosphate adjuvant and aluminum hydroxide adjuvant.

[0039]

[0043] Surprisingly, however, formulations with increased concentrations of HBsAg antigen (including S, Pre-S1 and Pre-S2 proteins) and the same concentrations of aluminum phosphate adjuvant demonstrated a substantial increase in the content of non-adsorbed antigen. An aluminum content of 500 μg / mL from the aluminum phosphate adjuvant is equivalent to 2.27 mg / mL of aluminum phosphate adjuvant. With an HBsAg concentration of 20 μg / mL and 500 μg / mL aluminum present as the aluminum phosphate adjuvant, the amount of non-adsorbed antigen was 54.8%. With an HBsAg concentration of 40 μg / mL and 500 μg / mL aluminum as the aluminum phosphate adjuvant, the amount of non-adsorbed antigen was 35.8%. With an HBsAg concentration of 60 μg / mL and 500 μg / mL aluminum as the aluminum phosphate adjuvant, the amount of non-adsorbed antigen was 47.4%. No reduction in antigen adsorption was observed in formulations containing the same HBsAg antigen concentration with aluminum hydroxide adjuvant. Indeed, when aluminum hydroxide adjuvant was used, the amount of non-adsorbed antigen remained below 5% even when the antigen concentration was doubled to 20 μg / mL (see Table 3). Thus, the use of aluminum phosphate adjuvant resulted in a marked and surprising increase in non-adsorbed antigen with increasing HBsAg concentration.

[0040]

[0044] Thus, in a preferred embodiment of the present disclosure, the immunogenic composition comprises 62.5-500 μg / mL aluminum as an aluminum phosphate adjuvant. In a particularly preferred embodiment, the immunogenic composition of the present disclosure comprises 500 μg / mL aluminum as an aluminum phosphate adjuvant and HBsAg antigen having 20 μg / mL-60 μg / mL of all three of the S, Pre-S1 and Pre-S2 proteins. In a particularly preferred embodiment, the immunogenic composition of the present disclosure comprises 500 μg / mL aluminum as an aluminum phosphate adjuvant and HBsAg antigen having 20 μg / mL of all three of the S, Pre-S1 and Pre-S2 proteins.

[0041]

[0045] Using in vivo studies in mice, the inventors of the present disclosure have demonstrated that the immunogenic composition of the present disclosure is effective in enhancing Th1 response in mice. This effect is unexpected, since no enhancement of Th1 response was observed in the HBsAg composition when aluminum hydroxide adjuvant was used, even when the antigen concentration was significantly increased. Specifically, as shown in Example 3, a higher concentration of 20 μg / mL of HBsAg antigen (including S, Pre-S1 and Pre-S2 proteins) with 500 μg / mL of aluminum as aluminum hydroxide adjuvant in mice did not enhance Th1 T cell immunity compared to a commercially available preventive vaccine (with the same antigen at 10 μg / mL and 500 μg / mL of aluminum as aluminum hydroxide adjuvant). However, as further described in Examples 4 and 5, when directly compared to a prophylactic formulation of the vaccine (having the same antigen at 10 μg / mL and 500 μg / mL aluminum from an aluminum hydroxide adjuvant), the preferred immunogenic composition of the present disclosure having 20 μg of antigen formulated with 500 μg / mL aluminum from an aluminum phosphate adjuvant elicited a significantly higher Th1 cell response. Furthermore, as described in Example 6, when directly compared to a prophylactic formulation of the vaccine (having the same antigen at 10 μg / mL and 500 μg / mL aluminum from an aluminum hydroxide adjuvant), all of the immunogenic compositions of the present disclosure having 20 μg, 40 μg, and 60 μg of antigen formulated with 500 μg / mL aluminum from an aluminum phosphate adjuvant elicited a higher Th1 cell response. These results indicate that simply altering the adjuvant:antigen ratio in the preferred immunogenic composition of the present disclosure was not sufficient to enhance Th1 T cell immunity. Rather, varying both the antigen concentration and the type of adjuvant (aluminum phosphate adjuvant rather than aluminum hydroxide adjuvant) was necessary to enhance Th1 T cell immunity. Enhancement of Th1 immunity was only seen in compositions with increasing amounts of non-adsorbed antigen.

[0042]

[0046] The present disclosure also provides a use of the immunogenic composition of the present disclosure for preparing a pharmaceutical composition for inducing or enhancing a Th1 cell immune response against hepatitis B infection in a patient. The present disclosure further provides a use of the immunogenic composition of the present disclosure for preparing a medicament for treating hepatitis B infection, particularly chronic hepatitis B infection, in a patient.

[0043]

[0047] The present disclosure also provides pharmaceutical compositions useful for therapeutic applications in individuals suffering from chronic hepatitis B infection.

[0044]

[0048] In certain embodiments, provided pharmaceutical compositions may be formulated for parenteral delivery, e.g., by injection. In these embodiments, the formulation may be suitable for intramuscular injection.

[0045]

[0049] In some embodiments, the pharmaceutical composition is provided in a liquid form suitable for injection. In some embodiments, the pharmaceutical composition is provided as a powder (e.g., lyophilized and / or sterilized), optionally under vacuum, which is reconstituted with an aqueous diluent (e.g., water, buffer, saline, etc.) before injection. In some embodiments, the pharmaceutical composition is diluted and / or reconstituted in water, gel, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, etc. In some embodiments, the powder must be mixed gently with the aqueous diluent (e.g., without shaking).

[0046]

[0050] In some embodiments, the pharmaceutical compositions provided include one or more pharma- ceutically acceptable excipients (e.g., preservatives, inert diluents, dispersing agents, surfactants and / or emulsifiers, buffers, etc.). Suitable excipients include, for example, water, saline, dextrose, sucrose, trehalose, glycerol, ethanol, or the like, and combinations thereof. Remington's The Science and Practice of Pharmacy, 21st ed., AR Gennaro, (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. Use of any conventional excipient vehicle is considered within the scope of the present disclosure, except insofar as it is incompatible with the substance or its derivatives, such as by producing some undesirable biological effect, or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition. In some embodiments, the pharmaceutical composition includes one or more preservatives. In some embodiments, the pharmaceutical composition does not include a preservative.

[0047]

[0051] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dose to be administered to a subject and / or a convenient fraction of such a dose, such as, for example, one-half or one-third of such a dose.

[0048]

[0052] The pharmaceutical compositions described herein will generally be administered in such amounts and for such periods of time as necessary or sufficient to induce or enhance a Th1 immune response in a subject. The dosing regimen may consist of a single dose or multiple doses over a period of time. The exact amount of the composition administered may vary between subjects and may depend on several factors. It will therefore be recognized that the exact dose used will generally be determined by the prescribing physician and will depend on the subject's weight and route of administration, as well as the subject's age and the severity of symptoms. In certain embodiments, a particular amount of the vaccine composition is administered as a single dose. In certain embodiments, a particular amount of the composition is administered as multiple doses.

[0049]

[0053] The present disclosure also provides a method of inducing or enhancing a T cell immune response against Hepatitis B infection in a mammal, comprising administering to the mammal a composition of the present disclosure. The immune response is preferably a Th1 response against Hepatitis B antigens. Administration can be performed by any means, for example, injection by intramuscular injection. Injection can be performed with a conventional syringe and needle or any other suitable device available to one of skill in the art.

[0050]

[0054] The present disclosure also provides a method of treating hepatitis B infection, particularly chronic hepatitis B infection, in a subject in need thereof, comprising administering a therapeutically effective amount of a composition of the present disclosure. Administration can be performed by any means, for example, injection by intramuscular injection. Injection can be performed with a conventional syringe and needle or any other suitable device available to one of skill in the art.

[0051]

[0055] If necessary, the method or use of the present disclosure may be performed in combination with one or more conventional therapeutic treatments for hepatitis B infection and / or hepatitis B mediated disease. The administration of the composition of the present disclosure may precede, be simultaneous with, or be subsequent to the administration of the composition of the present disclosure. The therapeutic treatment that may be combined with the composition of the present disclosure may be administered before or simultaneously with the administration of the immunogenic composition of the present disclosure for the purpose of reducing the amount of hepatitis B virus. Representative examples of hepatitis B treatments that may be combined with the composition of the present disclosure include, but are not limited to, polymerase inhibitors, RNase H inhibitors, nucleoside analogs, nucleotide analogs, TLR agonists, N-glycosylation inhibitors, siRNAs, antisense oligonucleotides, anti-hepatitis B antibodies, capsid inhibitors, core protein inhibitors, core assembly modulators, S antigen reducers or sequesterers including nucleic acid polymers, cccDNA inhibitors, interferons, and immune modulators. Such standard treatments may vary between patients, but the most common hepatitis B treatments include nucleotide or nucleoside analogs (such as lamivudine, entecavir, telbivudine, adefovir, dipivoxil, or tenofovir) with or without cytokines (e.g., IFNα, pegylated IFNα2a, and pegylated IFNα2b). However, newer treatments for reducing hepatitis B viral load and / or levels of secreted HBsAg present in plasma or serum, such as siRNA or S antigen sequestration agents (such as REP-2139, a nucleic acid polymer drug candidate produced by Replicor, Inc.), may be effectively combined with the compositions of the present disclosure. Hepatitis B treatments may be provided in a single dose or, alternatively, in multiple doses according to standard procedures, dosages, and regimens over the course of hours, days, weeks, and / or months.

[0052]

[0056] The compositions of the present disclosure may also include additional adjuvants.

[0053]

[0057] The present invention has been described in an illustrative manner, and numerous modifications and variations of the invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

[0054]

[0058] All disclosures of the patents, publications, and database entries cited above are specifically incorporated by reference herein in their entireties to the same extent as if each individual patent, publication, or entry was specifically and individually indicated to be incorporated by reference. EXAMPLES

[0055]

[0059] The following examples describe some typical methods of making and performing certain compositions described herein. It should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the compositions and methods described herein.

[0056] Example 1: Preparation of vaccine formulations

[0060] Hepatitis B surface antigen consisting of all three proteins, S, pre-S1 and pre-S2, was prepared in CHO cells according to the method described in US Pat. No. 5,242,812.

[0057]

[0061] Vaccine formulations containing aluminum hydroxide adjuvant were prepared as follows. Briefly, two different concentrations of HBsAg (10 mcg / mL and 20 mcg / mL) were mixed with aluminum hydroxide adjuvant (Alhydrogel®) with an aluminum content of 500 mcg / mL for a period of 16±4 hours at room temperature. More specifically, sucrose phosphate buffer and HBsAg were added to a sterile glass vial and mixed gently with a pipette. A small stir bar was added to a separate sterile glass vial, followed by the addition of 0.9% saline and Alhydrogel adjuvant while stirring at 100±50 rpm. While stirring the 0.9% saline and Alhydrogel, a mixture of PBS and HBsAg was slowly added using a 20-200 μL size pipette (see Table 1 below). A stopper or leak-proof lid was added to each vial and sealed securely. The Alhydrogel adjuvant vials were left to agitate at 100±50 rpm for 16±4 hours at room temperature (15-25° C.) Once agitation was completed, the vaccine formulations were stored at 2-8° C. until analysis or immunization.

[0058] [Table 3]

[0059]

[0062] Vaccine formulations containing aluminum phosphate adjuvant (Adjuphos®) were prepared as follows. Briefly, two different concentrations of HBsAg (10 μg / mL and 20 mcg / mL) were mixed with 500 μg / mL aluminum (Adjuphos) as the aluminum phosphate adjuvant and rotated at 8-12 rpm for 60 minutes at room temperature. More specifically, Adjuphos adjuvant was added to a sterile container followed by 10 mM 8% sucrose phosphate buffer. HBsAg was added to the same container and mixed slowly by aspirating with a pipette (see Table 2 below). The container was sealed, covered with aluminum foil, and then rotated at 8-12 rpm for 60 minutes at room temperature. Once mixing was complete, the vaccine formulations were stored at 2-8°C until analysis or immunization.

[0060] [Table 4]

[0061] Example 2: Evaluation of antigen adsorption to adjuvants

[0063] Hepatitis B surface antigen binding to aluminum hydroxide and aluminum phosphate adjuvants was measured as follows. Briefly, in a sterile biosafety cabinet, 500 μL of each vaccine formulation was aseptically transferred to a sterile polypropylene centrifuge tube after mixing by pipetting 10-20 times before transfer to ensure homogeneity of the solution. All aliquots were then centrifuged at 14,000 g for 120 min. The supernatant (450 μL) was carefully removed from the centrifuged tube containing the immunogenic formulation using a 20-200 μL pipette and transferred to a new sterile polypropylene centrifuge tube. The centrifuged tube containing the immunogenic formulation pellet was resuspended by adding an equal volume of buffer to the volume removed (450 μL). The tubes were appropriately labeled (centrifuged immunogenic formulation supernatant vs. resuspended pellet). The diluted pellet (containing bound HBsAg) and supernatant (containing free HBsAg) were stored in a refrigerator at 2-8°C. Table 3 below demonstrates the effect of HBsAg antigen concentration, choice of aluminum adjuvant and adjuvant dose on the amount of unbound / free HBsAg.

[0062] [Table 5]

[0063]

[0064] Test material 6 (TA6) was the commercially available prophylactic Sci-B-Vac vaccine, and there was no detectable non-adsorbed (free) HBsAg detectable in the supernatant. Varying the antigen-to-adjuvant ratio by increasing the antigen concentration four-fold (TA4) did not substantially change the amount of non-adsorbed antigen (2.5%). Similarly, varying the antigen-to-adjuvant ratio by decreasing the amount of aluminum hydroxide adjuvant (Alhydrogel) did not increase the amount of non-adsorbed antigen by more than 5% (TA1-3).

[0064]

[0065] When the concentration of HBsAg was kept at 10 μg / mL, changing the adjuvant used in the immunogenic composition from aluminum hydroxide adjuvant (Alhydrogel) (TA6) to aluminum phosphate adjuvant (Aduphos) (TA12) similarly failed to increase the amount of non-adsorbed HBsAg (0% detection). Surprisingly, however, by increasing the concentration of antigen two-fold (TA11) and / or decreasing the concentration of aluminum phosphate adjuvant (TA7-10), the amount of non-adsorbed HBsAg was substantially increased by more than 30%. In particular, the 20 μg / mL concentration of antigen demonstrated extremely high amounts of non-adsorbed HBsAg when formulated with 500 μg / mL of aluminum from the aluminum phosphate adjuvant. This adjuvant concentration, specifically 500 μg / mL, is widely used in commercially available HBsAg vaccines, albeit with aluminum hydroxide adjuvant rather than aluminum phosphate adjuvant.

[0065]

[0066] In summary, there is no non-adsorbed HBsAg in the commercially available prophylactic vaccine known as Sci-B-Vac (TA6), whereas an embodiment of the present disclosure (TA11) having 20 μg / mL HBsAg and 500 μg / mL aluminum as an aluminum phosphate adjuvant has more than 50% non-adsorbed HBsAg. Changes in both the type of aluminum-based adjuvant and the concentration of antigen were required to obtain amounts of non-adsorbed antigen greater than 30%.

[0066] Example 3: Evaluation of Th1 T cell immunity in mice after vaccination with two different immunogenic compositions containing HBsAg and aluminum hydroxide antigens, including S, Pre-S1 and Pre-S2

[0067] This example describes the evaluation of T cell responses in mice after immunization with two different immunogenic formulations (TA5 and TA6 in Table 3) containing different concentrations of antigen (HBsAg with S, Pre-S1 and Pre-S2 proteins) and 500 μg / mL aluminum from aluminum hydroxide adjuvant. Balb / c mice were vaccinated three times at weeks 0, 3, 10 (or 13) with 1 / 20 of the human dose formulation of vaccine (i.e., 0.5 μg of antigen from TA6 and 1.0 μg of antigen from TA5). Mice were sacrificed 6 days after the third vaccination and responses against pre-S1, pre-S2 and HBsAg S proteins were measured using overlapping peptide pools by enzyme-linked immunospot assay ("ELISPOT") as described below. Comparable anti-HBsAg antibody responses were induced by the two formulations (data not shown).

[0067]

[0068] IFN-γ ELISPOT analysis was performed to measure Th1 T cell responses as follows: Mice were divided into groups of 8 mice each immunized with the two test formulations described above. Each test formulation had the same concentration of aluminum hydroxide adjuvant but different concentrations of HBsAg antigen. Four mice per group were sacrificed and the spleens were removed. Spleens of individual mice were processed to make single cell suspensions. Red blood cells were lysed using a commercial buffer (BioLegend). Splenocytes were then cultured at 6×10 6 The spleen cells were resuspended at 1.5x10 splenocytes / mL. One day prior to spleen harvest and processing, ELISPOT plates were coated with interferon-gamma (IFN-γ) capture antibody. No peptide, actin peptide mix, Pre-S1 peptide mix, Pre-S2 peptide mix, HBsAg peptide mix, and phorbol 12-myristate 13-acetate and ionomycin (PMA / iono) were selected as stimuli. On the day of spleen harvest, stimuli were added to the designated ELISPOT plate wells. 1.5x10 5 3×10 splenocytes were added to PMA / iono wells. 5splenocytes were added to all other stimuli. The ELISPOT plates were then placed in a humidified 5% CO2 incubator at 37°C for 40-48 hours. After incubation, the plates were washed to remove splenocytes, stimuli and media, and IFN-γ capture antibody was added, followed by streptomycin-horseradish peroxidase (strep-HRP). The plates were finally developed with commercially available 3-amino-9-ethylcarbazole (AEC) substrate (BD BioSciences). The observed spots were counted using an ELISPOT plate reader, and the final data was reported as spot forming cells (SFC) per 1,000,000 splenocytes. Table 4 below shows the results of this immunogenicity study.

[0068] [Table 6]

[0069]

[0069] Table 4 demonstrates that higher amounts of HBsAg antigen in formulations containing aluminum hydroxide as an adjuvant did not show improvement in Th1 T cell responses. This result is consistent with the data in Table 2, which showed that both formulations did not have non-adsorbed antigen.

[0070] Example 4: Comparison of T cell immunity in mice following vaccination with a commercially available prophylactic vaccine known as Sci-B-Vac (10 μg / mL HBsAg with S, Pre-S1 and Pre-S2 and 500 μg / mL aluminum as an aluminum hydroxide adjuvant) and a formulation of the present disclosure containing 20 μg HBsAg and 500 μg / mL aluminum as an aluminum phosphate adjuvant. This example describes the evaluation of T cell responses in mice after vaccination with two different immunogenic compositions. The first composition was a commercially available prophylactic vaccine known as Sci-B-Vac (TA6 in Table 3) containing 10 μg / mL HBsAg with S, Pre-S1 and Pre-S2 proteins and 500 μg / mL aluminum as an aluminum phosphate adjuvant. The second composition was an immunogenic composition of the present disclosure (TA11 in Table 3) containing a two-fold higher concentration of the same HBsAg (20 μg / mL) and 500 μg / mL aluminum as an aluminum phosphate adjuvant. Balb / c mice were vaccinated three times at weeks 0, 3, 6 (or 8) with 1 / 20 of the human dose of each immunogenic composition (i.e., 0.5 μg of antigen for TA6 and 1.0 μg of antigen for TA11). Mice were sacrificed 6 days after the third vaccination and responses to pre-S1, pre-S2 and HBsAg proteins were measured using overlapping peptide pools by ELISPOT as described above. Comparable anti-HBsAg antibody responses were induced by the two formulations.

[0071] [Table 7]

[0072] As can be seen from Table 5, the immunogenic composition of the present disclosure containing 20 μg HBsAg and 500 μg / mL aluminum as an aluminum phosphate adjuvant stimulates a substantially greater Th1 cell response against Pre-S2 and S antigens than the commercially available prophylactic version of the vaccine formulated with a much lower concentration of the same antigen (10 μg / mL) and aluminum hydroxide adjuvant. This result is consistent with the data in Table 2, which shows a high percentage of unbound antigen (54.8%) in the immunogenic composition of the present disclosure (TA11) compared to no unbound antigen in the commercially available prophylactic HBsAg vaccine (TA6). The response appears to be greatest against Pre-S2. No response was detected against the preS1 antigen in either formulation.

[0073] Example 5: Comparison of individual IgG1 / IgG2a ratios in mice following vaccination with a commercially available prophylactic vaccine known as Sci-B-Vac (10 μg / mL HBsAg with S, Pre-S1 and Pre-S2 and 500 μg / mL aluminum as an aluminum hydroxide adjuvant) and a formulation of the present disclosure containing 20 μg HBsAg and 500 μg / mL aluminum as an aluminum phosphate adjuvant.

[0072] This example describes a comparison of individual IgG1 / IgG2a ratios in mice after vaccination with two different immunogenic compositions. The first composition was a commercially available prophylactic vaccine known as Sci-B-Vac (TA6 in Table 3) containing 10 μg / mL HBsAg with S, Pre-S1 and Pre-S2 proteins and 500 μg / mL aluminum as an aluminum hydroxide adjuvant. The second composition was an immunogenic composition of the present disclosure (TA11 in Table 3) containing a two-fold higher concentration of the same HBsAg (20 μg / mL) and 500 μg / mL aluminum as an aluminum phosphate adjuvant. Balb / c mice (n=8) were vaccinated three times at weeks 0, 3 and 6 with 1 / 20 of the human dose of each immunogenic composition (i.e., 0.5 μg of antigen for TA6 and 1.0 μg of antigen for TA11). Mice were sacrificed 6 days after the third vaccination and anti-HBs IgG1 and anti-HBs IgG2 were measured by ELISA as follows.

[0074] Anti-HBs IgG1: 96-well plates were coated with recombinant Hepatitis B surface antigen protein, Abcam (0.25 μg / mL in DPBS) overnight at 4° C. The next day, plates were blocked with 10% goat serum in ELISA wash buffer (0.05% Tween-20 in PBS) for 1 hour at 37° C. Plates were washed with wash buffer, then 2-fold dilutions of individual mouse sera were added; starting at 1:10,000 and ending at 1:128,000. Plates were incubated for 1.5 hours at 37° C., after which plates were washed and secondary antibody was added. Goat anti-mouse IgG1 (Bethyl) diluted 1:10,000 in 10% goat serum in ELISA wash buffer was added and plates were incubated for 1.5 hours at 37° C. TMB One component Microwell substrate was added to the plates, incubated for 10 minutes at room temperature, and then stop solution was added. Absorbance was read at 450 nm using a MAXline plate reader.

[0075] Anti-HBs IgG2a: 96-well plates were coated with recombinant Hepatitis B surface antigen protein, Abcam (0.25 μg / mL in DPBS) overnight at 4° C. The next day, plates were blocked with 10% goat serum in ELISA wash buffer for 1 hour at 37° C. Plates were washed with wash buffer, then 2-fold dilutions of individual mouse sera were added; starting at 1:5,000 and ending at 1:640,000. Plates were incubated at 37° C. for 1.5 hours, after which plates were washed and secondary antibody was added. Goat anti-mouse IgG2a (Bethyl) diluted 1:10,000 in 10% goat serum in ELISA wash buffer was added and plates were incubated at 37° C. for 1.5 hours. TMB One component Microwell substrate was added to the plates, incubated at room temperature for 10 minutes, and then stop solution was added. Absorbance was read at 450 nm using a MAXline plate reader.

[0076]

[0075] The results are shown in Table 6 below.

[0077] [Table 8]

[0078] As can be seen from Table 6, the immunogenic composition of the present disclosure containing 20 μg HBsAg and 500 μg / mL aluminum as an aluminum phosphate adjuvant stimulates a significantly lower IgG1 to IgG2a ratio. Since IgG2a stimulation is a marker for Th1 activity, the alteration in this ratio indicates that the composition containing 20 μg HBsAg and 500 μg / mL aluminum as an aluminum phosphate adjuvant elicits a stronger Th1 response than the composition with 20 μg HBsAg and 500 μg / mL aluminum as an aluminum phosphate adjuvant.

[0079] Example 6: Dose ranging study in mice following vaccination with a commercially available prophylactic vaccine known as Sci-B-Vac (10 μg / mL HBsAg with S, Pre-S1 and Pre-S2 and 500 μg / mL aluminum as an aluminum hydroxide adjuvant) and three different doses of a composition of the present disclosure containing 20 μg, 40 μg and 60 μg of HBsAg and 500 μg / mL aluminum as an aluminum phosphate adjuvant. This example describes a dose ranging study investigating Th1 activity in mice following vaccination with a commercially available prophylactic vaccine known as Sci-B-Vac (TA6 in Table 3) containing 10 μg / mL HBsAg with S, Pre-S1 and Pre-S2 proteins and 500 μg / mL aluminum as an aluminum hydroxide adjuvant, as well as three different doses of the same HBsAg immunogenic composition of the present disclosure and 500 μg / mL aluminum as an aluminum phosphate adjuvant. The three different doses were HBsAg (20 μg / mL) (TA11 in Table 3); HBsAg (40 μg / mL) (TA10 in Table 3) and HBsAg (60 μg / mL) (TA13 in Table 3).

[0080] Briefly, Balb / c mice (n=8 / group) were vaccinated intramuscularly three times at 3-week intervals with a mouse dose of each composition, specifically 3 μg, 2 μg, and 1 μg. Splenocytes were harvested from mice 7 days after the third vaccination and stimulated with overlapping peptide pools specific for preS1, Pre-S2, and HBsAg. IFN-γ-secreting T cell responses were assessed by ELISPOT after 48 hours of culture as follows: One day before spleen collection and processing, ELISpot plates (Millipore) were coated with 100 μL of interferon-gamma (IFN-γ) capture antibody (Mabtech) at a concentration of 15 μg / mL and incubated overnight at 4° C.

[0081] On the day of spleen collection and processing, coated ELISpot plates were washed 5 times with 200 μL sterile PBS and blocked with 100 μL R10 media for 1-2 hours. Once splenocytes were isolated and counted, R10 blocking media was removed and 50 μL splenocytes (300,000 cells) and 50 μL stimuli were plated onto the ELISpot assay plate. Splenocytes from each mouse were stimulated in duplicate with the following stimuli: PreS1 (final stimuli concentration = 13.5 μg / mL), Pre-S2 (final stimuli concentration = 5.5 μg / mL) and HBsAg (final stimuli concentration = 27 μg / mL), R10 as a negative control and phorbol 12-myristate 13-acetate and ionomycin PMA (20 ng / mL) / ionomycin (1 μg / mL) as positive controls. The ELISPOT plates were then placed in a humidified, 5% CO2 incubator at 37°C for 40-48 hours. After incubation, the plates were washed five times with 200 μL PBS-Tween to remove splenocytes, stimuli and vehicle, and 100 μL of IFN-γ capture antibody (Mabtech) at a concentration of 1 μg / mL was then added to each well. After 2 hours of incubation, the ELISpot plates were washed five times with PBS-Tween, and 100 μL of 1:1000 diluted streptomycin-horseradish peroxidase (strep-HRP) was added to each well. The plates were then incubated for an additional hour and developed by adding 100 μL of 3-amino-9-ethylcarbazole (AEC) substrate (BD BioSciences) for 30 minutes at room temperature. The observed spots were counted by ZellNet Consulting and the final data was reported as spot forming units (SFC) per 1,000,000 splenocytes. The Th1 activity of the different compositions measured by ELISPOT is shown in Table 7.

[0082] [Table 9]

[0083] As can be seen in Table 7, when compared with 10 μg / mL HBsAg with S, Pre-S1 and Pre-S2 proteins and 500 μg / mL aluminum as an aluminum hydroxide adjuvant (TA6 in Table 3), 20 μg, 40 μg and 60 μg doses of a composition comprising HBsAg with S, Pre-S1 and Pre-S2 proteins and 500 μg / mL aluminum as an aluminum phosphate adjuvant were able to elicit a higher number of Pre-S2 and HBsAg-specific IFN-γ secreting T cells by ELISPOT. Thus, each of the three doses of the immunogenic composition of the present disclosure induced a higher T cell response than a composition comprising 10 μg / mL HBsAg with S, Pre-S1 and Pre-S2 proteins and 500 μg / mL aluminum as an aluminum hydroxide adjuvant.

[0084] [Other embodiments]

[0081] Other embodiments of the present disclosure will be apparent to those skilled in the art from a consideration of this specification or practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the present disclosure being indicated by the following claims. The contents of any references referred to herein are incorporated herein by reference in their entirety.

[0085] [CROSS REFERENCE TO RELATED APPLICATIONS]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 760,439, filed November 13, 2018, the contents of which are incorporated herein by reference in their entirety.

Claims

1. (a) an HBsAg antigen comprising an S protein, a Pre-S1 protein and a Pre-S2 protein; and (b) Aluminum phosphate adjuvant 1. An immunogenic composition comprising: said composition comprising at least 20 μg / mL HBsAg antigen and wherein the amount of non-adsorbed antigen is at least 30%.

2. The immunogenic composition of claim 1, wherein the aluminum is present at a concentration of 500 μg / mL.

3. 3. The immunogenic composition of claim 1 or 2, wherein the HBsAg antigen is present at a concentration of 20 μg / mL.

4. 3. The immunogenic composition of claim 1 or 2, wherein the HBsAg antigen is present at a concentration of 40 μg / mL.

5. 3. The immunogenic composition of claim 1 or 2, wherein the HBsAg antigen is present at a concentration of 60 μg / mL.

6. An immunogenic composition according to any one of claims 1 to 5 for use in inducing a Th1 cell response in a mammal.

7. Use of the immunogenic composition according to any one of claims 1 to 5 for the manufacture of a medicament for inducing a Th1 cell response in a subject.

8. The use according to claim 7, wherein the subject is infected with Hepatitis B.

9. A pharmaceutical composition comprising the immunogenic composition of any one of claims 1 to 5 and a pharma- ceutically acceptable excipient.

10. 10. The pharmaceutical composition of claim 9 for use in treating a subject suffering from Hepatitis B.

11. A method of inducing a Th1 cell response in a mammal, comprising the step of administering an immunogenic composition according to any one of claims 1 to 5.

12. A method of treating Hepatitis B in a subject comprising administering to the subject a therapeutically effective amount of the immunogenic composition of any one of claims 1 to 5.

13. 13. The method of claim 12, wherein an additional Hepatitis B treatment is administered to the subject before, simultaneously with, or after administering the immunogenic composition to the subject.

14. 14. The method of claim 13, wherein the additional Hepatitis B treatment is a nucleoside inhibitor or pegylated interferon alpha.

15. 14. The method of claim 13, wherein the additional Hepatitis B therapy is a polymerase inhibitor, an RNase H inhibitor, a TLR agonist, an N-glycosylation inhibitor, an antisense oligonucleotide, an anti-Hepatitis B antibody, a capsid inhibitor, a core protein inhibitor, a core assembly modulator, an S antigen reducer or sequestrant, a nucleic acid polymer, a cccDNA inhibitor, an interferon, an immune modulator, or an siRNA.

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