阅读说明：本技术 用于治疗乙型肝炎的免疫原性组合物 (Immunogenic compositions for the treatment of hepatitis B ) 是由 大卫·伊万德·安德森 坦维尔·艾哈迈德 于 2019-11-13 设计创作，主要内容包括：本公开内容提供了可用于在患有乙型肝炎的对象中诱导The细胞应答的组合物和方法。如本文所述,本公开内容的组合物包含具有S、前S1和前S2蛋白的HBsAg和磷酸铝佐剂。在一个优选实施方案中,所述免疫原性组合物包含至少20μg/ml的HBsAg抗原,并且未吸附抗原的量为至少30％。(The present disclosure provides compositions and methods useful for inducing The cellular response in a subject having hepatitis b. As described herein, compositions of the disclosure comprise HBsAg with S, pre-S1 and pre-S2 proteins and an aluminum phosphate adjuvant. In a preferred embodiment, the immunogenic composition comprises at least 20 μ g/ml HBsAg antigen and the amount of unadsorbed antigen is at least 30%.)

1. An immunogenic composition comprising:

(a) an HBsAg antigen comprising the S protein, pre-S1 protein and pre-S2 protein; and

(b) an aluminium phosphate adjuvant is added to the adjuvant,

wherein the composition comprises at least 20 μ g/ml HBsAg antigen and the amount of unadsorbed antigen is at least 30%.

2. The immunogenic composition of claim 1, wherein aluminum is present at a concentration of 500 μ g/ml.

3. The immunogenic composition of claim 1 or 2 wherein the HBsAg antigen is present at a concentration of 20 μ g/ml.

4. The immunogenic composition of claim 1 or 2 wherein the HBsAg antigen is present at a concentration of 40 μ g/ml.

5. The immunogenic composition of claim 1 or 2 wherein the HBsAg antigen is present at a concentration of 60 μ g/ml.

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

7. Use of the immunogenic composition of 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 of claim 7, wherein the subject has been infected with hepatitis B.

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

10. The pharmaceutical composition of claim 9, for treating a subject having hepatitis b.

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

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

13. The method of claim 12, wherein an additional hepatitis b treatment is administered to the subject before, concurrently with, or after the administration of the immunogenic composition to the subject.

14. The method of claim 13, wherein the additional hepatitis b treatment is a nucleoside inhibitor or pegylated interferon alfa.

15. The method of claim 13, wherein the additional hepatitis b treatment is a polymerase inhibitor, RNase H inhibitor, TLR agonist, N-glycosylation inhibitor, antisense oligonucleotide, anti-hepatitis b antibody, capsid inhibitor, core protein inhibitor, core assembly modulator, S antigen reducing or chelating agent, nucleic acid polymer, ccc DNA inhibitor, interferon, immune modulator, or siRNA.

Technical Field

The present invention is in the field of immunogenic compositions, particularly those useful for treating hepatitis b infection.

Background

Hepatitis b is a viral infection that is the cause of acute and chronic liver disease. It is transmitted in humans by contact with blood or other body fluids from infected individuals. Hepatitis b infection is a widespread and significant health problem that causes more than 2.5 million people worldwide each year to suffer from chronic liver disease and nearly 100 million deaths (2017 global hepatitis report, world health organization). After infection, hepatitis b causes an acute phase disease with no symptoms in most people. Those exhibiting symptoms experience jaundice, fatigue, and abdominal pain, with a small percentage of patients experiencing acute liver failure, which can be fatal. After hepatitis b infection, the patient's immune system can clear the virus resulting in a cure, or the patient can develop chronic hepatitis b infection. The consequences of chronic hepatitis b are significant, since 30% of chronically infected adults eventually develop cirrhosis and/or liver cancer. The likelihood of developing chronic disease decreases with age, with infants having a 90% chance of chronic infection and healthy adults having a 5% -10% chance.

Although there has been an effort to provide vaccines for hepatitis b infection, there is still a need for improved treatments for chronic hepatitis b patients, in particular treatments capable of eliciting enhanced cellular immune responses in a subject.

Disclosure of Invention

Various embodiments are provided that relate to immunogenic compositions comprising an HBsAg antigen comprising 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.

In some embodiments, the disclosure provides immunogenic compositions comprising an HBsAg envelope antigen comprising S, pre-S1 and pre-S2 domains (e.g., 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 at least about 20 μ g/ml (e.g., at least about 30 μ g/ml, at least about 40 μ g/ml, at least about 50 μ g/ml, or at least about 60 μ g/ml) of an HBsAg envelope antigen comprising S, pre-S1, and pre-S2 domains, and an aluminum phosphate adjuvant, 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 a particularly preferred embodiment, the present disclosure provides a vaccine formulation comprising about 20 μ g/ml to 60 μ g/ml (e.g., about 20 μ g/ml, about 30 μ g/ml, about 40 μ g/ml, about 50 μ g/ml, or about 60 μ g/ml) of HBsAg antigen containing S, pre-S1, and pre-S2 domains, 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).

In some embodiments, the immunogenic compositions of the present disclosure comprise 62.5 μ g/ml to 500 μ g/ml of aluminum present as an aluminum phosphate adjuvant. In a preferred embodiment, the immunogenic composition of the present disclosure comprises 500 μ g/ml of aluminum as an aluminum phosphate adjuvant. In a particularly preferred embodiment, the immunogenic composition of the disclosure comprises 500 μ g/ml of aluminium as an aluminium phosphate adjuvant and 20 μ g/ml of HBsAg antigen with all three of the S, pre-S1 and pre-S2 proteins.

In another embodiment, the 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 μ g/ml of HBsAg envelope antigen comprising S, pre-S1 and pre-S2 proteins, and an aluminium phosphate adjuvant, wherein 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 from 20 μ g/ml to 60 μ g/ml of an HBsAg antigen comprising S, pre-S1 and pre-S2 proteins, and an aluminium phosphate adjuvant, wherein the amount of unbound antigen is at least 30%. In some embodiments, an immunogenic composition for eliciting a Th1 response in a mammal comprises 62.5 μ g/ml to 500 μ g/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 μ g/ml of aluminium as an aluminium phosphate adjuvant and 20 μ g/ml to 60 μ g/ml of HBsAg antigen containing 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 μ g/ml of aluminium as an aluminium phosphate adjuvant and 20 μ g/ml of HBsAg antigen containing S, pre-S1 and pre-S2 proteins.

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 μ g/ml of HBsAg envelope antigen comprising S, pre-S1 and pre-S2 proteins, and an aluminium phosphate adjuvant, wherein the amount of unbound antigen is at least 30%. In a particularly preferred embodiment, the present disclosure provides an immunogenic composition for the treatment of hepatitis b in a human subject in need thereof, the composition comprising from 20 μ g/ml to 60 μ g/ml of an HBsAg antigen comprising S, pre-S1 and pre-S2 proteins, and an aluminium phosphate adjuvant, wherein 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 μ g/ml to 500 μ g/ml of aluminum present as an aluminum phosphate adjuvant. In a preferred embodiment, the immunogenic composition for treating hepatitis b in a human subject in need thereof comprises 500 μ g/ml of aluminum as an aluminum phosphate adjuvant and 20 μ g/ml to 60 μ g/ml of HBsAg antigen containing S, pre-S1 and pre-S2 proteins. In a particularly preferred embodiment, the immunogenic composition for the treatment of hepatitis b in a human subject in need thereof comprises 500 μ g/ml of aluminium as an aluminium phosphate adjuvant and 20 μ g/ml of HBsAg antigen containing S, pre-S1 and pre-S2 proteins.

The present disclosure also includes the use of at least one immunogenic composition of the present disclosure in the manufacture of a pharmaceutical composition intended for the treatment of hepatitis b infection.

The present disclosure also provides pharmaceutical compositions comprising the immune compositions of the present disclosure for administration to a subject in need thereof.

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.

The present disclosure also provides methods 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.

In any aspect or embodiment described herein, a composition can comprise 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., a pre-S2 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), and a pre-S1 protein (e.g., a pre-S1 protein comprising or consisting of an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3). Some embodiments described herein may include about 75% to 90% by weight S protein, about 2% to 8% by weight pre-S1 protein, and about 5% to 15% by weight pre-S2 protein (e.g., 83 +/-3.3% S protein, 6 +/-3% pre-S1 protein, and 11 +/-3% pre-S2 protein).

Other features, objects, and advantages of the disclosure will be apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating some embodiments of the present disclosure, is given by way of illustration only, not limitation. Many variations and modifications within the scope of the disclosure will become apparent to those skilled in the art in light of the detailed description.

Sequence listing

The following is a list of sequences referenced herein:

SEQ ID NO: 1 is a small protein of the HBsAG amino acid sequence:

SEQ ID NO: 2 is a moderate protein of HBsAG amino acid sequence:

SEQ ID NO: 3 is a large protein of the amino acid sequence of HBsAG:

Detailed Description

The inventors of the present application have prepared an improved therapeutic hepatitis b vaccine that effectively induces a Th1 cellular immune response.

Hepatitis B virus is a member of the genus Orthohepadnavirus of the family Hepadnaviridae (Hepadnaviridae). It is a small enveloped DNA virus that contains a nucleocapsid and an outer envelope of Hepatitis B surface antigen (HBsAg). The HBsAg antigen consists of three related envelope proteins 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 their N-terminus due to the presence of three in-frame ATG initiation codons that divide the S ORF into three regions. The S or "small" envelope protein is the most abundant and minimal, 226 amino acids. The M or "intermediate" surface protein comprises the S protein and has an additional protein domain consisting of 55 amino acids called pre-S2. The protein called L or "large" protein consists of S, pre-S2 and a third protein domain called pre-S1 with 118 amino acids. The hepatitis b virus genome is susceptible to replication errors, with the result that there are many genotypes and genetic variants. At least ten different viral genotypes have been identified, as well as a number of mutations, some of which occur in the S and pre-S domains. Thus, there are many sequence variations in each of these three protein domains.

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

No effective treatment for hepatitis b infection has been found. The acute infection is treated by lying in bed. Chronic infections are often treated with antiviral agents, in particular nucleoside-based antiviral agents, such as tenofovir (tenofovir), lamivudine (1amivudine) or entecavir (entecavir) or with pegylated interferon alpha (PEG-IFN α). Antiviral agents can slow the progression of cirrhosis and reduce the incidence of liver cancer, but they have proven ineffective in achieving viral clearance from patients. In addition, they are expensive and they must be used continuously for the life of the patient to maintain effectiveness. PEG-IFN α is associated with side effects and elicits a sustained antiviral response in only about 30% of patients. Therefore, drug treatment for chronic hepatitis b is generally only effective in slowing the progression of the disease without a successful cure. In order to clear the hepatitis b virus and thereby avoid chronic conditions, patients must mount a potent and diverse immune response. In particular, a strong and specific T cell response is essential to achieve viral clearance of hepatitis B (Bertoletti and Rivino (2014) Curr Opin infection. Dis 27: 528-. This type of response is almost exclusively observed in adult patients with a mature immune system (Bertoletti and Gehring, (2013) PLOS Path.9: 1-4).

Given the significant health impact of hepatitis b infection, much attention has been focused on the prevention of this disease. Prophylactic vaccines against hepatitis b have been used for more than 30 years, and in many countries, children are generally vaccinated against hepatitis b. The first prophylactic vaccines against hepatitis b were developed in the end of the 20 th century 70 s using plasma from hepatitis b patients. The improved hepatitis b vaccine was used in the 80' S of the 20 th century and was produced by expressing the S domain of HBsAg in yeast cells using recombinant DNA technology. These newer vaccines quickly replaced the first vaccine and remain the primary commercial vaccine on the market today. More advanced hepatitis b vaccines were developed in the 90S of the 20 th century, which were produced in mammalian cells and contain all three domains of HBsAg, specifically S, pre-S1 and pre-S2. The presence of pre-S1 and pre-S2 domains is associated with enhanced immunogenic response, and also with inhibition of viral binding and infectivity (US 5,242,812). The biological functions of the various envelope proteins and the nature of the immune response to each component (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, while antibodies against the S domain appear after weeks. This, as well as studies using pre-S1 or pre-S2 domain antigens, suggested that the pre-S1 and pre-S2 antigens could be involved in inducing T cells to help produce antibodies against the S domain (Milich et al (1985) PNAS 828170-.

Commercial preventive hepatitis b vaccines have been shown to successfully induce strong antibody responses against the S protein domain of HBsAG in healthy subjects, particularly those that are young and whose immune system is not compromised. However, attempts to use these vaccines as therapeutic treatment for chronic hepatitis b patients have proven unsuccessful. Clinical trials to measure the therapeutic effect of hepatitis B vaccines comprising the HBsAG S domain alone and both the pre-S2 and S domains have shown that these vaccines do not achieve clearance of the hepatitis B virus (Pol et al, (2001) J.hepatol 34: 917-. The failure of immunogenic therapy to treat chronic hepatitis b infection is generally attributed to "immune tolerance", a phenomenon whereby the immune system of a hepatitis b patient becomes unresponsive to further exposure to hepatitis b antigens, particularly HBsAg. Immune tolerance in patients with chronic hepatitis B is associated with the massive production of hepatitis B antigen by the virus, which leads to the depletion of T cells, particularly hepatitis B specific CD8+ T cells (Boni, C.et al, (2007) J.Virol.81: 4215-. Depleted 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 load, destruction of antigen-specific T cells can result in the complete disappearance of these cells from the liver. The result is a reduced response of T cells to stimulation by hepatitis b antigen.

In addition to the challenge of immune tolerance, hepatitis b vaccines containing only the HBsAg S protein may not achieve viral clearance from chronic patients due to their weak ability to stimulate cellular immunity through T cell responses in most subjects. To more effectively stimulate T cell responses, therapeutic vaccination studies have been conducted in chronic hepatitis b patients using a hepatitis b vaccine containing all three of the pre-S1, pre-S2, and S protein domains. These studies indicate that there are hepatitis b specific T cell responses in some patients. However, this effect was transient and did not result in disease clearance, probably because the vaccine stimulated a Th2 response but not a CD8+ T lymphocyte response. (Jung et al, (2002) Vaccine 20: 3598-. Th2 cells, characterized by secretion of IL-4, IL-5 and IL-13 cytokines, promote antibody production and are generally associated with allergic responses, while the effects against viral infection are lower than Thl cells characterized by secretion of IFN- γ. Further attempts to induce successful immune responses using DNA vaccines have also failed to achieve sustained responses (Bertoletti and Gehring (2009) exp. rev, gastro. hep.3: 561-.

Many researchers conclude that a complete cure for hepatitis b still takes many years and by using combination therapy it is possible to achieve a so-called "functional cure" so that chronic patients acquire a sustained reduction of hepatitis b virus and other disease markers and a reduced incidence of liver cancer after cessation of therapy (Lok et al, (2017) j.hep.67: 847-. Since antiviral therapy and prophylactic vaccines alone are unable to achieve a functional cure, some researchers conclude that it may be necessary to combine a prophylactic vaccine with a hepatitis b treatment that reduces viral load. A reduction in viral load can reduce the level of immune tolerance and thereby improve the ability of vaccine therapy to induce an immune response capable of controlling infection (Zoulim, f.et al (2018) Cold Spring harb.perspect.med. (2015): 5: a 21501). For example, combinations of prophylactic vaccines with nucleoside inhibitors have been attempted. However, to date, the clearance of viruses from chronic hepatitis B patients has not been successfully achieved using combination therapy with commercially available hepatitis B vaccines (Vanderapeliere et al (2007) Vaccine 51: 8585-8597). In a vietnam study, an attempt was made to improve responsiveness by combining the newer vaccine Genhevac B (pre-S1, pre-S2, and S protein vaccine with aluminum hydroxide adjuvant) with a nucleoside inhibitor (lamivudine). This combination is superior to each treatment alone in reducing viral DNA levels in the patient. However, the effect does not persist after discontinuation of the vaccine therapy. Thus, such combination therapy fails to recruit the patient's immune system to effectively clear the virus (Hoa, (2009) Antimicrob. Agents and Chemo.53: 5134-5140).

More complex triple therapies have been proposed, including antibodies that block PD-1 activity, as well as nucleoside inhibitors and prophylactic hepatitis b vaccines. A study of this triple combination in the woodchuck model using a virus similar to hepatitis B was only effective in one third of the animals (Kosinka et al, (2015) supra: 103-114; Liu et al, virology, (2014) 29: 10-16). Moreover, the efficacy of this triple has not been demonstrated in humans, and it requires the possible indefinite use of expensive treatments. Thus, even in combination with other drugs, the use of conventional prophylactic vaccines for therapeutic purposes has not proven successful as a functional cure for hepatitis b in chronic patients. Therefore, in order to obtain a sustained response in chronic hepatitis b patients, improved methods must be found to enhance the ability of these patients to elicit T cell responses against the virus.

Newer strategies for treating chronic hepatitis b using gene silencing approaches have emerged. Some examples of such therapies include the use of Small Interfering RNA (SiRNA) to interfere with viral gene expression, particularly HBsAg. Like nucleoside inhibitors, these treatments have the potential to reduce viral load and levels of secreted HBsAg (Woodell et al, (2015) mol. Ther.21: 973-. However, the effectiveness of this combination has not been determined, and in view of the failure of early antiviral agent studies, effectiveness may depend on a combination comprising an immunological agent capable of stimulating a robust Th1 cell response.

Attempts have previously been made to enhance the response of T cells to hepatitis b vaccines by altering the vaccine formulation. Most of the commercially available hepatitis B vaccines, including the widely used hepatitis B vaccine EngerixAnd a hepatitis B vaccine comprising S/pre-S1 and pre-S2Are prepared by using an aluminum hydroxide adjuvant. Aluminum hydroxide adjuvant is one of a family of aluminum-containing adjuvants, sometimes informally referred to as "alum" (although the term "alum" is more accurately used to describe hydrated potassium aluminum sulfate (KAl (SO))₄)₂·12H₂O) which is used as an early adjuvant for diphtheria toxoid but was abandoned due to manufacturing difficulties). Aluminium-based adjuvants have been used in human vaccines since 1932, and although they have a long-term record of safety, the mode of action is not fully understood. It is generally believed that aluminum-based adjuvants enhance the immune response by activating dendritic cells.

Studies have been conducted to better understand and enhance the effectiveness of aluminum-based adjuvants. The two most commonly used aluminum-based adjuvants are referred to as "aluminum phosphate" and "aluminum hydroxide", with the aluminum hydroxide adjuvant being the most widely used commercially. The aluminum hydroxide adjuvant is not Al (OH)₃But crystalline aluminum oxyhydroxide (AlOOH) having a surface area greater than that of the crystalline aluminum hydroxide. The aluminum phosphate adjuvant is actually amorphous aluminum hydroxyphosphate (Al (OH)_x(PO4)_y) Wherein some of the hydroxyl groups in the aluminum hydroxide adjuvant are replaced with phosphate groups. The surface of the aluminum phosphate adjuvant is formed by Al-OH and Al-OPO₃The radicals are formed. Although they are chemically similar, the two adjuvants have different chemical properties. Aluminum hydroxide adjuvants have a crystal structure, a large surface area, and a positive charge at neutral pH (+30 mv). The aluminum phosphate adjuvant is amorphous and negatively charged (about-20 mv) at neutral pH. Also, the aluminium phosphate adjuvant appears to dissolve more readily after injection.

Antigens are "adsorbed" onto aluminium-based adjuvants, which means that they adhere to the adjuvant to form a layer on the surface. The antigen is adsorbed onto the surface of the aluminum-based adjuvant by electrostatic interactions, van der waals forces, and ligand (mainly phosphate) exchange. Ligand exchange is the strongest attractive force and can occur even in the presence of electrostatic repulsion. HBsAg is composed mainly of phospholipids containing phosphate groups strongly adsorbed on the hydroxylated mineral surface of the adjuvant by ligand exchange between phosphate groups in the antigen and surface hydroxyl groups in the aluminium-based adjuvant. Electrostatic attraction is not the primary adsorption force of HBsAg (Iver et al, (2004) Vaccine, 22: 1475-.

Traditionally, adsorption of antigen to aluminium-based adjuvants has been deliberately maximised, since adsorption is generally considered important for immune stimulatory effects, since the antigen remains at the injection site, creating a "depot" effect, prolonging the release of antigen to the immune system. Furthermore, complete adsorption of the antigen results in greater long-term stability of the vaccine formulation during long-term storage. EngerixContains 10. mu.g HBsAg per 250. mu.g aluminium from aluminium hydroxide adjuvant. This ratio of 0.04. mu.g HBsAg/. mu.g aluminium ensures complete adsorption. However, recently, the relationship between antibodies and T cell immunity, potency and adsorption has become less clear. Studies on the impact of the tightness of HBsAg binding to aluminium hydroxide on antibody production in immunized mice indicate that the antibody response is the lowest with the most compact vaccine formulation adsorbed antigen on adjuvant. (Clapp et al (2011) J.Pharm Sci 100(2) 388-. 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 response (Egan et al, (2009) Vaccine 27: 3175-3180). Similarly, a single antigen hepatitis b vaccine containing an aluminum phosphate adjuvant elicits a greater antibody response and is effective at lower adjuvant concentrations than a similar vaccine formulated with an aluminum hydroxide adjuvant (Fazeli et al, (2008) DARU 3: 143-. At least one study showed that the enhancement of antibody responses to adjuvants does not require adsorption of antigen by aluminum-based adjuvants, leading to the conclusion that inflammation, rather than adsorption, is associated with immunogenicity (Noe et al, (2010) Vaccine 28: 3588-. Little is known about the extent to which changes in aluminium composition and amounts affect Th2 and Th 1T cell immunity, but studies have reported that higher antibody responses are believed by those skilled in the art to promote enhanced Th2 responses rather than Th1 responses.

While aluminum-based adjuvants are well known to improve immunogenicity, they are also well known to act primarily to enhance antibody production and are therefore most effective in targeting pathogens that are killed or inhibited by interaction with antibodies. They are generally thought to be ineffective in eliciting Th1 responses, but to induce inflammatory Th2 responses by improving antigen uptake and attraction of Antigen Presenting Cells (APC). Therefore, aluminum-based adjuvants have not been used to find better Th1 cellular immune stimulators.

In order to improve the cellular or "innate" immune response, newer adjuvants have been added to hepatitis b vaccines. One such novel adjuvant is endotoxin, monophosphoryl lipid a (MPL), a bacterial lipopolysaccharide. WO 93/19780 discloses that HBsAg vaccines in combination with adjuvants stimulate T cell responses. No T cell response (as measured by IL-2, IFN-. gamma.and IL-4) was observed in Balb/c mice after immunization with HBsAg adsorbed on aluminum hydroxide. However, when 3-de-O-acylated monophosphoryl lipid A (3D-MPL) was added to the formulation, a Th1 cell response (as measured by IL-2 and IFN-. gamma.) was observed. Patent US 5,972,346, subsequently filed by the same research group, discloses improved humoral immunogenicity when aluminium phosphate is used instead of aluminium hydroxide in HBsAg/3D MPL vaccine formulations. The discovery results inDevelopment of commercial vaccines sold under the trade name. Subsequent studies showed that Fendrix elicited a Th1 cellular immune response. However, Fendrix is an expensive product and is only approved in europe for patients with renal defects over 15 years of age. Furthermore, in other studies, the effect of 3D-MPL adjuvant has been shown to be transient and limited primarily to injection sites and regional lymph nodes (De Pasquale et al, Vaccine 2015).

Recently, US 20160136264 discloses a hepatitis b vaccine consisting of a hepatitis b antigen fragment and an adjuvant consisting of an immunomodulatory DNA sequence containing CpG motifs, which is capable of mediating a Thl response in mice. However, safety issues for the use of CpG-based adjuvants in hepatitis b vaccines are addressed and no marketing approval has been obtained in all jurisdictions.

The inventors of the present application have found that an immunogenic composition comprising all three HBsAg proteins (S, pre-S1 and pre-S-2) and an aluminium phosphate adjuvant is effective in stimulating a Th1 cell response in a mammal when the composition has a significant amount of unbound antigen. Physicochemical analysis of the compositions of the disclosure showed that the adjuvant bound 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 aluminum hydroxide adjuvant. It is also significantly weaker than that seen in formulations containing HBsAg antigen and aluminium phosphate adjuvant with a lower ratio of HBsAg antigen to aluminium phosphate adjuvant.

Surprisingly, the compositions of the present disclosure induce an enhanced Th1 cell response in mammals in the absence of additional newer adjuvants known to stimulate cellular immunity (e.g., MPL or CpG). These Th1 cell responses were experimentally demonstrated using different immunological markers, in particular antigen-specific IFN-. gamma.responses and an IgG2a/IgG1 ratio. This was surprising in view of previous studies showing that conventional hepatitis b vaccines formulated with aluminum-based adjuvants alone do not have a Thl response.

Even more surprising, the immunogenic compositions of the present disclosure effectively enhance Th1 cell responses when using significantly reduced aluminum phosphate adjuvant concentrations relative to the amount of antigen in the composition, as compared to commercially available prophylactic vaccines. In particular, the compositions of the present disclosure are effective in enhancing Th1 responses when the ratio of aluminum phosphate adjuvant to antigen is reduced by 50% compared to the most widely used commercial prophylactic vaccine.

The vaccine of the present disclosure comprises hbsags that comprise all three of the S, pre-S1, and pre-S2 proteins. HBsAg may be derived from any genotype, strain or isolate of hepatitis B. Furthermore, the HBsAg may be derived from a native HBsAg or a 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 sections, biopsy, etc.), cultured cells, or tissue culture collected from an infected subject. HBsAg can also be produced in cells using recombinant techniques. 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. HBsAg antigens comprising S, pre-S1 and pre-S2 domains may be produced using the methods disclosed in US 5,242,812. Nucleotide sequences encoding different HBsAgs can be found, for example, in Genbank databases and published literature (e.g., Fukimori et al (1990)18Nuc. acid Res 4587; Vaudin et al (1988)69J. 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 20 μ g/ml or more of HBsAg antigen with all three of the S, pre-S1 and pre-S2 proteins. In another preferred embodiment, the immunogenic composition of the present disclosure comprises 20 μ g/ml to 60 μ g/ml HBsAg antigen with all three of the S, pre-S1 and pre-S2 proteins.

The vaccine formulations of the present disclosure further comprise an aluminum phosphate adjuvant. An example of an aluminum phosphate adjuvant suitable for use in the present disclosure isIt is an aluminum phosphate wet gel suspension manufactured by Brenntag.

As shown in example 2, HBsAg vaccine formulations using aluminium hydroxide adjuvant (including the commercial prophylactic vaccine formulations comprising all three of the S, pre-S1 and pre-S2 antigens, sold under the Sci-B-Vac trade mark) comprise less than 5% of unadsorbed antigen. As shown in table 3, the amount of non-adsorbed antigen of the preparation comprising the same amount of antigen as used in the prophylactic vaccine, 10 μ g/ml HBsAg antigen with all three of the S, Pre S1 and Pre-2 domains and aluminum phosphate adjuvant instead of aluminum hydroxide did not increase, and in fact, as shown in table 3, there was no non-adsorbed antigen in the preparation. This is surprising because it is known that aluminium phosphate adjuvants bind less strongly to HBsAg antigen than aluminium hydroxide adjuvants. However, similar levels of adsorption were observed with both the aluminium phosphate adjuvant and the aluminium hydroxide adjuvant at an HBsAg antigen concentration of 10. mu.g/ml.

However, surprisingly, formulations with increased concentrations of HBsAg antigen (containing S, pre-S1 and pre-S2 proteins) and the same concentration of aluminum phosphate adjuvant showed a substantial increase in the content of non-adsorbed antigen. The 500. mu.g/ml aluminum content from the aluminum phosphate adjuvant is equivalent to 2.27mg/ml aluminum phosphate adjuvant. The amount of non-adsorbed antigen was 54.8% with HBsAg concentration of 20. mu.g/ml and 500. mu.g/ml in the presence of aluminum as aluminum phosphate adjuvant. The amount of non-adsorbed antigen was 35.8% with HBsAg concentration of 40. mu.g/ml and 500. mu.g/ml of aluminum as an aluminum phosphate adjuvant. The amount of non-adsorbed antigen was 47.4% with HBsAg concentration of 60. mu.g/ml and 500. mu.g/ml of aluminum as an aluminum phosphate adjuvant. No reduced antigen adsorption was observed in formulations containing the same HBsAg antigen concentration with aluminum hydroxide adjuvant. In fact, when using an aluminum hydroxide adjuvant, the amount of unadsorbed antigen was below 5% even if the antigen concentration was doubled to 20. mu.g/ml (see Table 3). Thus, the use of an aluminium phosphate adjuvant at elevated HBsAg concentrations resulted in a significant and surprising increase in unadsorbed antigen.

Thus, in a preferred embodiment of the present disclosure, the immunogenic composition comprises 62.5 μ g/ml to 500 μ g/ml of aluminium as an aluminium phosphate adjuvant. In a particularly preferred embodiment, the immunogenic composition of the disclosure comprises 500 μ g/ml of aluminium as an aluminium phosphate adjuvant and 20 μ g/ml to 60 μ g/ml of HBsAg antigen with all three of the S, pre-S1 and pre-S2 proteins. In a particularly preferred embodiment, the immunogenic composition of the disclosure comprises 500 μ g/ml of aluminium as an aluminium phosphate adjuvant and 20 μ g/ml of HBsAg antigen with all three of the S, pre-S1 and pre-S2 proteins.

Using in vivo studies in mice, the inventors of the present disclosure have demonstrated that the immunogenic compositions of the present disclosure effectively enhance Th1 responses in mice. This effect was unexpected because in HBsAg compositions using aluminium hydroxide adjuvant, no enhancement of Th1 response was observed even though the antigen concentration was significantly increased. In particular, as shown in example 3, a higher 20 μ g/ml HBsAg antigen (comprising S, pre-S1 and pre-S2 proteins) concentration at 500 μ g/ml aluminium as aluminium hydroxide adjuvant did not enhance Th 1T cell immunity in mice compared to a commercial prophylactic vaccine with 10 μ g/ml of the same antigen and 500 μ g/ml aluminium as aluminium hydroxide adjuvant. However, as further described in examples 4 and 5, a preferred immunogenic composition of the present disclosure formulated with 20 μ g antigen and 500 μ g/ml aluminum from aluminum phosphate adjuvant elicited a significantly higher Th1 cell response when compared head-to-head with a prophylactic vaccine formulation (which had 10 μ g/ml of the same antigen and 500 μ g/ml aluminum from aluminum hydroxide adjuvant). Furthermore, as described in example 6, immunogenic compositions of the present disclosure formulated with 20 μ g, 40 μ g and 60 μ g of antigen and 500 μ g/ml of aluminum from aluminum phosphate adjuvant all elicited higher Th1 cell responses when compared head-to-head to a prophylactic vaccine formulation with 10 μ g/ml of the same antigen and 500 μ g/ml of aluminum from aluminum hydroxide adjuvant. These results indicate that adjuvants are simply altered in some preferred immunogenic compositions of the present disclosure: the antigen ratio was insufficient to enhance Th 1T cell immunity. In contrast, both antigen concentration and adjuvant type (aluminum phosphate adjuvant instead of aluminum hydroxide adjuvant) need to be changed to enhance Th 1T cell immunity. An increase in Th1 immunity was observed only in compositions with increased amounts of unadsorbed antigen.

The present disclosure also provides for the use of an immunogenic composition of the present disclosure for the preparation of a pharmaceutical composition for inducing or enhancing a Th1 cellular immune response against hepatitis b infection in a patient. The present disclosure also provides the use of an immunogenic composition of the disclosure for the manufacture of a medicament for the treatment of hepatitis b infection, in particular chronic hepatitis b infection, in a patient.

The present disclosure also provides pharmaceutical compositions useful for therapeutic applications in individuals having chronic hepatitis b infection.

In certain embodiments, the provided pharmaceutical compositions can be formulated for parenteral delivery, for example by injection. In some such embodiments, the formulation may be suitable for intramuscular injection.

In some embodiments, the pharmaceutical composition is provided in a liquid dosage form suitable for injection. In some embodiments, the pharmaceutical compositions are provided as powders (e.g., lyophilized and/or sterilized), optionally under vacuum, which are reconstituted with an aqueous diluent (e.g., water, buffer, saline solution, etc.) prior to injection. In some embodiments, the pharmaceutical composition is diluted and/or reconstituted in water, a gel, a sodium chloride solution, a sodium acetate solution, a benzyl alcohol solution, phosphate buffered saline, or the like. In some embodiments, the powder should be gently mixed with the aqueous diluent (e.g., not shaken).

In some embodiments, provided pharmaceutical compositions comprise one or more pharmaceutically acceptable excipients (e.g., preservatives, inert diluents, dispersing agents, surfactants and/or emulsifiers, buffers, and the like). 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 Edition, a.r. gennaro, (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses a variety of excipients used in formulating pharmaceutical compositions and known techniques for their preparation. Unless any conventional excipient medium is incompatible with a substance or derivative thereof, e.g., by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of a pharmaceutical composition, its use is contemplated within the scope of the present disclosure. In some embodiments, the pharmaceutical composition comprises one or more preservatives. In some embodiments, the pharmaceutical composition does not comprise a preservative.

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

The pharmaceutical compositions described herein are typically administered in an amount and for a time necessary or sufficient to induce or enhance a Th1 immune response, for example, 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 to be administered may vary from subject to subject and may depend on several factors. Thus, it will be understood that, in general, the precise dose employed will be determined by the prescribing physician and will depend not only on the weight of the subject and the route of administration, but also on the age of the subject and the severity of the symptoms. In certain embodiments, a particular amount of a vaccine composition is administered in a single dose. In certain embodiments, a particular amount of a composition is administered in more than one dose.

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 antigen. Administration can be by injection by any means, for example by intramuscular injection. The injection may be performed using a conventional syringe and needle, or any other suitable device available in the art.

The present disclosure also provides a method of treating hepatitis b infection, in particular 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 by injection by any means, for example by intramuscular injection. The injection may be performed using a conventional syringe and needle, or any other suitable device available in the art.

If desired, the methods or uses 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 diseases. Administration of the compositions of the present disclosure may be performed prior to, concomitantly with, or subsequent to administration of the compositions of the present disclosure. Therapeutic treatments that may be combined with the compositions of the present disclosure may be administered prior to or concomitantly with the administration of the immunogenic compositions of the present disclosure for the purpose of reducing the hepatitis b viral load. Representative examples of hepatitis b treatments that can be combined with the compositions 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 reducing or chelating agents comprising nucleic acid polymers, ccc DNA inhibitors, interferons, and immune modulators. Although such standard of care may vary from patient to patient, the most common hepatitis b treatments include nucleotides or nucleoside analogs (such as lamivudine, entecavir, telbivudine, adefovir, dipivoxil or tenofovir) with or without cytokines (e.g., IFN α, pegylated IFN α 2a and pegylated IFN α 2 b). However, newer treatments, such as siRNA or S antigen chelators (e.g., REP-2139, a nucleic acid polymer drug candidate produced by replior inc.) for reducing the hepatitis b viral load and/or secreted HBsAg levels present in plasma or serum can be effectively combined with the compositions of the present disclosure. Hepatitis b treatment can be provided in a single dose or, alternatively, in multiple doses over hours, days, weeks, and/or months according to standard protocols, dosages, and schedules.

The compositions of the present disclosure may also comprise additional adjuvants.

The present invention has been described in an illustrative manner, and many modifications and variations of the present 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.

The disclosures of the patents, publications, and database entries cited above are hereby expressly incorporated by reference in their entirety to the same extent as if each such individual patent, publication, or entry was specifically and individually indicated to be incorporated by reference.

Examples

The following examples describe some exemplary modes of making and practicing certain compositions described herein. It is to 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.

Example 1: preparation of vaccine formulations

Hepatitis B surface antigen consisting of all three of the S, pre-S1 and pre-S2 proteins was prepared in CHO cells according to the method described in US 5,242,812.

Vaccine formulations containing an aluminum hydroxide adjuvant were prepared as follows. Briefly, at room temperature, an aluminum hydroxide adjuvant having an aluminum content of 500mcg/ml will be usedThe two different concentrations of HBsAg (10meg/ml and 20mcg/ml) were stirred for a period of 16. + -.4 hours. More specifically, sucrose phosphate buffer and HBsAg were added to a sterile glass vial and gently mixed with a pipette. To a separate sterile glass vial was added a small stir bar followed by 0.9% saline and Alhydrogel adjuvant while stirring at 100 ± 50 rpm. A mixture of PBS and HBsAg was added slowly using a 20 μ l to 200 μ l sized pipette while stirring 0.9% saline and Alhydrogel (see table 1 below). A stopper or leak-proof cap is added to each vial and securely sealed. The AlhVprogel adjuvant vial was left to stir at 100 + -50 rpm for 16 + -4 hours at room temperature (15 deg.C to 25 deg.C). Once agitation is complete, the vaccine formulation is stored at 2 ℃ to 8 ℃ until analysis or immunization.

Table 1: immunogenic compositions comprising aluminum hydroxide adjuvant

Adjuvant comprising aluminum phosphate was prepared as followsThe vaccine formulation of (1). Briefly, HBsAg (10. mu.g/ml and 20mcg/ml) at two different concentrations using 500. mu.g/ml aluminum (Adjuphos) as aluminum phosphate adjuvant was spun at 8 to 12rpm for 60 minutes at room temperature. More specifically, add Adjuphos adjuvant to sterile container, then add 10mM phosphate 8% sucrose buffer. The HBsAg is added to the same container,and mixed slowly by pipette (see table 2 below). The container was sealed and covered with aluminum foil and then spun at 8 to 12rpm for 60 minutes at room temperature. Once agitation is complete, the vaccine formulation is stored at 2 ℃ to 8 ℃ until analysis or immunization.

Table 2: immunogenic formulations comprising aluminum phosphate adjuvants

HBsAg (concentration 200. mu.g/mL; diluted batch No. B-061-6), HBsAg (concentration 441. mu.g/mL; batch No. B-054-3),

example 2: evaluation of adjuvant adsorption to antigen

The binding of hepatitis b surface antigen to aluminum hydroxide adjuvant and aluminum phosphate adjuvant was measured as follows. Briefly, 500 μ L of each vaccine formulation was aseptically transferred to sterile polypropylene centrifuge tubes in a sterile biosafety cabinet, after 10 to 20 times mixing by pipetting before transfer, to ensure a homogeneous solution. All aliquots were then centrifuged at 14,000g for 120 minutes. The supernatant (450. mu.l) was carefully removed from the centrifuge tube containing the immunogenic formulation using a 20. mu.L to 200. mu.L pipette and transferred to a new sterile polypropylene centrifuge tube. The centrifuge tube containing the immunogenic formulation pellet was resuspended by adding a volume of buffer (450 μ Ι) equal to the volume removed. Tubes were appropriately labeled (centrifuged supernatant and resuspended pellet of immunogenic preparation). The diluted pellet (containing bound HBsAg) and supernatant (containing free HBsAg) were stored in a refrigerator at 2 ℃ to 8 ℃. Table 3 below shows the effect of HBsAg antigen concentration, aluminium adjuvant selection and adjuvant dose on the amount of unbound/free HBsAg.

Table 3: unadsorbed antigen content of tested HBsAg compositions

Test article 6(TA6) is a commercial prophylactic Sci-B-Vac vaccine and no detectable unadsorbed (free) HBsAg was detected in the supernatant. The antigen to adjuvant ratio was changed by increasing the antigen concentration 4-fold (TA4) without substantially changing the amount of unadsorbed antigen (2.5%). Similarly, changing the antigen to adjuvant ratio by reducing the amount of aluminum hydroxide adjuvant (Alhydrogel) did not increase the amount of unadsorbed antigen by more than 5% (TA1 to TA 3).

Changing the adjuvant used in the immunogenic composition from aluminium hydroxide adjuvant (Alhydrogel) (TA6) to aluminium phosphate adjuvant (Adjuphos) (TA12) similarly failed to increase the amount of unadsorbed HBsAg (0% detected) while the HBsAg concentration was maintained at 10 μ g/ml. However, surprisingly, increasing the antigen concentration 2-fold (TA11) and/or reducing the aluminum phosphate adjuvant concentration (TA7 to TA10) significantly increased the amount of unadsorbed HBsAg to over 30%. In particular, when formulated with 500. mu.g/ml aluminium from an aluminium phosphate adjuvant, the antigen at a concentration of 20. mu.g/ml showed a very high amount of unadsorbed HBsAg. This adjuvant concentration, specifically 500. mu.g/ml, has been widely used in commercial HBsAg vaccines, but is an aluminium hydroxide adjuvant rather than an aluminium phosphate adjuvant.

In summary, no HBsAg was adsorbed in the commercial prophylactic vaccine known as Sci-B-Vac (TA6), but in one embodiment of the present disclosure with 20 μ g/mL HBsAg and 500 μ g/mL aluminium as aluminium phosphate adjuvant (TA11), the unadsorbed HBsAg was more than 50%. To obtain an amount of unadsorbed antigen greater than 30%, the aluminum-based adjuvant type and antigen concentration must be varied.

Example 3: evaluation of Th 1T cell immunity in mice after vaccination with two different immunogenic compositions comprising HBsAg and aluminium hydroxide antigens with S, pre-S1 and pre-S2

This example describes the evaluation of T cell responses in mice after immunization with two different immunogenic formulations comprising different concentrations of antigen (HBsAg comprising S, pre-S1 and pre-S2 proteins) and 500 μ g/ml aluminium from aluminium hydroxide adjuvant (TA 5 and TA6 in table 3). Balb/c mice were vaccinated 3 times with vaccine formulation human dose of 1/20 (i.e. 0.5. mu.g of TA6 antigen and 1.0. mu.g of TA5 antigen) on weeks 0, 3, 10 (or 13). Mice were sacrificed on day 6 after the third vaccination and responses to pre-S1, pre-S2, and HBsAg S proteins were measured by enzyme linked immunospot assay ("ELISPOT") using overlapping peptide libraries as described below. Comparable anti-HBsAg antibody responses were induced with both formulations (data not shown).

An IFN- γ ELISPOT assay was performed to measure Th 1T cell responses as follows. The mice were divided into groups of 8 mice each and immunized with the two test formulations described above, each having the same concentration of aluminum hydroxide adjuvant but different concentrations of HBsAg antigen. Four mice per group were sacrificed and the spleens removed. Spleens from individual mice were treated to produce single cell suspensions. Erythrocytes were lysed using a commercially available buffer (BioLegend). The splenocytes were then plated at 6X 10⁶Individual splenocytes/mL were resuspended. The day before collection and treatment of the spleens, ELISPOT plates were coated with interferon-gamma (IFN- γ) capture antibody. None of the peptides, actin peptide mixture, pre-S1 peptide mixture, pre-S2 peptide mixture, HBsAg peptide mixture, and phorbol 12-myristate 13-acetate and ionomycin (PMA/iono) were selected as stimulants. On the day of spleen collection, stimulants were added to designated ELISPOT plate wells. Mixing 1.5X 10⁵Individual splenocytes were added to PMA/iono wells and 3X 10 cells were added⁵Individual splenocytes were added to all other stimulants. The ELISPOT plates were then placed in a humidified atmosphere at 37 ℃ and 5% CO₂The incubator is 40 to 48 hours. After incubation, the plates were washed to remove splenocytes, stimulants, and media, and an IFN- γ capture antibody was added, followed by streptomycin horseradish peroxidase (strep-HRP). Finally, the plate was developed using a commercially available 3-amino-9-ethylcarbazole (AEC) substrate (BD BioSciences). The observed spots were counted using an ELISPOT plate reader and the final data were reported as Spot Forming Cells (SFC) per 1 million splenocytes. Table 4 below shows the results of this immunogenicity study.

Table 4: th1 Activity measured by ELISPOT

Note that: GM is the geometric mean; standard error of SE ═

Table 4 shows that higher amounts of HBsAg antigen did not show an improved Th 1T cell response in formulations containing aluminium hydroxide as adjuvant. The results are consistent with the data in table 2, which indicates that neither formulation has unadsorbed antigen.

Example 4: comparison of T cell immunity in mice after vaccination with a commercial prophylactic vaccine named Sci-B-Vac (10. mu.g/ml HBsAg containing S, pre-S1 and pre-S2 and 500. mu.g/ml aluminium as aluminium hydroxide adjuvant) and a formulation of the present disclosure (containing 20. mu.g HBsAg and 500. mu.g/ml aluminium as aluminium 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 commercial prophylactic vaccine known as Sci-B-Vac, containing 10 μ g/mL HBsAg containing S, pre-S1 and pre-S2 proteins and 500 μ g/mL aluminium as aluminium phosphate adjuvant (TA6 in table 3). The second composition is an immunogenic composition of the disclosure comprising two-fold high concentration of the same HBsAg (20 μ g/mL) and 500 μ g/mL aluminum as an aluminum phosphate adjuvant (TA11 in table 3). Balb/c mice were vaccinated 3 times at week 0, 3, 6 (or 8) with human doses of 1/20 (i.e. 0.5 μ g of TA6 antigen and 1.0 μ g of TA11 antigen) per immunogenic composition. Mice were sacrificed on day 6 after the third vaccination and responses to pre-S1, pre-S2, and HBsAg proteins were measured by ELISPOT using overlapping peptide libraries as described above. Comparable anti-HBsAg antibody responses were induced with both agents.

Table 5: th1 Activity measured by ELISPOT

As can be seen from table 5, the immunogenic composition of the present disclosure comprising 20 μ g HBsAg and 500 μ g/ml aluminium as an aluminium phosphate adjuvant stimulated a significantly greater Th1 cell response against the pre-S2 and S antigens than the commercial prophylactic vaccine form formulated with much lower concentrations of the same antigen (10 μ g/ml) and aluminium hydroxide adjuvant. This result is consistent with the data in table 2, which shows a significant percentage of unbound antigen (54.8%) in the immunogenic composition of the disclosure (TA11) compared to the absence of unbound antigen in the commercial prophylactic HBsAg vaccine (TA 6). For the first S2, the response display is the largest. No response to the pre-S1 antigen was detected for either formulation.

Example 5: comparison of individual IgG1/IgG2a ratios in mice post-vaccination with a commercial prophylactic vaccine designated Sci-B-Vac (10. mu.g/ml HBsAg containing S, pre-S1 and pre-S2 and 500. mu.g/ml aluminum as aluminum hydroxide adjuvant) and a formulation of the present disclosure (containing 20. mu.g HBsAg and 500. mu.g/ml aluminum as aluminum phosphate adjuvant)

This example describes a comparison of individual IgG1/IgG2a ratios in mice after vaccination with two different immunogenic compositions. The first composition was a commercial prophylactic vaccine known as Sci-B-Vac, containing 10 μ g/mL HBsAg containing S, pre-S1 and pre-S2 proteins and 500 μ g/mL aluminium as an aluminium hydroxide adjuvant (TA6 in table 3). The second composition is an immunogenic composition of the disclosure comprising two-fold high concentration of the same HBsAg (20 μ g/m1) and 500 μ g/mL aluminum as an aluminum phosphate adjuvant (TA11 in table 3). Balb/c mice (n-8) were vaccinated 3 times at weeks 0, 3, 6 with a human dose of 1/20 (i.e. 0.5 μ g of TA6 antigen and 1.0 μ g of TA11 antigen) per immunogenic composition. Mice were sacrificed on day 6 after the third vaccination and anti-HBs IgG1 and anti-HB IgG2 were measured by ELISA as follows.

anti-HBs IgG 1: the 96-well plates were coated with recombinant hepatitis B surface antigen protein (Abcam) (0.25. mu.g/mL in DPBS) overnight at 4 ℃. The following day, plates were blocked with 10% goat serum in ELISA wash buffer (0.05% Tween-20 in PBS) for 1 hour at 37 ℃. The plates were washed with wash buffer and then 2-fold dilutions of individual mouse sera were added, starting at 1: 10,000 to 1: 1280,000. Plates were incubated at 37 ℃ for 1.5 hours, then plates were washed and secondary antibodies were added. Goat anti-mouse IgG1(Bethyl) diluted 1: 10,000 in 10% goat serum in ELISA wash buffer was added and the plates were incubated at 37 ℃ for 1.5 hours. A TMB One component Microwell substrate (TMB One component Microwell substrate) was added to the plate, incubated at room temperature for 10 minutes, and then stop solution was added. The absorbance was read at 450nm using a MAXline plate reader.

anti-HBs IgG2 a: 96-well plates were coated overnight at 4 ℃ with recombinant hepatitis B surface antigen protein (Abcam) (0.25. mu.g/m 1 in DPBS). The following day, plates were blocked with 10% goat serum in ELISA wash buffer for 1 hour at 37 ℃. The plates were washed with wash buffer and then 2-fold dilutions of individual mouse sera were added, starting at 1: 5,000 to 1: 640,000. Plates were incubated at 37 ℃ for 1.5 hours, then plates were washed and secondary antibodies were added. Goat anti-mouse IgG2a (Bethyl) diluted 1: 10,000 in 10% goat serum in ELISA wash buffer was added and the plates were incubated for 1.5 hours at 37 ℃. TMB component microwell substrate was added to the plate and incubated at room temperature for 10 minutes, followed by addition of stop solution. The absorbance was read at 450nm using a MAXline plate reader.

The results are shown in table 6 below.

Table 6: individual IgG1/IgG2 ratio

As can be seen in table 6, the immunogenic compositions of the present disclosure containing 20 μ g HBsAg and 500 μ g/ml aluminium as an aluminium phosphate adjuvant stimulated a significantly lower ratio of IgG1 to IgG2 a. Since IgG2a stimulation is a marker of Th1 activity, the rate of this change indicates that compositions comprising 20 μ g HBsAg and 500 μ g/ml aluminium as an aluminium phosphate adjuvant elicit a stronger Th1 response than compositions comprising 20 μ g HBsAg and 500 μ g/ml aluminium as an aluminium phosphate adjuvant.

Example 6: dose range study in mice after vaccination with a commercial prophylactic vaccine called Sci-B-Vac (10. mu.g/ml HBsAg containing S, pre-S1 and pre-S2 and 500. mu.g/ml aluminium as aluminium hydroxide adjuvant) and three different doses of a composition of the disclosure (containing 20. mu.g, 40. mu.g and 60. mu.g HBsAg and 500. mu.g/ml aluminium as aluminium phosphate adjuvant)

This example describes a dose range study that tested Thl activity in mice vaccinated with a commercial prophylactic vaccine known as Sci-B-Vac (which contained 10 μ g/mL HBsAg containing S, pre-S1 and pre-S2 proteins and 500gg/mL aluminium as aluminium hydroxide adjuvant) (TA6 in table 3) and an immunogenic composition of the disclosure (which contained three different doses of the same HBsAg and 500 μ g/mL aluminium as aluminium phosphate adjuvant). Three different doses were as follows: HBsAg (20. mu.g/ml) (TA11 in Table 3); HBsAg (40. mu.g/ml) (TA 10 in Table 3) and HBsAg (60. mu.g/ml) (TA 13 in Table 3).

Briefly, Balb/c mice (n-8/group) were vaccinated intramuscularly 3 times with mouse doses of each composition, 3 weeks apart, specifically 3 μ g, 2 μ g and 1 μ g. Splenocytes were harvested from mice 7 days after the third vaccination and stimulated with overlapping peptide pools specific for pre-S1, pre-S2, and HBsAg. After 48 hours of culture, IFN-. gamma.secreting T cell responses were evaluated by ELISPOT as follows. The day before collection and treatment of the spleens, ELISpot plates (Millipore) were coated with 100. mu.l of interferon-gamma (IFN-. gamma.) capture antibody (Mabtech) at a concentration of 15. mu.g/mL and incubated overnight at 4 ℃.

On the day of spleen collection and treatment, the coated ELISpot plates were washed 5 times with 200 μ Ι sterile PBS and blocked with 100 μ Ι R10 medium for 1 to 2 hours. Once splenocytes were isolated and counted, R10 blocking medium was removed and 50 μ l of splenocytes (300,000 cells) and 50 μ l of stimulating agent were plated onto ELISpot assay plates. Splenocytes from each mouse were stimulated in duplicate with the following stimulators: pre-S1 (final stimulation concentration of 13.5 μ g/m1), pre-S2 (final stimulation concentration of 5.5 μ g/mL) and HBsAg (final stimulation concentration of 27 μ g/mL), R10 as a negative control, and phorbol 12-myristate 13-acetate and ionomycin (PMA (20 ng/mL)/ionomycin (1 μ g/mL) as a positive control ELISpot plates were then placed in a humidified 37 ℃ and 5% CO₂The incubator is 40 to 48 hours. After incubation, the plates were washed 5 times with 200. mu.l PBS-Tween to remove splenocytes, stimulants and culture medium, and then 100. mu.l IFN-. gamma.capture at a concentration of 1. mu.g/ml was added to each wellAntibody (Mabtech). After 2 hours of incubation, ELISpot plates were washed 5 times with PBC-Tween, and 100. mu.l of streptomycin horseradish peroxidase (strep-HRP) diluted 1: 1000 was added to each well. The plates were then incubated for a further 1 hour, after which time they were developed for 30 minutes at room temperature by adding 100. mu.L of 3-amino-9-ethylcarbazole (AEC) substrate (BD BioSciences). The observed spots were counted by ZellNet Consulting and the final data were reported as spot forming units (SFC) per 1 million splenocytes. Table 7 shows Th1 activity of different compositions measured by ELISPOT.

Table 7: th1 Activity by ELISPOT

As can be seen from table 7, by ELISPOT, the compositions comprising HBsAg containing S, pre-S1 and pre-S2 proteins and aluminium as an aluminium phosphate adjuvant at 20 μ g and 40 μ g and 60 μ g doses were able to elicit a greater number of pre-S2 and HBsAg specific IFN- γ secreting T cells compared to 10 μ g/mL HBsAg containing S, pre-S1 and pre-S2 proteins and 500 μ g/mL aluminium as an aluminium hydroxide adjuvant (TA6 in table 3). Thus, each of the three doses of the immunogenic composition of the present disclosure induced a higher T cell response compared to a composition comprising 10 μ g/mL HBsAg containing S, pre-S1 and pre-S2 proteins and 500 μ g/mL aluminium as an aluminium hydroxide adjuvant.

Other embodiments

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