Methods and compositions for treating hepatitis b infection

Abstract

The invention relates to methods and compositions for treating hepatitis B infection. Disclosed is a biological nanoparticle (BNP) comprising a hepatophilic DNA virus envelope HBsAg-S fusion protein comprising one or more epitope regions wherein the epitope regions are selected from the group consisting of epitopes expressed in the Ring 1 region and the Ring 2 region of the HBsAg-S domain, and wherein the epitope regions comprise one or more of CTKPTDGNC (SEQ ID NO: 34) and / or CKTCTTPAQGNSMFPS (SEQ ID NO: 35).

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of May 9, 2019, application number 201980046550.8, and invention name “Methods and compositions for treating hepatitis B infection”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 669,663, filed May 10, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present technology relates to compositions and methods for treating hepatitis B infection, including chronic hepatitis B (CHB). Background Art

[0005] Reference in this specification to any previous publication (or information obtained therefrom) or reference to any known matter is not and should not be taken as an acknowledgment or acceptance or any form of implication that the previous publication (or information obtained therefrom) or known matter constitutes common knowledge in the field to which this specification relates.

[0006] Hepatitis B is the most common viral hepatitis, which may be life-threatening, has long-term complications, and is one of the major public health challenges in the world. At present, vaccines are the most effective tools to fight hepatitis B infection. Although the availability of vaccines reduces the number of new hepatitis B virus (HBV) infections, it does not benefit 257 million people who have been infected by the virus for a long time (WHO Fact Sheet July 18, 2018). It is estimated that between 2015 and 2030, 63 million new cases of chronic HBV infection will occur cumulatively, and 17 million HBV-related deaths (Nayagam et al. Lancet Infectious Dis 2016 16: 1399-1408).

[0007] Chronic infection with hepatitis B virus (HBV) leads to clinical consequences of liver disease, including cirrhosis and hepatocellular carcinoma (HCC). Despite the introduction of preventive hepatitis B vaccines 30 years ago, chronic hepatitis B (CHB) infection remains a global health problem (Lozano R et al. Lancet 2012; 380(9859): 2095-128), accounting for more than 50% of the world's liver cancer burden. If untreated, about one-third of chronic hepatitis B (CHB) individuals will die from severe liver disease, such as cirrhosis, hepatocellular carcinoma (HCC) and liver failure. In terms of quantity, an estimated 260 to 350 million people are infected with the virus worldwide, and 780,000 people die from HBV-related liver disease, including cirrhosis and liver cancer, each year (Lozano R et al. Lancet. 2012; 380(9859): 2095-128). Nucleoside (acid) analog (NA) therapy currently used for CHB effectively targets viral DNA inhibition (HBV DNA cannot be detected), but cannot eliminate HBV surface antigen (HBsAg), whose continued expression is associated with the risk of continued development of HCC (Fattovich G et al. Am J Gastroenterol. 1998; 93 (6): 896-900; Yuen MF et al. Gastroenterology 2008; 135 (4): 1192-1199). Complete cure of hepatitis B is defined by eradication of the virus and all its replicative intermediates (Revill P et al. Nature Reviews Gastroenterology and Hepatology, 2016, 13 (4): 239-248), which is currently considered an unrealistic result. The clinical endpoints of HBsAg loss and / or seroconversion to anti-HBs are the current goals of CHB therapy, but are rarely achieved. The basis for this clearance may be the selective pressure of an effective host antiviral (antibody-driven) response. However, it has been shown that the innate and adaptive immune responses of CHB patients are impaired, characterized by suboptimal antigen presentation, antigen-specific T cell exhaustion, and insufficient antibody production (Wang L et al. World J Hepatol. 2015, 7(30): 2980-91). Therefore, it is necessary to develop other treatments for CHB.

[0008] Currently, the preferred first-line treatment options are pegylated interferon α-2a (pegIFN-α), entecavir, and tenofovir based on superior antiviral efficacy and / or high resistance barriers. However, even with first-line treatment regimens, pegIFN-α is only effective in achieving sustained virological responses in 30% of HBeAg-positive cases and 40% of HBeAg-negative cases, and is often associated with severe side effects. On the other hand, nucleoside (acid) analogs are well tolerated and effectively inhibit HBV replication in the vast majority of patients treated. However, even the most effective nucleoside (acid) analogs rarely induce HBV surface antigen (HBsAg) seroconversion (a sign of a successful immunological response to HBV and complete and durable infection control or "functional cure"). Therefore, long-term and potentially lifelong NA treatment is required to continuously suppress HBV replication, which can be associated with a huge cost burden and limited by drug-related toxicity. Therefore, there is an urgent need to introduce treatment regimens that are safer and more effective in achieving functional cure.

[0009] Infectious HBV virions are spherical particles with a diameter of 42 nm, which consist of an icosahedral nucleocapsid that packages the viral DNA genome and a lipoprotein envelope containing three related transmembrane proteins (HBsAg), which are called HBsAg-large (HBsAg-L), HBsAg-medium (M) and HBsAg-small (S). The synthesis of the envelope proteins begins at three different in-frame translation start sites. Therefore, the envelope proteins have a shared region called the S-domain. HBsAg-S consists only of the S domain containing 226 amino acids (aa); HBsAg-M contains an additional N-terminal extension, a 55 amino acid preS2 domain; and HBsAg-L contains the preS2 domain and an additional 108 or 119 amino acids (genotype dependent) N-terminal extension, called the preS1 domain.

[0010] The ability of HBsAg-S to self-assemble in the presence of lipids on the endoplasmic reticulum (ER) results in the formation of secretable subviral particles (VLPs) that do not contain any other HBV viral components. The diameter of the HBsAg-S VLP is 22 to 25 nanometers (nm), highly compact, and it is estimated that one particle contains approximately one hundred HBsAg-S molecules. HBsAg-M also forms secretable VLPs. HBsAg-L also forms VLPs that are not fully secretable.

[0011] HBsAg particle formation is a complex process. The first step in particle formation is the co-translational insertion of the protein into the ER membrane with a short lumen-exposed N-terminal sequence, two transmembrane regions separated by a 57aa cytoplasmic loop, and a luminal extraluminal 70aa domain containing the major B-cell epitopes ('a'-determinants). HBsAg-S VLP has a highly compact structure due to the presence of a large number of intramolecular and intermolecular disulfide bonds within and between individual subunits.

[0012] VLP is the tool of leading innovative bionanotechnology carrier and vaccine development, and compared with traditional vaccines, they have many advantages. VLP does not contain viral genetic material, and has the high density display of viral structural protein, effectively triggers the key part of immune system (Buonaguro L et al., (2011), Expert Rev Vaccines 10:1569–1583 of B cell and / or T cell response; Jennings GT and Bachmann MF. (2009) Annu Rev Pharmacol Toxicol. 2009, 49:303-326; Pushko P et al., Intervirology 2013, 56:141–165).

[0013] Chimeric VLPs based on capsid proteins (e.g., HBV, human papillomavirus (HPV), and Qβ phage) have been engineered to express exogenous antigen sequences (including non-pathogen-related antigens, such as nicotine and angiotensin II for smoking cessation and hypertension, respectively) (Ambühl PM et al. J. Hypertension. 2007, 25: 63-72; Buonaguro L et al. Exp. Rev. Vaccines 2011, 10: 1569-1583; Cornuz J et al. Plos One 2008, 3: e2547). Compared to capsid VLPs consisting only of protein subunits, HBsAg-S VLPs based on HBV are composed of envelope proteins and lipids, and the ER is the cellular location of assembly. In order to present the antigen sequence to the immune system, HBsAg-S VLP has been modified to carry foreign epitopes (Delpeyroux F et al. Science 1986, 233: 472-475; Eckhart L. et al. J. Gen. Virology 1996, 77: 2001-2008; Fomsgaard A. et al. Scand. J. Immunol. 1998, 47: 289-295; Phogat S. et al. Virology 2008, 373: 72-84; Netter HJ et al. J. Virology 2001, 75: 2130-2141). VLP composed of HBsAg-L was developed as a delivery system for genes and drugs to human hepatocytes (Yamada T et al. Nature Biotechnology 2003, 21: 885-890). Duck hepatitis B virus envelope protein and other hepadnaviral envelope proteins have been modified to express target antigens as part of VLPs.

[0014] VLPs composed of the small envelope protein from HBV (HBsAg-S) are the antigenic components of successful protective vaccines (Jilg W et al. Lancet ii 1984, 1174-1175; Zuckerman JN. J Med Virology 2006, 78: 169-177). However, even with the availability of vaccines, hepatitis B remains a huge health problem.

[0015] An important issue in vaccine development is the declining capacity of the aging immune system and immunosenescence is associated with decreased vaccine efficacy in the elderly (Derhovanessian E and Pawelec G. Microbial Biotechnology 2012, 5:226-232; Pera A et al. Maturitas 2015, 82:50-55).

[0016] The endpoint of HBsAg loss in the absence or presence of seroconversion to anti-HBs is combined with undetectable levels of HBV DNA in serum, which is the goal of current CHB therapy, but it is rarely achieved. The basis of this removal is probably the selective pressure of effective host antiviral (antibody-driven) responses. However, it has been shown that the innate immune response and adaptive immune response in CHB patients are impaired, characterized by suboptimal antigen presentation, antigen-specific T cell exhaustion and insufficient antibody production (Wang L et al. World J Hepatology 2015, 7 (30): 2980-2991).

[0017] There is a need to develop effective B cell vaccines for the treatment of CHB. In particular, there is a need to develop therapeutic regimens that can achieve functional cure. Summary of the invention

[0018] In one aspect, the present disclosure provides a recombinant virus-like particle antigen (VLP-Ag) comprising a hepadnavirus envelope HBsAg-S fusion protein containing one or more antigenic epitope repeat regions, wherein the antigenic epitope repeat region is selected from the group consisting of antigenic epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain.

[0019] In some embodiments, one or more epitope repeat regions expressed in loop 1 are defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein X1 is K or R, X2 is T or M, X3 is T or I, X4 is PT or L, X5 is A or V, X6 is N or T, and X7 is F or Y; or wherein one or more epitope repeat regions are defined by the amino acid sequence PCX8TCX9X 10 X 11 Definition, where X8 is K or R, X9 is T or M, X 10 is T, I, or S, and X 11 It is P, T or L.

[0020] In some embodiments, one or more epitope repeat regions expressed in loop 2 consist of the consensus amino acid sequence CCCTKPX 12 DGNCX 13 Definition, where X 12 is T or S; and X13 It is T or S.

[0021] In some embodiments, one or more epitope repeat regions are selected from the group consisting of: PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTPAQGNSMFPS, CTKP(T / S)TDGNC, PC(K / R)TC(T / M)TP,

[0022] C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS、

[0023] PCRTCMTTAQGTSMYPSCCCTKPSDGNC, and

[0024] PCKTCTTPAQGNSMFPSCCCTKPTDGNC.

[0025] In some embodiments, the hepadnavirus envelope fusion protein comprises a spacer domain between the epitope repeat region and the envelope protein.

[0026] In one aspect, the present disclosure provides a nucleic acid encoding a VLP-Ag of the present technology. In one aspect, the present disclosure provides an expression vector comprising the nucleic acid.

[0027] In one aspect, the present disclosure provides a composition comprising the VLP-Ag of the present technology and a pharmaceutically acceptable carrier.

[0028] In one aspect, the present disclosure provides a method for treating hepatitis B infection in a subject in need thereof, comprising administering to the subject a recombinant VLP-Ag comprising a hepadnavirus envelope HBsAg-S fusion protein comprising one or more epitope repeat regions, wherein the epitope repeat region is selected from the group consisting of epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain.

[0029] In some embodiments, one or more epitope repeat regions expressed in loop 1 are defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein X1 is K or R, X2 is T or M, X3 is T or I, X4 is PT or L, X5 is A or V, X6 is N or T, and X7 is F or Y; or wherein one or more epitope repeat regions are defined by the amino acid sequence PCX8TCX9X 10 X 11 Definition, where X8 is K or R, X9 is T or M, X 10 is T, I, or S, and X 11 It is P, T or L.

[0030] In some embodiments, one or more epitope repeat regions expressed in loop 2 consist of the consensus amino acid sequence CCCTKPX 12 DGNCX 13 Definition, where X 12 is T or S; and X 13 It is T or S.

[0031] In some embodiments, one or more epitope repeat regions are selected from the group consisting of: PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTPAQGNSMFPS, CTKP(T / S)TDGNC, PC(K / R)TC(T / M)TP,

[0032] C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS、

[0033] PCRTCMTTAQGTSMYPSCCCTKPSDGNC, and

[0034] PCKTCTTPAQGNSMFPSCCCTKPTDGNC.

[0035] In some embodiments, the hepadnavirus envelope fusion protein comprises a spacer domain between the epitope repeat region and the envelope protein.

[0036] In one aspect, the present disclosure provides the use of a recombinant VLP-Ag in the manufacture of a medicament for treating hepatitis B infection in a subject in need thereof, wherein the VLP-Ag comprises a hepadnavirus envelope HBsAg-S fusion protein comprising one or more epitope repeat regions, wherein the epitope repeat regions are selected from the group consisting of epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain.

[0037] In some embodiments, one or more epitope repeat regions expressed in loop 1 are defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein X1 is K or R, X2 is T or M, X3 is T or I, X4 is PT or L, X5 is A or V, X6 is N or T, and X7 is F or Y; or wherein one or more epitope repeat regions are defined by the amino acid sequence PCX8TCX9X 10 X 11 Definition, where X8 is K or R, X9 is T or M, X 10 is T, I, or S, and X 11 It is P, T or L.

[0038] In some embodiments, one or more epitope repeat regions expressed in loop 2 consist of the consensus amino acid sequence CCCTKPX 12 DGNCX 13 Definition, where X 12 is T or S; and X 13 It is T or S.

[0039] In some embodiments, one or more epitope repeat regions are selected from the group consisting of: PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTPAQGNSMFPS, CTKP(T / S)TDGNC, PC(K / R)TC(T / M)TP,

[0040] C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS、

[0041] PCRTCMTTAQGTSMYPSCCCTKPSDGNC, and

[0042] PCKTCTTPAQGNSMFPSCCCTKPTDGNC.

[0043] In some embodiments, the hepadnavirus envelope fusion protein comprises a spacer domain between the epitope repeat region and the envelope protein.

[0044] In one aspect, the present disclosure provides a method for inducing an immune response against hepatitis B virus in a subject, comprising administering to the subject a recombinant VLP-Ag comprising a hepadnavirus envelope HBsAg-S fusion protein comprising one or more epitope repeat regions, wherein the epitope repeat region is selected from the group consisting of epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain.

[0045] In some embodiments, one or more epitope repeat regions expressed in loop 1 are defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein X1 is K or R, X2 is T or M, X3 is T or I, X4 is PT or L, X5 is A or V, X6 is N or T, and X7 is F or Y; or wherein one or more epitope repeat regions are defined by the amino acid sequence PCX8TCX9X 10 X 11 Definition, where X8 is K or R, X9 is T or M, X 10 is T, I, or S, and X 11 It is P, T or L.

[0046] In some embodiments, one or more epitope repeat regions expressed in loop 2 consist of the consensus amino acid sequence CCCTKPX12 DGNCX 13 Definition, where X 12 is T or S; and X 13 It is T or S.

[0047] In some embodiments, one or more epitope repeat regions are selected from the group consisting of: PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTPAQGNSMFPS, CTKP(T / S)TDGNC, PC(K / R)TC(T / M)TP,

[0048] C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS、

[0049] PCRTCMTTAQGTSMYPSCCCTKPSDGNC, and

[0050] PCKTCTTPAQGNSMFPSCCCTKPTDGNC.

[0051] In some embodiments, the hepadnavirus envelope fusion protein comprises a spacer domain between the epitope repeat region and the envelope protein.

[0052] In one aspect, the present disclosure provides the use of recombinant VLP-Ag in the manufacture of a composition for inducing an immune response against hepatitis B, wherein the VLP-Ag comprises a hepadnavirus envelope HBsAg-S fusion protein containing one or more antigenic epitope repeat regions, wherein the antigenic epitope repeat region is selected from the group consisting of antigenic epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain.

[0053] In some embodiments, one or more epitope repeat regions expressed in loop 1 are defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein X1 is K or R, X2 is T or M, X3 is T or I, X4 is PT or L, X5 is A or V, X6 is N or T, and X7 is F or Y; or wherein one or more epitope repeat regions are defined by the amino acid sequence PCX8TCX9X 10 X 11 Definition, where X8 is K or R, X9 is T or M, X 10 is T, I, or S, and X 11 It is P, T or L.

[0054] In some embodiments, one or more antigenic envelope repeat regions expressed in loop 2 consist of the consensus amino acid sequence CCCTKPX 12 DGNCX 13 Definition, where X12 is T or S; and X 13 It is T or S.

[0055] In some embodiments, one or more epitope repeat regions are selected from the group consisting of: PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTPAQGNSMFPS, CTKP(T / S)TDGNC, PC(K / R)TC(T / M)TP,

[0056] C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS、

[0057] PCRTCMTTAQGTSMYPSCCCTKPSDGNC, and

[0058] PCKTCTTPAQGNSMFPSCCCTKPTDGNC.

[0059] In some embodiments, the hepadnavirus envelope fusion protein comprises a spacer domain between the epitope repeat region and the envelope protein.

[0060] In one aspect, the present disclosure provides a kit for treating hepatitis B infection in a subject in need thereof, the kit comprising recombinant VLP-Ag, wherein the VLP-Ag comprises a hepadnavirus envelope HBsAg-S fusion protein comprising one or more epitope repeat regions, wherein the epitope repeat region is selected from the group consisting of epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain.

[0061] In some embodiments, one or more epitope repeat regions expressed in loop 1 are defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein X1 is K or R, X2 is T or M, X3 is T or I, X4 is PT or L, X5 is A or V, X6 is N or T, and X7 is F or Y; or wherein one or more epitope repeat regions are defined by the amino acid sequence PCX8TCX9X 10 X 11 Definition, where X8 is K or R, X9 is T or M, X 10 is T, I, or S, and X 11 It is P, T or L.

[0062] In some embodiments, one or more epitope repeat regions expressed in loop 2 consist of the consensus amino acid sequence CCCTKPX 12 DGNCX 13 Definition, where X 12 is T or S; and X 13 It is T or S.

[0063] In some embodiments, one or more epitope repeat regions are selected from the group consisting of: PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTPAQGNSMFPS, CTKP(T / S)TDGNC, PC(K / R)TC(T / M)TP,

[0064] C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS、

[0065] PCRTCMTTAQGTSMYPSCCCTKPSDGNC, and

[0066] PCKTCTTPAQGNSMFPSCCCTKPTDGNC.

[0067] In some embodiments, the hepadnavirus envelope fusion protein comprises a spacer domain between the epitope repeat region and the envelope protein.

[0068] In one aspect, the present disclosure provides a recombinant mRNA encoding a hepadnavirus envelope HBsAg-S fusion protein comprising one or more epitope repeat regions, wherein the epitope repeat region is selected from the group consisting of epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain.

[0069] In some embodiments, one or more epitope repeat regions expressed in loop 1 are defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein X1 is K or R, X2 is T or M, X3 is T or I, X4 is PT or L, X5 is A or V, X6 is N or T, and X7 is F or Y; or wherein one or more epitope repeat regions are defined by the amino acid sequence PCX8TCX9X 10 X 11 Definition, where X8 is K or R, X9 is T or M, X 10 is T, I, or S, and X 11 It is P, T or L.

[0070] In some embodiments, one or more epitope repeat regions expressed in loop 2 consist of the consensus amino acid sequence CCCTKPX 12 DGNCX 13 Definition, where X 12 is T or S; and X 13 It is T or S.

[0071] In some embodiments, one or more epitope repeat regions are selected from the group consisting of: PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTPAQGNSMFPS, CTKP(T / S)TDGNC, PC(K / R)TC(T / M)TP,

[0072] C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS、

[0073] PCRTCMTTAQGTSMYPSCCCTKPSDGNC, and

[0074] PCKTCTTPAQGNSMFPSCCCTKPTDGNC.

[0075] In some embodiments, the modified hepadnavirus envelope fusion protein comprises a spacer domain between the epitope repeat region and the envelope protein.

[0076] In one aspect, the present disclosure provides an expression vector comprising a recombinant mRNA of the present technology. In one aspect, the present disclosure provides a composition comprising a recombinant mRNA and a pharmaceutically acceptable carrier.

[0077] In one aspect, the present disclosure provides a method for treating hepatitis B infection in a subject in need thereof, comprising administering to the subject a recombinant mRNA encoding a hepadnavirus envelope HBsAg-S fusion protein comprising one or more epitope repeat regions, wherein the epitope repeat regions are selected from the group consisting of epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain.

[0078] In some embodiments, one or more epitope repeat regions expressed in loop 1 are defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein X1 is K or R, X2 is T or M, X3 is T or I, X4 is PT or L, X5 is A or V, X6 is N or T, and X7 is F or Y; or wherein one or more epitope repeat regions are defined by the amino acid sequence PCX8TCX9X 10 X 11 Definition, where X8 is K or R, X9 is T or M, X 10 is T, I, or S, and X 11 It is P, T or L.

[0079] In some embodiments, one or more epitope repeat regions expressed in loop 2 consist of the consensus amino acid sequence CCCTKPX 12 DGNCX 13 Definition, where X 12 is T or S; and X13 It is T or S.

[0080] In some embodiments, one or more epitope repeat regions are selected from the group consisting of: PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTPAQGNSMFPS, CTKP(T / S)TDGNC, PC(K / R)TC(T / M)TP,

[0081] C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS、

[0082] PCRTCMTTAQGTSMYPSCCCTKPSDGNC, and

[0083] PCKTCTTPAQGNSMFPSCCCTKPTDGNC.

[0084] In some embodiments, the modified hepadnavirus envelope fusion protein comprises a spacer domain between the epitope repeat region and the envelope protein.

[0085] In one aspect, the present disclosure provides the use of a recombinant mRNA in the manufacture of a medicament for treating a hepatitis B infection in a subject in need thereof, wherein the recombinant mRNA encodes a hepadnavirus envelope HBsAg-S fusion protein comprising one or more epitope repeat regions, wherein the epitope repeat regions are selected from the group consisting of epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain.

[0086] In some embodiments, one or more epitope repeat regions expressed in loop 1 are defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein X1 is K or R, X2 is T or M, X3 is T or I, X4 is PT or L, X5 is A or V, X6 is N or T, and X7 is F or Y; or wherein one or more epitope repeat regions are defined by the amino acid sequence PCX8TCX9X 10 X 11 Definition, where X8 is K or R, X9 is T or M, X 10 is T, I, or S, and X 11 It is P, T or L.

[0087] In some embodiments, one or more epitope repeat regions expressed in loop 2 consist of the consensus amino acid sequence CCCTKPX 12 DGNCX 13 Definition, where X 12 is T or S; and X 13 It is T or S.

[0088] In some embodiments, one or more epitope repeat regions are selected from the group consisting of: PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTPAQGNSMFPS, CTKP(T / S)TDGNC, PC(K / R)TC(T / M)TP,

[0089] C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS、

[0090] PCRTCMTTAQGTSMYPSCCCTKPSDGNC, and

[0091] PCKTCTTPAQGNSMFPSCCCTKPTDGNC.

[0092] In some embodiments, the hepadnavirus envelope fusion protein comprises a spacer domain between the epitope repeat region and the envelope protein.

[0093] In one aspect, the present disclosure provides a method for inducing an immune response against hepatitis B virus in a subject, comprising administering to the subject a recombinant mRNA encoding a modified hepadnavirus envelope HBsAg-S fusion protein comprising one or more epitope repeat regions, wherein the epitope repeat regions are selected from the group consisting of epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain.

[0094] In some embodiments, one or more epitope repeat regions expressed in loop 1 are defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein X1 is K or R, X2 is T or M, X3 is T or I, X4 is PT or L, X5 is A or V, X6 is N or T, and X7 is F or Y; or wherein one or more epitope repeat regions are defined by the amino acid sequence PCX8TCX9X 10 X 11 Definition, where X8 is K or R, X9 is T or M, X 10 is T, I, or S, and X 11 It is P, T or L.

[0095] In some embodiments, one or more epitope repeat regions expressed in loop 2 consist of the consensus amino acid sequence CCCTKPX 12 DGNCX 13 Definition, where X 12 is T or S; and X 13 It is T or S.

[0096] In some embodiments, one or more epitope repeat regions are selected from the group consisting of: PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTPAQGNSMFPS, CTKP(T / S)TDGNC, PC(K / R)TC(T / M)TP,

[0097] C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS、

[0098] PCRTCMTTAQGTSMYPSCCCTKPSDGNC, and

[0099] PCKTCTTPAQGNSMFPSCCCTKPTDGNC.

[0100] In some embodiments, the modified hepadnavirus envelope fusion protein comprises a spacer domain between the epitope repeat region and the envelope protein.

[0101] In one aspect, the present disclosure provides the use of a recombinant mRNA in the manufacture of a composition for inducing an immune response against hepatitis B, wherein the recombinant mRNA encodes a hepadnavirus envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein the antigenic epitope repeat region is selected from the group consisting of antigenic epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain.

[0102] In some embodiments, one or more epitope repeat regions expressed in loop 1 are defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein X1 is K or R, X2 is T or M, X3 is T or I, X4 is PT or L, X5 is A or V, X6 is N or T, and X7 is F or Y; or wherein one or more epitope repeat regions are defined by the amino acid sequence PCX8TCX9X 10 X 11 Definition, where X8 is K or R, X9 is T or M, X 10 is T, I, or S, and X 11 It is P, T or L.

[0103] In some embodiments, one or more epitope repeat regions expressed in loop 2 consist of the consensus amino acid sequence CCCTKPX 12 DGNCX 13 Definition, where X 12 is T or S; and X 13 It is T or S.

[0104] In some embodiments, one or more epitope repeat regions are selected from the group consisting of: PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTPAQGNSMFPS, CTKP(T / S)TDGNC, PC(K / R)TC(T / M)TP,

[0105] C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS、

[0106] PCRTCMTTAQGTSMYPSCCCTKPSDGNC, and

[0107] PCKTCTTPAQGNSMFPSCCCTKPTDGNC.

[0108] In some embodiments, the modified hepadnavirus envelope fusion protein comprises a spacer domain between the epitope repeat region and the envelope protein.

[0109] In one aspect, the present disclosure provides a kit for treating hepatitis B infection in a subject in need thereof, the kit comprising recombinant mRNA, wherein the recombinant mRNA encodes a hepadnavirus envelope HBsAg-S fusion protein comprising one or more epitope repeat regions, wherein the epitope repeat region is selected from the group consisting of epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain.

[0110] In some embodiments, one or more epitope repeat regions expressed in loop 1 are defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein X1 is K or R, X2 is T or M, X3 is T or I, X4 is PT or L, X5 is A or V, X6 is N or T, and X7 is F or Y; or wherein one or more epitope repeat regions are defined by the amino acid sequence PCX8TCX9X 10 X 11 Definition, where X8 is K or R, X9 is T or M, X 10 is T, I, or S, and X 11 It is P, T or L.

[0111] In some embodiments, one or more epitope repeat regions expressed in loop 2 consist of the consensus amino acid sequence CCCTKPX 12 DGNCX 13 Definition, where X 12 is T or S; and X 13 It is T or S.

[0112] In some embodiments, one or more epitope repeat regions are selected from the group consisting of: PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTPAQGNSMFPS, CTKP(T / S)TDGNC, PC(K / R)TC(T / M)TP,

[0113] C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS、

[0114] PCRTCMTTAQGTSMYPSCCCTKPSDGNC, and

[0115] PCKTCTTPAQGNSMFPSCCCTKPTDGNC.

[0116] In some embodiments, the modified hepadnavirus envelope fusion protein comprises a spacer domain between the epitope repeat region and the envelope protein.

[0117] In one aspect, the present disclosure provides a composition comprising one or more peptides selected from the group consisting of: TCTTPAQGNSMFPSC (SEQ ID NO: 17), TCTIPAQGTSMFPSC (SEQ ID NO: 18), TCTTPAQGTSMFPSC (SEQ ID NO: 19), CTKPTDGNCT (SEQ ID NO: 20), and CTKPSDGNCT (SEQ ID NO: 21).

[0118] In some embodiments, wherein the one or more peptides are conjugated to a carrier protein. In some embodiments, the carrier protein is keyhole limpet hemocyanin (KLH). In some embodiments, the one or more peptides are cyclic. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0119] In one aspect, the present disclosure provides a method for treating hepatitis B infection in a subject in need thereof, comprising administering to the subject one or more peptides selected from the group consisting of: TCTTPAQGNSMFPSC (SEQ ID NO: 17), TCTIPAQGTSMFPSC (SEQ ID NO: 18), TCTTPAQGTSMFPSC (SEQ ID NO: 19), CTKPTDGNCT (SEQ ID NO: 20), and CTKPSDGNCT (SEQ ID NO: 21).

[0120] In some embodiments, the one or more peptides are conjugated to a carrier protein. In some embodiments, the carrier protein is keyhole limpet hemocyanin (KLH). In some embodiments, the one or more peptides are cyclic. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1 Is a graphical representation showing the HBsAg epitope occupancy assay. 1. Magnetic beads; 2. Capture Ab: mouse anti-HBs monoclonal antibody (mAb) to the HBsAg external region; 3. Reference HBsAg sample. Ag only vs Ag bound to anti-HBs sample; 4. Reporter Ab: PE conjugated polyclonal anti-HBs. NCP: non-clearing property; CP: clearing property.

[0122] Figure 2 It is a graphical representation of predictive biomarkers for functional CHB cure (assay 1) and immune Ab response activity (assay 2). Assay 1 (upper panel): 1. Magnetic beads; 2. Capture Ab: multiplexed mouse anti-HBs mAb to HBsAg 'external region'; 3. Patient HBsAg sample; 4. Reporter Ab: PE-conjugated polyclonal anti-HBs. Assay 2 (lower panel): 1. Magnetic beads; 2. Capture Ab: dual mouse anti-HBs mAb to HBsAg 'external region'; 3. Patient sample: composite anti-HBs (containing HBsAg); 4. Reporter Ab: HRP-conjugated goat anti-human IgG Fc domain.

[0123] Figure 3 is a graphical representation showing CHB cured patients exhibiting HBsAg clearance profile (CP). CHB: chronic hepatitis B.

[0124] Figure 4 Is a graphical representation showing non-cured / non-responder patients exhibiting HBsAg non-clearance profile (NCP). RFU: Relative Fluorescence Unit.

[0125] Figure 5 Schematic diagram showing an overview of the proposed structure of the small hepatitis B surface (envelope) antigen (HBsAg-S), 226 amino acids long and ayw subtype (genotype D). The outer antigenic loop region between amino acids 100 and 160 of HBsAg-S is zoomed in: each amino acid is represented by a gray circle, cysteine ​​residues are shown as black circles, and potential -SS-disulfide bonds are highlighted. The glycosylation site (asparagine) at position 146 is shown in white, and the glycan structure is indicated. The positions for insertion of foreign sequences are located at amino acids 127 and 128.

[0126] Figure 6Schematic diagram of the proposed folding of the 226 amino acid long HBsAg-S protein showing BNP 1, BNP 2, BNP 3 and BNP 4. The outer antigenic loop region between amino acids 100 and 160 of HBsAg-S is magnified: each amino acid is represented by a gray circle, cysteine ​​residues are shown as black circles, and potential -SS-disulfide bonds are highlighted. The glycosylation site (asparagine) at position 146 is shown in white, and the glycan structure is indicated. The position for inserting exogenous sequences is located at amino acids 127 and 128, and the HBsAg-S sequence subtype ayw is used as a parent template. The antigenic sequence of another HBsAg-S source is inserted into the parent template at positions 127 and 128. The right panel shows the cure-associated loop sequences of BNP 1, BNP 2, BNP 3, and BNP 4 (SEQ ID NOs: 41-44, in order of appearance), with the HBsAg-S-specific antigen sequence in bold and the "GSGS" linker in italics and underlined. BNP-1 is the 'ayw' parent template with a trimeric PCKTCTTP (SEQ ID NO:28) insert (loop 1, genotype A specific, serotype adw); BNP-2 is the 'ayw' parent template with a trimeric PCRTCTTP (SEQ ID NO:33) insert (loop 1, genotype B / C, serotype ayw); BNP-3 is the 'ayw' parent template with a trimeric insert of the loop 2 sequence CTKPTDGNC (SEQ ID NO:34) (derived from serotype 'adw'); BNP-4 is the 'ayw' parent template with an insert (dimer) of the extended loop 1 sequence CKTCTTPAQGNSMFPS (SEQ ID NO:35) (serotype 'adw').

[0127] Figure 7 Schematic diagram showing the proposed folding of the 226 amino acid long HBsAg-S protein of BNP 5. The outer antigenic loop region of amino acids 100 to 160 of HBsAg-S is magnified: each amino acid is represented by a gray circle, cysteine ​​residues are shown as black circles, potential -SS-disulfide bonds are highlighted, and the "GSGS" linker is shown by a striped circle. The glycosylation site (asparagine) at position 146 is shown in white, and the glycan structure is indicated. Two additional loop 2 sequences are added to the wild-type loop to generate a repeating structure. The loop 2 CTKP (T / S) TDGNC (SEQ ID NO: 36) sequence is repeatedly framed. The right figure shows the sequence (SEQ ID NO: 45), with the HBsAg-S-specific antigen sequence in bold, the "GSGS" linker in italics, and underlined. BNP-5 is the parent 'ayw', parent template with a trimer loop 2 region. The 'ayw' loop 2 sequence is extended by two additional 'adw' loop 2 sequences.

[0128] Figure 8 Schematic diagram showing the proposed folding of the 226 amino acid long HBsAg-S protein of BNP 6. The outer antigenic loop region of amino acids 100 to 160 of HBsAg-S is magnified: each amino acid is represented by a gray circle, cysteine ​​residues are shown as black circles, potential -SS-disulfide bonds are highlighted, and the "GSGS" linker is shown by a striped circle. The glycosylation site (asparagine) at position 146 is shown in white, and the glycan structure is indicated. Two additional loop 1 PC (K / R) TC (T / M) TP (SEQ ID NO: 37) sequences and loop 2 CTKP (T / S) TDGNC (SEQ ID NO: 36) sequences are added within the corresponding loop sequences. The loop 1 and loop 2 sequences are repeatedly framed. The extended sequence (SEQ ID NOs: 46 and 47, in order of appearance) is shown on the right, and the HBsAg-S-specific antigen sequence is in bold, and the "GSGS" linker is italicized and underlined. Two additional loop 1 sequences were inserted (serotype adw) followed by the original loop 1 sequence of the parental template (serotype ayw). The original loop 2 sequence (parental serotype ayw) was followed by the two inserted loop 2 sequences, serotype adw.

[0129] Fig. 9 Schematic diagram showing the proposed folding of the 226 amino acid long HBsAg-S protein of BNP 7. Zoom in on the outer antigenic loop region between amino acids 100 and 160 of HBsAg-S: each amino acid is represented by a gray circle, cysteine ​​residues are shown as black circles, potential -SS-disulfide bonds are highlighted, and the "GSGS" linker is shown by a striped circle. The glycosylation site (asparagine) at position 146 is shown in white, and the glycan structure is indicated. Two additional loop 1 C(K / R)TC(T / M)T(P / T)AQG(N / T)SM(F / Y)PS (SEQ ID NO:38) sequences are added within the loop 1 sequence. Loop 1 repeats are boxed. The original amino acid positions of the wild-type HBsAg-S sequence are unchanged, and the additional amino acids are marked with ' and ". The right panel shows the extended sequence (SEQ ID NOs: 52, 48 and 49, in order of appearance), with the HBsAg-S-specific antigen sequence in bold, the "GSGS" linker in italics and underlined. Two extended loop 1 sequences (serotype adw) are inserted, amino acid sequences 121 to 136, followed by the original loop 1 sequence (serotype ayw).

[0130] Fig.10is a schematic diagram showing the proposed folding of the 226 amino acid long HBsAg-S protein of BNP 8. Zoomed in on the outer antigenic loop region between amino acids 100 and 160 of HBsAg-S: each amino acid is represented by a grey circle, cysteine ​​residues are shown as black circles, potential -SS-disulfide bonds are highlighted, and the "GSGS" linker is shown by a striped circle. The glycosylation site (asparagine) at position 146 is shown in white, and the glycan structure is indicated. Repeating loop 1 and loop 2 regions. The original amino acid positions of the wild-type HBsAg-S sequence are unchanged, and the additional amino acids of the repeated loop 1 and loop 2 are marked with '. BNP-8 contains the original loop 1 and loop 2 regions ('ayw', amino acids (aa) 120-147), followed by additional loop 1 and loop 2 'adw' sequences (aa 120-147).

[0131] Fig.11 It is a figure showing the HBsAg epitope curve (bioplex platform) analysis of CP-BNP 1, CP-BNP 2, CP-BNP 3 and CP-BNP 4 preparations and CP-BNP 1+3, CP-BNP 1+3+4 and CP-BNP 3+4 preparations. CP-BNP incorporates the loop 1 (CP-BNP 1, CP-BNP 2, CP-BNP 4) or loop 2 (CP-BNP 3) specific "clearing" epitope inserts displayed to induce targeted "clearing" anti-HBs antibody responses after immunization. HBsAg epitope curve analysis indicates that in all tested CP-BNP preparations and preparations, the epitope across both loop 1 and loop 2 is retained (within the normal range of + / -0.5 times) and / or enhanced (>0.5 times), indicating that the antigenicity of the target "clearing" epitope is correctly and completely displayed, and its strongest / widest epitope is retained in the BNP preparation to achieve.

[0132] Fig.12 is a graph showing the analysis of derived and purified monoclonal antibodies generated against cyclic peptides. The graph shows the CP curves (bioplex platform) analysis of selected generated monoclonal antibodies for anti-HBs antibody induction after immunization with loop 1 or loop 2 pan-genotypic / serotype cyclic peptide preparations. The generated monoclonal anti-HBs antibodies n=8 were analyzed for induction of IndCP (e.g., loss of epitope recognition at loop 1 and loop 2 epitopes) after pre-incubation with reference VLP antigens, indicating a "clearance" or CP-associated anti-HBs curve.

[0133] FIG. 13A to FIG. 13DIt is a chart showing the analysis of anti-HBs antibody reactivity after CP-BNP antigen immunization. After 3 immunizations with CP-BNP 1, CP-BNP 2, CP-BNP 3 and CP-BNP 4 preparations of CP-BNP 1+3, CP-BNP 1+3+4 and CP-BNP 3+4 preparations, animal blood (bloods 1-4, or averaged for all bloods) was subjected to standard enzyme immunoassay (EIA) analysis of serum. The specificity of the induced antibody response was analyzed for: recombinant WT HBsAg (VLP) ( Fig.13A ); HBsAg loop 1 specific cyclized peptide ( Fig. 13B ); and HBsAg loop 2-specific cyclized peptide ( Fig. 13C ). Fig.13D Anti-HBs reactivity was reported for WT-BNP, loop 1 peptide, and loop 2 peptide antigens. The strongest, most rapid, most extensive, or most conservative (to both VLP and loop 1 peptide and loop 2 peptide) anti-HBs antibody immunogenic responses were observed for CP-BNP formulations 1+3+4, 1+4+5, 5+7, and 5 that delivered target "clearance" epitopes of loop 1 and loop 2 (circular).

[0134] Fig.14 It is a chart showing the CP characteristics (bioplex platform) analysis of the anti-HBs antibody induction of the selected serum response after single or combined immunization with CP-BNP 1, CP-BNP 3, CP-BNP 4, CP-BNP 5 and CP-BNP 7 preparations. After the immunization scheme with CP-BNP preparations and preparations n=3 times, the anti-HBs antibody characteristics analysis was performed on the terminal blood serum. The HBsAg epitope curve pre-incubated with the anti-HBs positive individual mouse serum from the immunogenicity study was assessed for the induction of CP due to the "clearance" or CP-related anti-HBs in the serum. This targeted "clearance" epitope anti-HBs development was most consistently observed in mice (9-5, 13-2, 16-5 and 17-3) immunized with CP-BNP 1+4+5, CP-BNP 1+3+4, CP-BNP 5, or CP-BNP 5+7 preparations.

[0135] FIG. 15A to FIG. 15CIt is a graph showing the change of HBV DNA (log10 IU / mL) level relative to baseline (W10) after therapeutic vaccination of various CP-BNP combinations in a mouse model of CHB infection. 0.5 μg dose of CP-BNP 1+4+5 had the most rapid and absolute sustained reduction of HBV DNA relative to the same dose of placebo or WT-BNP. Other CP-BNP combinations also reduced HBV DNA levels, but compared with CP-BNP 1+4+5, they reduced HBV DNA levels at a slower rate and less absolute decline.

[0136] FIG. 16A to FIG. 16C It is a graph showing changes in HBsAg (log10 IU / mL) levels relative to baseline (W10) after therapeutic vaccination of various CP-BNP combinations in a mouse model of CHB infection. 0.5 μg dose of CP-BNP 1+4+5 has the fastest and absolute sustained HBsAg decline relative to the same dose of placebo or WT-BNP. Other CP-BNP combinations also reduce HBV DNA levels, but compared with CP-BNP 1+4+5, it reduces HBV DNA levels at a slower rate and less absolute decline.

[0137] FIG. 17A to FIG. 17B was shown to be transcribed in vitro and transfected into HEK293T ( Fig.17A ) and Huh7( Fig. 17B Figure of protein analysis of HBsAg BNP expression from RNA of WT-BNP and CP-BNP 4 after transfection in ) cell lines. Using anti-HBsAg antibody, HBsAg band (+ / - glycosylation, ~ 50%) was detected in WT-BNP (24 or 27kDa) and CP-BNP 4 (29 or 32kDa), produced after transfection of appropriate mRNA into two cell lines. Untreated and reagent test control transfections are negative for HBsAg. Controls include purified HBsAg (with Flag tag, 25 or 28kD) and samples from after transfection of WT-BNP DNA (with Flag tag, 25 or 28kD). mRNA transcripts encode WT-BNP or BNP4-specific HBsAg subunits in the absence of Flag tags. DETAILED DESCRIPTION

[0138] It should be understood that certain aspects, modes, embodiments, variations and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology. Definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by a person of ordinary skill in the art to which the technology belongs.

[0139] I. Summary

[0140] Functional hepatitis B cure is defined as HBsAg loss with or without seroconversion to anti-HBsAg antibodies ("anti-HBs") while maintaining serum HBV DNA undetectable (Revill P et al. Nature Reviews Gastroenterology and Hepatology. 2016, 13 (4): 239-248). HBsAg bionanoparticles (BNPs) with antigen-displayed epitopes associated with enhanced clearance properties (CP) are described herein to mimic the selective pressure of CP and promote effective and specific B cell anti-HBs immune responses for HBsAg clearance. The immunogenicity of BNP was initially verified in normal BALB / c mice, and then the results of therapeutic vaccination were evaluated in the newly developed and verified CBA Carter J (CBA / CaJ) mouse CHB model to drive HBsAg clearance and seroconversion (see, e.g., Chen HH et al. PNAS 112 (7): 2175-2180 (2015)). In some embodiments, the CP-expressing BNP therapeutic vaccine, when delivered to CHB patients, is intended to accelerate and drive functional hepatitis B cure. In some embodiments, the therapeutic function of CP-expressing BNP can also be enhanced by: highly glycosylated modification to enhance and accelerate B cell responses and promote HBsAg seroconversion, and / or modifying the BNP delivery backbone to induce a "stealth" state of BNP in CHB patients to potentially pre-existing, neutralizing, but not cleared circulating anti-HBs antibodies.

[0141] II. Definitions

[0142] The following terms are used herein and their definitions are provided for guidance only.

[0143] As used in this specification, the singular forms "a", "an", and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a property" includes a single property, as well as two or more properties; reference to "an epitope" includes a single epitope, as well as two or more epitopes; reference to "an antibody" includes a single antibody, as well as two or more antibodies; reference to "the present disclosure" includes both single and plural aspects of the present disclosure teachings; and so on.

[0144] The term "about" and generally used ranges (whether or not limited by the term about) mean that the numbers understood are not limited to the exact numbers recited herein, and are intended to refer to ranges substantially within the cited ranges without departing from the scope of the present technology. As used herein, "about" will be understood by one of ordinary skill in the art and will vary in the context of its use. If there is a use of a term that is unclear to one of ordinary skill in the art given the context of use, "about" means plus or minus 10% of the particular term.

[0145] The term "antigen" is used herein in its broadest sense to refer to a substance capable of reacting in an immune response and / or inducing an immune response. Reference to an "antigen" includes an antigenic determinant or epitope.

[0146] "Antigenic determinant" or "epitope" means the part of an antigen molecule to which a specific immune response is directed, and includes haptens. Typically, in animals, an antigen presents several or even many antigenic determinants simultaneously. A "hapten" is a substance that can specifically bind to an antibody but cannot or only poorly induce an immune response unless bound to a carrier. A hapten usually contains a single antigenic determinant or epitope.

[0147] As used herein, the term "biological nanoparticle" or "BNP" refers to a virus-like particle (VLP) that has been modified to contain or present one or more target insert epitopes. For the purposes of this disclosure, the term "biological nanoparticle" or "BNP" also refers to a "recombinant virus-like particle antigen" or "VLP-Ag".

[0148] As used herein, the term "effective amount" or "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount sufficient to achieve the desired biological, therapeutic and / or preventive effect, such as an amount that results in the prevention of a disease, disorder and / or its symptoms. In the context of therapeutic or preventive applications, the amount of the composition administered to the subject will depend on the type and severity of the disease and the characteristics of the individual, such as general health, age, sex, body weight and tolerance to the composition drug. It will also depend on the extent, severity and type of the disease or disorder. The skilled person will be able to determine the appropriate dosage based on these and other factors. In some embodiments, multiple doses are administered. Additionally or alternatively, in some embodiments, a variety of therapeutic compositions or compounds (e.g., immunogenic compositions, such as vaccines) are administered.

[0149] As used herein, the term "functional cure" refers to a functional cure of CHB and is defined as detectable loss of HBsAg in the presence or absence of naturally occurring or introduced anti-HBsAg antibodies (also referred to herein as "anti-HBs antibodies" or "anti-HBs"), including a population of antibodies that selectively bind to selected epitopes on HBsAg, which, when occupied by anti-HBs antibodies, result in clearance of HBsAg and ultimately HBV. HBV cannot be detected in completely cured subjects.

[0150] The term "immunogenic composition" is used herein to refer to a composition that will elicit an immune response in a mammal that has been exposed to the composition. In some embodiments, the immunogenic composition comprises at least one of eight CP-BNPs (e.g., BNP1, ​​BNP 2, BNP 3, BNP 4, BNP 5, BNP 6, BNP 7, BNP 8) and / or a CP epitope cyclic peptide.

[0151] In some embodiments, the immunogenic compositions described herein can be formulated for administration in a variety of ways. For example, in some embodiments, the immunogenic compositions are prepared for intravenous, intramuscular, subcutaneous, parenteral, oral, nasal or topical administration. Compositions can also be formulated for use in specific dosage forms. For example, in some embodiments, the immunogenic compositions can be formulated as liquids, gels, aerosols, ointments, creams, lyophilized formulations, powders, cakes, tablets or capsules. In other embodiments, the immunogenic compositions are formulated as controlled release formulations, delayed release formulations, extended release formulations, pulsatile release formulations and mixed immediate release formulations. In some embodiments, an immunogenic composition is provided in a liquid. In other embodiments, an immunogenic composition in lyophilized form is provided.

[0152] As used herein, the term "infected" refers to carrying a disease or pathogen, such as a virus. Infection can be intentional, such as by administering a virus or pathogen (e.g., by vaccination), or unintentional, such as by natural transfer of pathogens from one organism to another, or from a contaminated surface to an organism. In some embodiments, infection is induced in a model organism (e.g., a mouse model) by transfecting replicable DNA in vivo using a hydrodynamic injection method.

[0153] As used herein, "subject" and "patient" are used interchangeably and refer to an animal, such as a member of any vertebrate species. In some embodiments, the subject is a human.

[0154] As used herein, the term "treating" or "treatment" or "alleviation" refers to therapeutic treatment, wherein the purpose is to reduce, alleviate or slow the progression or advancement of the target pathological disorder or condition, and / or reverse the progression of the target pathological disorder or condition. For example, a subject's existing and / or persistent hepatitis B infection, including chronic hepatitis B (CHB) infection, is successfully "treated" if, after receiving a therapeutic amount of a composition of the present technology, the subject shows observable and / or measurable induction of antibodies that clear HBsAg, and / or detectable HBsAg loss, and / or reduced levels of HBsAg and / or HBV DNA according to the methods described herein.

[0155] As used herein, the term "vaccine" refers to a composition administered to a subject to produce or increase immunity to a particular disease. In some embodiments, the vaccine comprises a pharmaceutically acceptable adjuvant and / or a pharmaceutically acceptable carrier.

[0156] As used herein, "BNP 1" or "CP-BNP 1" refers to SEQ ID NO: 1, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1, such as the nucleotide sequence shown in SEQ ID NO: 9, a virus-like particle (VLP) or biological nanoparticle (BNP) comprising SEQ ID NO: 1, an immunogenic composition comprising SEQ ID NO: 1, or a vaccine comprising SEQ ID NO: 1.

[0157] As used herein, "BNP 2" or "CP-BNP 2" refers to SEQ ID NO:2, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2, such as the nucleotide sequence shown in SEQ ID NO:10, a VLP or BNP comprising SEQ ID NO:2, an immunogenic composition comprising SEQ ID NO:2, or a vaccine comprising SEQ ID NO:2.

[0158] As used herein, "BNP 3" or "CP-BNP 3" refers to SEQ ID NO:3, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:3, such as the nucleotide sequence shown in SEQ ID NO:11, a VLP or BNP comprising SEQ ID NO:3, an immunogenic composition comprising SEQ ID NO:3, or a vaccine comprising SEQ ID NO:3.

[0159] As used herein, "BNP 4" or "CP-BNP 4" refers to SEQ ID NO:4, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:4, such as the nucleotide sequence shown in SEQ ID NO:12, a VLP or BNP comprising SEQ ID NO:4, an immunogenic composition comprising SEQ ID NO:4, or a vaccine comprising SEQ ID NO:4.

[0160] As used herein, "BNP 5" or "CP-BNP 5" refers to SEQ ID NO:5, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:5, such as the nucleotide sequence shown in SEQ ID NO:13, a VLP or BNP comprising SEQ ID NO:5, an immunogenic composition comprising SEQ ID NO:5, or a vaccine comprising SEQ ID NO:5.

[0161] As used herein, "BNP 6" or "CP-BNP 6" refers to SEQ ID NO:6, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:6, such as the nucleotide sequence shown in SEQ ID NO:14, a VLP or BNP comprising SEQ ID NO:6, an immunogenic composition comprising SEQ ID NO:6, or a vaccine comprising SEQ ID NO:6.

[0162] As used herein, "BNP 7" or "CP-BNP 7" refers to SEQ ID NO:7, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:7, such as the nucleotide sequence shown in SEQ ID NO:15, a VLP or BNP comprising SEQ ID NO:7, an immunogenic composition comprising SEQ ID NO:7, or a vaccine comprising SEQ ID NO:7.

[0163] As used herein, "BNP 8" or "CP-BNP 8" refers to SEQ ID NO:8, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:8, such as the nucleotide sequence shown in SEQ ID NO:16, a VLP or BNP comprising SEQ ID NO:8, an immunogenic composition comprising SEQ ID NO:8, or a vaccine comprising SEQ ID NO:8.

[0164] As used herein, "clearance property (CP) epitope cyclic peptide 1" refers to SEQ ID NO: 17. As used herein, "CP epitope cyclic peptide 2" refers to SEQ ID NO: 18. As used herein, "CP epitope cyclic peptide 3" refers to SEQ ID NO: 19. As used herein, "CP epitope cyclic peptide 4" refers to SEQ ID NO: 20. As used herein, "CP epitope cyclic peptide 5" refers to SEQ ID NO: 21.

[0165] As used herein, antibodies within the anti-HBs response that lead to functional cure are referred to as "clearing antibodies". These antibodies define the "clearing properties" of antibodies that target specific HBsAg epitopes and ultimately lead to HBsAg clearance and functional cure. Epitopes are also referred to as "clearing epitopes", meaning that once occupied by antibodies in a host object, the object will produce or may produce a functional cure. Therefore, "clearing properties" can refer to the fingerprint of an unavailable epitope on HBsAg, or to a set or group of individual antibodies that occupy these epitopes and predict that a functional cure is achieved when they do occupy the epitope. The clearance properties of antibodies or epitopes on HBsAg represent biomarkers of the potential or possibility that the object receiving treatment will achieve a functional cure. Mentioning the "possibility" of a functional cure generally means that the possibility is 100%, that is, once the clearance properties of the antibody are detected, the object will reach a functional cure state or has achieved a functional cure in the absence of circulating HBsAg. However, as in any biological system, variability can occur. Thus, for the purposes of the present technology, reference to the "likelihood" of a functional cure means at least an 80% probability that a subject having the antibody clearance properties will achieve a functional cure. "At least 80%" means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0166] The "clearance profile of epitopes" or "CP of epitopes" on HBsAg is the presence of available or unavailable epitopes on HBsAg with the potential to be occupied by clearance antibodies. When the clearance profile of an epitope on HBsAg does not contain an epitope available for binding, the epitope is considered "unavailable", and this means that the epitope is occupied by antibodies and a functional cure will likely occur. Alternatively, if HBsAg is not detected, nor is any circulating HBV DNA detected, a functional cure has been achieved, and only the clearance profile of antibodies exists (or can be induced in an immune response following exposure to HBV).

[0167] The term "standardized" also encompasses "normalization" of the levels of HBsAg or HBsAg-Ab complexes and represents the optimal level of HBsAg or HBsAg-Ab complexes for the assay. "Blood-derived sample" includes serum.

[0168] III. Functional cure of chronic hepatitis B (CHB)

[0169] Clearance of serum HBsAg and / or seroconversion to anti-HBs and undetectable serum HBV DNA are considered functional cures for CHB because they not only allow cessation of therapy but are also associated with significantly reduced rates of cirrhosis and hepatocellular carcinoma (HCC) and increased overall survival (Fattovich G et al. Am J Gastroenterol. 1998; 93(6): 896-900; Yuen MF et al. Gastroenterology 2008, 135(4): 1192-1199; Simonetti J et al. Hepatology 2010, 51: 1531-1537). During spontaneous resolution of acute hepatitis B infection, synergistic interactions of HBV-specific T cells and B cells eliminate infected hepatocytes, inhibit viral replication via non-cytolytic pathways, and neutralize virions by producing virus-specific antibodies (anti-HBs). Chronic infection means that the host immune response cannot control HBV replication, and spontaneous clearance of HBsAg is rare and found to occur in only 1% to 2% of patients. Current antiviral therapy has not improved this rate, but the key factor associated with this spontaneous loss is a low serum level of HBsAg <100 IU / ml (Tseng TC et al. Gastroenterology 2011, 141(2):517-525, 25e1-2), and strategies designed to achieve this level will become part of the treatment armamentarium. The mechanism or mechanisms associated with HBsAg loss and / or seroconversion are not yet clear, but it has been demonstrated that an effective B cell response is essential for maintaining a functional hepatitis B cure, and that B-cell depletion therapy (e.g., rituximab, anti-CD 20) is associated with HBV reactivation, leading to liver failure and death (Shouval D. Semin Liver Dis. 2013; 33(2):167-177). Clinical and diagnostic observations show that there are B cell clones encoding anti-HBs with low affinity to homologous HBsAg in CHB patients (Gerlich WH. Clin Infect Dis. 2007; 44 (9): 1170-1172). The simultaneous expression of anti-HBs in HBsAg-positive CHB patients further indicates that the anti-HBs response is insufficient or inadequate (weak) in quantity and / or quality to overcome chronic infection, but does indicate that specific antibody production is not completely suppressed (Zhang JM et al. Clin Infect Dis. 2007; 44 (9): 1161-1169).

[0170] IV. Chronic Hepatitis B (CHB) Disease

[0171] Chronic hepatitis B (CHB) is an infectious disease that covers five recognizable and independent stages: Stage 1 is the immune tolerance (IT) stage, which covers the time from infection and establishment of chronic infection to the first signs of active disease. It is usually asymptomatic (no obvious liver disease), and these individuals acquired the infection at birth or shortly thereafter, and they are HBeAg positive. Stage 2 or immune clearance (IC) stage is an active period of the disease with significant progression in a person's liver disease. These patients are also HBeAg positive. In stage 3 or non-replication stage (NR), there is little or no evidence of active liver disease; low levels of virus can be identified, and the person is HBeAg negative. Stage 4 is a relapse or recurrence of liver disease activity and viral replication, and this stage is also called HBeAg-negative disease. This stage often eventually disappears on its own, and the patient usually then has cirrhosis. The final stage, stage 5, is identified as HBsAg loss and anti-HBs seroconversion. This is also considered the functional cure (FC) stage.

[0172] CHB includes at least 4 different diseases based on HBV genotype. Therefore, CHB can be classified into Asian CHB (genotype B and C), European CHB (genotype A-2 and D), African CHB (genotype A-1 and E) and Latin American CHB (genotype F and H), and each of these 4 groups has different races (Asians vs. Europeans / Caucasians vs. Africans vs. Latin Americans), age of acquisition (perinatal vs. early childhood vs. early adulthood) and transmission mode (mother to infant, child to child, cultural scar, iatrogenic / parenteral and sexual). Recombination between different genotypes (e.g., A and D, or B and C) is not common. CHB can be further classified according to HBeAg and disease status; HBeAg positive or HBeAg negative, with or without liver disease.

[0173] FC-P associated with FC and was demonstrated by loss of epitope recognition of loops 1 and 2 within the external loop region, which includes the “a” of HBsAg when mAb 5, mAb 6, mAb 7, and mAb 8 were used.

[0174] Determinants (see Figure 1 and Figure 2 ). The anti-HBs response after the cure outcome is "induced" against FC-P of the reference HBsAg sample. In some embodiments described herein, vaccination using a BNP platform comprising FC-P-associated epitopes (loop 1 + loop 2) generates an anti-HBs response associated with FC-P, thereby increasing the chance of cure.

[0175] V. HBsAg, anti-HBs and CP / F-CP

[0176] Measuring the epitope profile of the envelope protein (HBsAg) of HBV circulating in a patient's blood is a direct measure of the virus; it involves detecting foreign antigens found in a patient's blood. The logic or argument developed in CP is that an infected person will mount an immune response to the virus and the effect of the immune response selection pressure on the virus can be measured. The effect can be demonstrated by changes in the HBsAg epitopes on the viral envelope in that person. Measuring CP on a multiplex reporter panel ( Figure 2 ). FC-P is reported to be reactive to a 4-combination applied to a natural history cohort at St. Vincent's Hospital, Melbourne, where patients are HBeAg negative and have minimal or no disease activity, are in stage 4 and transition to stage 5, and the infected genotype is not restricted (i.e., genotype A, B, C, or D). In contrast, the measure of antibodies made by the patient is a direct measure of the host's response to the virus's significance; it is also found to circulate in the patient's blood. The logic or view developed in FC-P is that people who have been cured of CHB have successfully elicited an antibody response, the effect of which can be measured. The effect can be demonstrated by the ability of this antibody to induce epitope changes on the exogenously supplied HBsAg of the HBV group; that is, the patient's antibodies "induce" the response of the epitope curve to the supplied exogenous antigen. The "induced" FC-P is applicable to a natural history cohort of stage 4 / 5, HBeAg negative, genotype-unrestricted CHB patients, which is based on a 4-combination reporting group.

[0177] VI. HBsAg, VLPs and BNP

[0178] HBV encodes three HBsAg proteins that form the viral envelope; small (S), medium (M) and large (L) (Carman WF et al. J Hepatol. 1999; 31(2): 195-201; Seeger C et al. Hepadnaviruses. 2013). All share a common 226-residue HBsAg-S domain (Seeger C et al. Hepadnaviruses. In Fields Virology, 2013, 6th edition. Pages 2180-2221), while the N-terminal extensions of M and L encode the PreS2 and PreS1 domains, respectively. The HBsAg envelope protein is a key viral antigen; during infection, S, M and L are expressed and co-packaged in the ER membrane of the host cell to form the envelope of infectious HBV particles. The S protein is the only antigenic component of the hepatitis B vaccine, which is able to induce protective antibodies mainly against the major antigenic 'a' determinant cluster in the external loop region (residues 99-169). The S protein readily self-assembles into lipid-associated VLPs and forms the basis of current recombinant vaccines, which are prepared in yeast and are not glycosylated. The S protein has not yet been crystallized, but is believed to be a conformationally dynamic protein that contains a large number of cysteine ​​and proline residues that are expected to form discrete loop domains within the immunodominant 'a' determinant cluster (loop 1: aa 107-135; loop 2: aa139-149) (Stirk HJ et al. Intervirology. 1992, 33(3): 148-158). The cDNA sequence encoding the S protein is shown in SEQ ID NO: 22. The cDNA sequence encoding the S protein with an AgeI restriction site and a FLAG tag is shown in SEQ ID NO: 23. Changes within the 'a' determinant (variants and antibody occupancy) can modify HBsAg topology and directly affect the HBV neutralization phenotype (Carman WF et al. J Hepatol. 1999; 31(2): 195-201).

[0179] The cDNA sequence encoding the HBsAg-S protein having the amino acid sequence of SEQ ID NO: 50 is shown in SEQ ID NO: 22. The cDNA sequence encoding the S protein having the amino acid sequence of SEQ ID NO: 51 is shown in SEQ ID NO: 23. The HBsAg-S cDNA and amino acid sequences are provided in Table A below. Table A. HBsAg-S cDNA and amino acid sequences

[0180]

[0181]

[0182] HBsAg VLPs are very compact due to the large number of intramolecular and intermolecular disulfide bonds within and between the individual subunits (Seeger C et al. Hepadnaviruses. In "Fields Virology", 2013, 6th edition. Pages 2180-2221; Mangold CM et al. Arch Virol. 1997, 142(11):2257-2267), and clinical trials have been established that can be successfully modified to display inserted foreign antigens and medically relevant sequences as a bionanoparticle (BNP) delivery platform (Beaumont E et al. Vaccine. 2015, 33(8):973-976; Buonaguro L et al. Expert Rev Vaccines. 2011, 10(11):1569-1583; Cheong WS et al. Antiviral Res. 2009, 81(2):113-122; Moffat J et al. JM et al. Vaccine. 2013, 31(18):2310-2316; Netter HJ et al. J Virology 2001; 75(5):2130-2141; Phogat S et al. Virology. 2008, 373(1):72-84).

[0183] VII. Clearance epitopes of HBsAg loop 1 and loop 2

[0184] Clearance epitopes on HBsAg are those that are likely to result in functional cure when occupied by antibodies in a subject. The epitopes are located on each of loop 1 and loop 2 of HBsAg-S. For this assay, monoclonal antibodies (mAbs) were selected for multiple assays that target a range of epitopes on HBsAg-S. A group of mAbs named mAb5 and mAb6 target loop 1 epitopes. Two loop 1 epitopes defined by the following consensus amino acid sequences were screened:

[0185] CX1TCX2X3X4X5QGX6SMX7PC (SEQ ID NO: 24), wherein:

[0186] X1 is sK122 (genotype A1, A2, A6, B1, B2, B3, B6, C, F, G, H, I) or sR122 (genotype A3, A4, A5, B3, B4, B5, B7, B8, B9, C2, C4, D, E);

[0187] X2 is sT125 (genotype A, B, C, D, E, F, G, H, I) or sM125 (genotype D3, D5);

[0188] X3 is sT126 (genotype A, B, C, D, E, F, G, H, I) or sI126 (genotype C);

[0189] X4 is sP127 (genotype A, B, E, C, D, G, I) or sT127 (genotype C4, D2, D5) or sL127 (genotype E, F, H);

[0190] X5 is sA128(A, B, C, D, E, F, G, H, I) or sV128(D2);

[0191] X6 is sN131 (genotype A, G, I) or sT131 (genotype B, C, D, E, F, H);

[0192] X7 is sF134 (genotype A, B, C, E, F, H) or sY134 (genotype D, G, I);

[0193] and the consensus amino acid sequence PCX8TCX9X10X bound by mAb 10 11 (SEQ ID NO:25), wherein:

[0194] X8 is sK122 (genotype A1, A2, A6, B1, B2, B3, B6, C, F, G, H, I) or sR122 (genotype A3, A4, A5, B3, B4, B5, B7, B8, B9, C2, C4, D, E);

[0195] X9 is sT125 (genotype A, B, C, D, E, F, G, H, I) or sM125 (genotype D3, D5);

[0196] X 10 is sT126 (genotype A, B, C, D, E, F, G, H, I) or sI126 (genotype C) or sS126 or sA126 (common variants);

[0197] X 11 is sP127 (genotype A, B, E, C, D, G, I) or sT127 (genotype C4, D2, D5) or sL127 (genotype E, F, H);

[0198] Another group of mAbs (designated mAbs, 7, 8, 11, 12, 16, and 17) target an epitope on loop 2 defined by the following consensus amino acid sequence:

[0199] CCCTKPX 12 DGNCX 13 (SEQ ID NO:26)

[0200] in:

[0201] X 12 is sT140 (genotype A, B, C, D, G, H, I) or sS140 (genotype E or F); and

[0202] X 13 It is sT143 (genotype A, B) or sS143 (genotype C, D, E, F, G, H, I).

[0203] Without wishing to be bound by theory, it is proposed herein that when any one epitope of loop 1 is occupied, when an epitope of loop 2 is occupied, or when any one epitope of loop 1 is occupied together with an epitope of loop 2, the clearance properties of the antibody are achieved, resulting in functional clearance.

[0204] In embodiments, the occupied epitope is CKTCTTPAQGNSMFPSC (SEQ ID NO: 27); and / or PCKTCTTP (SEQ ID NO: 28); and CCCTKPTDGNCT (SEQ ID NO: 29).

[0205] In embodiments, the occupied epitope is CKTCTIPAQGTSMFPSC (SEQ ID NO:30); and / or PCKTCTTP (SEQ ID NO:28); and CCTKPSDGNCT (SEQ ID NO:31).

[0206] In an embodiment, the occupied epitope is CRTCTTPAQGTSMFPSC (SEQ ID NO:32); and / or PCKTCTTP (SEQ ID NO:28); and CCTKPSDGNCT (SEQ ID NO:31).

[0207] VIII. Clearance Properties - Bio-nanoparticle (CP-BNP) Therapeutic Vaccine Formulations

[0208] The disclosure of the present technology relates to the development of 8 CP-BNPs (SEQ ID NOs: 1-8) covering pan-genotype / serotype loop 1 and loop 2 HBsAg target epitopes, together with control wild-type (WT) BNP. The BNP backbone consists of non-infectious subviral particles (virus-like particles, or VLPs, which do not contain all the components of a complete virus) of the HBsAg-S envelope protein. In some embodiments, the HBV genotype is genotype A, A1, A2, A3, A4, A5, A6, B, B1, B2, B3, B4, B5, B6, B7, B8, B9, C, C1, C2, C4, D, D2, D3, D5, E, F, G, H or I. In some embodiments, the HBV genotype is genotype D. In some embodiments, the serotype is ayw or adw. In some embodiments, the serotype is ayw. Modification of VLPs to bionanoparticles (BNPs) to deliver target insert epitopes represents an active approach that stimulates the immune system to induce responses that can treat or cure (clear) persistent hepatitis B virus infections. Antibodies that can induce cures of existing and persistent hepatitis B infections will be generated. The CP epitope target (insert) sequences are listed in Table 1 below. BNPs (endogenous free) were produced and purified, and immunogenicity was assessed separately in formulations that considered combinations of loop 1 and loop 2 target epitopes. The formulations analyzed are listed in Table 2 below.

[0209] Table 1. Target HBsAg CP epitopes

[0210]

[0211] Table 2. CP-BNP preparations

[0212]

[0213]

[0214] The amino acid sequences of the eight CP-BNP constructs are provided in Table 3, Construct 1 (BNP 1) (SEQ ID NO: 1), Construct 2 (BNP 2) (SEQ ID NO: 2), Construct 3 (BNP 3) (SEQ ID NO: 3), Construct 4 (BNP 4) (SEQ ID NO: 4), Construct 5 (BNP 5) (SEQ ID NO: 5), Construct 6 (BNP 6) (SEQ ID NO: 6), Construct 7 (BNP 7) (SEQ ID NO: 7) and Construct 8 (BNP 8) (SEQ ID NO: 8).

[0215] Table 3. CP-BNP construct amino acid sequence

[0216]

[0217]

[0218]

[0219] Exemplary nucleotide sequences encoding eight CP-BNP amino acid constructs are provided in Table 4.

[0220] Table 4. Exemplary nucleotide sequences of CP-BNP constructs

[0221]

[0222]

[0223]

[0224]

[0225] IX. CP-peptide therapeutic vaccines

[0226] These epitope vaccines are also developed as an alternative delivery to pan-genotypic / serotypic cyclic peptide vaccines based on the HBsAg CP epitopes incorporated into the CP-BNP therapeutic vaccine as described in Table 1. The CP epitope cyclic peptides of the present technology are listed in Table 5.

[0227] Table 5. CP epitope-specific cyclized peptides / formulations

[0228]

[0229] Immunogenicity studies have shown that the lead cyclic peptide vaccine is an equimolar preparation of cyclic peptides CTKPTDGNCT (SEQ ID NO: 20) and CTKPSDGNCT (SEQ ID NO: 21) conjugated to keyhole limpet hemocyanin (KLH). Administration of the vaccine to mice is antigenic and induces an anti-HBs Ab response consistent with CP, indicating that anti-HBs Ab is "cleared". Analysis involves ELISA (immune peptide, control peptide, VLP antigen), inducible CP analysis, immunoprecipitation and protein blot.

[0230] X.mRNA vaccines

[0231] In some embodiments, the disclosure relates to the use of mRNA constructs to elicit an immune response in presence and / or persistence of hepatitis B, including chronic hepatitis B (CHB). In some embodiments, the mRNA encodes an epitope repeat region from an epitope expressed in loop 1 and loop 2 of the HBsAg-S domain. In some embodiments, the mRNA encodes BNP 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, the mRNA encodes a CP epitope cyclic peptide, such as those provided in Table 5.

[0232] XI. Modes of Administration and Effective Dosages

[0233] Immunogenic compositions (e.g., vaccines) as disclosed herein can be administered by any conventionally used or recommended route for vaccines (e.g., parenteral route), and can be administered in a variety of forms (e.g., injections). Vaccines can be administered by syringe or by a needle-free syringe for intramuscular, subcutaneous, or intradermal injection.

[0234] According to the present technology, the "effective amount" of the immunogenic composition is an effective amount sufficient to achieve the desired biological effect. It should be understood that in some embodiments, the effective dose will depend on the age, sex, health and weight of the recipient, the type of current treatment (if any), the frequency of treatment and the nature of the desired effect. The range of effective doses provided below is not intended to be limited and represents an exemplary dosage range. Therefore, in some embodiments, as understood and determined by those skilled in the art, the dose will be tailored to individual subjects. The dose of CP-BNP, CP epitope cyclic peptide or mRNA vaccine for adult mammals (e.g., humans) can be 0.01 μg to 10,000 μg or any range or value therein. In some embodiments, the dose can be 0.10 μg to 10,000 μg or any range or value therein. In some embodiments, the dose can be 1 μg to 10,000 μg or any range or value therein. In some embodiments, the dose can be 2 μg to 10,000 μg or any range or value therein. In some embodiments, the dose can be 3 μg to 10,000 μg or any range or value therein. In some embodiments, the dose may be 4 μg to 10,000 μg or any range or value therein. In some embodiments, the dose may be 5 μg to 10,000 μg or any range or value therein. In some embodiments, the dose may be 10 μg to 10,000 μg or any range or value therein. In some embodiments, the dose may be 15 μg to 10,000 μg or any range or value therein. In some embodiments, the dose may be 20 μg to 10,000 μg or any range or value therein. In some embodiments, the dose may be 25 μg to 10,000 μg or any range or value therein. In some embodiments, the dose may be 50 μg to 10,000 μg or any range or value therein. In some embodiments, the dose may be 100 μg to 10,000 μg or any range or value therein. In some embodiments, the dose may be 500 μg to 10,000 μg or any range or value therein. In some embodiments, the dosage may be from 1,000 μg to 10,000 μg, or any range or value therein. In some embodiments, the dosage may be from 5,000 μg to 10,000 μg, or any range or value therein.

[0235] XII. Treatment Methods

[0236] The following discussion is presented by way of example only and is not intended to be limiting.

[0237] One aspect of the present technology includes methods for treating existing and / or persistent hepatitis B (including chronic hepatitis B (CHB)) in subjects diagnosed with or suspected of having hepatitis B infection. In therapeutic applications, a composition or medicament comprising CP-BNP, CP epitope cyclic peptides and / or mRNA constructs of the present technology is administered to a subject suspected of or already having hepatitis B infection in an amount sufficient to cure or at least partially arrest the symptoms of the disease.

[0238] XIII. Determining the biological effects of CP-BNP, CP epitope cyclic peptide or mRNA of the present technology

[0239] Based on the results of the HBsAg immunoassays described herein, a clinician can decide to stop treatment when a functional cure has been achieved or will be achieved. Thus, the HBsAg immunoassays described herein can monitor treatment to determine whether an individual will achieve a functional cure, to determine whether an individual has achieved a functional cure, and to determine whether an individual has achieved a cure through natural defense mechanisms.

[0240] The regimen can be changed without departing from the essence of the present technology. The key endpoint is to determine the epitope fingerprints on HBsAg that have been occupied by individual antibody responses. If the fingerprints of the epitopes of ring 1 and ring 2 have been occupied, functional cure can be expected. If antibodies with the ability to bind to the fingerprint exist or are indicative after exposure to HBV, but HBsAg cannot be detected, functional cure can be achieved. At that time, treatment can be stopped.

[0241] Example

[0242] The following examples are provided only by way of illustration rather than by way of limitation. Those skilled in the art will readily recognize that various non-critical parameters that may be changed or modified to produce substantially identical or similar results. As defined by the appended claims, the embodiments should never be construed as limiting the scope of the present technology.

[0243] Example 1: Development of a Clearance Characterization Assay

[0244] A multiplex ("Bioplex") bead-based flow cytometry platform was used to develop an HBsAg epitope assay and to establish and map the HBsAg profile of epitopes that underlie HBsAg epitope clearance characteristics (see Figure 2). The "clearance properties of an epitope" are properties of an epitope targeted by an individual antibody. When all epitopes are bound by antibodies produced by an individual, clearance of HBsAg can be expected. The assay is derived from the assay of Lim et al. (2014) Hepatology 60(1) Suppl:1623,980A. In brief, the HBsAg multiplex immunoassay includes a set of fluorescently identified beads, each of which is conjugated to a different anti-HBs antibody that binds to HBsAg, followed by a polyclonal phycoerythrin (PE)-conjugated detection antibody. The HBsAg multiplex panel contains monoclonal antibodies (mAbs) against the external loop region of HBsAg, including the 'a' determinant and the C-terminal domain epitope spanning HBsAg residues 99-226. HBsAg epitopes are classified into the following domains (with monoclonal antibodies that bind to those domains): N-terminal (mAb 1); loop 1 (mAb 5, 6, 10); loop 2 (mAb 7, 8, 11, 12, 16, 17, 19); loop 1 / 2 combined (mAb 13, 14, 15); C-terminal (mAb 2, 3, 4); and conformational (mAb 9, 18). The intensity of PE associated with each bead provides a sensitive measure of antibody recognition of a specific HBsAg epitope within a sample. The epitope curve on HBsAg compared to the matched HBV strain backbone has been expressed as a fold change in antibody binding or epitope recognition after bioinformatics data analysis. The 95% CI of the normal range of changes in epitope recognition relative to the reference backbone has been determined to be + / –0.5 times change. Epitope gain recognition corresponds to a positive fold change (>0.5-fold), while a negative fold change (>-0.5-fold) corresponds to a loss or reduction in epitope binding. A graphical representation of the assay is shown in Figure 1 middle.

[0245] Example 2: Selection of mAbs

[0246] Selection and acquisition of HBsAg multiplex assays ( Figure 2The anti-HBs mAbs utilized in Figure 1 are provided to provide a broad coverage of epitopes reactive to the HBsAg antigen and C-terminal domain (residues 99-226) (covering both continuous and discontinuous epitopes), as well as epitope regions that are subject to high variability and also conserved recognition. The high level of epitope coverage provided by the mAbs enables analysis of HBsAg properties based on mapped epitope availability and is reported based on epitope regions: N-terminal (mAb 1); loop 1 (mAb 5, 6, 10); loop 2 (mAb 7, 8, 11, 12, 16, 17, 19); loop 1 / 2 combined (mAb 13, 14, 15); C-terminal (mAb 2, 3, 4); and conformational (mAb 9, 18). Interrogation of HBV strains (genotype AG) identified anti-HBs mAbs that exhibited conserved epitope recognition patterns (i.e., representing a conserved HBsAg fingerprint across genotypes and serotypes), which were mAb 3 and 4 (C-terminal), mAb 15 (loop 1 / 2 combined), and mAb 18 (conformational). The remaining anti-HBs mAbs were sensitive to changes in HBsAg properties as determined by altered epitope recognition patterns between HBV strains compared to the genotype A2, serotype adw2 vaccine strain.

[0247] Example 3: Optimizing Assays / Kinetics

[0248] HBsAg characteristic determination is developed and optimized using HBV A2 adw2 strain HBsAg (which forms the common basis of most universal HBV vaccines and therefore represents a relevant baseline or backbone for comparing HBsAg epitope recognition). The HBsAg source is mainly HBsAg patient serum, confirmed as wild-type A2 adw2 (compared with the consensus sequence) by HBV sequencing, and confirmed using recombinant wild-type A2 adw2 HBsAg from cell culture supernatant. Initial assay development is based on the reactivity of two anti-HBs mAbs (including by protein blotting, Elisa and fluorescence) well characterized in the laboratory to HBsAg, which are mAb10 (loop 1) and 18 (conformational). The prepared bead group labeled with a series of mAbs is incubated with a series of HBsAg concentrations. These concentrations represent the standardization of HBsAg. Identify the conditions corresponding to comfortable fluorescence readout (in the range of 10000-20000RFU), without causing beads / mAb-HBsAg complex overload and aggregation.Determine that the assay concentration of HBsAg is about 16IU / well (standardized level), and the HBsAg detected is as low as 1IU / well and as high as 100IU / well.The HBsAg of sample dilution series is incorporated in the assay of 8,16 and 32IU / well, to explain any slight error in the HBsAg concentration diagnostic assay.The optimal concentration of the anti-HBs mAb labeled to beads is optimized to mAb concentration, resulting in the fluorescence reactivity (15000-18000RFU) under the dynamic range of the instrument, and this is specifically / empirically determined for each mAb.By sequentially adding a single anti-HBs mAb conjugated to beads, the multiplicity of assay is established, thereby allowing identification of epitope competition caused by the epitope shared by mAb parts. To avoid measuring epitope competition between mAbs (i.e., to correctly record the HBsAg epitope profile), three sets of multiplexed anti-HBs beads / mAb were established: 4-plex, 5-plex, and 10-plex. HBsAg samples were analyzed in parallel (on the same plate) using these complexes, and the resulting data were combined to achieve a multiplexed readout of HBsAg properties ( Figure 2 (see above).

[0249] Example 4: Validation of assays on HBV strains / HBsAg characteristics

[0250] The assay was developed for HBsAg A2 adw2 (corresponding to common HBV vaccine strains), and thus reflects the HBsAg characteristics of the epitope recognition of multiple anti-HBs mAbs in the assay. The assay was validated for the following: HBsAg A2 adw2 from wild-type ex vivo serum HBsAg and recombinant HBsAg from in vitro cell culture to confirm the HBsAg characteristics of A2 adw2, which forms a vaccine strain background for comparison with other HBV strains (genotypes and serotypes) (which may be vaccine mismatched), and reported HBsAg variants (e.g., sG145R / A, sP120T / L, sD144E / A) with vaccine escape potential. The assay was further validated for HBsAg of different genotypes (AG) and serotypes derived from both patient serum and recombinant supernatants, which determined the HBsAg characteristics of each strain compared to the vaccine strain (A2 adw2) background. Reference sera representing each HBV strain were established to be continuously included in the assay as control data points in the study cohort. Genotype-specific changes in HBsAg properties between common genotypes (A, B, C, D) were significant at loop 1 (mAb6) and loop 2 (mAb8) epitopes, while serotypes were significant at loop 2 (mAb7) epitopes. HBV strains that were more divergent from vaccine strains (C4, E, and F) showed greater changes in HBsAg properties recognized by epitopes compared to vaccine strains.

[0251] Example 5: Characterization of clearance properties associated with 'clearing' anti-HBs responses

[0252] Figure 1 The application of the assay described in Example 1 is shown to characterize the clearance properties (CP) - associated with antibodies leading to functional cure. The assay determined HBsAg epitope occupancy using a panel of mAbs, where mAb 5 and mAb 6 target the following consensus sequence in loop 1:

[0253] CX1TCX2X3X4X5QGX6SMX7PC(SEQ ID NO:24)

[0254] in:

[0255] X1 is K or R;

[0256] X2 is T or M;

[0257] X3 is T or I;

[0258] X4 is P, T or L;

[0259] X5 is A or V;

[0260] X6 is N or T; and

[0261] X7 is F or Y.

[0262] mAb 10 binds to another epitope loop 1 within amino acids 120 to 127 of the consensus sequence:

[0263] PCX8TCX9X 10 X 11 (SEQ ID NO:25)

[0264] in:

[0265] X8 is K or R;

[0266] X9 is T or M

[0267] X 10 is T, I or S; and

[0268] X 11 It is P, T or L.

[0269] mAb 7, mAb 8, mAb 11, mAb 12, mAb 16, and mAb 17 bind to an epitope within amino acids 137 to 147 of the consensus sequence SEQ ID NO:26 in loop 2, wherein the consensus sequence is:

[0270] CCCTKPX 12 DGNCX 13 (SEQ ID NO:26)

[0271] in:

[0272] X 12 is T or S; and

[0273] X 13 It is T or S.

[0274] It is proposed that the population of antibodies occupying both SEQ ID NO:24 and / or SEQ ID NO:25 and SEQ ID NO:26 represents the clearance properties of antibodies indicative of a functional cure.

[0275] Example 6: Predictive biomarkers for functional CHB cure

[0276] Figure 2 An assay for detecting HBsAg with occupied epitopes (upper panel) and an assay for detecting HBsAg complexed with anti-HBs antibodies (lower panel) are described. The assay employs a multiplex immunoassay that detects occupancy of epitopes in loop 1 (consensus sequences SEQ ID NO: 24 and 25) and in loop 2 (consensus sequence SEQ ID NO: 26).

[0277] Example 7: HBsAg clearance characteristics in CHB cure

[0278] Figure 3 and Figure 4 is a graphical representation showing the HBsAg clearance profile (CP) in CHB cure. Figure 3 In functionally cured patients, an antibody response was generated that cleared the infection. Figure 4 In the non-cured / non-responding patients, no occupied HBsAg epitopes are carried. The term HBsAgCP means the clearance property of HBsAg epitopes. HBsAg-non-clearance property is called HBsAg-NCP.

[0279] Example 8: HBsAg clearance characteristics in CHB cure

[0280] Recognition of HBsAg epitopes by each assay mAb was recorded as recognition fold change; positive fold change involving enhanced or improved HBsAg epitope recognition, unchanged or normal range epitope recognition (within + / - 0.5 fold), and decreased HBsAg epitope recognition indicated by negative fold change. HBsAg CP is indicated by decreased HBsAg epitope recognition (e.g., due to epitope occupancy) at both loop 1 and loop 2 HBsAg epitopes.

[0281] Example 9: Identification of Predictive Clearance Profile Biomarkers

[0282] HBsAg characterization. Multiplex anti-HBs panel assay on bioplex ( Figure 2 , above) was originally developed based on the concept of diagnostic monitoring of HBsAg changes in CHB patients. For example, the assay can be used for patients receiving nucleotide analog (NA) therapy in whom HBV DNA cannot be detected but HBsAg is still positive, and for looking for the development of treatment-resistant mutations or vaccine escape variants. In addition, the assay can be used to try to understand the characteristics and changes of HBsAg that occur during the CHB disease stage, i.e., predictive biomarkers for CHB disease. The patient group (from the natural history group) that achieved functional cure of CHB is related to CP biomarker identification and will be further described below.

[0283] Results from HBsAg characterization assays led to the development of CP biomarkers (i.e., HBsAg is recognized by a subset of anti-HBs Abs in the assay). Results from these assays provide an understanding that: (a) functional cure is associated with anti-HBs responses; (b) "clearance" anti-HBs responses specifically target epitopes of / within loop 1 and loop 2 specific anti-HBs Abs in the assay; (c) patients have many different anti-HBs Abs, but only those targeting these two loop 1 and loop 2 epitopes (CP epitopes) are associated with achieving functional cure; and (d) enhanced and targeted display of these CP-related epitopes drives the generation of CP-related "clearance" anti-HBs Abs.

[0284] Natural History / Functional Cure Cohort: Anti-HBs Analysis Performed. This study involved six CHB patients identified in the St. Vincent Study (Natural History Cohort) who met the case definition of functional cure (FC). All six patients had discontinued antiviral therapy and had undetectable HBsAg and had successfully seroconverted anti-HBs. All six natural history cohort patients were anti-HBs positive in their serum samples (post-seroconversion) and this anti-HBs status was then analyzed for the ability to “induce” FC-P (functionally cured clearance property; based on anti-HBs analysis of functionally cured patients and a 4-plex assay panel) when incubated with a reference HBsAg (see Figure 1 FC-P was defined as loss of epitope binding at both loop 1 and loop 2 in the external loop region (including the "a" determinant of HBsAg) using four mAbs: mAb 5, mAb 6, mAb 7, and mAb 8: (mAb 5 and mAb 6 recognize the epitope in loop 1; mAb 7 and mAb 8 recognize the epitope in loop 2) (see Figure 2 , upper panel). One of the six natural history patients had a previously obtained serum sample that was still HBsAg-positive. The HBsAg-positive sample was epitope mapped using 4-plex anti-HBs Abs on the Bioplex platform and met the case definition for FC-characteristic (FC-P).

[0285] The derived HBsAg epitopes (see Table 1 for details) identified by 4-fold loop 1 or loop 2 anti-HBs Abs have been inserted into the HBsAg VLP vaccine delivery platform as multiple repeats to generate BNP / FC-P (CP-BNP). These BNP / FC-P vaccines are prepared in a single epitope delivery form or a variety of epitope mixture forms, which are administered to mice in an immunization manner. The ability of the resulting anti-HBs response inducing FC-P when incubated with reference HBsAg was analyzed, and FC-P was identified for the BNP immune mixture covering both loop 1 epitope and loop 2 epitopes. These BNP / FC-P immune preparations include the constructs detailed in Table 2. These vaccines tested so far include both loop 1 and loop 2 epitopes, and have resulted in the generation of antibodies associated with FC-P.

[0286] Example 10: VLP / BNP Production

[0287] Methods for protein production in mammalian cell lines and purification via ultracentrifugation, affinity, and gel filtration purification are standard protocols and are described below and in Hyakumura et al. Journal of Virology 2015, 89(22): 11312-11322 (2015).

[0288] In brief, to produce VLP or BNP, using polyethyleneimine (PEI) (Polysciences, Warrington, USA) or FectoPRO (PolyPlus Transfection USA), HEK293-T cells grown in Dulbecco's modified Eagle's medium DMEM (Gibco-BRL, Grand Island, New York) supplemented with GlutaMax-1 (Gibco-BRL), 10% fetal calf serum (FCS), penicillin and streptomycin (Gibco-BRL) are transfected with expression constructs. VLP / BNP are harvested from cell culture supernatant 5 to 8 days after transfection. VLP / BNP comprising N-terminal Flag tag is affinity purified from collected tissue culture supernatant, affinity purified on anti-Flag affinity resin column (CSIRO, Australia). Fusion protein is competitively eluted from anti-Flag affinity column in PBS with recombinant Flag peptide (0.4mgml; CSIRO, Australia). The VLP / BNP preparation peak of affinity purification in PBS is then purified by size exclusion on Superdex 200 or Superose 6 gel filtration columns (GE Healthcare). The preparation of final VLP / BNP purification is subjected to endotoxin test (Endosafe Test, Charles River, U.S.), and Amicon 50kDa cut-off centrifugal concentrator equipment is used to rotate and concentrate to <1ml. Alternatively, a table centrifuge is used to purify VLP / BNP from the tissue culture supernatant collected by rotating clarification, then supernatant is transferred in an ultracentrifuge tube, covered with 20% sucrose pad, and pellet is precipitated by ultracentrifugation. Abandon supernatant, and the VLP of agglomeration is resuspended in STE buffer (100mM NaCl, 10mM Tris, pH8, 1mM EDTA) for vaccination. The yield purity of VLP / BNP preparations was assessed by: i) A260 / 280 spectroscopy using a calculated VLP extinction coefficient of 37.26; ii) quantitative HBsAg serology (Elecsys HBsAG II kit; Roche); iii) SDS-PAGE followed by Coomassie staining and Western blotting (WB) using anti-HBs and anti-Flag detection antibodies; iv) HBsAg epitope profiling analysis on the Bioplex platform (in-house assay); and v) standard ELISA technique and detection by anti-HBs antibodies (in-house assay).

[0289] Example 11: CP-BNP epitope retention

[0290] Following BNP production and purification and quality assurance analysis of the yield and purity of the BNP preparations, CP-BNP was analyzed as a separate BNP preparation and also as a BNP preparation against HBsAg epitope profiling (Bioplex platform) to assess the antigenicity of loop 1 and loop 2 epitope presentation retention (i.e., displaying or presenting loop 1 and loop 2 epitopes) upon delivery, if necessary. Fig.11 As shown, the CP-BNP preparation retains the epitope insert of the present technology.

[0291] Example 12: Generation of CP epitope cyclic peptide

[0292] Cyclic CP epitope peptides. Cyclic peptides are designed to mimic the "clearing" epitopes of loop 1 and loop 2 targets identified in HBsAg, similar to the CP epitope inserts delivered in CP-BNP. Sequence variants within these epitopes depend on naturally occurring HBV genotypes and HBsAg serotype variants, and a variety of cyclic peptides are designed based on this consideration, thereby generating pan-genotype / pan-serotype equimolar preparations of loop 1 or loop 2 CP epitope cyclized peptides to be delivered as immune antigens in immunogenicity studies for the development of CP-related "clearing" anti-HBs antibodies. Order the designed peptides to be produced by Mimotopes (Australia) as having N-terminal free amines, C-terminal free acids, and cyclized by disulfide bonds at the original sequence cysteine ​​residues. Production is a purity of> 85% determined by HPLC and by mass spectrometry characteristics analysis. The cyclized peptides are coupled to KLH (keyhole limpet hemocyanin) via a glutaraldehyde linker for immunization, or coupled to BSA (bovine serum albumin) for screening assays. Exemplary cyclic peptides of the present technology are listed in Table 5 above.

[0293] Immunogenicity studies to generate CP-associated anti-HBs included immunization with either the Loop 1 mixture cyclic peptide or the Loop 2 mixture cyclic peptide formulations, administered to n=2 BALB / c and n=2 C57 / B6 mice for each peptide formulation. Mice were immunized with 3 x 2ug doses of the peptide mixture.

[0294] The antibody response of recombinant HBsAg (VLP) and HBsAg ring 1 or ring 2 peptide cyclized peptide (single peptide and the peptide preparation formula of collection) in the pre-fusion serum of harvest by standard ELISA scheme screening. Serum is also analyzed to generate the CP characteristic determination analysis (internal determination) of anti-HBs antibody induction on Bioplex platform. These data are used to inform which mice should be selected for spleen harvesting and fusion to develop monoclonal anti-HBs specific antibodies.

[0295] Initially, the antibody response specific to recombinant HBsAg (VLP) and to ring 1 and ring 2 cyclized peptides (single peptide and pooled peptide preparation formula) of the supernatant after fusion was screened by standard ELISA scheme. After SDS-PAGE and protein blotting, the clone supernatant was further analyzed to detect HBsAg to determine the affinity to linear epitopes. Compared with the pan-genotype / serotype HBsAg before incubation, the CP characteristic determination analysis (in-house determination) of the generation anti-HBs antibody induction of clone supernatant was analyzed on the Bioplex platform. This data is used to determine which clones should be selected for the development of monoclonal antibodies in other rounds, and clone supernatants are analyzed in each round, and include the analysis of the monoclonal anti-HBs antibodies (Table 6; Fig.12 ).

[0296] Table 6. Post-fusion clone selection for monoclonal antibody production

[0297]

[0298] Selected clones are in bold and Underline

[0299] # indicates weak response / reactivity Example 13: Study on the immunogenicity of CP-BNP and CP epitope cyclic peptides

[0300] This embodiment demonstrates the immunogenicity of CP-BNP and CP epitope cyclic peptides of the present technology. By the immunity of BALB / c strain mice, the immunogenicity of CP-BNP and CP-BNP preparations showing target ring 1, ring 2 or ring 1&2 "clearing" epitopes was evaluated (see Table 2 above). CP-BNP 1-4 and its preparations were evaluated in the first round of BNP immunogenicity studies (compared with WT-BNP background control). In the second immunogenicity study, these and evaluation CP-BNP 5-8 and its preparations will be evaluated again.

[0301] Immunogenicity study #1 combines the preparation shown in CP-BNP 1-4 and table 2 (compared with WT-BNP and PBS placebo control), and immunogenicity study IMM001 repeats the assessment carried out in the original study, and assesses the performance of CP-BNP5-8 and preparation 1+3+4 and 5+7 in addition.With the purified CP-BNP antigen immunization six week-old BALB / c mice (maximum 5 in each group) of 3x 2ug dosage at intervals of two weeks.Blood was drawn before each use of antigen, and then immunized once every other week, for 5 weeks.Mice were put to death at the last time point, and terminal blood was collected. Serum harvested at each bleeding time point was analyzed by the following methods to study the response of anti-HBs antibodies to CP-BNP immunization: i) using a diagnostic serological test (Elecsys Anti-HBs kit, Roche) to determine anti-HBs antibody titers (IU / L); ii) standard ELISA methods for detecting WT-VLP or HBsAg loop 1 or loop 2 peptides; and iii) anti-HBs antibody-induced CP characteristic assay analysis on the Bioplex platform (in-house assay). The loop 1 and loop 2 reactivity reported in this table is reported by HBsAg characteristic assay CP-BNP or analysis of the preparation to determine the retention display of one or more HBsAg CP epitopes associated with clearance (i.e., the target epitope of "clearing" anti-HBs response).

[0302] Fig.13A Anti-HBs antibody immunogenic responses of CP-BNP preparations and formulations are shown. Fig. 13B and Fig. 13C Anti-HBs antibody immunogenic responses to loop 1 and loop 2 of the CP epitope cyclic peptide are shown, respectively. Fig.13D Anti-HBs reactivity was reported for WT-BNP, loop 1 peptide, and loop 2 peptide antigens. Taken together, these results demonstrate that CP-BNP and CP epitope cyclic peptides of the present technology elicit HBV-specific immune responses and are useful in methods for inducing the production of anti-HBs.

[0303] Example 14: CP-BNP induces CHB clearance properties

[0304] This Example demonstrates that CP-BNP of the present technology is capable of inducing clearance properties in immunized subjects.

[0305] The anti-HBs antibody profile of terminal blood sera following n=3 immunization schedules with CP-BNP preparations (CP-BNP 1-8) and formulations (CP-BNP 1+3, 1+3+4, 1+4, WT+4, 5+7 or 1+4+5) was analyzed. Fig.14The results shown in demonstrate that CP-BNP of the present technology induces clearance properties (e.g., loss of epitope recognition of loop 1 and loop 2 epitopes). Therefore, these results indicate that compositions comprising CP-BNP of the present technology can be used in methods for treating chronic hepatitis B.

[0306] Example 15: mRNA encoding CP-BNP and CP epitope cyclic peptide induces CHB clearance properties

[0307] This example will demonstrate that mRNA encoding CP-BNP and CP epitope cyclic peptides of the present technology are capable of inducing clearance properties in immunized subjects.

[0308] Six week-old BALB / c mice (5 per group) were immunized with purified mRNA encoding CP-BNP or CP epitope cyclic peptides at intervals of 3x 2ug dosages for two weeks. Blood was drawn before each antigen application, and then blood was drawn at intervals of once a week for 5 weeks after immunization. Mice were put to death at the last time point and final blood was collected. The serum collected at each bleeding time point was analyzed by the following method to study the response of anti-HBs antibodies to CP-BNP immunity: i) anti-HBs antibody titer (IU / L) was measured using diagnostic serological tests (Elecsys Anti-HBs kit, Roche); ii) standard ELISA method for detecting WT-VLP or HBsAg ring 1 or ring 2 peptides; and iii) CP characteristic determination analysis (internal determination) of anti-HBs antibody induction on Bioplex platform.

[0309] The results will show that the mRNA constructs of the present technology induce clearance properties in immunized mice (e.g., loss of epitope recognition at loop 1 and loop 2 epitopes). Therefore, these results will demonstrate that compositions comprising the mRNA constructs of the present technology can be used in methods for treating chronic hepatitis B.

[0310] Example 16: CP-BNP reduces HBV DNA and HBsAg levels in a mouse model of chronic hepatitis B (CHB)

[0311] Materials and methods for producing test vaccine candidates (CP-BNP 1, 3, 4, 5, 7) were used for testing in a murine CHB model. Plasmids containing HBsAgS-specific cDNA inserts (WT or various inserts as described in Tables 3 and 4) were cloned into pCAGGS (with an N-terminal FLAG tag) and transfected into HEK293T cells using polyethyleneimine (PEI) as described above (Longo, 2013). Functional HBsAgS proteins (including chimeric HBsAgS proteins with other sequences inserted) were assembled into secretable virus-like particles, bionanoparticles (BNPs). Five days after transfection, cell culture media was harvested and centrifuged to remove cell debris. The cell culture media was centrifuged at high speed (ultracentrifugation) on a 20% sucrose cushion to pellet and partially purify BNP, and then resuspended in sodium chloride (NaCl)-Tris-EDTA (STE) buffer. Further purification of BNP was performed by ultracentrifugation using a sucrose (20%–50%) gradient in STE. Fractions containing VLPs were collected, pooled (as needed), buffer exchanged and concentrated in PBS (Hyakumura, 2015; Cheong, 2009; Patzer, 1986), and stored at -80°C until use. HBsAg VLPs were confirmed by ELISA or Western blot as previously described.

[0312] Murine CHB model. The CBA / caJ CHB mouse model was developed by hydrodynamic injection of HDI of HBV replicons (pAAV / HBV1.2), which allows DNA delivery to hepatocytes by intravenous injection into the tail vein (Kim, 2013; Yang, 2002; Huang, 2006). This in vivo transfection mode is necessary because hepatitis B virus (HBV) does not naturally infect mice. A single HDI containing a plasmid that replicates HBV DNA induces stable HBV, depending on the mouse strain used (for CBA mice, persists for >6 months and produces liver-specific HBV viral markers (up to 32 weeks)). Long-term expression of HBV in carrier mice does not cause liver damage, as evidenced by normal levels of liver damage indicators (alanine aminotransferase, ALT). After HDI, HBV DNA is measured in mouse serum to confirm that HBV persistence is established. Mice with persistent HBV were then used in immunization studies to test the ability of chimeric BNP with clearance-specific epitope repeats (CP-BNP) to induce therapeutic immune responses capable of interfering with and / or clearing HBV from the liver.

[0313] After establishment of chronic hepatitis B (approximately week 10), patients were treated with placebo (adjuvant control), control particles (Engerix B (a commercial HBV vaccine produced in yeast cells), wild-type BNP [WT-BNP])), or test vaccine candidates (CP-BNP1-7, in various combinations at equimolar ratios to final concentrations, e.g. FIG. 15A to FIG. 15C and FIG. 16A to FIG. 16C Groups of 6 to 8 CBA / caJ mice were treated with 100 mg / kg (listed) of 50 mg / kg BNP. The treatment schedule consisted of a mixture of different chimeric BNPs adjuvanted with aluminum hydroxide. Three doses of the vaccine were given subcutaneously with a 2-week interval between each dose. Submandibular bleeds were performed every 2 weeks until 26 weeks after HDI and tested to determine serum HBV DNA load (Sitnik, 2010) and HBsAg levels (according to the manufacturer's recommended protocol; qHBsAg Elecsys).

[0314] Mouse CHB Model Results – HBV DNA Levels. The HBV DNA level results described below are shown in FIG. 15A to FIG. 15CPlacebo (Group 1) did not significantly change HBV DNA levels throughout the duration of the study. WT-BNP was administered at 0.05μg (Group 2), 0.1μg (Group 3), or 0.5μg (Groups 4 and 12). WT-BNP at a dose of 0.05μg (Group 2) had no effect on HBV DNA levels, while doses of 0.1μg (Group 3) or 0.5μg (Groups 4 and 12) showed a dose-dependent decrease in HBV DNA levels. The combination of CP-BNP 1+4+5 at 0.5μg (Groups 8 and 13) showed the fastest and steepest decrease in HBV DNA, with a sustained effect from W12 (two weeks after the first dose) until the end of the study (W26). The maximum reduction was observed with 0.5 μg of CP-BNP 1+4+5, showing that HBV DNA had a 3log reduction relative to baseline W10 and that HBV DNA had at least a 2log reduction relative to WT-BNP (at the same dose) from W12 (maximum reduction was observed at W14) (>3log reduction). Using a dosage less than 0.5 μg, CP-BNP 1+4+5 also showed a dose-dependent reduction in HBV DNA, but a lower dose of 0.05 μg (group 6) or 0.1 μg (group 7) had a higher HBV DNA relative to the WT-BNP of the same dose. Other combinations of CP-BNP were also tested in this model, and it was found to be less effective than CP-BNP 1+4+5. For example, 0.5 μg of the combination of CP-BNP 1+3+4 and reduced combinations (CP-BNP 3+4 (Group 9), CP-BNP4+5 (Group 10), CP-BNP 5+7 (Group 11)) also resulted in reductions in HBV DNA levels, but at a slower rate and less absolute decline, with >1 log reductions beginning at W12 and maintained throughout the duration of the study. FIG. 15A to FIG. 15C The data presented in are also shown in tabular form as individual values, means, and mean changes from baseline in Tables 7, 8, and 9, respectively.

[0315] Table 7. Various HBV DNA data.

[0316]

[0317]

[0318] Table 8. Average HBV DNA data.

[0319]

[0320] Table 9. Mean changes from baseline HBV DNA data.

[0321]

[0322] Mouse CHB model results - HBsAg levels. The HBsAg level results described below are shown in FIG. 16A to FIG. 16C During the whole study period, placebo (group 1) did not significantly change HBsAg levels. WT-BNP was administered at 0.05μg (group 2), 0.1μg (group 3) or 0.5μg (groups 4 and 12). The WT-BNP 0.05μg (group 2) dose had no effect on HBsAg levels, while the 0.1μg (group 3) or 0.5μg (groups 4 and 12) doses showed a dose-dependent decrease in HBsAg levels. The combination of 0.5μg of CP-BNP 1+4+5 (groups 8 and 13) showed the fastest and steepest decrease in HBsAg, which had a sustained effect from W12 (two weeks after the first dose) until the end of the study (W26). The maximum reduction was observed with 0.5 μg of CP-BNP 1+4+5, showing a 3log reduction in HBsAg relative to baseline W10, and also with a>1log reduction in HBsAg relative to the WT-BNP of the same dose starting from W12, with the maximum reduction observed starting at W14. Using a dosage less than 0.5 μg, CP-BNP 1+4+5 also demonstrated a dose-dependent reduction in HBsAg, but a lower dose of 0.05 μg (group 6) was similar to the WT-BNP of the same dose, or 0.1 μg (group 7) had a higher HBsAg relative to the WT-BNP of the same dose. Other CP-BNP combinations were also tested in this model, and found to be less effective than CP-BNP 1+4+5, but with a slower rate and less absolute decline, it began to achieve>0.5log reduction at W12 and was maintained throughout the duration of the study.

[0323] FIG. 16A to FIG. 16C The data presented in are also shown in tabular form as individual values, means, and mean changes from baseline in Tables 10, 11, and 12, respectively.

[0324] Table 10. Individual HBsAg data.

[0325]

[0326]

[0327] Table 11. Mean HBsAg data.

[0328]

[0329] Table 12. Mean changes from baseline HBsAg data.

[0330]

[0331] FIG. 15A to FIG. 15C and FIG. 16A to FIG. 16C The results shown in show that the CP-BNP of the present technology effectively reduces HBV DNA viral load and HBsAg levels in the CHB model. Therefore, these results show that the composition comprising the CP-BNP of the present technology can be used in the method for treating chronic hepatitis B.

[0332] Example 17: Expression of proteins from mRNA and protein following transfection of nucleic acid encoding BNP into mammalian cells Vaccine candidate

[0333] Produce WT and CP-BNP4 candidate vaccines from mRNA. The pBluescript II SK (+) plasmid containing WT or BNP4 coding sequence is linearized, purified and confirmed via standard molecular biology techniques with EcoRV-HF restriction enzyme. The linearized plasmid is used as the template produced by the mRNA using in vitro transcription kit according to the recommendation of manufacturer (Life Technologies), and then purified (Life Technologies, Thermo Fisher). The resulting transcript is stored at -80 ° C until standby. Lipofectamine MessengerMAX reagent (ThermoFisher) is used to be transfected into HEK293T or Huh7 cells for 5 days. Then, culture medium is collected, and the above-mentioned sucrose ultracentrifugation method is used for processing. Alternatively, NP-40 lysis buffer is used to process cell lysate, and cell fragments are agglomerated. As previously mentioned, the sample is evaluated by western blot.

[0334] Protein expression results after transfection of BNP encoding nucleic acids (mRNA and DNA) into mammalian cell lines. HBsAg BNP expression from mRNA transfection was successfully achieved in two different cell lines (HEK293T and Huh7) as measured by Western blot analysis using anti-HBsAg antibodies. FIG. 17A to FIG. 17B Western blot identified multiple bands, including monomers of expected sizes (WT-BNP, 24 kD; CP-BNP 4, 29 kD) and glycosylated variants (WT-BNP, 27 kD; CP-BNP 4, 32 kD). In addition, HBsAg was detected after transfection with DNA constructs expressing WT-BNP (flag-tagged 15 and 28 kDa).

[0335] These results demonstrate that the mRNA constructs of the present technology are capable of expressing functional HBsAg-S subunits, as demonstrated by their ability to be secreted into cell culture supernatants, which is indicative of BNP formation. The CP-BNP of the present technology produced by RNA transfection can be used in compositions for treating chronic hepatitis B.

[0336] Equivalent solutions

[0337] The present technology is not limited by the specific embodiments described in this application, which are intended to be a single illustration of various aspects of the present technology. As will be apparent to those skilled in the art, many modifications and changes can be made to the present technology without departing from the spirit and scope of the present invention. In addition to the methods and devices listed herein, according to the foregoing description, it is apparent to those skilled in the art that the methods and devices that are functionally equivalent within the scope of the present technology are obvious to those skilled in the art. Such modifications and changes are intended to fall within the scope of the appended claims. The present technology is limited only by the appended claims and the full range of equivalents given by these claims. It should be understood that the present technology is not limited to specific methods, reagents, compound compositions or biological systems, which can certainly vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to be limited.

[0338] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0339] As will be understood by those skilled in the art, for any and all purposes, particularly in providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily identified as a full description, and the same range can be decomposed into at least equal half, one-third, one-quarter, one-fifth, a few tenths, etc. As a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. As will be understood by those skilled in the art, all languages, such as "at most", "at least", "greater than", "less than", etc., include quoted numbers, and refer to the ranges that can be subsequently decomposed into sub-ranges as discussed above. Finally, as will be understood by those skilled in the art, the range includes each individual member. Therefore, for example, a group with 1 to 3 units refers to a group with 1, 2 or 3 units. Similarly, a group with 1 to 5 units refers to a group with 1, 2, 3, 4 or 5 units, and so on.

[0340] All patents, patent applications, provisional applications, and publications mentioned or cited herein, including all figures and tables, are incorporated by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification.

[0341] Other implementations are set forth within the following claims.

[0342] References

[0343] WHO Fact Sheet, July 18, 2018

[0344] Nayagam et al. (2016) Lancet Infectious Dis 2016 16:1399-1408 Block et al. (2013) Antiviral Res. 98(1):27-34

[0345] Cheong WS et al.Chimeric virus-like particles for the delivery of aninserted conserved influenzy A-specific CTL epitope.2009Antiviral Res

[0346] 81(2):113-122.

[0347] Chothia et al. (1987) J. Mol. Biol. 196:901

[0348] Coligan et al. Current Protocols in Immunology, 1991-1997 Gefter et al. (1977) Somatic Cell Genet. 3: 231-236

[0349] Huang LR et al. An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. 2006PNAS103(47):17862-17867 Hyakumura M et al. Modification of asparagine-linked glycan density for the design of hepatitis V virus virus-like particles with enhanced immunogenicity.

[0350] 2015J Virol 89(22):11312-11322

[0351] Jones et al. (1986) Nature 321:522-525

[0352] Kim and Ahituv. The hydrodynamic tail vein assay as a tool for the study of liver promoters and enhancers. 2013 Methods Mol Biol;1015:279-289

[0353] Kohler et al. (1975) Nature 256:495-499

[0354] Kohler et al. (1976) Eur. J. Immunol. 6(7):511-519

[0355] Kozbor et al. (1986) Methods in Enzymology 121:140

[0356] Lim et al. (2014) Hepatology 60(1) supplement:1623,980A Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443 Longo PA et al. Transient mammalian cell transfection with polylethylenimine(PEI). 2013 Methods Enzymol 529:227-240

[0357] Patzer EJ et al. Cell culture derived recombinant HBsAg is highly immunogenic and protects chimpanzees from infection with hepatitis B virus. 1986 Nature Biotechnol 4:630-636

[0358] qHBsAg Elecsys from Roche Diagnostics. Immunoassay for the qualitative determination of hepatitis B surface antigen(HBsAg)(diagnostics.roche.com)Richmann et al. (1988) Nature 332:323-327

[0359] Shulman et al. (1978) Nature 276:269-270

[0360] Sitnik R. et al. A real-time quantitative assay for hepatitis B DNAvirus(HBV) developed to detect all HBV genotypes.2010Rev Inst Med Trop SaoPaulo 52(3):119-124

[0361] Toyama et al. (1987) Monoclonal Antibody, Experiment Manual, Kodansha Scientific Trowbridge Published (1982) J. Exp. Med. 148(1): 220-227 Verhoeyen et al. (1988) Science 239: 1534-1536

[0362] Volk et al. (1982) J. Virol. 42(1): 220-227

[0363] Volz et al. (2015) International Conference, Molecular Biology of Hepatitis B Virus, Session IX: Antiviral Therapy 0-205, Germany

[0364] Yang PL et al. Hydrodynamic injection of viral DNA: A mouse model ofacute hepatitis B virus infection. 2002PNAS 99(21):13825-13830

Claims

1. A biological nanoparticle (BNP) comprising a hepadnavirus envelope HBsAg-S fusion protein containing one or more antigenic epitope regions, wherein: The antigenic epitope region is selected from the group consisting of antigenic epitopes expressed in loop 1 and loop 2 regions of the HBsAg-S domain, and wherein the epitope region comprises one or more of CTKPTDGNC (SEQ ID NO: 34) and / or CKTCTTPAQGNSMFPS (SEQ ID NO: 35).

2. BNP according to claim 1, wherein, The one or more antigenic epitope regions comprise CKTCTTPAQGNSMFPS (SEQ ID NO: 35).

3. BNP according to claim 2, wherein, The one or more antigenic epitope regions include a first region containing CKTCTTPAQGNSMFPS (SEQ ID NO: 35) and a second region containing CKTCTTPAQGNSMFPS (SEQ ID NO: 35).

4. BNP according to claim 1, wherein, The one or more antigenic epitope regions comprise CTKPTDGNC (SEQ ID NO: 34).

5. BNP according to claim 4, wherein, The one or more antigenic epitope regions include a first region containing CTKPSDGNC (SEQ ID NO: 54), a second region containing CTKPTDGNC (SEQ ID NO: 34), and a third region containing CTKPTDGNC (SEQ ID NO: 34).

6. A biological nanoparticle (BNP) comprising a hepadnavirus envelope HBsAg-S fusion protein comprising an HBsAg-S template and an epitope insert, wherein the HBsAg-S template comprises the amino acids shown in SEQ ID NO: 50, and the epitope insert consists of the amino acids shown in SEQ ID NO: 44, wherein: The epitope insert replaces amino acid residues 125-128 of SEQ ID NO:

50.

7. A biological nanoparticle (BNP) comprising a hepadnavirus envelope HBsAg-S fusion protein comprising an HBsAg-S template and an epitope insert, wherein the HBsAg-S template comprises the amino acids shown in SEQ ID NO: 50, and the epitope insert consists of the amino acids shown in SEQ ID NO: 45, wherein: The epitope insert replaces amino acid residues 137-151 of SEQ ID NO:

50.

8. A composition comprising the bionanoparticle (BNP) as defined in any one of claims 1 to 7.

9. Use of the BNP according to any one of claims 1 to 7 or the composition according to claim 8 in the preparation of a medicament for therapy.

10. Use of the BNP according to any one of claims 1 to 7 or the composition according to claim 8 in the preparation of a medicament for treating or preventing hepatitis B infection.

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