Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/29—Hepatitis virus
  • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
  • A61K2039/5258—Virus-like particles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
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  • C12N2730/00—Reverse transcribing DNA viruses
  • C12N2730/00011—Details
  • C12N2730/10011—Hepadnaviridae
  • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
  • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2730/00—Reverse transcribing DNA viruses
  • C12N2730/00011—Details
  • C12N2730/10011—Hepadnaviridae
  • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
  • C12N2730/10123—Virus like particles [VLP]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2730/00—Reverse transcribing DNA viruses
  • C12N2730/00011—Details
  • C12N2730/10011—Hepadnaviridae
  • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
  • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2730/00—Reverse transcribing DNA viruses
  • C12N2730/00011—Details
  • C12N2730/10011—Hepadnaviridae
  • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
  • C12N2730/10171—Demonstrated in vivo effect

Definitions

• the present technology relates to compositions and methods for the treatment of hepatitis B infection, including chronic hepatitis B (CHB).
• CHB chronic hepatitis B
• Hepatitis B is the most common viral hepatitis, potentially life threatening, with long term complications and is one of the major public health challenges worldwide.
• vaccine is the most effective tool against hepatitis B infection. While the availability of a vaccine has reduced the number of new hepatitis B virus (HBV) infections, it does not benefit the 257 million people already chronically infected by the virus (WHO Fact Sheet 18 July 2018). It is estimated that a cumulative 63 million new cases of chronic HBV infection and 17 million HBV-related deaths to occur between 2015 and 2030 (Nayagam et al. Lancet Infectious Dis 2016 16:1399- 1408).
• HBV hepatitis B virus
• HCC hepatitis B virus
• HCC hepatocellular carcinoma
• Hepatitis B complete cure is defined by the eradication of the virus and all its replicative intermediates (Revill P, et al. Nature Reviews
• the preferred first-line treatment choices are pegylated-interferon alpha-2a (pegIFN-a), entecavir, and tenofovir, based on their superior antiviral efficacy and/or high resistance barrier.
• pegIFN-a is effective in achieving sustained virological response in only 30% of HBeAg-positive and 40% of HBeAg-negative cases and is usually associated with severe side-effects.
• nucleos(t)ide analogs are well tolerated and potently suppress HBV replication in the vast majority of treated patients. However, even the most potent nucleos(t)ide analogs rarely induce HBV surface antigen
• HBV seroconversion the hallmark of a successful immunologic response to HBV with complete and durable control of infection, or a“functional cure.”
• NA treatment is required to continuously suppress HBV replication, which may be associated with significant cost burden and limited by drug- associated toxicity. It is, therefore, a pressing need for the introduction of therapeutic regimens that are safer and effective in achieving a functional cure.
• the infectious HBV virion is a spherical particle 42 nm in diameter consisting of an icosahedral nucleocapsid in which the viral DNA genome is packaged, and a lipoprotein envelope containing three related transmembrane proteins (HBsAg) referred to as HBsAg large (HBsAg-L), HBsAg-middle (HBsAg-M) and HBsAg-small (HBsAg-S).
• HBsAg HBsAg large
• HBsAg-M HBsAg-middle
• HBsAg-S HBsAg-small
• HBsAg-S is composed only of the S-domain consisting of 226 amino acids (aa); HBsAg-M contains an additional N-terminal extensions, the 55 amino acid preS2 domain; and HBsAg-L contains the preS2 domain and an additional 108 or 119 amino acid (genotype dependent) N-terminal extensions called the preSl domain.
• HBsAg-S The capacity of HBsAg-S to self-assemble in the presence of lipid at the endoplasmic reticulum (ER) results in the formation of secretion competent subviral particles (VLPs), which do not contain any other HBV viral component.
• VLPs secretion competent subviral particles
• HBsAg-S VLPs are 22-25 nanometer (nm) in diameter, highly compact, and it is estimated that one particle contains approximately one hundred HBsAg-S molecules.
• HBsAg-M also forms secretion competent VLPs.
• HBsAg-L also forms VLPs which may not be fully secretion competent.
• HBsAg particle formation is an elaborate process.
• the first step in the particle formation is the cotranslational insertion of the protein into the ER membrane with a short luminal exposed N-terminal sequence, two transmembrane regions separated by a 57aa cytosolic loop, and a luminal external 70 aa domain containing the major B-cell epitopes (‘a’ -determinant).
• HBsAg-S VLPs represent a highly compact structure due to the large number of intra- and intermolecular disulfide bonds within and between the individual subunits.
• VLPs are tools of a leading innovative bionanotechnology vector and vaccine development, and they have a number of advantages over traditional vaccines. VLPs do not contain viral genetic material and represent high-density displays of viral structural proteins that efficiently trigger key parts of the immune system for B cell and/or T cell responses (Buonaguro L. et al ., (2011 ), Expert Rev Vaccines 10:1569- 1583; Jennings GT and Bachmann MF. (2009) Annu Rev Pharmacol Toxicol. 2009, 49:303-326; Pushko P, et al., Intervirology 2013, 56: 141-165).
• Chimeric VLPs based on the capsid proteins of e.g., HBV, human papilloma virus (HPV), as well as Ob phage have been engineered to express foreign antigenic sequences including non-pathogen associated antigens such as nicotine and angiotensin II for smoking cessation and to overcome hypertension, respectively (Ambiihl PM et al. ./. Hypertension. 2007, 25:63-72; Buonaguro L. et al. Exp. Rev. Vaccines 2011, 10: 1569-1583; Cornuz J. et al. PlosOne 2008, 3:e2547).
• HBV human papilloma virus
• Ob phage have been engineered to express foreign antigenic sequences including non-pathogen associated antigens such as nicotine and angiotensin II for smoking cessation and to overcome hypertension, respectively (Ambiihl PM et al. ./. Hypertension. 2007, 25:63-72; Buonaguro L.
• HBV based HBsAg-S VLPs are composed of envelope proteins and lipid, the ER being the cellular location for assembly.
• HBsAg-S VLPs have been modified to carry foreign epitopes
• VLPs composed of HBsAg-L were 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 proteins and other hepadnaviral envelope proteins have been modified to express antigens of interest as part of VLPs.
• VLPs composed of the small envelope proteins (HBsAg-S) derived from HBsAg-S.
• HBV are the antigenic components of a successful protective vaccine (Jilg W et al.
• hepatitis B still represents an enormous health problem.
• a significant issue in vaccine development is the diminished capacity of an aged immune system and immunosenescence being associated with a decreased
• the present disclosure provides a recombinant virus-like particle antigen (VLP-Ag) comprising a hepadnaviral envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein said antigenic epitope repeat regions are selected from the group consisting of antigenic epitopes expressed in the Loop 1 and Loop 2 regions of HBsAg-S domain.
• VLP-Ag virus-like particle antigen
• the one or more antigenic epitope repeat regions expressed in Loop 1 is defined by the amino acid sequence
• PCX8TCX9X10X11 wherein Xs is K or R, X9 is T or M, X10 is T, I or S, and X11 is P, T or L.
• the one or more antigenic epitope repeat regions expressed in Loop 2 is defined by the consensus amino acid sequence
• CCCTKPX12DGNCX13 wherein X12 is T or S; and X13 is T or S.
• the one or more antigenic epitope repeat regions is selected from the group consisting of PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTP AQGN SMFP S, CTKP(T/S)TDGNC, PC(K/R)TC(T/M)TP,
• the hepadnaviral envelope fusion protein comprises a spacer domain between the antigenic epitope repeat regions and the envelope protein.
• 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.
• the present disclosure provides a composition comprising a VLP-Ag of the present technology and a pharmaceutically acceptable carrier.
• the present disclosure provides a method of treating hepatitis B infection in a subject in need thereof, comprising administering to the subject a recombinant VLP-Ag comprising a hepadnaviral envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein said antigenic epitope repeat regions are selected from the group consisting of antigenic epitopes expressed in the Loop 1 and Loop 2 regions of HBsAg-S domain.
• the one or more antigenic epitope repeat regions expressed in Loop 1 is defined by the amino acid sequence
• PCX8TCX9X10X11 wherein Xs is K or R, X9 is T or M, X10 is T, I or S, and X11 is P, T or L.
• the one or more antigenic epitope repeat regions expressed in Loop 2 is defined by the consensus amino acid sequence
• CCCTKPX12DGNCX13 wherein X12 is T or S; and X13 is T or S.
• the one or more antigenic epitope repeat regions is selected from the group consisting of PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTP AQGN SMFP S, CTKP(T/S)TDGNC, PC(K/R)TC(T/M)TP,
• the hepadnaviral envelope fusion protein comprises a spacer domain between the antigenic epitope repeat regions and the envelope protein.
• 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 hepadnaviral envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein said antigenic epitope repeat regions are selected from the group consisting of antigenic epitopes expressed in the Loop 1 and Loop 2 regions of HBsAg-S domain.
• the one or more antigenic epitope repeat regions expressed in Loop 1 is defined by the amino acid sequence
• PCX8TCX9X10X11 wherein Xs is K or R, X9 is T or M, X10 is T, I or S, and X11 is P, T or L.
• the one or more antigenic epitope repeat regions expressed in Loop 2 is defined by the consensus amino acid sequence
• CCCTKPX12DGNCX13 wherein X12 is T or S; and X13 is T or S.
• the one or more antigenic epitope repeat regions is selected from the group consisting of PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTP AQGN SMFP S, CTKP(T/S)TDGNC, PC(K/R)TC(T/M)TP,
• the hepadnaviral envelope fusion protein comprises a spacer domain between the antigenic epitope repeat regions and the envelope protein.
• 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 hepadnaviral envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein said antigenic epitope repeat regions are selected from the group consisting of antigenic epitopes expressed in the Loop 1 and Loop 2 regions of HBsAg-S domain.
• the one or more antigenic epitope repeat regions expressed in Loop 1 is defined by the amino acid sequence
• PCX8TCX9X10X11 wherein Xs is K or R, X9 is T or M, X10 is T, I or S, and X11 is P, T or L.
• the one or more antigenic epitope repeat regions expressed in Loop 2 is defined by the consensus amino acid sequence
• CCCTKPX12DGNCX13 wherein X12 is T or S; and X13 is T or S.
• the one or more antigenic epitope repeat regions is selected from the group consisting of PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTP AQGN SMFP S, CTKP(T/S)TDGNC, PC(K/R)TC(T/M)TP,
• the hepadnaviral envelope fusion protein comprises a spacer domain between the antigenic epitope repeat regions and the envelope protein.
• the present disclosure provides the use of a recombinant VLP- Ag in the manufacture of a composition for inducing an immune response against hepatitis B, wherein the VLP-Ag comprises a hepadnaviral envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein said antigenic epitope repeat regions are selected from the group consisting of antigenic epitopes expressed in the Loop 1 and Loop 2 regions of HBsAg-S domain.
• the one or more antigenic epitope repeat regions expressed in Loop 1 is defined by the amino acid sequence
• the one or more antigenic epitope repeat regions expressed in Loop 2 is defined by the consensus amino acid sequence
• CCCTKPX12DGNCX13 wherein X12 is T or S; and X13 is T or S.
• the one or more antigenic epitope repeat regions is selected from the group consisting of PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTP AQGN SMFP S, CTKP(T/S)TDGNC, PC(K/R)TC(T/M)TP,
• the hepadnaviral envelope fusion protein comprises a spacer domain between the antigenic epitope repeat regions and the envelope protein.
• the present disclosure provides a kit for treating hepatitis B infection in a subject in need thereof comprising a recombinant VLP-Ag, wherein the VLP-Ag comprises a hepadnaviral envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein said antigenic epitope repeat regions are selected from the group consisting of antigenic epitopes expressed in the Loop 1 and Loop 2 regions of HBsAg-S domain.
• the one or more antigenic epitope repeat regions expressed in Loop 1 is defined by the amino acid sequence
• PCX8TCX9X10X11 wherein Xs is K or R, X9 is T or M, X10 is T, I or S, and X11 is P, T or L.
• the one or more antigenic epitope repeat regions expressed in Loop 2 is defined by the consensus amino acid sequence
• the one or more antigenic epitope repeat regions is selected from the group consisting of PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTP AQGN SMFP S, CTKP(T/S)TDGNC, PC(K/R)TC(T/M)TP,
• the hepadnaviral envelope fusion protein comprises a spacer domain between the antigenic epitope repeat regions and the envelope protein.
• the present disclosure provides a recombinant mRNA encoding a hepadnaviral envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein said antigenic epitope repeat regions are selected from the group consisting of antigenic epitopes expressed in the Loop 1 and Loop 2 regions of HBsAg-S domain.
• the one or more antigenic epitope repeat regions expressed in Loop 1 is defined by the amino acid sequence
• PCX8TCX9X10X11 wherein Xs is K or R, X9 is T or M, X10 is T, I or S, and X11 is P, T or L.
• the one or more antigenic epitope repeat regions expressed in Loop 2 is defined by the consensus amino acid sequence
• CCCTKPX12DGNCX13 wherein X12 is T or S; and X13 is T or S.
• the one or more antigenic epitope repeat regions is selected from the group consisting of PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTP AQGNSMFPS, CTKP(T/S)TDGNC, PC(K/R)TC(T/M)TP,
• the modified hepadnaviral envelope fusion protein comprises a spacer domain between the antigenic epitope repeat regions and the envelope protein.
• 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 the recombinant mRNA of and a pharmaceutically acceptable carrier.
• the present disclosure provides a method of treating hepatitis B infection in a subject in need thereof, comprising administering to the subject a recombinant mRNA encoding a hepadnaviral envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein said antigenic epitope repeat regions are selected from the group consisting of antigenic epitopes expressed in the Loop 1 and Loop 2 regions of HBsAg-S domain.
• the one or more antigenic epitope repeat regions expressed in Loop 1 is defined by the amino acid sequence
• PCX8TCX9X10X11 wherein Xs is K or R, X9 is T or M, X10 is T, I or S, and X11 is P, T or L.
• the one or more antigenic epitope repeat regions expressed in Loop 2 is defined by the consensus amino acid sequence
• CCCTKPX12DGNCX13 wherein X12 is T or S; and X13 is T or S.
• the one or more antigenic epitope repeat regions is selected from the group consisting of PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTP AQGN SMFP S, CTKP(T/S)TDGNC, PC(K/R)TC(T/M)TP,
• the modified hepadnaviral envelope fusion protein comprises a spacer domain between the antigenic epitope repeat regions and the envelope protein.
• the present disclosure provides the use of a recombinant mRNA in the manufacture of a medicament for treating hepatitis B infection in a subject in need thereof, wherein the recombinant mRNA encodes a hepadnaviral envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein said antigenic epitope repeat regions are selected from the group consisting of antigenic epitopes expressed in the Loop 1 and Loop 2 regions of HBsAg-S domain.
• the one or more antigenic epitope repeat regions expressed in Loop 1 is defined by the amino acid sequence
• PCX8TCX9X10X11 wherein Xs is K or R, X9 is T or M, X10 is T, I or S, and X11 is P, T or L.
• the one or more antigenic epitope repeat regions expressed in Loop 2 is defined by the consensus amino acid sequence
• CCCTKPX12DGNCX13 wherein X12 is T or S; and X13 is T or S.
• the one or more antigenic epitope repeat regions is selected from the group consisting of PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTP AQGN SMFP S, CTKP(T/S)TDGNC, PC(K/R)TC(T/M)TP,
• the hepadnaviral envelope fusion protein comprises a spacer domain between the antigenic epitope repeat regions and the envelope protein.
• 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 modified hepadnaviral envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein said antigenic epitope repeat regions are selected from the group consisting of antigenic epitopes expressed in the Loop 1 and Loop 2 regions of HBsAg-S domain.
• the one or more antigenic epitope repeat regions expressed in Loop 1 is defined by the amino acid sequence
• PCX8TCX9X10X11 wherein Xs is K or R, X9 is T or M, X10 is T, I or S, and X11 is P, T or L.
• the one or more antigenic epitope repeat regions expressed in Loop 2 is defined by the consensus amino acid sequence
• CCCTKPX12DGNCX13 wherein X12 is T or S; and X13 is T or S.
• the one or more antigenic epitope repeat regions is selected from the group consisting of PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTP AQGN SMFP S, CTKP(T/S)TDGNC, PC(K/R)TC(T/M)TP,
• the modified hepadnaviral envelope fusion protein comprises a spacer domain between the antigenic epitope repeat regions and the envelope protein.
• 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 hepadnaviral envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein said antigenic epitope repeat regions are selected from the group consisting of antigenic epitopes expressed in the Loop 1 and Loop 2 regions of HBsAg-S domain.
• the one or more antigenic epitope repeat regions expressed in Loop 1 is defined by the amino acid sequence CX1TCX2X3X4X5QGX6SMX7PC, wherein Xi is K or R, X 2 is T or M, X 3 is T or I, X 4 is P T or L, X5 is A or V, X 6 is N or T, and X7 is F or Y; or wherein the one or more antigenic epitope repeat regions is defined by the amino acid sequence
• PCX8TCX9X10X11 wherein Xs is K or R, X9 is T or M, X10 is T, I or S, and X11 is P, T or L.
• the one or more antigenic epitope repeat regions expressed in Loop 2 is defined by the consensus amino acid sequence
• CCCTKPX12DGNCX13 wherein X12 is T or S; and X13 is T or S.
• the one or more antigenic epitope repeat regions is selected from the group consisting of PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTP AQGN SMFP S, CTKP(T/S)TDGNC, PC(K/R)TC(T/M)TP,
• the modified hepadnaviral envelope fusion protein comprises a spacer domain between the antigenic epitope repeat regions and the envelope protein.
• the present disclosure provides a kit for treating hepatitis B infection in a subject in need thereof comprising a recombinant mRNA, wherein the recombinant mRNA encodes a hepadnaviral envelope HBsAg-S fusion protein comprising one or more antigenic epitope repeat regions, wherein said antigenic epitope repeat regions are selected from the group consisting of antigenic epitopes expressed in the Loop 1 and Loop 2 regions of HBsAg-S domain.
• the one or more antigenic epitope repeat regions expressed in Loop 1 is defined by the amino acid sequence
• PCX8TCX9X10X11 wherein Xs is K or R, X9 is T or M, X10 is T, I or S, and X11 is P, T or L.
• the one or more antigenic epitope repeat regions expressed in Loop 2 is defined by the consensus amino acid sequence
• CCCTKPX12DGNCX13 wherein X12 is T or S; and X13 is T or S.
• the one or more antigenic epitope repeat regions is selected from the group consisting of PCKTCTTP, PCRTCTTP, CTKPTDGNC, CKTCTTP AQGN SMFP S, CTKP(T/S)TDGNC, PC(K/R)TC(T/M)TP,
• the modified hepadnaviral envelope fusion protein comprises a spacer domain between the antigenic epitope repeat regions and the envelope protein.
• 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 I DNO: 20), and CTKPSDGNCT (SEQ ID NO: 21).
• the one or more peptides is conjugated to a carrier protein.
• the carrier protein is keyhole limpet hemocyanin (KLH).
• the one or more peptides is cyclic.
• the composition further comprises a pharmaceutically acceptable carrier.
• the present disclosure provides a method of 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 I DNO: 20), and CTKPSDGNCT (SEQ ID NO: 21).
• TCTTPAQGNSMFPSC SEQ ID NO: 17
• TCTIPAQGTSMFPSC SEQ ID NO: 18
• TCTTPAQGTSMFPSC SEQ ID NO: 19
• CTKPTDGNCT SEQ I DNO: 20
• CTKPSDGNCT SEQ ID NO: 21
• the one or more peptides is conjugated to a carrier protein.
• the carrier protein is keyhole limpet hemocyanin (KLH).
• the one or more peptides is cyclic.
• FIG. 1 is a diagrammatic representation showing HBsAg epitope occupancy assay.
• Magnetic bead 2. Capture Ab: mouse anti-HBs monoclonal Abs (mAbs) to HBsAg external region; 3. Reference HBsAg sample. Ag only vs Ag bound with anti- HBs sample; 4. Reporter Ab: PE conjugated polyclonal anti-HBs.
• NCP non clearance profile
• CP clearance profile.
• FIG. 2 is a diagrammatic representation of predictive biomarkers of functional CHB cure (assay 1) and immune Ab response activity (assay2).
• Assay 1 top panel: 1. Magnetic bead; 2. Capture Ab: multiplex mouse anti-HBs mAbs to HBsAg‘external region’; 3. Patient HBsAg sample; 4. Reporter Ab: PE conjugated polyclonal anti-HBs.
• Assay 2 (lower panel): 1. Magnetic bead; 2. Capture Ab: 2plex mouse anti-HBs mAbs to HBsAg‘external region’; 3. Patient sample: complexed anti- HBs (with HBsAg); 4. Reporter Ab: HRP conjugated goat anti-human IgG Fc domain.
• FIG. 3 is a diagrammatic representation showing CHB cure patients exhibiting an HBsAg clearance profile (CP).
• CHB Chronic hepatitis B.
• FIG. 4 is a diagrammatic representation showing non-cure/non-responder patients exhibiting an HBsAg non-clearance profile (NCP).
• RFET Relative
• FIG. 5 is a schematic showing an overview of the proposed structure of the small hepatitis B surface (envelope) antigen (HBsAg-S), 226 amino acids in length and subtype ayw (genotype D).
• the external antigenic loop region between amino acids 100 and 160 of HBsAg-S is enlarged: each amino acid is represented by a grey circle, the cysteine residues are shown as black circles, the potential -S-S- disulfide bonds highlighted.
• the glycosylation site (asparagine) at position 146 is shown in white, and the glycan structure is indicated.
• the position used to insert foreign sequence is located at amino acid 127 and 128.
• FIG. 6 is a schematic showing proposed folding of the HBsAg-S protein, 226 amino acids in length, for BNPs 1, 2, 3, and 4.
• the external antigenic loop region between amino acids 100 and 160 of HBsAg-S is enlarged: each amino acid is represented by a grey circle, the cysteine residues are shown as black circles, the potential -S-S- disulfide bonds highlighted.
• the glycosylation site (asparagine) at position 146 is shown in white, and the glycan structure indicated.
• the position used to insert foreign sequence is located at amino acids 127 and 128, the HBsAg-S sequence sub-type ayw is used as the parent template. Additional HBsAg-S derived antigenic sequences are inserted at position 127 and 128 into the parent template.
• Right panel shows the cure associated loop sequences for BNP 1, 2, 3 and 4 (SEQ ID NOs: 41-44, in order of appearance), the HBsAg-S-specific antigenic sequence is in bold, the“GSGS” linker in italic, and underlined.
• BNP-l is an‘ayw’ parent template with a trimer PCKTCTTP (SEQ ID NO: 28) insert (loopl, genotype A specific, serotype adw);
• BNP-2 is an‘ayw’ parent template with a trimer PCRTCTTP (SEQ ID NO: 33) insert (loop 1, genotype B/C, serotype ayw);
• BNP-3 is an‘ayw’ parent template with a trimer insert of the loop 2 sequence CTKPTDGNC (SEQ ID NO: 34) (derived from serotype‘adw’);
• BNP -4 is an‘ayw’ parent template with an insert (dimer) of the extended loop 1 sequence CKTCTTPAQGNSMFPS (SEQ ID NO: 35) (serotype‘adw’).
• FIG. 7 is a schematic showing proposed folding of the HBsAg-S protein, 226 amino acids in length, for BNP 5.
• the external antigenic loop region between amino acids 100 and 160 of HBsAg-S is enlarged: each amino acid is represented by a grey circle, the cysteine residues are shown as black circles, the potential -S-S- disulfide bonds highlighted, the“GSGS” linker by striped circles.
• the glycosylation site (asparagine) at position 146 is shown in white, and the glycan structure indicated.
• Two additional loop 2 sequences are added to the wild type loop to generate a repetitive structure.
• the loop 2 CTKP(T/S)TDGNC (SEQ ID NO: 36) sequence repeats are boxed.
• BNP-5 is an parent‘ayw’ parent template with a trimer loop 2 region.
• The‘ayw’ loop 2 sequence is extended by two additional‘adw’ loop 2 sequences.
• FIG. 8 is a schematic showing proposed folding of the HBsAg-S protein, 226 amino acids in length, for BNP 6.
• the external antigenic loop region between amino acids 100 and 160 of HBsAg-S is enlarged: each amino acid is represented by a grey circle, the cysteine residues are shown as black circles, the potential -S-S- disulfide bonds highlighted, the“GSGS” linker by striped circles.
• the glycosylation site (asparagine) at position 146 is shown in white, and the glycan structure indicated.
• Two additional loop 1 PC(K/R)TC(T/M)TP SEQ ID NO: 37
• loop 2 Two additional loop 1 PC(K/R)TC(T/M)TP
• CTKP(T/S)TDGNC SEQ ID NO: 36 sequences are added within the corresponding loop sequence.
• the loop 1 and loop 2 sequence repeats are boxed.
• Right panel shows the extended sequences (SEQ ID NOs: 46 and 47, in order of appearance), the HBsAg- S-specific antigenic sequences are in bold, the“GSGS” linker in italic and underlined.
• Two additional loop 1 sequences (serotype adw) are inserted followed by the original loop 1 sequence of the parent template (serotype ayw).
• the original loop 2 sequence parent serotype ayw
• the original loop 2 sequence parent serotype ayw
• two inserted loop 2 sequences serotype adw.
• FIG. 9 is a schematic showing proposed folding of the HBsAg-S protein, 226 amino acids in length, for BNP 7.
• the external antigenic loop region between amino acids 100 and 160 of HBsAg-S is enlarged: each amino acid is represented by a grey circle, the cysteine residues are shown as black circles, the potential -S-S- disulfide bonds highlighted, the“GSGS” linker by striped circles.
• the glycosylation site (asparagine) at position 146 is shown in white, and the glycan structure 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.
• the loop 1 repeats are boxed.
• the original amino acid positions for the wild type HBsAg-S sequence are unchanged, and the additional amino acids are marked with‘ and“.
• Right panel shows the extended sequences (SEQ ID NOs: 52, 48, and 49, in order of appearance), the HBsAg-S- specific antigenic sequences are in bold, the“GSGS” linker in italic and underlined.
• Two extended loop 1 sequences (serotype adw), amino acid sequence 121 to 136 were inserted followed by the original loop 1 sequence (serotype ayw).
• FIG. 10 is a schematic showing proposed folding of the HBsAg-S protein
• each amino acid is represented by a grey circle, the cysteine residues are shown as black circles, the potential -S-S- disulfide bonds highlighted, the“GSGS” linker is represented by striped circles.
• the glycosylation site (asparagine) at position 146 is shown in white, and the glycan structure indicated.
• the loop 1 and loop 2 regions were duplicated.
• the original amino acid positions for 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 region (‘ayw’, amino acid (aa) 120-147) followed by an additional loop 1 and loop 2‘adw’ sequence (aa 120-147).
• FIG. 11 is a chart showing the HBsAg epitope profile (bioplex platform) analysis of CP -BNP 1, 2, 3, and 4 preparations and CP -BNP 1+3, 1+3+4, and 3+4 formulations.
• the CP-BNPs incorporate displayed loop 1 (CP -BNP 1, 2, 4) or loop 2 (CP -BNP 3) specific“clearing” epitope inserts to induce targeted“clearing” anti-HBs antibody responses following immunization.
• HBsAg epitope profile analysis indicates that epitopes across both loop 1 and loop 2 are retained (within normal range of +/- 0.5 fold) and/or enhanced (>0.5 fold) in all tested CP -BNP preparations and formulations, indicating antigenicity for the target“clearing” epitopes are displayed correctly and intact, with the strongest/broadest epitope retention achieved in the BNP formulations.
• FIG. 12 is a chart showing a generated derived and purified monoclonal antibody analysis for the cyclic peptides.
• the chart shows the anti-HBs antibody induced CP profile (bioplex platform) analysis of selected generated monoclonal antibodies following immunization with loop 1 or loop 2 pan-genotypic/serotypic cyclised peptide formulations.
• IndCP e.g, loss of epitope recognition at both loop 1 and loop 2 epitopes
• FIGS. 13A-13D are charts showing the anti-HBs antibody reactivity analysis following CP -BNP antigen immunization.
• the induced antibody response was analysed for specificity to: recombinant WT HBsAg (VLP) (FIG. 13A); HBsAg loop 1 specific cyclised peptides (FIG.
• FIG. 13D reports the anti-Hbs reactivity for WT-BNP, loopl peptide and loop2 peptide antigens. The strongest, most rapid and broadest or most conserved (to VLP and both loop 1 and loop 2 peptides) anti-HBs antibody immunogenicity responses were observed for CP -BNP formulation 1+3+4, 1+4+5, 5+7 and 5 which delivered target“clearing” epitopes for both loop 1 and loop 2 (encircled). [0096] FIG.
• FIG. 14 is a chart showing the anti-HBs antibody induced CP profile (bioplex platform) analysis of selected sera responses following immunisation with single or combinations of CP-BNPs 1, 3, 4, 5, and 7 preparations.
• the HBsAg epitope profile of reference VLP antigen +/- pre-incubation with anti-HBs positive individual mouse sera from the immunogenicity study was assessed for the induction of a CP due to“clearing” or CP-associated anti-HBs in the sera. This targeted“clearing” epitope anti-HBs development was most consistently observed in mice (9-5, 13-2, 16-5 and 17-3) that received CP-BNP 1+4+5, 1+3+4, 5, or 5+7 formulation immunization.
• FIGS. 15A-15C are charts showing change in HBV DNA (loglO IU/mL) levels from baseline (W10) after therapeutic vaccination of various CP-BNP combinations in a murine model of CHB infection.
• CP-BNP 1+4+5 at 0.5 pg dose had the most rapid and absolute sustained decline in HBV DNA relative to either placebo or WT-BNP at the same dose.
• Other combinations of CP-BNPs also declined in HBV DNA levels, but did so at a slower rate and less absolute decline compared to CP-BNP 1+4+5.
• FIGS. 16A-16C are charts showing change in HBsAg (loglO IU/mL) levels from baseline (W10) after therapeutic vaccination of various CP-BNP combinations in a murine model of CHB infection.
• CP-BNP 1+4+5 at 0.5 pg dose had the most rapid and absolute sustained decline in HBsAg relative to either placebo or WT-BNP at the same dose.
• Other combinations of CP-BNPs also declined in HBsAg levels, but did so at a slower rate and less absolute decline compared to CP-BNP 1+4+5.
• FIGS. 17A-17B are images showing Western analysis of HBsAg BNP expression of WT-BNP and CP-BNP 4 from RNA after in vitro transcription and transfection into HEK293T (FIG.17A) and Huh7 (FIG. 17B) cell lines.
• HBsAg bands (+/- glycosylation, -50%) were detected in both WT-BNP (24 or 27 kDa) and CP-BNP 4 (29 or 32 kDa), produced after transfection of the appropriate mRNAs into both cell lines.
• Untreated and reagent assay control transfections were negative for HBsAg.
• Controls included purified HBsAg (with Flag tag, 25 or 28 kD) and samples from after transfection of WT-BNP DNA (with flag tag, 25 or 28 kD).
• the mRNA transcripts encode WT-BNP or BNP4-specific HBsAg subunits in the absence of a Flag tag.
• anti-HBs anti-HBsAg antibodies
• BNPs bio-nanoparticles
• CP-associated epitopes to mimic the selective pressure of a CP and promote efficient and specific B cell anti-HBs immune responses for HBsAg clearance.
• the immunogenicity of BNPs is initially validated in normal BALB/c mice, prior to evaluation of therapeutic vaccination outcomes to drive HBsAg clearance and seroconversion in a newly developed and validated CBA Carter J (CBA/CaJ) murine model of CHB (see, e.g. , Chen HH, et al. PNAS 112(7):2175- 2180 (2015)).
• CBA/CaJ CBA Carter J
• the aim of a CP-expressing BNP therapeutic vaccine when delivered to patients with CHB, is to accelerate and drive functional hepatitis B cure.
• the therapeutic function of CP-expressing BNPs can be further enhanced by hyperglycosylation modification to enhance and accelerate B cell responses and promote HBsAg seroconversion, and/or modification of the BNP delivery backbone to induce“stealth” status of the BNPs to potentially pre-existing, neutralising, but not clearing, circulating anti-HBs antibodies in CHB patients.
• an epitope includes a single epitope, as well as two or more epitopes,
• an antibody includes a single antibody, as well as two or more antibodies; reference to“the disclosure” includes a single and multiple aspects taught by the disclosure; and so forth.
• antigen is used herein in its broadest sense to refer to a substance that is capable of reacting in and/or inducing an immune response.
• Reference to an “antigen” includes an antigenic determinant or epitope.
• antigenic determinant or“epitope” is meant that part of an antigenic molecule against which a particular immune response is directed and includes a hapten.
• A“hapten” is a substance that can combine specificity with an antibody but cannot or only poorly induces an immune response unless bound to a carrier.
• a hapten typically comprises a single antigenic determinant or epitope.
• bio-nanoparticle or“BNP” refers to a virus-like particle (VLP) that has been modified to include or display one or more target insert epitopes.
• VLP virus-like particle
• the terms“bio-nanoparticle” or“BNP” also refer to“recombinant virus-like particle antigen” or“VLP-Ag.”
• the terms“effective amount” or“therapeutically effective amount” or“pharmaceutically effective amount” refer to a quantity sufficient to achieve a desired biological, therapeutic, and/or prophylactic effect, e.g ., an amount which results in the prevention of a disease, condition and/or symptom(s) thereof.
• the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to the composition drugs. It will also depend on the degree, severity and type of disease or condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
• multiple doses are administered.
• multiple therapeutic compositions or compounds e.g ., immunogenic compositions, such as vaccines
• the term“functional cure” refers to a functional cure of CHB and is defined by loss of detectable HBsAg with or without presence of naturally occurring or introduced anti-HBsAg antibodies (also referred to herein as“anti-HBs antibodies” or“anti-HBs”), which include a population of antibodies that selectively bind to select epitopes on HBsAg, which when occupied by anti-HBs antibodies, results in clearance of HBsAg and ultimately HBV. HBV is non-detectable in a fully cured subject.
• an 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.
• an immunogenic composition includes at least one of eight CP-BNPs (e.g., BNP 1, 2, 3, 4, 5, 6, 7, 8) and/or CP epitope cyclic peptides.
• the immunogenic compositions described herein may be formulated for administration in a number of forms.
• the immunogenic compositions are prepared for intravenous, intramuscular, subcutaneous, parenteral, oral, nasal, or topical administration.
• compositions may also be formulated for specific dosage forms.
• the immunogenic composition may be formulated as a liquid, gel, aerosol, ointment, cream, lyophilized formulation, powder, cake, tablet, or capsule.
• the immunogenic composition is formulated as a controlled release formulation, delayed release formulation, extended release formulation, pulsatile release formulation, and mixed immediate release formulation.
• the immunogenic composition is provided as a liquid.
• the immunogenic composition is provided in lyophilized form.
• the term“infected” refers to harboring a disease or pathogen, such as a virus. An infection can be intentional, such as by administration of a virus or pathogen ( e.g ., by vaccination), or unintentional, such as by natural transfer of the pathogen from one organism to another, or from a contaminated surface to the
• infection is induced in a model organism (e.g., murine model) by in vivo transfection of replication-competent DNA using a model organism (e.g., murine model) by in vivo transfection of replication-competent DNA using a model organism (e.g., murine model) by in vivo transfection of replication-competent DNA using a model organism (e.g., murine model) by in vivo transfection of replication-competent DNA using a model organism.
• a model organism e.g., murine model
• subject and“patient” are used interchangeably and refer to an animal, for example, a member of any vertebrate species. In some embodiments, the subject is a human.
• the terms“treating” or“treatment” or“alleviation” refers to therapeutic treatment, wherein the object is to reduce, alleviate, or slow down the progression or advancement of, and/or reverse the progression of the targeted pathological condition or disorder.
• a subject is successfully“treated” for an existing and/or persisting hepatitis B infection, including chronic hepatitis B (CHB) infection if, after receiving a therapeutic amount of the compositions of the present technology, according to the methods described herein, the subject shows observable and/or measurable induction of antibodies that clear HBsAg, and/or loss of detectable HBsAg, and/or reduced levels of HBsAg and/or HBV DNA.
• CHB chronic hepatitis B
• vaccine is used herein to refer to a composition that is
• vaccines include a pharmaceutically acceptable adjuvant and/or a pharmaceutically acceptable carrier.
• “BNP 1” or“CP-BNP 1” refers to SEQ ID NO: 1, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO 1, such as the nucleotide sequence set forth in SEQ ID NO: 9, virus-like particles (VLPs) or bio- nanoparticles (BNPs) comprising SEQ ID NO: 1, immunogenic compositions
• “BNP 2” or“CP-BNP 2” refers to SEQ ID NO: 2, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 2, such as the nucleotide sequence set forth in SEQ ID NO: 10, VLPs or BNPs comprising SEQ ID NO: 2, immunogenic compositions comprising SEQ ID NO: 2, or a vaccine comprising SEQ ID NO: 2.
• “BNP 3” or“CP-BNP 3” refers to SEQ ID NO: 3, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 3, such as the nucleotide sequence set forth in SEQ ID NO: 11, VLPs or BNPs comprising SEQ ID NO: 3, immunogenic compositions comprising SEQ ID NO: 3, or a vaccine comprising SEQ ID NO: 3.
• “BNP 4” or“CP-BNP 4” refers to SEQ ID NO: 4, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 4, such as the nucleotide sequence set forth in SEQ ID NO: 12, VLPs or BNPs comprising SEQ ID NO: 4, immunogenic compositions comprising SEQ ID NO: 4, or a vaccine comprising SEQ ID NO: 4.
• “BNP 5” or“CP-BNP 5” refers to SEQ ID NO: 5, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 5, such as the nucleotide sequence set forth in SEQ ID NO: 13, VLPs or BNPs comprising SEQ ID NO: 5, immunogenic compositions comprising SEQ ID NO: 5, or a vaccine comprising SEQ ID NO: 5.
• “BNP 6” or“CP-BNP 6” refers to SEQ ID NO: 6, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 6, such as the nucleotide sequence set forth in SEQ ID NO: 14, VLPs or BNPs comprising SEQ ID NO: 6, immunogenic compositions comprising SEQ ID NO: 6, or a vaccine comprising SEQ ID NO: 6.
• “BNP T or“CP-BNP T refers to SEQ ID NO: 7, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 7, such as the nucleotide sequence set forth in SEQ ID NO: 15, VLPs or BNPs comprising SEQ ID NO: 7, immunogenic compositions comprising SEQ ID NO: 7, or a vaccine comprising SEQ ID NO: 7.
• “BNP 8” or“CP-BNP 8” refers to SEQ ID NO: 8, nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 8, such as the nucleotide sequence set forth in SEQ ID NO: 16, VLPs or BNPs comprising SEQ ID NO: 8, immunogenic compositions comprising SEQ ID NO: 8, or a vaccine comprising SEQ ID NO: 8.
• “clearance profile (CP) epitope cyclic peptide 1” refers to SEQ ID NO: 17.
• “CP epitope cyclic peptide 2” refers to SEQ ID NO: 18.
• “CP epitope cyclic peptide 3” refers to SEQ ID NO: 19.
• “CP epitope cyclic peptide 4” refers to SEQ ID NO: 20.
• “CP epitope cyclic peptide 5” refers to SEQ ID NO: 21.
• a“clearance antibodies” the antibodies within an anti-HBs response which result in functional cure are referred to as“clearance antibodies.” These antibodies define a “clearance profile” of antibodies which target the specific HBsAg epitopes and which ultimately result in clearance of HBsAg and functional cure.
• the epitopes are referred to as“clearance epitopes” meaning once occupied by antibodies in the host subject, the subject will result or likely result in a functional cure.
• a“clearance profile” can refer to the fingerprint of non-available epitopes on HBsAg or the suite or population of an individual’s antibodies which occupy these epitopes and when they do occupy the epitopes are predictive of a functional cure being achieved.
• the clearance profile of antibodies or epitopes on HBsAg represent biomarkers of the potential or likelihood that a subject on treatment will achieve a functional cure.
• Reference to a“likelihood” of a functional cure generally means that the likelihood is 100%, that is, once a clearance profile of antibodies is detected, the subject will reach a state of functional cure or, in the absence of circulating HBsAg, has achieved a functional cure.
• reference to a“likelihood” of a functional cure means at least 80% probability that a subject with a clearance profile of antibodies will achieve a functional cure.
• By“at least 80% means 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
• The“clearance profile of epitopes” or“CP of epitopes” on HBsAg is the presence of available or non-available epitopes on HBsAg which have the potential to be occupied by the clearance antibodies.
• the clearance profile of epitopes on HBsAg comprise no epitopes available for binding, then the epitopes are deemed“non- available” and this means that the epitopes are occupied by antibodies and a functional cure will likely occur.
• a functional cure has been achieved and only the clearance profile of antibodies is present (or can be induced in an immune response following exposure to HBV).
• the term“standardized” also encompasses“normalization” of level of HBsAg or HBsAg-Ab complex and represents optimal level of HBsAg or HBsAg-Ab complex for the assay.
• A“blood-derived sample” includes serum.
• B-cell depleting therapies e.g ., rituximab, anti-CD 20
• CHB chronic hepatitis B
• Phase 1 is the immune tolerant (IT) phase covering the time from infection and establishment of chronic infection to the first signs of active disease. It is typically asymptomatic (no significant liver disease) and these individuals acquired infection at birth or shortly thereafter, and they are HBeAg- positive.
• Phase 2 or the immune clearance (IC) phase is a disease active phase with significant progression in the person’s liver disease. These patients are also HBeAg- positive.
• phase 3 or the non-replicative phase (NR) there is little or no evidence of active liver disease; viruses can be identified at low level, and the person is HBeAg- negative.
• Phase 4 is a relapse or recrudescence in liver disease activity and viral replication and this phase is also known as HBeAg-negative disease. This phase tends to eventually burn itself out and then patients often have cirrhosis.
• the final phase, phase 5, is identified as HBsAg loss and anti-HBs seroconversion. This is also recognised as the functional cure (FC) phase.
• CHB comprises at least 4 different diseases based on HBV genotypes.
• CHB can be considered under Asian CHB (genotypes B and C), European CHB (genotypes A-2 and D), African CHB (genotypes A-l and E) and Latin American CHB (genotypes F and H) and each of these 4 groups have different ethnicities (Asian versus European/Caucasian versus African versus Latino), age of acquisition (perinatal versus early childhood versus early adult hood) and mode of transmission (mother-to-baby, child-to-child, cultural scarification, iatrogenic/parenteral and sexual), respectively.
• CHB can be further stratified on the basis of HBeAg and disease status; either HBeAg-positive or HBeAg-negative, with or without liver disease.
• a FC-P is associated with FC and is demonstrated by loss of epitope recognition at loop 1 and loop 2 within the external loop region, which includes the“a” determinant of HBsAg when using mAbs 5, 6, 7, and 8 (see FIGS. 1 and 2). Anti-HBs responses following a cure outcome“induce” an FC-P against reference HBsAg
• vaccination using a BNP platform comprising FC-P associated epitopes generates an anti-HBs response associated with an FC-P enhancing the chance for cure.
• Measurement of the epitope profile of the envelope protein (HBsAg) of the HBV circulating in a patient’s blood is a direct measure of the virus; it comprises the detection of a foreign antigen found in the patient’s blood.
• the logic or argument developed in the CP is that an infected person will mount an immune response against the virus and the effects of that immune response selection pressure on the virus can be measured. The effect can be demonstrated as HBsAg epitope changes on the envelope of the virus in that person.
• the CP is measured across a multiplex reporting panel
• FC-P is reported against reactivity to a 4plex panel applied to a natural history cohort run at St. Vincent’s Hospital Melbourne, within which the patients were HBeAg negative and had minimal or no disease activity, were in phase 4 and
• genotypes A, B transitioning to phase 5, with the infecting genotype unrestricted (i.e., genotypes A, B,
• the measure of an antibody made by the patient is a direct
• FC-P The logic or argument developed in the FC-P is that a person who has cured their CHB has successfully mounted an antibody response, whose effect can be
• the effect can be demonstrated by the ability of that antibody to induce epitope changes onto exogenously supplied HBsAg of panels of HBV; that is, the patients’ antibody“induces” an epitope profile response on supplied, exogenous
• HBV encodes three HBsAg proteins that form the viral envelope; small (S), middle (M) and large (L) (Carman WF, et al. J Hepatol. 1999; 31(2): 195-201; Seeger C, et al. Hepadnaviruses. 2013). All share a common 226-residue HBsAg-S domain (Seeger C, el al. Hepadnaviruses. In“ Fields Virology 2013, 6th edition pp. 2180-2221), whilst N- terminal extensions of M and L encode the PreS2 and PreSl domains respectively.
• the HBsAg envelope proteins are key viral antigens; during infection, S, M, and L are expressed and co-packaged through the ER membrane of the host cell, for the formation of the envelope of infectious HBV particles.
• S proteins are the sole antigenic components of the hepatitis B vaccine able to induce protective antibodies mainly directed to the major antigenic‘a’ determinant of the external loop region (residues 99-169).
• S proteins readily self-assemble into lipid-associated VLPs, and form the basis of the current recombinant vaccines, which are made in yeast and are not glycosylated.
• the S protein has not been crystallized but is considered a conformationally dynamic protein containing numerous cysteine and proline residues, predicted to form discrete loop domains (loop l :aa 107-135; loop 2:aa 139-149) within the immunodominant‘a’ determinant (Stirk HJ, et al.
• the cDNA sequence encoding the S protein is set forth in SEQ ID NO: 22.
• the cDNA sequence encoding the S protein with an Agel restriction site and FLAG tag is set forth in SEQ ID NO: 23. Alterations (variants and antibody occupancy) within the‘a’ determinant can modify HBsAg topology and directly influence the HBV neutralization phenotype (Carman WF, et al. J Hepatol. 1999;
• the cDNA sequence encoding the HBsAg-S protein having the amino acid sequence of SEQ ID NO: 50 is set forth in SEQ ID NO: 22.
• the cDNA sequence encoding the S protein having the amino acid sequence of SEQ ID NO: 51 is set forth in SEQ ID NO: 23.
• the HBsAg-S cDNA and amino acid sequences are provided below in Table A.
• HBsAg VLPs are highly compact due to the large number of intra- and
• the clearance epitopes on HBsAg are those that when occupied by antibodies in a subject, the likely result is a functional cure.
• the epitopes are located on each of Loop 1 and Loop 2 of HBsAg-S.
• monoclonal antibodies In relation to the assay, monoclonal antibodies
• mAbs are selected for use in a multiplex assay which target a range of epitopes on
• HBsAg-S HBsAg-S.
• One set of mAbs designated mAb5 and 6 targets the Loop 1 epitopes.
• Two Loop 1 epitopes are screened defined by the consensus amino acid sequence:
• Xi is SK122 (Genotypes Al, A2, A6, Bl, B2, B3, B6, C, F, G, H, I) or sRl22 (Genotypes A3, A4, A5, B3, B4, B5, B7, B8, B9, C2, C4, D, E);
• X2 is sTl25 (Genotype A, B, C, D, E, F, G, H, I) or sMl25 (genotype D3,
• X3 is sTl26 (Genotypes A, B, C, D, E, F, G, H, I) or sll26 (Genotype C);
• X 4 is SP127 (Genotypes A, B, E, C, D, G, I) or sTl27 (genotype C4, D2, D5) or sLl27 (genotype E, F, H);
• Xs is SA128 (A, B, C, D, E, F, G, H, I) or sVl28 (D2);
• Xe is sNl3 l (Genotypes A, G, I) or sTl3 l (Genotypes B, C, D, E, F, H);
• X7 is sFl34 (Genotype A, B, C, E, F, H) or sYl34 (Genotype D, G, I); and the consensus amino acid sequence to which mAh 10 binds PCX8TCX9XIOX11 (SEQ ID NO: 25), wherein:
• Xs is SK122 (Genotypes Al, A2, A6, Bl, B2, B3, B6, C, F, G, H, I) or sRl22 (Genotypes A3, A4, A5, B3, B4, B5, B7, B8, B9, C2, C4, D, E);
• X9 is sTl25 (Genotype A, B, C, D, E, F, G, H, I) or sMl25 (genotype D3, Xio is sTl26 (Genotypes A, B, C, D, E, F, G, H, I) or sll26 (Genotype C) or sSl26 or sAl26 (common variants);
• Xu is sPl27 (Genotypes A, B, E, C, D, G, I) or sTl27 (genotype C4, D2, D5) or sLl27 (genotype E, F, H);
• Another set of mAbs (designated mAb, 7, 8, 11, 12, 16, and 17) targets an epitope on Loop 2 defined by the consensus amino acid sequence:
• X12 is sTl40 (Genotype A, B, C, D, G, H, I) or sSl40 (Genotype E or F) ;
• X13 is sTl43 (Genotype A, B) or sSl43 (Genotype C, D, E, F, G, H, I).
• the epitopes occupied are CKTCTTPAQGNSMFPSC
• the epitopes occupied are CKTCTIPAQGTSMFPSC (SEQ ID NO: 30); and/or PCKTCTTP (SEQ ID NO: 28); and CCTKPSDGNCT (SEQ ID NO: 30).
• the epitopes occupied are CRTCTTPAQGTSMFPSC (SEQ ID NO: 32); and/or PCKTCTTP (SEQ ID NO: 28); and CCTKPSDGNCT (SEQ ID NO: 32).
• the disclosure of the present technology relates to the development of 8 CP-BNPs (SEQ ID NOs: 1-8) encompassing the pan-genotypic/serotypic loop 1 and loop 2 HBsAg target epitopes, alongside a control wild-type (WT) BNP.
• the BNP backbone consists of HBsAg-S envelope protein non-infectious subviral particles (virus-like particles, or VLPs, that do not contain all the components of the complete virus).
• the HBV genotype is genotype A, Al, A2, A3, A4, A5, A6, B, Bl, B2, B3, B4, B5, B6, B7, B8, B9, C, Cl, C2, C4, D, D2, D3, D5, E, F, G, H, or I.
• the HBV genotype is genotype D.
• the serotype is ayw or adw. In some embodiments, the serotype is ayw.
• VLPs as bio-nanoparticles (BNPs) to deliver target insert epitopes
• BNPs bio-nanoparticles
• Antibodies will be generated which are able to induce a cure of an existing and persistent hepatitis B infection.
• the CP epitope target (insert) sequences are listed in Table 1 below.
• BNPs are produced and purified (endo-free), and assessed for immunogenicity individually and in formulations that consider combinations of loop 1 and loop 2 target epitopes. The formulations analysed are listed in Table 2 below.
• nucleotide sequences encoding the 8 CP-BNP amino acid constructs are provided in Table 4. Table 4. CP-BNP Construct Exemplary Nucleotide Sequences
• the lead cyclic peptide vaccine is an equimolar formulation of cyclic peptides CTKPTDGNCT (SEQ ID NO: 20) and CTKPSDGNCT (SEQ ID NO: 21), conjugated to keyhole limpet hemocyanin (KLH).
• KLH keyhole limpet hemocyanin
• Administration of this vaccine to mice was antigenic, raising an anti-HBs Ab response consistent with a CP, indicative of“clearing” anti-HBs Ab.
• Analysis involved ELISA (to immunizing peptides, control peptides, VLP antigen), Induced CP analysis,
• the present disclosure relates to the use of mRNA constructs to elicit an immune response in existing and/or persistent hepatitis B, including chronic hepatitis B (CHB).
• CHB chronic hepatitis B
• the mRNA encodes for an antigenic epitope repeat region from antigenic epitopes expressed in the loop 1 and loop 2 regions of the HBsAg-S domain.
• the mRNA encodes for BNP 1, 2, 3, 4, 5, 6, 7, or 8.
• the mRNA encodes for a CP epitope cyclic peptide, such as those provided in Table 5.
• An immunogenic composition e.g ., vaccine
• Vaccines may be administered by means of a syringe or by means of a needle-free injector for intramuscular, subcutaneous, or intradermal injection.
• the immunogenic composition is one that is sufficient to achieve a desired biological effect. It is understood that, in some embodiments, the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect wanted.
• the ranges of effective doses provided below are not intended to be limiting and represent exemplary dose ranges. Thus, in some embodiments, the dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art.
• the dosage of a CP-BNP, CP epitope cyclic peptide, or mRNA vaccine for a mammalian (e.g, human) adult can be from 0.01 pg to 10,000 pg, or any range or value therein.
• the dosage can be from 0.10 pg to 10,000 pg, or any range or value therein. In some embodiments, the dosage can be from 1 pg to 10,000 pg, or any range or value therein. In some embodiments, the dosage can be from 2 pg to 10, 000 pg, or any range or value therein. In some embodiments, the dosage can be from 3 pg to 10, 000 pg, or any range or value therein. In some embodiments, the dosage can be from 4 pg to 10,000 pg, or any range or value therein. In some embodiments, the dosage can be from 5 pg to 10,000 pg, or any range or value therein.
• the dosage can be from 10 pg to 10,000 pg, or any range or value therein. In some embodiments, the dosage can be from 15 pg to 10,000 pg, or any range or value therein. In some embodiments, the dosage can be from 20 pg to 10,000 pg, or any range or value therein. In some embodiments, the dosage can be from 25 pg to 10,000 pg, or any range or value therein. In some embodiments, the dosage can be from 50 pg to 10,000 pg, or any range or value therein. In some embodiments, the dosage can be from 100 pg to 10,000 pg, or any range or value therein.
• the dosage can be from 500 pg to 10,000 pg, or any range or value therein. In some embodiments, the dosage can be from 1,000 pg to 10,000 pg, or any range or value therein. In some embodiments, the dosage can be from 5,000 gg to
• One aspect of the present technology includes methods of treating existing and/or persistent hepatitis B, including chronic hepatitis B (CHB) in a subject
• compositions or medicaments comprising the CP-BNPs, CP epitope cyclic peptides, and/or mRNA constructs of the present technology are administered to a subject suspected of, or already suffering from, a hepatitis B infection in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease.
• the HBsAg immunoassay described herein can monitor treatment to determine if an individual will achieve a functional cure, determine if an individual has achieved a functional cure, and determine if an individual has achieved a cure through natural defence mechanisms.
• the protocol may be varied without departing from the essence of the present technology.
• the critical endpoint is the determination of the fingerprint of epitopes on HBsAg which have been occupied by an individual's antibody response. Where the fingerprint of epitopes at Loop 1 and Loop 2 has been occupied, then a functional cure can be expected. Where the antibodies which have the capacity to bind to this
• a multiplex (“Bioplex”) bead-based flow cytometric platform is used to develop an HBsAg epitope assay and establish and map the HBsAg profile of epitopes which constitute the clearance profile of HBsAg epitopes (see FIG. 2).
• The“clearance profile of epitopes” is a profile of epitopes targeted by an individual’s antibodies.
• the assay is derived from an assay by Lim et al. (2014) Hepatology 60 (1) supplement : 1623 , 980A. Briefly, the HBsAg multiplex
• immunoassay comprises panels of fluorescently identified beads each conjugated to a different anti-HBs antibody which binds to HBsAg, followed by a polyclonal phycoerythrin (PE)-conjugated detection antibody.
• the HBsAg multiplex panels comprise monoclonal antibodies (mAbs) directed against HBsAg external loop region including the‘a’ determinant, and C-terminal domain epitopes spanning residues 99- 226 of HBsAg.
• the HBsAg epitopes are categorized into the following domains (with the monoclonal antibodies that bind to those domains): N-terminal (mAh 1); Loop 1 (mAbs 5,6,10); Loop 2 (mAbs 7,8,11,12,16,17,19); Loop 1/2 combinational (mAbs 13,14,15); C-terminal (mAbs 2,3,4); and conformational (mAbs 9,18).
• the intensity of PE associated with each bead provides a sensitive measure of antibody recognition of the specific HBsAg epitope within the sample.
• the epitope profile on HBsAg in comparison to the matched HBV strain backbone has been expressed as fold change in antibody binding or epitope recognition following bioinformatics data analysis.
• the anti-HBs mAbs utilized in the HBsAg multiplex assay were selected and sourced to provide broad coverage of epitopes with reactivity against the HBsAg antigenic and C-terminal domains (residues 99-226), covering both continuous and discontinuous epitopes, and epitope regions subject to both high variability and also conserved recognition.
• the high level of epitope coverage provided by the mAbs enabled analysis of the HBsAg profile according to the mapped epitope availability, and reported according to epitope region: N-terminal (mAh 1); Loop 1 (mAbs 5,6,10); Loop 2 (mAbs 7,8,11,12,16,17,19); Loop 1/2 combinational (mAbs 13,14,15); C-terminal (mAbs 2,3,4); and conformational (mAbs 9,18).
• HBV strains Interrogation of HBV strains (genotypes A-G) identified anti-HBs mAbs which displayed conserved epitope recognition patterns (i.e., indicative of conserved HBsAg fingerprints across genotypes and serotypes), which were mAbs 3 and 4 (C-terminal), mAh 15 (Loop 1/2 combinational), and mAb 18 (conformational).
• the remaining anti- HBs mAbs were sensitive to variations in the HBsAg profile which are identified by altered patterns of epitope recognition between HBV strains, in comparison to the genotype A2, serotype adw2 vaccine strain.
• the HBsAg profile assay was developed and optimized using HBV A2 adw2 strain HBsAg, which forms common basis of the majority of generic HBV vaccines and thus represents a relevant baseline or backbone for comparison of HBsAg epitope recognition.
• the HBsAg source was primarily HBsAg patient sera, confirmed by HBV sequencing as wild-type A2 adw2 (compared to consensus sequences), with confirmation using recombinant wild-type A2 adw2 HBsAg from cell culture supernatant.
• HBsAg concentration of HBsAg was determined to be approximately 16 IU/well (the standardized level), with detection of HBsAg as low as lIU/well and up to 100 IU/well.
• a sample dilution series for HBsAg was incorporated in the assay of 8, 16, and 32 IU/well, to account for any slight inaccuracy in the diagnostic determination of HBsAg concentration.
• the optimal concentration of anti-HBs mAbs labeled to beads was optimized as the mAb concentration resulting in fluorescence reactivity with the dynamic range of the instrument (15000-18000 RFU), and this was
• the multiplexing of the assay was built by the sequential addition of individual anti-HBs mAbs conjugated to beads, which allowed the identification of epitope competition due to partial shared epitopes by mAbs.
• three sets of multiplex anti-HBs bead/mAbs were established, as a 4plex, 5plex, and lOplex.
• HBsAg samples were analyzed in parallel (on the same plate) with these plexes and resulting data combined to achieve a multiplex readout of the HBsAg profile (FIG. 2 top panel).
• the assay was developed against HBsAg A2 adw2, which corresponds to the common vaccine strain of HBV, and thus reflects the HBsAg profile of epitope recognition for the multiplexed anti-HBs mAbs in the assay.
• the assay was validated for both HBsAg A2 adw2 from wild-type ex vivo sera HBsAg and recombinant HBsAg from in vitro cell culture to confirm the HBsAg profile for A2 adw2, which formed a vaccine strain background for the comparison of other HBV strains (genotypes and serotypes), which may be vaccine mismatched, and for reported HBsAg variants with potential for vaccine escape ( e.g ., SG145R/A, SP120T/L, SD144E/A).
• the assay was further validated against HBsAg of different genotypes (A-G) and serotypes, sourced from both patient sera and recombinant supernatants, which established the HBsAg profile of each strain in comparison to the vaccine strain (A2 adw2) background.
• a reference panel of sera representative of each HBV strain was established for ongoing inclusion in the assay as control data points in study cohorts. Genotype specific variations in the HBsAg profile between the common genotypes (A, B, C, D) are apparent at Loop 1 (mAb6) and Loop 2 (mAb8) epitopes, whilst serotype at the Loop 2 (mAb7) epitope.
• the HBV strains which are more divergent from the vaccine strain (C4, E and F) show more exaggerated variation from the vaccine strain in their HBsAg profile of epitope recognition.
• Example 5 Characterizing clearance profile associated a‘clearing’ anti-HBs response
• FIG. 1 shows the application of the assay described in Example 1 to characterizing a clearance profile (CP)-associated with antibodies which result in a functional cure.
• the assay determines HBsAg epitope occupancy using a mAh panel wherein mAbs 5 and 6 target the consensus sequence in Loop 1 of:
• Xi is K or R
• X2 is T or M
• X3 is T, or I
• X 4 is P, T or L
• X5 is A or V
• Xe is N or T
• X7 is F or Y.
• mAh 10 binds to another epitope Loop 1 within amino acids 120 to 127 of the consensus sequence:
• X8 is K or R
• X9 is T or M
• X10 is T, I or S; and X11 is P, T or L.
• mAbs 7, 8, 11, 12, 16 and 17 bind to an epitope within amino acids 137 to 147 of consensus sequence SEQ ID NO: 26 in Loop 2 wherein the consensus sequence is:
• X12 is T or S; and X1 3 is T or S.
• FIG. 2 describes an assay which detects HBsAg with occupied epitopes (top panel) and an assay which detects HBsAg complexed to anti-HBs antibodies (lower panel).
• the assays employ a multiplex immunoassay which detects occupancy of epitopes at Loop 1 (consensus sequence SEQ ID NOs: 24 and 25) and at Loop 2 (consensus sequence SEQ ID NO: 26).
• FIG. 3 and FIG. 4 are diagrammatic representations showing HBsAg clearance profile (CP) in CHB cure.
• CP HBsAg clearance profile
• FIG. 3 functional cure patients develop an antibody response that clears infection.
• FIG. 4 non-cure/non-responder patients do not carry the occupied HBsAg epitopes.
• HBsAgCP means the clearance profile of HBsAg epitopes.
• a HBsAg-non-clearance profile is referred to as HBsAg- NCP.
• HBsAg CP is indicated by reduced HBsAg epitope recognition ( e.g ., due epitope occupancy) at both loop 1 and loop 2 HBsAg epitopes.
• HBsAg profile assay The multiplex anti-HBs panel assay (FIG. 2, top panel) on the bioplex was originally developed with the concept of diagnostic monitoring of CHB patients for HBsAg changes.
• the assay may be used in patients on nucleotide analog (NA) therapy where HBV DNA is undetectable but HBsAg remains positive, and in looking for the development of treatment resistance mutations, or vaccine escape variants.
• NA nucleotide analog
• the assay is useful for trying to understand the HBsAg profile and changes occurring during CHB disease phases, i.e., predictive biomarkers for CHB disease.
• a cohort of patients whom achieved CHB functional cure are relevant to the CP biomarker identification and are further detailed below.
• biomarker i.e ., recognition of HBsAg by subsets of anti-HBs Abs in the assay.
• the results from these assays provided an understanding that: (a) functional cure is related to an anti-HBs response; (b) the“clearing” anti-HBs response is specifically targeting 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 both the loop 1 and loop 2 epitopes (CP epitopes) are associated with achieving functional cure; and (d) enhanced and targeted display of these CP associated epitopes drives production of CP-associated“clearing” anti-HBs Abs.
• the FC-P was defined as a 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: mAh 5, mAh 6, mAh 7, and mAh 8: (mAh 5 and mAh 6 recognize epitopes in loop 1; mAh 7 and mAh 8 recognize epitopes in loop 2) (see FIG. 2, top panel).
• mAh 5, mAh 6, mAh 7, and mAh 8 (mAh 5 and mAh 6 recognize epitopes in loop 1; mAh 7 and mAh 8 recognize epitopes in loop 2) (see FIG. 2, top panel).
• One of the six natural history patients had serum samples previously available which were still HBsAg- positive.
• 4plex anti-HBs Abs on the Bioplex platform the HBsAg-positive sample was epitope mapped and fulfilled the case definition of the FC-profile (FC-P).
• BNP/FC-P VLP vaccine delivery platform to generate BNP/FC-P (CP-BNP).
• BNP/FC-P vaccines were prepared as either a single epitope delivery format or as multiple epitope mixtures which were administered as immunizations to mice.
• the resulting anti-HBs responses were analyzed for their ability to induce a FC-P when incubated with
• constructs detailed in Table 2. These vaccines tested to date comprised both loop 1 and loop 2 epitopes and resulted in the generation of FC-P associated antibody.
• Dulbecco’s modified Eagle’s medium DMEM (Gibco-BRL, Grand Island, NY)
• GlutaMax-l GlutaMax-l
• FCS fetal calf serum
• streptomycin Gibco-BRL
• PEI polyethylenimine
• FectoPRO PolyPlus
• VLPs/BNPs were harvested from the cell culture supernatant 5-8 days post transfection.
• VLPs/BNPs which include an N-terminal Flag tag were affinity purified from the collected tissue culture supernatant was affinity purified over anti-Flag affinity resin columns (CSIRO, Australia). Fusion proteins were either competitively eluted from the anti-Flag affinity column with recombinant Flag peptide (0.4mgml; CSIRO, Australia) in PBS.
• Affinity purified VLP/BNP preparations in PBS were then peak purified by size exclusion over Superdex 200 or Superose 6 gel filtration columns (GE Healthcare).
• VLP/BNP purified preparations were tested for Endotoxin (Endosafe Test, Charles River, ETSA) and spin concentrated to ⁇ 1 ml using Amicon 50kDa cut-off centrifugal concentrator devises.
• the VLPs/BNPs were purified from the collected tissue culture supernatant by spin clarification using a benchtop centrifuge, then the supernatant was transferred into an ultracentrifuge tube, underlaid with a 20% sucrose cushion, and the particles pelleted by ultracentrifugation.
• VLP/BNP preparation yield purity was assessed by: i) A260/280 spectra profile 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) with both anti-HBs and anti-Flag detection antibodies; iv) HBsAg epitope profile assay analysis on the Bioplex platform (in-house assay); and, v) standard ELISA techniques with detection by anti-HBs antibodies (in-house assay).
• CP-BNP were analysed for HBsAg epitope profile (Bioplex platform) as individual BNP preparations and also as BNP formulations to assess loop 1 and loop 2 epitope presentation retention (i.e., loop 1 and loop 2 epitopes are displayed or presented) as appropriate to antigenicity on delivery.
• HBsAg epitope profile Bioplex platform
• loop 1 and loop 2 epitope presentation retention i.e., loop 1 and loop 2 epitopes are displayed or presented
• Cyclised CP Epitope Peptides were designed to mimic the identified loop 1 and loop 2 target“clearing” epitopes within HBsAg, similar to the CP epitope inserts delivered in the CP-BNPs. Sequence variations within these epitopes exist dependant on naturally occurring HBV genotype and HBsAg serotype variations, and multiple cyclic peptides were designed based on this consideration, generating pan- genotypic/pan-serotypic equimolar formulations of loop 1 or loop 2 CP epitope cyclised peptides for delivery as immunizing antigens in immunogenicity studies for the
• CP-associated“clearing” anti-HBs antibodies The designed peptides were ordered for production by Mimotopes (Australia) with N-terminal free amine, C-terminal free acid, cyclised by disulphide bonds at native sequence cysteine residues. Production was to a purity of >85% by HPLC and determined by mass spectrometry profile analysis. Cyclised peptide was coupled via a glutaraldehyde linker to either KLH (keyhole limpet hemocyanin) for immunization or to BSA (bovine serum albumin) for screening assays. Exemplary cyclic peptides of the present technology are listed in Table 5 above.
• Post-fusion supernatant was screened initially by standard ELISA protocols for antibody responses specific to recombinant HBsAg (VLP) and to loop 1 and loop 2 cyclised peptides (individual peptides and pooled peptide formulations formulation). Clone supernatants were further analysed for detection of HBsAg following SDS-PAGE and Western Blot, to determine affinity for linear epitopes. The clone supernatants were analysed for the generation of anti-HBs antibody induced CP profile assay analysis on the Bioplex platform (in-house assay), with comparison to pre-incubation pan- genotypic/serotypic HBsAg. This data was used to determine which clones should be selected for further rounds of monoclonal antibody development, with analysis of clone supernatants at each round, and including analysis of final selected monoclonal anti-HBs antibodies (Table 6; FIG. 12).
• Example 13 CP -BNP and CP Epitope Cyclic Peptide Immunogenicitv Study
• This example demonstrates the immunogenicity of the CP-BNPs and CP epitope cyclic peptides of the present technology.
• the immunogenicity of CP-BNPs and CP-BNP formulations displaying target loop 1, loop 2, or loop 1 & 2“clearance” epitopes were assessed by immunization of B ALB/c strain mice.
• CP-BNPs 1-4 and formulations thereof were assessed in a first round BNP immunogenicity study. These will be reassessed alongside assessment of CP-BNPs 5-8 and formulations thereof in a second immunogenicity study.
• Immunogenicity study #1 incorporated CP-BNPs 1-4 and formulations as indicated in Table 2 (in comparison to WT-BNP and PBS placebo controls), whilst immunogenicity study IMM001 repeated assessment performed in the original study and additionally assessed performance of CP-BNPs 5-8 and formulations 1+3+4 and 5+7.
• Six week old BALB/c mice (up to 5 per arm) were immunised with 3 x 2ug doses of purified CP-BNP antigen at fortnightly intervals. Bleeds were taken prior to each antigen administration and then following immunisation at weekly intervals for 5 weeks. Mice were sacrificed at the final time point and terminal bleed collected.
• Sera harvested from each bleed time point was analysed to investigate the Anti-HBs antibody response to CP- BNP immunisation by: i) anti-HBs antibody titres (IU/L) using diagnostic serology tests (Elecsys Anti-HBs kit, Roche); ii) standard ELISA methodology for detection of WT-VLP or HBsAg loopl or loop2 peptides; and, iii) Anti-HBs antibody induced CP profile assay analysis on the Bioplex platform (in-house assay).
• FIG. 13A shows the anti-HBs antibody immunogenicity response for CP -BNP preparations and formulations.
• FIG. 13B and FIG. 13C show the loop 1 and loop 2, respectively, anti-HBs antibody immunogenicity responses for the CP epitope cyclic peptides.
• Example 14 CP-BNPs Induce a CHB Clearance Profile
• Example 15 mRNA Encoding CP-BNPs and CP Epitope Cyclic Peptides Induce a CHB Clearance Profile
• mice Six week old BALB/c mice (5 per arm) are immunized with 3 x 2ug doses of purified mRNA encoding CP-BNPs or CP epitope cyclic peptides at fortnightly intervals. Bleeds are taken prior to each antigen administration and then following immunization at weekly intervals for 5 weeks. Mice are sacrificed at the final time point and terminal bleed collected.
• Sera harvested from each bleed time point is analysed to investigate the anti-HBs antibody response to CP-BNP immunization by: i) anti-HBs antibody titres (IU/L) using diagnostic serology tests (Elecsys Anti-HBs kit, Roche); ii) standard ELISA methodology for detection of WT-VLP or HBsAg loop 1 or loop 2 peptides; and, iii) anti-HBs antibody induced CP profile assay analysis on the Bioplex platform (in-house assay).
• compositions comprising the mRNA constructs of the present technology are useful in methods of treating chronic hepatitis B.
• Example 16 CP-BNPs Reduce HBV DNA and HBsAg Levels in a Chronic Hepatitis B (CHB) Murine Model
• Plasmids containing HBsAgS-specific cDNA inserts were cloned into pCAGGS (with an N-terminal FLAG tag) and used to transfect HEK293T cells using polyethylenimine (PEI) as described previously (Longo, 2013).
• Functional HBsAgS proteins including chimeric HBsAgS proteins with inserted additional sequences assemble into secretion competent virus-like particles, bionanoparticles (BNPs).
• cell culture medium was harvested and centrifuged to remove cellular debris.
• the cell culture medium was centrifuged at high speed (ultra-centrifugation) over a 20% sucrose cushion to pellet and to partially purify the BNPs, then re-suspended in sodium chloride (NaCl)-Tris-EDTA (STE) buffer. Further purification of the BNPs was performed by ultracentrifugation using a gradient of sucrose (20 - 50%) 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.
• Murine CHB model The CBA/caJ CHB mouse model was developed by hydrodynamic injection HDI) of an HBV replicon (pAAV/HBVl.2), which allows for the delivery of DNA into hepatocytes through an intravenous injection in to the tail vein (Kim, 2013; Yang, 2002; Huang, 2006). This mode of in vivo transfection was necessary because hepatitis B virus (HBV) does not naturally infect mice.
• HBV hepatitis B virus
• a single HDI of a plasmid containing replication-competent HBV DNA induced stable HBV depending on the used mouse strain (for CBA mice persisted > 6 months with liver specific HBV viral marker production (up to 32 weeks)).
• HBV DNA was measured in the mouse serum to confirm that HBV persistence was established.
• Mice with a persistent HBV were then used for immunization studies to test for the ability of chimeric BNPs with clearance profile-specific epitope repeats (CP-BNP) to induce a therapeutic immune response able to interfere and/or clear the HBV from the liver.
• CP-BNP clearance profile-specific epitope repeats
• mice Following establishment of chronic hepatitis B (approximately week 10), groups of 6 - 8 CBA/caJ mice were treated with placebo (adjuvant control), control particles (Engerix B (marketed HBV vaccine manufactured in yeast cells), wild type BNP (WT-BNP])) or test vaccine candidates (CP-BNP 1 - 7, in various combinations at equimolar ratios with final concentrations as listed in FIGS. 15A-15C and 16A-16C).
• the treatment schedule included a mixture of different chimeric BNPs adjuvanted with aluminum hydroxide. Three doses of vaccine were given subcutaneously with 2 weeks between doses.
• Submandibular bleeds were performed every 2 weeks out to 26 weeks post-HDI, with testing to determine serum HBV DNA load (Sitnik, 2010) and HBsAg levels (per manufacturer’s recommended protocol; qHBsAg Elecsys).
• Murine CHB model results HBV DNA levels.
• the HBV DNA level results described below are shown in FIGS. 15A-15C.
• Placebo (arm 1) did not significantly change HBV DNA levels throughout the duration of the study.
• WT-BNP was dosed at 0.05 (arm 2), 0.1 (arm 3) or 0.5 (arm 4 and 12) pg.
• the WT-BNP at 0.05 pg (arm 2) dose had no effect on HBV DNA levels, while the 0.1 (arm 3) or 0.5 (arm 4 and 12) pg doses displayed a dose dependent decrease in HBV DNA levels.
• CP- BNP 1+4+5 also displayed a dose dependence decrease in HBV DNA, but the lower doses of 0.05 (arm 6) or 0.1 (arm 7) pg had higher HBV DNA relative to the same doses of WT- BNP.
• Other combinations of CP -BNPs were also tested in this model and found to be less efficacious than CP -BNP 1+4+5.
• Murine CHB model results HBsAg levels.
• the HBsAg level results described below are shown in FIGS. 16A-16C.
• Placebo arm 1 did not significantly change HBsAg levels throughout the duration of the study.
• WT-BNP was dosed at 0.05 (arm 2), 0.1 (arm 3) or 0.5 (arm 4 and 12) gg.
• the WT-BNP 0.05 gg (arm 2) dose had no effect on HBsAg levels, while the 0.1 (arm 3) or 0.5 (arm 4 and 12) ug doses displayed a dose dependent decrease in HBsAg levels.
• CP-BNP 1+4+5 also displayed a dose dependence decrease in HBsAg, but the lower dose of 0.05 gg (arm 6) was similar to WT-BNP at the same dose or 0.1 gg (arm 7) had higher HBsAg relative to the same dose of WT-BNP.
• Other combinations of CP-BNPs were also tested in this model and found to be less efficacious than CP -BNP 1+4+5.
• FIGS. 16A-16C are also shown in tabular format as individual, means, and mean changes from baseline in Tables 10, 11, and 12, respectively.
• Table 11 Mean HBsAg data.
• Table 12 Mean Changes from Baseline HBsAg data.
• Example 17 Production of Candidate Vaccines from mRNA and Protein Expression
• cell lysates were processed using NP-40 lysis buffer and cell debris pelleted. Samples were assessed by western blot as previously described.
• HBsAg BNP expression results following transfection of BNP -encoding nucleic acids (mRNA andDNA) into mammalian cell lines.
• HBsAg BNP expression from transfection of mRNA was successfully achieved in two different cell lines (HEK293T and Huh7) as measured by Western blot analysis using an anti-HBsAg antibody (FIGS. 17A-17B).
• the Western blot identified multiple bands, including expected size of the monomer (WT-BNP, 24 kD; CP -BNP 4, 29 kD) and glycosylated variants (WT-BNP, 27 kD; CP-BNP 4, 32 kD).
• WT-BNP expected size of the monomer
• CP -BNP glycosylated variants
• HBsAg was detected after transfection with a DNA construct that would express WT-BNP (with flag tag 15 and 28 kDa).
• a range includes each individual member.
• a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
• a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.
• HBsAg hepatitis B surface antigen
• Session IX Antiviral therapy , 0-205, Germany

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