vaccine

Abstract

The present disclosure provides an adenoviral vector comprising at least one modified capsid protein comprising an insertion or fusion of a first peptide partner, wherein the first peptide partner is covalently bonded to a second peptide partner that is linked by insertion or fusion to a decoy antigen; and wherein the decoy antigen comprises Hepatitis B virus PreS1 and / or Hepatitis B virus PreS2 or immunogenic fragments thereof. The present disclosure also provides methods of producing the adenoviral vector, as well as uses of the adenoviral vector in vaccines and pharmaceutical compositions.

Description

[0001] Invention Field This invention relates to adenovirus vaccines against hepatitis B, particularly vaccines for the treatment of chronic hepatitis B. The invention also relates to nucleic acids encoding those vaccines, cells containing those nucleic acids, and methods for the prevention or treatment of hepatitis B, particularly chronic hepatitis B. Background of the Invention Hepatitis B is a liver infection caused by the hepatitis B virus. The virus is transmitted through contact with infected bodily fluids (such as blood, saliva, semen, and vaginal fluid), with the most common route of transmission being mother-to-child transmission during the perinatal period.

[0003] Hepatitis B can be acute or chronic (CHB). Chronic HBV (CHB) has a high mortality rate, primarily resulting from cirrhosis and liver cancer. In 2019, the WHO estimated that 296 million people worldwide were infected with CHB, with 1.5 million new infections each year. In the same year, there were 820,000 deaths worldwide due to CHB.

[0004] Current HBV vaccines are effective as a preventative measure, but have very limited efficacy in treating individuals with pre-existing CHB infection. In most cases, lifelong antiviral therapy is required to suppress viral replication—there is currently no cure. Acute HBV infection is usually cleared by the host immune response, while CHB infection is characterized by dysfunctional anti-HBV cellular and humoral immunity.

[0005] It is generally believed that a “functional cure” for HBV (defined as the persistent loss of the hepatitis B surface antigen HBsAg) will require therapeutic strategies that can induce strong T cell (especially CD8+ T cell) responses against multiple HBV antigens and neutralizing humoral immunity against HBV. Invention Overview According to a first aspect, this disclosure provides an adenovirus vector comprising at least one modified capsid protein, the modified capsid protein comprising the insertion or fusion of a first peptide conjugate, wherein: the first peptide conjugate is covalently bonded to a second peptide conjugate; the second peptide conjugate is linked to a decorative antigen via insertion or fusion; and the decorative antigen comprises hepatitis B virus PreS1 and / or hepatitis B virus PreS2 or immunogenic fragments thereof.

[0007] In one embodiment, the modified capsid protein comprises a hexon or pIX protein. In another embodiment, the modified capsid protein comprises a hexon. In yet another embodiment, the modified capsid protein comprises pIX. The adenovirus vector can have any suitable genotype. The adenovirus vector can have any suitable serotype. The adenovirus vector serotype can be derived from any suitable species, such as human, chimpanzee, or gorilla. The adenovirus vector can be replicating or non-replicating. In addition to the modified capsid protein, the adenovirus vector may also have other modifications.

[0008] In another embodiment, the modified capsid protein comprises a hexagonal protein, and further, the modification of the hexagonal protein comprises fusing a first peptide spouse into a hypervariable region (HVR). The adenoviral hexagonal protein has seven HVR domains, HVR1 to HVR7. Suitably, any HVR domain can be used for insertion or fusion of the first peptide spouse. The selection of the hypervariable region can be determined by those skilled in the art according to their needs. In one embodiment, the hypervariable region comprises any one of HVR1, HVR2, or HVR5. In another embodiment, the hypervariable region comprises HVR1. In another embodiment, the hypervariable region comprises HVR2. In another embodiment, the hypervariable region comprises HVR5.

[0009] In another embodiment, the covalent bond between the first and second peptide conjugates in the adenoviral vector is an isopeptide bond. In one embodiment, the first and second peptide conjugates constitute a binding conjugate pair. The binding conjugate pair may be selected from: DogTag and DogCatcher, DogTag and SnoopTag, DogTag and SnoopTagJr, SnoopTag and SnoopCatcher, or SpyTag and SpyCatcher, or variations thereof. Other suitable binding conjugate pairs are known to those skilled in the art. In one embodiment, the binding conjugate pair is DogTag and DogCatcher. Suitably, the first peptide conjugate is DogTag, and the second peptide conjugate is DogCatcher.

[0010] In any embodiment of any aspect of this disclosure, the decorative antigens comprising hepatitis B virus PreS1 and / or hepatitis B virus PreS2 or immunogenic fragments thereof may be derived from any HBV strain or may be derived from a common sequence of multiple HBV strains. For example only, the reference strain NC_003977 (ayw) has been used herein as a proof-of-concept strain. Those skilled in the art will understand that the hepatitis B virus PreS1 and / or PreS2 decorative antigens refer to hepatitis B virus PreS1 and / or PreS2 polypeptides from any HBV strain that have been determined by sequence alignment to be equivalent to the same polypeptide from the reference strain NC_003977 (ayw). Furthermore, as described herein, the decorative antigens comprising hepatitis B virus PreS1 and / or hepatitis B virus PreS2 may be truncated, meaning that one or more amino acids are missing from the N-terminus and / or C-terminus relative to the full-length polypeptide. In any embodiment of any aspect of this disclosure, the immunogenic fragment of the decorative antigen comprising hepatitis B virus PreS1 and / or hepatitis B virus PreS2 refers to any portion of hepatitis B virus PreS1 and / or hepatitis B virus PreS2 that retains the ability to elicit an immune response when administered to a subject. In any embodiment of any aspect of this disclosure, the decorative antigen may comprise any region of HBsAg, such as the antigenic loop of HBV-S or an immunogenic fragment thereof, the antigenic loop of HBV-S or an immunogenic fragment thereof combined with HBV PreS1 and / or PreS2 or an immunogenic fragment thereof, or in lieu of those polypeptides.

[0011] In another aspect, the adenovirus vector further comprises a nucleic acid encoding one or more exogenous polypeptides, wherein one or more exogenous polypeptides comprise at least one hepatitis B virus polypeptide or an immunogenic fragment thereof. In one embodiment, the at least one hepatitis B virus polypeptide is selected from PreS1, PreS2, S-protein, or core, or an immunogenic fragment thereof. In another embodiment, one or more exogenous polypeptides comprise two or more hepatitis B virus polypeptides selected from PreS1, PreS2, S-protein, or core, or an immunogenic fragment thereof. In yet another embodiment, one or more exogenous polypeptides comprise all hepatitis B virus polypeptides PreS1, PreS2, S-protein, and core, or immunogenic fragments thereof. In one embodiment, at least one hepatitis B virus polypeptide comprises PreS1 or an immunogenic fragment thereof, and the decorative antigen also comprises hepatitis B virus PreS1 or an immunogenic fragment thereof; the encoded PreS1 or a fragment thereof and the decorative PreS1 or a fragment thereof may have the same amino acid sequence or different amino acid sequences. In other embodiments, other HBV peptides, such as HBeAg or HBV polymerase, may also be encoded. In other embodiments, peptides from sources other than HBV may also be encoded.

[0012] In any embodiment of any aspect of this disclosure, a hepatitis B virus polypeptide or an immunogenic fragment thereof may be derived from any HBV strain or may be a common sequence derived from multiple HBV strains. For example only, the reference strain NC_003977 (ayw) has been used herein as a proof-of-concept strain. Those skilled in the art will understand that a hepatitis B virus polypeptide, such as PreS1, PreS2, S-protein, or core, refers to a polypeptide derived from any HBV strain that has been identified by sequence alignment as equivalent to the same polypeptide from the reference strain NC_003977 (ayw). Furthermore, the hepatitis B virus polypeptides described herein may be truncated, meaning that one or more amino acids are missing from the N- and / or C-termini relative to the full-length polypeptide. In any embodiment of any aspect of this disclosure, an immunogenic fragment of a hepatitis B polypeptide refers to any portion of the hepatitis B polypeptide that retains the ability to elicit an immune response when administered to a subject.

[0013] In any embodiment of any aspect of this disclosure, a decorating antigen comprising hepatitis B virus PreS1 and / or hepatitis B virus PreS2 or an immunogenic fragment thereof, and, where present, at least one hepatitis B virus polypeptide or an immunogenic fragment thereof encoded by the nucleic acid of an adenovirus vector, may be derived from the same HBV strain, or may be derived from a common sequence derived from multiple HBV strains, or may be derived from more than one HBV strain, or any combination thereof. For example, in the case of decorating antigens, HBV PreS1 and PreS2 (both present) may both be derived from the same HBV strain, or from the same common HBV strain, or each from a different HBV strain. Similarly, where more than one hepatitis B virus polypeptide or an immunogenic fragment thereof is encoded by the nucleic acid of an adenovirus vector, each may be derived from the same HBV strain, or from the same common HBV strain, or each from a different HBV strain, or any combination thereof. Similarly, HBV PreS1 and / or PreS2 decorating antigens or immunogenic fragments thereof, and at least one hepatitis B virus polypeptide or immunogenic fragment thereof encoded by the nucleic acid of an adenovirus vector, can be derived from the same HBV strain, or from the same HBV co-existing strain, or each from different HBV strains, or any combination thereof. For example only, the reference strain NC_003977 (ayw) has been used herein as a proof-of-concept strain. Those skilled in the art will understand that hepatitis B virus polypeptides, such as PreS1, PreS2, S-protein, core, pre-core, pol, or x, refer to polypeptides from any HBV strain that have been determined by sequence alignment to be equivalent to the same polypeptide from the reference strain NC_003977 (ayw).

[0014] In another embodiment, when the adenovirus vector further comprises nucleic acid encoding one or more exogenous polypeptides, and wherein the one or more exogenous polypeptides comprise two or more hepatitis B virus polypeptides or immunogenic fragments thereof, the nucleic acid of the adenovirus vector may further comprise a sequence arranged to direct cleavage between the two or more hepatitis B virus polypeptides, optionally wherein the sequence arranged to direct cleavage is selected from P2A, F2A, or IRES. Suitably, other known cleavage-directing sequences may be used, such as sequences of other 2A self-cleaving peptides, such as T2A or E2A. Any IRES sequence may also be used. The sequence arranged to direct cleavage may be used to achieve cleavage between the two or more hepatitis B virus polypeptides using any mechanism.

[0015] In another embodiment, the nucleic acid of the adenoviral vector may contain additional non-coding or coding elements, including but not limited to promoters, enhancers, adapters, additional foreign genes, and terminators. Any suitable such sequence may be used.

[0016] On the other hand, vaccines containing adenovirus vectors are provided. A vaccine containing an adenovirus vector refers to an adenovirus vector provided in any suitable composition for administration to humans in need, and / or provided at any concentration (dose) of adenovirus vector suitable for eliciting an immune response upon administration. In addition to the adenovirus vector, the vaccine may also contain any other suitable components.

[0017] In another aspect, pharmaceutical compositions comprising vaccines and pharmaceutically acceptable buffers, excipients, carriers, adjuvants, or combinations thereof are provided.

[0018] On the other hand, adenovirus vectors or vaccines can be used to treat or prevent hepatitis B virus infection and / or hepatitis D virus infection.

[0019] On the other hand, adenovirus vectors can be used to prepare drugs for the treatment or prevention of hepatitis B virus infection and / or hepatitis D virus infection.

[0020] In the implementation plan, hepatitis B virus infection can be acute or chronic (CHB). Similarly, hepatitis D virus infection can be acute or chronic.

[0021] On the other hand, a method for treating patients with corresponding needs is provided, which includes administering a safe and effective amount of an adenovirus vector or vaccine. Suitablely, any method or schedule of administration may be used.

[0022] In another aspect, a method for generating an adenovirus vector is provided, the method comprising: introducing a nucleic acid encoding a first peptide conjugate into a nucleic acid encoding a capsid protein in an adenovirus genome; allowing expression of the adenovirus genome to obtain adenovirus particles; obtaining a second peptide conjugate linked to hepatitis B virus PreS1 and / or hepatitis B virus PreS2 or an immunogenic fragment thereof; and allowing the adenovirus particles and the second peptide conjugate linked to hepatitis B virus PreS1 and / or hepatitis B virus PreS2 or an immunogenic fragment thereof to mix under conditions allowing covalent binding of the first and second peptide conjugates. In one embodiment, the method further comprises introducing a nucleic acid encoding one or more exogenous polypeptides into the adenovirus genome, wherein the one or more exogenous polypeptides include at least one hepatitis B virus polypeptide or an immunogenic fragment thereof.

[0023] On the other hand, a method for manufacturing a vaccine is provided, the method comprising mixing an adenovirus vector with a pharmaceutically acceptable excipient. Any suitable excipient can be used.

[0024] Attached Figure Figure 1: A - Schematic diagram of an exemplary adenoviral vector showing a tag (first peptide partner) inserted into the capsid protein. B - Schematic diagram of an exemplary adenoviral vector showing the vector from A, where the PreS1 protein is coupled to the vector surface via an interaction between the catcher (second peptide partner) and the tag, and the GFP gene is inserted into the adenoviral genome. C - SDS-PAGE gel showing adenoviral vector particles with a DogTag inserted into a hexagonal protein, covalently decorated with decreasing concentrations of DogCatcher-PreS1 after overnight co-incubation. Coupling efficiency (as shown, expressed as hexagonal protein coupled with catcher-PreS1) was calculated from the relative band intensities of uncoupled hexagonal proteins and covalently coupled hexagonal protein-catcher-PreS1 species. D - In vitro infectivity of a GFP-expressing adenoviral vector decorated with decreasing concentrations of DogCatcher-PreS1 ligand in 293A cells (from the experiment shown in 1C). E - Mouse serum IgG antibody response to PreS1, measured by endpoint ELISA on day 20 (before booster) and day 35 (after booster) following administration of: undecorated adenovirus encoding the EGFP reporter gene (Ad(GFP)), PreS1-decorated adenovirus encoding the EGFP reporter gene (Ad(GFP):PreS1), or unconjugated DogCatcher-PreS1 protein with Alhydrogel® adjuvant (C-PreS1+alhydrogel). F - Splenic CD8+ T cell response to EGFP encoding EGFP was measured by overnight ex vivo interferon-γ (IFNγ)-ELISPOT assay, with the y-axis showing spotted cells per million spleen cells (SFC).

[0025] Figure 2: A - SDS-PAGE gel showing adenoviral vector particles covalently decorated with a hexagonal protein and DogTag inserted into the mammalian expression DogCatcher-PreS1 after overnight co-incubation. B - Calculation of relative band intensities of uncoupled hexagonal proteins and covalently coupled hexagonal protein-Catcher-PreS1 species using the coupling efficiency (expressed as hexagonal proteins coupled with Catcher-PreS1) at incremental concentrations (as shown in A). C - In vitro infectivity of GFP-expressing adenoviral vectors decorated with incremental DogCatcher-PreS1 ligands in 293A cells (as shown in A and B).

[0026] Figure 3: A - Schematic diagram of conjugated adenovirus vectors, showing the PreS1 protein coupled to the vector surface via a tag-catch pair via a hexagonal capsid protein, and optionally encoding HBV genes: HBV L encodes a large HBsAg polypeptide, while HBV CS encodes the HBV core and a small HBsAg polypeptide with intercalation cleavage sites. Sli = shark invariant chain, 2A = F2A (FMDV18 2A), GGS and GSGGS are linkers. B - Serum IgG antibody response to PreS1 as measured by endpoint ELISA after administration of: undecorated adenovirus encoding HBV CS (Ad(CS)), undecorated adenovirus encoding HBV L (Ad(L)), or the PreS1-decorated form of each adenovirus (Ad(CS):PreS1 and Ad(L):PreS1, respectively). In vitro anti-HBV serum neutralization of C-HepG2-NTCP cells is represented by a reduction in luminescence signal relative to the luciferase-expressing HBV virus control (“virus only”), expressed in relative light units (RLU). HepG2-NTCP cells were co-incubated with luciferase-expressing HBV virus and serum from the experiment shown in B, or serum from mice inoculated with undecorated adenovirus encoding GFP (Ad(GFP)).

[0027] Figure 4: A - Schematic diagram of the conjugated adenovirus vector, showing the PreS1 protein coupled to the vector surface and the optional encoding HBV gene. Sli = shark invariant strand, 2A = F2A (FMDV18 2A), GGS and GSGGS are linkers. BD - CD8+ T cell responses to the encoding gene in the spleen after administration of the following: adenovirus encoding HBV L and decorated with PreS1 at low capsid coverage (Ad(L):PreS1 LC), the same adenovirus decorated with PreS1 at high capsid coverage (Ad(L):PreS1 HC), adenovirus encoding HBV L followed by the HBV core (N-to-C-terminus) and decorated with PreS1 at high coverage (Ad(LC):PreS1HC), or adenovirus encoding the HBV core followed by the HBV L (N-to-C-terminus) and decorated with PreS1 at high coverage. For Figure 4C and Figure 4D All adenoviruses were decorated with PreS1 for high coverage. CD8+ T cell responses were assessed by overnight ex vivo IFNγ-ELISPOT assay using BS. 190-197 Peptides, C-core 93-100 Peptide or D-core P13LPeptides were used for measurement. E - Serum IgG antibody response to PreS1 in vaccinated mice, measured by endpoint ELISA on day 20 (before booster) and day 35 (after booster). F - In vitro anti-HBV serum neutralization in HepG2-NTCP cells, expressed as a reduction in luminescence signal relative to a luciferase-expressing HBV virus control (“virus only”), expressed in relative light units (RLU). HepG2-NTCP cells were co-incubated with luciferase-expressing HBV virus and serum from the experiments shown in BE or serum from mice inoculated with undecorated adenovirus encoding GFP (Ad(GFP)). G - Complete dilution series from the in vitro anti-HBV serum neutralization assay shown in F.

[0028] Figure 5 Western blot analysis of the expression of coding genes from ten different Ad-HBV vectors (designated AJ, as shown in Table 6) was designed to test the effect of incorporating nucleotide sequences arranged to guide cleavage between antigenic peptides.

[0029] Figure 6: A - Schematic diagram of the conjugated adenovirus vector, showing the PreS1 protein coupled to the vector surface and the optional coding gene. IRES = internal ribosome entry site cleavage sequence from encephalocarditis virus, F2A = FMDV18 2A, P2A = cleavage sequence of porcine cerebrovirus-1 2A, B - Serum IgG antibody response to PreS1 in mice inoculated with the construct in A, measured by endpoint ELISA, identified by the coding cassette on the X-axis. CE: T cell response to the coding gene in the spleen, measured by overnight ex vivo IFNγ-ELISPOT assay in the same mice.

[0030] Figure 7: A - Schematic diagram of the conjugated ChAd63 adenovirus vector, showing the PreS1 protein coupled to the vector surface and one or more optional coding genes, abbreviated as in other figures. B - Serum IgG antibody response to PreS1 as measured by endpoint ELISA. CE: T cell response to coding genes in spleen as measured by overnight ex vivo IFNγ-ELISPOT assay. Invention Details Hepatitis B virus Hepatitis B virus (HBV) belongs to the family Hepatotropic DNA Viridae (Family of Hepatotropic DNA Viruses) HepadnaviridaeHBV is a type of double-stranded DNA virus. Based on antigenic epitopes present on its envelope proteins, the virus is classified into four major serotypes (adr, adw, ayr, ayw). According to genomic sequence similarity, HBV strains are also classified into ten genotypes (AJ) and forty subgenotypes. Genotypes have different geographical distributions and also influence disease severity, course, likelihood of complications, and response to treatment.

[0032] HBV particles, or virions, also known as Dane particles, consist of an icosahedral nucleocapsid protein core surrounding viral DNA and DNA polymerase, which is enclosed by an outer lipid envelope containing embedded proteins. The viral proteins present in the nucleocapsid core are hepatitis B core antigen (HBcAg; HBV core protein) and hepatitis B virus DNA polymerase. Hepatitis B envelope antigen (HBeAg) is present between the nucleocapsid core and the lipid envelope but is not considered part of the virion and is secreted into the serum. The HBV-encoded oncogene X protein (HBx) is a small, non-structural protein that plays a role in HBV replication and HBV-related liver disease. Hepatitis B surface antigen (HBsAg; Australia antigen) is embedded in the viral envelope. HBsAg exists in three forms: small (S), medium (M), and large (L). All three forms are encoded by the same open reading frame and share a common 226-amino acid C-terminal domain, but are translated from one of three different start codons. The HBsAg L polypeptide (approximately 42 kDa) is translated from the most upstream (5'-) of the three start codons and contains the PreS1, PreS2, and SHBsAg domains. The HBsAg M polypeptide (approximately 33 kDa) is translated from the middle start codon and contains the PreS2 and SHBsAg domains. The HBsAg S polypeptide (approximately 24 kDa; approximately 80% of HBsAg is in this form) is translated from the most downstream (3'-) of the three start codons and contains only the SHBsAg domain. In nature, the PreS1 and PreS2 domains do not exist independently.

[0033] In this disclosure, the hepatitis B virus reference strain NC_003977 (ayw) has been used as a proof of concept. It should be understood that when an adenovirus vector contains more than one HBV nucleotide or amino acid sequence, any other HBV strain or combination of HBV strains may be appropriately used. Similarly, HBV common sequences can be used, where the common sequence is the calculated sequence of the most common residues (nucleotides or amino acids) found at each position in the sequence alignment. Those skilled in the art will understand that one or more strains used in an adenovirus vector can be varied to, for example, target HBV strains from one or more different geographic regions.

[0034] Variants, derivatives, and modifications of hepatitis B virus peptides can be prepared by any suitable method. Variants, derivatives, and functionally operable modifications can involve the addition, substitution, alteration, or deletion of amino acids.

[0035] Hepatitis B virus polypeptides can be defined by reference to a specific sequence, for example, by reference to the amino acid sequence of a reference strain of hepatitis B virus NC_003977 (ayw). This specific sequence can be 100% identical to the sequence of a wild-type hepatitis B virus polypeptide. Hepatitis B virus polypeptides can have a sequence identity of at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% with the wild-type sequence. Variants and derivatives of hepatitis B virus polypeptides can contain an amino acid sequence that is at least 90% or 95% similar to the wild-type sequence. Therefore, homologs of these entities can have at least 60% homology with the wild-type sequence, or at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology.

[0036] Hepatitis B disease Hepatitis B (the disease) caused by HBV infection affects the liver and can occur in both acute and chronic (CHB) forms.

[0037] Symptoms of acute hepatitis B include malaise, loss of appetite, nausea, vomiting, body aches, mild fever, and dark urine; jaundice may also occur. Acute hepatitis B lasts for several weeks and then subsides. Acute hepatitis B infection may be asymptomatic. CHB may also be asymptomatic, or, if left untreated, may be associated with chronic inflammation of the liver (chronic hepatitis), which can lead to cirrhosis. CHB increases the likelihood of developing hepatocellular carcinoma (HCC; liver cancer). In Europe, approximately 50% of hepatocellular carcinomas are associated with either hepatitis B or hepatitis C (caused by the hepatitis C virus).

[0038] HBV infection can occur through exposure to infected blood or other bodily fluids (including saliva, semen, and vaginal fluid). Most infections worldwide are related to mother-to-child transmission around birth (the perinatal period).

[0039] HBV infection lasting longer than six months is generally considered chronic. The development of chronic hepatobiliary (CHB) is associated with earlier age of HBV infection, with approximately 90% of those infected during the perinatal period later developing CHB. Infection after age 5 is associated with a risk of less than 10% developing CHB. Although CHB is often asymptomatic, approximately 25% of CHB cases eventually develop into cirrhosis and liver cancer.

[0040] HBV and HDV Hepatitis D virus (HDV) infection can only occur concurrently with or pre-existing HBV infection. This is because, in order to be infectious, the HDV viral envelope needs to include surface antigen proteins from HBV, among which the presence of HBsAg-1 is essential for HDV entry into cells.

[0041] Co-infection with HBV and HDV leads to a worse prognosis than HBV infection alone. In acute hepatitis B, there is a greater likelihood of developing liver failure with co-existing HDV infection. In chronic hepatitis B (CHB), cirrhosis progresses more rapidly, and the risk of developing liver cancer is increased.

[0042] Current treatment of CHB For a condition to be considered a “functional cure” for CHB, treatment should show a sustained or significant decrease in detectable serum HBsAg levels over time, such as a 1-log decrease in HBsAg levels from baseline at 24 weeks.

[0043] Currently, there are no commercially available vaccines that provide effective treatment for CHB. This is not to be bound by any theoretical framework, but rather is believed to be due to the severe dysfunction and low frequency of T and B cells targeting the S-protein in CHB, caused by HBV producing excessive amounts of S-containing subviral particles, leading to T and B cell exhaustion. Current treatments are based on long, often lifelong, courses of antiviral drugs such as tenofovir and entecavir. The costs associated with these drugs are a barrier to treatment in low-income settings.

[0044] In the presence of hepatitis B, treatment options for chronic hepatitis D include bulevirtide (Hepcludex; MyrB; Myrcludex-B), a 47-amino acid recombinant peptide derived from HBV PreS1 that acts as a competitive inhibitor by binding to the same cell surface receptor (sodium / bile acid cotransporter NTCP) used by both HBV and HDV to enter cells.

[0045] Current HBV vaccines Prophylactic HBV vaccines are usually based on recombinant hepatitis B virus surface antigen (HBsAg-S) rather than HBsAg-M or HBsAg-L, and therefore they lack PreS1 and / or PreS2.

[0046] Currently available prophylactic HBV vaccines have no therapeutic effect on individuals with chronic HBV infection, and therefore cannot control chronic HBV infection. Many T-cell inducible vaccines against HBV are under development with the aim of providing a treatment for HBV. See the review by Meng et al. (2020).

[0047] HepTcell from Altimmune TM HepTcell is a fully synthesized peptide product based on nine 32-40mer peptides derived from T-cell epitopes of the HBV protein. In a Phase I clinical trial, HepTcell peptides were administered as adjunctive therapy to entecavir or tenofovir in HBeAg-negative CHB patients, with and without adjuvant. TM It is generally well tolerated and, when delivered with adjuvant, delivers a T-cell response against HBV antigen, although the results do not mention the effect on HBsAg levels (Meng et al., 2020).

[0048] INO-1800 from Inovio is a DNA plasmid-based immunotherapy encoding hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg). In a Phase I clinical trial, it was delivered via electroporation to 90 CHB patients treated with nucleoside (nucleotide) analogues, but results have not yet been published (Meng et al., 2020).

[0049] GS-4774, from Globelmmune / Gilead, is a yeast-based vaccine engineered to express a chimera of HBV-encoded oncogene X protein (HBx), HBV surface protein (HBsAg), and HBV core protein (HBcAg). In a phase II clinical trial, it was administered in combination with the antiviral drug tenofovir disoproxil fumarate (TDF) to patients with chronic hepatitis B (CHB). The virus was found to be safe and well-tolerated, but it did not produce a significant reduction in HBsAg levels even when combined with the antiviral agent (Boni et al., 2019).

[0050] TG1050 from Transgene is a vaccine based on human adenovirus serotype 5 that expresses HBV polymerase as well as core and surface antigen domains. In a phase I clinical trial of CHB patients undergoing nucleoside (nucleotide) analogue NUC therapy, TG1050 was well tolerated but resulted in only a slight decrease in HBsAg levels (Zoulim et al., 2019).

[0051] WO2018 / 189522A1 describes a ChAdOx adenovirus vector HBV vaccine that encodes an HBV core, a modified HBV polymerase, and an HBV surface antigen (HBsAg) from an expression cassette, wherein the intergenic sequences are arranged to result in the expression of at least HBsAg as a protein separate from the HBV core and the modified HBV polymerase.

[0052] Adenovirus vector vaccine Adenoviruses (Ad) are non-enveloped, double-stranded DNA viruses with a genome of approximately 36 kilobases (kb). There are over 60 human adenovirus serotypes, grouped into classes A through G. Each group includes numerous adenovirus serotypes; for example, subgroup class C includes Ad5 and Ad2. Ad5 is the most widely studied serotype and the most widely used platform in oncolytic virus development. In oncolytic virus development, the goal is to target specific tissues and thus alter tropism. A major problem with the use of some adenovirus serotypes (including Ad5) in clinical settings is pre-existing immunity in humans. Some scientists have attempted to overcome pre-existing immunity by using adenoviruses first isolated from great apes (such as gorillas and chimpanzees). Examples of such vectors already used in clinical studies include ChAd3, ChAd155, and ChAdOx1. Such adenoviruses are expected to have lower seroprevalence in humans. Nevertheless, humans often have pre-existing immunity to such viruses, and of course, expecting more than one administration of the vector would raise concerns about pre-existing immunity.

[0053] Adenoviruses are typically 70–90 nm in size and have an icosahedral capsid shape. The capsid structure (also known as 'capsid proteins') comprises three major protein types: hexagonal proteins, fibrils, and penton bases. Additional minor proteins are present in the outer capsid, including VI, VIII, IX, IIIa, and IVa2 (also known as pVI, pVIII, pIX, pIIIa, and pIVa2). Hexagonal proteins are the major component of the adenoviral capsid, accounting for over 83% of the capsid proteins (Rux et al., 2003). Modifications to hexagonal proteins, including exchanges of hypervariable regions (HVRs) from different serotypes, have been shown to allow evasion of pre-existing neutralizing antibodies in some cases (Roberts et al., 2006). Adenoviral hexagonal proteins possess seven HVR domains, HVR1 through HVR7. Suitablely, any HVR domain can be engineered, for example, for inserting or fusing exogenous sequences, such as peptides. The selection of hypervariable regions to be engineered can be determined based on technical requirements; however, for example, HVR1, HVR2, or HVR5 can also be suitable as insertion or fusion sites.

[0054] Ad vectors (Ad) are commonly used in gene therapy, particularly as gene delivery vectors, because they have the ability to contain additional gene sequences. More than 2,000 gene therapy trials have been conducted using Ad. Adenoviral vectors allow the delivery of their carried transgenes into the host cell nucleus without integrating viral DNA into the host chromosome. When used as gene therapy vectors, Ad insert sequences are large, with usable capacities ranging from 8 to 36 kb.

[0055] Furthermore, due to its ability to induce both innate and adaptive immune responses, and its capacity to induce strong antigen-specific B-cell and T-cell immune responses, Ad has become a promising vaccine delivery medium. Adenovirus vectors are highly immunogenic and effective in antigen delivery.

[0056] Conventional adenovirus vector vaccines are among the most effective inducers of vaccine-specific T-cell immunity (especially CD8+ T cells) in humans, but humoral immunity is modest compared to other vaccine technologies, including recombinant protein / VLP and mRNA.

[0057] Several adenovirus vector-based vaccine candidates have been developed and further investigated in clinical trials; however, many of these have been unsuccessful. Merck's HIV-1 vaccine based on human adenovirus serotype 5 (Ad5) induced a CD8+ T-cell response but failed to prevent HIV infection. More recently, the SARS-CoV-2 pandemic and the use of adenovirus vaccines from Johnson & Johnson and Astra Zeneca (both expressing the "S" protein from SARS-CoV-2 in an adenovirus vector) have demonstrated the efficacy of adenovirus vector vaccines.

[0058] However, a major obstacle to the continued success of adenovirus-based vectors in human and animal treatments is the neutralization of the vector by adenovirus-specific antibodies. Natural adenovirus infection is high in both human and animal populations, and therefore the adaptive immune system can recognize and respond to the presence of adenovirus vectors by secreting neutralizing antibodies (NABs). Similarly, the innate immune system can also be responsible for assisting in the response to adenovirus vectors. For example, it is estimated that 50% to 90% of the adult population has pre-existing immunity to Ad5.

[0059] In our earlier applications PCT / GB2020 / 052774 (published as WO2021 / 084282A1) and PCT / GB2022 / 051137 (published as WO2022 / 234276A1) (both applications are incorporated herein by reference), we have demonstrated that the surface of an adenovirus vector can be decorated with an antigen of interest using a specific binding peptide pair, which, in addition to presenting the antigen to the immune system, allows the adenovirus vector to be shielded from anti-vector antibodies.

[0060] Adenoviruses can be replication-defective: certain genes are deleted from the genome to ensure that the adenovirus can no longer replicate when used as a therapeutic agent. For vaccination purposes, these vectors typically have deletions in the E1 gene (making the vector replication defective) and / or in the E3 gene (increasing the capacity for insert size). Other vectors can be generated by deleting a set of genes from the genome and are within the technical capabilities of those working with adenoviruses. This is an advantage for use in vaccines, where the purpose of the adenoviral vector is to present the antigen to the immune system in a highly immunogenic form while limiting cytotoxicity.

[0061] Adenovirus infectivity in cells expressing Coxsackievirus and its adenovirus receptor (CAR) is mediated by fibrin. An example of a cell line expressing the CAR receptor is the HEK293 cell. Fibrin binds to the CAR receptor on the cell surface, and this mediates the initial viral attachment. However, recent studies have shown that, instead of fibrin-mediated adenovirus entry, coagulation factor X (FX), present in human serum, can bind to hexaplex proteins of some adenovirus serotypes to facilitate viral entry into some cell types. An example of a cell line infected via hexaplex protein-mediated infection is SKOV3. It is believed that FX-mediated infection via adenovirus hexaplex proteins can enhance hepatic tropism of the adenovirus vector in vivo. Modifications to hexaplex proteins, such as the insertion of DogTags and conjugation to antigens, reduce hexaplex protein-mediated infectivity in cells. This is a desirable effect because the natural tropism of adenovirus can cause hepatotoxicity in patients when administered intravenously at very high doses. Reducing hexaplex protein-mediated infectivity to reduce hepatotoxicity would be beneficial in this invention.

[0062] The adenoviral hexagonal protein capsid protein is approximately 100 kDa in size, with 720 monomers per viral particle. These hexagonal protein monomers organize into trimers, resulting in 12 trimers on each of the 20 faces, leading to 240 trimers per viral particle. The hexagonal protein sequence contains hypervariable regions (HVRs) corresponding to loops on the viral outer surface, thus covering almost the entire surface of the virus. Each monomer has seven HVRs, designated HVR1-HVR7, which are serotype-specific. Because the loops are located on the outer surface of the virus, the hexagonal protein loops are major antigen recognition sites, i.e., targets of the host immune response. The lengths of the hexagonal proteins vary; for example, Ad2 is the longest known hexagonal protein, with a length of 968 amino acids (UniProt ID: P03277). Ad5, the most commonly used adenovirus for gene therapy, has a hexagonal protein of 952 amino acids in length (UniProt ID: P04133). Modifying hexagonal proteins containing serotype-specific epitopes (HVRs) appears to be a promising approach to overcoming host neutralization responses. Any type of HVR can be modified.

[0063] The adenovirus pIX protein is a minor capsid protein with a size of approximately 14.3 kDa. Each viral particle contains approximately 240 pIX monomers. The function of the pIX protein is to stabilize hexagonal proteins on the viral surface. The C-terminus of the pIX protein is exposed on the viral surface and is therefore an ideal site for the fusion of small and large peptides. Ad5 pIX has two domains linked by a flexible linker. The Ad5 pIX protein is 196 amino acids long (UniProt ID: Q2KS03).

[0064] In this disclosure, adenoviruses of genotypes Ad5 (serotype C, GenBank accession number AC_000008) and ChAd63 (serotype E, GenBank accession number CS479277) have been used as proof of concept. It should be understood that any other genotype of adenovirus or recombinant adenovirus may be used appropriately.

[0065] Adenoviral nucleic acids or peptides may have a sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with the wild-type sequence. Variants and derivatives of adenoviral peptides may contain amino acid sequences that are at least 90% or 95% similar to the wild-type sequence. Therefore, homologs of these entities may have at least 60% homology with them, or at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with them.

[0066] Modification of adenovirus capsid proteins can be genetic or non-genetic, including chemical. Genetic modification of capsid proteins can be achieved by incorporating antigens into the capsid. Alternatively, the viral particle surface can be directly modified. Modification of all three major capsid proteins has been previously demonstrated. However, the results of these modifications have been mixed, and significant obstacles remain regarding the insertion size offered by the most promising methods, particularly with respect to hexagonal proteins.

[0067] As used herein, “at least one modification” or “at least one modified capsid protein” means the insertion of a first peptide-coupler into an adenoviral capsid protein using any suitable means. For example, the first peptide-coupler may be inserted into the hexa-adjacent protein HVR loop of an adenovirus or fused to the adenoviral pIX minor capsid protein. Such modification can be performed genetically, for example, by gene fusion, or chemically.

[0068] Heteropeptide-binding coupler pairs Based on bacterial attachment proteins, the use of heteropeptide-binding couples (e.g., SpyCatcher and SpyTag (WO2011 / 098772)) has been established as a technique for the irreversible and spontaneous conjugation of recombinant proteins, etc., under physiological conditions. (Streptococcus pyogenes) Streptococcus pyogenesThe fibronectin-binding protein FbaB contains the CnaB2 adhesion protein domain. CnaB2 is stabilized by the spontaneous formation of isopeptide bonds through the spontaneous reaction of the Lys and Asp side chains. CnaB2 has been split into a 13-residue SpyTag peptide and a 116-residue SpyCatcher protein, each of which can be fused to two entities desired for binding via isopeptide bonds (Zakeri et al., 2012). Biological conjugation between entities that is impossible through genetic fusion between proteins can be achieved using peptide binding pairs, including for vaccine development (Brune et al., 2016; Thrane et al., 2016). Various Cater and Tag pairs are now available, some based on modifications of SpyCatcher and SpyTag, while others are based on similar chemistry from alternative bacterial proteins.

[0069] Proteins capable of spontaneously forming isopeptide bonds (so-called "isopeptide proteins") have been advantageously used to develop peptide-partner pairs (i.e., two-part linkers) that covalently bind to each other and provide irreversible interactions (see, for example, WO2011 / 098772 and WO2016 / 193746, both incorporated herein by reference, and WO2018 / 189517, WO2018 / 197854, WO2020 / 183198 and WO2022 / 214795, all incorporated herein by reference). In this regard, proteins capable of spontaneously forming isopeptide bonds can be expressed as separate fragments to provide a first peptide-partner and a second peptide-binding partner as a peptide-binding partner of the first peptide-partner, wherein these two fragments are covalently reconstructed via isopeptide bond formation. This covalent reconstruction links molecules or components fused to the second peptide-partner and the essential first peptide-partner. The isopeptide bonds formed by peptide couples are stable under conditions where non-covalent interactions would rapidly dissociate (e.g., long periods of time, such as weeks, high temperatures, at least 95°C, high forces, or harsh chemical treatments, such as pH 2–11, organic solvents, detergents, or denaturants).

[0070] Isopeptide bonds are amide bonds formed between a carboxyl / carboxamide group and an amino group, wherein at least one of the carboxyl or amino groups is outside the protein backbone (the protein's scaffold). Such bonds are chemically irreversible under typical biological conditions and are resistant to most proteases. Because isopeptide bonds are inherently covalent, they lead to some of the strongest measured protein-protein interactions.

[0071] In short, a two-part connector, namely a peptide partner pair (so-called peptide tag / binding partner or catcher pair), can be derived from a protein (isopeptide protein) capable of spontaneously forming isopeptide bonds, wherein the protein's domains are individually expressed to produce a peptide "tag" containing one of the residues involved in the isopeptide bond (e.g., aspartic acid or asparagine, or lysine), and a peptide partner or peptide binding partner (or "catcher") containing other residues involved in the isopeptide bond (e.g., lysine, or aspartic acid or asparagine) and at least one other residue required for the formation of the isopeptide bond (e.g., glutamate). Mixing the peptide tag and the binding / catcher partner results in the spontaneous formation of isopeptide bonds between the tag and the binding partner. Therefore, by incorporating the peptide tag and binding partner separately into different molecules or components, such as proteins, the molecules or components can be covalently linked by the isopeptide bonds formed between the peptide tag and the binding partner; that is, a connector is formed between the molecules or components incorporating the peptide tag and the binding partner.

[0072] The spontaneous formation of isopeptide bonds can be isolated and does not require the addition of any other entity. For some peptide tags and binding / catching couples, the presence of a third entity or auxiliary entity (such as a ligase) may be required to generate isopeptide bonds.

[0073] Variants, derivatives, and modifications of binding pairs can be made by any suitable method. Variants, derivatives, and functionally operable modifications can include the addition, substitution, alteration, or deletion of amino acids that maintain the same function in terms of their ability to form isopeptide bonds with the associated binding partner.

[0074] For some binding pairs, mediation by a third entity such as an enzyme is required. For example, SnoopLigase can be used to mediate bond formation between SnoopTagIR / SnoopTag and DogTag (Buldun et al., incorporated herein by reference). Therefore, pairing may require the assistance of enzymes such as ligases.

[0075] It should be understood that, as used herein, either the first peptide partner or the second peptide partner can be a peptide “tag”, while the other is a “binding partner / catcher”.

[0076] Appropriately, the first and second peptide wands form a peptide wand pair called SpyTag / SpyCatcher. Appropriately, the SpyCatcher component is DeltaN1 (ΔN1) SpyCatcher (as described in Li et al., 2014), which has a 23-amino acid truncation at the N-terminus compared to “SpyCatcher”.

[0077] In other embodiments, the first and second peptide conjugates form a peptide conjugate pair that is a mutant form of Spytag / SpyCatcher, exhibiting an increased reaction rate for isopeptide bond formation, such as those described, for example, in WO2018 / 197854 and Keeble et al. (2019). In some embodiments, these mutant forms can be used to link large proteins (e.g., >50 kDa or >100 kDa) and / or may be useful when slow reactions or steric hindrance may be a problem.

[0078] In some embodiments, the first and second peptide wands form a peptide wand pair, which is a modified form of RrgACatcher / RrgATag, referred to as DogCatcher / DogTag. These subsequent entities are described in Keeble et al. (2022) and WO2022 / 214795, both of which are incorporated herein by reference.

[0079] In other embodiments, the isopeptide protein that forms a peptide-couple pair may include SnoopTag / SnoopCatcher, such as that described in WO 2016 / 193746.

[0080] In some implementations, one or both isopeptide proteins that form peptide-couple pairs may have N-terminal or C-terminal truncation while still retaining the reactivity of the isopeptide bond.

[0081] SdyTag and SdyCatcher are constructed based on the native CnaB (CnaB) domain of a related fibronectin-binding protein from *Streptococcus dysgalactiae*. In some embodiments, the isopeptide proteins forming peptide-coupled pairs are SdyTag and SdyCatcher, or are based on modifications to these peptide-coupled pairs. Known modifications to SdyCatcher include those forming QueenCatcher, Mooncake, and Katl. Such modified forms of SdyCatcher can be paired with pre-existing or modified tags, including but not limited to SdyTag, SnoopTag, SpyTag, RumTag, RumTrunkTag, Clib9, PhoTag, EntTag, or BacTag. Such modifications are described in WO2021 / 224451, which is incorporated herein by reference.

[0082] Exemplary first and second peptide couples (peptide tag / binding couples; reaction pairs) are described in Table 1. This list is not exhaustive, and other peptide couples may be applicable for use in this invention. Table 1: Exemplary isopeptide binding pairs These entities are described in, for example, WO2011 / 098772, WO2016 / 193746, WO2018 / 197854, WO2018 / 189517, WO2020 / 183198, WO2022 / 214795 or Li et al. (2014), all of which are incorporated herein by reference.

[0083] Table 2: Exemplary amino acid sequences of various isopeptide conjugates Variants, derivatives, and modifications of binding pairs can be made by any suitable method. Variants, derivatives, and functionally operable modifications can include the addition, substitution, alteration, or deletion of amino acids that maintain the same function in terms of their ability to form isopeptide bonds with the associated binding partner.

[0084] Peptide pairs can be defined by a reference sequence. The sequence can be identical to the sequence listed for a peptide partner. A peptide partner can have a sequence with at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with the listed sequence. Variants and derivatives of the peptide partner can contain amino acid sequences that are at least 90% or 95% similar to the listed sequence. Therefore, homologs of these entities can have at least 60% homology with it, and at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with it.

[0085] For some binding pairs, a third entity, such as an enzyme, is required for mediation. For example, SnoopLigase can be used to mediate bond formation between SnoopTagJr and DogTag. Therefore, pairing may require the assistance of enzymes such as ligases.

[0086] antigen As used herein, an antigen refers to any molecule capable of inducing an immune response, such as a polypeptide or its immunogenic fragment. An antigen can be an allergen antigen, viral antigen, bacterial antigen, parasitic antigen, or fungal antigen. As used herein, “antigen” includes peptides and epitopes, their variants, and derivatives. As used herein, an antigen can refer to a full-length polypeptide expressed from a single gene or open reading frame, or it can refer to a truncated variant of that polypeptide. Truncation can be N- or C-terminal truncation, or, compared to a full-length polypeptide, an antigen can have both N- and C-terminal truncations. Furthermore, an antigen can have mutations compared to a wild-type (natural) protein, such mutations being, for example, the addition, deletion, rearrangement, or substitution of one or more amino acids.

[0087] As used in this article, B-cell epitopes are portions of antigens that are specifically recognized by antibodies and B cells of the immune system. As used in this article, T-cell epitopes are portions of antigens that are recognized by T cells.

[0088] Antigens can be viral antigens, but more specifically HBV antigens. HBV antigens include HBsAg, including any of the L, M, and S variants, as well as the PreS1 and / or PreS2 domains in the absence of the S domain. Other HBV antigens include the core protein. HBV antigens can be derived from any strain of HBV and can be mutated or truncated compared to wild-type antigens.

[0089] Adenovirus vaccines with decorative antigens The disclosure described herein allows for the simultaneous expression of target antigens for humoral immunity on the surface of the adenovirus capsid and the vector-encoded antigen, generating T-cell responses equivalent to those of conventional adenovirus vectors. In proof-of-concept studies, adenovirus vaccines derived from the receptor-binding domain (RBD) of SARS-CoV-2 S were demonstrated to produce ~50-fold higher SARS-CoV-2 neutralizing titers compared to conventional adenovirus vaccines encoding S (Dicks et al., 2022).

[0090] vaccine A vaccine is a preparation containing a fragment or the whole entity against which it can elicit an immune response. It is an entity capable of inducing an immune response, such as a protein, peptide, lipoprotein, glycoprotein, polysaccharide, or fragment thereof. For example, a vaccine may contain a microorganism or a portion thereof capable of inducing an immune response against said microorganism. A vaccine containing an immunogenic adenovirus vector according to the invention can be used against any pathogen against which an antigen, either exhibited or genetically encoded, is used to induce an immune response against that antigen.

[0091] Such vaccine compositions (or other immunogenic compositions) are formulated in a suitable delivery medium. Typically, the dosage of the immunogenic composition is within the range defined for a therapeutic composition. Optionally, the adenovirus vector or vaccine composition of this disclosure may be formulated to contain other components, including, for example, adjuvants, stabilizers, pH adjusters, preservatives, etc. Such components are well known to those skilled in the art of vaccines. Examples of suitable adjuvants include, but are not limited to, liposomes, alum, monophosphoryl lipid A, saponins such as Qs21, and any bioactive factors, such as cytokines, interleukins, chemokines, and optimal combinations thereof.

[0092] The adenovirus vector disclosed herein can be administered to target cells via intravenous, intradermal, intraarticular, intraperitoneal, intralesional, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, rectal, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, intracystic, mucosal, intraperitoneal, oral, local and / or aerosol, injection, infusion, continuous infusion, direct or via catheter and / or irrigation for local perfusion bath. Typically, the vaccine of this invention is administered intramuscularly.

[0093] The adenovirus vectors or vaccines disclosed herein may be used to treat or prevent HBV infection, or a combination of HBV and HDV (HBV infection and / or HDV infection).

[0094] Pharmaceutical Compositions and Uses The compositions disclosed herein may be incorporated into vaccine or therapeutic compositions. Suitable of the invention, the vaccine or immunogenic composition will contain an immunogenic dose of the particles of the present invention.

[0095] The pharmaceutical composition may comprise particles or compositions according to the invention, providing a pharmaceutically acceptable carrier. Suitable carriers are well known to those skilled in the art. In one embodiment, the pharmaceutical composition comprises a buffer, excipient, or carrier. Suitably, the pharmaceutical composition may comprise suitable excipients and formulations to maintain the stability of the composition. Suitably, the formulation may comprise an adjuvant. In one embodiment, the formulation may comprise AddaVax. TMOr similar squalene-based oil-in-water nanoemulsions with formulations similar to MF59®. Other suitable adjuvants include liposome-based adjuvants such as Matrix M and AS01. Other suitable adjuvants include aluminum-based formulations such as Alhydrogel®. In one embodiment, the formulation may contain EDTA, for example, at a concentration of 5 mM. Suitable excipients or formulations may depend on the nature of the particulate or immunogenic composition; for example, the choice of expression system may affect the stability, glycosylation, or folding of the protein in the composition, which in turn may affect the optimal formulation of the composition. Methods for determining suitable excipients, formulations, or adjuvants will be known to those skilled in the art.

[0096] This public information This disclosure provides candidate therapeutic HBV vectors and vaccines that (i) display the PreS1 antigen from HBV-1 on the adenovirus surface and optionally (ii) encode an antigen from HBV. PreS1 is the receptor-binding domain required for HBV host cell entry and is a key target for HBV neutralizing antibodies (NAbs). Current prophylactic HBV vaccines do not incorporate PreS1 because they are based on HBV HBsAg-S (a small subunit of the HBV surface antigen). PreS1 display on VLPs is known to generate effective NAb responses in mouse models of chronic HBV infection (Wang et al., 2002). Encoded antigens from HBV generate effective CD8+ and CD4+ T cell responses.

[0097] The candidate therapeutic HBV vaccine disclosed in this paper can induce robust IgG antibody responses against PreS1 and strong T cell responses against two full-length HBV proteins (HBsAg-L (L) and core (C)) in mice.

[0098] This disclosure demonstrates that the technology enables flexibility in the design of candidate vaccines; the magnitude of the immune response is independent of the encoded antigen orientation and PreS1 capsid coverage.

[0099] The disclosed transgenic construct design of HBV L_P2A_C with PreS1 capsid decoration via isopeptide binding to a partner body achieves robust anti-PreS1 IgG titers and strong T-cell responses against L and C antigens. Robust PreS1 antibody responses (such as those achieved herein) are expected to effectively neutralize HBV infection. Studies testing the ability of candidate vaccines to induce NAbs against different HBV strains are ongoing.

[0100] Different HBV strains can be incorporated into vectors and vaccines to increase the breadth of immunity provided and / or to target specific genotypes.

[0101] In view of this disclosure, various other aspects and embodiments of the invention will be apparent to those skilled in the art.

[0102] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0103] As used herein, “and / or” is considered to be a specific disclosure of each of two specified features or components, with or without the other. For example, “A and / or B” is considered to be a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were listed separately herein.

[0104] Unless the context otherwise indicates, the description and definition of the features listed above are not limited to any particular aspect or embodiment of the invention, and are equally applicable to all aspects and embodiments described.

[0105] Those skilled in the art will further understand that, although the invention has been described by way of example with reference to several embodiments, the invention is not limited to the disclosed embodiments, and alternative embodiments may be constructed without departing from the scope of the invention as defined in the appended claims.

[0106] As used herein to describe a polynucleotide, “recombinant” means a genomic, cDNA, semi-synthetic, or synthetically derived polynucleotide that, by virtue of its origin or manipulation, is wholly or partially unrelated to the polynucleotide associated with it in nature; and / or (2) linked to a polynucleotide different from its naturally occurring linker. The term “recombinant” as used with respect to proteins or polypeptides refers to a polypeptide produced by expressing a recombinant polynucleotide.

[0107] The term “comprising” encompasses both “including” and “consisting of”. For example, a composition that “comprising” X may consist of only X, or it may include other things, such as X+Y.

[0108] The term “about” for the numerical value x is optional and means, for example, x ± 10%.

[0109] Unless otherwise specified, a process or method that includes a series of steps does not require any particular order in which the steps are performed; any suitable order can be used, including simultaneously. For example, a process or method that includes the step of mixing two or more components does not require any particular mixing order. Therefore, the components can be mixed in any order. In the presence of three components, two components can be combined with each other, and then that combination can be combined with a third component, and so on.

[0110] Sequence identity between peptide sequences is preferably determined using the pairwise alignment algorithm of the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch 1970), with default parameters (e.g., vacancy opening penalty = 10.0, vacancy extension penalty = 0.5, using the EBLOSUM62 scoring matrix). This algorithm is conveniently implemented in the needle tool within the EMBOSS packages (Rice, Longden, and Bleasby 2000). Unless otherwise specified, sequence identity should be calculated over the entire length of the peptide sequence.

[0111] Any polypeptide sequence disclosed herein may have a sequence identity of at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% with its wild-type sequence. Variants and derivatives of the polypeptide may contain amino acid sequences that are at least 90% or 95% similar to the wild-type sequence. Thus, homologs of these entities may have at least 60% homology with it, or at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with it.

[0112] Example Example 1: PreS1 capsid demonstrates robust humoral immunity A schematic diagram of an adenovirus construct with an isopeptide tag fused to a capsid protein is shown in Figure 1A The Chinese side indicated that... Figure 1B A similar construct was shown, which has an exemplary GFP gene inserted into the adenovirus genome and HBV PreS1 conjugated to the surface of the adenovirus particle via a covalent bond between a tag (first peptide conjugate) and a catcher (second peptide conjugate).

[0113] A proof-of-concept replication-deficient (E1 / E3 deletion) adenovirus vector was constructed based on the Ad5 serotype, which has the ability to... GalKRecombinant genetically engineered insertions of the DogTag sequence into the surface loop of the hexagonal protein hypervariable 5 (HVR5) were performed (Dicks et al., 2022). For the insertion at HVR5, residues 269-281 of the native Ad5 hexagonal protein were deleted and replaced with a DogTag flanked by a GSGGSG flexible linker (DIPATYEFTDGKHYITNEPIPPK; SEQ ID NO: 22) (the amino acid sequence of this hexagonal protein construct is SEQ ID NO: 55; the nucleotide sequence is SEQ ID NO: 56). Similarly, another adenovirus genotype or another HVR loop, such as HVR1 or HVR2, can be chosen as the insertion site. The enhanced GFP gene was inserted into the E1 locus of the adenovirus genome, downstream of the early cytomegalovirus promoter (Dicks et al., 2022).

[0114] To generate the DogCatcher-PreS1 protein, the nucleotide sequence encoding the PreS1 region of HBV genotype Dayw (NC_003977) minus the starting methionine residue (equivalent to amino acids 2-108 of the HBV-1 antigen in Genbank NC_003977; amino acid sequence SEQ ID NO: 48) was cloned into the expression vector pET45(+)(EMD Millipore) for use in BL2L(DE3) Escherichia coli (…). Escherichia coli The protein was generated in (NEB). A variant of the DogCatcher nucleotide sequence from Keeble et al. (2022) (encoding the DogCatcher VL amino acid sequence SEQ ID NO: 10) was fused to the 5' end of the PreS1 gene, encoding a GSGGSGGS adapter between the DogCatcher and PreS1 sequences (DogCatcher-Adapter-PreS1 construct amino acid sequence SEQ ID NO: 57, nucleotide sequence SEQ ID NO: 58). The recombinant protein was purified using Ni-NTA affinity resin (Qiagen) according to standard protocol, dialyzed into PBS, and stored at -80°C.

[0115] Under conjugation-allowed conditions, adenovirus particles tagged with DogTag were mixed with DogCatcher-PreS1 protein. SDS-PAGE was used to determine the successful conjugation of PreS1 to the DogTag on the adenovirus particles via DogCatcher, and to determine the conjugation efficiency of DogCatcher-PreS1 at each concentration. Figure 1CThe coupling efficiency ranges from approximately 25% (0.3 μM DogCatcher-PreS1) to >90% (5 μM DogCatcher-PreS1).

[0116] Successful expression of GFP from the adenovirus transgene was confirmed by fluorescence microscopy. The in vitro infectivity of the DogCatcher-PreS1-decorated adenovirus vector in 293A cells was determined. Figure 1D ); with undecorated carrier (0 µM DGogCatcher-PreS1 (“Ad-DT only”), Figure 1D Compared to 0.7 µM conjugated DogCatcher-PreS1 (“DC-PreS1”) or lower conjugated carrier concentrations, infectivity remained unchanged.

[0117] Example 2: PreS1 capsid display using mammalian-expressed PreS1 In this embodiment, the same proof-of-concept Ad5 adenovirus vector as in Example 1 is used, which has a DogTag genetically inserted into the hexagonal protein HVR5 and enhanced GFP inserted into the adenovirus genome at the E1 locus.

[0118] In this embodiment and all subsequent embodiments, the DogCatcher-PreS1 protein is expressed in mammalian cells, although any suitable protein expression system can be used. To generate the DogCatcher-PreS1 protein, the nucleotide sequence encoding the HBV genotype Dayw PreS1 region (equivalent to HBV-L antigen amino acid 3-108, Genbank NC_003977) was cloned into the expression vector pcDNA3.4 for protein production in Chinese hamster ovary (CHO) cells. The starting methionine residue was removed compared to the native HBV genotype Dayw PreS1 region because this sequence was engineered to fuse with DogCatcher at its N-terminus, and the second native residue (glycine) was removed to avoid inhibiting protein production in mammalian cells, as it is myristylated in the native sequence. The DogCatcher nucleotide sequence (encoding the DogCatcherV1 amino acid sequence SEQ ID NO: 10) was genetically fused to the 5' end of the PreS1 gene, with a GSGGSGGS linker between the DogCatcher and PreS1 sequences (DogCatcher V1-linker-PreS1 amino acid sequence SEQ ID NO: 59; nucleotide sequence SEQ ID NO: 60). To facilitate secretion, the Igk-leader sequence METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 61) was introduced into the N-terminus of the fusion protein, and a C-terminal C-tag (EPEA; SEQ ID NO: 62) was added to enable affinity purification. DogCatcher-PreS1 was expressed in suspension ExpiCHO-S cells (Thermo Fisher); the protein was harvested from the culture supernatant, affinity purified using an AKTA chromatography system (GE Healthcare) with C-tag affinity resin (Thermo Fisher), and dialyzed into Tris buffered saline (TBS) at pH 7.4.

[0119] Under conjugation-allowed conditions, adenovirus particles tagged with DogTag were mixed with DogCatcher-PreS1 protein. SDS-PAGE was used to determine the successful conjugation of PreS1 with the DogTag on the adenovirus particles via DogCatcher at increasing concentrations. Figure 2A ), and determine the coupling efficiency of DogCatcher-PreS1 at each concentration ( Figure 2BThe conjugation efficiency ranged from approximately 20% (0.6 μM DogCatcher-PreS1) to >60% (DogCatcher-PreS1 concentrations from 3.5 μM to 5 μM). Successful expression of GFP from the adenovirus transgene was confirmed by fluorescence microscopy. The in vitro infectivity of the DogCatcher-PreS1-decorated adenovirus vector in 293A cells was determined. Figure 2C The infectivity of conjugated DogCatcher-PreS1 remained unchanged at all concentrations compared to the undecorated carrier.

[0120] Table 3: Mouse study design (homogeneous primary immunization - booster): The candidate vaccine based on this Ad5 serotype vector and unconjugated PreS1 were tested in mice using a homologous priming-boosting regimen. Table 3 shows the mouse immunization schedule. Mice were immunized on days 0 and 21 with unconjugated adenovirus expressing GFP, adenovirus expressing GFP and decorated with PreS1, or unconjugated DogCatcher-PreS1 recombinant protein.

[0121] Tail vein blood was collected on day 20 after initial immunization, and the study was terminated on day 35, with cardiac blood and spleen harvested. Ad(GFP): The effective PreS1 dose delivered by PreS1 is <0.2 μg.

[0122] result Serum IgG antibody response to PreS1 was measured by endpoint ELISA. Figure 1E The responses measured on day 20 after the initial immunization were compared with those measured on day 35 after the booster immunization. Data were analyzed in Prism v9. Normality of the data was checked using the Shapiro-Wilk test, and then analyzed by one-way ANOVA with post-test correction.

[0123] The response of CD8+ T cells to EGFP-encoded cells in the spleen was measured by overnight ex vivo IFNγ-ELISpot assay. Figure 1F ). 5x10 5 1 cell / well with 5 μg / ml EGFP 118-126 The peptides were incubated together. Spots were counted on an AID ELISpot reader v7 and analyzed in prism v9. The normality of the data was checked using the Shapiro-Wilk test and then analyzed by one-way ANOVA with post-test correction.

[0124] Conclusion: Immunization of mice with Ad modified with the PreS1 capsid generated a potent PreS1 IgG response, but not when mice were immunized with recombinant DogCatcher-PreS1 protein plus Alhydrogel® adjuvant. The PreS1-modified capsid did not suppress the splenic CD8+ T cell response against the encoded GFP compared to the unmodified vector. This is significant because inducing a potent CD8+ T cell response against the encoded transgenic antigen is a hallmark of adenovirus vector vaccines and is considered therapeutically beneficial in many cases, including for the treatment of chronic hepatitis B infection. Generally, techniques involving decorating or coating the surface of the adenovirus capsid (e.g., attaching polymers to achieve capsid shielding against unwanted interactants) tend to reduce vector infectivity and are therefore expected to significantly reduce CD8+ T cell immunogenicity against the encoded antigen.

[0125] Example 3: Optimization of anti-HBV PreS1 humoral immunity Using the adenovirus vectors as described in Examples 1 and 2, except that the GFP transgene is replaced with one of the following two coding cassettes: "HBV L" encodes a large HBsAg polypeptide (including PreS1, PreS2, and S proteins) from the HBV strain ayw genotype D (NC_003977), amino acid sequence SEQ ID NO: 45, and "HBV CS" encodes the HBV core (amino acid sequence SEQ ID NO: 50) and small HBsAg polypeptide (amino acid sequence SEQ ID NO: 47) also from the strain ayw genotype D (NC_003977), with an intercalated F2A (FMDV18 2A) cleavage sequence (amino acid sequence SEQ ID NO: 51) between the core and S. Both constructs also encode a shark invariant strand (Sli; SEQ ID NO: 63) at the N-terminus, including the shark invariant strand to enhance T cell responses. Figure 3A A schematic diagram of an adenovirus vector is shown, which has PreS1 covalently attached to the surface via a tag-catcher, and an HBV L or HBV CS transgenic cassette inserted into the adenovirus genome.

[0126] According to Example 2, the DogCatcher-PreS1 protein was expressed in mammalian cells.

[0127] Table 4: Mouse study design (homogeneous primary immunization - booster): The candidate vaccine was tested in mice using a homologous primitivism-boost regimen without adjuvant. Table 4 shows the immunization schedule for mice. Tail vein blood was collected on day 20 after primitivism, and the study was terminated on day 35, with heart blood and spleen harvested.

[0128] result For each vector in Table 4, the endpoint ELISA ( Figure 3B Serum IgG antibody response to PreS1 was measured. Responses measured after the initial immunization on D20 were compared with those measured after the booster immunization on D35. Data were analyzed in Prism v9. Normality of the data was checked using the Shapiro-Wilk test, and then analyzed by one-way ANOVA with post-test correction.

[0129] The neutralizing antibody response generated by inoculation with Ad(L), Ad(L):PreS1 and Ad(GFP) (according to Example 2) was measured on HepG2-NTCP (HBV-infectable) cells using an in vitro HBV neutralization assay. Figure 3C HBV virus expressing luciferase and serum from inoculated mice (1:250 dilution) were co-incubated with cells; neutralization is expressed as a reduction in luminescence signal (in relative light units, RLU) relative to the control (HBV virus expressing luciferase only). Figure 3C It can be seen that, compared with the control, vaccination with Ad(GFP) did not induce a reduction in luminescence / HBV neutralization, vaccination with Ad(L) induced only very limited HBV neutralization (HBV infection reduced by <4-fold), while vaccination with Ad(L):PreS1 provided strong neutralizing immunity (infection reduced by >80-fold).

[0130] Conclusion: Optimal PreS1 antibody response was achieved when PreS1 simultaneously displayed (:PreS1) and encoded (L). Crucially, PreS1 capsid display is essential for potent HBV neutralization.

[0131] Example 4: Demonstrating the strong efficacy of adenovirus vector encoding PreS1 and C and L in inducing anti-HBV T cell and antibody immunity. Effective combination The adenovirus vector Ad(L) with the coding cassette “HBV L” from Example 3 was used, along with two other vectors: the adenovirus vector Ad(CL) carrying cassette “HBV CL”, which encodes the HBV core protein (amino acid sequence SEQ ID NO: 45) upstream of the large HBsAg protein (amino acid sequence SEQ ID NO: 45), and the HBV core protein (amino acid sequence SEQ ID NO: 50) is separated from the large HBsAg protein (amino acid sequence SEQ ID NO: 45) by an F2A cleavage sequence (amino acid sequence SEQ ID NO: 51); and the adenovirus vector Ad(LC) carrying cassette “HBV LC”, which encodes the same protein, but has HBsAg-L upstream of the HBV core and also has an intermediate F2A sequence. Figure 4AA schematic diagram of these constructs is shown, in which PreS1 shows a surface decorated by covalent bonds between the tag (first peptide partner) and the catcher (second peptide partner).

[0132] All three vectors were decorated with DogCatcher-PreS1 at a density (“HC”) similar to that of the previous embodiments. In addition, the adenovirus vector Ad(L) (carrier “HBV L”) was subjected to a fourth treatment in which DogCatcher-PreS1 was conjugated at a lower density (“LC”).

[0133] LC = ~30% "Low" PreS1 capsid coverage (~215 copies / virus). HC = ~60% "High" PreS1 capsid coverage (~430 copies / virus).

[0134] Table 5: Mouse Study Design The candidate vaccine was tested in mice using a homologous primitivism-booster regimen (without adjuvant). Table 5 shows the immunization schedule for mice. Tail vein blood was collected on day 20 after primitivism, and the study was terminated on day 35, with heart blood and spleen harvested.

[0135] result The response of CD8+ T cells to gene-encoding substances in the spleen was measured by overnight in vitro IFNγ-ELISpot assay. 2.5 x 10⁻⁶ cells were used. 5 Cells / well with 5 μg / ml S 190-197 Peptide (CD8+ epitope) ( Figure 4B ),core 93-100 Peptide (CD8+ epitope) ( Figure 4C ) or core P13L Peptide (CD4+ epitope) ( Figure 4D The samples were incubated together. Spots were counted on an AID ELISpot reader v7 and analyzed in prism v9. The normality of the data was checked using the Shapiro-Wilk test and then analyzed by one-way ANOVA with post-test correction.

[0136] Serum IgG antibody response to PreS1 was measured by endpoint ELISA. Figure 4E The responses measured on day 20 (after the initial immunization) were compared with those measured on day 35 (after the booster immunization). Data were analyzed in Prism v9. The normality of the data was checked using the Shapiro-Wilk test, and then analyzed by one-way ANOVA with post-test corrections; no significant differences were found.

[0137] Using Example 3 / Figure 3CThe in vitro HBV neutralization assay described in [the text] measured the neutralizing antibody response induced by vaccination with Ad(LC):PreS1 and Ad(CL):PreS1 (both decorated with high capsid coverage PreS1) -- see [the text] Figure 4F Both vaccines induced potent neutralizing humoral immunity (>50-fold reduction in infection). A 4-fold serum dilution series from 1:250 to 1:256000 was also performed; the RLU signal decreased with increasing serum concentration, as shown in... Figure 4G middle.

[0138] Conclusions: Robust CD8+ and CD4+ T cell responses were obtained for both the L and C encoded antigens. The order of the encoded L and C antigens and the extent of PreS1 capsid coverage did not affect the strength of the T cell response. Robust anti-PreS1 IgG antibody titers were achieved. The order of the encoded L and C antigens and the extent of PreS1 capsid coverage did not affect the PreS1 ELISA titer. The order of the encoded L and C antigens had almost no effect on neutralizing titer; both Ad(LC):PreS1 and Ad(CL):PreS1 vaccines induced potent neutralizing immunity.

[0139] Example 5: Enhancing in vitro expression of HBV-L / S and HBV-C through construct design Numerous undecorated adenovirus vectors were generated by incorporating nucleotide sequences arranged to guide the cleavage between antigenic peptides, with the aim of producing significant expression of “unfused” HBV-L and HBV-C antigens. The constructs are similar to those used in Examples 1-4 and are summarized in Table 6 as follows, where core = HBV core, S = small HBsAg antigen, L = large HBsAg antigen, F2A = FMDV18 2A cleavage sequence, P2A = porcine cerebrovirus-1 2A cleavage sequence, IRES = cleavage sequence from the internal ribosome entry site of encephalomyocytovirus, Sli = shark invariant strand, and GSGGS, GSG, GGS are linkers.

[0140] Table 6: Adenovirus constructs 293A cells were infected with the vectors summarized in Table 6 at a MOI of 100 ifu for 16–18 hours, then harvested, lysed, and analyzed by Western blot. The antibodies used were mouse anti-HBsAg (Native antigen, clone 1837) at a dilution of 1:200 and mouse anti-core (SantaCruz, clone 10E11) at a dilution of 1:200, both of which were detected by goat anti-mouse alkaline phosphatase (Biorad, clone STAR117A) at a dilution of 1:1000. As controls, adenovirus hexagonal protein was detected by mouse anti-hexagonal protein (Invitrogen, clone 65H6) at a dilution of 1:500 and STAR117A at a dilution of 1:1000; β-actin was detected by rabbit anti-β-actin (Cell Signalling Technologies, clone D6A8) at a dilution of 1:500 and goat anti-rabbit alkaline phosphatase (Merck) at a dilution of 1:1000. See Western blot results for details. Figure 5 The detected proteins were labeled based on their predicted size. Different glycosylated forms of S are naturally produced, so double and quadruple bands were detected by staining S with S and L expression constructs.

[0141] Conclusion: Constructs D, E, G, and J produced significant expression of "unfused" HBV-L. Only construct D produced significant expression of "unfused" HBV-C. Therefore, P2A "cleavage" was superior to F2A, but less efficient than IRES, although downstream ORF expression was poor in IRES (constructor E).

[0142] Example 6: Compared to direct fusion, enhancing the resistance by including P2A or IRES sequences between ORFs. PreS1 antibody response Some vectors from Example 5 were selected for PreS1 decoration (conjugation) via DogTag-DogCatcher isopeptide binding. The vectors used are summarized in Table 7. Regarding the vectors in Example 5, these are equivalent to H (Ad(L-F2A-C)), J (Ad(C-F2A-L)), D (Ad(L-P2A-C)), E (Ad(L-IRES-C)), and F (Ad(LC)). Figure 6A A schematic diagram of a PreS1-conjugated vector is shown, illustrating the protein coupled to the vector surface via a tag-catcher isopeptide pair, and the encoded transgene.

[0143] Table 7: Mouse study design: Table 7 shows the immunization schedule for mice. Tail vein blood was collected on day 20 after primary immunization, and the study was terminated on day 35, with heart blood and spleen harvested.

[0144] Serum IgG antibody response to PreS1 was measured by endpoint ELISA. Figure 6B Data were analyzed in Prism v9. Normality of the data was checked using the Shapiro-Wilk test, followed by analysis using a one-way ANOVA with post-test correction. No significant differences were detected between vaccines at the pre-boost time point (day 20). At the post-boost time point (day 35), significant differences in endpoint titers were detected in serum from mice vaccinated with Ad(L-P2A-C):PreS1 and Ad(LC):PreS1, and Ad(L-IRES-C):PreS1 and Ad(LC):PreS1.

[0145] The T-cell response to gene encoding in the spleen was measured using an overnight in vitro IFNγ-ELISpot assay. 2.5 x 10⁻⁶ cells were used. 5 Cells / well with 5 μg / ml S 190-197 Peptide (CD8+ epitope; SEQ ID NO: 64) Figure 6C ) or core 93-100 Peptide (CD8+ epitope; SEQ ID NO: 65) Figure 6D ) or core P13L Peptide (CD4+ epitope; SEQ ID NO: 66) Figure 6E The samples were incubated together. Spots were counted on an AID ELISpot reader v7 and analyzed in prism v9. The normality of the data was checked using the Shapiro-Wilk test and then analyzed by one-way ANOVA with post-test correction.

[0146] Conclusion: P2A, IRES, and LC (without lysis) produced the highest median CD8+ response against HBsAg-L, but IRES produced the weakest CD8+ response against the HBV core, and LC (without lysis) produced a weak CD4+ response against the HBV core. Using the P2A sequence among ORFs produced robust CD8 T cell responses against both HBV-L and HBV-C antigens.

[0147] Example 7: PreS1 capsid on chimpanzee adenovirus serotype ChAd63 induces potent humoral immunity. An additional proof-of-concept replication-deficient (E1 / E3 deletion) adenovirus vector was constructed based on the ChAd63 chimpanzee adenovirus serotype, featuring a DogTag sequence genetically inserted into the surface loop of the hexagonal protein hypervariable 5 (HVR5) via GalK recombination engineering. For the insertion at HVR5, residues 256-264 of the native ChAd63 hexagonal protein were deleted and replaced with a DogTag (DIPATYEFTDGKHYITNEPIPPK; SEQ ID NO: 22) with a flexible linker flanked by GSGGSGGSGGSG (SEQ ID NO: 44).

[0148] The transgenic antigen (GFP or L-P2A-C as described above) was inserted into the ChAd63 adenovirus genome at the E1 locus, downstream of the immediate early cytomegalovirus promoter, and the vector was decorated with DogCatcher-PreS1 (SEQ ID NO:59). Figure 7A ).

[0149] The candidate vaccine based on this ChAd63 serotype vector was tested in mice using a homologous primate-boost regimen (without adjuvant). Table 8 shows the immunization schedule for mice. Tail vein blood was collected on day 20 after primate, and the study was terminated on day 35, with heart blood and spleen harvested. The vaccine tested was: undecorated ChAd63 expressing L and C (ChAd63(L-P2A-C)), in low-density (ChAd63(GFP):PreS1) vectors. 低 ) or high density (ChAd63(GFP):PreS1) 高 This displays PreS1 expressing GFP-encoded ChAd63 and expressing L and C antigens in a low-density (ChAd63(L-P2A-C):PreS1) format. 低 ) or high density (ChAd63(L-P2A-C):PreS1) 高 The ChAd63 of PreS1 is shown. The PreS1 capsid coverage in each group is shown in Table 8. The low and high densities in this example are equivalent to the high and low coverage (“HC” and “LC”) in Example 4.

[0150] Table 8: Mouse Study Design Serum IgG antibody response to PreS1 was measured by endpoint ELISA. Figure 7B The data were analyzed in Prism v9. The normality of the data was checked using the Shapiro-Wilk test, and then analyzed by one-way ANOVA with post-test correction.

[0151] The T-cell response to gene encoding in the spleen was measured using an overnight in vitro IFNγ-ELISpot assay. 2.5 x 10⁻⁶ cells were used. 5 Cells / well with 5 μg / ml S 190-197 Peptide (CD8+ epitope) ( Figure 7C ),core 93-100 Peptide (CD8+ epitope) ( Figure 7D ) or core P13L Peptide (CD4+ epitope) ( Figure 7E The samples were incubated together. Spots were counted on an AID ELISpot reader v7 and analyzed in prism v9. The normality of the data was checked using the Shapiro-Wilk test and then analyzed by one-way ANOVA with post-test correction.

[0152] Conclusion: PreS1 capsid decoration on ChAd63 induces a robust antibody response, with endpoint titers comparable in magnitude to those induced by Ad5. PreS1 antibody responses are comparable between vaccines decorated with low and high densities of PreS1, and between vectors encoding L-P2A-C and GFP. Compared to undecorated ChAd63 (L-P2A-C), PreS1-decorated vaccines induce >100-fold higher anti-PreS1 IgG titers after booster. PreS1-decorated vaccines encoding L and C antigens induce strong T-cell responses against both antigens.

[0153] sequence Hepatitis B virus (reference strain NC_003977 (ayw)) large HBsAg (HBsAg L) Amino acid sequence (SEQ ID NO: 45): MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQSGRQPTPLSPPLRNTHPQAMQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVLTTASPLSSIFSRIGDPALNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI HBsAg in hepatitis B virus (reference strain NC_003977 (ayw)) Amino acid sequence (SEQ ID NO: 46): MQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVLTTASPLSSIFSRIGDPALNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI Hepatitis B virus (reference strain NC_003977 (ayw)) small HBsAg (HBsAg S) Amino acid sequence (SEQ ID NO: 47): MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI Hepatitis B virus (reference strain NC_003977 (ayw)) PreS1 Amino acid sequence (SEQ ID NO: 48): MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQSGRQPTPLSPPLRNTHPQA Hepatitis B virus (reference strain NC_003977 (ayw)) PreS2 Amino acid sequence (SEQ ID NO: 49): MQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVLTTASPLSSIFSRIGDPALN Hepatitis B virus (reference strain NC_003977 (ayw)) core (C) Amino acid sequence (SEQ ID NO: 50): MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC F2A Amino acid sequence (SEQ ID NO: 51): VKQTLNFDLLKLAGDVESNPGP P2A Amino acid sequence (SEQ ID NO: 52): ATNFSLLKQAGDVEENPGP HBV-L-P2A-C Amino acid sequence (SEQ ID NO: 53): MSLLWGGVTVLAAMLIAGQVASVVFLVGSGGSGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQSGRQPTPLSPPLRNTHPQAMQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVLTTASPLSSIFSRIGDPALNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYIGSGATNFSLLKQAGDVEENPGPMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC HBV-L-P2A-C Nucleic acid sequence (SEQ ID NO: 54): Ad5-HVR5-DogTag hexagonal protein Amino acid sequence (SEQ ID NO: 55):MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFIPVDREDTAYSYKARFTLAVGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDEAATALEINLEEEDDDNEDEVDEQAEQQKTHVFGQAPYSGINITKEGIQIGVEGQTPKYADKTFQPEPQIGESQWYETEINHAAGRVLKKTTPMKPCYGSYAKPTNENGQGILVKQQNGKLESQVEMQFFSGSGGSGDIPATYEFTDGKHYITNEPIPPKGSGGSGPKVVLYSEDVDIETPDTHISYMPTIKEGNSRELMGQQSMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDSYDPDVRIIENHGTEDELPNYCFPLGGVINTETLTKVKPKTGQENGWEKDATEFSDKNEIRVGNNFAMEINLN ANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNTYDYMNKRVVAPGLVDCYINLGARWSLDYMDNVNPFNHHRNAGLRYRSMLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWAAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDGEGYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPYPLIGKTAVDSITQKKFLCDRTLRIPFSSNFMSMGALTDLQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVHRPHRGVIETVYLRTPFASAGNATT Ad5-HVR5-DogTag hexagonal protein Nucleic acid sequences including stop codons and sequences with lowercase DogTags. (SEQ ID NO: 56): DogCatcher Amino acid sequence (SEQ ID NO: 10): KLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQ DogCatcher-PreS1-Bacterial Expression Amino acid sequence (SEQ ID NO: 57): MAHHHHHHVGTGKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQGSGGSGGSGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPASTNRQSGRQPTPLSPPLRNTHPQA DogCatcher-PreS1-Bacterial Expression Nucleic acid sequence (SEQ ID NO: 58): ATGGCACATCACCACCACCATCACGTGGGTACCGGTAAGCTGGGTGAGATTGAATTCATCAAAGTGGACAAGACCGACAAAAAGCCGCTGCGTGGCGCAGTTTTTAGCCTGCAGAAACAACATCCGGACTACCCTGACATTTATGGTGCAATTGACCAGAATGGCACCTATCAGGATGTGCGTACCGGCGAAGACGGCAAACTTACGTTTACCAATCTGAGTGATGGCAAGTATCGTCTGATCGAAAATAGCGAACCGCCTGGTTATAAACCTGTTCAAAATAAACCGATCGTGAGCTTTCGTATTGTGGATGGTGAAGTTCGTGATGTTACCAGCATTGTTCCGCAGGGCTCTGGCGGAAGCGGCggatccGGTCAGAATCTGAGCACCAGCAATCCGCTGGGTTTCTTTCCGGATCATCAGTTAGATCCGGCATTTCGTGCAAATACCGCAAATCCTGATTGGGATTTTAACCCGAATAAAGATACCTGGCCTGATGCCAATAAAGTTGGTGCCGGTGCATTTGGTCTGGGTTTTACCCCTCCGCATGGTGGTCTGTTAGGTTGGAGTCCGCAGGCACAGGGTATTCTGCAGACCCTGCCTGCAAATCCGCCTCCGGCAAGCACCAATCGTCAGAGCGGTCGTCAGCCGACACCGCTGAGTCCGCCTCTGCGTAATACCCATCCGCAGGCCTAA DogCatcher-PreS1-Mammalian Expression Amino acid sequence (SEQ ID NO: 59): METDTLLLWVLLLWVPGSTGDGKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQGSGGSGGSQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQSGRQPTPLSPPLRNTHPQAEPEA DogCatcher-PreS1-Mammalian Expression Nucleic acid sequence (SEQ ID NO: 60): ATGGAAACCGACACACTGCTGCTGTGGGTGCTGCTTCTTTGGGTGCCCGGATCTACAGGCGACGGAAAGCTGGGCGAGATCGAGTTCATCAAGGTGGACAAGACCGACAAGAAGCCCCTGAGAGGCGCTGTGTTCAGCCTGCAGAAACAGCACCCTGACTACCCCGATATCTACGGCGCCATC GACCAGAACGGCACCTATCAGGATGTTCGGACAGGCGAGGATGGCAAGCTGACCTTCACCAATCTGAGCGACGGCAAGTACCGGCTGATCGAGAATAGCGAGCCTCCTGGCTACAAGCCCGTGCAGAACAAGCCCATCGTGTCCTTCAGAATCGTGGACGGCGAAGTGCGGGACGTGACCTCTA TAGTGCCTCAAGGCTCTGGCGGAAGCGGCggatccCAGAATCTGAGCACCTCTAATCCTCTGGGATTCTTCCCAGACCACCAGCTGGACCCTGCCTTCAGAGCCAATACCGCCAATCCTGACTGGGACTTCAACCCAACAAGGACACCTGGCCTGACGCCAACAAAGTTGGCGCTGGCGCTTT TGGCCTGGGCTTTACACCTCCTCATGGCGGACTGCTTGGATGGTCACCTCAGGCTCAGGCATCCTGCAAACCCTGCCTGCTAATCCTCCTCCTGCCTCCACCAACAGACAGAGCGGTAGACAGCCCACACCTCTGAGCCCTCCACTGAGAAACACACACCCTCAGGCTGAGCCCGAGGCCTAA Igk-leader sequence Amino acid sequence (SEQ ID NO: 61): MEDTLLWVLLLWVPGSTGD C-tag Amino acid sequence (SEQ ID NO: 62): PERIOD Shark Immutable Chain (Sli) Amino acid sequence (SEQ ID NO: 63): MSLLWGGVTVLAAMLIAGQVASVVFLV HBV S 190-197 peptides Amino acid sequence (SEQ ID NO: 64): VWLSVIWM HBV core 93-100 peptides Amino acid sequence (SEQ ID NO: 65): MGLKFRQL HBV core P13L peptides Amino acid sequence (SEQ ID NO: 66): PPAYRPPNAPILSTL References Boni C, Janssen HLA, Rossi M, et al . 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Claims

1. An adenovirus vector comprising at least one modified capsid protein, wherein the modification of the capsid protein includes insertion or fusion of a first peptide partner, wherein: The first peptide conjugate is covalently bonded to the second peptide conjugate; The second peptide spouse is linked to the decorative antigen via insertion or fusion; and The decorative antigen includes hepatitis B virus PreS1 and / or hepatitis B virus PreS2 or immunogenic fragments thereof.

2. The adenovirus vector according to claim 1, wherein the modified capsid protein comprises hexagonal protein or pIX.

3. The adenovirus vector according to claim 1 or claim 2, wherein the modified capsid protein comprises a hexagonal protein, and optionally further wherein the modification of the hexagonal protein comprises fusing the first peptide conjugate into the hypervariable region (HVR).

4. The adenovirus vector according to claim 3, wherein the hypervariable region includes HVR1, HVR2 or HVR5.

5. The adenovirus vector according to claim 3 or claim 4, wherein the hypervariable region includes HVR5.

6. The adenovirus vector according to any one of the preceding claims, wherein the covalent bond between the first peptide conjugate and the second peptide conjugate is an isopeptide bond.

7. The adenovirus vector according to any one of the preceding claims, wherein the first peptide conjugate and the second peptide conjugate constitute a binding conjugate pair, and wherein the binding conjugate pair is selected from: DogTag and DogCatcher; DogTag and SnoopTag; DogTag and SnoopTagJr; SnoopTag and SnoopCatcher; or SpyTag and SpyCatcher.

8. The adenovirus vector according to any one of the preceding claims, wherein the first peptide coupler and the second peptide coupler constitute a binding coupler pair, and wherein the binding coupler pair is DogTag and DogCatcher.

9. The adenovirus vector according to any one of the preceding claims, wherein the first peptide conjugate is DogTag and the second peptide conjugate is DogCatcher.

10. The adenovirus vector according to any one of the preceding claims, wherein the adenovirus vector further comprises a nucleic acid encoding one or more exogenous polypeptides, and wherein the one or more exogenous polypeptides include at least one hepatitis B virus polypeptide or an immunogenic fragment thereof.

11. The adenovirus vector according to claim 10, wherein the at least one hepatitis B virus polypeptide is selected from PreS1, PreS2, S-protein or core, or immunogenic fragments thereof.

12. The adenovirus vector according to claim 10, wherein the one or more exogenous polypeptides comprise two or more hepatitis B virus polypeptides, wherein the hepatitis B virus polypeptides are selected from PreS1, PreS2, S-protein or core, or immunogenic fragments thereof.

13. The adenovirus vector of claim 10, wherein the one or more exogenous polypeptides comprise all hepatitis B virus polypeptides PreS1, PreS2, S-protein and core, or immunogenic fragments thereof.

14. The adenovirus vector of claim 10, wherein the at least one hepatitis B virus polypeptide comprises PreS1 or an immunogenic fragment thereof, and wherein the decorating antigen comprises hepatitis B virus PreS1 or an immunogenic fragment thereof.

15. The adenovirus vector of claim 12 or claim 13, wherein the nucleic acid further comprises a sequence arranged to direct cleavage between the two or more hepatitis B virus polypeptides, optionally wherein the sequence arranged to direct cleavage is selected from P2A, F2A or IRES.

16. A vaccine comprising the adenovirus vector as described in any of the preceding claims.

17. A pharmaceutical composition comprising the vaccine of claim 16 and a pharmaceutically acceptable buffer, excipient, carrier, adjuvant, or combination thereof.

18. The adenovirus vector of any one of claims 1 to 15 or the vaccine of claim 16, for the treatment or prevention of hepatitis B virus infection and / or hepatitis D virus infection.

19. Use of the adenovirus vector according to any one of claims 1 to 15 in the preparation of a medicament for treating or preventing hepatitis B virus infection and / or hepatitis D virus infection.

20. A method of treating a patient with a corresponding need, comprising administering a safe and effective amount of the adenovirus vector of any one of claims 1 to 15 or the vaccine of claim 16.

21. A method for generating an adenovirus vector according to any one of claims 1 to 15, the method comprising: The nucleic acid encoding the first peptide spouse was introduced into the nucleic acid encoding the capsid protein in the adenovirus genome; Allow the adenovirus genome to be expressed to obtain adenovirus particles; Obtain a second peptide conjugate linked to hepatitis B virus PreS1 and / or hepatitis B virus PreS2 or their immunogenic fragments; The adenovirus particle is allowed to mix with the second peptide conjugate linked to hepatitis B virus PreS1 and / or hepatitis B virus PreS2 or their immunogenic fragments, provided that the first peptide conjugate and the second peptide conjugate are allowed to covalently bind.

22. The method of claim 21, further comprising introducing a nucleic acid encoding one or more exogenous polypeptides into the adenovirus genome, wherein the one or more exogenous polypeptides include at least one hepatitis B virus polypeptide or an immunogenic fragment thereof.

23. A method for preparing the vaccine of claim 16, the method comprising mixing an adenovirus vector of any one of claims 1 to 15 with a pharmaceutically acceptable excipient.

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