vaccines

Abstract

This disclosure provides an adenoviral vector comprising at least one modified capsid protein comprising the insertion or fusion of a first peptide partner, wherein the first peptide partner is covalently bonded to a second peptide partner that is joined to a decorating antigen by insertion or fusion; and wherein the decorating antigen comprises hepatitis B virus PreS1 and / or hepatitis B virus PreS2, or an immunogenic fragment thereof. The disclosure also provides a method of producing the adenoviral vector, and uses of the adenoviral vector in vaccines and pharmaceutical compositions.

Description

[0001] VACCINES

[0002] Field of the Invention

[0003] This invention relates to anti-hepatitis B adenoviral vaccines, particularly vaccines for treating chronic hepatitis B. The invention also relates to nucleic acids encoding those vaccines, cells comprising those nucleic acids, and methods of preventing or treating hepatitis B, particularly chronic hepatitis B.

[0004] Background of the Invention

[0005] Hepatitis B is an infection of the liver caused by the hepatitis B virus. The virus is spread via contact with infected body fluids e.g. blood, saliva, semen, vaginal fluid, with the most common transmission route being mother-to-child in the perinatal period.

[0006] Hepatitis B can be acute or chronic (CHB). Chronic HBV (CHB) has a high mortality rate, primarily from cirrhosis and cancer of the liver. In 2019, WHO estimated that 296 million people worldwide were living with CHB infection with 1 .5 million new infections occurring annually. In that same year, 820,000 deaths from CHB occurred worldwide.

[0007] Current HBV vaccines are effective as prophylactics, but have very limited efficacy in treating individuals with existing CHB infection. In most cases, life-long antiviral therapy is required to suppress virus replication - there is currently no cure. Acute HBV infections are typically cleared by the host immune response, whereas CHB infection is characterized by dysfunctional anti-HBV cellular and humoral immunity.

[0008] It is widely acknowledged that a ‘functional cure’ for HBV (defined as a sustained loss of hepatitis B surface antigen, HBsAg) will require therapeutic strategies able to induce robust T-cell (particularly CD8+ T cell) responses against multiple HBV antigens and neutralizing humoral immunity against HBV.

[0009] Summary of the Invention

[0010] According to a first aspect, this disclosure provides an adenoviral vector comprising at least one modified capsid protein, said modification to the capsid protein comprising the insertion or fusion of a first peptide partner, wherein: the first peptide partner is covalently bonded to a second peptide partner; the second peptide partner is joined to a decorating antigen by insertion or fusion; and the decorating antigen comprises hepatitis B virus PreS1 and / or hepatitis B virus PreS2, or an immunogenic fragment thereof.

[0011] In an embodiment, the modified capsid protein comprises hexon or pIX. In an embodiment, the modified capsid protein comprises hexon. In another embodiment, the modified capsid protein comprises pIX. The adenoviral vector may be of any suitable genotype. The adenoviral vector may be of any suitable serotype. The adenoviral vector serotype may be derived from any suitable species, for example human, chimpanzee or gorilla. The adenoviral vector may be replicative or non-replicative. The adenoviral vector may have other modifications in addition to the modified capsid protein.

[0012] In another embodiment, the modified capsid protein comprises hexon, and further wherein the modification to hexon comprises fusion of the first peptide partner into a hypervariable region (HVR). The adenovirus hexon protein has seven HVR domains, HVR1 to HVR7. Suitably, any of the HVR domains may be used for insertion or fusion of the first peptide partner. The choice of hypervariable region may be determined by the skilled person according to their requirements. In an embodiment, the hypervariable region comprises any 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.

[0013] In a further embodiment, the covalent bond between the first peptide partner and the second peptide partner in the adenoviral vector is an isopeptide bond. In an embodiment, the first peptide partner and the second peptide partner comprise a binding partner pair. The binding partner pair may be selected from: DogTag and DogCatcher, DogTag and SnoopTag, DogTag and SnoopTagJr, SnoopTag and SnoopCatcher, or SpyTag and SpyCatcher, or variants thereof. Other suitable binding partner pairs will be known to the skilled person. In an embodiment, the binding partner pair is DogTag and DogCatcher. Suitably, the first peptide partner is DogTag and the second peptide partner is DogCatcher.

[0014] In any embodiment of any aspect of this disclosure, the decorating antigen comprising hepatitis B virus PreS1 and / or hepatitis B virus PreS2, or an immunogenic fragment thereof, may be derived from any strain of HBV, or may be a consensus sequence derived from multiple strains of HBV. By way of example only, the reference strain NC_003977 (ayw) has been used as a proof-of-concept strain herein. A Hepatitis B virus PreS1 and / or PreS2 decorating antigen will be understood by the skilled person to mean a Hepatitis B virus PreS1 and / or PreS2 polypeptide from any strain of HBV that is determined by sequence alignment to be equivalent to the same polypeptide from reference strain NC_003977 (ayw). Furthermore, the decorating antigen comprising hepatitis B virus PreS1 and / or hepatitis B virus PreS2, as described herein may be truncated, meaning that one or more amino acid is absent from the N- and / or C-terminus relative to the full-length polypeptide. In any embodiment of any aspect of this disclosure, an immunogenic fragment of the decorating antigen comprising hepatitis B virus PreS1 and / or hepatitis B virus PreS2 means any portion of hepatitis B virus PreS1 and / or hepatitis B virus PreS2 that retains the capacity to provoke an immune response when administered to a subject. In any embodiment of any aspect of this disclosure, the decorating antigen may comprise any region of HBsAg, for example the antigenic loop of HBV-S or an immunogenic fragment thereof, either in combination with HBV PreS1 and / or PreS2 or an immunogenic fragment thereof, or instead of those polypeptides.

[0015] In another aspect, the adenoviral vector further comprises nucleic acid encoding one or more exogenous polypeptide, and wherein the one or more exogenous polypeptide comprises at least one hepatitis B virus polypeptide or an immunogenic fragment thereof. In an 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, the one or more exogenous polypeptide comprises two or more hepatitis B virus polypeptides selected from PreS1 , PreS2, S-protein or Core, or immunogenic fragments thereof. In another embodiment, the one or more exogenous polypeptide comprises all of hepatitis B virus polypeptides PreS1 , PreS2, S-protein and Core, or immunogenic fragments thereof. In an embodiment, the at least one hepatitis B virus polypeptide comprises PreS1 or an immunogenic fragment thereof, and the decorating antigen also comprises hepatitis B virus PreS1 or an immunogenic fragment thereof; the encoded PreS1 or fragment thereof and the decorating PreS1 or fragment thereof may have the same amino acid sequence as each other or different amino acid sequences. In further embodiments, other HBV polypeptides such as HBeAg or HBV polymerase may also be encoded. In other embodiments, polypeptides from sources other than HBV may also be encoded.

[0016] In any embodiment of any aspect of this disclosure, a hepatitis B virus polypeptide or immunogenic fragment thereof may be from any strain of HBV, or may be a consensus sequence derived from multiple strains of HBV. By way of example only, the reference strain NC_003977 (ayw) has been used as a proof-of-concept strain herein. A Hepatitis B virus polypeptide, for example PreS1 , PreS2, S- protein or Core, will be understood by the skilled person to mean a polypeptide from any strain of HBV that is determined by sequence alignment to be equivalent to the same polypeptide from reference strain NC_003977 (ayw). Furthermore, a Hepatitis B virus polypeptide as described herein may be truncated, meaning that one or more amino acid is absent from the N- and / or C-terminus relative to the full-length polypeptide. In any embodiment of any aspect of this disclosure, an immunogenic fragment of a hepatitis B polypeptide means any portion of a hepatitis B polypeptide that retains the capacity to provoke an immune response when administered to a subject.

[0017] In any embodiment of any aspect of this disclosure, the decorating antigen comprising hepatitis B virus PreS1 and / or hepatitis B virus PreS2, or an immunogenic fragment thereof, and where present the at least one hepatitis B virus polypeptide or an immunogenic fragment thereof encoded by the nucleic acid of the adenoviral vector, may be derived from the same strain of HBV, or may be derived from a consensus sequence derived from multiple strains of HBV, or may be derived from more than one strain of HBV, or any combination thereof. For example, in the case of the decorating antigen, the HBV PreS1 and PreS2 where both are present may both be derived from the same strain of HBV, or from the same consensus strain of HBV, or each from different strains of HBV. Likewise, in the case where more than one hepatitis B virus polypeptide or immunogenic fragment thereof is encoded by the nucleic acid of the adenoviral vector, each may be derived from the same strain of HBV, or from the same consensus strain of HBV, or each from different strains of HBV, or any combination thereof. Likewise, the HBV PreS1 and / or PreS2 decorating antigen or immunogenic fragment thereof, and the at least one hepatitis B virus polypeptide or immunogenic fragment thereof encoded by the nucleic acid of the adenoviral vector, may be derived from the same strain of HBV, or from the same consensus strain of HBV, or each from different strains of HBV, or any combination thereof. By way of example only, the reference strain NC_003977 (ayw) has been used as a proof-of-concept strain herein. A Hepatitis B virus polypeptide, for example PreS1 , PreS2, S-protein, Core, pre-core, pol orx, will be understood by the skilled person to mean a polypeptide from any strain of HBV that is determined by sequence alignment to be equivalent to the same polypeptide from reference strain NC_003977 (ayw).

[0018] In a further embodiment, where the adenoviral vector further comprises nucleic acid encoding one or more exogenous polypeptide, and wherein the one or more exogenous polypeptide comprises two or more hepatitis B virus polypeptides or immunogenic fragments thereof, the nucleic acid of the adenoviral 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 sequences known to direct cleavage may be used, such as sequences for other 2A self-cleaving peptides, for example T2A or E2A. Any IRES sequence may also be used. The sequence arranged to direct cleavage may use any mechanism to achieve cleavage between the two or more hepatitis B virus polypeptides.

[0019] In a further embodiment, the nucleic acid of the adenoviral vector may comprise further noncoding or coding elements, including but not limited to promoters, enhancers, linkers, additional exogenous genes, terminators. Any suitable such sequence may be used,

[0020] In another aspect, a vaccine comprising the adenoviral vector is provided. The vaccine comprising the adenoviral vector means the adenoviral vector provided in any suitable composition for administration to a human in need thereof, and / or at any concentration of adenoviral vector (dose) suitable to provoke an immune response on administration. The vaccine may comprise any suitable additional components in addition to the adenoviral vector.

[0021] In an additional aspect, a pharmaceutical composition comprising the vaccine and a pharmaceutically acceptable buffer, excipient, carrier, adjuvant, or combination thereof, is provided.

[0022] In another aspect, the adenoviral vector or the vaccine may be for use in the treatment or prophylaxis of Hepatitis B virus infection and / or Hepatitis D virus infection.

[0023] In another aspect, the adenoviral vector may be used in the manufacture of a medicament for the treatment or prophylaxis of Hepatitis B virus infection and / or Hepatitis D virus infection.

[0024] In embodiments, the Hepatitis B virus infection may be acute or chronic (CHB). Likewise the Hepatitis D virus infection may be acute or chronic.

[0025] In another aspect is provided a method of treating a patient in need thereof, comprising administering a safe and effective amount of the adenoviral vector or the vaccine. Suitably, any administration method or schedule may be used.

[0026] In another aspect is provided a method of producing the adenoviral vector, the method comprising: introducing a nucleic acid that encodes a first peptide partner into the nucleic acid encoding a capsid protein in an adenoviral genome; permitting expression of the adenoviral genome in order to obtain adenovirus particles; obtaining a second peptide partner attached to Hepatitis B virus PreS1 and / or hepatitis B virus PreS2, or immunogenic fragment thereof; allowing the adenovirus particles and the second peptide partner attached to a Hepatitis B virus PreS1 and / or Hepatitis B virus PreS2, or immunogenic fragment thereof, to mix under conditions that permit covalent binding of the first peptide partner and the second peptide partner. In an embodiment, the method further comprises introducing a nucleic acid that encodes one or more exogenous polypeptide into the adenoviral genome, wherein the one or more exogenous polypeptide comprises at least one hepatitis B virus polypeptide or an immunogenic fragment thereof.

[0027] In another aspect is provided a method of manufacturing the vaccine, the method comprising admixing the adenoviral vector with a pharmaceutically acceptable excipient. Any suitable excipient may be used.

[0028] Figures

[0029] Figure 1 : A - Schematic of an exemplary adenoviral vector showing a tag (first peptide partner) genetically inserted within the capsid protein. B - Schematic of exemplary adenoviral vector showing vector from A with PreS1 protein coupled to the vector surface via the interaction between the catcher (second peptide partner) and the tag, and a GFP gene inserted into the adenoviral genome. C - SDS- PAGE gel showing adenoviral vector particles with DogTag inserted into hexon covalently decorated with decreasing concentrations of DogCatcher-PreS1 upon overnight co-incubation. Coupling efficiency (as indicated, expressed as % hexon protein coupled to catcher-PreS1) was calculated from relative band intensities of uncoupled hexon and covalently coupled hexon-catcher-PreS1 species. D - In vitro infectivity in 293A cells of GFP-expressing adenoviral vectors decorated with decreasing concentrations of DogCatcher-PreS1 ligand (from experiment shown in 1C). E - Mouse serum IgG antibody responses to PreS1 measured by Endpoint ELISA at day 20 (pre-boost) and day 35 (post-boost) following administration of: undecorated adenovirus encoding an EGFP reporter gene (Ad(GFP)), PreS1- decorated adenovirus encoding an EGFP reporter gene (Ad(GFP):PreS1), or unconjugated DogCatcher- PreS1 protein plus Alhydrogel® adjuvant (C-PreS1 + alhydrogel). F - CD8+ T cell responses in the spleen to encoded EGFP were measured by overnight ex vivo interferon gamma (IFNy)-ELISPOT assay, the y-axis shows spot-forming cells (SFC) per million splenocytes.

[0030] Figure 2: A - SDS-PAGE gel showing adenoviral vector particles with DogTag inserted into hexon covalently decorated with increasing quantities of mammalian-expressed DogCatcher-PreS1 upon overnight co-incubation. B - Coupling efficiency (expressed as % hexon protein coupled to catcher- PreS1) with increasing concentration of catcher-PreS1 ligand, calculated from relative band intensities of uncoupled hexon and covalently coupled hexon-catcher-PreS1 species (shown in A). C - In vitro infectivity in 293A cells of GFP-expressing adenoviral vectors decorated with increasing amounts of DogCatcher-PreS1 ligand (as shown in A and B).

[0031] Figure 3: A - Schematic of conjugated adenoviral vector showing PreS1 protein coupled to the vector surface via the hexon capsid protein using a tag-catcher binding pair, and alternative encoded HBV gene(s): HBV L encodes the HBsAg Large polypeptide, whereas HBV C-S encodes HBV Core and the small HBsAg polypeptide with an intervening cleavage site. Sli = shark invariant chain, 2A = F2A (FMDV18 2A), GGS and GSGGS are linkers. B - Serum IgG antibody responses to PreS1 measured by Endpoint ELISA following administration of: undecorated adenovirus encoding HBV C-S (Ad(C-S)), undecorated adenovirus encoding HBV L (Ad(L)), or PreS1 -decorated versions of each adenovirus (Ad(C-S):PreS1 and Ad(L):PreS1 , respectively). C - In vitro anti-HBV serum neutralization in HepG2- NTCP cells, expressed as reduction in luminescence signal relative to luciferase-expressing HBV virus- only control (‘Virus only’) in relative light units (RLU). HepG2-NTCP cells were co-incubated with luciferase-expressing HBV virus, and either serum from the experiment shown in B as indicated or serum from mice vaccinated with undecorated adenovirus encoding GFP (Ad(GFP)).

[0032] Figure 4: A - Schematic of conjugated adenoviral vector showing PreS1 protein coupled to vector surface and alternative encoded HBV gene(s). Sli = shark invariant chain, 2A = F2A (FMDV18 2A), GGS and GSGGS are linkers. B-D - CD8+ T cell responses in the spleen to encoded genes following administration of: 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 then HBV Core (N- to C- terminus) and decorated with PreS1 at high coverage (Ad(L-C):PreS1 HC), or adenovirus encoding HBV Core then HBV L (N- to C-) and decorated with PreS1 at high coverage. For Figures 4C and 4D all adenovirus were decorated with PreS1 at high coverage. CD8+ T cell responses were measured by overnight ex vivo IFNy-ELISPOT assay using B - S190-197 peptide, C - Coregs-wo peptide or D - Corepm peptide. E - Vaccinated mouse serum IgG antibody responses to PreS1 were measured by Endpoint ELISA at day 20 (pre-boost) and day 35 (post-boost). F - In vitro anti-HBV serum neutralization in HepG2-NTCP cells, expressed as reduction in luminescence signal relative to luciferase-expressing HBV virus-only control (‘Virus only’) in relative light units (RLU). HepG2-NTCP cells were co-incubated with luciferase-expressing HBV virus, and either serum from the experiment shown in B-E as indicated or serum from mice vaccinated with undecorated adenovirus encoding GFP (Ad(GFP)). G - Full dilution series from the in-vitro anti-HBV serum neutralization assay shown in F.

[0033] Figure 5: Western blot analysis of encoded gene expression from ten different Ad-HBV vectors (designated A-J, outlined in Table 6) designed to test the effect of incorporating nucleotide sequences arranged to direct cleavage between the antigen polypeptides.

[0034] Figure 6: A - Schematic of conjugated adenoviral vector showing PreS1 protein coupled to vector surface and alternative encoded gene(s). IRES = Internal Ribosome Entry Site cleavage sequence from Encephalomyocarditis virus, F2A = FMDV18 2A, P2A = porcine teschovirus-1 2A cleavage sequence, B - Serum IgG antibody responses to PreS1 measured by Endpoint ELISA from mice vaccinated with the constructs in A, identified on the X-axis by the encoded cassette. C - E: T cell responses in the spleen to encoded genes were measured by overnight ex vivo IFNy-ELISPOT assay for the same mice.

[0035] Figure 7: A - Schematic of conjugated ChAd63 adenoviral vector showing PreS1 protein coupled to vector surface and alternative encoded gene(s), abbreviations as per other figures. B - Serum IgG antibody responses to PreS1 measured by Endpoint ELISA. C - E: T cell responses in the spleen to encoded genes were measured by overnight ex vivo IFNy-ELISPOT assay.

[0036] Detailed Description of the Invention

[0037] Hepatitis B virus

[0038] Hepatitis B virus (HBV) is a partially double-stranded DNA virus in the Hepadnaviridae family. The virus is divided into four major serotypes (adr, adw, ayr, ayw) based on antigenic epitopes present on its envelope proteins. HBV strains have also been divided into ten genotypes (A-J) and forty subgenotypes according to genome sequence similarities. The genotypes have distinct geographical distributions and also affect the disease severity, course, likelihood of complications, and response to treatment.

[0039] The HBV particle or virion, also called the Dane particle, is formed of an icosahedral nucleocapsid protein core enclosing the viral DNA and a DNA polymerase, the core being surrounded by an outer lipid envelope containing embedded proteins. 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 to form part of the virion and is secreted into serum. HBV-encoded oncogene X protein (HBx) is a small non-structural protein with a role in HBV replication and in HBV-associated liver disease. Hepatitis B surface antigen (HBsAg; Australia antigen) is embedded into the viral envelope. HBsAg occurs in three forms, small (S), medium (M) and large (L). All three forms are encoded from 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 (approx. 42 kDa) is translated from the most upstream (5’-) of the three start codons, and comprises the domains PreS1 , PreS2 and SHBsAg. The HBsAg M polypeptide (approx. 33 kDa) is translated from an intermediate start codon, and comprises the domains PreS2 and SHBsAg. The HBsAg S polypeptide (approx. 24 kDa; about 80% of HBsAg is in this form) is translated from the most downstream (3’-) of the three start codons and is composed of only SHBsAg. In nature, the PreS1 and PreS2 domains are not found in isolation.

[0040] In this disclosure the Hepatitis B virus reference strain NC_003977 (ayw) has been used as a proof-of-concept. It will be understood that any other strain of HBV, or combination of strains of HBV where an adenoviral vector comprises more than one HBV nucleotide or amino acid sequence, could suitably be used. Likewise, HBV consensus sequences may be used, where a consensus sequence is the calculated sequence of most frequent residues, either nucleotide or amino acid, found at each position in a sequence alignment. The skilled person would understand that the strain(s) used in the adenoviral vector could be varied in order to, for example, target HBV strains from one or more different geographical regions.

[0041] Variants, derivatives and modifications of Hepatitis B Virus polypeptides may be made by any suitable means. Variants, derivatives and functionally operative modifications may involve amino acid additions, substitutions, alterations or deletions.

[0042] Hepatitis B Virus polypeptides may be defined by reference to a sequence, for example by reference to the Hepatitis B virus reference strain NC_003977 (ayw) amino acid sequence. The sequence may be 100% identical to the sequence of a wild-type Hepatitis B Virus polypeptide. A Hepatitis B Virus polypeptide may have a sequence which has at least 60%, 70%, 80%, 85%, 90%, 95% or 99% sequence identity to the wild-type sequence. Variants and derivatives of a Hepatitis B Virus polypeptide may comprise an amino acid sequence that is at least 90% or 95% similar to the wild-type sequence. Homologues of these entities may therefore have at least 60% homology thereto, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% homology thereto.

[0043] Hepatitis B disease

[0044] Hepatitis B (disease), caused by infection with HBV, affects the liver, and can occur in acute and chronic (CHB) forms.

[0045] Acute hepatitis B symptoms include general ill-health, loss of appetite, nausea, vomiting, body aches, mild fever, and dark urine, and sometimes the development of jaundice. Acute hepatitis B lasts for a few weeks and then resolves. Acute hepatitis B infection may be asymptomatic. CHB may also be asymptomatic, or if untreated may be associated with chronic inflammation of the liver (chronic hepatitis), potentially leading to cirrhosis. CHB increases the likelihood of developing hepatocellular carcinoma (HCC; liver cancer). In Europe, about 50% of hepatocellular carcinomas are associated with hepatitis B or C (caused by hepatitis C virus) disease.

[0046] HBV infection can occur via exposure to infected blood or other body fluids including saliva, semen and vaginal fluid. The majority of infections world-wide are associated with maternal to child transmission around the time of birth (peri natally).

[0047] An HBV infection lasting longer than six months is usually considered chronic. The development of CHB is associated with earlier age of infection with HBV, with around 90% of those who are infected perinatally later developing CHB. Infection after the age of five is associated with less than 10% risk of developing CHB. Despite CHB frequently being asymptomatic, in about 25% of cases of CHB cirrhosis and liver cancer eventually develop.

[0048] HBV and HDV

[0049] Hepatitis D virus (HDV) infection can only occur with concomitant HBV infection, either simultaneous or pre-existing. This is because in order to be infectious the HDV viral envelope needs to include surface antigen proteins from HBV, with the presence of HBsAg-L being necessary for HDV entry into cells.

[0050] Coinfection 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. Whilst in CHB, there is a more rapid progression to liver cirrhosis with increased risk of developing liver cancer.

[0051] Current CHB treatments

[0052] To be considered a ‘functional cure’ for CHB, a treatment should display a loss or marked reduction in detectable HBsAg serum levels that is sustained over time, for example a 1 -log reduction in HBsAg levels from baseline at e.g. 24 weeks.

[0053] No currently commercially-available vaccines provide effective CHB treatment. Without wishing to be bound by any theory, this is believed to be because in CHB T cells and B cells against S-protein are very dysfunctional and low frequency, due to HBV generating an excess of sub-viral particles containing S, leading to T cell and B cell exhaustion. Current treatments are based on lengthy, often lifelong, courses of antiviral medications, such as tenofovir and entecavir. The cost associated with those medications is a barrier to treatment in low-income settings.

[0054] Treatment options for chronic hepatitis D in the presence of hepatitis B include Bulevirtide (Hepcludex; MyrB; Myrcludex-B) which is a 47-amino acid recombinant peptide derived from HBV PreS1 , which acts as a competitive inhibitor by binding to the same cell-surface receptor (the sodium / bile acid cotransporter NTCP) that both HBV and HDV also use to gain access to cells.

[0055] Current HBV vaccines

[0056] Prophylactic HBV vaccines are typically based on recombinant Hepatitis B surface small antigen (HBsAg-S), and not HBsAg-M or-L, therefore they lack PreS1 and / or PreS2.

[0057] Currently available prophylactic HBV vaccines have no therapeutic effect on chronically infected HBV individuals and hence they fail to control chronic HBV infection. A number of T-cell inducing vaccines against HBV are in development with the aim of providing treatment of HBV. See Meng et al. (2020) for a review.

[0058] HepTcell™, from Altimmune is a completely synthetic peptide product based on nine 32-40mer peptides derived from HBV protein T-cell epitopes. In a Phase I clinical study HepTcell peptides were administered both with and without adjuvant as an add-on therapy to entecavir or tenofovir in patients with HBeAg-negative CHB. HepTcell™ was generally well-tolerated, and when delivered with adjuvant T cell responses against HBV antigens were delivered, although the results were silent on the effect on HBsAg levels (Meng et al., 2020).

[0059] 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 by electroporation in ninety nucleos(t)ide analogue-treated CHB patients, but the results have not been made available (Meng et al., 2020).

[0060] 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 to patients with CHB with the antiviral tenofovir disoproxil fumarate (TDF). The virus was found to be safe and well tolerated but did not produce significant decreases in levels of HBsAg even when combined with the antiviral (Boni et al., 2019).

[0061] TG1050 by Transgene is a human adenovirus serotype 5 based vaccine that expresses HBV polymerase and domains of core and surface antigen. In a Phase I clinical trial in CHB patients under nucleoside(d)tide analog NUC therapy, TG1050 was well tolerated but resulted in only a minor decrease in HBsAg levels (Zoulim et al., 2019).

[0062] WO2018 / 189522A1 describes a ChAdOx adenovirus-vectored HBV vaccine that encodes HBV Core, a modified HBV polymerase, and HBV surface antigen (HBsAg) from an expression cassette, with an intergenic sequence arranged to cause expression of at least the HBsAg as a separate protein from the HBV core and the modified HBV polymerase.

[0063] Adenovirus-vectored vaccines

[0064] Adenoviruses (Ad) are a non-enveloped double stranded DNA virus with a genome of approximately 36 kilobases (kb). There are over 60 human adenovirus serotypes grouped into species A to G. Each group comprises of a number of adenoviral serotypes, for example, the subgroup species C includes Ad5 and Ad2. Ad5 is the most extensively studied serotype, and the most widely used platform for the development of oncolytic viruses. In the development of oncolytic viruses, it is desirable to be able to target particular tissues, and therefore the tropism may be altered. A major issue with using some adenovirus serotypes, including Ad5, in clinical settings is the pre-existing immunity in humans. Some scientist have tried to overcome the pre-existing immunity by using adenovirus first isolated from the great apes, such as Gorilla and Chimpanzees. Examples of such vectors that have been used in clinical studies include ChAd3, ChAd155, and ChAdOxl . Such adenoviruses are hoped to have a lesser sero-prevalence in humans. Nonetheless humans often have pre-existing immunity to such viruses and of course the issue of pre-existing immunity may arise should it be desired to administer the vector on more than one occasion.

[0065] Adenoviruses are typically 70-90 nm in size with an icosahedral capsid shape. The outer capsid structure, also known as ‘capsid protein’ comprises three major types of protein (hexon, fiber and penton base). There are additional minor proteins in the outer capsid including VI, VIII, IX, Illa and IVa2 (also known as pVI, pVIII, pIX, pllla and plVa2). Hexon is the major component of the adenoviral capsid accounting for more than 83% of the capsid protein (Rux et al., 2003). Hexon modification has been shown to allow for circumvention of pre-existing neutralising antibodies in some circumstances, including the swapping of hyper variable regions (HVR) from different serotypes (Roberts et al., 2006). The adenovirus hexon protein has seven HVR domains, HVR1 to HVR7. Suitably, any of the HVR domains may be engineered, for example used for insertion or fusion of exogenous sequences, e.g. peptides. The choice of hypervariable region to be engineered may be determined depending on technical requirements, but, for example, HVR1 , HVR2 or HVR5 may be equally suitable as insertion or fusion sites.

[0066] Adenoviruses (Ads) are commonly used for gene therapy, in particular as gene delivery vectors, due to their capacity for inclusion of additional genetic sequences. Over 2,000 gene therapy trials have been conducted using Ads. Adenoviral vectors allow for the transmission of the transgene they carry into the host nucleus but do not integrate viral DNA into the host chromosome. The insert size for Ads when used as gene therapy vectors is large, with a capacity of 8-36 kb possible.

[0067] Additionally, Ads have emerged as a promising vaccine delivery vehicle due to their ability to induce both innate and adaptive immune responses; having the capacity to induce potent antigenspecific B and T cell immune responses. Adenoviral vectors are highly immunogenic and are efficient in delivering antigens.

[0068] Conventional adenovirus vectored vaccines are among the most potent inducers of vaccinespecific T cell immunity (particularly CD8+ T cells) in humans, but humoral immunity is modest compared to other vaccine technologies including recombinant protein / VLPs and mRNA.

[0069] Several adenoviral vector based vaccine candidates were developed and pursued further in clinical trials, however, many of these were not successful. Adenovirus of the human serotype 5 (Ad5) based vaccine, developed against HIV-1 by Merck, induced CD8+ T cell responses but failed to prevent HIV infections. More recently the utility of Adenoviral vectored vaccines have been demonstrated by the SARS-CoV-2 pandemic and the use of adenoviral vaccines from Johnson and Johnson and Astra Zeneca which both express the “S” protein from SARS-CoV-2 in adenoviral vectors. A major obstacle to the continued success of adenoviral based vectors in human and animal therapy is the neutralisation of the vector by adenovirus-specific antibodies. Natural infection by adenovirus is high in human and animal populations, and therefore the adaptive immune system may recognise and respond to the presence of adenoviral vectors by the secretion of neutralising antibody (NAB). Similarly, the innate immune system may also be responsible for assisting the response to adenoviral vectors. It is estimated that 50% to 90% of the adult population has pre-existing immunity to Ad5 for example.

[0070] In our earlier applications PCT / GB2020 / 052774, published as WO2021 / 084282A1 , and PCT / GB2022 / 051137, published as WO2022 / 234276A1 , (both applications herein incorporated by reference), we have demonstrated that it is possible to decorate the surface of an adenoviral vector with an antigen of interest by using specific binding peptide pairs, which as well as presenting of the antigen to the immune system, also allows shielding of the adenoviral vector from anti-vector antibodies.

[0071] Adenovirus can be replication-defective: certain genes are deleted from the genome in order to ensure that when the adenovirus is used as a therapeutic, it is no longer capable of replication. For vaccination purposes these vectors typically have deletions in E1 (renders vector replication defective) and / or E3 genes (increases capacity for insert size). Other vectors may result from the deletion of a set of genes from the genome, and is within the skills of those working with adenoviruses. This is an advantage for use in vaccines, where the aim of the adenoviral vector is to present the antigen to the immune system in a format that makes it highly immunogenic, while limiting cytotoxicity.

[0072] Adenovirus infectivity in cells that express the Coxsackievirus and adenovirus receptor (CAR) is mediated via the fiber protein. An example of a cell line that expresses the CAR receptor is HEK293 cells. Fiber binds to the CAR receptor on the surface of cells and this mediates the initial attachment of the virus. However, it was recently demonstrated that instead of a fiber-mediated entry of the adenovirus, Factor X (FX) - a coagulation factor present in human serum can bind to the hexon proteins of some adenovirus serotypes to facilitate the entry of the virus in some cell types. An example of a cell line that mediates infection via the hexon protein is SKOV3. It is believed that FX mediated infection via the adenovirus hexon can enhance liver tropism of adenovirus vectors in vivo. Modifications of the hexon protein such as insertion of DogTag and coupling to an antigen reduces hexon-mediated infectivity of the cells. This is a desirable effect as the natural tropism of adenovirus when injected intravenously can cause liver toxicity in patients at very high doses. Reduction of hexon-mediated infectivity to reduce liver toxicity would be advantageous to the present invention.

[0073] Adenovirus hexon capsid protein is approximately 100 kDa in size, with 720 monomers per virion. Hexon monomers organise into trimers so that 12 lie on each of the 20 facets, resulting in 240 trimers per virion. Hexon sequences contain hypervariable regions (HVR) corresponding to loops on the external surface on the virus and therefore cover almost the entire surface of the virus. Each monomer has seven HVRs identified as HVR1-HVR7 which are serotype specific. As the loops are on the external surface of the virus, hexon loops are the main antigen recognition site, a target for host immune responses. Hexon protein varies in length, for example, Ad2 is the longest known hexon protein with a length of 968 amino acids (UniProt ID: P03277). Ad5, the most commonly used adenovirus for gene therapy has a hexon of length of 952 amino acids (UniProt ID: P04133). Modifying hexon HVRs which contain the serotype-specific epitope seems to be a promising approach to overcome the host neutralisation response. Any one of the HVRs could be modified.

[0074] Adenovirus pIX protein is a minor capsid protein which is approximately 14.3 kDa in size. There are approximately 240 pIX monomers per virion. The pIX protein functions to stabilise the hexons on the viral surface. The C-terminus of the pIX protein is exposed on the surface of the virus and is therefore a desirable site for fusion of small and large peptides. Ad5 pIX has two domains connected by a flexible linker. The Ad5 pIX protein has a length of 196 amino acids (UniProt ID: Q2KS03).

[0075] In this disclosure adenoviruses of genotype Ad5, a species C serotype (GenBank accession AC_000008), and ChAd63, a species E serotype (GenBank accession CS479277), have been used as proofs of concept. It will be understood that any other genotype of adenovirus, or recombinant adenovirus, could suitably be used.

[0076] An Adenovirus nucleic acid or polypeptide may have a sequence which has at least 60%, 70%, 80%, 85%, 90%, 95% or 99% sequence identity to the wild-type sequence. Variants and derivatives of an adenovirus polypeptide may comprise an amino acid sequence that is at least 90% or 95% similar to the wild-type sequence. Homologues of these entities may therefore have at least 60% homology thereto, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% homology thereto.

[0077] Modification to the adenovirus capsid proteins can be genetic or non-genetic, including chemical. The capsid proteins can be genetically modified through the incorporation of antigens into the capsid. Alternatively, the viral particle surface may be directly modified. Modification of all three major capsid proteins has been demonstrated previously. However, the results from these modifications has been mixed, and there is a major obstacle in the size of the insert that the most promising approaches offer, particularly regarding modification of hexon.

[0078] “At least one modification” or “at least one modified capsid protein” as used herein refers to the inclusion of a first peptide partner insertion into the adenoviral capsid protein using any appropriate means. For example, the insertion of the first peptide partner into the adenoviral hexon HVR loops or the fusion of a first peptide partner to the adenoviral pIX minor capsid protein. This modification may be made genetically through gene fusion, for example, or chemically. Isopeptide binding partner pairs

[0079] Use of isopeptide binding partner pairs, such as SpyCatcher and SpyTag (WO2011 / 098772), based upon attachment proteins from a bacterium, has been established as a technology to irreversibly and spontaneously conjugate recombinant proteins and the like under physiological conditions. The fibronectin binding protein, FbaB, from Streptococcus pyogenes contains a CnaB2 adhesin domain. CnaB2 is stabilized by a spontaneous reaction of Lys and Asp side chains to form an isopeptide bond. CnaB2 has been split into the 13-residue SpyTag peptide and the 116-residue SpyCatcher protein, that each may be fused to two entities that it is desired to bind via an isopeptide bond (Zakeri et al., 2012). Bio-conjugation between entities that would be impossible to achieve through genetic fusion between proteins can work using peptide binding pairs, including for vaccine development (Brune et al., 2016; Thrane et al., 2016). Various Catcher and Tag pairs are now available, some based upon modifications of SpyCatcher and SpyTag, and others based upon similar chemistry from alternative bacterial proteins.

[0080] Proteins that are capable of spontaneous isopeptide bond formation (so-called “isopeptide proteins”) have been advantageously used to develop peptide partner pairs (i.e. two-part linkers) which covalently bind to each other and provide irreversible interactions (see e.g. WO2011 / 098772 and WO2016 / 193746 both herein incorporated by reference, together with WO2018 / 189517, WO2018 / 197854, WO2020 / 183198, and WO2022 / 214795, all incorporated herein by reference). In this respect, proteins which are capable of spontaneous isopeptide bond formation may be expressed as separate fragments, to give a first peptide partner and a second peptide partner which is the peptide binding partner for the first peptide partner, where the two fragments are capable of covalently reconstituting by isopeptide bond formation. This covalent reconstitution links molecules or components fused to the second peptide partner and the requisite first peptide partner. The isopeptide bond formed by the peptide partner pair is stable under conditions where non-covalent interactions would rapidly dissociate, e.g. over long periods of time (e.g. weeks), at high temperature (to at least 95°C), at high force, or with harsh chemical treatment (e.g. pH 2-11 , organic solvent, detergents or denaturants).

[0081] Isopeptide bonds are amide bonds formed between carboxyl / carboxamide and amino groups, where at least one of the carboxyl or amino groups is outside of the protein main-chain (the backbone of the protein). Such bonds are chemically irreversible under typical biological conditions and they are resistant to most proteases. As isopeptide bonds are covalent in nature, they result in some of the strongest measured protein-protein interactions.

[0082] In brief, a two-part linker, i.e. a peptide partner pair (a so-called peptide tag / binding partner or catcher pair) may be derived from a protein capable of spontaneously forming an isopeptide bond (an isopeptide protein), wherein the domains of the protein are expressed separately to produce a peptide “tag” that comprises one of the residues involved in the isopeptide bond (e.g. an aspartate or asparagine, or a lysine) and a peptide or peptide binding partner (or “catcher”) that comprises the other residue involved in the isopeptide bond (e.g. a lysine, or an aspartate or asparagine) and at least one other residue required to form the isopeptide bond (e.g. a glutamate). Mixing the peptide tag and binding / catcher partner results in the spontaneous formation of an isopeptide bond between the tag and binding partner. Thus, by separately incorporating the peptide tag and binding partner into different molecules or components, e.g. proteins, it is possible to covalently link said molecules or components together via an isopeptide bond formed between the peptide tag and binding partner, i.e. to form a linker between the molecules or components incorporating the peptide tag and binding partner.

[0083] The spontaneous formation of the isopeptide bond may be in isolation, and not require the addition of any other entity. For some peptide tag and binding / catcher partner pairs, the presence of a third or helper entity, such as a ligase, may be required in order to generate the isopeptide bond.

[0084] Variants, derivatives and modifications of the binding pairs may be made by any suitable means. Variants, derivatives and functionally operative modifications may involve amino acid additions, substitutions, alterations or deletions that retain the same function in relation to the ability to form an isopeptide bond with the relevant binding partner.

[0085] For some of the binding pairs, mediation by a third entity such as an enzyme is required. For example, SnoopLigase may be used to mediate the bond formation between SnoopTagJr / SnoopTag and DogTag (Buldun et al., incorporated herein by reference). Thus, the pairing may require the assistance of an enzyme such as a ligase.

[0086] It will be understood that as used herein, either the first peptide partner or the second peptide partner may be the peptide “tag” and the other is the “binding partner / catcher”.

[0087] Suitably, the first and second peptide partners form the peptide partner pair termed SpyTag / SpyCatcher. Suitably, the SpyCatcher component is DeltaNI (AN1) SpyCatcher (as described in Li et al., 2014) which has a 23 amino acid truncation at the N-terminus compared to “SpyCatcher”.

[0088] In other embodiments, the first and second peptide partners form a peptide partner pair which is a mutated version of SpyTag / SpyCatcher displaying an increased rate of reaction for isopeptide bond formation such as, for example, those described in WO2018 / 197854 and in Keeble et al. (2019). In some embodiments, these mutated forms may be useful for the attachment of large proteins (e.g. >50 kDa or >100 kDa) and / or where slow reactions or steric hindrance may be an issue.

[0089] In some embodiments, the first and second peptide partners form a peptide partner pair which is a modified version of RrgACatcher / RrgATag named DogCatcher / DogTag. These latter entities are described in. Keeble et al. (2022) and WO2022 / 214795, both publications are incorporated herein by reference.

[0090] In other embodiments, the isopeptide proteins forming the peptide partner pair may include SnoopTag / SnoopCatcher, described, for example in WO 2016 / 193746.

[0091] In some embodiments, one or both of the isopeptide proteins forming the peptide partner pair may have N- or C-terminal truncations, whilst still retaining the reactivity of the isopeptide bond. SdyTag and SdyCatcher were constructed based on the native Cna protein B-type (CnaB) domain from a related fibronectin-binding protein in Streptococcus dysgalactiae. In some embodiments, the isopeptide proteins forming the peptide partner pair are SdyTag and SdyCatcher, or are based on modifications to these peptide partner pairs. Known modifications to the SdyCatcher include the modifications to form QueenCatcher, Mooncake and Katl. Such modified versions of SdyCatcher may 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 herein incorporated by reference.

[0092] Exemplary first and second peptide partner pairs (peptide tag / binding partner pairs; reactive pairs) are described in Table 1 ; this list is not exhaustive and further peptide partner pairs may be suitable for use in the present invention:

[0093] Table 1 : Exemplary isopeptide binding pairs

[0094] These entities are described, for example, in WO2011 / 098772, WO2016 / 193746, WO2018 / 197854, WO2018 / 189517, WO2020 / 183198, WO2022 / 214795, or Li et al. (2014), all incorporated herein by reference. Table 2: Exemplary amino acid sequences for various isopeptide partners

[0095]

[0096] Variants, derivatives and modifications of the binding pairs may be made by any suitable means.

[0097] Variants, derivatives and functionally operative modifications may involve amino acid additions, substitutions, alterations or deletions that retain the same function in relation to the ability to form an isopeptide bond with the relevant binding partner.

[0098] The peptide pairs may be defined by reference to a sequence. The sequence may be identical for the sequence listed for the peptide partner. The peptide partner may have a sequence which has at least 60%, 70%, 80%, 85%, 90%, 95% or 99% sequence identity to the listed sequence. Variants and derivatives of the peptide partner may comprise an amino acid sequence that is at least 90% or 95% similar to the listed sequence. Homologues of these entities may therefore have at least 60% homology thereto, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.

[0099] For some of the binding pairs, mediation by a third entity such as an enzyme is required. For example, SnoopLigase may be used to meditate the bond formation between SnoopTagJr and DogTag. Thus, the pairing may require the assistance of an enzyme such as a ligase.

[0100] Antigen

[0101] An antigen as used herein refers to any molecule that is capable of inducing immune responses, such as a polypeptide or an immunogenic fragment thereof. An antigen can be an allergenic antigen, viral antigen, bacterial antigen, parasitic antigen or fungal antigen. “Antigen” as used herein includes peptides and epitopes, variants and derivatives thereof. Antigen as used herein may refer to a full-length polypeptide expressed from a single gene or open reading frame, or it may refer to a truncated variant of that polypeptide. The truncation may be an N- or a C-terminal truncation, or the antigen may have both N- and C-terminal truncations compared to the full-length polypeptide. Further, an antigen may have a mutation compared to the wild-type (native) protein, such mutation being, for example, an addition, a deletion, a rearrangement or a substitution of one or more amino acid.

[0102] As used herein, a B cell epitope is the part of an antigen that is recognised by the immune system specifically by antibodies and B cells. As used herein, a T cell epitope is the part of the antigen that is recognised by T cells.

[0103] The antigen may be a viral antigen, more particularly an HBV antigen. HBV antigens include HBsAg, including any of the L, M and S variants, and also the PreS1 and / or PreS2 domains in the absence of the S domain. Other HBV antigens include the Core protein. HBV antigens may be from any strain of HBV, and may have mutations or truncations compared to the wild-type antigen. Adenoviral vaccines with decorating antigens

[0104] The disclosure described herein allows target antigens for humoral immunity to be displayed on the adenovirus capsid surface, as well as simultaneous expression of vector encoded antigens, generating eguivalent T cell responses to conventional adenovirus vectors. In a proof of concept study, adenoviral vaccines displaying the receptor binding domain (RBD) from SARS-CoV-2 S generated ~50- fold higher SARS-COV-2 neutralisation titres compared to conventional adenovirus vaccines encoding S (Dicks et al., 2022).

[0105] Vaccines

[0106] A vaccine is a preparation that comprises a fragment or entire entity against which it is possible to raise an immune response. It is an entity such as a protein, peptide, lipoprotein, glycoprotein, polysaccharide or fragments thereof that are capable of inducing an immune response. For example, the vaccine may comprise micro-organisms or a part thereof capable of inducing an immune response against said micro-organism. A vaccine comprising an immunogenic adenoviral vector in accordance with the invention can be used against any pathogen for which the antigen displayed or encoded by the transgene for the induction of an immune response against the antigen

[0107] Such vaccine compositions (or other immunogenic) are formulated in a suitable delivery vehicle. Generally, doses for the immunogenic compositions are within the ranges defined for therapeutic compositions. Optionally, an adenoviral vector or vaccine composition of the disclosure may be formulated to contain other components, including, for example, adjuvants, stabilizers, pH adjusters, preservatives and the like. Such components are well known to those skilled in the art of vaccines. Examples of suitable adjuvants include, without limitation, liposomes, alum, monophosphoryl lipid A, saponins, such as Qs21 , and any biologically active factor, such as a cytokine, an interleukin, a chemokine and optimally combinations thereof.

[0108] The adenoviral vector of the present disclosure can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, rectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, intrapericardially, orally, locally and / or using aerosol, injection, infusion, continuous infusion, localized perfusion bathing target cells directly or via a catheter and / or lavage. Typically the vaccines of the invention are administered intramuscularly.

[0109] The adenoviral vector or vaccine herein disclosed may be used to treat or prevent infection with HBV, or HBV in combination with HDV (HBV infection and / or HDV infection). Pharmaceutical composition and use

[0110] The compositions of this disclosure may be incorporated into a vaccine or therapeutic composition. Suitably, a vaccine or immunogenic composition will comprise particles of the invention in an immunogenic dose.

[0111] A pharmaceutical composition may comprise a particle or composition in accordance with the invention provided with a pharmaceutically acceptable carrier. Suitable carriers are well known to those skilled in the art. In one embodiment a pharmaceutical composition comprises a buffer, excipient or carrier. Suitably a pharmaceutical composition may comprise suitable excipients and formulations to maintain stability of the composition. Suitably the formulation may comprise an adjuvant. In one embodiment, the formulation may comprise AddaVax™ or a similar squalene-based oil-in-water nanoemulsion with a formulation similar to MF59®. Other suitable adjuvants include liposome-based adjuvants such as Matrix M and AS01. Other suitable adjuvants include aluminium-based formulations such as Alhydrogel®. In one embodiment the formulation may comprise EDTA, for example at a concentration of 5 mM. Suitable excipients or formulations may depend on the properties of the particle or immunogenic composition; for example, the choice of expression system may affect the stability, glycosylation or folding of the proteins of the composition, which may in turn affect the optimal formulation of the composition. Methods of determination of a suitable excipient, formulation or adjuvant will be known to those skilled in the art.

[0112] Present disclosure

[0113] This disclosure provides candidate therapeutic HBV vectors and vaccines that (i) display the PreS1 antigen from HBV-L on the adenoviral surface, and optionally (ii) encode antigens from HBV. PreS1 is the receptor binding domain required for HBV host cell entry and a key target for HBV neutralizing antibodies (NAbs). Current prophylactic HBV vaccines do not incorporate PreS1 because they are based on HBV HBsAg-S (small subunit of HBV surface antigen). PreS1 display on VLPs is known to generate efficacious NAb responses in a mouse model of chronic HBV infection (Wang et al., 2020). The encoded antigens from HBV generate potent CD8+ and CD4+ T cell responses.

[0114] The candidate therapeutic HBV vaccines disclosed herein are capable of inducing robust IgG antibody responses against PreS1 and strong T cell responses against two full-length encoded HBV proteins (HBsAg-L (L) and Core (C)) in mice.

[0115] This disclosure demonstrates that the technology enables flexibility in the design of the vaccine candidates; the magnitudes of immune responses are shown to be independent of encoded antigen orientation and PreS1 capsid coverage.

[0116] The disclosed transgenic construct design of HBV L_P2A_C with PreS1 capsid decoration via isopeptide binding partners achieves robust anti-PreS1 IgG titers, and strong T cell responses against both L and C antigens. Robust PreS1 antibody responses (such as those achieved here) would be expected to effectively neutralise HBV infection. Studies to test the capacity of the candidate vaccines to induce NAb against different HBV strains is ongoing.

[0117] It is possible to incorporate different strains of HBV into the vectors and vaccines to increase the breadth of immunity provided and I or to target the vaccine to specific genotypes.

[0118] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0119] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0120] “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0121] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0122] It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments. It is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.

[0123] "Recombinant" as used herein to describe a polynucleotide means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and / or (2) is linked to a polynucleotide other than that to which it is linked in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.

[0124] The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

[0125] The term "about" in relation to a numerical value x is optional and means, for example, x±10%.

[0126] Unless specifically stated, a process or method comprising a series of steps does not require any specific order of carrying out the steps, any suitable order may be used including contemporaneously. For example a process or method comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc. Sequence identity between polypeptide sequences is preferably determined by pairwise alignment algorithm using the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch 1970), using default parameters (e.g. with Gap opening penalty = 10.0, and with Gap extension penalty = 0.5, using the EBLOSUM62 scoring matrix). This algorithm is conveniently implemented in the needle tool in the EMBOSS package (Rice, Longden and Bleasby 2000). Sequence identity should be calculated over the entire length of a polypeptide sequence unless otherwise stated.

[0127] Any polypeptide sequence disclosed herein may have a sequence which has at least 60%, 70%, 80%, 85%, 90%, 95% or 99% sequence identity to its wild-type sequence. Variants and derivatives of a polypeptide may comprise an amino acid sequence that is at least 90% or 95% similar to the wild-type sequence. Homologues of these entities may therefore have at least 60% homology thereto, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% homology thereto.

[0128] Examples

[0129] Example 1 : PreS1 capsid display generates robust humoral immunity

[0130] A schematic of an adenoviral construct with an isopeptide tag fused to the capsid protein is represented in Figure 1A. Figure 1 B shows a similar construct with an exemplary GFP gene inserted into the adenoviral genome and HBV PreS1 conjugated to the adenovirus particle surface via the covalent bond between the tag (first peptide partner) and the catcher (second peptide partner).

[0131] A proof-of-concept replication defective (E1 / E3 deleted) adenoviral vector was constructed based on the Ad5 serotype with a DogTag sequence genetically inserted in hexon hypervariable 5 (HVR5) surface loop by GalK recombineering (Dicks et al., 2022). For insertion at HVR5, native Ad5 hexon residues 269-281 were deleted and replaced with DogTag (DIPATYEFTDGKHYITNEPIPPK; SEQ ID NO: 22) flanked by GSGGSG flexible linkers (amino acid sequence for this hexon construct is SEQ ID NO: 55; nucleotide sequence is SEQ ID NO: 56). Equally, another adenovirus genotype, or another HVR loop could have been selected as the insertion site, for example HVR1 or HVR2. An enhanced GFP gene was inserted into the adenoviral genome at the E1 locus, downstream of the immediate-early cytomegalovirus promoter (Dicks et al., 2022).

[0132] To generate DogCatcher-PreS1 protein, the nucleotide sequence encoding HBV genotype D ayw (NC_003977) PreS1 region minus the initial methionine residue (equivalent to HBV-L antigen amino acids 2-108 of Genbank NC_003977; amino acid sequence SEQ ID NO: 48) was cloned into expression vector pET45(+) (EMD Millipore) for protein production in BL21 (DE3) Escherichia coll (NEB). The variant DogCatcher nucleotide sequence from Keeble et al. (2022) (encoding DogCatcher V1 amino acid sequence SEQ ID NO: 10) was fused at the 5’-end of the PreS1 gene, with a GSGGSGGS linker encoded between the DogCatcher and PreS1 sequences (DogCatcher-linker-PreS1 construct amino acid sequence SEQ ID NO: 57, nucleotide sequence SEQ ID NO: 58). Recombinant proteins were purified using Ni-NTA affinity resin (Qiagen) according to standard protocols, dialyzed into PBS, and stored at -80 degrees C.

[0133] The DogTag-tagged adenovirus particles and DogCatcher-PreS1 proteins were mixed under conditions permissive to conjugation. Successful conjugation of PreS1 via DogCatcher to DogTag on the adenoviral particle at various concentrations of DogCatcher-PreS1 was determined by SDS-PAGE and coupling efficiency at each concentration of DogCatcher-PreS1 was determined (Figure 1 C). Coupling efficiency ranged from ~25% (0.3 pM DogCatcher-PreS1) to >90% (5 pM DogCatcher-PreS1).

[0134] Successful expression of GFP from the adenovirus transgene was determined by fluorescence microscopy. In vitro infectivity of 293A cells by the DogCatcher-PreS1 decorated adenoviral vectors was determined (Figure 1 D); at concentrations of 0.7 pM conjugated DogCatcher-PreS1 (‘DC-PreS1 ’) or lower decorated vector infectivity remained unchanged compared to undecorated vector (0 pM DogCatcher-PreS1 (‘Ad-DT only’), Figure 1 D).

[0135] Example 2: PreS1 capsid display usinq mammalian expressed PreS1

[0136] In this example, the same proof-of-concept Ad5 adenoviral vector with DogTag genetically inserted in hexon HVR5 and enhanced GFP inserted into the adenoviral genome at the E1 locus as Example 1 was used.

[0137] In this example, and all subsequent examples, DogCatcher-PreS1 protein was expressed in mammalian cells, although any suitable protein expression system may be used. To generate DogCatcher-PreS1 protein, the nucleotide sequence encoding HBV genotype D ayw PreS1 region (equivalent to HBV-L antigen amino acids 3-108, Genbank NC_003977) was cloned into expression vector pcDNA3.4 for protein production in Chinese hamster ovary (CHO) cells. Compared to the native HBV genotype D ayw PreS1 region, the initial Methionine residue was removed as the sequence was designed to be fused with DogCatcher at its N terminus, and the second native residue (Glycine) was removed in order not to inhibit protein production in mammalian cells because it is myristoylated in the native sequence. The DogCatcher nucleotide sequence (encoding DogCatcher V1 amino acid sequence SEQ ID NO: 10) was genetically fused at 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 at 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); protein was harvested from culture supernatant, affinity purified using C-tag affinity resin (Thermo Fisher) using an AKTA chromatography system (GE Healthcare), and dialyzed into Tris-buffered saline (TBS) pH7.4.

[0138] The DogTag-tagged adenovirus particles and DogCatcher-PreS1 proteins were mixed under conditions permissive to conjugation. Successful conjugation of PreS1 via DogCatcher to DogTag on the adenoviral particle at increasing concentrations of DogCatcher-PreS1 was determined by SDS-PAGE (Figure 2A) and coupling efficiency at each concentration of DogCatcher-PreS1 was determined (Figure 2B). Coupling efficiency ranged from ~20% (0.6 pM DogCatcher-PreS1) to >60% (concentrations of DogCatcher-PreS1 from 3.5 pM to 5 pM). Successful expression of GFP from the adenovirus transgene was determined by fluorescence microscopy. In vitro infectivity of 293A cells by the DogCatcher-PreS1 decorated adenoviral vectors was determined (Figure 2C); at all concentrations of conjugated DogCatcher-PreS1 infectivity remained unchanged compared to undecorated vector.

[0139] Table 3: Mouse study design (homologous prime-boost):

[0140] Candidate vaccines based on this Ad5 serotype vector and unconjugated PreS1 were tested in mice in a homologous prime-boost regimen. Table 3 shows the mouse immunization schedule. Mice were vaccinated on day 0 and day 21 with either unconjugated adenovirus expressing GFP, adenovirus expressing GFP and decorated with PreS1 , or unconjugated DogCatcher-PreS1 recombinant protein.

[0141] Post-prime tail vein blood was taken on day 20 and the study was terminated on day 35 with cardiac blood and spleens harvested. Effective PreS1 dose delivered by Ad(GFP):PreS1 < 0.2pg.

[0142] Results

[0143] The serum IgG antibody responses to PreS1 were measured by Endpoint ELISA (Fig. 1 E). Responses measured post-prime on Day 20 were compared to those measured post-boost on Day 35. The data were analysed in prism v9. Data were checked for normality using the Shapiro-Wilk test and then analysed by one-way ANOVA with post-test correction.

[0144] CD8+ T cell responses in the spleen to encoded EGFP were measured by overnight ex vivo IFNy-ELISpot assay (Fig. 1 F). 5x105cells / well were incubated with 5 pg / ml EGFP118-126 peptide. Spots were counted on an AID ELISpot reader v7 and analysed in prism v9. Data were checked for normality using the Shapiro-Wilk test and then analysed by one-way ANOVA with post-test correction.

[0145] Conclusions: Potent PreS1 IgG responses were generated when mice were immunized with PreS1 capsid decorated Ad, but not with recombinant DogCatcher-PreS1 protein plus Alhydrogel® adjuvant. Capsid decoration with PreS1 did not inhibit splenic CD8+ T cell responses against encoded GFP compared to the undecorated vector. This is important, since induction of potent CD8+ T cell responses against encoded transgene antigens are a hallmark of adenovirus vector vaccines and are considered therapeutically beneficial in many settings including treatment of chronic Hepatitis B infection. Typically, technologies that have involved decorating or coating the adenovirus capsid surface of adenovirus (such as attachment of polymers to achieve capsid shielding from undesirable interactors) have tended to reduce vector infectivity and would therefore be predicted to significantly lower CD8+ T cell immunogenicity against encoded antigens.

[0146] Example 3: Optimising anti-HBV PreS1 humoral immunity

[0147] The adenoviral vector as in Examples 1 and 2 was used, except the GFP transgene was replaced with one of two encoded cassettes: ‘HBV L’ encodes the large HBsAg polypeptide (encompassing PreS1 , PreS2 and S protein) from HBV strain ayw genotype D (NC_003977), amino acid sequence SEQ ID NO: 45, and ‘HBV C-S’ encodes HBV Core (amino acid sequence SEQ ID NO: 50) and the small HBsAg polypeptide (amino acid sequence SEQ ID NO: 47) also from strain ayw genotype D (NC_003977), with an intervening F2A (FMDV18 2A) cleavage sequence (amino acid sequence SEQ ID NO: 51) between Core and S. Both constructs also encoded shark invariant chain (Sli; SEQ ID NO: 63) N-terminally, included to enhance T cell responses. Figure 3A shows a schematic of an adenoviral vector with PreS1 conjugated to the surface via a tag-catcher covalent bond, and either HBV L or HBV C- S transgenic cassette inserted into the adenovirus genome.

[0148] DogCatcher-PreS1 protein was expressed in mammalian cells as per Example 2.

[0149] Table 4: Mouse study design (homologous prime-boost):

[0150] Vaccine candidates were tested in mice in a homologous prime-boost regimen with no adjuvant. Table 4 shows the mouse immunization schedule. Post-prime tail vein blood was taken on day 20 and the study was terminated on day 35 with cardiac blood and spleens harvested

[0151] Results

[0152] Serum IgG antibody responses to PreS1 were measured by Endpoint ELISA (Fig. 3B) for each of the vectors in Table 4. Responses measured post-prime on D20 were compared to those measured post-boost on D35. The data were analysed in prism v9. Data were checked for normality using the Shapiro-Wilk test and then analysed by one-way ANOVA with post-test correction.

[0153] Neutralizing antibody responses generated by vaccination with Ad(L), Ad(L):PreS1 , and Ad(GFP) (as per Example 2) were measured using an in vitro HBV neutralization assay on HepG2-NTCP (HBV infectable) cells (Fig. 3C). HBV virus expressing luciferase and serum from vaccinated mice (diluted 1 :250) was co-incubated with the cells; neutralization was expressed as reduction in luminescence signal (in relative light units, RLU) relative to control (luciferase-expressing HBV virus only). From Fig. 3C it can be seen that vaccination with Ad(GFP) did not induce a reduction in luminescence / HBV neutralization compared to control, vaccination with Ad(L) induced only very modest HBV neutralization (<4-fold reduction in HBV infection), whereas vaccination with Ad(L):PreS1 provided potent neutralizing immunity (>80-fold reduction in infection).

[0154] Conclusion: Optimum PreS1 antibody responses were achieved when PreS1 is both displayed (:PreS1) and encoded (L). Crucially, capsid display of PreS1 is required for potent HBV neutralization.

[0155] Example 4: Adenoviral vector displaying PreS1 and encoding C and L induces a potent combination of anti-HBV T-cell and antibody immunity

[0156] Adenoviral vector Ad(L) with encoded cassette ‘HBV L’ from Example 3 was used, along with two additional vectors: Adenoviral vector Ad(C-L) carried the cassette ‘HBV C-L’ which encoded the HBV Core protein (amino acid sequence SEQ ID NO: 50) upstream of and separated by an F2A cleavage sequence (amino acid sequence SEQ ID NO: 51) from the large HBsAg protein (amino acid sequence SEQ ID NO: 45), whereas Adenoviral vector Ad(L-C) carried the cassette ‘HBV L-C’ which encoded the same proteins but with HBsAg-L upstream of HBV Core, also with an intervening F2A sequence. Figure 4A shows a schematic of these constructs with PreS1 shown decorating the surface via the covalent bond between the tag (first peptide partner) and the catcher (second peptide partner). All three vectors were decorated with DogCatcher-PreS1 at a similar density to the previous examples (‘HC’), in addition a fourth treatment of Adenoviral vector Ad(L) (carrying cassette ‘HBV L’) with DogCatcher-PreS1 conjugated at lower density (‘LC’).

[0157] LC = ~30% ‘low’ PreS1 capsid coverage (~215 copies / virion). HC = ~60% ‘high’ PreS1 capsid coverage (~430 copies / virion).

[0158] Table 5: Mouse study design

[0159] Vaccine candidates were tested in mice in homologous prime-boost regimen (no adjuvant). Table 5 shows the mouse immunization schedule. Post-prime tail vein blood was taken on day 20 and the study was terminated on day 35 with cardiac blood and spleens harvested.

[0160] Results

[0161] CD8+ T cell responses in the spleen to encoded genes were measured by overnight ex vivo IFNy-ELISpot assay. 2.5x105cells / well were incubated with 5pg / ml S190-197 peptide (CD8+ epitope) (Fig. 4B), Core93-ioo peptide (CD8+ epitope) (Fig. 4C), or Corepi3L peptide (CD4+ epitope) (Fig. 4D). Spots were counted on an AID ELISpot reader v7 and analysed in prism v9. Data were checked for normality using the Shapiro-Wilk test and then analysed by one-way ANOVA with post-test correction.

[0162] Serum IgG antibody responses to PreS1 were measured by Endpoint ELISA (Fig. 4E). Responses measured post-prime on D20 are compared to those measured post-boost on D35. The data were analysed in prism v9. Data were checked for normality using the Shapiro- Wilk test and then analysed by one-way ANOVA with post-test correction, no significant differences were found.

[0163] Neutralizing antibody responses generated by vaccination with Ad(L-C):PreS1 and Ad(C- L):PreS1 (both decorated with PreS1 at high capsid coverage) were measured using the in vitro HBV neutralization assay described in Example 3 / Fig. 3C - see Fig. 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; reduction in RLU signal with increasing serum concentration is shown in Fig. 4G.

[0164] Conclusion: Robust CD8+ T cell and CD4+ T cell responses were obtained to both L and C encoded antigens. Neither order of encoded L and C antigens nor extent of PreS1 capsid coverage affected the magnitude of the T cell response. Robust anti-PreS1 IgG antibody titres were achieved. Neither order of encoded L and C antigens nor extent of PreS1 capsid coverage affected PreS1 ELISA titres. Order of encoded L and C antigens had little effect on neutralization titres, both Ad(L-C):PreS1 and Ad(C- L):PreS1 vaccines induced potent neutralizing immunity.

[0165] Example 5: Increasing In vitro expression of HBV-L / S and HBV-C by construct design

[0166] A number of undecorated adenoviral vectors were generated with the aim of generating significant expression of ‘unfused’ HBV-L and HBV-C antigens, by incorporating nucleotide seguences arranged to direct cleavage between the antigen polypeptides. The constructs were similar to those used in Examples 1-4, and are summarized in Table 6 and as follows, where Core = HBV Core, S = small HBsAg antigen, L = large HBsAg antigen, F2A = FMDV18 2A cleavage seguence, P2A = porcine teschovirus-1 2A cleavage seguence, IRES = Internal Ribosome Entry Site cleavage seguence from Encephalomyocarditis virus, Sli = shark invariant chain, and GSGGS, GSG, GGS are linkers.

[0167] Table 6: Adenoviral constructs

[0168] 293A cells were infected with the vectors summarized in Table 6 at an MOI of 100 ifu for 16-18 hours before being harvested, lysed and analysed by Western blot. Antibodies used were mouse anti- HBsAg (Native antigen, clone 1837) used at 1 :200 and mouse anti-Core (SantaCruz, clone 10E11) used at 1 :200, both detected with goat anti-mouse alkaline phosphatase (Biorad, clone STAR117A) used at 1 :1000. As controls, Adenovirus hexon was detected with mouse anti-hexon (Invitrogen, clone 65H6) used at 1 :500 and detected with STAR117A at 1 :1000, and B-Actin was detected with rabbit anti-B-actin (Cell Signalling Technologies, clone D6A8) used at 1 :500 and detected with goat anti-rabbit alkaline phosphatase (Merck) used at 1 :1000. See Fig. 5 for the Western blot results. Detected proteins are labelled based on their predicted sizes. Different glycosylated versions of S are produced naturally, hence the doublet and quadruplicate bands detected with S staining of S and L expressing constructs.

[0169] Conclusion: Constructs D, E, G, and J generated significant expression of ‘unfused’ HBV-L. Of these, only construct D generated significant expression of ‘unfused’ HBV-C. P2A ‘cleavage’ therefore appeared to be superior to F2A, but not as efficient as IRES, but expression of the ORF downstream of IRES was poor (construct E).

[0170] Example 6: Anti-PreS1 antibody responses post-boost improved via inclusion of P2A or IRES sequences between ORFs compared to direct fusion

[0171] Some of the vectors from Example 5 were selected for decoration (conjugation) with PreS1 via DogTag-DogCatcher isopeptide binding. The vectors used are summarized in Table 7. With respect to 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(L-C)). Fig. 6A shows a schematic of the PreS1 -conjugated vectors showing protein coupled to the vector surface via a tag-catcher isopeptide pair, and the encoded transgenes.

[0172] Table 7: Mouse study design:

[0173] Table 7 shows the mouse immunization schedule. Post-prime tail vein blood was taken on day 20 and the study was terminated on day 35 with cardiac blood and spleens harvested.

[0174] Serum IgG antibody responses to PreS1 were measured by Endpoint ELISA (Fig. 6B). The data were analysed in prism v9. Data were checked for normality using the Shapiro- Wilk test and then analysed by one-way ANOVA with post-test correction. At the pre-boost timepoint (day 20), no significant differences were detected between vaccines. At the post-boost timepoint (day 35), significant differences in Endpoint titre were detected between sera from mice vaccinated with Ad(L-P2A-C):PreS1 and Ad(L- C):PreS1 , and Ad(L-IRES-C):PreS1 and Ad(L-C):PreS1 .

[0175] T cell responses in the spleen to encoded genes were measured by overnight ex vivo IFNy- ELISpot assay. 2.5x105cells / well were incubated with 5pg / ml S190-197 peptide (CD8+ epitope; SEQ ID NO: 64) (Fig. 6C), or Coregs wo peptide (CD8+ epitope; SEQ ID NO: 65) (Fig. 6D), or Corepi3L peptide (CD4+ epitope; SEQ ID NO: 66) (Fig. 6E). Spots were counted on an AID ELISpot reader v7 and analysed in prism v9. Data were checked for normality using the Shapiro-Wilk test and then analysed by one-way ANOVA with post-test correction.

[0176] Conclusion: P2A, IRES and L-C (no cleavage) generated the highest median CD8+ responses against HBsAg-L, but IRES generated the weakest CD8+ response against HBV Core, and L-C (no cleavage) generated a weak CD4+ response against HBV Core. Use of P2A sequence between ORFs generated robust CD8 T cell responses against both HBV-L and HBV-C antigens.

[0177] Example 7: PreS1 capsid display on chimpanzee adenovirus serotype ChAd63 induces potent humoral immunity

[0178] A further proof-of-concept replication defective (E1 / E3 deleted) adenoviral vector was constructed based on the ChAd63 chimpanzee adenovirus serotype with a DogTag sequence genetically inserted in hexon hypervariable 5 (HVR5) surface loop by GalK recombineering. For insertion at HVR5, native ChAd63 hexon residues 256-264 were deleted and replaced with DogTag (DIPATYEFTDGKHYITNEPIPPK; SEQ ID NO: 22) flanked by GSGGSGGSGGSG (SEQ ID NO: 44) flexible linkers.

[0179] Transgene antigens (either GFP, or L-P2A-C as described above) were inserted into the ChAd63 adenoviral genome at the E1 locus, downstream of the immediate-early cytomegalovirus promoter, and vectors were decorated with DogCatcher-PreS1 (SEQ ID NO: 59) (Fig. 7A).

[0180] Candidate vaccines based on this ChAd63 serotype vector were tested in mice in a homologous prime-boost regimen (no adjuvant). Table 8 shows the mouse immunization schedule. Post-prime tail vein blood was taken on day 20 and the study was terminated on day 35 with cardiac blood and spleens harvested. Vaccines tested were: undecorated ChAd63 expressing encoded L and C (ChAd63(L-P2A- C)), GFP-expressing ChAd63 displaying PreS1 at low density (ChAd63(GFP):PreS1iow), or high density, (ChAd63(GFP):PreS1high), and ChAd63 expressing encoded L and C antigens and displaying PreS1 at low density (ChAd63(L-P2A-C):PreS1 iow), or high density (ChAd63(L-P2A-C):PreS1high). PreS1 capsid coverage in each group is shown in Table 8. Low and high density in this example are equivalent to high coverage and low coverage (‘HC’ and ‘LC’) in Example 4. Table 8: Mouse study design

[0181] Serum IgG antibody responses to PreS1 were measured by Endpoint ELISA (Fig. 7B). The data were analysed in prism v9. Data were checked for normality using the Shapiro- Wilk test and then analysed by one-way ANOVA with post-test correction.

[0182] T cell responses in the spleen to encoded genes were measured by overnight ex vivo IFNy- ELISpot assay. 2.5x105cells / well were incubated with 5pg / ml S190-197 peptide (CD8+ epitope) (Fig. 7C), Core93-ioo peptide (CD8+ epitope) (Fig. 7D), or Corepi3L peptide (CD4+ epitope) (Fig. 7E). Spots were counted on an AID ELISpot reader v7 and analysed in prism v9. Data were checked for normality using the Shapiro-Wilk test and then analysed by one-way ANOVA with post-test correction.

[0183] Conclusion: PreS1 capsid decoration on ChAd63 induced potent antibody responses, with endpoint titres comparable in magnitude to those induced using Ad5. PreS1 antibody responses were comparable between vaccines decorated with PreS1 at low and high density, and between vectors encoding L-P2A-C and GFP. PreS1 decorated vaccines induced >100-fold higher anti-PreS1 IgG titres compared to undecorated ChAd63(L-P2A-C) post-boost. PreS1 decorated vaccines encoding L and C antigens induced strong T cell responses against both antigens. Sequences

[0184] Hepatitis B virus (reference strain NC 003977 (ayw)) Large HBsAq (HBsAq L)

[0185] Amino acid sequence (SEQ ID NO: 45):

[0186] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGW

[0187] SPQAQGILQTLPANPPPASTNRQSGRQPTPLSPPLRNTHPQAMQWNSTTFHQTLQDPRVRGLYFPAGG

[0188] SSSGTVNPVLTTASPLSSIFSRIGDPALNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLG

[0189] GTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSST

[0190] TSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWF

[0191] VGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI

[0192] Hepatitis B virus (reference strain NC 003977 (ayw)) Medium HBsAq

[0193] Amino acid sequence (SEQ ID NO: 46):

[0194] MQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVLTTASPLSSIFSRIGDPALNMENITSGFLGPLLVL QAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFI LLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWA FGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI

[0195] Hepatitis B virus (reference strain NC 003977 (ayw)) Small HBsAq (HBsAq S)

[0196] Amino acid sequence (SEQ ID NO: 47):

[0197] MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCP GYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKP SDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILS PFLPLLPIFFCLWVYI

[0198] Hepatitis B virus (reference strain NC 003977 (ayw)) PreS1

[0199] Amino acid sequence (SEQ ID NO: 48):

[0200] MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGW SPQAQGILQTLPANPPPASTNRQSGRQPTPLSPPLRNTHPQA

[0201] Hepatitis B virus (reference strain NC 003977 (ayw)) PreS2

[0202] Amino acid sequence (SEQ ID NO: 49):

[0203] MQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVLTTASPLSSIFSRIGDPALN

[0204] Hepatitis B virus (reference strain NC 003977 (ayw)) Core (C)

[0205] Amino acid sequence (SEQ ID NO: 50):

[0206] MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLAT WVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPIL STLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC F2A

[0207] Amino acid sequence (SEQ ID NO: 51):

[0208] VKQTLNFDLLKLAGDVESNPGP

[0209] P2A

[0210] Amino acid sequence (SEQ ID NO: 52):

[0211] ATNFSLLKQAGDVEENPGP

[0212] HBV-L-P2A-C

[0213] Amino acid sequence (SEQ ID NO: 53):

[0214] MSLLWGGVTVLAAMLIAGQVASVVFLVGSGGSGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPN

[0215] KDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPPASTNRQSGRQPTPLSPPLRNTH

[0216] PQAMQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVLTTASPLSSIFSRIGDPALNMENITSGFLGPL

[0217] LVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFII

[0218] FLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPS

[0219] SWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCL

[0220] WVYIGSGATNFSLLKQAGDVEENPGPMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALES

[0221] PEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRE

[0222] TVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQ

[0223] C

[0224] HBV-L-P2A-C

[0225] Nucleic acid sequence (SEQ ID NO: 54):

[0226] ATGTCTTTGCTGTGGGGCGGAGTTACAGTGCTGGCCGCCATGCTGATTGCTGGACAGGTGGCCTCT

[0227] GTGGTGTTTCTCGTTGGAAGCGGCGGATCCGGCCAGAACCTGTCCACCTCTAATCCACTGGGATTC

[0228] TTCCCAGACCACCAGCTGGACCCTGCCTTCAGAGCCAATACCGCCAATCCTGACTGGGACTTCAAC

[0229] CCCAACAAGGACACCTGGCCTGACGCCAACAAAGTTGGCGCTGGCGCTTTTGGCCTGGGCTTTACA

[0230] CCTCCTCATGGCGGACTGCTTGGATGGTCACCTCAGGCTCAGGGCATCCTGCAAACCCTGCCAGCT

[0231] AATCCTCCTCCTGCCTCCACCAACAGACAGAGCGGTAGACAGCCCACACCTCTGAGCCCTCCACTG

[0232] AGAAACACACACCCTCAGGCCATGCAGTGGAACAGCACCACCTTCCACCAGACACTGCAGGACCCT

[0233] AGAGTGCGGGGCCTGTATTTTCCAGCCGGCGGATCTAGCAGCGGCACCGTGAATCCTGTGCTGAC

[0234] CACAGCCTCTCCACTGTCCTCCATCTTCAGCAGAATCGGCGACCCCGCTCTGAACATGGAGAATAT

[0235] CACCAGCGGCTTTCTGGGCCCTCTGCTGGTTCTGCAGGCCGGCTTCTTTCTGCTGACCCGCATCCT

[0236] GACAATCCCTCAGAGCCTGGATAGCTGGTGGACCAGCCTGAATTTTCTCGGCGGCACCACAGTGTG

[0237] CCTGGGACAGAATAGCCAGTCTCCTACCTCCAATCACAGCCCCACAAGCTGCCCTCCAACCTGTCC

[0238] TGGCTACAGATGGATGTGCCTGCGGCGGTTCATCATCTTTCTGTTCATCCTGCTGCTGTGCCTGATC

[0239] TTCCTGCTGGTGCTGCTGGACTACCAGGGCATGCTGCCTGTGTGTCCTCTGATCCCTGGCAGCAGC

[0240] ACAACAAGCACAGGCCCTTGCAGAACCTGCATGACAACAGCCCAGGGCACCAGCATGTACCCTAGC

[0241] TGCTGTTGCACCAAGCCAAGCGACGGCAACTGCACATGCATCCCCATTCCTAGCAGCTGGGCCTTC

[0242] GGCAAGTTTCTGTGGGAATGGGCCTCCGCCAGATTCAGCTGGCTGTCTCTCCTGGTGCCATTCGTG

[0243] CAGTGGTTCGTGGGACTGAGCCCTACAGTGTGGCTGAGCGTGATCTGGATGATGTGGTACTGGGG CCCTAGCCTGTACTCTATTCTGAGCCCCTTCTTGCCCCTGCTGCCTATCTTCTTTTGCCTGTGGGTG

[0244] TACATCGGCAGCGGCGCCACAAATTTCAGCCTGCTGAAACAGGCCGGCGACGTGGAAGAGAACCC

[0245] TGGACCTATGGACATCGACCCCTACAAAGAATTTGGCGCCACCGTGGAACTGCTGAGCTTCCTGCC

[0246] TAGCGACTTCTTCCCTTCCGTGCGGGATCTGCTGGATACAGCCAGCGCACTGTATAGAGAGGCCCT

[0247] GGAAAGCCCCGAGCACTGTTCTCCACATCACACAGCCCTGAGACAGGCCATCCTGTGTTGGGGCG

[0248] AACTGATGACACTGGCCACATGGGTCGGAGTGAACCTGGAAGATCCCGCCAGCAGAGATCTGGTG

[0249] GTGTCCTACGTGAACACCAACATGGGCCTGAAGTTCCGGCAGCTGCTGTGGTTCCACATCTCCTGC

[0250] CTGACCTTCGGCCGGGAAACCGTGATCGAGTACCTGGTGTCTTTCGGCGTGTGGATCAGAACCCCT

[0251] CCAGCCTACAGACCTCCTAACGCTCCCATCCTGAGCACCCTGCCTGAGACAACAGTCGTGCGGAGA

[0252] AGAGGCAGAAGCCCCAGAAGAAGAACCCCATCTCCTCGGAGGCGGAGATCTCAGTCCCCTCGGAG

[0253] AAGAAGATCCCAGAGCAGAGAAAGCCAGTGCTGA

[0254] Ad5-HVR5-DoqTaq Hexon

[0255] Amino acid sequence (SEQ ID NO: 55):

[0256] MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDVTTDRSQRLTLRFI

[0257] PVDREDTAYSYKARFTLAVGDNRVLDMASTYFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCEWDE

[0258] AATALEINLEEEDDDNEDEVDEQAEQQKTHVFGQAPYSGINITKEGIQIGVEGQTPKYADKTFQPEPQIGE

[0259] SQWYETEINHAAGRVLKKTTPMKPCYGSYAKPTNENGGQGILVKQQNGKLESQVEMQFFSGSGGSGDI

[0260] PATYEFTDGKHYITNEPIPPKGSGGSGPKVVLYSEDVDIETPDTHISYMPTIKEGNSRELMGQQSMPNRP

[0261] NYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSIGDRTRYFSMWNQAVDS

[0262] YDPDVRIIENHGTEDELPNYCFPLGGVINTETLTKVKPKTGQENGWEKDATEFSDKNEIRVGNNFAMEIN

[0263] LNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNTYDYMNKRVVAPGLVDCYINLGARWSLDYMDNV

[0264] NPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDL

[0265] RVDGASIKFDSICLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNW

[0266] AAFRGWAFTRLKTKETPSLGSGYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNE

[0267] FEIKRSVDGEGYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTK

[0268] YKDYQQVGILHQHNNSGFVGYLAPTMREGQAYPANFPYPLIGKTAVDSITQKKFLCDRTLWRIPFSSNFM

[0269] SMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVHRPHRGVIETVYLRTPFSAGN

[0270] ATT

[0271] Ad5-HVR5-DoqTaq Hexon

[0272] Nucleic acid sequence including The STOP codon, the lower-case text is the sequence for DoqTaq

[0273] (SEQ ID NO: 56):

[0274] ATGGCTACCCCTTCGATGATGCCGCAGTGGTCTTACATGCACATCTCGGGCCAGGACGCCTCGGAG

[0275] TACCTGAGCCCCGGGCTGGTGCAGTTTGCCCGCGCCACCGAGACGTACTTCAGCCTGAATAACAA

[0276] GTTTAGAAACCCCACGGTGGCGCCTACGCACGACGTGACCACAGACCGGTCCCAGCGTTTGACGC

[0277] TGCGGTTCATCCCTGTGGACCGTGAGGATACTGCGTACTCGTACAAGGCGCGGTTCACCCTAGCTG

[0278] TGGGTGATAACCGTGTGCTGGACATGGCTTCCACGTACTTTGACATCCGCGGCGTGCTGGACAGG

[0279] GGCCCTACTTTTAAGCCCTACTCTGGCACTGCCTACAACGCCCTGGCTCCCAAGGGTGCCCCAAAT

[0280] CCTTGCGAATGGGATGAAGCTGCTACTGCTCTTGAAATAAACCTAGAAGAAGAGGACGATGACAAC GAAGACGAAGTAGACGAGCAAGCTGAGCAGCAAAAAACTCACGTATTTGGGCAGGCGCCTTATTCT GGTATAAATATTACAAAGGAGGGTATTCAAATAGGTGTCGAAGGTCAAACACCTAAATATGCCGATA AAACATTTCAACCTGAACCTCAAATAGGAGAATCTCAGTGGTACGAAACTGAAATTAATCATGCAGCT GGGAGAGTCCTTAAAAAGACTACCCCAATGAAACCATGTTACGGTTCATATGCAAAACCCACAAATG AAAATGGAGGGCAAGGCATTCTTGTAAAGCAACAAAATGGAAAGCTAGAAAGTCAAGTGGAAATGCA ATTTTTCTCAGGCAGCGGAGGATCCGGCgatattccggctacatatgaatttaccgatggtaaacattatatcaccaatgaaccg ataccgccgaaaGGCTCTGGCGGAAGCGGCCCTAAAGTGGTATTGTACAGTGAAGATGTAGATATAGAA ACCCCAGACACTCATATTTCTTACATGCCCACTATTAAGGAAGGTAACTCACGAGAACTAATGGGCC AACAATCTATGCCCAACAGGCCTAATTACATTGCTTTTAGGGACAATTTTATTGGTCTAATGTATTACA ACAGCACGGGTAATATGGGTGTTCTGGCGGGCCAAGCATCGCAGTTGAATGCTGTTGTAGATTTGC AAGACAGAAACACAGAGCTTTCATACCAGCTTTTGCTTGATTCCATTGGTGATAGAACCAGGTACTTT TCTATGTGGAATCAGGCTGTTGACAGCTATGATCCAGATGTTAGAATTATTGAAAATCATGGAACTGA AGATGAACTTCCAAATTACTGCTTTCCACTGGGAGGTGTGATTAATACAGAGACTCTTACCAAGGTA AAACCTAAAACAGGTCAGGAAAATGGATGGGAAAAAGATGCTACAGAATTTTCAGATAAAAATGAAA TAAGAGTTGGAAATAATTTTGCCATGGAAATCAATCTAAATGCCAACCTGTGGAGAAATTTCCTGTAC TCCAACATAGCGCTGTATTTGCCCGACAAGCTAAAGTACAGTCCTTCCAACGTAAAAATTTCTGATAA CCCAAACACCTACGACTACATGAACAAGCGAGTGGTGGCTCCCGGGTTAGTGGACTGCTACATTAA CCTTGGAGCACGCTGGTCCCTTGACTATATGGACAACGTCAACCCATTTAACCACCACCGCAATGCT GGCCTGCGCTACCGCTCAATGTTGCTGGGCAATGGTCGCTATGTGCCCTTCCACATCCAGGTGCCT CAGAAGTTCTTTGCCATTAAAAACCTCCTTCTCCTGCCGGGCTCATACACCTACGAGTGGAACTTCA GGAAGGATGTTAACATGGTTCTGCAGAGCTCCCTAGGAAATGACCTAAGGGTTGACGGAGCCAGCA TTAAGTTTGATAGCATTTGCCTTTACGCCACCTTCTTCCCCATGGCCCACAACACCGCCTCCACGCT TGAGGCCATGCTTAGAAACGACACCAACGACCAGTCCTTTAACGACTATCTCTCCGCCGCCAACAT GCTCTACCCTATACCCGCCAACGCTACCAACGTGCCCATATCCATCCCCTCCCGCAACTGGGCGGC TTTCCGCGGCTGGGCCTTCACGCGCCTTAAGACTAAGGAAACCCCATCACTGGGCTCGGGCTACGA CCCTTATTACACCTACTCTGGCTCTATACCCTACCTAGATGGAACCTTTTACCTCAACCACACCTTTA AGAAGGTGGCCATTACCTTTGACTCTTCTGTCAGCTGGCCTGGCAATGACCGCCTGCTTACCCCCA ACGAGTTTGAAATTAAGCGCTCAGTTGACGGGGAGGGTTACAACGTTGCCCAGTGTAACATGACCA AAGACTGGTTCCTGGTACAAATGCTAGCTAACTACAACATTGGCTACCAGGGCTTCTATATCCCAGA GAGCTACAAGGACCGCATGTACTCCTTCTTTAGAAACTTCCAGCCCATGAGCCGTCAGGTGGTGGA TGATACTAAATACAAGGACTACCAACAGGTGGGCATCCTACACCAACACAACAACTCTGGATTTGTT GGCTACCTTGCCCCCACCATGCGCGAAGGACAGGCCTACCCTGCTAACTTCCCCTATCCGCTTATA GGCAAGACCGCAGTTGACAGCATTACCCAGAAAAAGTTTCTTTGCGATCGCACCCTTTGGCGCATC

[0281] CCATTCTCCAGTAACTTTATGTCCATGGGCGCACTCACAGACCTGGGCCAAAACCTTCTCTACGCCA ACTCCGCCCACGCGCTAGACATGACTTTTGAGGTGGATCCCATGGACGAGCCCACCCTTCTTTATG TTTTGTTTGAAGTCTTTGACGTGGTCCGTGTGCACCGGCCGCACCGCGGCGTCATCGAAACCGTGT ACCTGCGCACGCCCTTCTCGGCCGGCAACGCCACAACATAA DoqCatcher

[0282] Amino acid sequence (SEQ ID NO: 10):

[0283] KLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIEN

[0284] SEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQ

[0285] DoqCatcher-PreS1 - bacterial expression

[0286] Amino acid sequence (SEQ ID NO: 57)

[0287] MAHHHHHHVGTGKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGKLT

[0288] FTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQGSGGSGGSGQNLSTSNPLGFFPD

[0289] HQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQTLPANPPP

[0290] ASTNRQSGRQPTPLSPPLRNTHPQA

[0291] DoqCatcher-PreS1 - bacterial expression

[0292] Nucleic acid sequence (SEQ ID NO: 58):

[0293] ATGGCACATCACCACCACCATCACGTGGGTACCGGTAAGCTGGGTGAGATTGAATTCATCAAAGTG

[0294] GACAAGACCGACAAAAAGCCGCTGCGTGGCGCAGTTTTTAGCCTGCAGAAACAACATCCGGACTAC

[0295] CCTGACATTTATGGTGCAATTGACCAGAATGGCACCTATCAGGATGTGCGTACCGGCGAAGACGGC

[0296] AAACTTACGTTTACCAATCTGAGTGATGGCAAGTATCGTCTGATCGAAAATAGCGAACCGCCTGGTT

[0297] ATAAACCTGTTCAAAATAAACCGATCGTGAGCTTTCGTATTGTGGATGGTGAAGTTCGTGATGTTACC

[0298] AGCATTGTTCCGCAGGGCTCTGGCGGAAGCGGCggatccGGTCAGAATCTGAGCACCAGCAATCCGC

[0299] TGGGTTTCTTTCCGGATCATCAGTTAGATCCGGCATTTCGTGCAAATACCGCAAATCCTGATTGGGA

[0300] TTTTAACCCGAATAAAGATACCTGGCCTGATGCCAATAAAGTTGGTGCCGGTGCATTTGGTCTGGGT

[0301] TTTACCCCTCCGCATGGTGGTCTGTTAGGTTGGAGTCCGCAGGCACAGGGTATTCTGCAGACCCTG

[0302] CCTGCAAATCCGCCTCCGGCAAGCACCAATCGTCAGAGCGGTCGTCAGCCGACACCGCTGAGTCC

[0303] GCCTCTGCGTAATACCCATCCGCAGGCCTAA

[0304] DoqCatcher-PreS1 - mammalian expression

[0305] Amino acid sequence (SEQ ID NO: 59):

[0306] METDTLLLWVLLLWVPGSTGDGKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDV

[0307] RTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQGSGGSGGSQNLSTS

[0308] NPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGILQ

[0309] TLPANPPPASTNRQSGRQPTPLSPPLRNTHPQAEPEA

[0310] DoqCatcher-PreS1 - mammalian expression

[0311] Nucleic acid sequence (SEQ ID NO: 60):

[0312] ATGGAAACCGACACACTGCTGCTGTGGGTGCTGCTTCTTTGGGTGCCCGGATCTACAGGCGACGG

[0313] AAAGCTGGGCGAGATCGAGTTCATCAAGGTGGACAAGACCGACAAGAAGCCCCTGAGAGGCGCTG

[0314] TGTTCAGCCTGCAGAAACAGCACCCTGACTACCCCGATATCTACGGCGCCATCGACCAGAACGGCA

[0315] CCTATCAGGATGTTCGGACAGGCGAGGATGGCAAGCTGACCTTCACCAATCTGAGCGACGGCAAGT

[0316] ACCGGCTGATCGAGAATAGCGAGCCTCCTGGCTACAAGCCCGTGCAGAACAAGCCCATCGTGTCCT

[0317] TCAGAATCGTGGACGGCGAAGTGCGGGACGTGACCTCTATAGTGCCTCAAGGCTCTGGCGGAAGC GGCggatccCAGAATCTGAGCACCTCTAATCCTCTGGGATTCTTCCCAGACCACCAGCTGGACCCTGC

[0318] CTTCAGAGCCAATACCGCCAATCCTGACTGGGACTTCAACCCCAACAAGGACACCTGGCCTGACGC

[0319] CAACAAAGTTGGCGCTGGCGCTTTTGGCCTGGGCTTTACACCTCCTCATGGCGGACTGCTTGGATG

[0320] GTCACCTCAGGCTCAGGGCATCCTGCAAACCCTGCCTGCTAATCCTCCTCCTGCCTCCACCAACAG

[0321] ACAGAGCGGTAGACAGCCCACACCTCTGAGCCCTCCACTGAGAAACACACACCCTCAGGCTGAGC CCGAGGCCTAA

[0322] Iqk-leader sequence

[0323] Amino acid sequence (SEQ ID NO: 61):

[0324] METDTLLLWVLLLWVPGSTGD

[0325] C-taq

[0326] Amino acid sequence (SEQ ID NO: 62)

[0327] EPEA

[0328] Shark invariant chain (Sli)

[0329] Amino acid sequence (SEQ ID NO: 63)

[0330] MSLLWGGVTVLAAMLIAGQVASVVFLV

[0331] HBV S190-197 peptide

[0332] Amino acid sequence (SEQ ID NO: 64)

[0333] VWLSVIWM

[0334] HBV Core93-ioo peptide

[0335] Amino acid sequence (SEQ ID NO: 65)

[0336] MGLKFRQL

[0337] HBV CorePi3L peptide

[0338] Amino acid sequence (SEQ ID NO: 66)

[0339] PPAYRPPNAPILSTL

[0340] References

[0341] Boni C, Janssen HLA, Rossi M, et al. Combined GS-4774 and Tenofovir Therapy Can Improve HBV- Specific T-Cell Responses in Patients With Chronic Hepatitis. Gastroenterology. 2019;157(1):227- 241. e7. doi:10.1053 / j.gastro.2019.03.044.

[0342] Brune, K., Leneghan, D., Brian, I. et al. Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization. Sci Rep 6, 19234 (2016). https: / / doi.org / 10.1038 / srep19234

[0343] Buldun et al., SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation. Journal of the American Chemical Society 2018 140 (8), 3008-3018 https: / / pubs.acs.org / doi / 10.1021 / jacs.7b13237

[0344] Dicks MDJ, Rose LM, Russell RA, et al. Modular capsid decoration boosts adenovirus vaccine-induced humoral immunity against SARS-CoV-2. Mol Ther. 2022;30(12):3639-3657. doi:10.1016 / j.ymthe.2022.08.002

[0345] Keeble AH et al., Approaching infinite affinity through engineering of peptide-protein interaction, PNAS December 26, 2019 116 (52) 26523-26533 https: / / doi.org / 10.1073 / pnas.1909653116

[0346] Keeble AH et al. DogCatcher allows loop-friendly protein-protein ligation. Cell Chemical Biology Volume 29, Issue 2, 17 February 2022, Pages 339-350. e10, (https: / / doi.Org / 10.1016 / j.chembiol.2021.07.005)

[0347] Li, L., Fierer, J. O., Rapoport, T. A. & Howarth, M. Structural analysis and optimization of the covalent association between SpyCatcher and a peptide Tag. J. Mol. Biol. 426, 309-317 (2014)

[0348] Meng Z, Chen Y, Lu M. Advances in Targeting the Innate and Adaptive Immune Systems to Cure Chronic Hepatitis B Virus Infection. Front Immunol. 2020;10:3127. Published 2020 Feb 7. doi:10.3389 / fimmu.2019.03127

[0349] Roberts, D., Nanda, A., Havenga, M. et al. Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity. Nature 441 , 239-243 (2006). https: / / doi.org / 10.1038 / nature04721

[0350] Rux JJ, Kuser PR, Burnett RM. Structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic, molecular modeling, and sequence-based methods. J Virol. 2003;77(17):9553-9566. doi: 10.1128 / jvi.77.17.9553-9566.2003

[0351] Thrane S, Janitzek CM, Matondo S, et al. Bacterial superglue enables easy development of efficient virus-like particle based vaccines. J Nanobiotechnology. 2016;14:30. Published 2016 Apr 27. doi:10.1186 / sl 2951 -016-0181-1

[0352] Wang, W., Zhou, X., Bian, Y. et al. Dual-targeting nanoparticle vaccine elicits a therapeutic antibody response against chronic hepatitis B. Nat. Nanotechnol. 15, 406-416 (2020). https: / / doi.Org / 10.1038 / S41565-020-0648-y Zakeri B, Fierer JO, Celik E, et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A. 2012;109(12):E690-E697. doi:10.1073 / pnas.1115485109

[0353] Fabien Zoulim, Claire Fournier, Frangois Habersetzer, et al. Safety and immunogenicity of the therapeutic vaccine TG1050 in chronic hepatitis B patients: a phase 1 b placebo-controlled trial, Human Vaccines & Immunotherapeutics, (2020) 16:2, 388-399, DOI: 10.1080 / 21645515.2019.1651141

Claims

Claims1 . An adenoviral vector comprising at least one modified capsid protein, said modification to the capsid protein comprising the insertion or fusion of a first peptide partner, wherein: the first peptide partner is covalently bonded to a second peptide partner; the second peptide partner is joined to a decorating antigen by insertion or fusion; and the decorating antigen comprises hepatitis B virus PreS1 and / or hepatitis B virus PreS2, or an immunogenic fragment thereof.

2. The adenoviral vector as claimed in claim 1 , wherein the modified capsid protein comprises hexon or pIX.

3. The adenoviral vector as claimed in claim 1 or claim 2, wherein the modified capsid protein comprises hexon, and optionally further wherein the modification to hexon comprises fusion of the first peptide partner into a hypervariable region (HVR).

4. The adenoviral vector as claimed in claim 3, wherein the hypervariable region comprises HVR1 , HVR2 or HVR5.

5. The adenoviral vector as claimed in claim 3 or claim 4, wherein the hypervariable region comprises HVR5.

6. The adenoviral vector as claimed in any preceding claim, wherein the covalent bond between the first peptide partner and the second peptide partner is an isopeptide bond.

7. The adenoviral vector as claimed in any preceding claim, wherein the first peptide partner and the second peptide partner comprise a binding partner pair, and wherein the binding partner pair is selected from:DogTag and DogCatcher;DogTag and SnoopTag;DogTag and SnoopTagJr;SnoopTag and SnoopCatcher; orSpyTag and SpyCatcher.

8. The adenoviral vector as claimed in any preceding claim, wherein the first peptide partner and the second peptide partner comprise a binding partner pair, and wherein the binding partner pair is DogTag and DogCatcher.

9. The adenoviral vector as claimed in any preceding claim, wherein the first peptide partner is DogTag and the second peptide partner is DogCatcher.

10. The adenoviral vector as claimed in any preceding claim, wherein the adenoviral vector further comprises nucleic acid encoding one or more exogenous polypeptide, and wherein the one or moreexogenous polypeptide comprises at least one hepatitis B virus polypeptide or an immunogenic fragment thereof.11 . The adenoviral vector as claimed in claim 10, wherein the at least one hepatitis B virus polypeptide is selected from PreS1 , PreS2, S-protein or Core, or an immunogenic fragment thereof.

12. The adenoviral vector as claimed in claim 10, wherein the one or more exogenous polypeptide comprises two or more hepatitis B virus polypeptides selected from PreS1 , PreS2, S-protein or Core, or immunogenic fragments thereof.

13. The adenoviral vector as claimed in claim 10, wherein the one or more exogenous polypeptide comprises all of hepatitis B virus polypeptides PreS1 , PreS2, S-protein and Core, or immunogenic fragments thereof.

14. The adenoviral vector as claimed in 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 adenoviral vector as claimed in 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 adenoviral vector of any preceding claim.

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

18. The adenoviral vector as claimed in any of claims 1 to 15 or a vaccine as claimed in claim 16 for use in the treatment or prophylaxis of Hepatitis B virus infection and / or Hepatitis D virus infection.

19. Use of an adenoviral vector of any of claims 1 to 15in the manufacture of a medicament for the treatment or prophylaxis of Hepatitis B virus infection and / or Hepatitis D virus infection.

20. A method of treating a patient in need thereof, comprising administering a safe and effective amount of the adenoviral vector of any one of claims 1 to 15 or the vaccine of claim 16.21 . A method of producing the adenoviral vector of any of claim 1 to claim 15, the method comprising: introducing a nucleic acid that encodes a first peptide partner into the nucleic acid encoding a capsid protein in an adenoviral genome; permitting expression of the adenoviral genome in order to obtain adenovirus particles; obtaining a second peptide partner attached to Hepatitis B virus PreS1 and / or Hepatitis B virus PreS2, or immunogenic fragment thereof;allowing the adenovirus particles and the second peptide partner attached to a Hepatitis B virus PreS1 and / or Hepatitis B virus PreS2, or immunogenic fragment thereof, to mix under conditions that permit covalent binding of the first peptide partner and the second peptide partner.

22. The method as claimed in claim 21 , the method further comprising introducing a nucleic acid that encodes one or more exogenous polypeptide into the adenoviral genome, wherein the one or more exogenous polypeptide comprises at least one hepatitis B virus polypeptide or an immunogenic fragment thereof.

23. A method of manufacturing the vaccine of claim 16, the method comprising admixing the adenoviral vector of any of claims 1 to 15 with a pharmaceutically acceptable excipient.

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