Avian adenovirus subunit vaccine and method for producing same

Abstract

The present invention provides a FAdV subunit vaccine comprising at least a chimeric avian adenovirus (FAdV) fiber protein and an adjuvant, which may be used to ameliorate or prevent adenoviral gizzard erosion (AGE), inclusion body hepatitis (IBH) or hepatitis-hydropericardium syndrome (HHS) in birds. The present invention further relates to a method for producing a FAdV subunit vaccine, comprising expressing a chimeric FAdV fiber protein in an expression system, purifying said fiber protein, and combining said fiber protein with an adjuvant to obtain said FAdV subunit vaccine.

Description

[Technical field]

[0001] The field of the invention relates to avian adenovirus (FAdV) fiber subunit vaccines and methods for their production. [Background technology]

[0002] FAdVs from the genus Aviadenovirus in the family Adenoviridae are nonenveloped double-stranded DNA viruses classified into five species (Triaviadenovirus A to Triviadenovirus E, FAdV-A to FAdV-E) based on genomic characteristics and into 12 subserotypes (FAdV-1 to -8a, -8b to -11) defined by cross-neutralization (Harrach et al., 2012; Hess, 2020).

[0003] The FAdV-associated avian diseases hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH) share characteristic pathogenicity and immune mechanisms and are overall significantly different from those of adenoviral gizzard erosion (AGE). One of the underlying reasons seems to be the closer molecular relationship between the causative types of HHS (FAdV-4, FAdV-C species) and IBH (FAdV-2 / -11, FAdV-D; FAdV-8a and -8b, FAdV-E) compared to the genetically distant FAdV-1 (species FAdV-A) carrying AGE (Schachner et al., 2018; Hess, 2020).

[0004] Both HHS and IBH are characterized by hemorrhagic and dystrophic necrotic changes in the liver and kidneys, with intranuclear inclusions. The characteristic pathological finding is an enlarged, dystrophic liver with a yellowish color and gritty texture. More rarely, macroscopically visible necrotic foci can be detected in the liver. The kidneys are enlarged, pale, mottled, and with multiple hemorrhages. Atrophy of the bursa of Fabricius may be observed. Ecchymoses and streaks of skeletal muscles are often observed. Microscopically, extensive dystrophic changes and necrosis of the liver parenchyma are detected. Basophilic or eosinophilic inclusions are detected in the nuclei of hepatocytes.

[0005] Diagnosis is based on typical gross lesions and medical history. In most cases, the primary lesion is prominent spotty or streaky hemorrhage in the liver. In addition to these lesions, HHS is characterized by a yellowish straw-colored fluid within the hydropericard. Outbreaks occur primarily in broiler chickens, most commonly between 1 and 8 weeks of age. In contrast to HHS, various serotypes, mainly serotypes 2 to 11, have been reported to cause IBH outbreaks.

[0006] Clinical signs can be observed already a few hours before the occurrence of death. They consist of pale comb and downy hair, depression and lethargy. IBH is characterized by a sudden onset and a rapid increase in mortality that reaches a peak value by the 3rd-4th day and returns to the normal range by the 6th-7th day. Overall mortality is usually less than 10%, but can reach up to 30%, and in the case of HHS up to 70%.

[0007] Despite the global distribution and severe economic impact of FAdV-associated diseases, commercial immunization strategies mainly rely on the use of autologous vaccines. Licensed products are mainly available to protect birds against HHS and were developed after the first emergence. In Australia, a live vaccine based on FAdV-8b was already developed in 1989 to control IBH (Steer et al. 2011). In recent years, the lack of available vaccines has been addressed by the experimental development of alternative vaccination antigens, including specific live and inactivated vaccines (Schonewille et al., 2010; Kim et al., 2014; Gupta et al., 2018; Popowich et al., 2018; Steer-Cope et al., 2019; Grafl et al., 2020) as well as subunit vaccines (Shah et al., 2012; Schachner et al., 2014; Wang et al., 2018; Schachner et al., 2018; Wang et al., 2019). Most studies have used structural proteins of the FAdV capsid in subunit vaccine formulations based on their immunogenic potential and role in conferring antigenicity of the virus (Norrby, 1969). In particular, recombinant penton base and fiber derived from FAdV-4 have proven successful in protecting chickens against the related disease, HHS (Shah et al., 2012; Schachner et al., 2014; Chen et al., 2018; Wang et al., 2019). Finally, fiber 2 was incorporated into a recombinant Newcastle disease virus that can be used to protect birds against HHS, albeit only after intramuscular vaccination (Tian et al., 2020).

[0008] WO2015 / 024932A1 discloses a subunit vaccine comprising the fiber 2 protein of FAdV-C or an immunogenic fragment thereof, which can be used to prevent HHS in birds.

[0009] WO2015 / 024929A2 discloses a subunit vaccine comprising a fiber protein selected from the fiber-2 protein of FAdV-C, the fiber-2 protein of FAdV-A, the fiber proteins of FAdV-B, FAdV-D and FAdV-E, or immunogenic fragments thereof, which can be used to prevent HHS, IBH or gizzard erosion in birds.

[0010] CN109721642B relates to a bivalent FAdV subunit vaccine and its preparation method. This document discloses the use of full-length fiber proteins of serotype 4 and serotype 8 of FAdV as immunogens for the preparation of a bivalent subunit vaccine.

[0011] However, due to simultaneous occurrence and mixed infections with diverse FAdV strains in the field, vaccines with an extended spectrum of protection are still needed ( Gomis et al., 2006 ; Mittal et al., 2014 ; Schachner et al., 2016 ; Niu et al. 2018 ). Summary of the Invention [Problem to be solved by the invention]

[0012] It is therefore an object of the present invention to provide FAdV vaccines with an extended spectrum of protection, which should furthermore be producible with reduced complexity and / or increased efficiency, in particular under Good Manufacturing Practice (GMP) conditions, compared to prior art FAdV vaccines.

[0013] The present invention provides an FAdV subunit vaccine (for birds, preferably poultry, especially chickens) comprising at least a chimeric FAdV fiber protein and an (immune-effective) adjuvant.

[0014] The vaccine is preferably for use in the amelioration or prevention of AGE, IBH or HHS in birds, preferably poultry, especially chickens.

[0015] In another aspect, the present invention relates to a method for vaccinating birds against FAdV infection comprising the steps of obtaining a vaccine of the present invention and administering said vaccine to a bird.

[0016] In yet another aspect, the present invention relates to a method for producing a FAdV subunit vaccine, comprising the steps of expressing a chimeric FAdV fiber protein in an expression system, purifying the fiber protein, and combining the fiber protein with an adjuvant (preferably with further vaccine components such as a pharma- ceutically acceptable carrier and / or a pharma-ceutically acceptable diluent, in particular as disclosed herein), to obtain a FAdV subunit vaccine.

[0017] During the course of the present invention, it was surprisingly found that FAdV subunit vaccines using chimeric fiber proteins are sufficiently immunogenic to provide an extended spectrum of protection (compared to, for example, FAdV subunit vaccines based on a single FAdV strain). The use of chimeric fiber proteins has the added advantage of simplifying vaccine production compared to expressing and combining several different recombinant fiber proteins (e.g., from multiple serotypes) in a single vaccine, since chimeric fiber protein expression in a single cell line (harboring the chimeric fiber gene) in a single bioreactor is readily possible, the number of mixing steps during production can be reduced, and the 1:1 stoichiometry can be tightly controlled (chimeric fibers are typically recombinantly expressed from a single gene). This advantage is particularly important if the FAdV vaccine is to be produced on an industrial scale and / or under GMP conditions (required for veterinary medicines in most countries).

[0018] FAdV diseases with distinct clinical manifestations caused by specific serotypes are an economic burden for the poultry industry on a worldwide scale. The growing awareness of antigenically diverse FAdV types cocirculating in the field, and the demand for their control, explains why many studies have been devoted recently to elucidating the broad-protectivity of candidate antigens, with not very promising results so far. As a general paradigm, immunity generated against a specific FAdV type does not confer protection against other types. This conclusion is supported by the observation of a shift to outbreaks caused by other virus types after the implementation of vaccination regimes against previously dominant virus types (Steer et al., 2011; Venne, 2013; Wang et al., 2018; Bertran et al. 2021; Mo 2021).

[0019] FAdV fiber is a surface antigen characterized by a high level of diversity between FAdV species and serotypes. Example 1 demonstrates cross-protection between different IBH-causing serotypes by the introduction of a novel chimeric fiber protein that combines both predicted epitopes from FAdV-8a and FAdV-8b fibers. In Example 2, a novel chimeric fiber (called "crecFib-4 / 11") was generated that retains the molecular properties of both FAdV-4 and FAdV-11. The new construct was then tested in vivo for its efficacy in immunizing chickens against different FAdV-associated diseases, such as HHS and IBH, and performed well.

[0020] Outside the present context, chimeric fiber proteins based on human adenovirus (HAdV) fiber proteins are disclosed in U.S. Pat. No. 7,741,099. Such chimeric HAdV fiber proteins have an entirely different purpose, namely, to circumvent pre-existing anti-adenovirus immunity in the human host, for example when using chimeric HAdV capsids as vectors for gene therapy. This purpose is, of course, opposite to the purpose of the vaccine of the present invention, namely, to induce anti-adenovirus immunity in the host (i.e., bird). Thus, such chimeric HAdV fiber proteins in the prior art are neither used as subunit vaccines nor together with immunologically effective adjuvants, but rather both.

[0021] According to a preferred embodiment, a chimeric FAdV fiber protein comprises an N-terminal fiber protein fragment from a first FAdV serotype and a C-terminal fiber protein fragment from a second (ie, different) FAdV serotype.

[0022] For an even broader spectrum of protection, it is preferred that the first FAdV serotype and the second FAdV serotype belong to different FAdV species (e.g., the first FAdV serotype belongs to FAdV-C (i.e. FAdV-4) and the second FAdV serotype belongs to FAdV-D, or vice versa).

[0023] It is particularly preferred that the first FAdV serotype belongs to the FAdV species FAdV-C and the second FAdV serotype belongs to an FAdV species selected from FAdV-B, FAdV-D and FAdV-E (e.g. FAdV-E), or vice versa.

[0024] In another embodiment, it is preferred that the first FAdV serotype belongs to the FAdV species FAdV-A and the second FAdV serotype belongs to a FAdV species selected from FAdV-B, FAdV-D and FAdV-E, or vice versa.

[0025] In yet another embodiment, it is preferred that the first FAdV serotype belongs to the FAdV species FAdV-C and the second FAdV serotype belongs to the FAdV species FAdV-A, or vice versa.

[0026] In another preferred embodiment, the first FAdV serotype belongs to a FAdV species selected from FAdV-B, FAdV-D and FAdV-E (particularly FAdV-E) and the second FAdV serotype belongs to a FAdV species selected from FAdV-B, FAdV-D and FAdV-E (particularly FAdV-E), or vice versa, preferably two different species are selected.

[0027] According to another preferred embodiment, the first FAdV serotype is FAdV-4 and the second FAdV serotype is selected from FAdV-2, FAdV-11, FAdV-8a and FAdV-8b (in particular FAdV-11), or vice versa.

[0028] In another embodiment, it is preferred that the first FAdV serotype is FAdV-1 and the second FAdV serotype is selected from FAdV-2, FAdV-11, FAdV-8a and FAdV-8b, or vice versa.

[0029] In yet another embodiment, it is preferred that the first FAdV serotype is FAdV-4 and the second FAdV serotype is FAdV-1, or vice versa.

[0030] According to yet another preferred embodiment, the first FAdV serotype is selected from FAdV-2, FAdV-11, FAdV-8a and FAdV-8b (in particular FAdV-8b) and the second FAdV serotype is selected from FAdV-2, FAdV-11, FAdV-8a and FAdV-8b (in particular FAdV-8a), or vice versa, with the proviso that preferably two different FAdV serotypes are selected.

[0031] The vaccines of the present invention are typically cross-protective, i.e. they induce (at least partial) immune protection against at least two FAdV serotypes (from the same or different FAdV species) in birds, preferably poultry, more preferably chickens.

[0032] It is highly preferred that both the N-terminal and C-terminal fiber protein fragments of the chimeric FAdV fiber protein are immunogenic in birds, such as chickens.

[0033] To this end, it is highly preferred that the N-terminal fiber protein fragment contains at least one, preferably at least two, in particular at least three (B cell) epitopes, and the C-terminal fiber protein fragment contains at least one, preferably at least two, in particular at least three (B cell) epitopes. Such epitopes can be experimentally confirmed or predicted using methods known in the art, for example DiscoTope 2.0 software (Vindahl Kringelum et al, 2012).

[0034] During the course of this invention, it was found that chimeric FAdV fiber proteins having a "specificity switch" within the fiber knob domain (see Examples 1 and 2, especially Figure 1) are particularly suitable for the purposes of the present invention. Thus, in another preferred embodiment, the N-terminal fiber protein fragment comprises the fiber shaft domain and the N-terminal portion of the fiber knob domain, and the C-terminal fiber protein fragment comprises the C-terminal portion of the fiber knob domain.

[0035] In the present invention, fragments of fiber proteins (particularly fiber 2) of FAdV-C or FAdV-A, or fragments of fiber proteins of FAdV-B, FAdV-D or FAdV-E may be used as fiber protein fragments (e.g., N-terminal or C-terminal fiber protein fragments) of chimeric FAdV fiber proteins. Many FAdV fiber protein sequences are known to those skilled in the art, for example those of reference strain KR5 (HE608152) or CFA20 (AF160185) or strain ON1 (GU188428 = NC_015323) or any other FAdV-C field isolates, such as isolates IV37, K99-97, K388-95, K88-95, K31, Peru53, Peru54, c344, K1013, AG234, C2B, 09-584, 09-8846, 09-2602, 922-1, Da60, K1013QT and INT4 (Marek et al. (disclosed by et al., 2012); and CELO (FAdV-A; Q64787), 340 (FAdV-B), A2-A (FAdV-D; AC000013), HG (FAdV-E; GU734104); corresponding to UniProt entries H8WG65, H8WG69, H8WG72, H8WG77, H8WG70, H8WG73, H8WG66, H8WG76, H8WG60, H8WG61, H8WG62, H8WG75, H8WG67, H8WG78, H8WG63, H8WG68, H8WG64, H8WG74, H8WG71, H8WQZ7, H8WQZ2, H8WQW9, Q0GH78, O55281, and F2VJI5. Further examples of FAdV fiber proteins are listed, for example, in Table 3 of WO2015 / 024929A2 and in Figures 5a to 5i of WO2015 / 024929A2.

[0036] In particular, any of the FAdV fiber fragments disclosed in Examples 1 and 2 may be used in the present invention (more specifically, the translated amino acid sequences), preferably in the combinations and orders disclosed in these examples.

[0037] Thus, it is preferred that the N-terminal fiber protein fragment comprises an amino acid sequence that is 70% or more identical, preferably 80% or more identical, more preferably 90% or more identical, even more preferably 95% or more identical, or even 97.5% or more identical, particularly 99% or more identical, or even completely identical to any one of the following sequences: FAdV-8a fiber N-terminal fragment (SEQ ID NO: 1): MATSTPHAFSFGQIGSRKRPAGGDGERDASKVPKMQTPAPSATANGNDELDLVYPFWLQNGSTGGGGGGGSGGNPSLNPPFLDPNGPLTVQNNLLKVNTTAPIAVTNKAL TLSFDAESLELTNQQQLAVKIDPEGPLKATTEGIQLSVDPTTLEVDDVDWELTVNLDPNGPLDSSATGITVRVDDTLLIDDDGSGQGKELGVHLNPDGPITADQNGLDLE IDNQTLKITPGSAGGVLSVQLKPQGGLNSQSDGIQVVTQNSIEVDNGALDVKVVANGPLSTTPNGLTLNYDTGDFTVNAGTLSILRNPSLVANAYLTSGARTLQQFTAKG ENSSQFSFPCAYYLQQWLSDGLIFSSLYLKLDRTRFTGMSSDPSYQNARYFTFWVGGGAAMNLSQLSTPTITPSTTEWTAFAPALNYSGAPAFVYDANPVFTIYFQPNTGR FAdV-8b fiber N-terminal fragment (SEQ ID NO:2): MATSTPHAFSFGQIGSRKRPAGGDGERDASKVPKMQTPAPSATANGNDELDLVYPFWLQNGSTGGGGGGGSGGNPSLNPPFLDPNGPLTVQNNLLKVNTTAPIAVTNKAL TLAYESDSLELTNQQQLAVKIDPEGPLKATTEGIQLSVDPTTLEVDDVDWELTVKLDPDGPLDSSATGITVRVDETLLIEDDGSGQGKELGVNLNPTGPITADDQGLDLE IDNQTLKVNSVTGGGVLAVQLKSQGGLTAQTDGIQVNTQNSITVTNGALDVKVAANGPLESTDTGLTLNYDPGDFTVNAGTLSIIRDPALVANAYLTSGASTLQQFTAKS ENSSQFSFPCAYYLQQWLSDGLIFSSLYLKLDRAQFTNMPTGANYQNARYFTFWVGAGTSFNLSTLTEPTITPNTTQWNAFAPALDYSGAPPFIYDASSVVTIYFEPTSGR FAdV-4 fiber-2 N-terminal fragment (SEQ ID NO:3): MLRAPKRRHSENGQPESEAGPSPAPIKRAKRMVRASQLDLVYPFDYVADPVGGLNPPFLGGSGPLVDQGGQLTLNVTDPIIIKNRSVDLAHDPSLDVNAQG QLAVAVDPEGALDITPDGLDVKVDGVTVMVNDDWELAVKVDPSGGLDSTAGGLGVSVDDTLLVDQGELGVHLNQQGPITADSSGIDLEINPNMFTVNTSTGS GVLELNLKAQGGIQAGSSGVGVSVDESLEIVNNTLEVKPDPSGPLTVSANGLGLKYDSNTLAVTAGALTVVGGGSVSTPIATFVSGSPSLNTYNATIVNSSSHPFSCAYYLQQWNVQGLLFTSLYVKLDSTTMGTRPGDNSSANAKWFTFWVSAYLQQCNPSGIQAGTVSPSTAALADFEPMANRSVSSPWTYSANAYQPSSG FAdV-11 fiber N-terminal fragment (SEQ ID NO: 4): MAKSTPFTFSMGQHSSRKRPADSENTQNASKVAKTQTSATRAGVDGNDDLNLVYPFWLQNSTSGGGGGGGSGGNPSLNPPFIDPNGPLYVQNSLLYVKTTAPIEVENKSLALAYDSSLDVDAQ NQLQVKVDAEGPIRISPDGLDIAVDPSTLEVDDEWELTVKLDPAGPISSSSAGINIRVDDTLLIEDDDTAQVKELGVHLNPNGPITADQDGLDLEVDPQTLTVTTSGATGGVLGVLLKPSGG LQTNIQGIGVAVADTLTISSNTVEVKTDPNGSIGSSSNGIAVVTDPAGPLTTSSNGLSLKLTPNGSIQSSSTGLSVQTDPAGPITSGANGLSLSYDTSDFTVSQGMLSIIRNPSTYPDAYLE SGTNLLNNYTAYAENSSNYKFNCAYFLQSWYSNGLVTSSLYLKINRDNLNSLPSGQLSENAKYFTFWVPTYESMNLSNVATPTITPSSVPWGAFLPAQNCTSNPAFKYYLTQPPSIYFEPESG

[0038] Alternatively, or in addition, it is preferred that the C-terminal fiber protein fragment comprises an amino acid sequence that is 70% or more identical, preferably 80% or more identical, more preferably 90% or more identical, even more preferably 95% or more identical, or even 97.5% or more identical, particularly 99% or more identical, or even completely identical to any one of the following sequences: FAdV-8a fiber C-terminal fragment (SEQ ID NO:5): VDTYLPVLTGDWKTSSTYNPGTITLVVRNATIQLQSQSTFTTSVCYNFRCQNSGIFNNNATSGTLTLGPIFYSCPALSTADVS FAdV-8b fiber C-terminal fragment (SEQ ID NO:6): LESYLPVLTDNWSQTYNPGTVTLCVKTVRVQLRSQGTFSTLVCYNFRCQNTGIFNSNATAGTMTLGPIFFSCPALSTANAP FAdV-11 fiber C-terminal fragment (SEQ ID NO: 7): SVQTFQPVLTGDWDTNTYNPGTVQVCILPQTVVGGQSTFVNMTCYNFRCQNPGIFKVAASSGTFTIGPIFYSCPTNKLTQP FAdV-4 fiber C-terminal fragment (SEQ ID NO: 8): EFQVFTPVVTGAWNPGNIGIRVLPVPVTASGDRYTLLCYSLQCTNSSIFNPANSGTMIVGPVLYSCPAASVP

[0039] In particularly preferred embodiments, the chimeric FAdV fiber protein comprises an amino acid sequence that is 70% or more identical, preferably 80% or more identical, more preferably 90% or more identical, even more preferably 95% or more identical, or even 97.5% or more identical, particularly 99% or more identical, or even completely identical to any one of the following sequences: FAdV-8a / 8b chimera (SEQ ID NO: 9): MATSTPHAFSFGQIGSRKRPAGGDGERDASKVPKMQTPAPSATANGNDELDLVYPFWLQNGSTGGGGGGGSGGNPSLNPPFLDPNGPLTVQNNLLKVNTTAPIAVTNKALTLSFDAESLELTNQQQLAVK IDPEGPLKATTEGIQLSVDPTTLEVDDVDWELTVNLDPNGPLDSSATGITVRVDDTLLIDDDGSGQGKELGVHLNPDGPITADQNGLDLEIDNQTLKITPGSAGGVLSVQLKPQGGLNSQSDGIQVVTQNS IEVDNGALDVKVVANGPLSTTPNGLTLNYDTGDFTVNAGTLSILRNPSLVANAYLTSGARTLQQFTAKGENSSQFSFPCAYYLQQWLSDGLIFSSLYLKLDRTRFTGMSSDPSYQNARYFTFWVGGGAAM NLSQLSTPTITPSTTEWTAFAPALNYSGAPAFVYDANPVFTIYFQPNTGRLESYLPVLTDNWSQTYNPGTVTLCVKTVRVQLRSQGTFSTLVCYNFRCQNTGIFNSNATAGTMTLGPIFFSCPALSTANAP FAdV-8b / 8a chimera (SEQ ID NO: 10): MATSTPHAFSFGQIGSRKRPAGGDGERDASKVPKMQTPAPSATANGNDELDLVYPFWLQNGSTGGGGGGGSGGNPSLNPPFLDPNGPLTVQNNLLKVNTTAPIAVTNKALTLAYESDSLELTNQQQLAVKI DPEGPLKATTEGIQLSVDPTTLEVDDVDWELTVKLDPDGPLDSSATGITVRVDETLLIEDDGSGQGKELGVNLNPTGPITADDQGLDLEIDNQTLKVNSVTGGGVLAVQLKSQGGLTAQTDGIQVNTQNSI TVTNGALDVKVAANGPLESTDTGLTLNYDPGDFTVNAGTLSIIRDPALVANAYLTSGASTLQQFTAKSENSSQFSFPCAYYLQQWLSDGLIFSSLYLKLDRAQFTNMPTGANYQNARYFTFWVGAGTSFNL STLTEPTITPNTTQWNAFAPALDYSGAPPFIYDASSVVTIYFEPTSGRVDTYLPVLTGDWKTSSTYNPGTITLVVRNATIQLQSQSTFTTSVCYNFRCQNSGIFNNNATSGTLTLGPIFYSCPALSTADVS FAdV-4 / 11 chimera (SEQ ID NO: 11): MLRAPKRRHSENGQPESEAGPSPAPIKRAKRMVRASQLDLVYPFDYVADPVGGLNPPFLGGSGPLVDQGGQLTLNVTDPIIIKNRSVDLAHDPSLDVNAQGQLAVAVDPEGALDIPTPDGLDV KVDGVTVMVNDDWELAVKVDPSGGLDSTAGGLGVSVDDTLLVDQGELGVHLNQQGPITADSSGIDLEINPNMFTVNTSTGSGVLELNLKAQGGIQAGSSGVGVSVDESLEIVNNTLEVKPDP SGPLTVSANGLGLKYDSNTLAVTAGALTVVGGGSVSTPIATFVSGSPSLNTYNATIVNSSSHPFSCAYYLQQWNVQGLLFTSLYVKLDSTTMGTRPGDNSSANAKWFFTFWVSAYLQQCNPSG IQAGTVSPSTAALADFEPMANRSVSSPWTYSANAYYQPSSGSVQTFQPVLTGDWDTNTYNPGTVQVCILPQTVVGGQSTFVNMTCYNFRCQNPGIFKVAASSGTFTIGPIFYSCPTNKLTQP FAdV-11 / 4 chimera (SEQ ID NO: 12): MAKSTPFTFSMGQHSSRKRPADSENTQNASKVAKTQTSATRAGVDGNDDLNLVYPFWLQNSTSGGGGGGGSGGNPSLNPPFIDPNGPLYVQNSLLYVKTTAPIEVENKSLALAYDSSLDVDAQNQLQVKVDAEGPIRISPD GLDIAVDPSTLEVDDEWELTVKLDPAGPISSSSAGINIRVDDTLLIEDDDTAQVKELGVHLNPNGPITADQDGLDLEVDPQTLTVTTSGATGGVLGVLLKPSGGLQTNIQGIGVAVADTLTISSNTVEVKTDPNGSIGSS SNGIAVVTDPAGPLTTSSNGLSLKLTPNGSIQSSSTGLSVQTDPAGPITSGANGLSLSYDTSDFTVSQGMLSIIRNPSTYPDAYLESGTNLLNNYTAYAENSSNYKFNCAYFLQSWYSNGLVTSSLYLKINRDNLNSLPS GQLSENAKYFTFWVPTYESMNLSNVATPTITPSSVPWGAFLPAQNCTSNPAFKYYLTQPPSIYFEPESGEFQVFTPVVTGAWNPGNIGIRVLPVPVTASGDRYTLLCYSLQCTNSSIFPANSGTMIVGPVLYSCPAASVP

[0040] The vaccine according to the invention preferably comprises an adjuvant (in an immunologically effective amount) selected from the group consisting of Freund's complete adjuvant, Freund's incomplete adjuvant, aluminium hydroxide, Bordetella pertussis, saponin, muramyl dipeptide, ethylene vinyl acetate copolymer, oil, vegetable oil or mineral oil, in particular peanut oil or silicone oil, and combinations thereof.

[0041] Adjuvants are substances that enhance the immune response to the immunogen, specifically the chimeric FAdV fiber protein. Adjuvants can include aluminum hydroxide and aluminum phosphate, saponins, such as Quil A, water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions. Emulsions can be based on light liquid paraffin oil (European Pharmacopoeia type), in particular; isoprenoid oils, such as squalane or squalene; oils resulting from the oligomerization of alkenes, in particular isobutene or decene; esters of acids or alcohols containing linear alkyl groups, more specifically vegetable oils, ethyl oleate, propylene glycol di(caprylate / caprate), glyceryl tri(caprylate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearate esters. Oils are used in combination with emulsifiers to form emulsions. Emulsifiers are preferably non-ionic surfactants, in particular sorbitan, mannides (e.g. anhydromannitol oleate), glycerol, polyglycerol, propylene glycol and optionally ethoxylated esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, as well as polyoxypropylene-polyoxyethylene copolymer blocks, in particular Pluronic® products, especially L121. For example, adjuvanted vaccines are prepared in the following manner: 50 v / v to 90 v / v of an aqueous phase containing the immunogen is emulsified with 1% w / v to 10% w / v of anhydromannitol oleate, 1% w / v to 10% w / v of oleic acid ethoxylated with 11EO (ethylene oxide) and 5% v / v to 40% v / v of light liquid paraffin oil (European Pharmacopoeia type) with the aid of an emulsifying turbo mixer. An alternative method for preparing the emulsion consists of emulsifying a mixture of 1% w / v to 10% w / v squalane, 1% w / v to 10% w / v Pluronic® L121, 0.05% w / v to 1% w / v of an ester of oleic acid with anhydrosorbitol ethoxylated with 20 EO, and 50% v / v to 95% v / v of the immunogen in an aqueous phase by passing through a high pressure homogenizer.It is also possible to formulate with synthetic polymers (e.g. homo- and copolymers of lactic and glycolic acid, which are used to produce microspheres that encapsulate immunogens, such as biodegradable microspheres). Further examples of adjuvants are compounds selected from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride with alkenyl derivatives. Advantageous adjuvant compounds are polymers of acrylic or methacrylic acid, in particular cross-linked with polyalkenyl ethers of sugars or polyalcohols. These compounds are known under the term carbomers and are, for example, acrylic polymers cross-linked with polyhydroxylated compounds having at least three hydroxyl groups, preferably up to eight hydroxyl groups, in which the hydrogen atoms of at least three hydroxyls are replaced by unsaturated aliphatic radicals having at least two carbon atoms. Preferred radicals are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. Particularly suitable are the products sold under the name Carbopol® (BF Goodrich, Ohio, USA). They are crosslinked with allyl sucrose or allyl pentaerythritol. Among them, Carbopol® 974P, 934P and 971P can be mentioned. Among the copolymers of maleic anhydride and alkenyl derivatives, the copolymers EMA® (Monsanto), which are copolymers of maleic anhydride and ethylene, linear or crosslinked, for example with divinyl ether, are preferred. These polymers are dissolved in water, resulting in an acidic solution, preferably at physiological pH, which is neutralized in order to obtain an adjuvant solution in which the immunogenic, immunological or vaccine composition itself is incorporated. The carboxyl groups of the polymer are then partly in the COO-form.

[0042] In another preferred embodiment, a solution of the adjuvant is prepared in distilled water, preferably in the presence of sodium chloride, the resulting solution being at an acidic pH. This stock solution is diluted by adding several aliquots at once to the desired amount (to obtain the desired final concentration) or a substantial portion thereof of water with added NaCl, preferably physiological saline (NaCl 9 g / l), with simultaneous or subsequent neutralization (pH 7.3 to pH 7.4), preferably with NaOH. This solution, at physiological pH, can be used as is for mixing with the immunogen and can be stored, inter alia, in lyophilized, liquid or frozen form.

[0043] The vaccine according to the invention preferably comprises a pharma- ceutically acceptable diluent and / or carrier, preferably selected from the group consisting of water for injection, saline, tissue culture medium, propylene glycol, polyethylene glycol, vegetable oils, in particular olive oil, and injectable organic esters such as ethyl oleate.

[0044] The vaccine of the present invention is preferably for use in the amelioration or prevention of AGE, IBH or HHS in birds, preferably poultry, especially chickens.

[0045] In other words, the vaccine of the present invention is preferably for use in immunizing birds, preferably poultry, especially chickens, against FAdV infection (especially infection with the first and / or second FAdV serotypes).

[0046] For the prophylactic use or method of the present invention, the vaccine is administered to the bird at the appropriate time in an effective amount. Typical administration methods are intravenous, subcutaneous, intramuscular, oral, intraovo, intranasal, eye drop or intracavitary administration. Preferably, vaccination in chickens is carried out up to the 19th week.

[0047] According to another preferred embodiment, the vaccine is administered to the bird at least twice, with boosters having been found to be particularly useful in raising antibodies against the chimeric fiber protein.

[0048] The chimeric FAdV fiber protein may be produced by any suitable expression system. Preferably, the production is carried out in a eukaryotic expression system. Particularly preferred expression systems are the baculovirus expression system, bacterial expression systems such as the E. coli expression system, the Pichia pastoris expression system. However, practically any suitable expression system or vector can be used for the production of the vaccine provided by the invention. By way of example, the suitable expression or vector system can be selected according to the conditions and needs of each particular case from among plasmids, bacmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacteriophage P1-based artificial chromosomes (PACs), cosmids or viruses, which may further comprise, for example, a heterologous origin of replication in bacteria or yeast so as to be amplified in bacteria or yeast, as well as a marker that can be used to select transfected cells that is different from the gene or genes of interest. These expression systems or vectors can be obtained by conventional methods known to those skilled in the art.

[0049] Once expressed, the chimeric FAdV fiber protein may then be purified by any suitable protein purification method known in the art, for example, affinity chromatography if the chimeric FAdV fiber protein bears a His tag or another affinity tag, which may be cleaved prior to formulating the final vaccine dosage form.

[0050] The vaccine according to the invention can be produced in industrial quantities. The individual vaccine doses given to animals can be within the ranges applicable to other vaccines. Preferably, the chimeric FAdV fiber protein is contained in the vaccine in an amount of 0.1 μg / ml to 10 mg / ml, preferably 1 μg / ml to 1 mg / ml, in particular 10 μg / ml to 100 μg / ml.

[0051] The vaccine according to the present invention, especially when provided as a commercial vaccine product, preferably comprises a pharma- ceutically acceptable diluent. Suitable vehicles can be both aqueous and non-aqueous. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.

[0052] According to a preferred embodiment, the vaccine according to the invention is formulated as a dosage form, ie ready-formulated to be administered without further distribution / formulation / separation steps.

[0053] Vaccines of the invention typically do not contain nucleic acid and / or typically do not contain a viral vector (ie, are vector-free) or do not contain a viral capsid.

[0054] As used herein, "UniProt" refers to the Universal Protein Resource. UniProt is a comprehensive resource for protein sequence and annotated data. UniProt is a collaboration between the European Bioinformatics Institute (EMBL-EBI), the SIB Swiss Institute of Bioinformatics and the Protein Information Resource (PIR). Over 100 people across the three institutes are involved through different tasks such as database management, software development and support. Website: http: / / www.uniprot.org /

[0055] Entries in the UniProt database are identified by their accession codes (referred to herein, for example, as "UniProt accession codes" or "UniProt" for short, followed by the accession code), typically six-letter alphanumeric codes (e.g., "H8WG77"). Unless otherwise specified, accession codes used herein refer to entries in UniProt's Protein Knowledge Base (UniProtKB). Unless otherwise stated, the UniProt database status for all entries referenced herein (or in WO2015 / 024929A2) is as of February 10, 2021 (UniProt / UniProtKB release 2021_01).

[0056] In the context of this application, sequence variants (referred to in UniProt as "natural variants") are explicitly included when referring to UniProt database entries.

[0057] "Percent (%) amino acid sequence identity" or "X% identical" (such as "70% identical") with respect to a reference polypeptide or protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of one in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, Megalign (DNASTAR) or the "needle" pairwise sequence alignment application of the EMBOSS software package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are calculated using sequence alignment with the computer program "needle" from the EMBOSS software package (publicly available from the European Molecular Biology Laboratory; Rice et al., 2000).

[0058] The needle program can be accessed at the website http: / / www.ebi.ac.uk / Tools / psa / emboss_needle / or downloaded for local installation as part of the EMBOSS package from http: / / emboss.sourceforge.net / . It runs on many widely used UNIX operating systems, including Linux.

[0059] To align two protein sequences, the needle program is preferably run with the following parameters:

[0060] Command line: needle-auto-stdout-asequence SEQUENCE_FILE_A-bsequence SEQUENCE_FILE_B-datafile EBLOSUM62-gapopen 10.0-gapextend 0.5-endopen 10.0-endextend 0.5-aformat3 pair-sprotein1-sprotein2(Align_format:pair Report_file:stdout)

[0061] The % amino acid sequence identity of a given amino acid sequence A to, with, or to a given amino acid sequence B (or in other words, a given amino acid sequence A having or containing a particular % amino acid sequence identity to, with, or to a given amino acid sequence B) for a given amino acid sequence B is calculated as follows:

[0062] 100 times the fraction X / Y

[0063] where X is the number of amino acid residues scored as identical matches by the sequence alignment program needle in that program's alignment of A with B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. If "the sequence of A is more than N% identical to the entire sequence of B", then Y is the entire sequence length of B (i.e., the total number of amino acid residues in B). Unless otherwise stated, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the needle computer program.

[0064] The present invention is further illustrated by the following figures and examples, but is not limited thereto. [Brief description of the drawings]

[0065] [Figure 1] (a) The crecFib knob domain, represented diagrammatically as a two-dimensional ribbon model (adapted from El Bakkouri et al., 2008). Arrows represent β-strands (designated A–J in linear order from N-terminus to C-terminus of the knob sequence) connected by lines representing intervening loops. Structures encoded by sequences of discriminable serotypes are shown in different colors opposite the chimeric exchange sites (the “specificity switch” indicated by the dashed line at the left end of the G strand). (b) Coordinates of the β-strands of the fiber knob (black bars), candidate epitopes (blue bars, epitopes predicted by DiscoTope 2.0; red bars, epitopes previously reported by Wang et al., 2018) and the specificity switch annotated on a Wu-Kabat plot of the FAdV-E fiber sequence. The degree of variation, indicated by the bar size at each residue position, has been calculated as the Wu-Kabat index from available fibers representing all types (N=29) of the species. The amino acid positions of the multiple sequence alignment are annotated along the horizontal axis. The candidate epitopes match well with less strictly conserved residues, but the specificity switch is located at an intraspecies consensus site. (c) Comparison of two crecFib knobs (8a / 8b and 8b / 8a) in similarity modeling of amino acid sequences (A and B) and their predicted surface electrostatics in the lower panel (molecules shown in side view). In the upper panel, only one strand of the fiber trimer is colored in orange shades for the sequence portion derived from FAdV-8a and in green shades for the sequence portion derived from FAdV-8b. The muted shades indicate the three epitopes predicted in this study. The same epitopes are highlighted with black boxes in the electrostatic surface charge image with a color transition from red (negative charge) to blue (positive charge) and compared to the corresponding sites in the cognate native knobs of FAdV-8a and FAdV-8b (C). [Diagram 2]Antibody development after immunization with crecFib constructs. The header of each panel indicates the construct used for immunization, which also corresponds to the coating antigen for the ELISA to determine the weekly OD from 1 to 8 weeks post vaccination (wpv). If detectable, the mean neutralization titers are plotted in the respective color for the viruses against which the sera were tested (including the chimeric template and the challenge strains of this study from FAdV-8a and FAdV-8b). [Figure 3-1] Western blot analysis to assess the reactivity of the crecFib constructs alongside monospecific control fibers from FAdV-8a, FAdV-8b and FAdV-7 (all FAdV-E species). The order of antigens is the same for all membranes, as labeled for the first membrane: 1, Fib-8a; 2, Fib-8b; 3, crecFib-8a / 8b; 4, crecFib-8b / 8a; 5, Fib-7. However, each membrane was incubated with a different antiserum, as specified in the header of each membrane. The corresponding serotype is indicated in brackets. Molecular weight sizes are indicated by standards in the first lane in (a). In all subsequent blots, only the relevant 50 kDa-70 kDa range is marked. Signals that are present but difficult to distinguish from background are boxed. (a) Confirmation of monomeric fiber via the polyhistidine tag. (b) Detection of crecFib by antisera raised against FAdV-8a and FAdV-8b derived constructs and FAdV-7 as an outlier. (c) Detection of crecFib by antisera from vaccinated birds and the reversed-order construct. CrecFib antisera also recognize all monospecific control fibers. (d) Detection of crecFib and control fibers by antisera against field isolates, including the two challenge strains in this study carrying either one (FAdV-B) or two (FAdV-A) fiber genes, the natural recombinant strain and an exemplary strain derived from the exogenous FAdV-E. [Figure 3-2] Same as above. [Figure 4]Experimental design of protection study 1 and protection study 2. The procedures performed on the birds (vaccination, booster, challenge) are shown on the timeline (as days of survival and days post challenge = dpc). The individual treatment schemes for each group are specified below. Asterisks indicate blood sampling and crosses indicate sequential killing by necropsy and organ sampling. [Diagram 5] Pre-challenge antibody development in protection study 1 (prime-boost vaccination with crecFib-8a / 8b or crecFib-8b / 8a). (a) Mean OD values ​​for each treatment (groups receiving the same treatment were pooled) measured by crecFib-8a / 8b ELISA (A) and crecFib-8b / 8a ELISA (B) at 21 days of age (18 dpv), 28 days of age (7 dpb), and 35 days of age (14 dpb). (b) Neutralizing antibody titers at 35 days of age (14 dpb = just before challenge) against FAdV-8a (strains TR59 and 11-16629) and FAdV-8b (strains 764 and 13-18153). [Figure 6] Protection endpoints assessed after FAdV-8b challenge in protection study 1 (3 dpc, 5 dpc and 7 dpc). (i) Organ body weight (BW) ratios for liver (A) and spleen (B). Lowercase letters above bars indicate significant differences (vs. negative control, "a"; vs. challenge control, "b"). (ii) Plasma AST (significantly different from negative control, "a"; significantly different from challenge control, "b"). (iii) Viral load in liver (A) and pancreas (B). Significant differences from challenge control are indicated with an asterisk. [Figure 7]Antibody development (ELISA and VNT) immediately prior to challenge (21 d) and immediately following challenge (7 dpc and 14 dpc) in protection study 2 (comparison of V8b / 8a and VV8b / 8a regimens against challenge with either FAdV-8a or FAdV-8b). Only identically challenged groups were compared with each other; (A) summary of birds challenged with FAdV-8a (strain 11-16629) and (B) summary of birds challenged with FAdV-8b (strain 13-18153). The top panel shows neutralization against the virus type used for challenge and the bottom panel shows neutralization against the respective heterologous virus type. [Figure 8-1] Protection endpoints in protection study 2 (assessed at 3 dpc, 5 dpc, 7 dpc and 14 dpc). Groups are designated V or VV according to the single or prime boost regimen with crecFib-8b / 8a and C followed by the respective challenge virus (either FAdV-8a or FAdV-8b). N indicates negative control. (i) Organ body weight (BW) ratios for liver (A), spleen (B) and bursa of Fabricius (C). Lower case letters above bars indicate significant differences (relative to negative control, "a"; relative to corresponding challenge control, "b"). (ii) Plasma AST (significantly different to negative control, "a"; significantly different to challenge control, "b"). (iii) Viral load in liver (A), pancreas (B), spleen (C) and bursa of Fabricius (D). Significant differences relative to corresponding challenge control are indicated with an asterisk. Data excludes single vaccination regimens at 7 and 14 dpc due to small sample sizes (shown in §). [Figure 8-2] Same as above. [Figure 9]Organ-BW mean values ​​after challenge. Means and standard deviations of liver-BW ratio (A), spleen-BW ratio (B), bursa-BW ratio (C) and thymus-BW ratio (D) for each experimental group in the post-challenge period. Significant differences relative to the negative control are indicated by the letter "a" and significant differences relative to the respective challenge control are indicated by the letter "b". Significance was assessed at p ≤ 0.05. No significant differences were observed for thymus-BW in the FAdV-11 lines at any of the measurement time points. [Figure 10] Mean AST values ​​after challenge. Mean and standard deviation of plasma AST for each experimental group during the post-challenge period. Significant differences from the negative control are indicated by the letter "a" and significant differences from the respective challenge control are indicated by the letter "b". Significance was assessed at p ≤ 0.05. [Figure 11] Viral load in major target organs after challenge. Mean and standard deviation of viral load (expressed in log10) for each experimental group in the post-challenge period in liver (A), spleen (B) and bursa of Fabricius (C). Asterisks indicate significant differences compared to the respective challenge controls. Significance was assessed at p≦0.05. [Figure 12] Antibody development after vaccination. Mean titers (OD) of the experimental groups tested by crecFib-4 / 11 ELISA throughout the protection study. Values ​​given for pre-challenge time points (14d and 21d) are cumulative for the three vaccinated groups (I, II, III) and the two challenge control groups (IV, V). Cut-off values ​​are marked in red. [Figure 13] Development of neutralizing antibodies after vaccination. Mean and standard deviation of VNT titers (log2) pre-challenge (21 d) and at 5 dpc-7 dpc: vaccinated groups and FAdV-4 challenge controls against FAdV-4 challenge strain (A) and vaccine strain (B); vaccinated groups and FAdV-11 challenge controls against FAdV-11 challenge strain (C) and vaccine strain (D). Headers indicate the number of birds showing nAb (some individuals could not be tested due to insufficient serum volume). No nAb were observed for the vaccine only group at any measurement time point. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0066] Example 1: Cross-protective subunit vaccine using chimeric fibers based on two FAdV-E serotypes Control of IBH, an economically important disease for chicken production worldwide, is complicated by a pathogenesis that involves multiple different FAdV types. Fiber protein is effective in inducing protective antibodies in vaccinated chickens.

[0067] In this study, we designed chimeric proteins in which the N-distal and C-distal fiber portions containing putative epitopes were exchanged between different serotypes, FAdV-8a and FAdV-8b.

[0068] Remarkably, the chimeric fiber vaccine induced protective responses against challenge with both serotypes in chickens.

[0069] Materials and Methods In silico design for recombinantly expressed chimeric fibers ("crecFibs") The open reading frame of FAdV fiber was split into amino (N)-distal and carboxy (C)-distal segments, amplified separately from different template strains, and seamlessly fused via Gibson assembly cloning to reconstitute a novel full-length fiber (hereafter referred to as "crecFib") (Fig. 1a, 1b). As a result, the crecFib construct contains a crossover between heterologous sequences of serotypes FAdV-8a and FAdV-8b at the junction of the N-distal and C-distal segments. The junction (termed the "specificity switch") was engineered at the consensus residue position aa441-aa442 in the pairwise sequence alignment of FAdV-8a and FAdV-8b fibers and mapped within the proposed fiber head ("knob") domain. Fitting in silico predicted epitopes on both sides, this strategy was expected to combine the antigenic specificities of both constitutive serotypes into a single antigen. By engineering a specificity switch inside the knob, we also sought to maintain sequence integrity at the putative shaft knob boundary (Hong et al, 1996), which represents a key element for fiber trimerization.

[0070] Candidate epitopes were inferred from (i) previously reported epitopes in fiber 2 of FAdV-4 (FAdV-C) (Wang P et al., 2018) and (ii) in silico predictions using DiscoTope 2.0 software (Vindahl Kringelum et al., 2012), whereby fiber 2 of the FAdV-1 (FAdV-A) reference strain CELO (El Bakkouri, 2008), the closest related fiber knob for which a molecular model is currently available, was used for homology modeling. Positional homology between members of the different types was determined by multiple sequence alignments made with MegAlign software (DNASTAR, Madison, WI, USA). Homology modeling was also used to assign structural domains (tail, shaft, knob) of the FAdV fiber based on existing information (Hess et al., 1995).

[0071] Sequence of the crecFib construct CrecFib-8a / 8b: N-terminus: nt1-1323 from the FAdV-8a reference strain ((TR59)KT862810) C-terminus: nt 1324-1569 from the FAdV-8b reference strain ((764)KT862811) CTGGAAAGCTATCTCCCCGTCTTACCGATAACTGGAGCCAAACCTACAACCCCGGCACCGTCACCCTGTGTGTAAAAACGGTAAGGGTTCAATTGAGATCACAAGGAACCTTCAGCACTCTAGTCTGTTACAATTTCCGCTGTCAGAACACGGGCATTTTTAACAGCAACGCTACAGCGGGAACCATGACACTTGGACCTATCTTCTTTAGTTGTCCTGCCTTAAGCACCGCCAACGCTCCTTAA Full sequence:

[0072] CrecFib-8b / 8a: N-terminus: nt1-1323 from FAdV-8b reference strain (764) KT862811) C-terminus: nt 1324-1575 from the FAdV-8a reference strain ((TR59)KT862810) GTGGACACATACCTGCCGGTTCTCACCGGCGACTGGAAAACTTCTTCCACTTACAATCCAGGAACAATAACACTCGTCGTAAGGAACGCTACGATCCAGCTGCAATCACAGAGCACGTTCACAACCAGTGTGTGTTACAACTTCCGATGTCAAAACTCTGGGATTTTCAACAACAACGCCACCTCGGGAACGCTCACCCTAGGTCCGATCTTTTACAGTTGTCCGGCCCTAAGTACTGCGGACGTGTCATAA Full sequence:

[0073] Cloning and expression of crecFib constructs Based on the FAdV reference strain selected as expression template, the N-distal and C-distal fiber segments identified above were amplified using primer pairs featuring overhangs with flanking sequences and between the two segments themselves from EheI / StuI digested pFAST BAC expression vector (Invitrogen, Vienna, Austria). Ligation into linearized pFAST BAC vector was performed using Gibson Assembly Master Mix (NEB, Ipswich, MA) according to the manufacturer's instructions. For each chimeric combination, two constructs with reversed order of specificity were generated and named crecFib-8a / 8b and crecFib-8b / 8a, respectively.

[0074] Correct insertion of the segments into the vector was confirmed by Sanger sequencing across the multiple cloning site (LGC Genomics, Berlin, Germany).

[0075] Recombinant proteins were expressed in Fall Armyworm Sf9 cells and purified via their polyhistidine tags on affinity chromatography columns as previously described (Schachner et al., 2014), and their concentrations were determined by Bradford assay (Thermo Fisher Scientific, Vienna, Austria).

[0076] Evaluation of immunogenicity and in vitro reactivity spectrum of crecFib Specific pathogen-free (SPF) chickens (Valo BioMedia GmbH, Osterholz-Scharmbeck, Germany), hatched and housed in our facility, were immunized intramuscularly (im) with crecFib-8a / 8b (n=5) or crecFib-8b / 8a (n=5). Three birds in each group received 50 μg and two birds 100 μg of recombinant protein mixed 1:1 with GERBU adjuvant P (GERBU Biotechnik GmbH, Heidelberg, Germany). Post-immunization sera were collected weekly for parallel monitoring by ELISA and virus neutralization tests.

[0077] Additional immune sera were provided for comparison purposes. Briefly, these sera were derived from SPF chickens injected with whole virus or immunized intramuscularly with monospecific FAdV fiber (i.e. recombinantly expressed fiber based on the sequence of a single serotype). Whole viruses for antiserum production were plaque purified three times (3-fold) and characterized by at least partial analysis of the hexon and fiber genes, and in most cases by whole genome sequencing and cross-neutralization tests as previously described (Schachner et al., 2016). Whenever available, our test setup included sera against inactivated adjuvanted FAdV (prepared in 1% formaldehyde and administered in a 1:1 mixture with GERBU adjuvant) and live virus to evaluate possible differential recognition of denatured antigens in immunoblots.

[0078] crecFib-based enzyme-linked immunosorbent assay (ELISA) Sera from birds immunized with each type of crecFib were tested on ELISA plates coated with the corresponding crecFib following the protocol described in Feichtner et al., 2018.

[0079] Viral (cross)neutralization (VN) test Neutralizing antibodies (nAb) in sera were determined in a microtiter assay on primary chicken embryo liver (CEL) cells. Serial two-fold serum dilutions (1:8 to 1:16,384) were incubated with 100 TCID50 of virus. The crecFib antisera were tested against FAdV-8a and FAdV-8b reference strains (expression templates) and two field isolates from each serotype that served as challenge strains in protection studies. For comparison purposes, anti-fib sera with monotypic specificity (Fib-8a and Fib-8b sera) were included in certain settings. In addition, each microtiter plate contained positive (cells + virus) and negative (cells only) control wells. After 5 days at 37°C in 5% CO2, wells were examined for cytopathic effect (CPE) and titers were defined by the highest serum dilution that inhibited CPE.

[0080] Immunofluorescence (IF) staining After virus cross-neutralization tests against FAdV-8a and FAdV-8b reference strains as described above, cells were fixed by adding ice-cold methanol for 5 min. After successive washing steps and 1 h blocking with 3% BSA, in-house generated polyclonal rabbit α-FAdV (diluted 1:500 in PBS) was added to each well overnight. After removing the rabbit antiserum and washing, the plates were incubated with 1:200 diluted Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen, Life Technologies, Carlsbad, CA, USA) for 1 h in the dark. After another wash, cells were stained with 1:1000 4'6-diamidino-2-phenylindole solution (DAPI; Roche Diagnostics GmbH, Vienna, Austria) for 5 min, subjected to a final wash and covered with PBS.

[0081] Internalization of viral particles was examined and recorded with a Zeiss Axiovert 200 M fluorescence microscope (Zeiss, Jena, Germany) coupled to a FLEXACAM C1 camera and Leica Application Suite X (LAS X) (Leica Microsystems GmbH, Wetzlar, Germany).

[0082] Western blot The crecFib constructs were screened side-by-side with the most closely related in-house expressed monospecific fibers (strains Fib-8a / TR59, Fib-8b / 764 and Fib-7 / YR36, which are reference type representatives of FAdV-E) using polyclonal immune sera. To minimize variation in the ratio of reactants, the concentration of recombinant protein loaded was adjusted to 7.5 μg per lane and detection sera with similar ELISA titers (2.5 ≤ OD ≤ 3.0) against the homologous type of recombinant fiber and neutralization titers within the log2 range, where applicable, were used. Briefly, recombinant purified proteins were separated by 12% SDS-PAGE and transferred to BioTrace PVDF transfer membranes (Pall, Vienna, Austria) using the Trans-Blot Turbo transfer system (Bio-Rad, Vienna, Austria). After blocking with 3% (w / v) skim milk, the membranes were incubated separately with polyclonal sera, preabsorbed with 1% Sf9 cell powder and diluted 1:2000. As a control for the presence and size of monomeric fibers, one membrane was incubated with an anti-polyhistidine antibody (Sigma-Aldrich, Vienna, Austria). After incubation with secondary rabbit anti-chicken-IgG-HRP (Sigma-Aldrich, Vienna, Austria) or goat anti-mouse-IgG(H+L)-HRP (Bio-Rad, Vienna, Austria) (for controls) and intermediate washes, the blots were developed with Clarity Western ECL substrate (Bio-Rad, Vienna, Austria). Visualization was performed using a ChemiDoc Imager (Bio-Rad, Vienna, Austria).

[0083] Protection studies with crecFib constructs To sequentially address (i) that the crecFib construct confers in vivo protection and (ii) whether the protection induced by crecFib is appropriate for the broad coverage of the IBH complex, a two-round vaccination-challenge study was performed. An overview of both experimental designs is summarized in Figure 4.

[0084] Clinical trial 1: Protective efficacy of crecFib-8a / 8b and crecFib-8b / 8a In the first study, two groups of SPF broiler chickens (n=12) were prime-boost vaccinated with either crecFib-8a / 8b or reversed crecFib-8b / 8a, followed in each case by challenge with FAdV-8b. SPF broilers were obtained from the Animal Health Service (Deventer, The Netherlands) and housed in separate isolator units (HM2500, Montair, The Netherlands). Vaccination consisted of 50 μg of each crecFib formulated in a 40% (w / v) antigen-oil based adjuvant phase administered intramuscularly, followed by im challenge with 106.2 TCID50 of FAdV-8b (strain 13-18153). An additional group served as challenge control, injected with a PBS / adjuvant mixture instead of vaccination, and a negative control received only sterile PBS following the same scheme. Blood was collected from boosters every week until challenge and at 3, 5, and 7 days (dpc) after challenge. Four birds per group were sacrificed and necropsied at 3 and 5 dpc, and the remaining birds were similarly necropsied at 7 dpc. Protection endpoints included daily assessment of clinical signs in the post-challenge period, organ weight (BW) ratios for liver and spleen, aspartate transaminase (AST) content in plasma as previously described (Matos et al., 2016), and viral load quantification in liver and pancreas by a qPCR protocol adapted from Gunes et al., 2012.

[0085] Clinical trial 2: Broad protective efficacy of crecFib-8b / 8a applied with different vaccination regimens In this setting, we proceeded with only one of the chimeras, crecFib-8b / 8a, this time testing its protective efficacy against challenge with both virus types of interest (FAdV-8a or FAdV-8b). Furthermore, a prime-boost vaccination regime was compared with a single injection regime using a group of 20 SPF broilers. Each vaccination involved the administration of 50 μg of crecFib-8b / 8a, formulated in a 40% (w / v) antigen-oil based adjuvant phase, intramuscularly. An intramuscular challenge was performed with 106.2 TCID50 of FAdV-8a (strain 11-16629) or FAdV-8b (strain 13-18153), negative control birds were again administered PBS instead.

[0086] Blood was collected weekly from 2 weeks of age until challenge and then at each of the following sampling time points: 3 dpc, 5 dpc, 7 dpc and 14 dpc. Up to 5 birds / flock were killed and necropsied at 3 dpc, 5 dpc, 7 dpc and 14 dpc along with any individuals that died from infection.

[0087] Protection endpoints included organ-to-BW ratios in liver, spleen and bursa of Fabricius, AST content in plasma, and viral loads in liver, pancreas, spleen and bursa of Fabricius.

[0088] Statistical analysis of the data sets was performed using the Shapiro-Wilk test along with visual inspection of histograms and normal QQ plots to verify the assumption of normal distribution. Mean values ​​of organ-to-BW ratios, plasma AST and viral load in target organs of vaccinated groups were compared with negative controls and their corresponding challenged control groups by unpaired Student's t test. Data sets that did not meet the assumption of normality were analyzed by pairwise comparisons with the Mann-Whitney U test. In all cases, a p value ≤ 0.05 was considered statistically significant. Statistical analysis was performed using SPSS version 26 (IBM SPSS Statistics; IBM Corp., Armonk, NY, USA).

[0089] result In silico design and recombinant expression of crecFib constructs In silico epitope analysis of the fiber knobs of TR59 and 746 suggested assigned sites in the CD loop (G.SSD, N.PTG), β-strand F / FG loop (V.DANP, I.DASS), and HI loop (QSQ, RSQ) of the knobs based on homology modeling. According to a three-dimensional model constructed based on the chimeric knob sequence, all predicted epitopes were localized externally and apically on the molecule (Fig. 1c). Furthermore, the two aforementioned sites in the β-strand F / FG loop and the HI loop are discontinuous in the primary sequence but conformationally in contact, revealing a coherent surface patch formed by residues encoded at the N- and C-distal ends of the chimeric junction.

[0090] Both chimeric proteins were successfully recovered from the soluble fraction of infected Sf9 cells, as confirmed by bands with the appropriate monomer size in Western blots. The yields of purified chimeric fibers were approximately 13.4 mg (crecFib-8a / 8b) and 14.8 mg (crecFib-8b / 8a) per liter of Sf9 cell culture.

[0091] Immunogenicity and in vitro reactivity spectrum of crecFib crecFib antibody induction detected by ELISA By homologous antigen-ELISA, birds immunized with crecFib-8a / 8b showed a flat increase in OD magnitude, with only sporadic occurrences of OD > 1 in single individuals at three consecutive time points between 5 and 7 weeks post vaccination (wpv). All other birds did not exceed OD 0.5 at any time point during the 8-week monitoring period (Figure 2). In contrast, birds immunized with the reversed order, crecFib-8b / 8a, developed a sharp increase in OD against the homologous ELISA antigen as early as 2 wpv, at which point five out of five birds already showed an OD close to 3 (mean OD 3.00 ± 0.18), independent of antigen dose (50 vs. 100 μg). The OD of crecFib-8b / 8a remained at a constant level in all five individuals until the end of the monitoring period at 8 wpv, with mean weekly ODs ranging from 2.76 ± 0.29 to 3.39 ± 0.12.

[0092] Neutralizing activity of crecFib antiserum For crecFib-8b / 8a antisera, neutralization was first present at 2 wpv, with all birds showing low to moderate titers (log24–7) against at least one of the constitutive types (Figure 2). In fact, already 4 out of 5 individuals had developed bilateral nAbs against both types at this time point. Neutralization titers of crecFib-8b / 8a antisera continuously increased during the monitoring period; from 5 wpv onwards, all birds had titers ≥log26 against the complete set of test strains, with one bird reaching the maximum of the measurement range (log214) against FAdV-8b. At the individual bird level, the neutralization responses showed an overall balanced distribution, with similar titers against both types, regardless of whether the corresponding virus was the template (reference) or a field strain.

[0093] Immunofluorescence staining in a virus neutralization setting comparing the crecFib-8b / 8a antiserum directly side-by-side with anti-Fib-8a or Fib-8b antisera on the same microtiter plate demonstrated that only the chimeric serum was able to efficiently inhibit both FAdV-8a (strain TR59) and FAdV-8b (strain 764) infection in vitro. The monospecific Fib-8a and Fib-8b antisera showed neutralizing activity against their cognate serotypes with similar titers compared to the crecFib antiserum, but none of them was able to inhibit the opposite serotype.

[0094] Fiber recognition in Western blots Independent of their genetic background and their monospecific or chimeric composition, all recombinant fibers investigated were detected by immune sera representing the complete spectrum of FAdV types defined by fiber specificity (Figure 3).

[0095] Mutual recognition was possible between reactive partners with different fiber expression (fiber 1, fiber 2 of FAdV-A and FAdV-C versus single fibers of other FAdV species) and even between chimeric counterparts with reversed template order (e.g., the crecFib-8b / 8a antiserum was able to recognize crecFib-8a / 8b).

[0096] Notably, no differences were observed in the ability of antisera raised against live (native) FAdV and inactivated adjuvanted virus preparations, nor against subunit-directed sera, to recognize fiber monomers in immunoblots.

[0097] Clinical Trials Clinical trial 1: Protective efficacy of crecFib-8a / 8b and crecFib-8b / 8a An overview of the in vivo experimental design is shown in Figure 4. VV 8a / 8b C 8bThe group, designated VV, was vaccinated twice with crecFib-8a / 8b at 3 and 21 days of age and subsequently challenged with FAdV-8b at 36 days of age (= 15 days post-booster, dpb). 8b / 8a C 8b The second group, designated C, was vaccinated with the reversed order, crecFib-8b / 8a, at the same time points and then also challenged with FAdV-8b. A challenge control mock-vaccinated with PBS / adjuvant mixture prior to FAdV-8b challenge was designated C. 8b The negative control was administered only with sterile PBS at each time point and designated as N.

[0098] The onset of vaccine-induced antibody development was not detected by ELISA until after the booster (7 dpb = 28 days of age), and VV 8a / 8b In birds, only low levels were observed (mean OD 0.51 ± 0.65), indicating the onset of an increase. 8b / 8ab The peak detectable level was already reached in N and C (3.14 ± 0.73) (Fig. 5). 8b Birds (before challenge) never exceeded an OD of 0.07 ± 0.01. Just before challenge (14 dpb), VV 8a / 8b Although the antibodies reached a moderate OD (1.45±0.95), none of the sera showed neutralizing activity (SN titer ≦log28). 8b / 8a At OD levels remained fairly constant (2.60 ± 0.85) and all sera, except for one bird, showed neutralizing activity, ranging from low titers against at least one of the tested serotypes (3 birds) to titers at the highest end of the measured range against both serotypes (FAdV-8a and FAdV-8b reference and challenge viruses) in the remaining birds (log214). A general trend was observed of higher titers against FAdV-8a (earlier onset, based on sera with only unilateral neutralization). In addition, nAbs against FAdV-8b never diverged by more than 3 titers between the reference and challenge strains, whereas nAbs against FAdV-8a diverged up to 5 titers with a more pronounced reactivity against the challenge compared to the reference strain.

[0099] After the challenge, 2 Cs were found 4dpc-5dpc. 8b A mild depression was noted in the bird (anorexia, hunchbackedness, ruffled feathers). During necropsy at this time, the bird had a significantly increased liver:BW ratio. The increased liver:BW ratio was also consistent with the VV 8a / 8b C 8b and V.V. 8b / 8a C 8b This occurred late at 7 dpc (Figure 6). 8b The group also experienced a significant increase in spleen:BW (7 dpc), which was consistent with the VV 8b / 8a C 8b Not present in VV 8a / 8b C 8b is moderate but not significant.

[0100] The highest plasma AST was C 8b was recorded at 3dpc and 5dpc, and was significant at 5dpc, but VV 8a / 8b C 8b and V.V. 8b / 8a C 8b remained comparable to N (Fig. 6). At 7 dpc, plasma AST levels were no longer distinguishable between groups.

[0101] Liver viral load vs. C 8b A decrease in VV 8a / 8b C 8b and V.V. 8b / 8a C 8b Recognized in both VV 8b / 8a C 8b The lowest and most significant levels were consistently detected in the pancreas at 5 dpc (Fig. 6). 8b / 8a C 8b It was completely absent at 3 dpc and significantly decreased at 5 dpc and 7 dpc. 8a / 8b C 8b In the present study, the pancreatic viral load was significantly decreased at 5 days after administration.

[0102] Clinical trial 2: Broad protective efficacy of crecFib-8b / 8a applied with different vaccination regimens Two groups were administered a prime-boost vaccination regimen at 1 and 7 dpv. One group was challenged with FAdV-8a at 22 dpv (VV 8b / 8a C 8a ), and the other was challenged with FAdV-8b (VV 8b / 8a C 8b This was then combined with a single vaccination regimen at 1 day of age (group V 8b / 8a C 8a , V 8b / 8a C 8b ) compared with a mock vaccinated challenge control group (C 8a , C 8b ), and a negative control group (N) that received PBS similar to the prime-boost regimen.

[0103] At 21 days of age, just prior to challenge, birds in each vaccination regimen had developed antibody levels detectable by ELISA. 8b / 8a ) had a higher OD, with only 1 individual out of 40 having an OD < 3 (mean OD 3.3 ± 0.1), whereas the single vaccine regimen (V 8b / 8a ) had overall lower and more unevenly distributed titers (2.65 ± 0.79) (Figure 7). After challenge and regardless of the type of challenge virus, V 8b / 8a and V.V. 8b / 8a The OD of V reached (and then remained at) the highest measurable endpoint. 8b / 8a and V.V. 8b / 8a In contrast, unvaccinated challenged control birds showed a steady slope of titers to 2.15 ± 1.02 by 14 dpc (C 8a ), or a sharp increase to 2.02 ± 0.95 at 7 dpc (C 8b ) and remained stable at that time.

[0104] 36 out of 40 VV 8b / 8aIn the prechallenge Ab birds, the prechallenge Ab birds had neutralizing activity against both FAdV-8a and FAdV-8b, whereas the remaining four birds showed only one-sided neutralizing activity against one of the serotypes. 8b / 8a Birds showed a rarer presence of prechallenge nAbs, with 11 of 40 birds having unilateral nAbs and 7 birds being completely absent of nAbs. Furthermore, titer levels were higher for VV 8b / 8a Compared to V 8b / 8a Although the neutralization was lower in the V- and V-specific antibodies, the regimens were similar in eliciting stronger neutralization against FAdV-8a (V- and V-specific antibodies against FAdV-8a). 8b / 8a log23.28±3.22 vs VV 8b / 8a log25.58±2.00, and V 8b / 8a log22.88±2.44 vs. VV 8b / 8a log24.30±3.13).

[0105] FAdV-8a challenge was performed in naive birds (C 8a ) resulted in a relatively rapid increase in nAb levels in the vaccinated groups, reaching higher levels at 7 dpi compared to either of the vaccinated groups. 8b / 8a C 8a In the study, overall higher titers were seen against FAdV-8b than against FAdV-8a, but V 8b / 8a C 8a By 14 dpc, birds in all three groups reached similar mean titers to FAdV-8a. FAdV-8b challenge produced similar mean titers (log210.70 ± 1.34) to FAdV-8a challenge at 7 dpc compared with naive birds (C 8b ) but not in vaccinated birds (V 8b / 8a C 8b , V.V. 8b / 8a C 8b ) did not significantly exceed the mean titer of V 8b / 8a C 8b and V.V. 8b / 8a C 8b continued to develop nAbs to FAdV-8a, but the mean titers to FAdV-8b remained consistently higher.

[0106] The criterion for clinical morbidity after challenge was one chick (C 8a ) and 4 birds (C 8b ) mild depression, and C 8b An additional dead bird was recorded 4 dpc in the vaccinated group. Not surprisingly, among all birds in the vaccinated group, 100% of the birds were dead, with 100% of the birds lacking prechallenge nAb. 8b / 8a C 8b In only one individual, a transient depressive state occurred between 2 and 3 dpc.

[0107] The mean liver:BW and spleen:BW ratios were most affected at 3 and 5 dpc, increasing significantly in both challenge controls (Figure 8). Although plateauing, this effect was consistent with C 8a At 7 days after birth, the spleen was 8a and C 8b In contrast, the liver:BW and spleen:BW ratios of the vaccinated / challenged groups were significantly higher than those of the vaccinated / challenged groups at 14 dpc. 8b / 8a C 8b The liver remained comparable to the negative control (N) throughout the experiment, with the exception of the V. The capsule:BW ratio was most affected at 5 dpc, with the same trend shifting to 14 dpc. 8b / 8a C 8b All groups, except for , were significantly decreased relative to N.

[0108] Furthermore, C 8a and C 8b had the highest plasma AST levels of all groups from 3 dpc to 7 dpc. 8b / 8a C 8b and V 8b / 8a C 8a ) were found. Moreover, vaccinated groups consistently showed lower viral loads in target organs compared to their challenged controls, while vaccination even prevented detectable infection at some time points. With a few exceptions, C 8a and C 8bLiver, pancreas and spleen samples from all time points were positive (C 8a In only one bird, the liver was negative at 7 dpc, and the pancreas and spleen of another bird were negative at 14 dpc; 8b (In one study, two livers were negative at 14 dpc.) In contrast, viral DNA was not detected in the VV 8b / 8a C 8a In the study, the virus was detected only in the liver of one bird at 5 dpc and in the liver and pancreas of another bird at 14 dpc, and the viral load significantly decreased from 3 to 7 dpc. 8b / 8a C 8a Similar results were achieved in , with the pancreas and spleen of only one bird testing positive at the earliest time point (3 dpc). 8b / 8a C 8b In our study, mean viral loads were significantly reduced from 5 to 7 dpc in the liver and at all time points in the pancreas and spleen; in fact, all target organs remained completely negative at 5 and 14 dpc. 8b / 8a C 8b In the spleen at 3-5 dpc and in the liver and pancreas at 5 dpc, a significant decrease in viral load occurred. The bursa of Fabricius was the organ with the lowest mean viral load, whereas C 8a In all but two samples, C 8b Positivity was still seen at 3-7 dpc in 4, 3 and 1 samples from all vaccinees. Of all vaccinees, only 3 individuals challenged with FAdV-8b who did not have prechallenge nAbs were positive at 3 dpc. At 14 dpc, viral DNA was generally no longer detectable in the bursa.

[0109] Example 2: Vaccination with FAdV-chimeric fiber protein simultaneously protects chickens against HHS and IBH During the past decades, FAdV-associated diseases have become an increasing concern for poultry farming worldwide. Various immunization strategies against FAdVs have been experimentally investigated, focusing in particular on subunit vaccines against HHS caused by FAdV serotype 4, and IBH caused by serotypes 2, 8a, 8b, and 11. In this study, we extended the innovative concept of recombinant chimeric fiber proteins to design a novel chimera carrying both epitopes from serotypes belonging to FAdV-4 and FAdV-11 and to investigate its efficiency in simultaneously protecting chickens against HHS and IBH. Specific pathogen-free chickens were vaccinated with the novel chimeric fiber and subsequently challenged with either strains causing HHS or IBH. The development of neutralizing antibodies was restricted against FAdV-11 and absent against FAdV-4. Nevertheless, surprisingly, vaccinated birds showed reduced clinical signs, limited hepatomegaly, and lower levels of AST upon virus challenge compared to the respective challenged controls. Furthermore, the vaccine prevented atrophy of lymphoid organs affected by HHS, such as the thymus and bursa of Fabricius, and significantly reduced viral load in target organs. Clinical protection was associated with high levels of prechallenge antibodies measured on ELISA plates coated with vaccination antigens. In conclusion, we demonstrated that the chimeric fiber vaccine concept can be extended across viral species boundaries. Our vaccine represents the first FAdV subunit vaccine that can achieve comprehensive protection against different FAdV-associated diseases.

[0110] Materials and Methods Design and expression of chimeric fibers In this study, we designed chimeric fiber proteins that carry epitopes from FAdV-4 (fib-2) and FAdV-11. Briefly, the fiber open reading frame (ORF) was split into amino (N)- and carboxy (C)-distal segments, amplified separately from different template strains, and seamlessly fused via Gibson assembly cloning to reconstitute a novel full-length fiber. The junction between the N- and C-distal segments, which represents a crossover between heterologous sequences of distinct serotypes, is hereafter referred to as the specificity switch. The switch was engineered at an intertype consensus motif corresponding to positions aa441-aa442 or aa491-aa492 in the pairwise sequence alignment of FAdV-4 fiber 2 and FAdV-11 fibers, which maps to the proposed G strand within the fiber head ("knob") domain. Flanked by putative B-cell epitopes, this strategy allowed the incorporation of epitope regions from both constitutive serotypes into the final chimeric product. Furthermore, an internal modification of the knob could be reconciled with the maintenance of sequence integrity at the putative shaft-knob boundary, which has a reported function in fiber trimerization (Hong & Engler, 1996). Candidate epitopes were inferred from previously reported epitopes in fiber 2 of FAdV-4 (Wang et al., 2018) and in silico predictions using DiscoTope 2.0 software (Vindahl Kringelum et al., 2012), utilizing fiber 2 of the FAdV-1 reference strain CELO (El Bakkouri et al., 2008), the closest related fiber for which a molecular model is currently available. Positional homology between members of the different types was subsequently determined based on multiple sequence alignments made with MegAlign software (DNASTAR, Madison, WI, USA). Homology mapping was also used to assign structural domains (tail, shaft, knob) of the FAdV fiber based on existing information ( Hess et al., 1995 ).Based on the FAdV reference strain selected as expression template, the N-distal and C-distal fiber segments as defined above were amplified using a primer pair featuring overhangs with flanking sequences and between the two segments themselves from EheI / StuI digested pFAST BAC expression vector (Invitrogen, Vienna, Austria). Detailed information on the cloning strategy is shown in Table 1. Ligation into the linearized pFAST BAC vector was performed using Gibson Assembly Master Mix (NEB, Ipswich, MA) according to the manufacturer's instructions, and the resulting construct was named crecFib-4 / 11.

[0111] Table 1. Chimeric FAdV fiber proteins used in this study. Overhangs at the 5' end of the primer sequences with adjacent vector sequences (FP1 primer and RP2 primer) or corresponding template sequences (RP1 primer and FP2 primer) are represented by underlined nucleotides.

[0112] [Table 1]

[0113] Correct insertion of the segments into the vector was confirmed by Sanger sequencing across the multiple cloning site (LGC Genomics, Berlin, Germany).

[0114] Recombinant proteins were expressed in Fall Armyworm Sf9 cells and purified via their polyhistidine tags on affinity chromatography columns as previously described (Schachner et al., 2014), and their concentrations were determined by Bradford assay (Thermo Scientific, Vienna, Austria).

[0115] Sequence of the crecFib construct crecFib-4 / 11 (used for Fib-4 fiber 2) N-terminus: nt1-1221 from FAdV-4 reference strain (KR5) (HE608152) C-terminus: nt 1468-1713 derived from FAdV-11 field strain (strain 13 / 14796) TCCGTGCAAACTTTCCAACCCGTATTGACAGGAGATTGGGATACCAACACCTACAACCCAGGAACCGTTCAAGTCTGCATACTGCCTCAAACCGTTGTGGGAGGCCAGTCGACCTTTGTTAACATGACATGTTATAACTTCCGGTGTCAAAATCCTGGAATATTCAAGGTTGCTGCTAGTAGCGGCACATTCACTATCGGACCCATTTTCTACTCCTGTCCAACCAACAAATTAACACAACCCTAA Full sequence:

[0116] Virus preparation Field isolates AG234 and 08-18926 (GenBank accession numbers MK572849 and MK572871) were analyzed by next-generation sequencing and virus neutralization tests (VNT), identified as members of FAdV-types 4 and 11, respectively (Schachner et al., 2019), and used as challenge strains during protection studies. Strains were 3-fold plaque purified and propagated on primary chicken embryo liver (CEL) cells (Schachner and Sellers, 2008). Viral titers were determined by end-point titration (Reed and Muench, 1938).

[0117] Clinical Trials One hundred and twenty SPF chicks were hatched, individually tagged, and divided into six groups (n=20) designated as follows: vaccine only, vaccine vs. FAdV-4, vaccine vs. FAdV-11, FAdV-4 challenge control, FAdV-11 challenge control, and negative control (Table 2).

[0118] Table 2. Animal study design.

[0119] [Table 2]

[0120] Each group was housed separately in an isolator unit (HM2500, Montair, The Netherlands). Vaccination consisted of an injection in the lateral tibialis muscle of 0.5 ml containing 50 μg of crecFib-4 / 11 homogenized in an oil-based adjuvant, performed on 1-day-old birds of the vaccinated group, challenge control birds were injected with phosphate-buffered saline (PBS) mixed with adjuvant, and negative control birds received only sterile PBS. A booster injection was administered in the three vaccinated groups on the 7th day of life (6 dpv) as well, challenge and negative control birds received injections of adjuvant mixed with PBS and PBS only, respectively, as described above. Blood was collected at weekly intervals from the second week of life until challenge, and then at each of the following sampling time points: 3 dpc, 5 dpc, 7 dpc, and 14 dpc. Birds were challenged at 22 days of age (15 dpb) with FAdV-4 strain AG234 (group vaccine vs. FAdV-4, FAdV-4 challenge control) and FAdV-11 strain 08-18926 (group vaccine vs. FAdV-11, FAdV-11 challenge control). Birds were monitored daily for clinical signs during the post-challenge period. Up to five birds per group were killed and necropsied at 3 dpc, 5 dpc, 7 dpc and 14 dpc, and individuals euthanized due to severe clinical morbidity were immediately necropsied and sampled.

[0121] clinical chemistry Immediately prior to euthanasia, blood was collected from the jugular vein of each bird from the infected group and the negative control into heparin tubes (VACUETTE®, Greiner Bio-One, Kremsmünster, Austria) to determine plasma aspartate transaminase (AST) content as previously described ( Matos et al., 2016 ).

[0122] Quantitative polymerase chain reaction (qPCR) from tissues of target organs Tissue samples for viral load quantification, including liver, spleen and bursa of Fabricius, were collected from infected and negative control birds and stored at −20°C until processing. DNA extraction was performed using the DNeasy Blood&Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, and DNA was subsequently analyzed with an adapted qPCR assay based on the 52K gene ( Gunes et al., 2012 ).

[0123] Enzyme-linked immunosorbent assay (ELISA) and virus neutralization test (VNT) of chimeric fibers Sera collected during the experiment were tested on ELISA plates coated with crecFib-4 / 11 according to the protocol described by Feichtner et al. (2018). The cut-off OD value was calculated from the sera of negative control birds by calculating the arithmetic mean + 3 times the standard deviation. Samples from 21-, 27- and 29-day-old birds (prechallenge, 5 dpc and 7 dpc, respectively) were also investigated in VNT against the strain serving as template for fiber expression (vaccine strain) and the field isolate used as challenge strain during protection studies (challenge strain) as previously described (Schachner et al., 2014).

[0124] statistical analysis Preliminary analysis of the data sets was performed using the Shapiro-Wilk test along with visual inspection of histograms and normal QQ plots to verify the assumption of normal distribution. Mean values ​​of organ-to-BW ratios, plasma AST and viral load in target organs of the vaccinated groups were compared with the negative controls and their respective challenge control groups by unpaired Student's t-test. The Mann-Whitney U-test was used for data sets that did not meet the assumption of normality. In all cases, a p-value ≤ 0.05 was considered statistically significant. Statistical analysis was performed using the software package SPSS version 26 (IBM SPSS Statistics; IBM Corp., Armonk, NY, USA).

[0125] result Clinical Trials In the FAdV-4 challenged controls, clinical signs started at 2 dpc with 2 birds with mild depression and progressed across the group with 13 affected birds at 3 dpc, 3 of which showed severe depression. At 4 dpc, there were 9 affected birds, 2 of which had to be euthanized due to their inability to move and consume feed; at 5 dpc, 3 birds still showed milder clinical signs (Table 3). In the FAdV-11 challenged controls, only one bird was affected with mild depression at 4 dpc. Of the vaccinated groups, only one individual challenged with FAdV-4 showed signs of depression at 3 dpc. No clinical signs were recorded in the vaccine only and negative control groups throughout the experiment. Parameters recorded for birds euthanized at 4 dpc were included in the 5 dpc cluster for statistical analysis.

[0126] Table 3. Daily clinical signs for each experimental group in the post-challenge period. Number of affected individuals is shown in brackets. No clinical signs were recorded before 2 dpc and after 5 dpc.

[0127] [Table 3]

[0128] The mean liver-to-body weight (BW) ratio was significantly increased in the FAdV-4 challenged controls from 3 dpc to 7 dpc and in the vaccine vs. FAdV-4 group from 3 dpc to 5 dpc compared to the negative controls, whereas the spleen-to-BW ratio remained affected up to 7 dpc in both groups (Figures 9A, 10B). The FAdV-4 challenged controls also showed a significant decrease in the organ-to-BW ratio of the bursa of Fabricius at 3 dpc and 5 dpc, and the thymus-to-BW ratio at 5 dpc and 7 dpc, compared to the negative controls, unlike the respective vaccinated groups (Figures 9C, 10D). The FAdV-11 challenged controls showed a significant increase in the liver-to-BW ratio at 3 dpc and 7 dpc, as well as a significant increase in the spleen-to-BW ratio from 3 dpc to 7 dpc, whereas the vaccine vs. FAdV-11 group showed a significant effect of the spleen-to-BW ratio only at 5 dpc.

[0129] plasma AST Plasma AST was significantly increased from 3 dpc to 7 dpc in the FAdV-4 challenged controls and from 3 dpc to 5 dpc in the vaccine vs. FAdV-4 group compared to the negative controls, although values ​​in vaccinated birds at these time points were significantly lower compared to the FAdV-4 challenged controls (Figure 10). It was significantly increased at 7 dpc in the FAdV-11 challenged controls compared to the negative controls, and at 14 dpc in the vaccine vs. FAdV-11 group.

[0130] Viral load in target organs Within the FAdV-4 lineage, the mean viral load of vaccinated birds was significantly lower than challenge controls at 5 dpc in the liver, 3 dpc in the spleen and at both time points in the bursa of Fabricius (Figure 11). No significant differences were recorded in the organs analyzed between the FAdV-11-infected groups at any time point. At 14 dpc, it was possible to detect viral DNA only in the spleens of birds from the FAdV-4-infected group.

[0131] Antibody response: ELISA and VNT The cut-off value for the crecFib-4 / 11 ELISA was calculated at OD = 0.12. At 14 days of age (7 dpb), the mean OD across vaccinated birds (n = 60) was OD = 0.48 ± 0.48, with 81.7% of birds (49 / 60) above the cut-off (Figure 12). The day before challenge (21 days of age, 14 dpb), the mean OD value of vaccinated birds increased to 2.43 ± 0.87, with all birds above the cut-off. Adjuvant-injected birds (n = 40) had mean OD values ​​of 0.07 ± 0.03 and 0.05 ± 0.01 at 14 and 21 days of age, respectively. The mean OD of the vaccine-only group decreased at 3 dpc (1.70 ± 1.09) and then increased continuously throughout the experiment, reaching OD = 3.46 ± 0.01 at 36 days of age (29 dpb). After challenge, the mean OD of the vaccine vs. FAdV-4 group increased to 3.09 ± 0.42 at 3 dpc, reached the highest end of measurable values ​​(3.32 ± 0.28) at 5 dpc, and then remained constant at that plateau until the end of the experiment. In comparison, a rapid rise in antibodies measured by ELISA was observed in FAdV-4 challenged control birds from 5 dpc to 14 dpc (mean OD = 0.25 ± 0.15 to 2.57 ± 0.44), but they remained lower than the vaccinated group. Vaccinated birds challenged with FAdV-11 also rapidly reached a plateau after infection (mean OD 3.24 ± 0.13 at 3 dpc to 3.39 ± 0.13 at 14 dpc), whereas the FAdV-11 challenged controls showed low levels of antibody development (peak at 14 dpc and mean OD 0.28 ± 0.13). Sera from negative control birds remained below the cutoff value throughout the experiment.

[0132] On the day before challenge, only four of the vaccinated birds showed any nAb to FAdV-11, of which one reacted to both vaccine and challenge strains (titers: 3 log2 and 8 log2, respectively), one reacted only to the vaccine strain (titers: 5 log2), and two reacted to the challenge strain (titers: 3 log2 and 9 log2) (Figure 13). In contrast, there was no neutralizing activity against any of the FAdV-4 strains, and the vaccine only group showed no nAb expression at any of the time points tested. The rise in nAbs against FAdV-4 began after challenge in infected birds, with vaccinated and challenged controls showing similar values ​​against both the challenge strain (mean titers: 5.07 ± 2.09 log2 vs. 4.57 ± 1.83 log2 at 5 dpc, and 7.56 ± 1.51 log2 vs. 6.90 ± 0.99 log2 at 7 dpc) and the vaccine strain (2.53 ± 2.56 log2 vs. 3.64 ± 2.37 log2 at 5 dpc, and 6.67 ± 1.87 log2 vs. 6.10 ± 0.99 log2 at 7 dpc). The experimental groups infected with FAdV-11 showed a similar pattern against the challenge strains, but titers in vaccinated birds were slightly lower than challenge controls (mean: 3.79 ± 2.78 log2 vs. 5.36 ± 2.68 log2 at 5 dpc and 5.90 ± 1.10 log2 vs. 7.00 ± 1.63 log2 at 7 dpc), and mean titers were generally lower against the vaccine strain (0.29 ± 1.07 log2 vs. 2.80 ± 2.10 log2 at 5 dpc and 0.50 ± 1.29 log2 vs. 2.30 ± 2.11 log2 at 7 dpc). Interestingly, a small number of vaccinated birds infected with FAdV-4 developed nAbs against FAdV-11 after challenge, with 3 individuals showing nAbs against both strains and 1 individual only against the vaccine strain between 5 dpc and 7 dpc. Data on the number of individuals showing nAbs for each group are shown in Figure 13.

[0133] conclusion The vaccine with the chimeric fiber protein crecFib-4 / 11 was able to prevent clinical signs upon FAdV-4 infection in all but one immunized bird, in contrast to the challenge control, in which several birds were affected at various time points (including severe depression that led to the euthanasia of two individuals). Despite the presence of hepatomegaly in both FAdV-4-infected groups at the early stage of infection, the vaccinated group experienced a faster recovery within one week after challenge. This trend was reflected by plasma AST analysis, another indicator of liver health, as well as a significant reduction in the viral load in the liver of vaccinated birds compared to the FAdV-4-challenged control for the same period. In contrast, splenomegaly was recorded in both vaccinated and non-vaccinated birds. However, when considering this parameter, the dual role of the spleen as a lymphoid organ and target of infection must be taken into account, since the magnitude of the immune response may have played a role in influencing the spleen size in vaccinated birds. Indeed, the viral load in the spleen was generally lower in the vaccinated group. Furthermore, the vaccine prevented HHS-induced atrophy of other major lymphoid organs, such as the thymus and bursa of Fabricius, possibly by limiting viral spread, as supported by significantly lower viral loads in the bursa of vaccinated groups compared with FAdV-4-challenged controls.

[0134] The impact of IBH infection with FAdV-11 was generally milder than that of HHS. Among all FAdV-11 infected birds, only one challenged control showed signs of mild depression. However, the deleterious effect of the virus was evident from the significant increase in liver-BW and spleen-BW ratios in the challenged control group, sometimes paired with a pathological increase in AST, a lesion that was consistently prevented by the crecFib-4 / 11 vaccine, with the only exception of splenomegaly observed in the vaccinated group at 5 dpc.

[0135] The clinical protection provided by crecFib-4 / 11 against both HHS and IBH was coupled with the induction of high and uniform levels of systemic antibodies against the vaccination antigens measured by in-house fiber ELISA immediately prior to challenge, with all vaccinated birds expressing OD values ​​above the calculated cut-off value. Overall levels in the vaccinated / challenged groups reached the highest end of measurable values ​​immediately after challenge and plateaued until the end of the experiment. The inclusion of a vaccine-only group allowed monitoring of antibody development over the entire period, and showed that ELISA-measured antibodies in vaccinated / unchallenged birds overlapped with those in the vaccinated / challenged groups, confirming that such efficient humoral responses were not necessarily due to a booster effect by live virus. Indeed, antibodies against crecFib-4 / 11 never reached the highest measurable OD in the challenge controls. However, a consistent lack of neutralizing antibodies (nAbs) was observed in the vaccinated groups prior to challenge, with very few birds showing neutralization against FAdV-11 and none against FAdV-4. Neutralization against FAdV-11 also appeared after infection with FAdV-4 in a minority of vaccinated birds, indicating a delayed emergence of vaccine nAbs or a possible viral booster effect. In contrast, challenged control birds consistently showed neutralizing activity against each of the infecting viruses after challenge, with all survivors showing nAbs one week after infection.

[0136] In summary, subunit vaccination with crecFib-4 / 11 protected chickens against clinical signs and severe outcomes of HHS while suppressing pathological morbidity from both HHS and IBH, thus again confirming that chimeric fibers represent an efficient protection strategy to provide broad coverage against FAdV, not only at the heterotypic level as previously demonstrated in Example 1, but also across species boundaries.

[0137] Non-patent literature Bertran K, et al (2021) A 10-Year retrospective study of inclusion body hepatitis in meat-type chickens in Spain (2011-2021). Viruses 13:2170 Chen L, et al (2018) Immunogenicity and protective efficacy of recombinant fiber-2 protein in protecting SPF chickens against fowl adenovirus 4. Vaccine 36:1203-1208 De Luca C, et al (2020) Fowl adenovirus (FAdV) fiber-based vaccine against inclusion body hepatitis (IBH) provides type-specific protection guided by humoral immunity and regulation of B and T cell response. Vet Res 51:143 El Bakkouri M, et al (2008) Structure of the C-terminal head domain of the fowl adenovirus type 1 short fibre. Virology 378:169-176 Feichtner F, et al (2018) Development of sensitive indirect enzyme-linked immunosorbent assays for specific detection of antibodies against fowl adenovirus serotypes 1 and 4 in chickens. Avian Pathol 47:73-82 Gomis S, et al (2006) Inclusion body hepatitis as a primary disease in broilers in Saskatchewan, Canada. Avian Dis 50:550-555 Grafl B, et al (2020) Successful reproduction of adenoviral gizzard erosion in 20-week-old SPF layer-type chickens and efficacious prophylaxis due to live vaccination with apathogenic fowl adenovirus serotype 1 strain (CELO). Vaccine 38:143-149 Gunes A, et al (2012) Real-time PCR assay for universal detection and quantitation of all five species of fowl adenoviruses (FAdV-A to FAdV-E). J Virol Methods 183:147-153 Gupta A, et al (2017) Immunogenicity and protective efficacy of virus-like particles and recombinant fiber proteins in broiler-breeder vaccination against fowl adenovirus (FAdV)-8b. Vaccine 35:2716-2722 Gupta A, et al (2018) Inactivated and live bivalent fowl adenovirus (FAdV8b + FAdV11) breeder vaccines provide broad-spectrum protection in chicks against inclusion body hepatitis (IBH). Vaccine 36:744-750

[0138] Harrach B, et al, Family Adenoviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses, 9th ed. Elsevier Academic Press, San Diego Henry Dunand CJ, et al (2016) Both neutralizing and non-neutralizing human H7N9 influenza vaccine-induced monoclonal antibodies confer protection. Cell Host Microbe 19:800-813 Hess M, et al (1995) The avian adenovirus penton: two fibres and one base. J Mol Biol 252:379-385 Hess M (2020) Aviadenovirus infections. In: Swayne DE, Boulianne M, Logue CM, McDougald LR, Nair V, Suarez DL (eds) Diseases of Poultry, 14th ed. Wiley-Blackwell, Hoboken Hong JS, et al (1996) Domains required for assembly of adenovirus type 2 fiber trimers. J Virol 70:7071-7078 Hu Z, et al (2020) Hemagglutinin-specific non-neutralizing antibody is essential for protection provided by inactivated and viral-vectored H7N9 Avian Influenza vaccines in chickens. Front Vet Sci 6:482 Kim M, et al (2014) An inactivated oil-emulsion fowl adenovirus serotype 4 vaccine provides broad cross-protection against various serotypes of fowl adenovirus. Vaccine 32:3564-3568 Marek et al (2012) Two fiber genes of nearly equal lengths are a common and distinctive feature of fowl adenovirus C members. Vet. Microbiol. 156: 411-417) Matos M, et al (2016) Selected clinical chemistry analytes correlate with the pathogenesis of inclusion body hepatitis experimentally induced by fowl aviadenoviruses. Avian Pathol 45:520-529 Mittal D, et al (2014) Characterization of fowl adenoviruses associated with hydropericardium syndrome and inclusion body hepatitis in broiler chickens. Virusdisease 25:114-119

[0139] Mo J (2021) Historical investigation of fowl adenovirus outbreaks in South Korea from 2007 to 2021: A comprehensive review. Viruses 13:2256 Norrby E (1969) The relationship between the soluble antigens and the virion of adenovirus type 3. IV. Immunological complexity of soluble components. Virology 37:565-576 Pallister JA, et al (1993) Serological relationships within the group E fowl adenoviruses. Intervirology 36:84-90 Popowich S, et al (2018) Broad spectrum protection of broiler chickens against inclusion body hepatitis by immunizing their broiler breeder parents with a bivalent live fowl adenovirus vaccine. Res Vet Sci 118:262-269 Reed LJ, Muench H (1938) A simple method of estimating fifty percent endpoints. Am J Hyg 27:493-497 Schachner A, et al (2014) Recombinant FAdV-4 fiber-2 protein protects chickens against hepatitis-hydropericardium syndrome (HHS). Vaccine 32:1086-1092 Schachner A, et al (2016) Detailed molecular analyses of the hexon loop-1 and fibers of fowl aviadenoviruses reveal new insights into the antigenic relationship and confirm that specific genotypes are involved in field outbreaks of inclusion body hepatitis. Vet Microbiol 186:13-20 Schachner A, et al (2018) Fowl adenovirus-induced diseases and strategies for their control - a review on the current global situation. Avian Pathol 47:111-126 Schachner A, et al (2019) Fowl adenovirus (FAdV) recombination with intertypic crossovers in genomes of FAdV-D and FAdV-E, displaying hybrid serological phenotypes. Viruses 11:1094 Schat K, et al (2008) Cell culture methods. In: Dufour-Zavala L, Swayne DE, Glisson JR, Pearson JE, Reed WM, Jackwood MW, Woolcock PR (eds) A laboratory manual for the isolation and identification of avian pathogens, 5th ed. OmniPress Inc, Madison Schonewille E, et al (2010) Specific-pathogen-free chickens vaccinated with a live FAdV-4 vaccine are fully protected against a severe challenge even in the absence of neutralizing antibodies. Avian Dis 54:905-10

[0140] Shah MS, et al (2012) A subunit vaccine against hydropericardium syndrome using adenovirus penton capsid protein. Vaccine 30:7153-7156 Steer PA, et al (2011) Application of high-resolution melting curve analysis for typing of fowl adenovirus in field cases of inclusion body hepatitis. Aust Vet J 89:184-192 Steer-Cope PA, et al (2019) Vaccination with FAdV-8a induces protection against inclusion body hepatitis caused by homologous and heterologous strains. Avian Pathol 48:396-405 Tian KY, et al (2020) Protection of chickens against hepatitis-hydropericardium syndrome and Newcastle disease with a recombinant Newcastle disease virus vaccine expressing the fowl adenovirus serotype 4 fiber-2 protein. Vaccine 38:1989-1997 Venne D (2013) Field data of the changing clinical picture over time of inclusion body hepatitis in Canada with an emphasis on diagnosis, prevention and trials on supportive treatments. In: Proceedings of the 24th Annual Australian Poultry Science Symposium, Sydney, New South Wales, Australia, February 2013. The Poultry Research Foundation and the World´s Poultry Science Association, pp 222-229 Vindahl Kringelum J, et al (2012) Reliable B cell epitope predictions: impacts of method development and improved benchmarking. PLoS Comput Biol, doi 10.1371 / journal.pcbi.1002829 Wang J, et al (2018) Variant serotypes of fowl adenovirus isolated from commercial poultry between 2007 and 2017 in some regions of China. Avian Dis 62:171-176 Wang X, et al (2018b) Immune protection efficacy of FAdV-4 surface proteins fiber-1, fiber-2, hexon and penton base. Virus Res 245:1-6 Wang P, et al (2018) A novel monoclonal antibody efficiently blocks the infection of serotype 4 fowl adenovirus by targeting fiber-2. Vet Res 49:29; doi 10.1186 / s13567-018-0525-y Wang X, et al (2019) Penton-dodecahedron of fowl adenovirus serotype 4 as a vaccine candidate for the control of related diseases. Vaccine 37:839-47

Claims

1. An avian adenovirus (FAdV) subunit vaccine comprising at least a chimeric FAdV fiber protein and an adjuvant.

2. 2. The vaccine of claim 1, wherein the chimeric FAdV fiber protein comprises an N-terminal fiber protein fragment derived from a first FAdV serotype and a C-terminal fiber protein fragment derived from a second FAdV serotype.

3. The vaccine of claim 2 , wherein the first FAdV serotype and the second FAdV serotype belong to different FAdV species.

4. 4. The vaccine of claim 3, wherein the first FAdV serotype belongs to the FAdV species FAdV-C and the second FAdV serotype belongs to a FAdV species selected from FAdV-B, FAdV-D and FAdV-E, or vice versa; or the first FAdV serotype belongs to the FAdV species FAdV-A and the second FAdV serotype belongs to a FAdV species selected from FAdV-B, FAdV-D and FAdV-E, or vice versa; or the first FAdV serotype belongs to the FAdV species FAdV-C and the second FAdV serotype belongs to the FAdV species FAdV-A, or vice versa.

5. 5. The vaccine of claim 2, wherein the first FAdV serotype is FAdV-4 and the second FAdV serotype is selected from FAdV-2, FAdV-11, FAdV-8a and FAdV-8b, or vice versa; or the first FAdV serotype is FAdV-1 and the second FAdV serotype is selected from FAdV-2, FAdV-11, FAdV-8a and FAdV-8b, or vice versa; or the first FAdV serotype is FAdV-4 and the second FAdV serotype is FAdV-1, or vice versa.

6. The vaccine of any one of claims 2 to 4, wherein the first FAdV serotype is selected from FAdV-2, FAdV-11, FAdV-8a and FAdV-8b and the second FAdV serotype is selected from FAdV-2, FAdV-11, FAdV-8a and FAdV-8b, or vice versa, with the proviso that two different FAdV serotypes are selected.

7. The vaccine of any one of claims 1 to 6, wherein the N-terminal fiber protein fragment comprises the N-terminal portion of the fiber shaft domain and the fiber knob domain, and the C-terminal fiber protein fragment comprises the C-terminal portion of the fiber knob domain.

8. 8. The vaccine of any one of claims 1 to 7, wherein the N-terminal fiber protein fragment comprises an amino acid sequence that is 70% or more identical, preferably 80% or more identical, more preferably 90% or more identical, even more preferably 95% or more identical, or even 97.5% or more identical, particularly 99% or more identical, or even completely identical to the sequence specified in any one of SEQ ID NOs: 1 to 4.

9. 8. The vaccine of any one of claims 1 to 7, wherein the C-terminal fiber protein fragment comprises an amino acid sequence that is 70% or more identical, preferably 80% or more identical, more preferably 90% or more identical, even more preferably 95% or more identical, or even 97.5% or more identical, particularly 99% or more identical, or even completely identical to the sequence specified in any one of SEQ ID NOs: 5 to 8.

10. The vaccine of any one of claims 1 to 9, wherein the vaccine is cross-protective.

11. A vaccine according to any one of claims 1 to 10 for use in immunising birds, preferably poultry, especially chickens, against FAdV infection.

12. The vaccine according to any one of claims 1 to 10 for use in ameliorating or preventing adenoviral gizzard erosion (AGE), inclusion body hepatitis (IBH) and / or hepatitis-hydropericardium syndrome (HHS) in birds, preferably poultry, especially chickens.

13. Obtaining a vaccine according to any one of claims 1 to 10, and administering said vaccine to a bird.

13. A method for vaccinating birds against FAdV infection comprising:

14. 1. A method for producing a FAdV subunit vaccine, comprising: expressing the chimeric FAdV fiber protein in an expression system; purifying the fiber protein; and combining said fiber protein with an adjuvant to obtain said FAdV subunit vaccine. The method includes: A method, wherein preferably said FAdV subunit vaccine is a vaccine according to any one of claims 1 to 10.

15. 15. The method of claim 14, wherein the expression system is selected from a baculovirus expression system, a bacterial expression system, and a yeast expression system.

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