Fowl adenovirus vaccine

Abstract

Disclosed is a vaccine comprising fiber (2) protein of Fowl Adeno-virus C (FAdV-C) or an immunogenic fragment thereof for use in preventing hepatitis-hydropericardium Syndrome (HHS) in birds, preferably in poultry, especially in broilers.

Claims

1. A vaccine for use in preventing hepatitis-hydropericardium syndrome (HHS) in birds comprising fiber-2 protein of Fowl Adenovirus C (FAdV-C), wherein the vaccine is further defined as a subunit vaccine comprising an immuno-effective amount of an adjuvant.

2. The vaccine of claim 1, wherein the adjuvant is Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide, Bordetella pertussis, saponin, muramyl dipeptide, ethylene vinyl acetate copolymer, oil, a vegetable oil or a mineral oil.

3. The vaccine of claim 2, wherein the adjuvant is peanut oil or silicone oil.

4. The vaccine of claim 1, wherein the fiber-2 protein of FAdV-C has a protein sequence of any one of SEQ ID NO: 9 through SEQ ID NO: 33.

5. The vaccine of claim 4, wherein the sequence of the fiber-2 protein of FAdV-C is the protein sequence of SEQ ID NO: 23.

6. The vaccine of claim 1, further comprising a pharmaceutically acceptable diluent and/or carrier.

7. The vaccine of claim 6, wherein the pharmaceutically acceptable diluent and/or carrier comprises a water-for-injection, physiological saline, tissue culture medium, propylene glycol, polyethylene glycol, vegetable oil, or an injectable organic ester.

8. The vaccine of claim 1, wherein the fiber-2 protein of FAdV-C is contained in an amount of 0.1 g/ml to 10 mg/ml.

9. The vaccine of claim 8, wherein the fiber-2 protein of FAdV-C is contained in an amount of 1 g/ml to 1 mg/ml.

10. The vaccine of claim 9, wherein the fiber-2 protein of FAdV-C is contained in an amount of 10 to 100 g/ml.

11. The vaccine of claim 1, consisting of: fiber-2 protein of FAdV-C, in an amount of 0.1 g to 10 mg; and a pharmaceutically acceptable carrier and/or diluent and/or adjuvant.

12. The vaccine of claim 11, wherein the fiber-2 protein of FAdV-C is in an amount of 1 g to 1 mg.

13. The vaccine of claim 12, wherein the fiber-2 protein of FAdV-C is in an amount of 10 to 100 g.

14. A method for preventing HHS in birds, comprising administering to the birds a vaccine of claim 1.

15. The method of claim 14, wherein the birds are in a parent flock.

16. The method of claim 14, wherein the birds are poultry.

17. The method of claim 16, wherein the poultry are broilers.

18. A kit comprising an isolated fiber-2 protein of FAdV-C immobilized on a solid surface or an immunogenic fragment of a fiber-2 protein of FAdV-C immobilized on a solid surface.

19. The kit of claim 18, further comprising a detection component that can detect the binding of an antibody to the immobilized fiber-2 protein of FAdV-C or the immobilized immunogenic fragment thereof.

Description

(1) The invention is further illustrated by the following examples and figures, yet without being restricted thereto.

(2) FIG. 1. Survival rates of birds of group I (Fib-1 vaccinated), group II (Fib-2 vaccinated) and group III (Hex L1 vaccinated), together with groups IV (positive control) and V (negative control), after infection with virulent FAdV strain AG234.

(3) FIG. 2. Pathologic lesions as manifested by focal necroses in the liver and pericardial sac filled with straw-coloured fluid in a bird from the positive control group IV that died 3 days post challenge (d.p.c.).

(4) FIG. 3. Results of antibody investigation as detected by (a) commercial FAdV Group-1 ELISA (results indicated as Sample to Positive (S/P) ratio of the mean OD value of maximum ten tested sera from each group, starting measurements on day 21 (before challenge), (b) Serum neutralization test (SNT) (results indicated as log.sub.2 transformed mean titers of maximum ten tested sera from each group, starting measurements on day 21; titres3 were considered negative), and (c) custom-made ELISA using recombinant Fib-2 protein (results indicated as mean OD values measured from sera of all Fib-2 vaccinated birds as well as positive and negative control birds, starting on day 7).

(5) FIG. 4. Immunoblots of purified recombinant KR5 proteins incubated with chicken sera collected on 21st day of life (preabsorbed with insect cell powder, diluted 1:2000). A, lanes 1, 2 and 3 purified Fib-1 incubated with sera from Fib-1 vaccinated birds. B, lanes 1, 2 and 3 purified Fib-2 incubated with sera from Fib-2 vaccinated birds. C, lanes 1, 2 and 3 purified Hex L1 incubated with sera from Hex L1 vaccinated birds. D, lane 1 purified Hex L1, lane 2 purified Fib-1, lane 3 purified Fib-2, incubated with serum from a bird of the positive control group (vaccinated with purified, non-infected insect cell material). E, lane 1 purified Hex L1, lane 2 purified Fib-1, lane 3 purified Fib-2, incubated with serum from a bird of the negative control group (non-vaccinated). Proteins are detected by serum antibodies as bands migrated to estimated molecular weight sizes of 51 kDa (Fib-1), 56 kDa (Fib-2) and 36 kDa (Hex L1).

(6) FIG. 5. Alignment of fiber proteins according to the present invention (Fib-2 of FAdV-C). Alignment is shown for (SEQ ID NO: 9) FIBER-2_PERU53, (SEQ ID NO: 10) FIBER-2_PERU54, (SEQ ID NO: 11) FIBER-2_C344, (SEQ ID NO: 12) FIBER-2_K1013QT, (SEQ ID NO: 13) FIBER-2_K1013, (SEQ ID NO: 14) FIBER-2_K31, (SEQ ID NO: 15) FIBER-2_K88-95, (SEQ ID NO: 16) FIBER-2_IV37, (SEQ ID NO: 17) FIBER-2_K99-97, (SEQ ID NO: 18) FIBER-2_C2B, (SEQ ID NO: 19) FIBER-2_09-584, (SEQ ID NO: 20) FIBER-2_09-8846, (SEQ ID NO: 21) FIBER-2_09-2602, (SEQ ID NO: 22) FIBER-2_DA60, (SEQ ID NO: 23) FIBER-2_KR5, (SEQ ID NO: 24) FIBER-2_ON1_GU188428, (SEQ ID NO: 25) FIBER-2_922-1, (SEQ ID NO: 26) FIBER-2_INT4, (SEQ ID NO: 27) FIBER-2_AG234, (SEQ ID NO: 28) FIBER-2_K388-95, (SEQ ID NO: 29) FIBER-2_CELO AC000014, (SEQ ID NO: 30) FIBER-2?_TADV-1_GU936707, (SEQ ID NO: 31) FIBER_A2-A_AC000013, (SEQ ID NO: 32) FIBER_HG_GU734104, and (SEQ ID NO: 33) FIBER_340.

EXAMPLES

(7) In the examples of the present invention, fiber-1, fiber-2 and the loop-1 region of hexon of an FAdV-C reference strain (KR5), were recombinantly expressed in the baculovirus system. In a vaccination trial, the efficacy of these capsid components to induce protective immunity in chickens was assessed by challenging birds with virulent FAdV. Hence, this is the first study of its kind to employ both fiber proteins individually in an in vivo experiment with the aim to further elucidate the functional significance of the investigated FAdV capsid proteins in the infection process and to address their potential use as candidate subunit vaccines for the control of HHS.

(8) 1. Materials and Methods

(9) 1.1. Virus Propagation and DNA Extraction

(10) FAdV-C (=FAdV-4) reference strain KR5 and the challenge virus AG234 were propagated on primary chicken-embryo liver (CEL) cells according to a protocol described by Schat and Sellers, A Laboratory Manual for the Isolation and Identification of Avian Pathogens, (2008), 195-203). Viral titer was determined according to the method of Reed and Muench (Am. J. Hyg. 27 (1938), 493-497) by endpoint titration. DNA extraction from cell culture supernatant was carried out with the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany).

(11) 1.2. Cloning and Initial Protein Expression

(12) Primers were designed on the basis of the complete genomic KR5 sequence (GenBank accession number HE608152) and contained 5-terminal restriction sites for cloning into the pFastBac transfer vector (Invitrogen, Vienna, Austria) (Table 1). The entire encoding regions for fiber-1 and fiber-2 (nucleotides 30438 to 31739 and 31723 to 33162, respectively) and the hexon loop-1 region (nucleotides 20481 to 21366) were amplified from the FAdV-C reference strain KR5 using a proofreading DNA polymerase (Invitrogen, Vienna, Austria). Following intermediate cloning into the pCR4Blunt-TOPO vector (Invitrogen) and digestion with BamHI/StuI (Fib-1), StuI/XbaI (Fib-2) and NcoI/XhoI (Hex L1) fragments were ligated into the cleaved pFastBac vector at the respective restriction sites. After determining the correct insertion of each product into pFastBac by sequencing, the construct was transformed into competent E. coli DH10Bac cells (Invitrogen, Vienna, Austria). Recombinant baculovirus DNA was isolated from transformed colonies using the S.N.A.P. Miniprep Kit (Invitrogen, Vienna, Austria). The genes of interest were expressed in Spodoptera frugiperda Sf9 cells (Invitrogen, Vienna, Austria) as His-tag fusion proteins according to the manufacturer's protocol.

(13) 1.3. Identification of Recombinant Proteins

(14) To verify expression of the recombinant proteins and to optimize the expression conditions, SDS-PAGE was performed on the soluble and membrane-bound fractions of the cell lysate, collected from infected Sf9 monolayer cultures at different time intervals (24, 48, 72, 96 h) post-infection. Recombinant proteins were identified by immunoblot using anti polyhistidine antibody (Sigma-Aldrich, Vienna, Austria). Non-infected Sf9 cells were processed in the same way to serve as negative control.

(15) 1.4. Expression and Purification of Recombinant Proteins

(16) For expression, Sf9 suspension cultures (50 ml) were infected with amplified recombinant baculovirus at an MOI of 3. Cultures collected after 72 h inoculation in a shaking incubator were concentrated by centrifugation for 5 min at 3500 rpm. The resulting cell pellet was disrupted by resuspension in lysis buffer (containing 20 mM sodium phosphate, 0.5 M NaCl, 20-40 mM imidazole, 0.2 mg/ml lysozyme, 20 g/ml DNAse, 1 mM MgCl.sub.2, 1 mM PMSF and proteinase inhibitors) and sonication, with subsequent incubation on ice for 1 h. Clarified supernatants obtained by centrifugation of the crude cell lysates at 14000 rpm for 20 min at 4 C. were used for purification on affinity chromatography columns (His GraviTrap, GE Healthcare, Freiburg, Germany). Hexon L1 protein presented as insoluble material in the pellet fraction was solubilised with phosphate buffer containing 8 M urea. The 0.45 m-filtered sample was loaded on columns equilibrated with phosphate buffer containing 8 M urea, and the protein was eluted after step-washing the columns with decreasing concentrations of urea. Samples from each purification fraction were subsequently analyzed for presence of the proteins of interest by SDS-PAGE and immunoblotting.

(17) Prior to in vivo administration, the recombinant proteins were transferred into sterile PBS (Gibco/Invitrogen, Vienna, Austria) by buffer exchange in Slide-A-Lyzer 7K Dialysis Cassettes (Thermo Scientific, Vienna, Austria). Protein Hex L1 was additionally processed through Amicon Ultra-15 size exclusion spin columns (Millipore, Vienna, Austria) to remove eluted insect cell proteins and to concentrate the target protein. Protein concentrations were determined by Bradford assay (Thermo Scientific, Vienna, Austria).

(18) 1.5. Animal Experiment

(19) A total of 112 SPF (specific pathogen-free) chickens (VALO, Lohmann Tierzucht GmbH, Cuxhaven, Germany) were divided into five groups that were housed separately in isolator units (Montair Andersen bv, HM 1500, Sevenum, Netherlands). At first day of life, a 500 l injection was administered intramuscularly to each animal, containing 50 g of the recombinant protein, with group I (n=26) receiving fiber-1 (Fib-1), group II (n=28) receiving fiber-2 (Fib-2) and group III (n=26) receiving hexon loop-1 (Hex L1), mixed 1:1 with GERBU Adjuvant LQ #3000 (GERBU Biotechnik GmbH, Heidelberg, Germany; a sterile aqueous suspension of lipid particles with excipients and emulsifiers).

(20) Equally, birds of group IV (n=23) were injected with purified and dialysed material from non-infected insect cells to serve as a positive control. Birds of group V (n=9) were treated as a negative control and received an injection of 500 l sterile PBS.

(21) At day 21 of life, animals of groups I to IV were intramuscularly challenged with 200 l of 10.sup.7 50% tissue culture infective dose (TCID.sub.50)/ml of the virulent FAdV-C virus AG234. Birds of the negative control group were administered the same amount of sterile PBS intramuscularly.

(22) Upon challenge, the birds were monitored daily for clinical signs. Necropsy was performed on all animals that died or had to be euthanized in the course of the study. Samples taken at regular intervals included blood (collected on days 7, 11, 14, 21, 28, 35 and 42) for detection of antibodies and cloacal swabs (collected on days 21, 28 and 35) or tissue from the large intestine (taken on day 42) for detection of virus excretion at regular intervals.

(23) All remaining birds were killed at the termination of the experiment on day 42 of life.

(24) The trial and all of the included procedures on experimental birds were discussed and approved by the institutional ethics committee and licensed by the Austrian government (license number BMWF-68.205/0196-II/3b/2012).

(25) 1.6. Antibody Response

(26) Commercial FAdV Enzyme-Linked Immunosorbent Assay (ELISA)

(27) Commercially available FAdV Group 1 Antibody Test Kit was obtained from BioChek (Reeuwijk, Holland) to test antibody levels in sera of each group before (day 21) and after challenge (days 28, 35 and 42).

(28) Serum Neutralization Test (SNT)

(29) Test sera were inactivated at 56 C. for 30 min. CEL cells were prepared from 14-day-old chicken embryos and plated in 96-well plates (Sarstedt, Wiener Neudorf, Austria) with a density of 110.sup.6 cells/ml. The assay was performed according to a constant virus diluted serum method using 100 TCID.sub.50/100 l KR5. The plates were inoculated at 37 C. in 5% CO.sub.2 and investigated for CPE after 5 days.

(30) Fib-2 ELISA

(31) After predetermining optimal virus- and serum-dilutions by checker-board titrations, 96-well ELISA plates (Nunc Medisorb, Roskilde, Denmark) were coated with 100 l recombinant affinity-purified Fib-2 protein per well, diluted in coating buffer (0.015 M Na.sub.2CO.sub.3, 0.035 M NaHCO.sub.3, pH 8.4) to a final concentration of 0.05 g/ml. After 24 h, plates were washed and 100 l of the test sera, diluted 1:100 in blocking buffer (Starting Block T20 PBS, Thermo Scientific), were added to each well for 1 h. Following a washing step, 100 l Goat-Anti-Chicken-IgG-HRP (Southern Biotechnology, Birmingham, USA) diluted 1:5000 in PBS-0.05% v/v Tween 20 (Calbiochem, Darmstadt, Germany) were added to each well and incubated for 1 h. After another washing step, 100 l TMB (tetramethylbenzidine) substrate (Calbiochem, Darmstadt, Germany) were added to each well and the plates were incubated for 15 min in the dark. The reaction was stopped with 100 l 0.5 M sulphuric acid/well and the optical density (OD) of each well was measured with an ELISA reader (Sunrise-Basic, Tecan, Grdig, Austria) at a wavelength of 450 nm.

(32) On each plate, a positive and a negative control were included. All sera were tested in duplicate and the OD is indicated as the mean value of the duplicates. A tentative cut-off value was established as the arithmetic mean of all OD values plus three times the standard deviation determined from serum samples from the negative control group.

(33) 1.7. Western Blot Analysis

(34) Purified recombinant Fib-1, Fib-2 and Hex L1 proteins were boiled for 5 min in sample buffer containing 4% SDS and 10% mercaptoethanol, separated by 12% SDS-PAGE and electrotransferred onto BioTrace PVDF Transfer Membrane (Pall, Vienna, Austria). After 3 h of blocking with 3% (w/v) skim milk, the membrane was cut into strips which were incubated separately in the test sera (preabsorbed with 1% Sf9 cell powder, diluted 1:2000) for 1 h. After several washes with PBS-0.05% Tween 20, the membrane strips were incubated for 1 h with rabbit anti-chicken IgG-HRP conjugate (Sigma-Aldrich, Vienna, Austria) diluted 1:2500, followed by several washes and incubation with Clarity Western ECL substrate (Bio-Rad Laboratories GmbH, Vienna, Austria). Visualization was performed on x-ray film (Super RX, Fuji, Japan) after exposure for 12 sec.

(35) 1.8. Real-Time (Rt) PCR from Cloacal Swabs and Intestine

(36) Excretion of challenge virus was investigated from cloacal swabs taken on days 7 and 14 post challenge (p.c.) and tissue samples taken from the large intestine at termination of the study (day 21 p.c.) from five birds of each group, using an rt PCR assay based on the 52K gene, following DNA extraction with a commercial system (Qiagen, Hilden, Germany) (Gnes et al., J. Virol. Meth. 183 (2012), 147-153).

(37) 2. Results

(38) 2.1. Expression of Proteins

(39) Characteristic morphologic changes were exhibited by Sf9 cell cultures within 48-96 h after inoculation with recombinant baculovirus. Recombinant proteins were detected by SDS-PAGE and Western blot as bands migrated to estimated molecular weight sizes of 51 kDa (Fib-1), 56 kDa (Fib-2) and 35 kDa (Hex L1) with peak expression around 72 h after inoculation. Furthermore, expression analysis showed that large fractions of Fib-1 and Fib-2 were expressed as soluble proteins in the supernatant, whereas Hex L1 protein was preferentially found in the pellet.

(40) 2.2. Protection of Recombinant Proteins Against Virulent FAdV

(41) Following challenge, clear-cut differences in severity of clinical signs and mortality rates were noticed between individual groups (FIG. 1). The difference in mortality between the groups was found to be highly significant by chi-square analysis (.sup.2=46; p<0.01) and significant differences were also indicated in the pairwise comparison of mortality between the Fib-2 vaccinated group and all other challenged groups (Bonferroni corrected chi-square test).

(42) Onset of mortality was recorded on day 3 p.c., in coincidence with the overall peak of mortality. Dead birds were observed until day 5 p.c., and after that no more animals died. After infection with the virulent virus, birds of group IV (positive control) showed severe clinical depression as manifested by huddling together with ruffled feathers, and 18 out of 23 animals (78%) died. In contrast, birds in group II (Fib-2 vaccinated) displayed no apparent clinical symptoms and only one dead animal out of 28 on day 3 p.c. after the challenge was recorded. Birds of group I (Fib-1 vaccinated) partially showed clinical symptoms and 10 out of 26 animals died resulting in an overall mortality of 38%. In group III (Hex L1 vaccinated), severity of clinical affection was comparable to the positive control group, and 19 out of 26 animals (73%) died. Necropsy revealed severe lesions in heart and liver of all animals found dead or those which had to be euthanized during the experiment. Characteristic findings included straw-colored fluid in the pericardial sac and focal necrosis in the livers (FIG. 2).

(43) Surviving animals of clinically affected groups experienced full recovery by 26 days of life. No more lesions were recorded in any of the surviving animals at termination of the experiment on day 42 of life. In group V (negative control), no clinical signs were observed at any time of the experiment and no pathological lesions were noticed at termination of the study.

(44) 2.3. Detection of Antibodies

(45) Commercial FAdV ELISA and SNT

(46) No antibodies were detected with the commercial ELISA and the SNT prior to challenge at day 21 in any of the groups (FIGS. 3a and 3b). Following challenge, birds of groups I-IV developed an increase in antibody levels detectable by both commercial ELISA and SNT. In the vaccinated groups, antibodies measured by commercial ELISA increased until 7 d.p.c. and after that gradually declined, whereas antibody levels in the positive control group display a continuous increase until termination of the experiment. Development of neutralizing antibodies p.c. continuously increased in groups I-IV with highest titres obtained in non-vaccinated birds

(47) No antibodies were detected in negative control animals at any of the tested time points during the experiment.

(48) Fib-2 ELISA

(49) To investigate a specific antibody response against Fib-2 prior to and after challenge a custom-made ELISA using recombinant purified protein was developed. Starting measurements in Fib-2 vaccinated birds on day 7, the ELISA first detected an increase in mean OD value above the determined cut-off on day 11 and peaked at 7 d.p.c. (FIG. 3b). Until termination of the experiment, mean Fib-2 antibody levels declined only slightly. Of note, the antibody response of the bird that did not survive challenge was only 0.21 and differed significantly from all other birds.

(50) Birds of the positive control group were tested negative for Fib-2 antibodies on day 21. Survivors, however, developed a strong anti-Fib-2 response p.c., reaching the level of vaccinated birds by the end of the experiment.

(51) Sera obtained from the negative control group before and after challenge were tested negative in the Fib-2 ELISA (FIG. 3c), similarly to sera from Fib-1 and Hex L1 vaccinated groups.

(52) 2.4. Western Blot

(53) Immunoblots with sera from three birds of each group I-III obtained on day 21 after administration of recombinant proteins confirmed the presence of antibodies against Fib-1, Fib-2 and Hex L1, respectively (FIG. 4). No antibodies were detected in sera from one bird of the positive and negative control group when tested against each of the purified recombinant proteins in the immunoblot.

(54) 2.5. Virus Excretion

(55) No virus excretion was detected in any of the samples taken from negative control animals (Table 2). Following challenge, viral excretion was noticed in all tested birds of groups I-IV, at 7 d.p.c with no evident difference in viral load between protein-vaccinated and positive control birds. Shedding was verified until termination of the experiment and the majority of birds were recorded positive for virus excretion in the faeces. The large intestine of half of the infected birds was positive at termination of the study, with positive birds in each of the groups I-IV.

(56) 3. Discussion

(57) While human adenoviruses are well studied on a molecular basis for their use as vaccine and gene therapy vectors, current understanding of FAdV-host interaction and molecules involved is still limited. Interaction between capsomer and host cell has been established as the critical factor in formation of host immunity, rendering adenovirus capsid proteins interesting candidates for subunit vaccine development. In regard to the prevention of HHS, E. coli expressed penton base was recently proposed as a potential subunit antigen. In the present study, the efficacy of fiber subunit immunization derived from FAdV-C was investigated by utilizing for the first time the novel finding of two distinct fiber-encoding genes in FAdV-C. In addition, hexon loop-1, a surface-exposed structure with immunogenic potential, was investigated.

(58) The choice of the baculovirus expression system was based on evidence for possible post-translational modifications of such adenovirus proteins.

(59) Upon challenge with the virulent strain AG234, different degrees of protection were observed in chickens vaccinated with recombinant FAdV capsid proteins. Although Hex L1-specific antibodies were detected prior to challenge, this protein could not be proven as an effective subunit antigen in our study. In comparison, an immune response directed against Fib-2 is highly efficacious as it prevents any clinical signs of disease. This could indicate a key role of the Fib-2 protein in the initial steps of infection, possibly by mediating attachment to host cell receptors. Cellular attachment via binding of fiber to the ubiquitously present coxsackievirus-adenovirus receptor (CAR) is a well-known mechanism in human adenoviruses. However, knowledge about CAR-fiber interaction is primarily derived from in vitro studies and the role of CAR as primary receptor for adenovirus entry into the host cell is increasingly questioned. In this context, binding to primary receptors specific for avianbut not mammalian,cells was suggested to be mediated by the short fiber of CELO. Previous phylogenetic data show a higher degree of relatedness of FAdV-C Fib-2 with the short fiber gene of CELO and the single fiber gene found in other FAdV species, as compared to Fib-1. Based on these informations, together with the actual finding of highly efficacious immune response directed against FAdV-C Fib-2, Fib-2 could serve as the primary ligand for induction of a host-cell dependent infection pathway.

(60) Antibodies raised against Fib-2 following vaccination were detected with the exception of one bird, indicating a correlation with protection, in contrast to the commercial ELISA which failed to detect antibodies before challenge. Obviously, the type specificity of the fiber antigen results in a binding incompatibility of the induced antibodies within the commercial ELISA test system. The results obtained from SNT indicate that antibodies directed against Fib-2 do not possess neutralizing capacity, which is in agreement with previously reported observations of weak or lacking serum neutralization activity elicited by fiber if administered as an isolated virus component.

(61) The challenge virus was detected in cloacal swabs of groups I-IV alike, demonstrating that vaccination does not prevent virus excretion and shedding, even in birds protected from clinical disease. This finding is supported by a previous study that reports excretion of challenge virus even in birds clinically fully protected by a live attenuated FAdV vaccine (Schonewille et al., Avian Dis. 54 (2010), 905-910).

(62) In summary, identification of virulent strains of FAdV-C as causative agents of HHS together with the limitations faced by currently employed inactivated vaccines argue for the development of next-generation immunization strategies. The findings presented in the present invention shows high efficacy of recombinant Fib-2 protein for the development of an effective and safe subunit vaccine.

(63) Tables

(64) TABLE-US-00004 TABLE1 Primersused. Primer name Sequence(5-3) Position Purpose KR5-b 5-GGATCCATGTCGG 30438-30453.sup.a Amplificationofthefiber-1geneof Fib-1f CCCTAATCG-3 strainKR5andcloningintothe pFastBacvector KR5-b 5-AGGCCTTTAGGGG 31725-31739.sup.a Amplificationofthefiber-1geneof Fib-1r CTCGGAGC-3 strainKR5andcloningintothe pFastBacvector KR5-b 5-AGGCCTATGCTCC 31723-31738.sup.a Amplificationofthefiber-2geneof Fib-2f GAGCCCCTA-3 strainKR5andcloningintothe pFastBacvector KR5-b 5-TCTAGATTACGGG 33146-33162.sup.a Amplificationofthefiber-2geneof Fib-2r ACGGAGGCTG-3 strainKR5andcloningintothe pFastBacvector FAVf 5-AATTCCATGGACA 20481-20502.sup.a Amplificationofthehexonloop-1 AGTTCAGGCAGACGGT generegionofstrainKR5and CGT-3 cloningintothepFastBacvector FAVr 5-TAACTCGAGCTAG 21347-21366.sup.a Amplificationofthehexonloop-1 TGATGCCGGGACATCA generegionofstrainKR5and T-3 cloningintothepFastBacvector 52K-fw 5-ATGGCKCAGATGG 13075-13093.sup.b Amplificationofthe52kgenein CYAAGG-3 rt-PCR 52K-rv 5-AGCGCCTGGGTCA 13250-13232.sup.b Amplificationofthe52kgenein AACCGA-3 rt-PCR .sup.a Position is indicated for the complete genomic KR5 sequence (HE608152). .sup.b Position is indicated for the complete genomic CELO sequence (U46933).

(65) TABLE-US-00005 TABLE 2 Detection of viral excretion in cloacal swab samples (taken on days 21, 28 and 35) and tissue from the large intestine (taken on day 42) by real-time PCR from five birds of each group. Results are shown as number of positive samples/number of samples tested. group I group II group III group IV group V d .sup.a (Fib-1) (Fib-2) (Hex L1) (positve control) (negative control) 21 28 5/5 5/5 5/5 5/5 0/5 35 3/5 4/5 4/5 4/5 0/5 42 1/5 3/5 5/5 1/5 0/5 .sup.a Day of life

(66) TABLE-US-00006 TABLE 3 List of examples of fiber proteins useable according to the present invention: Fowl adenovirus 4 isolate Punjab 1 fiber gene, complete cds 1,386 bp linear DNA DQ864436.1 GI:112735223 Fowl adenovirus 4 isolate Punjab 2 fiber gene, complete cds 1,386 bp linear DNA DQ864434.1 GI:112735219 Fowl adenovirus 4 short fiber gene, complete cds 1,482 bp linear DNA AY340863.1 GI:33359662 Fowl adenovirus 10 short fiber protein gene, complete cds 1,496 bp linear DNA AF007579.1 GI:2674070 Fowl adenovirus 4 isolate Kr-Yeoju short fiber gene, complete cds 1,425 bp linear DNA HQ709232.1 GI:318040046 Fowl adenovirus 4 isolate Kr-Gunwi short fiber gene, complete cds 1,425 bp linear DNA HQ709231.1 GI:318040044 Fowl adenovirus 4 isolate Kr-Andong short fiber gene, complete cds 1,425 bp linear DNA HQ709230.1 GI:318040042 Fowl adenovirus 4 isolate Kr-Changnyeong short fiber gene, complete cds 1,425 bp linear DNA HQ709229.1 GI:318040040 Fowl adenovirus partial sf gene for short fiber protein, isolate OTE 1,197 bp linear DNA FN557186.1 GI:315455213 Fowl adenovirus partial sf gene for short fiber protein, isolate 08-5769 1,197 bp linear DNA FN557185.1 GI:315455211 Fowl adenovirus partial sf gene for short fiber protein, isolate 08-3622 1,197 bp linear DNA FN557184.1 GI:315455209 Fowl adenovirus 4 isolate Bareilly fiber protein gene, complete cds 1,437 bp linear DNA FJ949088.1 GI:238683632 Fowl adenovirus C pVIII gene, U-exon gene, fiber-1 gene, fiber-2 gene and ORF22, isolate C2B 4,345 bp linear DNA HE608154.1 GI:381214073 Fowl adenovirus C pVIII gene, U-exon gene, fiber-1 gene, fiber-2 gene and ORF22, isolate AG234 4,321 bp linear DNA HE608153.1 GI:381214067 Fowl adenovirus C complete genome, isolate KR5 45,810 bp linear DNA HE608152.1 GI:381214017 Fowl adenovirus C partial fiber-2 gene, isolate K388-95 1,395 bp linear DNA FR872927.1 GI:381214013 Fowl adenovirus C partial fiber-2 gene, isolate 09/8846 1,440 bp linear DNA FR872926.1 GI:381214011 Fowl adenovirus C partial fiber-2 gene, isolate 09/584 1,440 bp linear DNA FR872925.1 GI:381214009 Fowl adenovirus C partial fiber-2 gene, isolate 09/2602 1,329 bp linear DNA FR872924.1 GI:381213952 Fowl adenovirus C partial fiber-2 gene, isolate K99-97 1,340 bp linear DNA FR872923.1 GI:381213950 Fowl adenovirus C partial fiber-2 gene, isolate Peru54 1,421 bp linear DNA FR872922.1 GI:381213948 Fowl adenovirus C partial fiber-2 gene, isolate Peru53 1,416 bp linear DNA FR872921.1 GI:381213946 Fowl adenovirus C partial fiber-1 gene, isolate K1013 1,184 bp linear DNA FR872898.1 GI:381213900 Fowl adenovirus C partial fiber-1 gene, isolate 922/1 1,311 bp linear DNA FR872897.1 GI:381213898 Fowl adenovirus C partial fiber-1 gene, isolate C2B 1,302 bp linear DNA FR872896.1 GI:381213896 Fowl adenovirus C partial fiber-1 gene, isolate Da60 1,302 bp linear DNA FR872895.1 GI:381213894 Fowl adenovirus C partial fiber-1 gene, isolate KR5 1,302 bp linear DNA FR872894.1 GI:381213892 Fowl adenovirus C partial fiber-1 gene, isolate INT4 (QT-cell passaged AG234) 1,188 bp linear DNA FR872893.1 GI:381213890 Fowl adenovirus C partial fiber-1 gene, isolate AG234 1,302 bp linear DNA FR872892.1 GI:381213888 Fowl adenovirus C partial fiber-1 gene, isolate K31 1,181 bp linear DNA FR872891.1 GI:381213886 Fowl adenovirus 4 isolate Kr-Yeoju short fiber gene, complete cds 1,425 bp linear DNA HQ709232.1 GI:318040046 Fowl adenovirus 4 isolate Kr-Gunwi short fiber gene, complete cds 1,425 bp linear DNA HQ709231.1 GI:318040044 Fowl adenovirus 4 isolate Kr-Andong short fiber gene, complete cds 1,425 bp linear DNA HQ709230.1 GI:318040042 Fowl adenovirus 4 isolate Kr-Changnyeong short fiber gene, complete cds 1,425 bp linear DNA HQ709229.1 GI:318040040 Fowl adenovirus partial sf gene for short fiber protein, isolate OTE 1,197 bp linear DNA FN557186.1 GI:315455213 Fowl adenovirus partial sf gene for short fiber protein, isolate 08-5769 1,197 bp linear DNA FN557185.1 GI:315455211 Fowl adenovirus partial sf gene for short fiber protein, isolate 08-3622 1,197 bp linear DNA FN557184.1 GI:315455209 Fowl adenovirus 4 isolate Bareilly fiber protein gene, complete cds 1,437 bp linear DNA FJ949088.1 GI:238683632 Fowl adenovirus 4 short fiber gene, complete cds 1,482 bp linear DNA AY340863.1 GI:33359662 Fowl adenovirus 4 isolate Punjab 1 fiber gene, complete cds 1,386 bp linear DNA DQ864436.1 GI:112735223 Fowl adenovirus 4 isolate Punjab 2 fiber gene, complete cds 1,386 bp linear DNA DQ864434.1 GI:112735219 Fowl adenovirus 10 short fiber protein gene, complete cds 1,496 bp linear DNA AF007579.1 GI:2674070
The nature of the sequence, the FAdV species/serotypes, the-length of the sequence, the GenBank accession number and the version is indicated for each of the sequences.

(67) TABLE-US-00007 TABLE 4 List of species in the genus Aviadenovirus: Falcon adenovirus A Falcon adenovirus 1 [AY683541] (FaAdV-1) Fowl adenovirus A Fowl adenovirus 1 (CELO) [U46933 = (FAdV-1) _ AC_000014] Fowl adenovirus B Fowl adenovirus 5 (340) [AF508952] (FAdV-5) Fowl adenovirus C Fowl adenovirus 4 (ON1) [GU188428 = (FAdV-4) NC_015323] Fowl adenovirus 10 (CFA20) [AF160185] (FAdV-10) Fowl adenovirus D Fowl adenovirus 2 (P7-A) [AF339915] (FAdV-2) Fowl adenovirus 3 (75) [AF508949] (FAdV-3) Fowl adenovirus 9 (A2-A) [AF083975 = (FAdV-9) AC_000013] Fowl adenovirus 11 (380) [AF339925] (FAdV-11) Fowl adenovirus E Fowl adenovirus 6 (CR119) [AF508954] (FAdV-6) Fowl adenovirus 7 (YR36) [AF508955] (FAdV-7) Fowl adenovirus 8a (CFA40) [AF155911] (FAdV-8a) Fowl adenovirus 8b (764) [AF508958] (FAdV-8b) Goose adenovirus Goose adenovirus 1 (GoAdV-1) Species names are in italic script; names of types and isolates ( ) are in roman script. Sequence accession numbers [ ] and assigned abbreviations ( ) are also listed.