WO2003039593A1

WO2003039593A1 - Fowl adenovirus vaccine

Info

Publication number: WO2003039593A1
Application number: PCT/EP2002/012529
Authority: WO
Inventors: Guntram Paul; Michael Bernard Hess
Original assignee: Akzo Nobel N.V.
Priority date: 2001-11-08
Filing date: 2002-11-07
Publication date: 2003-05-15

Abstract

The present invention is concerned with an attenuated, orally immunogenic fowl adenovirus of serotype 4, that is able to produce a cytopathic effect (CPE) in cells of a QT 35 cell line, and to vaccines containing such viruses.

Description

Fowl Adenovirus vaccine

The present invention is concerned with an attenuated fowl adenovirus (FAV) of serotype 4, with a vaccine containing said virus, a method for preparation of said virus and the use of such a virus for the protection of poultry.

Avian adenoviruses can be classified in three groups (I to III) which do not share a common antigen ("group antigen") that enable to distinguish them from each other (Monreal, G.; Poultry Science Rev.4, 1-27, 1992). Group I adenoviruses can be isolated from different avian species, i.e. from chickens, geese, ducks and turkeys (Wiegand et al; Intervirology, 18, 169-176, 1982). Group II adenoviruses, comprise a small number of viruses, such as the Turkey Haemorrhagic Enteritis virus. Group III adenoviruses comprise the Egg Dropping Syndrome (EDS) causing virus as the only serotype.

The fowl adenoviruses (FAV) of group I are widely distributed in poultry flocks. Serological classification of FAV results in 12 serotypes (Monreal, G.; Archiv fϋr Geflϋgelkunde, 48, 245-250, 1984). The main method to type avian adenoviruses is the neutralisation assay. The official guideline is given by the International Committee on Taxonomy of Viruses (ITCV) which defines two strains as serological different if the heterologous titre via the homologous titre is >16 in both directions. If the titre ratio is between 8 and 16, serotype assignment can be done if substantial biophysical or biochemical differences exist Hess et al. (Avian Pathology, 27, 196- 199, 1998) and Erny et al. (Archives of Virology, 140, 491-501,1995).

In addition to serological typing FAV can be classified by analysis of their DNA into 5 DNA types.

The exact role of FAV serotypes, as etiological agent of specific diseases (except for quail bronchitis that is caused by FAV 1) has not been clarified yet. In connection with classical inclusion body hepatitis (lBH) different FAV serotypes were isolated.

A new chicken disease was first reported in 1987 in Pakistan and was called "Angara disease" or hydropericardium syndrome (HPS). Several outbreaks of HPS were reported from South America (e.g. Mexico, Ecuador and Peru) with severe losses (Shane; Avimex: 7^th Symposium, Mexico City, 1995). HPS is seen primarily in broilers at an age 3 to 5 weeks, although outbreaks have also been reported in layers and breeders. This disease is characterised by an accumulation of straw coloured fluid in the pericardial sac and an enlarged discoloured liver with foci of haemorrhage and/or necrosis.

Recently FAV of serotype 4 was identified as etiological agent of HPS (Mazaheri et al; Avian Pathology, 27, 269-276,1998). It was shown that adenoviruses which were isolated from field outbreaks of HPS in Ecuador and Pakistan (strains K1013, K31) belong to FAV of serotype 4 (Voβ, M; Lohmann Symposium on the Hydropericardium Syndrome, Cuxhaven, Germany, 1989).

In order to reduce economic losses by hydropericardium syndrome, vaccines containing FAV of serotype 4 have been developed. Currently, only inactivated vaccines are used to prevent HPS in chickens (Afzal, M. et al; Veterinary Record 126, 59-60, 1990). The commercially available inactivated vaccine Nobilis® FAV (Intervet International, Boxmeer, The Netherlands) contains the Mexican field strain VC that was grown on chicken embryo liver cells and is applied subcutaneously or intramuscularly to broilers at 8-10 day of age if they have maternal antibodies or at day one of age without maternal antibodies. For breeders a single vaccination at 16- 18 weeks of age is recommended.

However, inactivated vaccines generally induce a lower level of immunity, requiring additional immunisations, thus disadvantageously require an adjuvant and are expensive to produce. Another disadvantage of inactivated vaccines is the necessary administration via the parenteral (injectable) route. This requires extensive handling of the animals and is not convenient to use in young, e.g. one- day old chicken for an early protection.

Currently available non- attenuated wild/type FAV of serotype 4 cannot be used in a live form due to their pathogenicity, whereas the available natural a-virulent FAV of serotype 4 strains induce only a low immunity after administration via oral or intranasal route. It is described in the prior art that, if birds without serum neutralising antibody to FAV are infected orally or intranasally, they do not develop precipitating antibodies (McFerran et al.; American Journal of Veterinary Research 36, 527-529, 1975). After oral or intranasal vaccination of day-old chicks with 7 log₁₀/ml using various strains of FAV representing different serotypes, all chickens vaccinated with FAV serotype 2, FAV serotype 3 and FAV serotype 8 strains developed antibodies three weeks after vaccination, but those vaccinated with KR-5, the reference strain of FAV of serotype 4, (Cook, Avian Pathology 12, 35-43, 1983) failed to develop an immune response.

The reference strain KR-5 was described by Kawamura et al (National Institute of Animal Health Quarterly (Tokyo), 4, 183-193, 1964).

As a substrate for growth of avian adenoviruses primary embryonic liver cells from chicken and ducks (e.g. chicken embryo liver -CEL), embryonic kidney cells from ducks and chickens have been described (Nonaka, F; J. of the Japan Veterinary Medical Association 37, 510-515, 1984) . Recently the propagation of FAV serotype 8 (DNA type E) on a continuous chicken hepatoma cell line (CH-SAH, Scholz et al: J. Virol. Methods, 43, 273-286, 1993) has been described (Alexander et al., J. Virol. Methods, 74, 9-14, 1998).

However, until now the propagation of FAV of serotype 4 on avian continuous cell lines has not been reported. In particular it was reported in the prior art (Cowen et al; Avian Diseases 32: 282-297, 1988) that cells of an avian continuous cell line (QT 35 cells) are not susceptible for avian adenoviruses including FAV of serotype 4.

It would be preferred for the production of commercial FAV vaccines (both live and inactivated) to grow viruses on a continuous cell line because of the inherent advantages of such a cell line over primary cell cultures or embryonated eggs.

The use of primary cell culture and embryonated eggs for production purposes is not optimal because the quality is variable and the process of harvesting requires a large number of a-septic handling, resulting in high risk of contamination. Furthermore chicken embryos and primary cell culture are composed of heterogeneous cell populations and embryonated eggs are subject to hormonal influences.

In view of the above there is a need for FAV of serotype 4 that can be prepared on an avian continuous cell line. Moreover, it would be highly advantageous to have a live attenuated FAV of serotype 4 vaccine available. Such a live attenuated FAV vaccine would be also very desirable as the basis for a vector vaccine that can be obtained by inserting a heterologous nucleic acid sequence encoding a polypeptide heterologous to FAV in an insertion region of the FAV genome.

Surprisingly it has now been found that fowl adenoviruses of serotype 4 (FAV) can be adapted to grow on a QT 35 cell line and that such viruses produce a cythopathic effect (CPE).

Therefore, the present invention provides an attenuated, fowl adenovirus (FAV) of serotype 4 characterised in that is able to produce a CPE in cells of a QT 35 cell line and to induce protective immunity after oral application to birds.

The propagation of FAV in continuous cell lines is advantageous because it avoids the problems associated with the use of primary cell cultures or embryonated eggs. The quality of continuous cell lines is more constant than in primary cell cultures because the use of a continuous cell line is less laborious and less a-septic handling have to be carried out. Therefore, risks for contamination are lower. That is especially important if the use of such strains in live vaccines is intended. A further advantage of the use of a continuous cell line for production purposes is that they can be made available easily and continuously by simple procedures. Embryonated eggs or primary cell cultures however, must be derived each time from specific-pathogen-free (SPF) sources to assure their freedom from interfering antibodies and/or vertically transmitted viruses.

The majority of avian continuous cell lines consist of lymphoblastoid cells, which are obtained from animals with lymphoid leucosis or Marek's disease. Continuous cell lines are serially replicating "immortalised" cells. Several continuous tissue culture cell lines that derived from chemically induced tumours of Japanese quail are described in prior art. The QT 35 cell line is such a stabilised cell line. The development of QT 35 cell line has been described by Moscovici et al Cell 1J.: 95- 103, 1977. The QT 35 cell-line was established from a methylcholanthrene - induced fibrosarcoma of Japanese quail. A sample of this cell line is available from ATCC (Rockville, MD, US) under No. 10967 and from ECACC, Salisbury, Wiltshire UK under Ref. No. 93120832.

QT 35 cells can be grown by various culture methods suitable for anchorage dependent cells. For example, the cells can be grown in roller bottles, in cell cubes and on microcarriers e.g. composed of or containing gelatine, plastic or glass. In addition there is a multiplicity of other stationary systems for the culture of anchorage-dependent cells. A common feature of the latter systems is that they have a very large surface for cell attachment.

The cells can be grown using a variety of cell culture media or combinations of cell media known in the art. Optionally these cell media or combinations of cell media can be supplemented with readily available energy sources (in particular sugars, such as glucose, fructose ribose) and /or with amino acid sources such as proteins (e.g. milk proteins and/or serum proteins). Cell culture growth or maintenance medium (medium 199/F10) as described in Cho (Cho et al; Avian Diseases 27: 261-270), 1983) with antibiotics and foetal calf serum (5%) was used in the current invention.

For the subcultivation of the QT 35 cells it is possible to use proteolytic enzymes such as trypsin and collagenase. Cells can be frozen in the presence of cryoprotectors, such as Dimethyl sulfoxide (DMSO) or glycerol. Freezing of the cells as well as the cell associated virus can be performed by establishing a gradual decrease in temperature of e.g. 1 °C per minute to the desired storage temperature which is preferably the temperature of liquid nitrogen.

In the prior art QT 35 cells are described to be suitable, to grow strains of various families of avian viruses, e.g. birnavirus, coronavirus, paramyxovirus, herpesvirus, reovirus, and retrovirus, but not for the growth of FAV (Cowen et al; Avian Diseases 32: 282-297, 1988). It was established by Cowen et al that virus of the FAV family are not able to produce a CPE in cells of a QT 35 cell line (Table 3). Cowen et al used QT 35 cells from passage no. 57 in an F10-199 growth or maintenance medium and maintained them in a humidified CO₂ (5%) incubator at 37°C.

However, the present inventors provide a method that allows propagation and serial passaging of FAV of serotype 4 in cells of a QT 35 cell line. The present inventors adapted FAV of serotype 4 to QT 35 cells of "young stock" instead of using the high passage QT 35 cells that were used by Cowen et al. By "young stock" a QT 35 cell line is meant that was passaged not more than 10 times continuously after it was taken from a frozen stock. Maximal 10 cell passages were performed, because the QT 35 cells of higher passages show a decreasing permissiveness for the production of a CPE as a result of FAV propagation.

The cultivation of QT 35 cells and the adaptation of FAV of serotype 4 to the QT 35 cell line is described in more detail in Example 1. Example 1a) discloses the propagation of FAV of serotype 4 on QT 35 cells. In a first step FAV of serotype 4 from CEL (chicken embryo liver cell) propagation was passaged on appropriate QT 35 cells until a CPE was observable.

By "able to produce a CPE in cells of a QT 35 cell line" it is meant that after 1 to 10 passages a cytopathic effect is visible i. e.. cells become round and refractile and detach from the surface of the culture vessel or any lesions indicating cytopathic changes.

FAV according to the invention can be grown on a QT 35 cell line with yields high enough for an economically production of vaccine, as it is shown in examplel c). Virus yields are usually expressed in "infectious virus particles per unit volume" (EID₅₀/ml, TCID₅₀/ml).

Additionally it has been demonstrated that FAV of serotype 4, that are serially passaged in QT 35 cells display an attenuated phenotype and induce protective immunity after oral administration to birds.

Surprisingly the present invention have also found that serial passaging of FAV on QT 35 cells result in a generation of attenuated FAV that can safely administered orally to one-day old chickens. Even more surprisingly it has been also found that such attenuated FAV strains according to the current invention are able to induce a protective immunity after administration to chickens via the oral route. The attenuated FAV of serotype 4 according to the invention can be obtained by serial passaging of virulent wild-type FAV of serotype 4 in the appropriate QT 35 cells. With increasing number of passages the virulence decreases.

A live vaccine virus is rendered a- virulent through attenuation and is still able to multiply in the host. The aim of attenuation is to weaken or eliminate the virulence of the organism, without affecting its immunogenicity. Attenuation means that at least 90 % of the one-day old birds survive the oral administration of the virus according to the invention at a dose that would induce 100% mortality in one-day old birds after administration of virulent wild type virus to one-day old birds at the same route of administration. Example 1 b) provides more details about the attenuation of FAV of serotype 4 on QT 35 cells.

In the present examples evidence is provided that confirm the poor immunogenicity of natural a-pathogenic FAV of serotype 4. It was demonstrated that the attenuated FAV of serotype 4 that are able to produce a CPE in QT 35 cells induce a protective immunity in birds in contrast to the natural a-pathogenic FAV of serotype 4.

With "induce protective immunity " it is meant that after oral administration of an effective dosage of FAV of serotype 4 according to the invention to one-day old SPF birds, a survival rate of at least 50%, preferably at least 60%, more preferably of 100% is achieved after challenge 3 weeks after oral administration with a virulent FAV of serotype 4 that would cause at the same dose 100% mortality in unvaccinated birds. Effective dosage for FAV of serotype 4 ranges from 10³ to 10⁸ TCIDβo/ml, preferably 10⁴ to 10⁷TCID₅₀/ml.

Such an attenuated FAV of serotype 4 according to the invention is obtainable by serial passaging of virulent FAV of serotype 4 in cells of a young stock QT 35 cell line. The number of passages required can easily be established empirically and may depend on the type of strain and specific conditions used. Typically, the attenuated FAV of serotype 4 according to the invention is obtainable after 20 to 60 passages, preferably 30 to 45 passages. It is furthermore shown by the inventors that as a result of the attenuation process a deletion of 2-3 kb is introduced in the genome of the FAV 4. The size of the genome of non-passaged parent FAV is usually about 44 kb.

Therefore, a particularly preferred attenuated FAV 4 according to the present invention is a FAV 4 as defined above that is further characterised by a deletion of (about) 2-3 kb in the genome of the FAV 4.

In Examples 2 and 3 it was shown that the administration of the FAV of serotype 4 (FAQ) according to the invention to susceptible chickens did not cause any disease signs after oral administration. Example 4 provides evidence that FAV of serotype 4 according to the invention (FAQ) causes after oral administration a protective immunity in susceptible chickens and is therefore a suitable vaccine candidate.

In Example 5 it was shown, that FAV according to the invention (FAQ) is a- pathogenic when given orally. It was also shown that FAV of serotype 4 according to the invention (FAQ) induces a much better serological response and causes a higher protection rate after intramuscular challenge with a virulent FAV of serotype 4, compared to an inactivated FAV of serotype 4 vaccine or to an live natural a- pathogenic FAV of serotype 4.

In a preferred embodiment the current invention discloses a FAV virus of serotype 4 characterised in that it is in a live form. Live viruses are applicable as vaccines e.g. via drinking water or aerosol and therefore allow the application to a large number of one- day old animals at the same time and mimic a natural infection more closely than vaccination with an inactivated virus. Live vaccines are considered to induce immunity within a couple of days that in most vaccines a longer immunity. The administration of a live virus vaccine therefore would lead to an early protection of the animals and reduces efforts and costs. For the use of a virus strain in live vaccines it is rendered avirulent or the virulence is reduced through attenuation and is still able to multiply in the host.

A special use for live viruses are vector vaccines.

FAV of serotype 4 according to the invention can also be used as a vector to heterologous nucleic acid fragments. Therefore the present invention provides a FAV of serotype 4 characterised in that the genome of the virus comprises a heterologous nucleic acid fragment encoding a polypeptide.

Such a vector vaccine offers the possibility to immunise an animal at the same time against FAV and in addition against other avian pathogens by expression of antigens of said pathogens within infected cells of the immunised host. Alternatively, the FAV vector according to the invention may solely be used for the purpose of immunising animals against another avian pathogen. The favourable safety properties of the FAV vector make it especially suitable as a prophylactic agent for those diseases for which no appropriate safe live vaccine is available. A FAV vector vaccine can be obtained by inserting a heterologous nucleic acid sequence encoding a polypeptide heterologous to FAV in an insertion region of the FAV genome. Vaccination with such a vector vaccine is preferably then followed by replication of the FAV within the inoculated host, expressing in vivo FAV polypeptides, and (the) heterologous polypeptide(s). An immune response will subsequently be elicited against FAV and the heterologous polypeptide(s).

The term "polypeptide" refers to a molecular chain of amino acids with a biological activity, does not refer to a specific length of the product and if required can be modified in vivo or in vitro, for example by glycosylation, amidation, carboxylation or phosphorylation; thus inter alia peptides, oligopeptides and proteins are included within the definition of polypeptide. Such heterologous polypeptide are for example antigenic polypeptides or polypeptides for pharmaceutical or diagnostic application.

The prerequisite for a useful FAV vector is that the heterologous nucleic acid sequence is incorporated in a permissive position or region of the genome FAV sequence, i.e. a position or region which can be used for the incorporation of a heterologous sequence without disrupting essential functions of FAV such as those necessary for infection or replication. Such a region is called insertion region.

In WO 94/24268 a recombinant vector comprising a recombinant avian adenovirus is described. This recombinant avian adenovirus is based on FAV strains of serotype 11. In WO 94/24268 suitable insertion regions for an FAV strain of serotype 11 and the characterisation of major late promoter (MLP) and splice leader sequences (LS) and non-essential regions of a FAV genome are disclosed. Such non-essential regions may be located outside of the essential 4- 33 kb region e.g. at the right end of the genome at map units 97 to 99.9. Recently the construction of an recombinant adenovirus vector based on FAV serotype 8 strain has been described (Johnson et al; Developmental and Comparative Immunology 24, 343-254, 2000). The recombinant FAV serotype 8 was constructed by inserting an expression cassette consisting of the FAV major late promoter/splice leader sequences, the chicken interferon-γ gene and SV 40 polyA into sites in the right hand end of the FAV serotype 8 genome.

It will be understood that for the DNA sequence of the FAV genome, natural variations can exist between individual FAV viruses. These variations may result in deletions, substitutions, insertions, inversions or additions of one or more nucleotides, which possibly influence the position of one or more restriction sites.

An essential requirement for the expression of the heterologous nucleic acid sequence in a cell infected with the FAV vector according to the invention is an adequate promoter probably linked to the heterologous nucleic acid sequence. It is obvious to those skilled in the art that the choice of a promoter extends to any eukaryotic, prokaryotic or viral promoter capable of directing gene transcription in cells infected by FAV according to the current invention. Examples of such promoters are those of cytomegalovirus (CMV), SV40 promoters, human adenovirus or the avian adenoviral major late promoter. The major late promoter lies near 16-17 map units on the adenovirus genetic map and contains a classical TATA sequence motif (Johnson et al, Virology 164, 1-14, 1988).

Well-known procedures for inserting DNA sequences into a cloning vector can be used, for example introducing specific restriction cutting site (s) and/or by homologous recombination using transfection plasmids.

In one method of construction of a FAV vector, a restriction enzyme site preferably being one that does not cleave the host genome selected for construction, is inserted into a non-essential and preferably non-coding region of the host genome. The viral genome thus is provided with a unique restriction enzyme site in a non-essential region to allow insertion of heterologous nucleotide sequences by simple restriction enzyme cleavage and ligation. This method has the added advantage of enabling, if preferred, deletion of portions of the non-essential region to allow the insertion of greater portions of DNA.

In an alternative method of construction of a suitable vector the non- essential region to be altered to incorporate foreign DNA could be constructed via homologous recombination. By this method the non-essential region of the FAV genome is cloned, a portion of it is deleted and foreign DNA together with promoter, leader and poly adenylation sequences is inserted preferably by homologous recombination between flanking sequences. This is accomplished by first constructing a recombinant DNA molecule for recombination with FAV. Such a molecule may be derived from any suitable plasmid, cosmid or phage, plasmids being most preferred, and contains a heterologous nucleic acid sequence, if desired operably linked to a promotor. Said nucleic acid sequence and promotor are introduced into a fragment of genome FAV DNA containing insertion-region sequences as defined herein subcloned in the recombinant DNA molecule. The insertion region sequences, which flank the heterologous nucleic acid sequence, should be of appropriate length as to allow in vivo homologous recombination with the viral FAV genome to occur.

The heterologous nucleic acid sequence to be incorporated into the genome of FAV according to the present invention can be derived from any source, e.g. viral, prokaryotic, eukaryotic or synthetic. Preferably the nucleotide sequence is capable of expression as an antigenic polypeptide although it may be also an immunopotentiator. The nucleotide sequence is conveniently foreign to the host vector.

Furthermore, nucleic acid sequences encoding polypeptides for pharmaceutical or diagnostic application, in particular immune modulators such as lymphokines, interferons or cytokines may be incorporated into the insertion region. Examples of nucleic acid sequences encoding polypeptides for pharmaceutical or diagnostic application are avian cytokine , growth factors, interleukines and chicken γ-interferon.

The current invention provides a FAV of serotype 4 virus according to the invention characterised in that the heterologous nucleic acid sequence to be incorporated into the genome of FAV according to the present invention encodes an antigen of an avian pathogen. The nucleic acid sequence to be incorporated into the genome of FAV according to the present invention can be derived from a pathogen, preferably an avian pathogen, which after insertion into the FAV genome can be applied to induce immunity against important diseases e.g. intestinal infections caused by parasites; for example coccidiosis causing agents e.g. Eimeria spp., or respiratory viruses, for example infectious bronchitis virus. Other infectious organisms against which immunity may be desirable include those that target internal organs such as the bursa of Fabricius, for example, infectious bursal disease. Preferably, nucleic acid sequences derived from Eimeria spp., Infectious Bronchitis virus, Infectious bursal disease (Gumboro disease) virus, Mareks disease virus, Newcastle disease virus, Egg Drop syndrome virus, Infectious Laryngotracheitis virus, Mycoplasma spp., Chicken Anaemia agent, Avian Influenza, Reo virus, Avian Retro virus, Avian Encephalomyelitis virus, Haemorrhagic Enteritis Virus of Turkeys, Salmonella spp. or E. coli spp. are contemplated for incorporation into the insertion region of the FAV genome.

If desired, a construct can be made which contains two or more different heterologous nucleic acid sequences derived from e.g. the same or different pathogens. A live attenuated FAV as described above, expressing one or more different heterologous polypeptides of specific pathogens can be used as a monovalent or multivalent vaccine for avian animals, susceptible to FAV and these pathogens. An animal vaccinated with such a FAV will be immune for a certain period to subsequent infection of FAV and pathogen(s).

Furthermore this invention provides a vaccine capable of inducing protection in birds, characterised in that it comprises a virus as defined above and a pharmaceutically acceptable carrier or diluent and a method for the preparation of the vaccine.

A vaccine containing FAV according to the invention can be prepared and marketed in a form of a suspension or in a lyophilised form and additionally contains a pharmaceutically acceptable carrier or diluent customary for such compositions. Carriers include stabilisers, preservatives and buffers. Suitable stabilisers are, for example SPGA carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried milk serum, albumin, or casein) or degradation products thereof. Suitable buffers are for example alkali metal phosphates. Suitable preservatives are thiomersal, merthiolate and gentamicin. Diluents include water, aqueous buffer (such as buffered saline) and polyols (such as glycerol). If desired the live vaccine according to the invention may contain an adjuvant. Suitable compounds or compositions for this purpose include aluminium hydroxide, -phosphate or -oxide, oil-in-water or water-in-oil emulsion based on, for example a mineral oil, such as Bayol® or Marcol® or a vegetable oil such as vitamin E acetate and saponins.

A vaccine according to the present invention can be administered in principle to birds by any suitable means. Exemplary means for administration are post hatch mass applications; e.g. oral administration via drinking water or in feed, aerosol, via eye drop or intranasally. Other ways of administration are parenteral routes e.g. by intramuscular, intraperitoneal or subcutaneous injection or pre- hatch administration (in ovo vaccination).

Poultry are conveniently inoculated with vaccines according to the invention at any age. In view of early protection an application to the birds as early as possible in the life is desirable. Where chickens are concerned, broilers may be vaccinated at day - old or in ovo. Breeders and layers may be vaccinated regularly up to point of lay and later.

For the application of vaccines in general mass application is considered to be the most effective way of administration of vaccines. A mass application allows the vaccination of a number of birds of any age simultaneously. Mass application routes can be also used in very young chicken were a parenteral (injectable) vaccination of the bird is very difficult.

The post hatch mass applications do not require a handling of the birds and therefore avoids stress-induced losses. For the oral application the vaccine is e.g. added to the drinking water or feed and than actively consumed by the animal. In the aerosol application the vaccine is distributed in a certain area via a fine aerosol that is absorbed by the animal via the mucosal tissue of the respiratory (and to a minor extent) upper digestive tract. For some avian pathogens (e.g. Coccidiosis causing protozoa e.g. Eimeria spp.) the oral administration mimics the natural route of infection and is therefore especially effective to induce protective immunity against this pathogen. For these pathogens the oral administration of the vaccine is the most preferred embodiment. For other avian pathogens (e.g. respiratory diseases) the spray, intranasal or eye drop administration mimics effectively the natural way of infection. Alternatively the vaccine might be administered by any suitable means that is considered an effective way of administration for the vector virus and/or the heterologous polypeptide that is encoded by the nucleic acid insert in the FAV genome.

The vaccine according to the invention comprises an effective dosage of the FAV of serotype 4 according to the invention as the active component, i.e. an amount of immunising FAV material that will induce immunity in the vaccinated birds against challenge by a virulent FAV virus. Immunity is defined herein, as the induction of a significant higher level of protection in a population of birds, after vaccination, compared to an unvaccinated group.

Depending on the site and manner of administration, the species, age and condition of the subject, the used virus dose might be in a range from 10² to 10°, preferably 10³ to 10⁷ TCID₅₀ /ml (i.e. one Tissue Culture Infectious Dose 50 is the dose at which 50% of the infected tissue cultures show a CPE).

A vaccine according to the current invention can be used in combination with other live vaccine strains. The combined administration of more than one vaccine strain is advantageous for economical reasons, because it requires either fewer handling of the birds or fewer vaccine inoculations in the egg.

Preferably, the combination vaccine additionally comprises one or more vaccine strains of Mareks disease virus (MDV) infectious bronchitis virus (IBV), Gumboro disease, Newcastle disease (NDV), Haemorrhagic Enteritis Virus of Turkeys, Chicken Anemia Virus egg drop syndrome (EDS) virus, turkey rhinotracheitis virus (TRTV) avian influenza or reovirus.

Although, the FAV vaccine according to the present invention may be used effectively in chickens, also other poultry that are susceptible to one of the avian pathogens as turkeys, guinea fowl, ostrich, pigeons and partridges may be successfully vaccinated with the vaccine. Chickens include broilers, reproduction stock and laying stock. The vaccine of the invention may of course be combined with vaccines against other avian pathogens at the time of administration.

Another aspect of the invention is a method for the preparation of the virus as disclosed above, characterised in that a virulent FAV of serotype 4 is serially passaged on a QT 35 cell line and subsequently harvested from the cell line. The FAV of serotype 4 according to the invention can be obtained by conventional methods known in the art.

Briefly, cells of a QT 35 cell line are inoculated with virulent FAV of serotype 4 and propagated until the virus replicated to a desired titre after which the FAV containing material is harvested from the cell line. Alternatively the virulent FAV of serotype 4 can be attenuated by serial passaging on QT 35 cells and than propagated on CEL cells.

More in particular, confluent monolayer QT 35 cells were prepared at a final concentration of 0.85 x 10⁵/cm₂. The cells were cultured in medium 199/F10 containing 5% foetal calf serum. Per cm₂ of confluent QT35 cells 10μl of virulent wild- type FAV of serotype 4 was added. After incubation up to 8 days until the appearance of a CPE typically for FAV, i.e. cells become round and retractile and detach from surface of culture vessel the virus can be harvested and used to inoculate CEL cells.

Examples

Example 1a) Adaptation of FAV of serotype 4 to QT 35 cells

I Preparation of QT 35 cells

0.85 x 10⁵ QT 35 cells were cultured per cm² of a culture dish or flask. The cells are suspended at 37°C and 5 % CO₂ for one day in cell culture medium (medium 199/F10) with antibiotics and foetal calf serum (5%) added until the cells form a confluent monolayer.

QT 35 cells derived from stock material frozen in liquid nitrogen were used only for a limited passage rate. Maximal 10 cell passages were performed.

II Adaptation of FAV of serotype 4 on QT 35 cells

The medium of confluent QT 35 cell cultures was discarded and per cm² of a culture dish or flask 10μl of fowl adenovirus (FAV, wild-type strain VC) containing inoculum was added. After one hour of incubation fresh cell culture medium (medium 199/F10) with antibiotics and foetal calf serum (5%) is added.

The cell cultures were incubated until the appearance of a visible cytopathic effect (CPE) typically for FAV, i.e. cells become round and refractile and detach from surface of culture vessel. After the appearance of CPE or after up to eight days if no CPE has appeared the supernatant was collected. The supernatant was frozen at - 20 °C or directly used to inoculate fresh QT 35 confluent monolayer cell cultures. The FAV after propagation on QT 35 (FAQ/1 ) was than used in the following experiments.

Table 1 : Adaptation of FAV strains of all 12 serotypes in QT 35 cell line

+++ CPE characterized by detachment of most cells from surface of culture vessel

++ CPE in the complete confluent cell monolayer

CPE in some parts of the confluent cell monolayer no CPE

Example 1b) Attenuation of FAV of serotype 4 on QT 35 cells

Attenuation was tested after 47 passages of FAV of serotype 4 (VC) on QT 35 cells including 3 steps of plaque-purification. Passages were performed as described in example 1a.

For each passage the collected culture supernatant of the pre-passage was clarified by centrifugation for 10 minutes at 100 x g before inoculation of the cell line as described in example 1a. Example 1c) Infectious virus titre of FAQ produced on QT 35 cells Table 2

Example 1d) Adaptation and attenuation of FAV of serotype 4 on QT 35 cells

The same methods for the adaptation and attenuation of FAV 4 strain VC as described above were used to adapt and attenuate FAV 4 strain INT. Using these methods this strain was passaged until it reached a level of 40 passages (FAQ/2).

Example 1e) Molecular characterisation of FAQ strains

The size of the FAV 4 genomes of the wild type viruses and the viruses that have been passaged on QT35 cells was determined. DNA extraction and gel electrophoresis were carried out as follows: isolates were grown in chick embryo liver or on QT35 cells for 3-4 days until greater than 80% CPE could be seen. After three freeze-thaw cycles 100 ml of infected cells were mixed with 50 ml of chloroform and centrifuged for 20 min at 4000 g to remove cellular debris. The supernatant was centrifuged at 100000 g for 90 min and the pellet was resuspended in Tris-EDTA buffer containing 1 mg proteinase K/ml. DNA was purified by phenol/chloroform extraction and precipated with ethanol. DNA content was measured fluorometrically with the VersaFluor™ Fluorometer System (Bio-Rad Laboratories). Briefly, lambda DNA was used for calibration together with the Hoechst Dye 33258 (bisbenzimide). Measurement was performed with the filter set EX 360/40 and EM 460/10 as described in the manual. Purified FAV genomic DNAs were subjected to electrophoresis on a 0.5% agarose gel.

The size of the whole FAV 4 genome is about 44 kb. It is shown in Figure 1 that the genomes of the passaged FAV 4 comprises a deletion of about 2-3 kb. Figure 1 : Gel electrophoresis of genomic DNA isolated from passaged and non- passaged FAV 4.

Lane 1 : Marker (High molecular weight DNA: Invitrogen, cat. No. 15618-010)

Lane 2: Strain INT, non-passaged

Lane 3: Strain INT, 40 passages (FAQ/2)

Lane 4: Strain VC, non-passaged

Lane 5: Strain VC, 41 passages (FAQ/1 )

Lane 6: Marker (same as lane 1)

Example 2 Safety of FAV according to the invention (FAQ) in one-day old chicken after i.m. and oral administration

Material and methods:

Each ten one-day old SPF chickens were inoculated with FAV according to the invention (FAQ/1) of 47^th QT 35 passage after one additional passage either on CEL

(logio 8.8 TCIDso /ml) or QT 35 cells (log₁₀ 7.4 TCID₅₀ /ml). The material was inoculated via oral (0.5 ml per bird) or i.m. (0.3 ml per bird) route.

The birds were observed for signs of clinical disease for two weeks. Then blood samples were taken from all birds and thereafter the birds were sacrificed and examined post-mortem. Results of clinical and post-mortem examination:

No signs of any disease were observed in the birds inoculated via oral route. In the groups of i.m. inoculation two birds died in the group that had received 47. QT 35 passage + 1 CEL passage material and one bird died in the group that received 47.QT 35 + 1 QT 35 passage material. The mortality rate (%) in day-old chickens after oral or i.m. inoculation of FAV of serotype 4 according to the invention (FAQ) is summarised in figure 2.

During post mortem examination of birds sacrificed two weeks after i.m. inoculation liver lesions were found in two birds of the 47.QT 35 passage +1 CEL passage group and in one bird of the 47.QT 35 passage÷ 1 QT 35 passage group. All animals of the i.m. inoculated groups showed depression in growth and were after the experiment according to their age too small.

Figure 2: Mortality rate (%) in day-old chickens after oral or i.m. inoculation of FAV of serotype 4 according to the invention (FAQ)

Example 3 Safety of FAV in one-day old chicken after oral administration

Material and methods: Five groups each of ten one day old chickens were inoculated orally with 1 ml per chicken of 10⁵ or 10³ of FAV according to the invention (FAQ/1) or FAV of serotype 4 (wild type-VC) or left not inoculated. The five groups were housed in five different isolators and clinically observed for three weeks.

Results:

All chickens in the two wild-type groups died within 10 days post inoculation. All other chickens did not show any signs of disease. The mortality rate is shown in figure 3.

Figure 3: Mortality rate (%) in chickens after oral inoculation of two different dose FAV according to the invention (FAQ) or wild-type FAV of serotype 4

Example 4 Efficacy of FAQ in one-day old chickens after oral administration

Material and methods: Five groups each of ten one-day old chickens were inoculated orally with 1 ml per chicken of 10⁵ or 10³ of FAQ/1 or FAV of serotype 4 wild type (strain VC) or left not inoculated (controls). The five groups were housed in five different isolators. Three weeks after vaccination all remaining chickens were i.m. challenged withl ml per chicken of 10⁷wild type FAV of serotype 4.

Results: All chickens vaccinated with a dose of 10⁵ FAV according to the invention (FAQ) were 100%. protected against i.m. challenge with wild-type FAV 4. 60% of the chickens that were vaccinated with a dose of 10³ FAQ were protected against challenge with wild-type FAV 4. All unvaccinated control chickens died after challenge with wild-type FAV 4. The results are summarised in figure 4.

Figure 4: Mortality rate (%) after challenge with wild-type FAV 4 three weeks post oral vaccination of day-old chickens with two different dose of FAQ / unvaccinated control.

Example 5 Efficacy of different live FAV virus strains compared to inactivated virus

Materials and methods: 50 SPF chickens were allotted to four groups + 1 control group of 10 each. At day one of age each ten chickens of each group were vaccinated with an inactivated vaccine (im) or three different live viruses (orally):

A) FAQ/1 (48e passage on QT 35 cells) at 7.6 log₁₀ TCID₅₀ / ml ,

B) wild type FAV of serotype 4 VC (pathogenic) at 8.5 log ₀ TCID₅₀ / ml, C) KR-5 live natural apathogenic FAV of serotype 4 at 8.0 log₁₀ TCID₅₀ / ml, D) inactivated FAV of serotype 4 .

Three weeks after vaccination the chickens were challenged intramuscularly by inoculation with 0.3 ml of virulent FAV of serotype 4 (VC) and kept for further three weeks. Blood samples were taken from all chickens three weeks post vaccination and three weeks post challenge. Serum samples were examined for the absence/presence of antibodies to FAV in the FAV indirect ELISA. Throughout the experiment, daily all chicken were observed individually for the occurrence of clinical disease according to standard procedures.

Results

Observations post vaccination before challenge: None of the chickens in the groups A, C and D showed any signs of clinical disease. All chickens in the group B, that were vaccinated with a virulent wild type FAV of serotype 4 died within one week after vaccination. All chickens of group B showed typical signs of Hydropericardium- Hepatitis syndrome.

Observations post challenge: In group A (FAQ) all chickens survived. 30 % of the chickens in group C (KR-5) survived the challenge. 40% of the chickens in group D (inactivated) survived the challenge. All chickens that had not survived or had to be killed due to severe signs of diseases showed typical signs of HPS disease during post mortem examination. All chickens that survived the challenge were killed at the end of the experiment at two weeks post challenge, but did not show any abnormalities during gross-macroscopical examination. The survival rates are illustrated in figure 5. Serology: At 21 days post vaccination sero-conversion to FAV was observed in 30 % of chickens in group D (inactivated vaccine) compared to 90 % in group C (KR-5) or 100% in group A (FAQ) of the chickens that were vaccinated with live vaccines. The mean titer was lowest in group D, which was inoculated by the inactivated vaccine with 7.1 log₂. Higher titers were achieved by live vaccination. A titer of 8.7 log₂ was found in group C (KR-5). After vaccination with FAQ (group A) the mean titer was 11.3 log₂.

The highest mean titer after vaccination was achieved by FAQ. FAQ gave efficient protection against HPS. KR-5 that induced a high percentage of antibody responders gave much less protection of 30 % that is even less than the 40 % protection rate achieved with the inactivated vaccine. FAQ is safe given orally and induces a much better serological response and causes a very high protection rate compared to an inactivated FAV of serotype 4 vaccine or to the natural avirulent FAV KR-5.

Figure 5: Protection rate (%) against wild type FAV of serotype 4 (VC) challenge after vaccination with FAV of serotype 4 according to the invention (FAQ), natural apathogenic FAV of serotype 4 (KR-5) and inactivated vaccine (FAV inac).

Percentage of protected chicken

Example 6 Safety and efficacy of FAQ/2 vaccine

Day-old chicks were infected orally with the virus dose (FAQ/2) given in Table 3. Each virus titre was determined on the respective substrate. For the vaccination experiment the viruses which were grown on different substrates were compared. The surviving birds in Isolators 1 and 2 were used as "vaccinates" for a second experiment (challenge). The infection volume was 1ml of INT (10⁷) (CEL-material) given intramuscularly and the birds were challenged at day 21 which is 3 weeks after vaccination. In both experiments the birds which died were necropsied and presence of viral antigen was demonstrated by PCR. Details of the animal experiment are also mentioned in Tables 3 and 4.

It is shown that the FAV 4 strain attenuated by serial passaging on QT35 cells is safe for young chicks after oral administration, whereas the non-passaged parent strain INT induces dose-dependent mortality (Table 3).

Table 3: Mortality of day old chicks vaccinated orally with FAQ/2.

^a infected birds contact birds ^c contact birds ^d- no mortality Birds vaccinated with 10⁵⁰ TCID₅₀ of FAQ/2 were completely protected. Partial protection was obtained after vaccination with a lower dose. All birds that survived infection with the non-passaged parent strain survived challenge with virulent virus

(Table 4).

All birds which died were necropsied and presence of FAV 4 antigen in the liver was confirmed for all dead birds by PCR.

Table 4: Results of challenge experiment

Claims

1. An attenuated fowl adenovirus of serotype 4 (FAV 4), characterised in that it is able to produce a cytopathic effect (CPE) in cells of a QT 35 cell line and to induce protective immunity after oral application to birds.

2. A virus according to claim 1 , characterised in that the genome of the attenuated FAV 4 comprises a deletion of 2-3 kb.

3. A virus according to claim 1 or 2, characterised in that it is in a live form.

4. A virus according to any of the claims 1 to 3, characterised in that the genome of the virus comprises a heterologous nucleic acid fragment encoding a polypeptide.

5. A virus according to claim 4, characterised in that the polypeptide is an antigen of an avian pathogen.

6. A virus according to claim 5, characterised in that the avian pathogen is selected from Eimeria spp., Infectious Bronchitis virus, Gumboro disease virus, Marek's disease virus, Newcastle disease virus, Egg Drop Syndrome virus, Infectious

Laryngotracheitis virus, Mycoplasma spp., Chicken Anaemia agent, Avian Influenza virus, Reo virus, Avian Retro virus, Avian Encephalomyelitis virus, Haemorrhagic Enteritis Virus of Turkeys, Salmonella spp. and E. coli spp.

7. A virus according to claim 4, characterised in that the nucleic acid fragment encodes a cytokine, a growth factor or a chicken γ-interferon.

8. A vaccine that is capable of inducing protection in birds against an avian pathogen, characterised in that it contains a virus according to any of the claims 1 to 7 and a pharmaceutical suitable carrier or a diluent.

9. A method for preparation of the virus according to any of the claims 1 to 7, characterised in that a virulent FAV of serotype 4 is serially passaged on a QT 35 cell line and subsequently harvested from the cell line.

10. A method for preparation of the vaccine according to claim 8, characterised in that a virus according to any of claims 1 to 7 is mixed with a pharmaceutical suitable carrier or a diluent.

1. Use of the virus according to any of the claims 1 to 7 for the manufacture of a vaccine for the protection of birds against diseases that are caused by an avian pathogen.