WO1999031249A1 - Nucleic acids encoding hev structural proteins, hev structural proteins encoded thereby and uses thereof - Google Patents

Abstract

Infectious diseases in farm animals are one of the most important economic factors in the poultry industry. One of the principal diseases in birds, is caused by infection with Hemorrhagic Enteritis Virus (HEV), which suppresses the immune system. The present invention relates to nucleic acid molecules encoding structural proteins of Hemorrhagic Enteritis Virus (HEV) which are capable of eliciting in an animal, particularly in birds, protective immunity against HEV. More specifically, the invention relates to nucleic acid sequences which encode the hexon, the penton base or the fiber proteins of HEV. Peptides encoded by the disclosed nucleic acid molecules and any vector or DNA construct comprising them are also within the scope of the invention. A vaccine may comprise as active ingredient a nucleic acid molecule according to the invention, a protein encoded thereby, a DNA construct comprising the nucleic acid molecule, or any mixture of the same.

Description

NUCLEIC ACIDS ENCODING HE VSTRUCTURAL PROTEINS, HEV STRUCTURAL PROTEINS ENCODED THEREBYAND USES THEREOF

FIELD OFTHE INVENTION The present invention relates to nucleic acids encoding structural proteins of Hemorrhagic enteritis virus (HEV) which are capable of eliciting in an animal protective immunity particularly against HEV. The invention also relates to recombinant vectors and DNA constructs comprising the nucleic acid molecules of the invention, to proteins encoded thereby, and to various uses of these nucleic acids and proteins.

BACKGROUND OF THE INVENTION

Infectious diseases in farm animals are one of the most important economic factors in the poultry industry. The minimalization of losses from diseases, by means of effective vaccines, plays a major part in achieving profit in today's intensive poultry industry. The health of domesticated animals depends on management, on a proper vaccination system and on the availability of effective vaccines.

Further, some diseases suppress elements of the immune system, and thereby decrease its activity. One of the principal diseases which suppress the immune system in birds is caused by infection with Hemorrhagic Enteritis Virus (HEV). HEV belongs to the Adenoviridae family, which is divided into two groups: the mammalian adenoviruses and the avian adeno viruses (genus Aviadenovirus). HEV belongs to serotype II of the avian adenoviruses, which consists of double-stranded DNA genome species. The HEV is a non-enveloped DNA virus, with a diameter of about 70-90nm and having an icosahedral symmetry. The virus replicates in the host cell nucleus and consists of 11 proteins, encoded by its DNA segment. The molecular weights of the HEV proteins range from 97 kD to 14 kD [Nazerian K.L., et al, Avian Dis. 35:572-578 (1991)]. The 97 kD polypeptide is the structural hexon protein, a monomer of the major outer capsid. Another structural protein is the penton base protein, having a predicted size of about 50 kD. A different protein anchored by the penton base protein is the fiber protein. This fiber protein consist of a tail and a globular head, which plays an important role in the first attachment of the virus to the cell receptor.

The HE virus is known to infect and destroy B cells and macrophages in birds, particularly domesticated turkeys. Since B cells play an important role in the primary immune response, afflicted birds suffer mostly from weight loss. The rate of mortality is high and, since the immune response is damaged, the surviving birds exhibit high vulnerability to other diseases. Moreover, infection with HEV reduces the effectiveness of response to various vaccines. As a result of lowered resistance, an outbreak of the HEV infection may further lead to outbreaks of other diseases. Naturally, such events result in heavy financial loss to the breeders.

Infection of birds by the HE virus is especially prevalent during the ages 7-9 weeks [Domermuth CH. & Gross W.B., Diseases of Poultry, Iowa State University press, 8th Edition pp. 511-516, (1984)]. Younger birds are protected by maternal antibodies [Van den Hurk, J.V., Avian Dis. 30:662-671 (1986); Harris J.R. & Domermuth C.H., Avian Dis. 21: 120-122 (1977); Fadly, A.M. & Nazerian K., Avian Dis. 33: 778-786 (1989)].

The current approach in production of vaccines against HEV is by using inactivated virus for the vaccine or producing live vaccines of low virulence.

Antibodies effective in neutralizing the HE virus are produced principally against certain polypeptides. The main polypeptide, as tested by monoclonal antibodies [Nazerian et at. (1991) ibid.], is the structural hexon protein. A second structural protein, the penton, was also found to stimulate the production of antibodies, however, to a smaller extent. P.L. Stewart et al. [Stewart, P.L., et al, EMBO J. 16: 1189-1198 (1997)] describe an epitope for neutralizing antibodies on human adenovirus penton base protein. The fiber protein was found to stimulate the production of neutralizing antibodies against the HEV [Wohlfart, C.U., et al, J. Virology 56:896-903 (1985)]. These antibodies recognize conformational, but not linear epitops of the fiber [Fender, P., et al, Virology 214:110-117 (1995)]. Although this protein is involved in the attachment of the virus to the cell, antibodies produced against it did not exhibit a neutralization effect. Nevertheless, in other adenoviruses, a neutralizing effect was observed with antibodies raised against the fiber protein [Wohlfart et. al. (1985) ibid.].

Immunization against the HE virus is currently performed by injection of an inactivated virus vaccine to young birds at the age of 3 weeks. In the past years, an attenuated virus was isolated and used for vaccination against the HEV [Fadly & Nazerian (1989) ibid.]. The use of attenuated viruses, and even inactivated viruses, for the preparation of vaccines may entail some danger. For example, there always exists the possibility that inactivation has not been complete, or that mild attenuated virus will revert to virulence. Further, the reliance on tissue culture, embryonated specific pathogen free (SPF) eggs or on live turkeys providing spleen tissue for vaccine virus, and propagation in large quantities, involves heavy expenditure.

Since the price of a single bird is relatively low, the cost of production and delivery of the vaccine becomes critical. Naturally, if the cost of the production of the vaccine is too high, its use will not be economically feasible. Thus, the preferred vaccines are those of low cost.

Over the recent years, a development in the production of vaccines has been reached at from several directions. First, the effectiveness of sub-unit virus vaccines has been tested. For developing a sub-unit vaccine, polypeptides of the entire virus are first isolated by any suitable means, each examined for their ability to stimulate neutralizing antibodies against the specific disease. The nucleotide sequence of the effective segment/s is determined and the gene is expressed for mass production of the stimulatory protein. For example, a viral protein (VP2) of Infectious Bursal Disease virus (IBDV) was expressed in a baculovirus expression system and was capable of conferring in birds full protection against the disease [Pitcovski, J., et al, Avian Dis. 40:753-761 (1996)]. Many other sub-unit vaccines have been found to be efficient, including a vaccine against hepatitis B in humans [Mackett, M. & Williamson, J.D., Human Vaccines and Vaccinations, pp. 159-176; Bios Scientific Publishers LTD. UK (1995)].

Fowlpox virus (FPV) has been found to be a suitable vector for the expression of foreign proteins. The large size of the FPV genome, permitting the introduction of foreign DNA, without affecting its reproductive ability, and the limited reproduction of the virus to fowl, makes it an efficient vector which can be used without endangering humans. Furthermore, attenuated FPV has been used on poultry farms since the 20's and is still widely used by poultry growers.

In general, since the FPV has a large genome, the target gene cannot be inserted into it directly. Rather, an intermediate vector (plasmid) is required. Such a plasmid contains a DNA segment of FPV, encoding the enzyme tymidine kinase (TK), which is not essential to the reproduction of the virus. The target gene is inserted into this section of the DNA, under the control of a promotor that will be expressed in the FP virus, as a result of which the gene encoding TK is damaged. The plasmid, together with the viral DNA, are joined to the target cell, which is TK",originating from the bird. In the course of the development of the virus in the cell, an interchange between the plasmid segments and the viral segments takes place, giving rise to the recombinant virus, in which an inessential area is formed in the area containing the target gene. A virus of this sort is TK", and can be positively selected in a growth substrate that supports cells and TK" viruses.

The efficiency of immunization by such recombinant FP virus was reported in connection to a number of disease-causing viruses. For example, the infection of birds by the recombinant FP virus, expressing the protein haemagglutinin of Avian Influenza, provided protection of birds exposed to this disease [Taylor, J., et al, Vaccine 6:504-508 (1988); Tripathy, D.N. & Wittek, R., Avian Dis. 34:218-220 (1990)]. Immunization of birds with recombinant FPV, into which the gene of the HN protein of the Newcastle Disease virus (NDV) was inserted, induced the production of antibodies against the disease [Edbauer, C, et al, Virology 179:901- 904 (1990)]. Furthermore, these birds, immunized with the above mentioned recombinant virus, developed resistance to NDV without any decrease in their resistance to FPV [Ogawa, R., et al, Vaccine 8:486-490 (1990)]. Since such recombinant viruses are not expensive and are found to be effective in conferring immunity to birds [Schnitzlein W.M., et al, Virus Res. 10:65-76 (1988)], they may be potential suitable systems for the production of fowl vaccines. This method of vaccination is said to allow the building of various "recombinants" for the production fowl vaccines. Recombinants of this sort are cheap and effective vaccinating agents for birds.

In the case of HEV, to date, immunization against the virus is in the form of live and inactivated vaccines, which are expensive and involve mass infection of birds in order to isolate the virus from their spleens.

The efficiency of live vectors like FPV in the production of vaccines, and the fact that adenoviruses are found to be efficient expression vectors, recombinant vaccination vehicles and a potential tool in gene therapy [Kozarsky, K.F. & Wilson, J.M., Current Opinion in Genetics and Development 3:499-503 (1993); Callebaut, P., et al, J. Gen. Virol. 77:309-313 (1996)] led the inventors to the idea of producing avian-specific recombinant vaccines in the form of sub-unit vaccines, comprising polypeptide units of the HEV, that have immunity-conferring properties, or DNA sequences encoding the polypeptide units, cloned into an expression vector, such as the above described FPV, which express these polypeptide units.

SUMMARY OF THE INVENTION

The present invention relates to nucleic acid molecules encoding structural proteins of Hemorrhagic enteritis virus (HEV), which proteins are capable of eliciting in an animal, preferably domesticated birds, protective immunity against HEV. The nucleic acid molecules according to the invention are preferably genomic DNA or cDNA.

The present invention also relates to a protein capable of eliciting in an animal protective immunity against HEV, encoded by a nucleic acid molecule of the invention or immunologically active homologues and fragments of such protein.

The invention further relates to a recombinant polypeptides capable of eliciting in an animal protective immunity against HEV, comprising at least one protein of the invention. The recombinant polypeptide may further comprise additional proteins or peptides capable of eliciting protective immunity against pathogens other than HEV.

In a different aspect, the invention is concerned with a recombinant vector comprising the nucleic acid molecule of the invention.

Further, a DNA construct for the expression of a protein product in a host cell, comprising an expression vector and at least one exogenous nucleic acid molecule encoding a structural protein of HEV, which structural protein is capable of eliciting in an animal protective immunity against HEV, is within the scope of the invention. The exogenous nucleic acid molecule also may comprise the recombinant vector of the invention.

The DNA construct according to the invention optionally further comprises at least one additional exogenous nucleic acid molecule encoding a protein or peptide product capable of eliciting in an animal protective immunity against a specific pathogen, other than HEV.

In a further aspect, the invention relates to a vaccine for immunizing an animal against HEV comprising as active ingredient an effective immunizing amount of at least one protein of the invention or at least one DNA construct of the invention, or at least one nucleic acid molecule of the invention or mixtures of the same. In addition, methods of immunizing animals against HEV by administering to the animals an effective immunizing amount of the vaccine of the invention are also within the scope of the invention.

Finally, antibodies directed against the proteins of the invention and different uses thereof also constitute part of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 Gel agarose of HEV DNA

M - Marker, Lambda DNA cut with Hindll; lane 1 - Upper band HEV

DNA

Figure 2 The plasmids used as a shuttle to insert the hexon gene into Pichia These plasmids may be used also to insert the penton gene into Pichia a) pHIL-Sl; b) pPIC3K

Figure 3 Hexon and penton proteins expressed in E. coli

SM Size Marker; lane 1 - hexon protein expressed in E. coli (pellet) indicating the C-terminal fraction (hexon Ct) and N-terminal fraction

(hexon Nt) of the protein expressed ; lane 2 - hexon protein expressed in E.coli (supernatant); lane 3 - hexon protein expressed in E.coli (pellet); lane 4 - negative control E.coli; lane 5 - negative control E.coli; lane 6 - penton base protein expressed in E.coli (supernatant); lane 7 - penton base protein expressed in E.coli (pellet) indicated by the corresponding arrow.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a nucleic acid molecule encoding a structural protein of Hemorrhagic enteritis virus (HEV), which protein is capable of eliciting in an animal, preferably a domesticated bird, protective immunity against HEV. The nucleic acid molecule according to the invention can be a genomic DNA molecule or cDNA.

In one embodiment, the nucleic acid molecule of the invention may comprise a nucleotide sequence substantially as set forth in is SEQ ID NO: l or immunologically functional homologues and fragments thereof. According to this embodiment, the nucleic acid molecule encodes the hexon protein of HEV.

The nucleic acid molecule of the invention may comprise a nucleotide sequence substantially as set forth in SEQ ID NO:2 or immunologically functional homologues and fragments thereof. This molecule encodes a different structural protein of HEV, the penton base protein of HEV. Alternatively, the nucleic acid molecule may comprise a nucleotide sequence substantially as set forth in SEQ ID NO: 3 or immunologically functional homologues and fragments thereof. This nucleic acid molecule encodes an HEV fiber protein.

In addition, the invention relates to proteins capable of eliciting in an animal, preferably a domesticated bird, protective immunity against HEV, and to immunologically active homologues or fragments thereof. The proteins encoded by the nucleic acid molecules of the invention are preferably intended for protecting birds against HEV infection.

The protein according to one embodiment of the invention comprises the amino acid sequence substantially as set forth in SEQ ID NO:4, or immunologically active homologues and essential fragments thereof. In particular, the protein consists of the amino acid sequence substantially as set forth in SEQ ID NO:4 or of immunologically active homologues and essential fragments thereof.

In a second embodiment, the protein of the invention comprises the amino acid sequence substantially as set forth in SEQ ID NO: 5 or immunologically active homologues and essential fragments thereof. More specifically, the protein consists of the amino acid sequence substantially as set forth in SEQ ID NO:5, or of immunologically active homologues and essential fragments thereof. In a further embodiment, the protein of the invention comprises the amino acid sequence substantially as set forth in SEQ ID NO:6 or immunologically active homologues and essential fragments thereof. However, the protein may have the amino acid sequence substantially as set forth in SEQ ID NO: 6 or immunologically active homologues and essential fragments thereof

Any variations of the nucleic acid sequence and/or the amino acid sequence of the invention are also within the scope of the invention. By the term 'variations' it is meant all deletions, substitutions and/or insertions of nucleic acid/s or amino acid/s in the DNA and/or amino acid sequences herein defined, respectively, by which the immunological functionality of the products against at least HEV infection is preserved.

Also within the scope of the invention is a recombinant polypeptide capable of eliciting in an animal, preferably a domesticated bird, protective immunity against HEV and comprising at least one protein of the invention. The at least one protein is a structural protein of HEV and, more preferably, a protein selected from the group consisting of the hexon protein, the penton base protein and the fiber protein of HEV. As noted hereinbefore, under normal conditions, the fiber protein is known to mediate the first stage of virus-cell attachment. Thus, when used in sub-unit vaccination, alone or in combination with other immunologically active proteins, the fiber protein may block the attachment of the virus to the target cells, in addition to stimulating the production of the specific antibodies. Thus, one aim of the invention is to construct a recombinant polypeptide in which the fiber protein is hybridized to other immunologically active proteins of the HEV, thereby obtaining a fusion protein, which has multiple effect.

Within the recombinant polypeptides, which can also be referred to as fusion proteins, of the invention, other HEV proteins, fragments of proteins or peptides may also be integrated into said recombinant polypeptide, as long as the protein product obtained by such hybridization is capable of providing protection against the virulent strains in the field.

In addition, hybridizations between HEV proteins or peptides of the invention, with amino acid sequence/s from other pathogens, or with immunologically active homologues and essential fragments thereof, are possible, provided that said other amino acid sequence/s have some therapeutical purpose and that the produced protein or recombinant polypeptide are immunologically functional, in an animal, against at least infection by HEV. Examples for preferred pathogenic agents, but not limited thereto, are infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), egg drop syndrome adenovirus (EDS), infectious bronchitis (IB), Marek disease virus (MDV), avian influenza and fowl pox virus, or any type of bacterial or parasitic disease such as salmonella infection, coccidia or bacteria causing cholera such as pasteurella multocida.

It is understood that any nucleic acid molecule encoding the proteins of the invention, also constitutes part of the invention. The sequences of the virus provided herein enable the isolation, cloning and production of protein products which preferably are used in the preparation of sub-unit vaccines.

The use of hybrid proteins for sub-unit vaccination, such as hybrids of the proteins of the present invention, can have several advantages. For example, a proteinous sub-unit vaccination involves a limited number of antigens, these being only the ones essential to stimulate response and lead to the formation of antibodies capable of neutralizing the virus, therefore, allowing optimal immunity formation, while preventing exposure of the animal to non-relevant antigens of the virulent field virus, which, in turn, may also be immunosuppressive.

In a different aspect, the invention is concerned with a recombinant vector comprising the nucleic acid molecule of the invention, said molecule being a genomic DNA or a cDNA. According to one embodiment, the recombinant vector comprises the nucleic acid sequence substantially as set forth in SEQ ID NO: l or functional homologues and fragments thereof. Alternatively, the recombinant vector comprises the nucleic acid sequence substantially as set forth in SEQ ID NO:2 or functional homologues and fragments thereof. Nonetheless, the recombinant vector may comprise the nucleic acid sequence substantially as set forth in SEQ ID NO: 3 or functional homologues and fragments thereof. Such recombinant vectors will later be cloned into a suitable expression system.

In addition, the invention is concerned with a DNA construct for expression of a protein product in a host cell, comprising an expression vector and at least one exogenous nucleic acid molecule encoding a structural protein of HEV, preferably the nucleic acid molecule of the invention. The structural protein is capable of eliciting in an animal protective immunity against HEV and is preferably selected from the group consisting of the hexon protein, penton base protein and fiber protein of HEV. In particular, the said structural proteins elicit protective immunity against HEV, in birds.

In one embodiment according to the invention, at least one exogenous nucleic acid molecule within the DNA construct of the invention comprises the nucleotide sequence substantially as set forth in SEQ ID NO: l or functional homologues and fragments thereof.

Also, a DNA construct, wherein said at least one exogenous nucleic acid molecule comprises the nucleotide sequence substantially as set forth in SEQ ID NO:2 or functional homologues and fragments thereof is part of the invention. Alternatively, said at least one exogenous nucleic acid molecule within said DNA construct comprises the nucleotide sequence substantially as set forth in SEQ ID NO:3 or functional homologues and fragments thereof. In a further embodiment, the said at least one exogenous nucleic acid molecule is the recombinant vector of the invention.

The DNA construct according to the invention optionally further comprises at least one additional exogenous nucleic acid sequence encoding a protein or peptide product which are capable of eliciting in animals protective immunity against a specific pathogen, other than HEV. These additional nucleic acid sequences are inserted into the expression vectors in reading frame to enable expression thereof, in a host. The different sequences may be either separated by termination and initiation sequences or they may form a single reading frame and thus produce a single "fusion protein". The said other pathogens refer to such agents that, in normal conditions, may lead to a viral infection, a bacterial infection or any other type of harmful infection. Examples for such pathogens, without being limited thereto, are infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), egg drop syndrome adenovirus (EDS), infectious bronchitis (IB), Marek disease virus (MDV), avian influenza and fowl pox virus, or any type of bacterial disease such as salmonella, coccidia and bacteria causing cholera such as pasteurella multocida.

In general, introduction of the nucleic acid molecule or the recombinant vector of the present invention into an animal, via the DNA construct of the invention, is of viral infection. Infection of this sort offers several advantages over other listed methods. Higher efficiency is obtained due to the infectious nature of viruses. Moreover, viruses are very specific and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target vectors, in vivo, to specific cells or tissues.

The expression vector comprised within the DNA construct of the present invention may be selected from the group consisting of fowlpox virus, vaccinia virus, MDV, HEV, baculovirus, bacteria, yeast, plants and plant cells. Examples of other vectors include viruses such as retroviruses, cosmids, liposomes and other DNA viruses. Notwithstanding the above, other expression vectors are known, or can be constructed, by those skilled in the art. Evidently, such expression vectors will contain all expression elements necessary to achieve the desired transcription of the sequences to be expressed. Other beneficial characteristics can also be contained in the vectors, such as mechanisms for recovery of the nucleic acids in a different form. Phagemids are a specific example of such beneficial vectors because they can be used either as plasmids or as bacteriophage vectors. Other additional features which can be added to the vector may ensure its safety and/or enhance its therapeutic efficacy. Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus which will ensure that if, for example, a mutation arises that produces altered forms of the viral vector or recombinant sequence, cellular transformation will not occur. Features that limit expression to particular cell types can also be included, for example, promoter and regulatory elements that are specific for the desired cell type. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events. Further, signal sequences such as initiation and termination sequences, as known to the man skilled in the art can be introduced into said construct. The vectors can also contain elements for use in either prokaryotic or eukaryotic host systems. One of ordinary skilled in the art will know which host systems are compatible with a particular vector.

In one embodiment of the invention, the expression vector is of yeast origin. In this embodiment the construct obtained is preferably used for mass production of the proteins of the invention. In particular, the expression vector in the DNA construct of the invention, is the yeast plasmid pHIL-Sl, having the restriction map as set forth in Fig. 2(A). In a second embodiment, the expression vector is the yeast plasmid pPIC3K having the restriction map as set forth in Fig. 2(B). Nevertheless, other expression systems, capable of expressing the desired nucleotide sequence are acceptable.

In general, the nucleic acid molecule and the recombinant vectors of the invention can be introduced into cells or tissues by any of a variety of known methods within the art, which include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. A host transformed with the nucleic acid molecule or with the recombinant vector of the invention also constitutes part of the invention. The host cell preferably being a bacterial cell, an yeast cell, an insect cell, a plant cell, a mammalian cell, a bird cell or any other suitable cell, capable of expressing the nucleic acid molecule or recombinant vector of the invention. In one preferred embodiment, the host is the Pichia pastor is yeast cell.

In a different aspect, the invention relates to a vaccine for immunizing an animal against HEV comprising as active ingredient an effective immunizing amount of at least one protein according to the invention. For multiple-purpose vaccination, the vaccines of the invention may optionally further comprise at least one additional protein, peptide or any other immunological active ingredient which is capable of eliciting protective immunity against specific pathogens, other than HEV.

Alternatively, the vaccine of the invention comprises as active ingredient an effective immunizing amount of the nucleic acid molecule of the invention either in its naked form or as part of an appropriate plasmid. The vaccine can also comprise the DNA construct of the invention. Nonetheless, the vaccine my comprise any suitable and veterinarily acceptable combinations of the above detailed ingredients.

In any case, said vaccine is preferably a sub-unit type vaccine. As known to a man well versed in the art, since a sub-unit vaccine contains only part of the viral components, the virus would not replicate in the host and thus the danger inherent in using a live or inactivated vaccine, like an outbreak of the disease as a result of reversion of the virus to virulence, or its incomplete inactivation, is eliminated.

The sub-unit vaccine comprising the products of the invention, can be used directly, by inoculating animals. The proteins are capable of inducing the production of neutralizing antibodies in the receiving animal and thus may provide protection against the specific infections and diseases. As noted above for the preparation of a vaccine, the complete protein or nucleotide sequence or only essential fragments thereof, alone or as a combination thereof may be used. Further, a DNA construct according to the invention may be constructed in a way to contain more than one copy of the encoding vector, thus producing, for example, multiple copies of the desired protein. The vaccine is preferably used for immunizing birds.

The nucleic acid molecules, the proteins, and the DNA constructs of the invention, may be administered to the animal in various ways. It can be administered as the product itself or in its veterinarily acceptable salt form, and can be administered alone or as an active ingredient in a vaccine, combined with pharmaceutically acceptable carriers, diluents, adjuvants and vehicles which generally are referred to inert, non-toxic solid or liquid fillers, diluents or encapsulating material, not reacting the active ingredients of the invention.

The nucleic acid molecule, the proteins and the DNA constructs of the invention or their vaccine form, are administered and dosed in accordance with good veterinary practice, taking into account the clinical condition of the individual animal, the site and method of administration, scheduling of administration, the animal's age, body weight, diet, and other factors, well known to medical veterinarians.

The veterinarily 'effective amount' for purposes herein is that determined by such considerations as are known in the art. The amount must be sufficient to stimulate the immune system and confer immunity against HEV and other desired pathogens. Apart from the immunity conferred to the animal vaccinated according to the invention, the vaccination according to the invention confers immunity to progeny of the immunized animal, via maternal antibodies.

Administration may be orally, subcutaneously or parenterally, including intravenous, intramuscular, intraperitoneally and intranassal administration as well as intrathecal and infusion techniques. Most preferred methods are oral administration and intravenous, intramuscular or subcutaneuos injection. Following injection, the therapeutic product, and specifically the DNA construct of the invention, will circulate until it recognizes the host cell with the appropriate target specificity for infection. The vaccine of the invention, may be provided in various forms such as lysates of cells expressing the protein of the invention, partially or completely purified proteins, as recombinant vectors or as said DNA constructs.

The doses may be single doses or multiple doses and vaccination may be effected at any age from day one. However, when the animals protected by the vaccines of the invention are birds, a single administration at the age of three weeks is preferably performed.

Thus, the present invention also relates to methods of immunizing animals against HEV. According to one method, the animals are immunized by administering an effective immunizing amount of at least one nucleic acid molecule or at least one DNA construct of the invention. Alternatively, the animals are immunized by administering thereto an effective immunizing amount of at least one protein of the invention. According to one method, birds are preferably protected by the products of the invention, especially domestic birds.

In addition, the products, i.e. the nucleic acid molecule/s, the protein/s, the polypeptide/s, the recombinant vector/s and the DNA construct/s of the invention, alone or in its vaccine form may be administered to the animal, as eye-drops via the ocular route, or by course of spraying or aerosol. Also mass vaccination via drinking water may be performed. However, the preferred method for vaccination, when birds are the subject to be immunized, is by injecting them at the age of three weeks.

Furthermore, the products of the invention alone or in their vaccine form may be administered directly to an embryo or through maternal immunity. All possible uses of the products of the present invention, in the preparation of vaccines for immunizing animals, especially birds, against HEV, are also within the scope of the invention.

Further, within the scope of the invention are antibodies, either monoclonal, polyclonal or recombinant, directed against the proteins of the invention, which are specific immunogens. The proteins may be prepared either synthetically, based on the sequence disclosed herein after, or recombinantly by cloning techniques of the natural or recombinant gene product and/or portions thereof and after which they may be isolated and used as the immunogen. These immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art as described generally by Harlow et al. [Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY (1988)] and by Borrebaeck et al. [Borrebaeck, Antibody Engineering - A Practical Guide, W.H. Freeman and Co., (1992)]. Antibody fragments may also be prepared from the antibodies by methods known to those skilled in the art and include the Fab, F(ab')2 and Fv fragments.

In general, for producing polyclonal antibodies a host, such as a rabbit or goat, is immunized with the immunogen or immunogen fragment, generally with an adjuvant and, if necessary, coupled to a carrier, antibodies to the immunogen are collected from the sera. Further, the polyclonal antibody can be absorbed such that it is monospecific. That is, the sera can be absorbed against related immunogens so that no cross-reactive antibodies remain in the sera rendering it monospecific.

For producing monoclonal antibodies, the technique involves hyperimmunization of an appropriate donor with the immunogen, generally a mouse, and isolation of splenic antibody producing cells. These cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody. The cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use.

For producing recombinant antibody messenger RNA's from antibody producing B- lymphocytes of animals, or hybridoma are reverse-transcribed to obtain complimentary DNA's (cDNA's). Antibody cDNA, which can be full or partial length, is amplified and cloned into a phage or a plasmid. The cDNA, can be a partial length of heavy and light chain cDNA, separated or connected by a linker. The antibody, or antibody fragment, is expressed using a suitable expression system to obtain recombinant antibody. Antibody cDNA can also be obtained by screening pertinent expression libraries.

The antibody can be bound to a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known [for a general discussion of conjugation of fluorescent or enzymatic moieties see Johnstone & Thorpe, Immunochemistry in Practice, Blackwekk Scientific Publications, Oxford, 1982]. The detectable moieties contemplated with the present invention can include but are not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferittin, alkaline phosphatase, β-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, ^J4C and iodination. The antibodies produced against the immunogens of the present invention, may have different uses. Within one scope of the present invention, these antibodies are used for detecting of the presence of anti-HEV antibodies in a serum drawn from an infected animal, such as domestic birds.

One mode of employing said antibodies in said detection comprises the steps of

(a) coating polystyrene microtiter plates with at least one protein of the invention;

(b) adding to the coated plates sera obtained from infected birds and incubating said sera in said plate for several hours at 37°C; (c) washing the wells; (d) further incubating said wells of step (c) with rabbit anti-turkey Ig conjugated to alkaline phosphatase; (e) washing said wells of step (d); (f) adding a labeling substrate to each well of said plate/s, preferably nitrophenyl phosphate and washing with a washing buffer; and (g) reading the wells in an ELISA reader. Such a method is advantageous for the identification of anti HEV antibodies.

A different method of detecting the presence of HEV in a sample employs the Polymerase Chain Reaction (PCR) technology, whereby short primers (12 to 30 nucleotides) comprising part of the DNA sequences of the invention, as depicted in SEQ ID NOs: l and 2 are used for intensifying the DNA fragments of HEV in the sample and thus enabling to identify small amounts of the virus. Also, for this purpose, the ImmunoComb technology (to ORGENICS) may be utilized. Said technology is based on concentrating an antigen or an antibody on a plastic surface which undergoes a special excitation treatment. Subsequently, a color is revealed whose intensity is directly correlated to the concentration of the antibody or the antigen tested. ImmunoComb is a plastic card shaped like a comb on which purified antigens are attached.

The invention will now be described in an illustrative manner, and it is to be understood that the terminology which will be used is intended to be in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present invention are possible in light of the above teaching. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

EXAMPLES Standard methods

Standard molecular biology techniques known in the art and not specifically described are generally followed as in J. Sambrook et al. [Sambrook, J., et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989, 1992)], and in Ausubel et al. [Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989)]. Polymerase chain reaction (PCR) is carried out generally according to M.A. Innis et al. [PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA (1990)]. Reactions and manipulations involving other nucleic acid techniques, unless states otherwise, are performed as generally described in Sambrook et al. {ibid.) and methodologies as set froth in several United States patents, such as US Patent No. 4,666,828 and US Patent No. 4,683,202, incorporated herein by reference. In situ (in cell) PCR in combination with Flow Cytometry can be used for detection of cells containing specific DNA and mRNA sequences [Testoni, et al, Blood 87:3822 (1996)].

Standard methods in immunology known in the art and not specifically described are generally followed as in Stites et al. (Eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (Eds), Selected Methods in Cellular Immunology, H. Freeman and Co., New York (1980).

In general, ELISA's are the preferred immunoassays employed to assess a specimen. ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in the assays. Where appropriate other immunoassays, such as radioimmunoassays (RIA) can be used as are known to those in the art. Available immunoassays are extensively described in the patent and scientific literature such as in United States Patents Nos. 3,791,932; 5,011,771 and 5,281,521, as well as in Sambrook J. et al. (1989) ibid.

General methods for recombinant protein purification may be obtained from Marshak et al. [Marshak et al, Strategies for Protein Purification and Characterization, A laboratory course manual, CSHL Press, (1996)].

Example 1

Isolation of HEV infected birds

Turkeys were exposed to a virulent strain of HEV. Five days later, the turkeys were sacrificed and their spleens were removed. To the spleens, distilled water, twice the volume of the tissue, was added, and the tissue was ground and homogenized for 5 minutes. In order to rapture the cells, the tissue homogenate was frozen and thawed three times at -70°C and 30°C, respectively, followed by centrifugation at 10,000xg, at 4°C, for 20 min. The supernatant was mixed with trichlorotrifluoroethane (1 :3) and centrifuged at 5000xg, at 4°C, for 30 min. The supernatant was collected and added on top of the following density gradient: 12ml of 46.2% (w/v) cesium chloride (CsCl) (density of 1.35g/ml); 12 ml of 35% (w/v) CsCl (density of 1.24g/ml); 6 ml of 1M sucrose. The gradient with the supernatant was centrifuged for 24 hours at 85,000xg, at 4°C, with SW28 rotor. The virus was isolated from a white ring that was formed between the two CsCl layers. This virus band was collected, diluted in Tris EDTA (TE), and repelleted by centrifugation at 26000xrpm for 2 hours. The pellet was collected, resuspended in distilled water and dialyzed against TE. The virus was stored at -20°C until use.

Purification of HEV DNA

The isolated virus was incubated for 3 hours in a solution containing 0.01M Tris, 0.01M NaCl, 0.01M EDTA, 0.5% SDS, and 50 μg/ml proteinase K. Following incubation, the DNA of HEV was electrophoresed on 0.8% agarose gel. The DNA of the virus, of the size of 26 Kb, was visualized by Ethidium Bromide (Figure 1).

Cloning of segments of HEV DNA into the plasmid pBS for sequencing

The HEV DNA was cut by restriction enzymes EcoRI and Pstl. Each of these enzymes cut the DNA into seven fragments. Each fragment was isolated from an agarose gel, ligated into pBS (Stratagene) that was previously digested with the same enzymes, respectively. These plasmids were transformed into E. coli. XL1- blue cells and white colonies that grew on Luria Bertani medium (LB), containing ampicillin (100 μg/ml) and X-Gal (200 μg/ml) were isolated. Plasmid DNA was extracted from clones containing fragments of different sizes (0.9-8.5 kb for HEV DNA cut by EcoRI and 0.7-12 kb for Pstl fragments) and was used for DNA sequencing. HEV DNA that was contained in plasmids E1-E7 (harboring EcoRI fragments 1 (0.7 kb) to 7 (8.5 kb)), and P1-P7 (harboring Pstl fragments respectively), was sequenced by the thermocycling sequencing method using ABI 377 Perkin Elmer DNA sequencer. The sequence analysis was initiated from both ends of the fragments, using two commercially available primers corresponding to the 5' and the 3 ' ends of the pBS multiple cloning site (Universal Primers, New- England biolabs). 250-350bp were resolved in a typical sequencing reaction. Synthetic primers corresponding to sequences near ends of the progressing DNA sequences were synthesized to facilitate further DNA sequence analysis. The sequences corresponding to the hexon protein, to the penton base protein and to the fiber protein of the HE virus are depicted in SEQ ID NO: l, SEQ ID NO:2 and SEQ ID NO:3, respectively. The sequences of the HEV hexon and penton base proteins share partial sequence homology, both at the DNA level and at the protein level, with those of other strains. When compared to the proteins of the fowl adenovirus CELO, 45% homology was observed for the fiber protein and 53% homology for the hexon protein. Yet, there are some differences in the sequences of both proteins that are substantially unique to this HEV strain, that may reflect its unique virulence.

Example 2

Production of large amounts of hexon, penton base and fiber proteins, using the yeast Pichia pastoris expression system (PPES)

The Pichia pastoris expression vector system is widely applicable as an alternative to prokaryotic and other eukaryotic systems for the expression of heterologous proteins. A variety of recombinant proteins are already produced by this system [Digan, M.E. et al, Biotechnology 7: 160-164 (1989); Kniskern B.J., et al, Vaccine 12: 1021-1025 (1994)]. Post-transcriptional and post-translational modifications such as RNA-splicing, glycosylation, phosphorylation, assembly of multimeric proteins and signal recognition have been observed in the PPES system [Cregg, J.M., et al, BioTech. 5:479-485 (1987); Romanos M.A., et al, Vaccine 9:901-906 (1991)]. The hexon and penton base protein were also expressed in large amounts, using the PPES system.

Cloning hexon and penton base proteins into the PPES plasmids

The plasmids pHILSl and pPIC3K presented in Figure 2(A) and 2(B) were used for cloning both the penton base and the hexon genes. In the case of the hexon protein, these plasmids were digested with the restriction enzymes EcoRI. and BamHI. The hexon gene was then propagated by PCR, using the primers hexstart and hexstop of 5' and 3' (respectively) of the hexon gene and then cloned into the appropriate vector.

The hexstart primer:

EcoRI Hexon 's start

5' GCG GAATTC ATG GAC ATA TCA AAT GCT AC 3'

The hexstop primer:

EcoRI Smal BmaHI Hexon 's end

5' ATA GAA TTC CCG GGA TCC TTA TAC TGA AGC AGT TCC AG

3' The isolated fragments were ligated to the PPES, previously digested with EcoRI and BamHI. Clones that included the inserted hexon DNA were identified following PCR with primers of the plasmid and internal primers of the hexon gene. In positive clones, the site of the junction between the plasmid and the insert was sequenced to make sure that the insert is in the reading frame of the vector.

In the same manner the penton base gene was cloned into the PPES plasmids digested with the restriction enzymes Kpnl and Pstl. The primers used for isolation of the penton segment from the viral DNA are preferably the following:-

The penstart primer:

Kpnl site Penton's start

CGC GGTACCC ATG GAATC TTC GAA CAC TGC

The penstop primer:

Pstl site Penton's end

AAA CTG CAG TTA TTG CAA AGT TTT GC Using these primers, the penton gene was propagated by PCR and cloned into the appropriate vector. The restriction enzymes sites at the ends of the primers enable cloning of the gene into a bacterial expression system.

The proteins that were expressed were characterized by SDS-PAGE. Figure 3 presents the SDS-PAGE results in which the size marker bands indicate 104 Kd, 81 Kd, 47 Kd, 34 Kd, 28 Kd. Two bands of the hexon protein are indicated, the C- terminal and N-teminal thereof, having the total size of ~100Kd as expected for the hexon protein. The size of the penton was 47 kD also as expected. Antibodies that were raised against the whole virus identified the recombinant penton base in immunoblot analysis.

Basically, the same methods as described hereinbefore were used in order to produce fiber protein. The restriction enzymes were Kpnl and Xbal, the primers used are as follows:-

The fiberstart:

Kpnl site Fiber's start

5 'AT GGTACC GATGCATCACCATCACCATCAC AGAATTGGC AAAGG ATTAAAG3 ' The fiberstop:

Xbal Fiber's end

5 'CG TCTAGA TCA GCC TAT CAA ACA AG 3 '

Isolation of recombinant yeast carrying hexon, penton base or fiber nucleic acid molecules and protein production

The identification of yeast cells carrying the hexon or penton base or fiber genes, induction of cells to produce these gene products and identification of positive colonies is performed according to the instructions in: Pichia Expression Kit of "Invitro" corporation, USA. In principle, the identification of colonies expressing a recombinant protein, in a Pichia expression system comprises the steps:- (a) preparing minimal dextrane (MD) and minimal methanol (MM) cells and placing a Hybond-N membrane on the MD plate; (b) transferring transformed colonies to the membrane on the MD plate; (c) incubating the colonized MD plate at 30°C for two days; (d) transferring the colonized membrane from the MD plate to the MM plate and further incubation of the colonies, at 30°C for two days; (e) removing the membrane from the MM plate and washing the same three times, 10 min. each, with TNT buffer (150mM NaCl, lOmM Tris, 0.05% Tween, pH 8.0); (f) incubating for 30 min. the membrane in a milk buffer (lOmM Na₂HP0₄, 150mM NaCl, pH 7.0 dissolved in skim milk); (g) washing the membrane, twice, with TNT buffer, each for 5 min.; (h) incubating the membrane with suitable antibodies against the expected protein, diluted in the milk buffer (preferably, 1 :500 dilution) and shaking the same for 2 hrs at room temperature; after which the membrane is washed twice with TNT buffer, each wash 10 min.; (i) incubating the membrane for 1 hr, at room temperature with antibodies against the first antibodies attached to an enzyme (preferably peroxidase), the antibodies diluted in the milk buffer (1 : 1000 dilution) and washing the membrane twice, with TNT buffer, each wash 10 min.; (j) reacting the membrane with a suitable substrate to produce color around the colonies which will indicate the secretion of the expected protein, when the enzyme used is peroxidase, the substrate is diaminobenzidine+H2θ2 in 50mM Tris, pH 7.6.

Determination of recombinant hexon protein production

The production of desired recombinant HEV proteins of the invention, in infected host cells such as yeast, is determined by two procedures:- by staining with Coomassie blue or subjecting the cells to western blot analysis. Uninfected cells serve as a control.

In principle, yeast cells are first infected with pHILS 1 plasmid carrying the hexon gene and grown at 30°C over night and then induced to produce the foreign protein by expressing the foreign gene. Four days post induction, the cells are harvested and broken by glass beads and centrifuges. The supernatant is separated on a 10% SDS polyacry amide gel electrophoresis (PAGE). The gels are either stained by Coomassie blue or subjected to western transfer analysis. Uninfected yeast cells serve as the control.

Partial purification of recombinant HEV recombinant proteins

Partial purification of the hexon, the penton base and the fiber proteins is performed using the ammonium sulfate precipitation procedure [Guide to Protein Purification. Methods in Enzymology volume 182].

In principle, the fraction obtain from the previous step, which contains the recombinant proteins, is diluted in lysis buffer, and chilled on ice. Ammonium sulfate powder is than gradually added to a final concentration of about 25%. Following 1 hour of gentle shake on ice, the proteins are precipitated by spinning for about 30 min. The pellet are resuspended in phosphate buffered saline (PBS) while ammonium sulfated is added to the supernatant to a final concentration of 40% and precipitating protein recovered.

Example 3

Production of antibodies against HEV in birds vaccinated with hexon protein or penton base protein, produced in yeast Two methods can be used to define the quantity and sort of antibodies produced following injection of hexon or penton base recombinant proteins.

a) Enzyme-Linked Immunosorbent Assay (ELISA)

Standardization of the ELISA test: positive antiserum are obtained from infected birds, breeder flocks and rabbit infected with whole virus. Negative control sera are prepared preferably from birds tested previously and determined as non- responders. The positive antigens are virus isolated from spleens of birds infected with HEV and the recombinant hexon or penton base protein. Negative controls are extracts of wild type yeast cells. The antigens diluted in coating buffer (e.g. 0.05M buffer carbonate-bicarbonate, pH=9.6) are incubated in polystyrene microtiter plates for about 1 hr at 37°C or overnight at 4°C, thus providing coated plates. After rinsing the plate three times with, preferably, PBS containing 0.05% Tween 20, the antisera obtained as described above, is double diluted in PBS with

0.1% BSA and added in at least duplicates and in subsequential dilutions to the antigen coated wells. After several hours of incubation at 37°C, the plates are washed in the same rinsing solution and rabbit anti-chicken Ig conjugated to alkaline phosphatase is added. The plates are then further incubated for several hours, at 37°C. After rinsing the plates, a substrate, nitrophenyl phosphate, is added to the wells which produced a yellow color therein, the intensity of which is in proportion to the amount of turkey anti-HEV antibodies. Light absorbencies is read in an ELISA reader.

b) Polyacrylamide gel electrophoresis (PAGE) and Western blotting For polyacrylamide gel electrophoresis (PAGE) and Western blotting, protein preparations are boiled for 3 min., in a sample buffer containing 3% sodium dodecyl sulfate (SDS) and 5% mercaptoethanol. In case the protein preparation contained the hexon, 6 M urea are also added. Polypeptides are analyzed in 12% w/v polyacrylamide slab gels, using the discontinuous SDS gel system [Laemmli, U.K., Nature 227:680-685 (1970)]. In most cases, two slab gels are electro- phoresed simultaneously. One is stained with Coomassie Brilliant Blue R, and the second is electrotransferred on to nitrocellulose filter using a semi-dry system.

The filters are cut into 5mm strips and incubated separately in about 1 :200 dilution of the relevant sera. After several washes in PBS, the filters are incubated with, preferably, 1 : 1000 dilution of rabbit anti-turkey IgG peroxidase conjugated

(Sigma), followed by incubation with 3,3Xdiamino benzidine(Sigma). Example 4

Protection achieved by recombinant structural HEV proteins vaccination against virulent HEV

The efficiency of antibodies produced against the recombinant hexon or penton base proteins, to protect birds against HEV, is determined. Twenty one days old birds are bled and divided into four groups. Each group is injected intramuscularly at 21 days of age with a different antigen that is emulsified in an adjuvant (for example in

Freund's complete adjuvant or Freund incomplete adjuvant). The total volume for injection is preferably 0.5 ml per bird. The first group is inoculated with preferably 50 μg of recombinant hexon or penton base or fiber protein, the second group acts as a positive control and thus is injected with a preferred commercial vaccine of killed HEV virus such as DAMIN (BLT., ABIC, ISRAEL]. Wild type Pichia pastoris yeast cells and PBS are injected as negative controls in the third and fourth group, respectively. About two weeks after the second injection, the birds are bled and then infected with the virulent strain. Preferably three days post inoculation the birds are weighed and bled for testing pathologic changes and virus presence.

Example 5

Persistence of antibodies in a maternal flock

A group of about 30 birds are vaccinated against HEV at preferably one month of age with an inactivated vaccine. At about five months of age the birds are divided into five groups of about five birds each and injected with either recombinant hexon or penton base or fiber proteins or a combination of the same (group 1 to 6 respectively), commercial inactivated HEV vaccine (group 3), PBS (group 4) or unvaccinated (group 5). At different time points, the antibody level are tested by an ELISA system. Birds that are injected with the recombinant hexon or penton base protein showed higher level of anti-HEV antibodies which persisted for a longer period of time than birds vaccinated with the commercial vaccine. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Abie Ltd.

(B) STREET: P. O. Box 8077, Kiryat Nordau, Industrial zone

(C) CITY: Natanya

(E) COUNTRY: ISRAEL

(F) POSTAL CODE (ZIP) : 42504

(G) TELEPHONE: 972-9-8639777 (H) TELEFAX: 972-9-8354129

(ii) TITLE OF INVENTION: Nucleic Acids Encoding HEV Structural

Proteins, HEV Structural Proteins Encoded Thereby and Uses Thereof

(iii) NUMBER OF SEQUENCES: 6

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: IL 122626

(B) FILING DATE: 16-DEC-1997

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2720 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATGGACATAT CAAATGCTAC GCCAAAACTT GATATATTCC ACATAGCTGG ACCAGATGCT 60

TCAGAATATC TTTCAGAAAA TCTCGTTAAT TTCATCTCCA GTACAGAATC GTATTTTCCA 120

ATTAATAAAA AATTTAGAGA AACAATTGTA GCACCAACAA AAGGTGTGAC GACAGAACAA 180

TCTCAGAAAT TGCAAGTTAA AATTGTTCCA ACTTTGACAC AAGATTTAGA AAATAGTTTT 240

ACTGCTAGAT TTACTATTGC TGTTGGCGAT GGTCGGGTTT TGGATATGGG AAGTACGTAT 300

TTTGATATTA GGGGTAACAT TGATCGGGGA CCTTCATTTA AGCCATATGG TGGTACAGCA 360

TATAATCCTC TAGCTCCAAG GTCAGCTCAA TTTAATAATA TTAAAACTGT GGGTGGTAAA 420 ACATATTTGA CTGCTCAAGC TACTAAATTT TTTTCAACAT CTGGAAATGG TTGTGCAGCT 480

GCTAATACTG AAGCAAGTTC ATTTACAAAT TTAGTTCCTT CACCTAATAC TGGTTCAGCA 540

GAAAGTTCTT TTGATCCTAC AACAGAGGGA GCTAGTTGCA GAGCTATAAC ACTTGGCAGT 600

TCTGTAACAG ATGCAACTTG TTATGGAGCT TATACACCTA TTCAAAATGC TAATGGTTCA 660

ATTTTACCTC CATCTGTTAC GCCTGATAAA AAATTTGCCG ATGCTGGTAA ATCTGGCAGT 720

GTTACATGTA CTGCTGCTAT TTGTTGTGAT AATGTTACTG TACAATATCC AGATACTAGA 780

ATAGTTGCTT ATGACTCTAC TGATAAAATA GCAACTAGAA TGGGTAACAG AATTAATTAT 840

ATTGGATTTA GAGATAATTT TATAGGTTTG ATGTATTATG ATAATGGTGC ACATAGTGGT 900

TCTTTGGCTA CAGAAACAGG AGATATAAAT TTGGTAGAAC AATTGCAAGA TAGAAATACA 960

GAAATTAGTT ATCAATATAT GTTAGCGGAT TTGATGAGTA GGAATCATTA TTATAGTCAG 1020

TGGAATCAAG CTGTAGATGA TTATGATTTA AATGTTAGAG TACTTACAAA TATTGGTTAT 1080

GAAGAGGGTC CTCCAGGTTA CTGTTATCCA AGCACAGGCA TGGGCAACTA TCCTAATACT 1140

GTCATGTCGG TTGGGACATT AGTGGATAAT AATGGTACAA CTGCTACAAC AACGTCAAAT 1200

ACTGTAGCTG TGATGGGTTT TGGCAGTGTT CCTACTATGG AAATTAACGT TCAAGCTTAT 1260

TTGCAAAAAT GTTGGATGTA TGCTAACATT GCAGAATATT TACCTGATAA GTATAAAAAA 1320

GCTATTCAAG GTACTAGTGA AACTGATCCA ACAACTTATA GTTATATGAA TAGTAGGCTT 1380

CCTAATGTGA ATATGGCTGA TCTCTTTACA CATATTGGCG GGCGTTATAG TTTGGATGTA 1440

ATGGATAATG TTAATCCTTT TAATCATCAT AGAAATAGAG GTTTGCAATA TAGAAGTCAA 1500

ATTTTGGGTA ATGGTAGAAA TGTCCGTTTT CATATTCAGG TACCTCAGAA ATTTTTTGCT 1560

ATTAAGAATC TATTGTTACT TCCTGGAACT TATAGTTATG AATGGTGGTT CAGGAAAGAT 1620

CCAAACTTAG TACTACAGTC TACGTTGGGA AATGATTTAA GAAAAGATGG AGCAAGCATT 1680

CAGTTTAGCA GTATTAGTCT TTATGCGAGT TTTTTTCCTA TGGATCACGC TACTTGTAGT 1740

GAGCTTATTT TAATGCTTAG AAACGATCAA AATGATCAAA CTTTTATGGA TTATATGGGT 1800

GCAAAGAATA ATTTGTATTT AGTTCCTGCT AATCAAACTA ATGTTCAGAT TGAAATACCT 1860

TCTAGAGCTT GGACAGCATT TAGAGGCTGG AGTTTTAACC GAATTAAAAC TGCTGAGACA 1920

CCAGCTGTGT GGTCTACTTA TGATCTTAAT TTTAAATATT CTGGCTCAAT ACCTTATCTA 1980

GATGGTACAT TTTATCTTTC TCACACTTTT AACTCTATGT CTATTTTGTT TGATTCAGCA 2040

ATAACATGGC CAGGTAATGA TAGAATGTTA GTTCCGAATT TTTTTGAAAT AAAAAGAGAG 2100

ATAGATACGG AGGGATACAC TACTAGTCAG TCTAATATGA CTAAAGATTG GTATTTGATT 2160

CAAATGTCTG CAAATTATAA CCAGGGGTAT CACGGTTATA GTTTTCCAGC AGATAAAGTA 2220

TACAGACAGT ATGATTTTAT GTCAAATTTT GATTCTATGT CTGTTCAAGT ACCCCGGTCA 2280 GGTCTGGCAT TTTTGTTTAA TGAAAATTAT AACTTGATAG TAAATAATTC AGGATTTTTG 2340

CCCAGTAGGA CGGCTCCAAT TGCTGGAGTT AATGAAGGCC ATCCTTATCC AGCAAACTGG 2400

CCAGCGCCAT TAATAGGTAA TAGTCCTGAT AGTGTTGTTA CAGTTAGGAA ATTTTTATGT 2460

GATAAGTATT TATGGACAAT ACCTTTTTCA AGCAATTTTA TGAATATGGG TGAATTGACT 2520

GACCTTGGAC AGAGTTTGCT GTATACTGAG TCTGCACATA GTTTGCAAAT AACATTTAAT 2580

GTTGATCCAA TGCCTGAGCC TACGTACATT TACTTACTTT ATAGTGTTTT TGATTGTGTT 2640

AGGGTCAATC AACCTAACAA AAATTACTTA TCTGCAGCTT ATTTCAGAAC TCCTTTTGCT 2700

ACTGGAACTG CTTCAGTATA 2720 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1347 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

ATGGAATCTT CGAACACTGC CACTAGAATT TTTGCTCCAA CGGAAGGGAG AAACAGTATA 60

ATTTACAGCA ACTTGCCTCC TGTTCAAGAT ACAACCAAAA TATTTTATAT AGATAACAAG 120

GCCATTGATA TAGAGTCATA TAATCAAGAG AAAGATCATT CTAATTATTA TACTAATATA 180

ATTCAAACAC AGAACATTTC AACTATTGAT TCAAGTATAC AGCAAATTCA GTTAGATGAA 240

AGGTCTAGAT GGGGAGGAGA ACTACATACA AGCTTAGTAA CATCTGTTAT GAATTGTACT 300

AAACATTTTA ATTCAGATAG ATGTTTAGTG AAAATTCAGA CTATTAAGAG TCCACCTACA 360

TTTGAATGGA AAGAATTGAA AATACCTGAG GGAAACTATG TTTTAAATGA GTTTATTGAT 420

TTATTAAATG AAGGTATTAC TTCTTTATAC CTTCAGTATG GCAGGCAACA GGGTGTACTT 480

GAAGAAGACA TAGGAATAAA ATTTGATACT CGCAATTTTG AAATTGGTAA AGATCCAACT 540

ACTAATCTTG TTACTCCTGG TAAATACTTG TTTAAGGGTT ATCATGCTGA TATAATACTT 600

CTTCCTGGTT GGGCTATTGA TTTTTCTTTT TCTAGATTGG GTAACATTTT AGGTATTAGA 660

AAACGTGAGA CTTATAAAGC TGGCTTTTTG ATTGAATATG ATGACTTGAC AAATGGTAAT 720

ATTCCACCAC TGTTGGATGT TGCTAACTAT AAGTCTACAA GTCAAGCTAA ACCATTATTA 780

CAGGATCCAT CTGGCAGATC TTACCACGTT ATGGATAGTG ATTCTAACAG ACCTGTGACT 840

GCATATAGGT CTTTTGTTTT GTCATATAAC AATGAAGGTG CTGCAAAATT AAAGTTTTTG 900 ATGTGTATGA GTGATATAAC GGGGGGTCTC AATCAGCTGT ATTGGTGTTT GCCTGATTCT 960

TATAAACCGC CAGTATCTTT TAAGCAAGAA ACGCAAGTAG ATAAACTGCC TGTTGTTGGT 1020

ATGCAACTTT TTCCTTTTGT CTCTAAATCT GTGTATTCTG GTGCTGCTGT TTACACACAG 1080

TTAATTGAAC AGCAGACTAA TTTGACACAA ATTTTTAACA GATTTCATGA TAATGAAATT 1140

TTAAAACAAG CTCCATATGT GAATCAAGTT TTATTGGCTG AAAATGTGCC CATAAATGTT 1200

AATCAGGGAA CAATACCAAT ATTTTCAACT CTTCCAGGAG TACAGAGAGT GGTTGTGGAA 1260

GACGATAGGA GAAGAACTGT ACCCTACGTT ACCAAGTCAC TTGCTACAGT ATATCCGAAG 1320

GTTTTGTCTA GCAAAACTTT GCAATAA 1347 (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1179 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

ATGAGAATTG GCAAAGGATT AAAGTTTGAA AATGGTAATC TAGTTGTATC AGATCAACAG 60

TATAATGTTA CACCACCTTT AATTGCAGAT CAGTCAACAT TAGGTTTAAA GTATAATCCG 120

GATGTTCTTT CTTTAACACA TTCAGGTGCT TTAACTTTGC CAACTATTCA ACATCCCCTC 180

CAGGCTTCAG CTGGAAAATT TGAACTTGCT TTGTCATCAG GTTTAAAATC TGATGATCAA 240

GGTTTAACTT TAGATTTGGA TCCTGTATTT TCTACAGAAT CATCAAAATT TTTGCTTAAT 300

TGTTCATTGC CGTTAGATAA GAATAGTGAC AAGTTAACGT TAAAATTTGG TAATGGTCTT 360

GGATTGAATA ATGACCAGCT AGAGAATACT ATGACTTATA ATCTTCCTTT AAAACGTGAT 420

GGAACTAATG TTAGTCTTTC ATTTGGAACT AATTTCAAAA TATTGAATGA GATGTTAGAT 480

TTAAATCTTG TGGCACCTAT GTCTAATTCA GCAGGAGGAT TAGCATTGCA ATTTAAAAGC 540

CCTTTGTCAG CAGATGATGG TATTTTATCA ATTAAAACAG ATACATCTTT GGGTATAACA 600

GGAAATAAAT TAGGAATAAG ATTGGCCCCT AACAGTGGTC TGCAAATAAC ACCAAATGGT 660

CTAGCAGTTA GTGTTAATGC TGTGCAAATT CTAAGTAGTC CTTTAATTAC TGCAGCGTCT 720

ATAGGCCCAC CAACAACAAT GGTTACTGGA ACAGTGTCAC CGGGCAGAGC AACAAATGGT 780

CAATTTGTAA CCAAAACTGC TAAAGTTTTA CGTTATAAAT TTGTGAGATG GGATGCTCTG 840

TTAATCATAC AGTTTATAGA TAACATAGGT GTAATAGAAA ACCCTACCTT TTATCGTAAC 900 AAAAGTATTG AATTAAGATC TGCTGATTTC TTGAGTCCTA CGTTAAATAA TACATATATA 960

GTGCCATTGA ATGGAGGGGT AAGGGTAGAA TCACCTACTA TTCCTGTACA ATTAGAAGTT 1020

ATACTTGAAA ACAATTCCTC TTTCATTCAA GTAGGGTTTG TTAGGTTAAC AGTTAAGAAT 1080

GGTAACCCTC ATATGATTAT TCAGTGTAAT CCTGTACCTG GGAATATTAA AATGATAAAG 1140

ATAAAATCTG TAATGCTTTT TACTTGTTTG ATAGGCTGA 1179 (2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 905 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Asp lie Ser Asn Ala Thr Pro Lys Leu Asp lie Phe His lie Ala 1 5 10 15

Gly Pro Asp Ala Ser Glu Tyr Leu Ser Glu Asn Leu Val Asn Phe lie 20 25 30

Ser Ser Thr Glu Ser Tyr Phe Pro lie Asn Lys Lys Phe Arg Glu Thr 35 40 45 lie Val Ala Pro Thr Lys Gly Val Thr Thr Glu Gin Ser Gin Lys Leu 50 55 60

Gin Val Lys lie Val Pro Thr Leu Thr Gin Asp Leu Glu Asn Ser Phe 65 70 75 80

Thr Ala Arg Phe Thr lie Ala Val Gly Asp Gly Arg Val Leu Asp Met 85 90 95

Gly Ser Thr Tyr Phe Asp lie Arg Gly Asn lie Asp Arg Gly Pro Ser 100 105 110

Phe Lys Pro Tyr Gly Gly Thr Ala Tyr Asn Pro Leu Ala Pro Arg Ser 115 120 125

Ala Gin Phe Asn Asn lie Lys Thr Val Gly Gly Lys Thr Tyr Leu Thr 130 135 140

Ala Gin Ala Thr Lys Phe Phe Ser Thr Ser Gly Asn Gly Cys Ala Ala 145 150 155 160

Ala Asn Thr Glu Ala Ser Ser Phe Thr Asn Leu Val Pro Ser Pro Asn 165 170 175

Thr Gly Ser Ala Glu Ser Ser Phe Asp Pro Thr Thr Glu Gly Ala Ser 180 185 190 Cys Arg Ala lie Thr Leu Gly Ser Ser Val Thr Asp Ala Thr Cys Tyr 195 200 205

Gly Ala Tyr Thr Pro lie Gin Asn Ala Asn Gly Ser lie Leu Pro Pro 210 215 220

Ser Val Thr Pro Asp Lys Lys Phe Ala Asp Ala Gly Lys Ser Gly Ser 225 230 235 240

Val Thr Cys Thr Ala Ala lie Cys Cys Asp Asn Val Thr Val Gin Tyr 245 250 255

Pro Asp Thr Arg lie Val Ala Tyr Asp Ser Thr Asp Lys lie Ala Thr 260 265 270

Arg Met Gly Asn Arg lie Asn Tyr lie Gly Phe Arg Asp Asn Phe lie 275 280 285

Gly Leu Met Tyr Tyr Asp Asn Gly Ala His Ser Gly Ser Leu Ala Thr 290 295 300

Glu Thr Gly Asp He Asn Leu Val Glu Gin Leu Gin Asp Arg Asn Thr 305 310 315 320

Glu He Ser Tyr Gin Tyr Met Leu Ala Asp Leu Met Ser Arg Asn His 325 330 335

Tyr Tyr Ser Gin Trp Asn Gin Ala Val Asp Asp Tyr Asp Leu Asn Val 340 345 350

Arg Val Leu Thr Asn He Gly Tyr Glu Glu Gly Pro Pro Gly Tyr Cys 355 360 365

Tyr Pro Ser Thr Gly Met Gly Asn Tyr Pro Asn Thr Val Met Ser Val 370 375 380

Gly Thr Leu Val Asp Asn Asn Gly Thr Thr Ala Thr Thr Thr Ser Asn 385 390 395 400

Thr Val Ala Val Met Gly Phe Gly Ser Val Pro Thr Met Glu He Asn 405 410 415

Val Gin Ala Tyr Leu Gin Lys Cys Trp Met Tyr Ala Asn He Ala Glu 420 425 430

Tyr Leu Pro Asp Lys Tyr Lys Lys Ala He Gin Gly Thr Ser Glu Thr 435 440 445

Asp Pro Thr Thr Tyr Ser Tyr Met Asn Ser Arg Leu Pro Asn Val Asn 450 455 460

Met Ala Asp Leu Phe Thr His He Gly Gly Arg Tyr Ser Leu Asp Val 465 ^ 470 475 ^*** 480

Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Arg Gly Leu Gin 485 490 495

Tyr Arg Ser Gin He Leu Gly Asn Gly Arg Asn Val Arg Phe His He 500 505 510 Gln Val Pro Gin Lys Phe Phe Ala He Lys Asn Leu Leu Leu Leu Pro 515 520 525

Gly Thr Tyr Ser Tyr Glu Trp Trp Phe Arg Lys Asp Pro Asn Leu Val 530 535 540

Leu Gin Ser Thr Leu Gly Asn Asp Leu Arg Lys Asp Gly Ala Ser He 545 550 555 560

Gin Phe Ser Ser He Ser Leu Tyr Ala Ser Phe Phe Pro Met Asp His 565 570 575

Ala Thr Cys Ser Glu Leu He Leu Met Leu Arg Asn Asp Gin Asn Asp 580 585 590

Gin Thr Phe Met Asp Tyr Met Gly Ala Lys Asn Asn Leu Tyr Leu Val 595 600 605

Pro Ala Asn Gin Thr Asn Val Gin He Glu He Pro Ser Arg Ala Trp 610 615 620

Thr Ala Phe Arg Gly Trp Ser Phe Asn Arg He Lys Thr Ala Glu Thr 625 630 635 640

Pro Ala Val Trp Ser Thr Tyr Asp Leu Asn Phe Lys Tyr Ser Gly Ser 645 650 655

He Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Ser His Thr Phe Asn Ser 660 665 670

Met Ser He Leu Phe Asp Ser Ala He Thr Trp Pro Gly Asn Asp Arg 675 680 685

Met Leu Val Pro Asn Phe Phe Glu He Lys Arg Glu He Asp Thr Glu 690 695 700

Gly Tyr Thr Thr Ser Gin Ser Asn Met Thr Lys Asp Trp Tyr Leu He 705 710 715 720

Gin Met Ser Ala Asn Tyr Asn Gin Gly Tyr His Gly Tyr Ser Phe Pro 725 730 735

Ala Asp Lys Val Arg Gin Tyr Asp Phe Met Ser Asn Phe Asp Ser Met 740 745 750

Ser Val Gin Val Pro Arg Ser Gly Leu Ala Phe Leu Phe Asn Glu Asn 755 760 765

Tyr Asn Leu He Val Asn Asn Ser Gly Phe Leu Pro Ser Arg Thr Ala 770 775 780

Pro He Ala Gly Val Asn Glu Gly His Pro Tyr Pro Ala Asn Trp Pro 785 790 795 800

Ala Pro Leu He Gly Asn Ser Pro Asp Ser Val Val Thr Val Arg Lys 805 810 815

Phe Leu Cys Asp Lys Tyr Leu Trp Thr He Pro Phe Ser Ser Asn Phe 820 825 830

Met Asn Met Gly Glu Leu Thr Asp Leu Gly Gin Ser Leu Leu Tyr Thr 835 840 845

Glu Ser Ala His Ser Leu Gin He Thr Phe Asn Val Asp Pro Met Pro 850 855 860

Glu Pro Thr Tyr He Tyr Leu Leu Tyr Ser Val Phe Asp Cys Val Arg 865 870 875 880

Val Asn Gin Pro Asn Lys Asn Tyr Leu Ser Ala Ala Tyr Phe Arg Thr 885 890 895

Pro Phe Ala Thr Gly Thr Ala Ser Val 900 905

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 448 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Glu Ser Ser Asn Thr Ala Thr Arg He Phe Ala Pro Thr Glu Gly 1 5 10 15

Arg Asn Ser He He Tyr Ser Asn Leu Pro Pro Val Gin Asp Thr Thr 20 25 30

Lys He Phe Tyr He Asp Asn Lys Ala He Asp He Glu Ser Tyr Asn 35 40 45

Gin Glu Lys Asp His Ser Asn Tyr Tyr Thr Asn He He Gin Thr Gin 50 55 60

Asn He Ser Thr He Asp Ser Ser He Gin Gin He Gin Leu Asp Glu 65 70 75 80

Arg Ser Arg Trp Gly Gly Glu Leu His Thr Ser Leu Val Thr Ser Val 85 90 95

Met Asn Cys Thr Lys His Phe Asn Ser Asp Arg Cys Leu Val Lys He 100 105 110

Gin Thr He Lys Ser Pro Pro Thr Phe Glu Trp Lys Glu Leu Lys He 115 120 125

Pro Glu Gly Asn Tyr Val Leu Asn Glu Phe He Asp Leu Leu Asn Glu 130 135 140

Gly He Thr Ser Leu Tyr Leu Gin Tyr Gly Arg Gin Gin Gly Val Leu 145 150 155 160

Glu Glu Asp He Gly He Lys Phe Asp Thr Arg Asn Phe Glu He Gly 165 170 175 Lys Asp Pro Thr Thr Asn Leu Val Thr Pro Gly Lys Tyr Leu Phe Lys 180 185 190

Gly Tyr His Ala Asp He He Leu Leu Pro Gly Trp Ala He Asp Phe 195 200 205

Ser Phe Ser Arg Leu Gly Asn He Leu Gly He Arg Lys Arg Glu Thr 210 215 220

Tyr Lys Ala Gly Phe Leu He Glu Tyr Asp Asp Leu Thr Asn Gly Asn 225 230 235 240

He Pro Pro Leu Leu Asp Val Ala Asn Tyr Lys Ser Thr Ser Gin Ala 245 250 255

Lys Pro Leu Leu Gin Asp Pro Ser Gly Arg Ser Tyr His Val Met Asp 260 265 270

Ser Asp Ser Asn Arg Pro Val Thr Ala Tyr Arg Ser Phe Val Leu Ser 275 280 285

Tyr Asn Asn Glu Gly Ala Ala Lys Leu Lys Phe Leu Met Cys Met Ser 290 295 300

Asp He Thr Gly Gly Leu Asn Gin Leu Tyr Trp Cys Leu Pro Asp Ser 305 310 315 320

Tyr Lys Pro Pro Val Ser Phe Lys Gin Glu Thr Gin Val Asp Lys Leu 325 330 335

Pro Val Val Gly Met Gin Leu Phe Pro Phe Val Ser Lys Ser Val Tyr 340 345 350

Ser Gly Ala Ala Val Tyr Thr Gin Leu He Glu Gin Gin Thr Asn Leu 355 360 365

Thr Gin He Phe Asn Arg Phe His Asp Asn Glu He Leu Lys Gin Ala 370 375 380

Pro Tyr Val Asn Gin Val Leu Leu Ala Glu Asn Val Pro He Asn Val 385 390 395 400

Asn Gin Gly Thr He Pro He Phe Ser Thr Leu Pro Gly Val Gin Arg 405 410 415

Val Val Val Glu Asp Asp Arg Arg Arg Thr Val Pro Tyr Val Thr Lys 420 425 430

Ser Leu Ala Thr Val Tyr Pro Lys Val Leu Ser Ser Lys Thr Leu Gin 435 440 445

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 392 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met Arg He Gly Lys Gly Leu Lys Phe Glu Asn Gly Asn Leu Val Val 1 5 10 15

Ser Asp Gin Gin Tyr Asn Val Thr Pro Pro Leu He Ala Asp Gin Ser 20 25 30

Thr Leu Gly Leu Lys Tyr Asn Pro Asp Val Leu Ser Leu Thr His Ser 35 40 45

Gly Ala Leu Thr Leu Pro Thr He Gin His Pro Leu Gin Ala Ser Ala 50 55 60

Gly Lys Phe Glu Leu Ala Leu Ser Ser Gly Leu Lys Ser Asp Asp Gin 65 70 75 80

Gly Leu Thr Leu Asp Leu Asp Pro Val Phe Ser Thr Glu Ser Ser Lys 85 90 95

Phe Leu Leu Asn Cys Ser Leu Pro Leu Asp Lys Asn Ser Asp Lys Leu 100 105 110

Thr Leu Lys Phe Gly Asn Gly Leu Gly Leu Asn Asn Asp Gin Leu Glu 115 120 125

Asn Thr Met Thr Tyr Asn Leu Pro Leu Lys Arg Asp Gly Thr Asn Val 130 135 140

Ser Leu Ser Phe Gly Thr Asn Phe Lys He Leu Asn Glu Met Leu Asp 145 150 155 160

Leu Asn Leu Val Ala Pro Met Ser Asn Ser Ala Gly Gly Leu Ala Leu 165 170 175

Gin Phe Lys Ser Pro Leu Ser Ala Asp Asp Gly He Leu Ser He Lys 180 185 190

Thr Asp Thr Ser Leu Gly He Thr Gly Asn Lys Leu Gly He Arg Leu 195 200 205

Ala Pro Asn Ser Gly Leu Gin He Thr Pro Asn Gly Leu Ala Val Ser 210 215 220

Val Asn Ala Val Gin He Leu Ser Ser Pro Leu He Thr Ala Ala Ser 225 230 235 240

He Gly Pro Pro Thr Thr Met Val Thr Gly Thr Val Ser Pro Gly Arg 245 250 ^"255

Ala Thr Asn Gly Gin Phe Val Thr Lys Thr Ala Lys Val Leu Arg Tyr 260 265 270

Lys Phe Val Arg Trp Asp Ala Leu Leu He He Gin Phe He Asp Asn 275 280 285 Ile Gly Val He Glu Asn Pro Thr Phe Tyr Arg Asn Lys Ser He Glu 290 295 300

Leu Arg Ser Ala Asp Phe Leu Ser Pro Thr Leu Asn Asn Thr Tyr He 305 310 315 320

Val Pro Leu Asn Gly Gly Val Arg Val Glu Ser Pro Thr He Pro Val 325 330 335

Gin Leu Glu Val He Leu Glu Asn Asn Ser Ser Phe He Gin Val Gly 340 345 350

Phe Val Arg Leu Thr Val Lys Asn Gly Asn Pro His Met He He Gin 355 360 365

Cys Asn Pro Val Pro Gly Asn He Lys Met He Lys He Lys Ser Val 370 375 380

Met Leu Phe Thr Cys Leu He Gly 385 390

Claims

1. A nucleic acid molecule encoding a structural protein of Hemorrhagic enteritis virus (HEV) which protein is capable of eliciting in an animal protective immunity against HEV.

2. A nucleic acid molecule according to claim 1 wherein said animal is a bird.

3. A nucleic acid molecule according to claim 1 being genomic DNA or cDNA molecule.

4. A nucleic acid molecule according to claim 3 comprising the nucleotide sequence substantially as set forth in SEQ ID NO :1 and immunologically functional homologues and fragments thereof.

5. A nucleic acid molecule according to claim 4 encoding the hexon protein of HEV.

6. A nucleic acid molecule according to claim 3 comprising the nucleotide sequence substantially as set forth in SEQ ID NO:2 and immunologically functional homologues and fragments thereof.

7. A nucleic acid molecule according to claim 6 encoding the penton base protein of HEV.

8. A nucleic acid molecule according to claim 3 comprising the nucleotide sequence substantially as set forth in SEQ ID NO: 3 and immunologically functional homologues and fragments thereof.

9. A nucleic acid molecule according to claim 8 encoding the fiber protein of HEV.

10. A protein capable of eliciting in an animal protective immunity against HEV, which protein is encoded by a nucleic acid molecule according to claim 1 and immunologically active homologues and fragments thereof.

11. A protein capable of eliciting in animal protective immunity against HEV, which protein is encoded by the nucleic acid molecule of claim 4 and immunologically active homologues and fragments thereof.

12. A protein capable of eliciting in an animal protective immunity against HEV, which protein is encoded by the nucleic acid molecule of claim 6 and immunologically active homologues and fragments thereof.

13. A protein capable of eliciting in an animal protective immunity against HEV, which protein is encoded by the nucleic acid molecule of claim 8 and immunologically active homologues and fragments thereof.

14. A protein according to claim 10, wherein said animal is a bird.

15. A protein according to claim 11 comprising an amino acid sequence substantially as set forth in SEQ ID NO:4 or immunologically active homologues and essential fragments thereof.

16. A protein according to claim 15 having an amino acid sequence substantially as set forth in SEQ ID NO:4 or immunologically active homologues and essential fragments thereof.

17. A protein according to claim 12 comprising an amino acid sequence substantially as set forth in SEQ ID NO: 5 or immunologically active homologues and essential fragments thereof.

18. A protein according to claim 17 having an amino acid sequence substantially as set forth in SEQ ID NO: 5 or immunologically active homologues and essential fragments thereof.

19. A protein according to claim 13 comprising an amino acid sequence substantially as set forth SEQ ID NO: 6 or immunologically active homologues and essential fragments thereof.

20. A protein according to claim 19 having an amino acid sequence substantially as set forth in SEQ ID NO: 6 or immunologically active homologues and essential fragments thereof.

21. A recombinant polypeptide capable of eliciting in an animal protective immunity against HEV comprising at least one protein according to claim 10.

22. A recombinant polypeptide according to claim 21, wherein said animal is a bird.

23. A recombinant polypeptide according to claim 21, wherein said at least one protein is selected from the group consisting the hexon protein, the penton base protein and the fiber protein of HEV.

24. A recombinant polypeptide according to claim 23 further comprising an additional protein or peptide, or immunologically active homologues or essential fragments of said additional protein or peptide, which recombinant polypeptide is capable of eliciting in an animal protective immunity against a pathogen other than HEV.

25. A recombinant polypeptide according to claim 24 wherein said animal is a bird.

26. A recombinant polypeptide according to claim 24, wherein said pathogen is IBDV, NDV, EDS, IB, MDV, avian influenza virus, fowl pox virus, salmonella, coccidia or bacteria causing cholera such as pasteurella multocida.

27. A recombinant vector comprising at least the nucleic acid molecule of claim 1.

28. A recombinant vector according to claim 27 wherein said nucleic acid molecule is a genomic DNA or a cDNA.

29. A recombinant vector according to claim 28 wherein said nucleic acid sequence is substantially as set forth SEQ ID NO: 1 or functional homologues and fragments thereof.

30. A recombinant vector according to claim 28 wherein said nucleic acid sequence is substantially as set forth SEQ ID NO:2 or functional homologues and fragments thereof.

31. A recombinant vector according to claim 28 wherein said nucleic acid sequence is substantially as set forth SEQ ID NO: 3 or functional homologues and fragments thereof.

32. A DNA construct for the expression of a protein product in a host cell, comprising an expression vector and at least one exogenous nucleic acid molecule encoding a structural protein of Hemorrhagic enteritis virus (HEV), which structural protein is capable of eliciting in an animal protective immunity against HEV.

33. A DNA construct according to claim 32 wherein said animal is a bird.

34. A DNA construct according to claim 32, wherein said exogenous nucleic acid molecule comprises the nucleic acid molecule of claim 1.

35. A DNA construct according to claim 32, wherein said at least one exogenous nucleic acid molecule comprises the nucleotide sequence substantially as set forth SEQ ID NO: l or functional homologues and fragments thereof.

36. A DNA construct according to claim 32, wherein said at least one exogenous nucleic acid molecule comprises the nucleotide sequence substantially as set forth in SEQ ID NO:2 or functional homologues and fragments thereof.

37. A DNA construct according to claim 32, wherein said at least one exogenous nucleic acid molecule comprises the nucleotide sequence substantially as set forth in SEQ ID NO:3 or functional homologues and fragments thereof.

38. A DNA construct according to claim 32, wherein said structural protein is selected from the group consisting of the hexon protein, the penton base protein and the fiber protein of HEV.

39. A DNA construct according to claim 32, wherein said at least one exogenous nucleic acid molecule is the recombinant vector of claim 22.

40. A DNA construct according to claim 32 which optionally further comprises at least one additional exogenous nucleic acid sequence encoding a protein or peptide product, said protein of peptide being capable of eliciting in an animal protective immunity against a specific pathogen other than HEV.

41. A DNA construct according to claim 40 wherein said pathogen is IBDV, NDV, EDS, IB, MDV, avian influenza virus, fowl pox virus, salmonella, coccidia or bacteria causing cholera such as pasteurella multocida.

42. A DNA construct according to claim 32, wherein said expression vector is selected from the group consisting of fowlpox virus, vaccinia virus, Marek disease virus, baculovirus, bacteria, yeast and plant.

43. A DNA construct according to claim 42, wherein said expression vector is yeast.

44. A DNA construct according to claim 43, wherein said expression vector is the yeast plasmid pHIL-Sl having the restriction map as set forth in Fig. 2(A).

45. A DNA construct according to claim 43 wherein said expression vector is the yeast plasmid pPIC3K having the restriction map as set forth in Fig. 2(B).

46. A host cell transformed with the nucleic acid molecule of claim 1 or with the recombinant vector of claim 27.

47. A host cell according to claim 46 which is a bacteria cell, a yeast cell, an insect cell or a plant cell.

48. A host cell according to claim 47, which is a Pichia pastoris yeast cell.

49. A vaccine for immunizing an animal against HEV comprising as active ingredient an effective immunizing amount of at least one protein or polypeptide according to claim 10.

50. A vaccine according to claim 49 optionally further comprising at least one additional protein or peptide which protein or peptide are capable of eliciting in said animal protective immunity against a specific pathogen other than HEV.

51. A vaccine for immunizing animals against HEV comprising as active ingredient an effective immunizing amount of the DNA construct of claim 32.

52. A vaccine for immunizing animals against HEV comprising as active ingredient at least one nucleic acid molecule according to claim 1, or at least one protein according to claim 10, or at least one DNA construct according to claim 32, or a mixture of the same.

53. A vaccine according to claim 49 for immunizing birds.

54. A method of immunizing an animal against HEV by administering to said animal an effective immunizing amount of the vaccine according to claim 49.

55. A method according to claim 54 of immunizing birds.

56. A method according to claim 55 which comprises a single administration of the vaccine of claim 49 at the age of three weeks.

57. A method of according to claim 54 which comprises repeated administrations of the vaccine according to claim 49.

58. A method according to claim 54, wherein said vaccine is administered to the birds by injection.

59. A method according to claim 54, wherein said vaccine is administered to the birds via drinking water.

60. A method according to claim 54, wherein said vaccine is administered to the birds via_the ocular route, as eye drops.

61. A method according to claim 54, wherein said vaccine is administered to the birds by spraying or aerosol.

62. Use of the nucleic acid molecule of claim 1 in the preparation of a vaccine for immunizing birds against HEV.

63. Use of the protein of claim 10 in the preparation of a vaccine for immunizing a bird against HEV.

64. Use of the recombinant vector of claim 27 in the preparation of a vaccine for immunizing a bird against HEV.

65. Use of the DNA construct of any claim 32 in the preparation of a vaccine for immunizing a bird against HEV.

66. An antibody directed against the protein of claim 10.

67. Use of the antibodies of claim 66 in the detection of the presence of anti-HEV antibodies in a serum obtained from a sick animal.