REOVIRUS VACCINE BASED ON SIGMA C PROTEIN SEQUENCE

TECHNICAL FIELD The present invention relates to novel sigma C proteins from avian reovirus isolates from Israel, peptides derived therefrom, and to broad activity anti-reovirus vaccines.

BACKGROUND ART Avian reovirus (ARV) is a disease agent that causes economic losses in the poultry industry. ARV belongs to the Orthoreovirus genus in the family Reoviridae and was first isolated from birds in 1954 (Fahey and Crawley, 1954). Since then, many variants with broad antigenic diversity have been isolated in many countries. Avian reovirus is the main cause of viral arthritis and tendosynovitis (Robertson and Wilcox, 1986). Damage is also caused by infection of the liver, heart and intestine, and by immunosupression. Birds are most susceptible at young ages (Rosenberger et al, 1989). The available vaccines are the attenuated vaccine strain si 133, developed in 1980, and inactivated vaccines developed in 1985 and 1994. Protection of offspring is conferred by antibodies transferred from maternal flocks that have been vaccinated with the si 133 vaccine (TriReo: Fort Dodge) (Rekik and Silim, 1992; Shapouri et al, 1995; Varela, R., and J. Benavente). In Israel, a vaccine was developed in 1994 using a local isolate (strain 641). This vaccine consists of an inactivated virus injected to maternal flocks. Despite vaccination, birds are not fully protected. The avian reovirus genome consists of 10 fragments of double-stranded

RNA of 1 to 4 kb that are divided into large (L), medium (M) and small (S) in accordance to their size. The virus is non-enveloped with a capsid of 70 to 80 nm in diameter (Jokik, 1983; Spandidis and Graham, 1976). The RNA sequence of some Avian reovirus strains has been determined, and the proteins encoded by avian

reovirus RNA have been identified (Cashdollar, et al, 1984; McCrae and Joklik, 1978; Schnitzer, 1985; Wickramasinghe et al, 1993). Sigma C protein was found to be encoded in the third open reading frame (ORF) of the Sl fragment (Schnitzer, 1985; Wickramasinghe et al, 1993). This protein is located on the surface of the capsid (Wickramasinghe et al, 1993) and functions in the identification and binding of the virus to the target cell.

Sigma C is a homotrimer with a tertiary structure consisting of two domains: the "head" domain which is located at the C terminal end of the protein and the "shaft" domain at the N terminal part. Sigma C is a relatively small protein of 326 amino acids (Schnitzer et al, 1982) and its relative amount on the viral capsid is small, as compared to other viral proteins. The importance of sigma C manifests itself at two levels; first, it is the most variable protein of the reovirus (Liu and Giambrone, 1997) and second, it induces the production of neutralizing antibodies.

No difference in infectivity and disease rates were found between offspring derived from flocks with high or low antibody titers raised by vaccination. This may be due to inefficiency of the vaccine due to differences in sigma C or other proteins relevant for induction of protective antibodies (Vasserman et al, 2004).

SUMMARY OF INVENTION The present invention relates, in one aspect, to a peptide selected from a peptide derived from a conserved region of a sigma C protein of an avian reovirus isolate, and an analog of said peptide.

In another aspect, the present invention provides a fusion polypeptide comprising the sequences of at least two peptides derived from reovirus sigma C proteins, wherein each of the at least two peptides has from 5 to 23 amino acid residues, each peptide is bound to the other peptide via a linker comprising 1-3 amino acid residues, the fusion polypeptide comprises at least two copies of each peptide and has a total of 4-16, preferably 6, 8, 10, 12, more preferably, 14, peptide sequences.

The present invention also provides an immunogenic composition comprising at least one fusion polypeptide of the invention, with or without an adjuvant.

In another aspect, the present invention relates to a sigma C protein (SCP) of an avian reovirus isolate (RI) from Israel, wherein said RI belongs to the Group I, II or III of reovirus as herein defined, and said RIs are selected from ISR521, ISR526. ISR5215, ISR5220, ISR5225, ISR5226 (Group I), ISR522, ISR5217, ISR5221, ISR5222, ISR5223 (Group II); and ISR524, ISR527, ISR528, ISR529, ISR5211, ISR5213 (Group III), or a fraction or fragment of said reovirus SCP. The present invention further provides vaccines comprising one or more of said peptides, fusion polypeptides or SCP and a vaccine comprising at least four inactivated reovirus isolates.

BRIEF DESCRIPTION OF DRAWINGS Fig. 1 depicts a phylogenetic tree of avian reovirus strains according to the variability in sigma C amino acid sequences. The strains in the squares are isolates from Israel. The Roman numbers I-IV designate the four reovirus isolate (RJ) groups as defined herein.

Fig. 2 depicts an antigenic chromatogram for prediction of antigenic sites on the sigma C protein. Peak- antigenic region; Star- Conserved regions and sites conserved among isolates.

Fig. 3 shows a bar graph representing an ELISA in which the wells were coated with whole reovirus 1 133.

Figs. 4A-4B depict a Western blot showing production in E. coli cells of the fusion polypeptides herein designated R-PEP Al and R-PEP A2. Fig. 4A: SDS- PAGE of extract fractions of E. coli expressing R-PEP Al and R-PEP A2. Circles indicate the specific recombinant polypeptides. Fig 4B: immunoblot of the gel shown in 3 A using anti-histidine antibodies. Lane 1, R-PEP Al in pellet obtained after spin at 4000rpm; Lane 2, R-PEP Al in pellet obtained after spin at 12000rpm; Lane 3, R-PEP Al in supernatant obtained after spin at 12000rpm; Lane 4, size

marker (Ladder Plus, Fermentas); Lane 5, R-PEP A2 in pellet obtained after spin at 4000rpm; Lane 6, R-PEP A2 in pellet obtained after spin at 12000rpm; Lane 7, R- PEP A2 in supernatant obtained after spin at 12000rpm; Lane 8, empty; Lane 9, Negative control, an irrelevant protein expressed in E.coli; Lane 10, empty. Figs. 5A-5B depict immunoblots of R-PEP Al and R-PEP A2, respectively, as antigens, exposed to chicken sera from chicken immunized with R-PEP Al (Lane 6); R-PEP A2 (Lane 5); R-PEP A1+A2 (Lane 4); reovirus (Lane 3); sigma C peptide (Lane 2). Lane 8, size markers; Lane 7, detection with anti-histidine antibodies; Lane 1 , Negative control, an irrelevant protein expressed in E.coli. Arrow points at specifically labeled fusion polypeptide.

Fig. 6 shows a dot blot of reovirus (strain si 133) as antigen exposed to chicken sera from chicken immunized with R-PEP Al (Row 1); R-PEP A2 (Row 2); R-PEP A 1+A2 (Row 3); sigma C peptide (Row 4); reovirus (Row 5); Negative control (Row 6).

MODES FOR CARRYING OUT THE INVENTION

It has been found, in accordance with the present invention, that the highly variable and immunogenic avian reovirus sigma C protein amino acid sequence contains conserved and immunogenic domains that are common among all avian reovirus isolates identified in Israel and elsewhere.

The term "conserved domain" refers herein to a domain conserved among the virus variants; namely, the amino acid sequences of the domains are similar but not necessarily absolutely identical to each other. It has also been found herein that at least some of the conserved domains comprise amino acid sequences with characteristics of predicted antigenic sites.

It has further been found, in accordance with the present invention, that the genetically variable avian reovirus strains, or isolates, infecting birds in Israel and elsewhere can be divided into four groups according to the variability in the amino acid sequence of the sigma C protein expressed by the isolates. One member of one of the groups represents only a fraction of the avian reovirus isolates in nature;

however, one member of each of the four groups taken together represent many, if not most, variants existing in nature. The four groups of reovirus isolates (RI) are referred to herein as RI Groups I to IV.

The four groups of avian reovirus sigma C proteins (SCP) were defined by comparing the sequences of the proteins expressed by the different isolates using the software found at http://www.dnastar.com and http://www.ebi.ac.uk/clustalw/. Other algorithms and different software known in the art of bioinformatics, such as those found at http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.ht ml could be used to perform the comparison. The defining feature of each SCP group is that the amino acid sequences of the different sigma c proteins of said SCP group has at least 75% identity. The four SCP groups disclosed in Table 2 herein below are based on the sigma C proteins of 19 isolates from Israel and elsewhere as defined using the software mentioned above. The composition of the groups may vary slightly depending on the algorithm and software used to analyze the sequences. The important concept is that each group represents a fraction of the viral population and that single representatives taken from each one of the groups together represent the whole viral population.

The composition of each of the four groups is not limited to the sequences disclosed in the table, but comprises all amino acid sequences that are at least 75% identical to the sequences of each of the groups in Table 2. Thus, for example, SCP of Group IV is not limited to the two sequences SEQ ID NO: 1 and SEQ ID NO: 2, but comprises also additional sequences (that have not been identified) that are at least 75% identical to the sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

In one aspect, the invention provides a peptide selected from a peptide derived from a conserved region of a sigma C protein (SCP) of an avian reovirus isolate (RI), and an analog of said peptide. As used herein, the term "peptides of the invention" include both the SCP peptides and their analogs. The peptide has from 5 to 30, preferably, 6, 7, 8, 9, 10, 12, 14, 16, 19 amino acid residues.

The peptide of the invention is preferably derived from a SCP of an RI from Israel that belongs to one of the four Groups I to IV according to the phylogenetic

tree depicted in Fig. 1 herein. The RI from Israel is preferably selected from ISR521, ISR526. ISR5215, ISR5220, ISR5225, ISR5226 (Group I), ISR522, ISR5217, ISR5221, ISR5222, ISR5223 (Group II); ISR524, ISR527, ISR528, ISR529, ISR521 1 , ISR5213 (Group III); and 59103, S l 133 (Group IV), more preferably ISR5215, ISR5223, ISR528 and 59103, but other isolates are also contemplated by the present invention.

In one preferred embodiment, the peptides of the invention are selected from the peptides of SEQ ID NOs: 3-28 presented in Table 3 herein, more preferably the peptides of SEQ ID NOs: 10, 1 1 , 13-20 and 22. The invention also relates to an analog of a peptide derived from a conserved region of a sigma C protein of an avian reovirus isolate, preferably the peptides of SEQ ID NOs: 29-33 presented in Table 4 herein .

The peptide of the invention may be attached to: (i) a carrier protein such as E. coli enterotoxin LT or bovine serum albumin (BSA); (ii) to microbeads carrying an enhancer molecule such as LT; or (iii) to biotin and then to avidin, wherein the avidin may be comprised within microbeads. These attached peptides are suitable for their use in immunogenic compositions and vaccines and the invention also provides an immunogenic composition or vaccine comprising one or more of said peptides attached to (i), (ii) or (iii) as defined above or any other suitable attachment for their use in immunogenic compositions and vaccines.

In view of the finding of the conserved domains of the sigma C proteins of the new isolates, it was envisaged to synthesize a synthetic polypeptide comprising such conserved domains representing all variants of avian reovirus for use in a vaccine that can induce a protective immune response in birds against infection with any one of the many variants.

The synthetic polypeptide, referred to herein as a fusion polypeptide, is designed to elicit an optimal immune response. This is achieved by including at least two copies of each domain sequence within the sequence of the fusion polypeptide and by ensuring that the fusion polypeptide acquires a linear configuration in solution. The linear configuration is achieved by inserting a linker

composed of 1-3 amino acids, preferably two lysine residues, linking each two peptide sequences.

Thus, in another aspect, the present invention provides a fusion polypeptide comprising the sequences of at least two peptides derived from reovirus sigma C proteins, wherein each of the at least two peptides has from 5 to 23, preferably 6 to 20, amino acid residues, each peptide is bound to the other peptide via a linker comprising 1-3 amino acid residues, the fusion polypeptide comprises at least two copies of each peptide and has a total of 4-16, preferably 6, 8, 10, 12, more preferably, 14, peptide sequences. The linker between two peptide sequences in the fusion polypeptide is preferably composed of two amino acids and is more preferably Lys-Lys to ensure a linear configuration of the fusion polypeptide in solution.

Any reovirus SCP peptide may be used to construct the fusion polypeptide. Preferably, the conserved peptides of SEQ ID NOs. 3 to 33 presented in Tables 3 and 4 herein are used, more preferably the peptides of SEQ ID NO: 10, 1 1, 13-20 and 22.

In one embodiment, the fusion polypeptide comprises 6-8, preferably 7 different peptides, each peptide appearing twice, thus the fusion polypeptide has a total of 12- 16, preferably 14 peptide sequences, each two peptides linked by Lys- Lys. In one preferred embodiment, the fusion polypeptide has the Lys-Lys residue also at the N- and C-termini.

In one preferred embodiment, the fusion polypeptide is of SEQ ID NO:34, herein designated R-PEP Al, which comprises two sequences in tandem of each of the peptides of SEQ ID NOs: 13-17, 20 and 22, and comprises the sequence Lys-Lys at the N- and C-termini and linking each two peptides.

The fusion polypeptide R-PEP Al can be represented by the structure:

Lys-Lys-Xl - Lys-Lys-Xl - Lys-Lys-X2- Lys-Lys-X2- Lys-Lys-X3- Lys-Lys- X3- Lys-Lys-X4- Lys-Lys-X4-Lys-Lys-X5- Lys-Lys-X5- Lys-Lys-X6- Lys-Lys- X6- Lys-Lys-X7- Lys-Lys-X7- Lys-Lys

wherein Xl is the peptide of SEQ ID NO:22, X2 is the peptide of SEQ ID NO: 13, X3 is the peptide of SEQ ID NO: 14, X4 is the peptide of SEQ ID NO: 15, X5 is the peptide of SEQ ID NO: 16, X6 is the peptide of SEQ ID NO:20, and X7 is the peptide of SEQ ID NO: 17. In another preferred embodiment, the fusion polypeptide is of SEQ ID

NO:35, herein designated R-PEP A2, which comprises two sequences in tandem of each of the peptides of SEQ ID NOs: 10, 11, 15, and 17-20, and comprises the sequence Lys-Lys at the N- and C-termini and linking each two peptides.

The fusion polypeptide R-PEP A2 can be represented by the structure: Lys-Lys-X8- Lys-Lys-X8- Lys-Lys-X9- Lys-Lys-X9- Lys-Lys-X 10- Lys-

Lys-Xl O- Lys-Lys-X4- Lys-Lys-X4-Lys-Lys-Xl 1- Lys-Lys-X 1 1- Lys-Lys-X6- Lys-Lys-X6- Lys-Lys-X7- Lys-Lys-X7- Lys-Lys wherein X8 is the peptide of SEQ ID NO: 10, X9 is the peptide of SEQ ID NO: 18, XlO is the peptide of SEQ ID NO: 1 1 , X4 is the peptide of SEQ ID NO: 15, Xl 1 is the peptide of SEQ ID NO: 19, X6 is the peptide of SEQ ID NO:20, and X7 is the peptide of SEQ ID NO: 17.

The present invention also provides an immunogenic composition comprising at least one fusion polypeptide of the invention, with or without an adjuvant. In preferred embodiments, the immunogenic composition comprises the fusion polypeptide R-PEP Al , R-PEP A2 or a mixture thereof, with or without an adjuvant. Any suitable adjuvant can be used such as Freund's complete or incomplete adjuvant, LT, aluminum hydroxide, water-in-oil, oil-in-water, etc.

The fusion polypeptide of the invention can be synthesized by methods well known in the art either by peptide chemistry technology or by recombinant techniques starting from specially designed polynucleotides encoding the fusion polypeptide sequence. Thus, the invention also provides a polynucleotide encoding a fusion polypeptide as defined herein. In preferred embodiments, the polynucleotide is of the SEQ ID NO:36 or 37 and code for the fusion polypeptides R-PEP Al or R-PEP A2, respectively.

For expression of the fusion polypeptides, bacterial or eukaryotic cells, such as yeast or plant cells, may be transformed with the polynucleotide molecule and the expressed fusion polypeptide may be purified using the purification tag inserted into the polypeptide and/or by other protein purification methods well known in the art. Preferably, the fusion polypeptide is expressed in a bacterial cell, more preferably E. coli.

As mentioned above, in a preferred embodiment the fusion polypeptide comprises at least two copies of each one of at least two conserved immunogenic domains, wherein said at least two copies of each conserved immunogenic domain appear in tandem. The repeating occurrences of a certain domain are preferably ordered in tandem, since two adjacent copies of said domain may bind to the same multivalent antibody molecule and thus enhance the immunogenicity of said domain by increasing the avidity of the binding.

For use in immunogenic compositions/vaccines, the fusion polypeptide may be combined with a carrier protein such as, but not limited to, E. coli heat labile enterotoxin (LT), bovine serum albumin and flagellin. The carrier protein may be fused to the N- or the C terminus of the fusion polypeptide or it may flank the fusion polypeptide on both ends. The carrier protein may be fused to the fusion polypeptide by recombinant techniques, i.e. the fusion polypeptide and the carrier protein are encoded by a single nucleic acid sequence and are expressed as a continuous polypeptide. Alternatively, the fusion polypeptide and the carrier protein may be produced separately and then chemically attached to each other.

The preferred carrier protein for all aspects of the present invention is the LT protein. It confers the function of carrier and is also known as a powerful adjuvant in injection and oral administration that is capable of eliciting strong systemic IgG and local IgA responses as well as cytotoxic helper T-cell response. Since the reovirus is a mucosal antigen, it is preferential to stimulate the immune response of the mucosal system. IgA antibodies developed by oral vaccinations in mucosal tissues are the main deterrents against the challenge offered by mucosal antigens. The LT protein is a hexamer that consists of two subunits: the 27-kDa catalytic A

domain (LTA) anchored in a ring of five identical 1 1.6-kDa B subunits (LTBs). The Al fragment is toxic and catalyzes the transfer of an ADP-ribose from NAD to stimulatory α-subunits of G proteins (Gsα). To use LT as an immunostimulator in animals, its toxicity may be neutralized by mutations, such as the LTK63 mutant of the A subunit (Partidos et al.,1996). Either one of the A and B subunits may be used in conjunction with the fusion polypeptide of the invention.

The fusion polypeptide may also be attached to physiologically acceptable microbeads, which may carry enhancer molecules such as LT in addition to the fusion polypeptide. Alternatively, the fusion polypeptide may be bound indirectly to the carrier via antibodies or biotinylated fusion polypeptides may be bound to the carrier via avidin or streptavidin.

Microparticles intended for use in the present invention preferably have a size in the range from 10 nm to 200 μm. The size chosen for a particular microparticle will depend on the active agent to be delivered, and the intended route of administration. For oral delivery, particles are conveniently in the size range 0.5 to 5.0μm. For subcutaneous delivery, a suitable size is less than lOOμm. Microparticles for parenteral delivery conveniently have a size of less than 200μm, preferably less than 150μm.

In another embodiment, the fusion polypeptide may comprise a purification tag, which is preferably a His-tag (e.g. His ₆ ), but may also be a (His-Asn) ₆ tag, a Flag tag, or any other tag that may facilitate the purification of the fusion polypeptide. A sole tag can be used or different tags can be used in combination. The tag(s) can be fused to the C-terminus or the N-terminus of the fusion polypeptide. As stated above, the optimal induction of a protective immune response may be obtained by presenting to immuno-reactive cells such as B and T-cells at least two epitopes that may interact with the multivalent receptors on the cells' surface. In order to improve the chance of the preferably two copies of an immunogenic domain occurring in tandem to be presented in a configuration enabling multivalent

interaction with e.g. an antibody displayed on a B-cell, the polypeptide has to be in a linear configuration and not folded upon itself.

Two features of the fusion polypeptide ensure that this is the case. First, two lysine residues are interspersed in between each domain in the amino acid sequence of the fusion polypeptide. The positively charged lysine residues repulse each other and ensure that the polypeptide chain does not fold. However, this is true as long as the length of the polypeptide chain does not exceed a certain limit. According to calculations done by the inventors, looking at certain conserved and immunogenic domains of the sigma C protein, it seems that not more than about 7 couples of different domains could be accommodated within a theoretically linear chimeric polypeptide, i.e. the maximum size of such a protein is approximately 5OkD. Methods for determining the conformation of peptides in solution are well known in the art and facilitate easy screening for multiple variations of the chimeric peptide and selection of linear peptides for continued development. For example, circular dichroism is a simple and fast method used to determine the secondary structure of proteins (Bierzynsk, 2001).

A further finding in accordance with the present invention is that even if the chimeric protein is packed in inclusion bodies of E. coli cells that are used to vaccinate birds, it is capable of eliciting a beneficial immune response. For example, the fusion polypeptide of the invention is capable of eliciting a specific B-cell and/or T-cell response in a bird. Specifically, T-helper cells and cytotoxic T cells are induced.

In another aspect, the present invention relates to a sigma C protein (SCP) of an avian reovirus isolate (RI) from Israel, wherein said RI belongs to the Group I, II or III of reovirus as herein defined, and said RIs are selected from ISR521, ISR526. ISR5215, ISR5220, ISR5225, ISR5226 (Group I), ISR522, ISR5217, ISR5221, ISR5222, ISR5223 (Group II); and ISR524, ISR527, ISR528, ISR529, ISR521 1 , ISR5213 (Group III), or a fraction or fragment of said reovirus SCP.

In preferred embodiments, the invention provides the recombinant SCPs of SEQ ID NO: 38 of RI ISR5223, SEQ ID NO:39 of RI ISR5215 and SEQ ID NO:40 of RI ISR528.

The invention also provides a polynucleotide encoding a reovirus sigma C protein of the invention. In preferred embodiments, the polynucleotide is of SEQ ID NOs. 41 , 42 or 43, which code for the SCPs of SEQ ID NOs: 38, 39, and 40, respectively.

In accordance with the present invention, we have also determined the partial sequences of sigma C proteins of avian reovirus from several new field isolates from Israel. These partial sequences, herein termed 'fractions' or 'fragments', are also encompassed by the present invention. In preferred embodiments, these fragments include SEQ ID NO:44 (ISR521 ), SEQ ID NO:45 (ISR522), SEQ ID NO:46 (ISR524), SEQ ID NO:47 (ISR525), SEQ ID NO:48 (ISR526), SEQ ID NO:49 (ISR527), SEQ ID NO:50 (ISR528), SEQ ID NO:51 (ISR529), SEQ ID NO:52 (ISR5210), SEQ ID NO:53 (ISR521 1), SEQ ID NO:54 (ISR5212), SEQ ID NO:55 (ISR5213), SEQ ID NO:56 (ISR5215), SEQ ID NO:57 (ISR5217), SEQ ID NO:58 (ISR5219), SEQ ID NO:59 (ISR5220), SEQ ID NO:60 (ISR5221 ), SEQ ID NO:61 (ISR5222), SEQ ID NO:62 (ISR5223), SEQ ID NO:63 (ISR5224), SEQ ID NO:64 (ISR5225), SEQ ID NO:65 (ISR5226), SEQ ID NO:66 (ISR5229) and SEQ ID NO:67 (ISR5231). The sigma C proteins of the new isolates ISR 523, ISR5216 and ISR 5218 were found to be identical to the sequences of ISR522 (SEQ ID NO:5), ISR5215 (SEQ ID NO:59) and ISR5217 (SEQ ID NO:57), respectively. The partial sequences of the strains 59103 (SEQ ID NO: 1) and si 133 (SEQ ID NO:2) are known.

INDUSTRIAL APPLICABILITY

In one preferred aspect, the invention provides a vaccine comprising at least one recombinant sigma C protein (SCP) of an avian reovirus isolate (RI) from Israel of each of Groups I, II, III or IV of reovirus as herein defined, optionally together with an adjuvant.

In one embodiment, the RI is selected from ISR521, ISR526. ISR5215, ISR5220, ISR5225, ISR5226 (Group I), ISR522, ISR5217, ISR5221, ISR5222, ISR5223 (Group II); ISR524, ISR527, ISR528, ISR529, ISR5211, ISR5213 (Group III); and 59103 and Sl 133 (Group IV). The vaccine may comprise one recombinant SCP or a mixture of 2, 3 or 4 or more SCPs, each representative of one of the groups I, II, III or IV. In one preferred embodiment, the vaccine comprises at least one representative of each group I, II, III and IV. In a more preferred embodiment, the vaccine comprises a mixture of the (i) recombinant SCP of Group I of the SEQ ID NO: 38; (ii) recombinant SCP of Group II of the SEQ ID NO: 39; (iii) recombinant SCP of Group III of the SEQ ID NO: 40; and (iv) recombinant SCP of Group IV of SEQ ID NO: 68.

In another aspect, the invention relates to a reovirus vaccine comprising at least one of the new reovirus isolates ISR521, ISR526. ISR5215, ISR5220, ISR5225, ISR5226, ISR522, ISR5217, ISR5221, ISR5222, ISR5223, ISR524, ISR527, ISR528, ISR529, ISR521 1 , and ISR5213 in inactivated form, or a mixture thereof that may include also known strains such as 59103 and si 133. The vaccine may optionally contain an adjuvant.

In one embodiment, the vaccine composition comprises one variant of each RI group; each variant being the most representative variant of its group, i.e. each variant expresses a sigma C protein that has the highest identity to the sigma C proteins expressed by the known members of the group.

In a preferred embodiment, the vaccine comprises at least four inactivated reovirus isolates (RI), each RI belonging to one of the Groups I, II, III or IV of reovirus as herein defined, optionally together with an adjuvant. Preferably, the vaccine comprises RIs ISR521 , ISR526. ISR5215, ISR5220, ISR5225, ISR5226 (Group I), ISR522, ISR5217, ISR5221, ISR5222, ISR5223 (Group II); ISR524, 1SR527, ISR528, ISR529, ISR521 1 , ISR5213 (Group III); and 59103, Sl 133 (Group IV). In a more preferred embodiment, the vaccine comprises a mixture of the isolates ISR5215 (Group I), ISR5223 (Group II), ISR528 (Group III), and 59103 (Group IV).

The production of the inactivated avian reovirus isolates is carried out by methods well-known in the art, preferably in Vero cells or in embryonic chick eggs, e.g., in allantoic cavity of 11-day-old embryonated Specific Pathogen Free (SPF) hen eggs. In a further aspect, the invention provides the new reovirus isolates 1SR521,

ISR526. ISR5215, ISR5220, ISR5225, ISR5226, ISR522, ISR5217, ISR5221 , ISR5222, ISR5223, ISR524, ISR527, ISR528, ISR529, ISR5211, and ISR5213.

The sigma C protein is located on the outer part of the capsid and functions in the identification and binding of the virus to the target cell. Thus, similarly to the vaccine disclosed above that confers protection in birds against all avian reovirus variants due to the representation in the recombinant fusion polypeptides of the vaccine of all variable regions of the sigma C protein, it is also contemplated by the present invention a vaccine comprising all variable regions of the sigma C protein presented on the capsid of inactivated viral particles. The defining feature of each RI group is that the sigma C protein amino acid sequence expressed by isolates of said RI group has at least 75% identity with the sigma C protein amino acid sequences of other isolates of the same RI group. The four RI groups disclosed in Table 2 herein below are based on the sigma C proteins expressed by 19 isolates from Israel and elsewhere. The composition of each of the four groups is not limited to the variants/isolates disclosed in the table, but comprises all variants expressing sigma C protein amino acid sequences that are at least 75% identical to the sigma C polypeptides expressed by variants of each of the groups in Table 2. Thus, for example, RJ group 4 is not limited to the two isolates 59103 and S l 133, but comprises also variants (that have not been isolated) that express sigma C proteins that are at least 75% identical to the sequences of sigma C proteins expressed by 59103 and S l 133.

The invention also contemplates a DNA or RNA vaccine comprising a polynucleotide of the invention or an expression vector comprising said (deoxy)polynucleotide.

The immunogenic compositions/vaccines of the present invention, whether they comprise at least one inactivated RI, at least one reovirus recombinant sigma C protein, a fusion polypeptide comprising conserved domains of the reovirus sigma

C protein, or a conserved peptide are capable of conferring protection in mammals, including humans, or birds, against all known avian reovirus variants.

The invention further relates to a method for inducing an avian immune response conferring protection against reovirus, which comprises administering a vaccine or immunogenic composition according to the invention. The composition may be administered by any suitable route of administration such as by injection, intradermally or orally to birds via the drinking water.

The invention will now be illustrated by the following non-limitative examples.

EXAMPLES Materials and Methods (i) Avian reovirus propagated in Vero cells. Vero cells were grown in minimal essential medium (MEM), and fungizone (1.25 μg/ml) at 37 ⁰ C in a 5% CO ₂ incubator. Avian reovirus propagates in Vero cells. Upon development of 80% cytopathic effect, the cell culture was centrifuged after three freeze-thaw cycles. The supernatant containing the viruses was collected and stored at -70°C. (H) Virus propagated in embryonic chicken eggs. Reovirus was injected into the yolk sack of SPF embryonic chicken eggs (SPAFAS, USA) at 6-8 days. The virus was extracted from the allantoic fluid after 4 days.

(Hi) Isolation of virus, RNA extraction, reverse transcription and PCR amplification. Virus was isolated from the tendon of infected birds. The viral RNA was extracted (protocols were performed according to QlAamp Viral RNA- Mini Kit, Qiagen, USA). The sigma C gene was amplified by RT-PCR: 1 μl of purified dsRNA was denatured in boiling water for 10 min, chilled on ice for 5 min, and then used as template; dsRNA was used to generate cDNA by reverse transcription and polymerase chain reaction. PCR reactions were subjected to 35 cycles consisting of denaturation for 1 min at 94°C, annealing for 2 min at 42 ⁰ C, and

extension for 1 min at 72°C, and one final extension cycle at 74°C for 15 min. The PCR product, namely, the sigma C protein, was purified (protocols were performed according to QIAGEN Gel Extraction Kit) and sequenced.

(iv) Design, production and cloning of genes coding for sigma c recombinant protein.

Partial sequences of sigma c of isolates 5215, 5223, 528 and 59103 were determined. To each sequence, flanking regions were added to the 5' and 3' end in order to ensure correct folding and maintain protein structure. Furthermore, the added sequence introduced restriction sites to enable cloning into pQE30 plasmid, inserting the coding sequence in the correct position relative to the promoter and in frame. The designed genes were manufactured by GenScript (USA) and supplied in pQE30 plasmid. The plasmid is transformed into E. coli JM 109 and the produced proteins purified and used for vaccination.. The sequences of the full recombinant proteins and the nucleic acids encoding them are disclosed throughout the present specification. The restriction sites introduced into the four recombinant proteins disclosed herein were Kpnl and Pstl.

Example 1. New Israeli reovirus isolates

RNA sequences of sigma C of 30 isolates of avian reovirus from infected birds in Israel were determined and the amino acid sequences were deduced (SEQ

ID NOs: 44-67) and compared to the sequence of the vaccine strain and published sequences from the around the world. The four groups defined below in the present invention are based on 19 out of the 30 isolates.

High variability was found on the amino acid sequence level. According to an analysis based on comparison of the amino acid sequences of sigma C proteins of avian reovirus from Israel and other published isolates from the rest of the world, the phylogenetic tree of avian reoviruses according to sigma C sequences depicted in Fig. 1, and the percent of identity in sigma C among the conserved domains of the Israeli ARV isolates shown in Table 1, it was found that the Israeli ARV

isolates may be divided into four groups, herein identified as ISR RI Groups I to IV (ISR stands for Israel Reovirus and RI for Reovirus isolate). Table 2 shows the various RIs belonging to each of the Groups I to IV (each isolate, with exception of the RIs of Group IV, are designated by a number starting with the algorism '52') and the SEQ ID NO: (SIN) of the partial sequence of the sigma C protein (SCP) identified in each of the isolates. Thus, Group I comprises the RIs ISR521, ISR526. ISR5215, ISR5220, ISR5225, ISR5226; Group II comprises the RIs ISR522, ISR5217, ISR5221 , ISR5222, ISR5223; Group III comprises the RIs ISR524, ISR527, ISR528, ISR529, ISR5211, ISR5213; and Group IV comprises the RIs 59103 and Sl 133. These ARV strains, with the exception of ISR59103 and ISR 1 133 of Group IV, have never been disclosed before.

These results lead us to formulate the new vaccine strategies of the invention based on the whole inactivated viruses and on the sigma C recombinant proteins.

EXAMPLE 2. Whole inactivated virus vaccine.

A vaccine based on the whole inactivated viruses comprising four isolates representatives of each of the four Groups I to IV, the RIs ISR5215 (Group I), ISR5223 (Group II), ISR528 (Group III) and ISR59103 (Group IV), all marked in bold in Table 2, is used as Anti Reovirus Broad Activity (ARBA). The viruses are propagated separately in Vero cells, inactivated by overnight incubation in 0.3-0.5% formalin and mixed with adjuvant. The experiment includes seven groups of birds: 1. Positive control (si 133); 2. Negative control (PBS); 3-6. The four representative viruses; and 7. A mixture of the four representative viruses as detailed above. The vaccine administered to Group 7 contains and displays all common conserved region sequences of sigma C. It may be used, for example, for intramuscular injection along with 0.5 ml of commercial Freund's incomplete adjuvant, but other adjuvants may be used. The birds are grown from day 1 in a positively pressured isolator. SPF chickens are vaccinated twice, at the age of 3 and 7 weeks or a similar schedule. Blood is collected at, for example, 7 and 9 weeks of age and sera is tested

Table 1. Percent of amino acid sequence identity in s >igma C among Israeli ARV isolates.

by Virus Neutralization (VN) in embryonate eggs and in Vero cells, by ELISA for antibody titer, or other methods.

The birds are then challenged with a homologous virus or a mixture of viruses from the 4 genetic groups.

Table 2. Reovirus Isolates (RI) from Israel arranged in four RI groups and SEQ ID NO (SIN) of sigma C proteins (SCP) arranged in four corresponding SCP groups, according to the phylogenetic tree (Fig. 1).

EXAMPLE 3. Recombinant sigma C proteins and vaccine comprising them

The deduced amino acid sequence of sigma C was determined. For use in the vaccine, the complete sequence of the representative sigma C protein (SCP) of each of the Groups I, II and III, namely, the strains ISR5215 (Group I), ISR5223 (Group IT) and ISR528 (Group III) was deduced. Their amino acid sequences are represented by SEQ ID NOs: 56, 62 and 50, respectively. The recombinant sigma C proteins derived from these sequences, were designated as SCP Group I (SEQ ID NO:39), SCP Group II (SEQ ID NO:38), SCP Group III (SEQ ID NO:40). The full sequence of the SCP of RI 59103, previously designated R99, (Group IV) is represented by SEQ ID NO: 1. This sequence was determined at the laboratory of the main inventor and has been submitted to GenBank, accession no. AY332520 (Vasserman et al, 2004). The recombinant SCP derived from SEQ ID NO: 1 (SCP Group IV) is represented by SEQ ID NO: 68.

For the preparation of the recombinant sigma C proteins, four polynucleotide sequences (SEQ ID NOs: 41-43 and 69) encoding for the proteins were designed by the inventors as described above in Materials and Methods (iv). The desired genes for expression in E. coli were manufactured by GenScript (USA) and supplied in pUC57 plasmid. The gene was transferred to pAL plasmid and transformed into E. coli JM 109 (Invitrogen, San Diego, CA). The expression of the recombinant proteins in E. coli was carried out by the inventors as described (Vasserman et al., 2004). The recombinant sigma C protein was isolated from the bacterial cells and purified. For the preparation of the vaccine, the four purified recombinant sigma C proteins of SEQ ID NOs: 38-40 and 68 are mixed and used with or without an adjuvant. The vaccine contains and display all common conserved region sequences of sigma C. In one example, SPF chicks (SPAFAS, USA) are divided into groups and each group was vaccinated twice with partially purified protein (lmg/ml) intramuscularly or orally (50-150 μg/dose).

Example 4. New peptides derived from avian sigma C proteins and analogs thereof

In spite of the variability of the sigma C proteins of the various isolates, some regions were detected that were conserved in all isolates. Following sequencing of 30 isolates in Israel and comparison to published sequences, sites were detected on the sigma C proteins that are both common to all sequences and predicted as antigenic.

Table 3 shows the sequences of 26 new peptides of the invention of SEQ ID NOs: 3-28 with amino acid sequences comprised within the conserved regions of sigma C proteins of avian reovirus isolates.

In addition, one of the sequences was analyzed using the program found at http://bio.dfci.harvard.edu/Tools/antigenic.pl to search for predicted antigenic sites, and it was found that some of the conserved domains are encompassed by the sequences predicted as antigenic (Table 4 and Fig. 2).

"Start and end of the specific peptide on the sigma C protein.

Underlined sequences are homologous to sequences of some of the conserved domains shown in Table 3)

The peptides presented in Tables 3 and 4 may be produced synthetically or using recombinant techniques and may be used for vaccinating birds to confer broad protection.

Example 5. Vaccines based on peptides derived from avian sigma C proteins 5.1 Design of vaccines

The peptides from Table 3 or 4 as well as analogs thereof can be used for preparation of different vaccines, for example: (i) Vaccine comprising a peptide from Table 3 or 4 bound to a carrier protein such as E. coli enterotoxin LT or bovine serum albumin (BSA).

(ii) Vaccine comprising a mixture of 2 or more peptides from Table 3 or 4 bound to a carrier protein such as LT or BSA.

(iii) Vaccine comprising a peptide from Table 3 or 4 chemically synthesized with biotin attached to the N-terminus as done for peptides of SEQ ID NOs: 3-12 in Table 3 and then bound to avidin that serves as a carrier to enhance immune response.

(iv) Vaccine comprising one or more peptides from Table 3 or 4 bound to microbeads carrying an enhancer molecule, such as avidin, in which case the peptide is biotinylated, or LT.

5.2. Inimunogenicity of the peptides.

The antigenicity of representative peptides was proven in the following experiment.

The peptides of SEQ ID NOs: 3, 5, 8 and 11 were attached to a carrier protein, in this case BSA, to form a peptide conjugate. The peptides of SEQ ID

NOs: 3 and 5, which contain a where attached to BSA via their cystein residue and a

Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (Sulfo-

SMCC. Pierce) linker according to the manufacturer's instructions.

The peptides of SEQ ID NOs: 8 and 11, which lack a cystein residue, where attached to BSA via their free carboxyl terminus and an adipic acid dihydrazide

(ADH, Sigma) after activation of the carboxyl group with N-(3-

Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, Sigma). In this case 4 mg BSA was dissolved in 1 ml phosphate buffer (pH 5.6) to which 60 mg

ADH and 48 mg EDC were added. The solution was incubated for 3 hrs at room temperature and the excess reagents were removed by filtering through an Amicon

Ultra 3,500MWCO cartridge according the manufacturers instructions. At this point

1 mg peptide and 10 mg EDC were added to the solution.

The peptide conjugates, emulsified in Freund's complete adjuvant, were injected intramuscularly in four groups of ten 3 weeks old chicks, and after two weeks a boost of peptide conjugates in Freund's incomplete adjuvant was injected.

An ELISA using plates coated with avian reovirus 1 133 showed that the peptide-conjugates evoked production of specific antibodies against the reovirus

(Fig. 3). In particular, it can be seen that a mixture of the sera against the four different peptides produced a higher signal than each one of the sera against a specific peptide.

Example 6. Fusion polypeptides comprising peptides derived from avian sigma C proteins and production thereof

Two fusion polypeptides, herein designated R-PEP Al and R-PEP A2, comprising sequences of reovirus sigma C protein peptides depicted in Table 3,

were prepared. Each peptide appears twice in the polypeptide in order to amplify the immunogenicity of the molecule, and each two peptides are separated by two lysine residues to enable a linear structure. In addition, the fusion polypeptide comprises two lysine residues at the N-terminus and at the C-terminus. R-PEP A 1 is a polypeptide comprising the sequences of 7 reovirus sigma C peptides arranged from the N-terminus to the C-terminus in the following order: the peptides 20, 1 1, 12, 13, 14, 18 and 15 as presented in Table 3. Each sequence appears twice, and sequence pair is intercalated by two lysine residues. R-PEP Al has the following sequence (SEQ ID NO: 34):

R-PEP A2 is a polypeptide comprising the sequences of 7 reovirus sigma C peptides arranged in the same manner as described for R-PEP Al . The peptides appearing in R-PEP A2 are 8. 16, 9. 13, 17. 18 and 15 as presented in Table 3 R- PEP A2 has the following sequence (SEQ ID NO: 35):

KKDPYECSKKBK^gSKKTSYSEEAtLMQFRWKKlSySEEAQLMQFRWKKRTDYMMSKKR Restriction sites were incorporated in the 5' and 3' ends of the R-PEP gene

(SEQ ID NO: 36 for R-PEP Al , and SEQ ID NO: 37 for R-PEP A2) to enable the incorporation of enhancer protein(s) (e.g LT). The general design of the gene is: KpnI-6Xhis-BamHI-R-PEP-BglII-6Xhis-Stop-PstI, where Kpnl, BamHI, BgIII and Pstl designates restriction enzyme recognition sequences, His is the codon for Histidine (CAT) and Stop designates TAA. Six histidine residues are added to the N or C terminal part of R-PEP for the purpose of purification on a nickel column (e.g. Quagen Ni-NTA Agarose resin). The Optimization Region for R-PEP Alwas 31 - 552, and for R-PEP A2 it was 31 - 483. The GC Range for both proteins was 30 - 70. For The R-PEP gene was designed according to the E.coli codon usage.

For the production of the recombinant fusion polypeptides, the polynucleotides of SEQ ID NO: 36 and SEQ ID NO: 37, encoding R-PEP Al and

R-PEP A2, respectively, were inserted into the expression vector PQE-30 (Qiagen) at the Kpnl and Pstl restriction sites, and E.coli cells were transformed with the purified expression vector using methods well known in the art.

Extracts of the transformed bacteria were fractionated by centrifugation and detected by SDS-PAGE as shown in Fig. 4A. The proteins were transferred to Hybond-c Nitrocellulose membrane (Amersham), stained with anti-histidine antibodies and detected by Western blot with anti-histine antibodies and visualized with diaminobenzidine tablets (Sigma) (Fig. 4B). The labeled bands (arrow) correspond to the expected molecular weights of the two fusion polypeptides which is 20 kDa and 17.7 kDa for R-PEP Al and R-PEP A2, respectively.

The results demonstrate that both R-PEP Al and R-PEP A2 are efficiently expressed at their full length in the insoluble fraction of E.coli cells.

EXAMPLE 7. Immunogenicity of R-PEP Al and R-PEP A2.

Five groups of birds (four each) were vaccinated with either R-PEP AL R- PEP A2, R-PEP A1+A2, si 133 vaccine strain, or sigma C, and a control group of four birds was not vaccinated. Antibody production to the various antigens was tested by immunoblot. As can be seen in Figs. 5 A and 5B, each one of R-PEP Al and R-PEP A2 is recognized by sera from birds immunized with the respective antigen and by sera from birds immunized with R-PEP A1+A2, chicken anti-reovirus (si 133) and chicken anti- sigma C (produced in the laboratory of the inventors), but not with sera from birds immunized with the specific peptide not used as antigen in the immunoblot. Thus, the immunblot containing the R-PEP Al peptide did not bind anti-R-PEP A2 sera, and vice versa. This shows that the R-PEP Al and R-PEP A2 peptides are immunogenic and that the sera raised against these antigens are specific.

In a dot-blot assay performed with reovirus (si 133) used as antigen, it was shown that the anti-R-PEP Al and R-PEP A2 sera specifically recognize the whole

reovirus as does the anti-R-PEP A1+A2, anti-reovirus (si 133) and anti-recombinant sigma C sera (Fig. 6).

Vaccines comprising R-PEP Al and/or R-PEP A2 are injected intramuscularly along with 0.5 ml of commercial Freund's incomplete adjuvant, but other adjuvants may be used. The birds are grown from day 1 in a positively pressured isolator. SPF chickens are vaccinated twice, at the age of 3 and 7 weeks or a similar schedule. Blood is collected at, for example, 7 and 9 weeks of age and sera is tested by Virus Neutralization (VN) in embryonate eggs and in Vero cells, by ELISA for antibody titer, or other methods.

REFERENCES

Atherton, E.; Sheppard, R.C. (1989). Solid Phase peptide synthesis: a practical approach. Oxford, England: IRL Press

Bierzynsk (2001) Acta Biochem Polonica 48 1091-99. Cashdollar, L. W., Chmelo, R., Esparza, G., Hudson, G. R., and W.K. Jokik.

Molecular cloning of the complete genome of reovirus serotype 3. Virology 133 : 191- 196. 1984.

Fahey, J. E., and J. F. Crawley. Studies on chronic respiratory disease of chickens. II Isolation of a virus. Can. J. Comp. Med. 18: 13-21. 1954. Jokik, W. K. The Reoviridae. Plenum Press, New York. 1983.

Liu, H. J., and J. J. Giambrone. Amplification, cloning and sequencing of the σC-encoded gene of avian reovirus. J. Virological Meth. 63:203-208. 1997.

Liu, H. J., Giambrone, J. J., and B. L. Nielsen. Molecular characterization of avian reoviruses using nested PCR and nucleotide sequence analisis. J. Virological Meth. 65: 159-167. 1997.

McCrae, M. A., and W. K. Joklik. The nature of the polypeptide encoded by each of the 10 double-stranded RNA segments of reovirus type 3. Virology 89:578- 593. 1978.

Rekik, M. R., and A. Silim. Comparison of a vaccine strain and field isolates of avian reovirus by Tl -oligonucleotide mapping. Avian Dis. Apr-Jun;36(2):237- 46.1992.

Robertson, M. D., and G. E. Wilcox. Avian reovirus. Vet. Bull. 56: 154-174. 1986.

Rosenberger, J. K., Sterner, F. L, Botts, S., Lee, K. P., and A. Margolin. In vitro and in vivo characterization of avian reoviruses. I. Pathogenicity and antigenic relatedness of several avian reovirus isolates. Avian Dis. 33:535-544. 1989.

Schnitzer, T. J. Protein coding assignment of the S gene of avian reovirus S 1 133.Virology 141 : 167- 170. 1985.

Schnitzer, T. J., Ramos, T., and V. Gouvea. Avian reovirus polypeptides: analysis of intracellular virus specified products, virions, top component, and cores. J. Virol. 43(3): 1006-1014. 1982.

Shapouri, M. R. S., Kane, M., Letarte, M., Bergeron, J., Arella, M., and A. Silim. Cloning, sequencing and expression of the Sl gene of avian reovirus. J. Gen. Virol. 76: 1515-1520. 1995.

Spandidis, D. A., and A. F. Graham. Physical and chemical characterization of an avian reovirus. J. Virol. 19:968-976. 1976.

Varela, R., and J. Benavente. Protein coding assignment of avian reovirus S l 133 strain. J. Virol. 68:6775-6777. 1994.

Vasserman Y, Eliahoo D, Hemsani E, Kass N, Ayali G, Pokamunski S, Pitcovskiad J. The influence of reovirus sigma C protein diversity on vaccination efficiency. Avian Dis. 2004 Apr-Jun;48(2):271-8.

Wickramasinghe, R., Meanger, J., Enriquez, C. E., and G. E.Wilcox. Avian reovirus proteins associated with neutralization of virus infectivity. Virology Jun: 194(2):688-696. 1993