AU721451B2

AU721451B2 - Recombinant infectious laryngotracheitis virus and uses thereof

Info

Publication number: AU721451B2
Application number: AU53690/96A
Authority: AU
Inventors: Mark D. Cochran; Martha A Wild
Original assignee: Syntro Corp
Current assignee: Syntro Corp
Priority date: 1995-03-23
Filing date: 1996-03-21
Publication date: 2000-07-06
Anticipated expiration: 2016-03-21
Also published as: WO1996029396A1; JPH11503009A; AU5369096A; EP0822980A4; EP0822980A1; JP3964458B2; CA2216139C; CA2216139A1

Description

WO 96/29396 PCTIUS96/03916 RECOMBINANT INFECTIOUS LARYNGOTRACHEITIS VIRUS AND USES THEREOF This application is a continuation-in-part of U.S. Serial No. 08/126,597, filed September 24, 1993 which is hereby incorporated by reference into this application.

Within this application several publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification preceding the claims. The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION Infectious laryngotracheitis virus is a herpesvirus that causes a respiratory illness of varying virulence in chickens. Live attenuated ILTV vaccines are available to protect against the disease, but several reports have implicated vaccine viruses in the possible recurrence and spread of the disease (65 and 72), limiting vaccination to use in uninfected birds early in an outbreak. In order to design a more efficacious. attenuated vaccine, the genomic organization of the ILTV virus has been studied.

The ability to isolate viral DNA and clone this isolated DNA into bacterial plasmids has greatly expanded the approaches available to make viral vaccines.

The methods used to make the present invention involve modifying cloned viral DNA sequences by insertions, deletions and single or multiple base changes.

The modified DNA is then reinserted into the viral genome to render the virus non-pathogenic. The resulting live virus may then be used in a vaccine to elicit an immune response in a host animal and to protect the animal against a disease.

WO 96/29396 PCT/US96/03916 -2- One group of animal viruses, the herpesviruses or Herpetoviridae, is an example of a class of viruses amenable to this approach. These viruses contain 100.000 to 200,000 base pairs of DNA as their genetic material. Importantly, several regions of the genome have been identified that are nonessential for the replication of virus in vitro in cell culture. Modifications in these regions of the DNA may lower the pathogenicity of the virus, attenuate the virus. For example, inactivation of the thymidine kinase gene renders human herpes simplex virus non-pathogenic and pseudorabies virus of swine nonpathogenic Removal of part of the repeat region renders human herpes simplex virus nonpathogenic A repeat region has been identified in Marek's disease virus that is associated with viral oncogenicity A region in herpesvirus saimiri has similarly been correlated with oncogenicity Removal of part of the repeat region renders pseudorabies virus non-pathogenic Patent No.

4,877,737, issued October 31, 1989). A region in pseudorabies virus has been shown to be deleted in naturally-occurring vaccine strains 8) and it has been shown that these deletions are at least partly responsible for the lack of pathogenicity of these strains.

It is generally agreed that herpesviruses contain non-essential regions of DNA in various parts of the genome. Some of these regions are associated with virulence of the virus, and modification of them leads to a less-pathogenic virus, from which a vaccine may be derived.

Infectious laryngotracheitis virus (ILTV), an alpha herpesvirus is an important pathogen of poultry in the USA, Europe, and Australia, responsible for egg production losses and death It causes an acute disease of chickens which is characterized by respiratory depression, gasping and expectoration of bloody exudate. Viral replication is limited to cells of the respiratory tract wherein infection of the trachea gives rise to tissue erosion and hemorrhage.

WO 96/29396 PCTfUS96103916 In chickens, no drug has been effective in reducing the degree of lesion formation or in decreasing clinical signs. Vaccination of birds with various modified forms of the ILT virus derived by cell passage and/or tedious regimes of administration have been used to confer acceptable protection in susceptible chickens. Due to the limited degree of attenuation of current ILTV vaccines care must be taken to assure that the correct level of virus is maintained; enough to provide protection, but not enough to cause disease in the flock (11- 21). Furthermore, these viruses may revert back to virulence, causing disease rather than providing protection against it.

ILTV has been analyzed at the molecular level. Restriction maps of the ILTV genome have been reported (22-26). The DNA sequence of several genes have been identified, thymidine kinase (27, 28), glycoprotein gB (27. 29. ribonucleotide reductase (27. 31). capsid p40 (31, 32).

Furthermore. Shepard, et al. (53) disclosed that several genes located in the unique long region of the infectious laryngotracheitis virus genomic DNA are non-essential for viral replication.

Applicants have unexpectedly found that the unique short region of the ILT virus genomic DNA contains genes that are associated with ILTV virulence and that a deletion in those genes leads to an attenuated ILTV. Particularly, it was found that a deletion in the glycoprotein G (gG) gene of the ILT virus results in an attenuated virus, which is useful as a vaccine against subsequent attack by a virulent ILTV strains.

Applicants also found that a deletion in the glycoprotein I (gl) gene of the unique short region also attenuates the ILTV. Furthermore, it is contemplated that a deletion in the US2 gene, the UL-47 like gene, and the glycoprotein gene of the unique short region will also attenuate the ILTV.

ILTV can become latent in healthy animals which makes them potential carriers WO 96/29396 PCT[US96/03916 -4of the virus. For this reason, it is clearly advantageous to be able to distinguish animals vaccinated with non-virulent virus from animals infected with diseasecausing wild-type or naturally-occurring virus. The development of differential vaccines and companion diagnostic tests has proven valuable in the management of pseudorabies disease A similar differential marker vaccine would be of great value in the management of ILTV caused disease. The construction of differential diagnostics has focused on the deletion of glycoproteins.

Theoretically, the glycoprotein chosen to be the diagnostic marker should have the following characteristics: the glycoprotein and its gene should be nonessential for the production of infectious virus in tissue culture; the glycoprotein should elicit a major serological response in the animal; and (3) the glycoprotein should not be one that makes a significant contribution to the protective immunity.

The ILT virus has been shown to specify at least four major glycoproteins as identified by monoclonal antibodies 205K, 115K, 90K and 60K). Three glycoproteins seem to be antigenically related 205K, 115K, and (34-36).

Three major ILT virus glycoproteins, gB (29, 30), gC (27, 51), and g60 (34, 53) have been described in the literature. These three genes have been sequenced and two of the ILTV genes have been shown to be homologous to the HSV glycoproteins gB, and gC.

Of these, it is known that the ILTV gB gene is an essential gene and would not be appropriate as deletion marker genes. Furthermore, the gC gene of herpesviruses has been shown to make a significant contribution to protective immunity as a target of neutralizing antibody (56) and as a target of cellmediated immunity Therefore, the gC gene is not desirable as a deletion marker gene.

As to other glycoprotein encoding, genes cited above, it is not known whether WO 96/29396 PCTIUS96/03916 or not they would be suitable candidates for deletion in order to construct a recombinant ILT virus which can be used as a diagnostic vaccine.

Applicants have unexpectedly found that there are two glycoprotein encoding S 5 genes located within the unique short region of the ILT viral genome which could be safely deleted in order to construct a recombinant ILT virus that can be used as a diagnostic vaccine. These are the glycoprotein gG gene and the glycoprotein gI gene. By genetically engineering an ILT virus with a deletion in the glycoprotein G gene or the glycoprotein I gene, a ILT virus is produced which does not express any glycoprotein G or glycoprotein I. None of the prior arts teach or suggest that these two genes in the unique short region of the virus are appropriate candidates for deletion in order to create a diagnostic ILT virus vaccine. Although several of the herpesviruses have been genetically engineered, no examples of recombinant ILTV have been reported.

The ability to engineer DNA viruses with large genomes, such as vaccinia virus and the herpesviruses, has led to the finding that these recombinant viruses can be used as vectors to deliver vaccine antigens and therapeutic agents for animals. The herpesviruses are attractive candidates for development as vectors because their host range is primarily limited to a single target species (37) and they have the capacity for establishing latent infection (38) that could provide for stable in vivo expression of a foreign gene. Although several herpesvirus species have been engineered to express foreign gene products, recombinant infectious laryngotracheitis viruses expressing foreign gene products have not been constructed. The infectious laryngotracheitis viruses described above may be used as vectors for the delivery of vaccine antigens from microorganisms causing important poultry diseases. Other viral antigens which may be included in a multivalent vaccine with an ILTV vector include infectious bronchitis virus (IBV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), and Marek's disease virus (MDV). Such multivalent recombinant viruses would protect against ILT disease as well as other diseases. Similarly the infectious laryngotracheitis viruses may be used as vectors for the delivery of therapeutic WO 96/29396 PCT/US96/03916 -6agents. The therapeutic agent that is delivered by a viral vector of the present invention must be a biological molecule that is a by-product of ILTV replication. This limits the therapeutic agent in the first analysis to either DNA.

RNA or protein. There are examples of therapeutic agents from each of these classes of compounds in the form of anti-sense DNA, anti-sense RNA (39), ribozymes suppressor tRNAs interferon-inducing double stranded RNA and numerous examples of protein therapeutics, from hormones. e.g., insulin, to lymphokines, interferons and interleukins, to natural opiates.

The discovery of these therapeutic agents and the elucidation of their structure and function does not necessarily allow one to use them in a viral vector delivery system, however, because of the experimentation necessary to determine whether an appropriate insertion site exists.

ILTV is classified as an alpha herpesvirus with a type D genome (78) composed of a unique long region and a unique short region flanked by inverted repeats.

A genomic restriction map of an Australian ILTV isolate (SA-2) was described by Johnson et al. Using this map, Guo et al. (62) isolated and sequenced a DNA fragment from the USDA challenge strain which appeared to be derived from the unique short region. Applicants map the USDA challenge strain of ILTV, and reports characteristics of the putative genes present in the unique short region. The map disclosed herewith indicates that the sequence identified by Guo et al. (62) is part of the short repeat sequence, and is not from the unique short. Other reports (69 and 70) describe the sequences of two genes, one homologous to PRV gG and the other unlike other reported herpesvirus genes. These two genes were mapped to the unique long region of SA-2.

However, these sequences are identical to sequences identified in this application as being from the unique short region. The data in this application indicate that the overall organization of the short region of ILTV is similar to other herpesviruses.

WO 96/29396 PCTfUS96/03916 -7- SUMMARY OF THE INVENTION The present invention provides a recombinant, attenuated infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene. This attenuated virus is useful as a vaccine against infectious laryngotracheitis virus.

The present invention also provides a recombinant, attenuated infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the US2 gene, UL47-like gene, ORF4 gene or glycoprotein g60 gene.

The present invention also provides a method for distinguishing chickens or other poultry vaccinated with a recombinant infectious laryngotracheitis virus which produces no glycoprotein gG from those infected with a naturallyoccuring infectious laryngotracheitis virus.

WO 96/29396 PCT/US96/03916 -8- BRIEF DESCRIPTION OF THE FIGURES Figures 1A-1H: The nucleotide sequence of 13,473 base pairs of contiguous DNA from the unique short region of the ILT virus. This sequence contains the entire 13,098 base pair unique short region as well as 273 base pairs of repeat region at one end and 102 base pairs of repeat region at the other end. The nucleotide sequences of Figures 1A-1H begin with the internal repeat sequence and end within the terminal repeat sequence. The unique short region begins at base pair 274 of this Figure. Sequence ID NO:59 contains the nucleotide sequence of 18,912 base paris of contiguous DNA from the unique short and repeat regions of the ILT virus. This sequence contains the entire 13,094 base pair unique short region as well as 2909 base paris of internal repeat region and 2909 base paris of short terminal repeat region. The nucleotide sequences begin with the internal repeat sequence and end within the terminal repeat sequence. The unique short region begins at base pair 2910.

Figure 2: Asp718 I restriction enzyme map of the infectious laryngotracheitis virus (ILTV) USDA 83-2 genome. The upper diagram identifies the unique long internal repeat unique short and terminal repeat (TR) sections found in the ILTV genome. A map of the Asp718 I restriction endonuclease sites in the ILTV genome is shown below. Letters A through 0 identify Asp718 I restriction endonuclease fragments with representing the largest fragment. Fragment is the 2.5 kb Asp718 I fragment, fragment is the 5164 bp Asp718 I fragment. and fragment WO 96/29396 PCT/US96/03916 -9is the 8.0 kb Asp718 I fragment. The fragments marked with asterisks contain a hypervariable region of approximately 900 bp that is repeated from one to 12 times. Since no one size predominates, these fragments appear in submolar amounts that are not well resolved on an ethidium bromide stained gel. The position of these repeats is indicated in the Figures by the crooked dashed lines.

Figure 3: Open reading frames within the unique short region of infectious laryngotracheitis virus (ILTV) USDA 83-2. The 13,473 base pairs of the short region of ILTV contains the entire 13,098 base pair unique short region as well as 273 base pairs of repeat region at one end and 102 base pairs of repeat region at the other end. The unique short region contains 13 methionine initiated open reading frames (ORF) of greater than or equal to 110 amino acids (excluding smaller nested ORFs). All 13 ORFs were aligned to the Entrez release 6.0 virus division of the Genbank DNA database utilizing the IBI MacVector Protein to DNA alignment option (default settings). Eight of the ORFs exhibited significant homology to one or more other virus genes: unique short (US2). protein kinase unique long 47-like (UL47-like). and glycoproteins gG, g60, gD, gI, and gE.

Figures 4A-4B: Detailed description of the DNA insertion in Homology Vector 472-73.27. Diagram showing the orientation of DNA fragments assembled in plasmid 472-73.27. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 21. 22 and 23). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The WO 96/29396 PCTfUS96/03916 location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, infectious laryngotracheitis virus (ILTV), human cytomegalovirus immediate early (HCMV IE), pseudorabies virus (PRV), lactose operon Z gene (lacZ), Escherichia coli coli), polyadenylation signal (poly and base pairs (BP).

Figures Detailed description of the DNA insertion in Homology Vector 501-94. Diagram showing the orientation of DNA fragments assembled in plasmid 501-94. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 24, 26, and 27). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets indicate the remnants of sites which were destroyed during construction.

The following abbreviations are used, infectious laryngotracheitis virus (ILTV). human cytomegalovirus immediate early (HCMV IE). pseudorabies virus (PRV), lactose operon Z gene (lacZ), Escherichia coli coli), polyadenylation signal (poly A), thymidine kinase and base pairs (BP).

Figures 6A-6B: Detailed description of the DNA insertion in Homology Vector 544-55.12. Diagram showing the orientation of DNA fragments assembled in plasmid 544-55.12. The origin of each fragment WO 96/29396 PCTIUS96/03916 -11is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 28, 29, 30, and 31). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets indicate the remnants of sites which were destroyed during construction.

The following abbreviations are used, infectious laryngotracheitis virus (ILTV), herpes simplex virus type 1 (HSV- pseudorabies virus (PRV), -glucuronidase gene (uidA), Escherichia coli (E.

coli), polyadenylation signal (poly and base pairs (BP).

Figures 7A-7C: Detailed description of the DNA insertion in Homology Vector 562-61.1F. Diagram showing the orientation of DNA fragments assembled in plasmid 562-61.1F. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 32, 33. 34 35. 36 and 37). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction.

The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets indicate the remnants of sites which were destroyed during construction.

The following abbreviations are used, infectious laryngotracheitis virus (ILTV), herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV), p-glucuronidase gene (uidA), Escherichia coli (E.

coli), polyadenylation signal (poly and base pairs (BP).

Figures 8A-8C: Detailed description of the DNA insertion in Homology Vector WO 96/29396 PCTfUS96/03916 -12- 560-52.F1. Diagram showing the orientation of DNA fragments assembled in plasmid 560-52.F1. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 38.

39, 40, 41, and 42). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, infectious laryngotracheitis virus (ILTV) herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV), P--glucuronidase gene (uidA), Escherichia coli coli), polyadenylation signal (poly A), unique long 47 (UL47-like), open reading frame 4 (ORF4).

glycoprotein G and base pairs (BP).

Figures 9A-9B: Detailed description of the DNA insertion in Homology Vector 579-14.G2. Diagram showing the orientation of DNA fragments assembled in plasmid 579-14.G2. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 43, 44, 45, and 46). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, infectious laryngotracheitis virus (ILTV), herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV), 1-glucuronidase gene (uidA), Escherichia coli (E.

WO 96/29396 PCTfUS96/03916 -13coli), polyadenylation signal (poly and base pairs (BP).

Figures 10A-10B: Detailed description of the DNA insertion in Plasmid Vector 544-39.13. Diagram showing the orientation of DNA fragments assembled in plasmid 544-39.13. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 47, 48, 49, and 50). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, pseudorabies virus (PRV), P-glucuronidase gene (uidA), Escherichia coli coli), herpes simplex virus type 1 (HSV-1), polyadenylation signal (poly A), and base pairs (BP).

Figures 11A-I IC: Detailed description of the DNA insertion in Plasmid Vector 388-65.2. Diagram showing the orientation of DNA fragments assembled in plasmid 388-65.2. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 51, 52, 53, and 54). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets indicate the WO 96/29396 PCTIUS96/03916 Figure 12: -14remnants of sites which were destroyed during construction. The following abbreviations are used, human cytomegalovirus immediate early (HCMV IE), lactose operon Z gene (lacZ).

Escherichia coli coli), pseudorabies. virus (PRV).

polyadenylation signal (poly and base pairs (BP).

The genome of the ILTV virus, identifying the unique long unique short internal repeat and terminal repeat (TR) is shown. The BamHI, Asp718I, Noti, and Sfil restriction maps of the virus are drawn underneath, with the highly repetitive region of the short repeats indicated by a set of wavy lines. The position of the cosmids used to determine the map of ILTV are drawn beneath the restriction map. Note that cosmid 2F 12 contains two non-contiguous sections. Three probes used to characterize the ILTV genome are indicated as PI, P2, and P3. PI is a 0.9 kb NotI fragment found at the terminus of the unique long region, P2 is the 856 bp HindII fragment found in multiple copies within the short repeat, and P3 Is a 6.6 kb Notl fragment used to identify the fragments at the end of the terminal repeat.

The region sequenced. and the positions of the Asp7l8I, BamHI, Notl. and Sfil sites are shown. The and extent and orientation of the open reading frames found in the ILTV unique short and the flanking short repeat regions are indicated.

Southern blot showing the repetition of an 856 bp element within the short repeat. Genomic ILTV DNA digested with Sfil HindIII Noti Asp7181 or BamHI was probed with an 856 bp HindIII fragment from the short repeat.

Positions of molecular weight markers are indicated.

Figure 13: Figure 14: WO 96/29396 PCTIUS96/03916 Figure 15: Figure 16: Depiction of the position of the 856 bp repeat region in the USDA strain, compared to the same region from the SA-2 strain as described by Johnson et al. Three repeats are arbitrarily shown in the USDA strain, the region is not repeated in SA2.

B=BamHI, H=HindIII, R=856 bp repeat.

Southern blot identifying fragments from the internal and terminal repeat that hybridized to a 6.6 kb NotI fragment containing the junction of the unique long and the internal repeat. Genomic ILTV DNA digested with NotI Asp7181 and BamHI was probed with the 6.6 kb NotI fragment.

Positions of molecular weight markers are indicated.

The relationship of herpesvirus UL47 proteins to each other and to the ILTV UL47 homolog in a conserved region. Amino acids shared between ILTV UL47 and the other UL47 proteins are in boldface type. Pairwise comparisons have been made between the sequences as shown. A vertical bar indicates an identical amino acid, two dots indicate a positive probable acceptable mutation rate and one dot indicates a neutral probable acceptable mutation rate Figure 17: WO 96/29396 PCTfUS96/03916 -16- DETAILED DESCRIPTION OF THE INVENTION The present invention provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the glycoprotein gG gene. Said deletion attenuates the virus, rendering it suitable for use as a vaccine against infectious laryngotracheitis virus. A preferred embodiment of this invention is a recombinant infectious laryngotracheitis designated S-ILT-014 (ATCC Accession No. 2427). The S- ILT-014 virus has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. on September 22, 1993 under ATCC Accession No. 2427). Another preferred embodiment of this invention is a recombinant infectious laryngotracheitis virus designated S-ILT- 002.

For purposes of this invention, "a recombinant infectious laryngotracheitis virus" is a live infectious laryngotracheitis virus which has been generated by the recombinant methods well known to those of skill in the art, the methods set forth in DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS in Materials and Methods, and the virus has not had genetic material essential for the replication of the infectious laryngotracheitis virus deleted.

The present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene and a deletion in the US2 gene. One preferred embodiment of this invention is a recombinant infectious laryngotracheitis virus designated S-ILT-009.

The present invention further provides a recombinant laryngotracheitis virus WO 96/29396 PCTIUS96/03916 -17comprising the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene and a deletion in the ORF4 gene.

The present invention further provides a recombinant infectious laryngotracheitis virus which comprises the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene and a deletion in the UL47-like gene.

The present invention further provides a recombinant infectious laryngotracheitis virus which comprises the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene, a deletion in the ORF4 gene, and a deletion in the UL47-like gene. A preferred embodiment of this invention is a recombinant infectious laryngotracheitis virus designated S- ILT-015.

The present invention further provides a recombinant infectious laryngotracheitis virus which comprises the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene and a deletion in the glycoprotein g60 gene. A preferred embodiment of this invention is a recombinant infectious laryngotracheitis virus designated S-ILT-017.

The present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene and a deletion in the glycoprotein gl gene.

The present invention further provides a recombinant infectious laryngotracheitis virus which comprises the infectious laryngotracheitis viral genome containing a deletion in the glycoprotein gG gene and a deletion in the thymidine kinase (TK) gene.

The present invention further provides a recombinant infectious WO 96/29396 PCT/US96/03916 -18laryngotracheitis virus comprising the infectious laryngotracheitis virus genome which contains a deletion in the unique short region of the viral genome, wherein the deletion in the glycoprotein gG gene, and which also contains an insertion of a foreign gene. The foreign gene is inserted into a non-essential site of the infectious laryngotracheitis viral genome in such a way that it is capable of being expressed in a recombinant infectious laryngotracheitis infected host cell.

For purposes of this invention, "a non-essential site" of the infectious laryngotracheitis viral genome is a region of the viral genome which is not necessary for viral infection and replication.

The following non-essential sites of the infectious laryngotracheitis viral genome are preferred sites for inserting a foreign gene into the virus the thymidine kinase (TK) gene, the US2 gene, the UL47-like gene, the ORF4 gene, the glycoprotein gG gene. the glycoprotein g60 gene, and the glycoprotein gI gene.

The foreign gene, which is inserted into a non-essential site in the infectious laryngotracheitis viral genome. may encode a screenable marker, such as E. coli B-galactosidase or E. coli B-glucuronidase.

The foreign gene which is inserted into a non-essential site in the infectious laryngotracheitis viral genome, may encode an antigenic polypeptide which, when introduced into the host cell, induces production of protective antibodies against an avian disease causing agent from which the antigen is derived or derivable. Antigenic polypeptide which includes, but is not limited to: marek's disease virus (MDV) gA. marek's disease virus gB, marek's disease virus gD, Newcastle disease virus (NDV) HN, Newcastle disease virus F, infectious laryngotracheitis virus (ILT) gB, infectious laryngotracheitis virus gl, infectious laryngotracheitis virus gD, infectious bursal disease virus (IBDV) VP2, infectious bursal disease virus VP3, infectious bursal disease virus VP4, WO 96/29396 PCT/US96/03916 -19infectious bursal disease virus polyprotein, infectious bronchitis virus (IBV) spike, infectious bronchitis virus matrix, avian encephalomyelitis virus, avian reovirus, avian paramyxovirus. avian influenza virus, avian adenovirus. fowl pox virus, avian coronavirus, avian rotavirus, chick anemia virus, Salmonella spp. E. coli, Pasteurella spp., Bordetella spp., Eimeria spp., Histomonas spp..

Trichomonas spp.. Poultry nematodes, cestodes, trematodes, poultry mites/lice.

and poultry protozoa.

In one embodiment of the recombinant infectious laryngotracheitis virus the foreign DNA sequence encodes a cytokine. In another embodiment the cytokine is chicken myelomonocytic growth factor (cMGF) or chicken interferon (cIFN). Cytokines include, but are not limited to: transforming growth factor beta, epidermal growth factor family, fibroblast growth factors, hepatocyte growth factor, insulin-like growth factors, B-nerve growth factor.

platelet-derived growth factor, vascular endothelial growth factor, interleukin 1, IL-1 receptor antagonist, interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6. IL-6 soluble receptor, interleukin 7, interleukin 8, interleukin 9, interleukin 10. interleukin 11, interleukin 12, interleukin 13.

angiogenin, chemokines. colony stimulating factors, granulocyte-macrophage colony stimulating factors, erythropoietin, interferon, interferon gamma, leukemia inhibitory factor. oncostatin M. pleiotrophin, secretory leukocyte protease inhibitor, stem cell factor, tumor necrosis factors, and soluble TNF receptors. These cvtokines are from humans, bovine, equine, feline, canine, porcine or avian. Recombinant ILT virus expressing cytokines is useful to enhance the immune response when combined with vaccines containing anitgens of disease causing microorganisms.

Recombinant infectious laryngotracheitis virus expressing cytokines is used to enhance the immune response either alone or when combined with vaccines containing cytokines or antigen genes of disease causing microorganisms.

Antigenic polypeptide of a human pathogen which are derived from human WO 96/29396 PCT/US96/03916 herpesvirus include, but are not limited to: hepatitis B virus and hepatitis C virus hepatitis B virus surface and core antigens, hepatitis C virus, human immunodeficiency virus, herpes simplex virus-1, herpes simplex virus-2. human cytomegalovirus, Epstein-Barr virus, Varicella-Zoster virus, human herpesvirus- 6, human herpesvirus-7, human influenza, measles virus, hantaan virus.

pneumonia virus, rhinovirus, poliovirus, human respiratory syncytial virus, retrovirus, human T-cell leukemia virus, rabies virus, mumps virus, malaria (Plasmodiumfalciparum), Bordetella pertussis, Diptheria. Rickettsiaprowazekii.

Borrelia berfdorferi, Tetanus toxoid, malignant tumor antigens.

The antigenic polypeptide of an equine pathogen is derived from equine influenza virus, or equine herpesvirus. In one embodiment the antigenic polypeptide is equine influenza neuraminidase or hemagglutinin. Examples of such antigenic polypeptide are: equine influenza virus type A/Alaska 91 neuraminidase and hemagglutinin, equine influenza virus type A/Prague 56 neuraminidase and hemagglutinin, equine influenza virus type A/Miami 63 neuraminidase. equine influenza virus type A/Kentucky 81 neuraminidase and hemagglutinin, equine herpesvirus type 1 glycoprotein B, and equine herpesvirus type I glycoprotein D, Streptococcus equi, equine infectious anemia virus, equine encephalitis virus, equine rhinovirus and equine rotavirus.

The antigenic polypeptide of an equine pathogen is derived from bovine respiratory syncytial virus or bovine parainfluenza virus, and is capable of being expressed in a host infected by the recombinant infectious bovine rhinotracheitis virus. For example, the antigenic polypeptide is derived from bovine respiratory syncytial virus attachment protein (BRSV bovine respiratory syncytial virus fusion protein (BRSV bovine respiratory syncytial virus nucleocapsid protein (BRSV bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza virus type 3 hemagglutinin neuraminidase.

The foreign gene may be put under control of an endogenous upstream infectious laryngotracheitis virus promoter, or it may be put under control of WO 96/29396 PCT/US96/03916 -21a heterologous upstream promoter. The heterologous upstream promoter may be derived from the HCMV IE promoter, the PRV gX promoter, and BHV-1.1 VP8 promoter.

The present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion or other alteration in the unique short region of the viral genome, wherein the deletion or alteration is in the glycoprotein gG gene, so that upon replication, the recombinant virus produces no glycoprotein gG.

The following recombinant viruses are preferred embodiments of this invention: A recombinant infectious laryngotracheitis virus designated S-ILT-002, S-ILT- 014, S-ILT-009, S-ILT-015, and S-ILT-017.

The present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion or other alteration in the unique short region of the viral genome. wherein the deletion or alteration is in the glycoprotein gI gene, so that upon replication, the recombinant virus produces no glycoprotein gI.

The present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion or other alteration in the unique short region of the viral genome, wherein the deletion or alteration is in the glycoprotein gG gene and in the glycoprotein gl gene. so that upon replication, the recombinant virus produces no glycoprotein gG and no glycoprotein gl.

The present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the US2 gene, UL47-like gene, glycoprotein gene. It is contemplated that a deletion in any one of these genes will attenuate the virus, rendering it suitable to be used as a vaccine against infectious WO 96/29396 PCTIUS96/03916 -22laryngotracheitis virus.

The present invention further provides a recombinant infectious laryngotracheitis virus which comprises a foreign gene inserted within the unique short region of the infectious laryngotracheitis viral genome, provided.

however, that the insertion is not in the protein kinase gene, the glycoprotein gD gene, the glycoprotein gE gene and the ORF10 gene. The foreign gene is inserted in such a way that it is capable of being expressed in the recombinant infectious laryngotracheitis virus infected host cell. Preferred insertion sites are the US2 gene. the UL47-like gene, the ORF4 gene and the glycoprotein gene.

A foreign gene may be inserted within any one of these sites in such a way that it may be expressed in a host cell which is infected which the recombinant infectious laryngotracheitis virus of the present invention.

The foreign gene thus inserted may encode a screenable marker, such as E. coli B-galactosidase or E. coli B-glucuronidase.

The foreign gene thus inserted may encode an antigenic polypeptide which, when introduced into the host cell. induces production of protective antibodies against an avian disease causing agent from which the antigen is derived or derivable. Such antigenic polypeptide may be derived or derivable from infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus. Such antigenic polypeptide may also be derived or derivable from avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian influenza virus, avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, chick anemia agent, Salmonella spp. E. coli, Pasterurella spp., Bordetella spp. Eimeria spp. Histomonas spp., Trichomonas spp, Poultry nematodes, cestodes, trematodes, poultry mites/lice, poultry protozoa.

WO 96/29396 PCTIUS96/03916 -23- The foreign gene thus inserted may be put under control of an endogenous upstream infectious laryngotracheitis virus promoter, or it may be put under control of a heterologous upstream promoter. The heterologous upstream promoter may be the HCMV IE promoter, the PRV gX promoter or BHV-1.1 VP8 promoter.

The present invention further provides a vaccine for infectious laryngotracheitis virus which comprises a suitable carrier and an effective immunizing amount of any of the recombinant infectious laryngotracheitis virus of the present invention. This vaccine may contain either inactivated or live recombinant virus.

Suitable carriers for the recombinant virus are well known in the art and include proteins, sugars, etc. One example of such a suitable carrier is a physiologically balanced culture medium containing one or more stabilizing agents such as hydrolyzed proteins, lactose, etc. Preferably, the live vaccine is created by taking tissue culture fluids and adding stabilizing agents such as stabilizing, hydrolyzed proteins. Preferably, the inactivated vaccine uses tissue culture fluids directly after inactivation of the virus.

The present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the glycoprotein gG gene. A preferred embodiment of this invention is a vaccine which comprises a suitable carrier and an effective immunizing amount of any one of the following viruses: recombinant infectious laryngotracheitis viruses designated S-ILT-014, S-ILT-002, S-ILT- 009, S-ILT-015 and S-ILT-017.

The present invention further provides a multivalent vaccine for infectious laryngotracheitis virus and for one or more of other avian diseases which WO 96/29396 PCT/US96/03916 -24comprises an effective immunizing amount of a recombinant virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region, wherein the deletion is in the glycoprotein gG gene. and an insertion of a foreign gene into a non-essential site of the viral genome.

The foreign gene encodes an antigenic polypeptide which induces host cell production of protective antibodies against an avian disease causing agent from which the antigen is derived or derivable.

The foreign gene may be derived or derivable from infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus, avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian influenza virus. avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, chick anemia agent, Salmonella spp., E. coli, Pasteurella spp., Bordetella spp.. Eimeria spp., Histomonas spp., Trichomonas spp., poultry nematodes, cestodes, trematodes, poultry mites/lice, poultry protozoa.

The present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome containing a deletion or other alteration in the unique short region of the viral genome, wherein the deletion or alteration is in the glycoprotein gG gene, so that upon replication, the recombinant virus produces no glycoprotein gG. A preferred embodiment" of this invention is a vaccine which comprises a suitable carrier and an effective immunizing amount of any one of the following viruses: recombinant infectious laryngotracheitis viruses designated S-ILT-014, S-ILT- 002, S-ILT-009. S-ILT-015 and S-ILT-017.

The present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion or other alteration in the unique short region of the WO 96/29396 PCTIUS96/03916 viral genome. wherein the deletion or alteration is in the glycoprotein gl gene so that upon replication, the recombinant virus produces no glycoprotein gl.

The present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion or other alteration in the unique short region of the viral genome. wherein the deletion or alteration is in the glycoprotein gG gene and the glycoprotein gI gene so that upon replication, the recombinant virus produces no glycoprotein gG and glycoprotein gI.

The present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the US2 gene, UL47-like gene, or glycoprotein gene.

The present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the US2 gene, ORF4 gene, UL47-like gene, or glycoprotein g60 gene, and insertion of a foreign gene into a non-essential site in the viral genome.

The foreign gene may be derived or derivable from infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease WO 96/29396 PCTfUS96/03916 -26virus, avian encephalomyelitis virus, avian reovirus. avian paramyxovirus, avian influenza virus, avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, chick anemia agent, Salmonella spp., E. coli, Pasteurella spp., Bordetella spp., Eimeria spp., Histomonas spp., Trichomonas spp.. poultry nematodes, cestodes, trematodes, poultry mites/lice, poultry protozoa.

The present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains an insertion of a foreign gene into a non-essential site in the viral genome. The foreign gene encodes an antigenic polypeptide which induces host cell production of protective antibodies against an avian disease causing agent from which the antigen is derived or derivable.

The foreign gene may be derived or derivable from infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus, avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian influenza virus. avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, chick anemia agent. Salmonella spp. E. coli, Pasterurella spp., Bordetella spp. Eimeria spp. Histomonas spp., Trichomonas spp, Poultry nematodes, cestodes. trematodes, poultry mites/lice, poultry protozoa.

The present invention further provides a method of immunizing an animal against infectious laryngotracheitis virus which comprises administering to chickens or other poultry an effective immunizing dose of any of the vaccines of the present invention.

The present invention further provides a method for distinguishing chickens or other poultry which are vaccinated with an effective immunizing amount of a recombinant virus which produces no glycoprotein gG from those which are infected with a naturally-occurring infectious laryngotracheitis virus. This method comprises analyzing a sample of body fluid from the chickens or other WO 96/29396 PCT/US96/03916 -27poultry for the presence of glycoprotein gG of the infectious laryngotracheitis virus and at least one other antigen normally expressed in chickens or other poultry infected by a naturally-occurring infectious laryngotracheitis virus. The presence of antigen which is normally expressed in chickens.or other poultry infected by a naturally-occurring infectious laryngotracheitis virus and the absence of glycoprotein gG in the body fluid is indicative of being vaccinated with the recombinant vaccine and not infected with a naturally-occurring infectious laryngotracheitis virus. The presence of glycoprotein gG and the antigen in the body fluid may be determined by detecting in the body fluid antibodies specific for the antigen and glycoprotein gG.

The present invention further provides a method for distinguishing chickens or other poultry which aie vaccinated with an effective immunizing amount of a recombinant infectious laryngotracheitis virus which produces no glycoprotein gI from those which are infected with a naturally-occurring infectious laryngotracheitis virus. This method comprises analyzing a sample of body fluid from the chickens or other poultry for the presence of glycoprotein gI of the infectious laryngotracheitis virus and at least one other antigen normally expressed in chickens or other poultry infected by a naturally-occurring infectious laryngotracheitis virus. The presence of the antigen which is normally expressed in chickens or other poultry infected by a naturallyoccurring infectious laryngotracheitis virus and the absence of glycoprotein gl in the body fluid is indicative of being vaccinated with the recombinant vaccine and not infected with a naturally-occurring infectious laryngotracheitis virus.

The presence of the antigen and glycoprotein gI in the body fluid may be determined by detecting in the body fluid antibodies specific for the antigen and glycoprotein gl.

The present invention further provides a method for distinguishing chickens or other poultry which are vaccinated with an effective immunizing amount of a recombinant virus which produces no glycoprotein gG and no glycoprotein gI from those which are infected with a naturally-occurring infectious WO 96/29396 PCTIUS96/03916 -28laryngotracheitis virus. This method comprises analyzing a sample of body fluid from the chickens or other poultry for the presence of glycoprotein gG and gl of the infectious laryngotracheitis virus and at least one other antigen normally expressed in an animal infected by a naturally-occurring infectious laryngotracheitis virus. The presence of the antigen which is normally expressed in chickens or other poultry by a naturally-occurring infectious laryngotracheitis virus and the absence of glycoprotein gG and gI in the body fluid is indicative of being vaccinated with the vaccine and not infected with a naturally-occurring infectious laryngotracheitis virus. The presence of the antigen and glycoprotein gG and gI in the body fluid may be determined by detecting in the body fluid antibodies specific for the antigen and glycoprotein gG and gI.

The present invention further provides a homology vector for producing a recombinant infectious laryngotracheitis virus by inserting a foreign DNA into the unique short region of the infectious laryngotracheitis genomic DNA, which comprises a double-stranded DNA molecule consisting essentially of a doublestranded foreign gene, which is flanked on either side by the double-stranded DNA homologous to the DNA located in the unique short region of the genomic DNA. provided, however, that the flanking sequences are not homologous to the glycoprotein gD gene, the glycoprotein gE gene, the protein kinase gene, and the ORF10 gene. The foreign gene may encode a screenable marker, such as E coli B-galactosidase or E. coli B-glucuronidase.

The present invention further provides a homology vector for producing a recombinant infectious laryngotracheitis virus by deleting DNA which encodes a screenable marker, which has been inserted into the infectious laryngotracheitis virus genomic DNA, which comprises a double stranded DNA molecule consisting essentially of a double-stranded DNA to be deleted, which is flanked on each side by a double stranded DNA homologous to the infectious laryngotracheitis virus glycoprotein gG gene, glycoprotein gI gene, US2 gene, or UL-47 like gene. Preferred embodiments of this invention are the homology WO 96/29396 PCTIUS96/03916 -29vectors designated Homology Vector 544-55.12, Homology Vector 562-61.1 F, Homology Vector 472-73.27, Homology Vector 560-52.F1 and Homology Vector 579-14.G2.

This invention provides an isolated nucleic acid molecule encoding a US gene (SEQ ID NOs:60 and 70), AvSp gene (SEQ ID NOs: 61 and 71), US2 gene (SEQ ID NO:62), PK gene (SEQ ID NO:63), UL47 gene (SEQ ID NO:64), gG gene (SEQ ID NO:65), ORF5 gene (SEQ ID NO: 66), gD gene (SEQ ID NO:67), gl gene (SEQ ID NO:68), gE gene (SEQ ID NO:69), or ORF9 gene (SEQ ID This invention provides an isolated polypeptide encoded by the US10 gene (SEQ ID NOs:60 and 70). AvSp gene (SEQ ID NOs: 61 and 71), US2 gene (SEQ ID NO:62). PK gene (SEQ ID NO:63), UL47 gene (SEQ ID NO:64), gG gene (SEQ ID NO:65), ORF5 gene (SEQ ID NO: 66), gD gene (SEQ ID NO:67), gl gene (SEQ ID NO:68), gE gene (SEQ ID NO:69), or ORF9 gene (SEQ ID WO 96/29396 PCT/US96/03916 EXPERIMENTAL DETAILS Materials and Methods PREPARATION OF INFECTIOUS LARYNGOTRACHEITIS VIRUS STOCK SAMPLES. Infectious laryngotracheitis virus stock samples were prepared by infecting primary chicken embryo kidney cells (CEK; obtained from Spafas, Inc.) or primary chicken kidney cells (CK; obtained from chicks hatched from fertile eggs supplied by Hyvac) (50) in 225 cm 2 flasks with ml of viral stock containing 10'-106 pfu in IX Eagle's Basal Medium (modified) with Hank's salts (BME), 10% bromoethylamine(BEI)-treated fetal bovine serum (FBS), 1% glutamine stock, 2% pennicillin/streptomycin (P/S) stock, and 1% sodium bicarbonate stock (these components are obtained from Irvine Scientific or an equivalent supplier, and hereafter the growth medium is referred to as complete BME medium). Viral stocks were then harvested days later. Infected media and cells were resuspended in complete medium containing 20% sterile whole milk and stored frozen at -70 0

C.

PREPARATION OF INFECTIOUS LARYNGOTRACHEITIS VIRUS DNA. Four to five days after viral infection, cells and media were scraped from each flask into 15 ml conical centrifuge tubes and pelleted at 1700 x g for minutes at 4°C. Because as much as 50% of the virus may be in the media, the supernatants were saved and treated as will be described below. The cell pellets were resuspended in I ml PBS per tube, combined and centrifuged again at 1700 x g for 5 minutes. The pellets were resuspended in 1 ml/flask of a buffer containing 10 mM Tris-HCl pH 7.5, 1 mM EDTA, and 1.5 mM MgCl 2 and were incubated for 15 minutes at 4°C. Twenty five pis of 20% NP40 per flask was added, and the mixture was then homogenized in a dounce homogenizer using an A pestle. The preparation was centrifuged at 1700 x g for 10 minutes at 4°C and the supernatant was retained. Ten 4l of 0.5 M EDTA. 50 ul of SDS, and 25 pl of 10 mg/ml proteinase K was added to the supernatant (per original flask). In some cases, this was then combined with virus obtained from WO 96/29396 PCTfUS96/03916 -31the cell media supernatants (see above). The mixture was then treated at for 1-16 hours, followed by two extractions with phenol saturated with 100 mM Tris-HCl, pH 8. DNA in the aqueous phase was then precipitated with added 3 M sodium acetate (1/10th volume) and 2.5 vols of 100% ethanol.

To obtain virus from the media, the cell media supernatants were centrifuged at 23,500 x g for 30 minutes, and drained well. The pellet was resuspended in the above proteinase K-containing mixture as described. The DNA pellets were resuspended in 20 uil TE/flask and could be used at this point for further experiments or treated further to remove RNA with pancreatic RNase A.

followed by phenol extraction and ethanol precipitation to obtain the DNA.

To prepare viral DNA minipreps, infected 10 cm. dishes were scraped into conical centrifuge tubes and centrifuged 5 minutes at 1000 x g. Cell media supernatants were kept and treated as above. The cell pellets were each resuspended in 0.5 ml of 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% and incubated 10 minutes at room temperature. Ten pl of 10 mg/ml RNase A was added, and the preparation was centrifuged 5 minutes at 1000 x g. Twentyfive pl of 20 SDS and 25 4l of 10 mg/ml proteinase K was added to the supernatant, and the entire preparation was added to the viral pellet from the cell media if it was used. The mixture was incubated at 55-65°C for one hour, extracted with buffer-saturated phenol and precipitated by the addition of 1 ml of ethanol. The DNA pellet was resuspended in 20 pl of TE and stored at 4°C.

POLYMERASE FILL-IN REACTION. DNA was resuspended in buffer containing 50 mM Tris pH 7.4, 50 mM KC1, 5 mM MgCl 2 and 400 micromolar each of the four deoxyribonucleotides. Ten units of Klenow DNA polymerase (Gibco BRL) were added and the reaction was allowed to proceed for 15 minutes at room temperature. The DNA was phenol extracted and ethanol precipitated as above.

DNA SEQUENCING. Sequencing was performed using the Sequenase Kit (US WO 96/29396 PCT/US96/03916 -32- Biochemicals) and a 3 S-dATP (New England Nuclear). Reactions using both the dGTP mixes and the dITP mixes were performed to clarify areas of compression. Alternatively, compressed areas were resolved on formamide gels.

Templates were double-stranded plasmid subclones or single stranded M I3 subclones, and primers were either made to the vector just outside the insert to be sequenced, or to previously obtained sequence. Sequence obtained was assembled and compared using Dnastar software. Manipulation and comparison of sequences obtained was performed with IBI MacVector, Superclone and Supersee Align programs from Coral Software.

MOLECULAR BIOLOGICAL TECHNIQUES. Techniques for the manipulation of bacteria and DNA, including such procedures as digestion with restriction endonucleases, gel electrophoresis, extraction of DNA from gels, ligation, phosphorylation with kinase, treatment with phosphatase, growth of bacterial cultures, transformation of bacteria with DNA, and other molecular biological methods are described (42, 43). The polymerase chain reaction (PCR) was used to introduce restriction sites convenient for the manipulation of various DNAs In general amplified fragments were less than 500 base pairs in size and critical regions of amplified fragments were confirmed by DNA sequencing. Except as noted. these techniques were used with minor variation.

SOUTHERN BLOTTING OF DNA. The general procedure for Southern blotting was taken from Maniatis el al. (1982) and Sambrook, et.al.(198 9 (42, 43). DNA was blotted to nylon membrane (Biorad Zetaprobe) in 0.4M NaOH and prehybridized for 5 minutes in a solution containing 0.25 M Na2HPO pH 7.2, 1 mM EDTA, 7% SDS at 65°C. Labeled probe was added that had been labeled by random priming using a GeniusTM non-radioactive labeling kit from Boehringer-Mannheim. Hybridization was overnight at 65°C. Filters were washed twice with 40 mM NaHPO pH 7.2, 1 mM EDTA, 5% SDS and then twice with 40 mM NaHPO 4 pH 7.2, 1 mM EDTA, 1% SDS for 30 minutes each at 65*C. Detection of bound probe was performed using the Boehringer WO 96/29396 PCTfUS96/03916 -33- Mannheim GeniusTM non-radioactive detection kit.

DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS. The method is based upon the CaCl 2 procedure of Chen and Okayama (1987) (45) with the following modifications. Generation of recombinant ILT virus is dependent upon homologous recombination between ILT viral DNA and the plasmid homology vector containing the desired foreign DNA flanked by the appropriate herpesvirus cloned sequences. Plasmid DNA (10-40 mg) was added to 250 ml of a solution having a final concentration of 0.25 M CaCl,. An equal volume of a buffer containing 50 mM MOPS (pH 6.95), 280 mM NaCI, and 1.5 mM NaHPO 4 was added to the DNA/CaCl, solution. After 10 minutes at room temperature, the mixture was added dropwise to a 6 cm dish of CEK cells on maintenance media, and placed at 39 0 C for 4 to 5 hours. The cells were rinsed once with PBS, once with 20% glycerol in PBS for 2 minutes, rinsed again with PBS and fed with maintenance media. 1.5 ml of ILT viral stock was added to the media, and the cells were incubated overnight. The next day, fresh maintenance media was added, and the cells were incubated for two more days.

The transfection stock was harvested, aliquoted, and frozen at PROCEDURE FOR GENERATING ILTV SUBGENOMIC DNA FRAGMENTS. The ability to generate herpesviruses by cotransfection of cloned overlapping subgenomic fragments has been demonstrated for pseudorabies virus If deletions and/or insertions are engineered directly into the subgenomic fragments prior to the cotransfection, this procedure results in a high frequency of viruses containing the genomic alteration, greatly reducing the amount of screening required to purify the recombinant virus. The procedure of overlapping cosmids to map restriction enzyme sites was employed.

A library of subclones containing overlapping ILTV subgenomic fragments was generated as follows. USDA ILTV Strain 83-2 has been designated S-ILT-001.

Approximately 20 4g of ILTV DNA (obtained from S-ILT-001) in 0.5 ml of WO 96/29396 PCTIUS96/03916 -34mM Tris-HCl pH 8.0, 1 mM EDTA (TE) was sheared by passing it twice through a 25 guage needle as previously described The DNA was centrifuged through a 15-40% glycerol gradient in 50 mM Tris-HCl pH 8.0, 1 mM EDTA, and 0.3 M NaCi for 5.5 hours at 274,000 x g. Fractions were analyzed on a 0.3% agarose gel, and those containing DNA of 35-50 kb were pooled, diluted twofold with TE, and precipitated with one tenth volume of 3 M sodium acetate and 2.5 volumes of ethanol. The tubes were centrifuged for one hour at 109,000 x g at 10°C Pellets were resuspended, transferred to microfuge tubes, and precipitated with one tenth volume of 3 M sodium acetate and 2.5 volumes of ethanol. The DNA was resuspended in TE. DNA ends were made blunt ended by the POLYMERASE FILL-IN REACTION. The DNA was purified by extraction with both buffer saturated phenol and ether, precipitated with sodium acetate and ethanol as above, and resuspended in TE. Half of this material was ligated with 3 mg of vector, pSY1626, by the DNA ligation reaction. The vector used was pSY1626, which was made as follows. Cosmid pHC79 (Gibco BRL) was cut with HindlII and AvaI to remove the tetracycline gene, and the ends were filled in with Klenow polymerase (FILL IN REACTION). The polylinker from pWE15 (Stratagene) was ligated into this vector. The polylinker was isolated by digestion with EcoRI, the ends were filled in with Klenow polymerase (FILL IN REACTION), and the fragment was purified on a LMP-agarose gel. DNA ligation was performed in the presence of melted agarose. The resulting cosmid, pSY1005, was modified at the EcoRI site to create pSY1626 by blunt-ended insertion of a 1.5 kb HindIII-BamHI fragment from pNEO (P-L Biochemicals) containing the neomycin resistance gene. pSY1626 was cut and made blunt at the BamHI site. and ligated with sheared ILTV fragments as described above. The ligation mixture was packaged using Gigapack XL (Stratagene) according to the manufacturers instructions.

The packaging mixture was added to AGI cells (Stratagene) grown in the presence of maltose, and colonies were selected on LB plates containing kanamycin. Cosmid subclones containing ILTV DNA were identified by comparing restriction enzyme maps of individual cosmid clones to each other and to ILVTV genomic DNA to obtain a contiguous sequence of ILTV WO 96/29396 PCT/US96/03916 genomic DNA.

SCREEN FOR RECOMBINANT ILTV EXPRESSING ENZYMATIC MARKER GENES. When the E. coli p-galactosidase or p-glucuronidase (uidA) marker gene was incorporated into a recombinant virus the plaques containing the recombinants were visualized by a simple assay. The enzymatic substrate was incorporated (300 pg/ml) into the agarose overlay during the plaque assay. For the lacZ marker gene the substrate Bluogal T (halogenated indolyl-p-D-galactosidase, Gibco BRL) was used. For the uidA marker gene the substrate X-Glucuro Chx (5-bromo-4-chloro-3-indolyl-p-D-glucuronic acid Cyclohexylammonium salt, Biosynth AG) was used. Plaques that expressed active marker enzyme turned blue. The blue plaques were then picked onto fresh cells and purified by further blue plaque isolation. In recombinant virus strategies in which the enzymatic marker gene was removed, the assay involves plaque purifying white plaques from a background of parental blue plaques.

Viruses were typically purified with five to ten rounds of plaque purification.

SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT ILTV USING BLACK PLAQUE ASSAYS. To analyze expression of foreign antigens expressed by recombinant ILT viruses, monolayers of CEK cells were infected with recombinant ILT virus, overlaid with nutrient agarose media and incubated for 3-5 days at 39 0 C. Once plaques have developed, the agarose overlay was removed from the dish, the monolayer rinsed once with PBS, fixed with 100% methanol for 10 minutes at room temperature and the cells air dried.

After re-hydrating the plate with PBS, the primary antibody was diluted to the appropriate dilution with PBS plus Blotto and incubated with the cell monolayer for 2 hours to overnight at room temperature. Unbound antibody was removed from the cells by washing four times with PBS at room temperature. The appropriate secondary antibody conjugate was diluted 1:500 with PBS and incubated with the cells for 2 hours at room temperature. Unbound secondary antibody was removed by washing the cells three times with PBS at room temperature.The monolayer was rinsed in color development buffer (100mM WO 96/29396 PCT/US96/03916 -36- Tris pH 9.5/ 100mM NaCI/ 5mM MgC12), and incubated 10 minutes to overnight at room temperature with freshly prepared substrate solution (0.3 mg/ml nitro blue tetrazolium 0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphatase in color development buffer).The reaction was stopped by replacing the substrate solution with TE (10mM Tris, pH7.5/ 1 mM EDTA). Plaques expressing the correct antigen stain black.

PURIFICATION OF ILTV gG FROM ILT VIRUS OR RECOMBINANT VIRUSES EXPRESSING ILTV gG. ILTV gG was purified from the media of cells infected with either wild type ILTV or with FPV or SPV vectors expressing ILTV gG. Cells were allowed to go to complete cytopathic effect (CPE), the media was poured off, and cell debris was pelleted in a table-top centrifuge. The media was concentrated in an Amicon concentrator using a ultrafiltration membrane at 15 psi. The concentrate was dialyzed against 20 mM Tris-HCI, pH 7.0 and loaded onto a DEAE-Sephacel (Pharmacia) column equilibrated with the same buffer. The material was eluted using a salt gradient from 0 to 1.5 M NaCI in 20 mM Tris-HCl, pH 7.0. Three ml fractions were collected and assayed by Western blot. A peptide antibody against ILTV gG was used to identify fractions containing ILTV gG. Fractions were pooled and further concentrated in a Centricon-10 microconcentrator (Amicon).

GROWTH OF CHICKEN KIDNEY CELLS AND ILT VIRUS. An ILTV virus, designated fowl laryngotracheitis challenge virus, lot number 83-2, was obtained from the National Veterinary Services Laboratories, USDA/APHIS, Ames, Iowa. ILTV viruses were grown in primary chicken kidney cells (CK) obtained by dissection of kidneys from 6-9 day old SPF chicks, obtained from Hy-Vac Laboratory Eggs Co. Fresh kidney cells were minced and disassociated with 5 mg/ml trypsin and were then pelleted and resuspended at 1.3 x 106 cells/ml. Growth media (GM) was IX Eagle's Basal Medium (modified) with Hank's salts, with added 10% binary ethyleneimine-treated fetal bovine serum (FBS), 2 mM glutamine. 200 units/ml penicillin, 200 mg/ml streptomycin, and 8.9 mM sodium bicarbonate After resuspension. cells were plated and WO 96/29396 PCT/US96/03916 -37incubated at 39"C. Cells were rinsed and fed after 24 hours with maintenance media which is GM with 1% FBS. CKs were inoculated with ILTV at 0.01 to 0.1 MOI and viral stocks were harvested 4-5 days later by scraping and sonicating. Titers were typically 10'-106 pfu/ml.

PREPARATION OF VIRAL DNA. Cells and media from infected flasks were pelleted at 1700 g for 5' at 4"C. Supernatant and cell pellet were initially treated separately. Virion particles were centrifuged out of the supernatant at 23,500 g for 30 minutes. The original cell pellet was rinsed with PBS and spun again. This pellet was resuspended in 1 ml/flask of a buffer containing 10 mM Tris-HCl pH 7.5, 1 mM EDTA, and 1.5 mM MgCl 2 and incubated 15', 4°C.

To this was added 25 ul/flask of 20% NP40, and the mixture was dounce homogenized using an A pestle. The preparation was centrifuged at 1700 g, 4"C, and the supernatant was retained and the pellet discarded. To the supernatant was added (per original flask) 10 pl of 0.5 M EDTA, 50 .l of SDS, and 25 pl of 10 mg/ml proteinase K. This mixture was used to resuspend the pellet of viral particles obtained by high speed centrifugation of the first supernatant. The mixture was treated at 65°C for 1-16 hours, extracted twice with buffer-saturated phenol, and precipitated with added salt and ethanol. The resulting DNA pellet was resuspended in 100 4l TE/flask. This was treated further to remove RNA with pancreatic RNase A, followed by phenol extraction and ethanol precipitation to obtain the DNA.

CREATION OF THE COSMID LIBRARY. The cosmid library of ILTV DNA was created following the protocol of van Zijl et al., Approximately gg of ILTV DNA in 0.5 ml of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (TE) was sheared by passing it twice through a 25 gauge needle. The DNA was centrifuged through a 15-40% glycerol gradient in 50 mM Tris-HCl, pH 1 mM EDTA, 0.3 M NaCl for 5.5 h at 274,000 g. Fractions were analyzed on a 0.3% agarose gel, and those containing DNA of 35-50 kb were pooled, diluted twofold with TE, and precipitated with added salt and ethanol. The tubes were spun 1 h at 10 0 C and 109,000 g. Pellets were resuspended and WO 96/29396 PCT/US96/03916 -38reprecipitated with added salt and ethanol. The DNA was resuspended in TE and the ends were made blunt by treatment with T4 DNA polymerase for 2 h at 15"C, in the presence of appropriate buffer and 25 pM dNTP, followed by treatment with Klenow polymerase for 16 h at 15'C using 0.25 mM dNTP.

The DNA was extracted with phenol and then ether, precipitated with added salt and ethanol, and resuspended in TE. This material was ligated overnight with 3 pg of cosmid vector pSY1626. Cosmid pSY1626 was made by digesting cosmid pHC79 (BRL) with HindII and Aval to remove the tetracycline gene.

The remaining fragment and the EcoRI digested polylinker from (Stratagene) were filled in with Klenow polymerase and ligated together. The resulting cosmid vector, pSY1005, was modified at the EcoRI site to create pSY1626 by blunt-ended insertion of a 1.5 kb HindlIll-BamHI fragment from pNEO (P-L Biochemicals) containing the kanamycin resistance gene. PSY1626 was cut and made blunt at the BamH1 site for use as the cosmid vector. The ligation mixture was packaged using Gigapack XL (Stratagene) according to the manufacturer's directions. Colonies were selected on LB plates containing kanamycin.

SEQUENCING. Manual sequencing was performed using 35 S-dATP (NEN) with the BRL Sequenase Kit which uses the dideoxyribonucleotide chain termination method described by Sanger et al. Reactions using both dGTP and dITP mixes were performed to clarify areas of compression.

Alternatively, compressed areas were resolved on 8% acrylamide gels that were in formamide. Automatic fluorescence sequencing was performed using an Applied Biosystems (ABI) 373A DNA Sequencer. Subclones were made to facilitate sequencing. Internal primers were synthesized on an ABI 392 DNA synthesizer. Sequence was obtained for both strands and was assembled using DNAstar software. Manipulation and comparison of sequences was performed with DNAstar programs, Superclone and Supersee programs from Coral Software. Comparisons with GenBank were performed at the NCBI using the BLAST network service (58).

WO 96/29396 PCT/US96/03916 -39- HOMOLOGY VECTOR 501-94. The plasmid 501-94 was constructed for the purpose of deleting a portion of the thymidine kinase (TK) gene coding region from the ILT virus It incorporates the HCMV IE promoter and a screenable marker, the E. coli lacZ gene, flanked by ILT virus DNA. The HCMV IE promoter-E. coli lacZ gene is inserted in the opposite transcriptional orientation to the ILTV TK gene. Upstream of the marker gene is an approximately 1087 base pair fragment of ILTV DNA which includes the first 77 amino acid codons of the ILTV TK gene. Downstream of the lacZ gene is an approximately 675 base pair fragment of ILTV DNA which includes amino acid codons at the 3' end of the ILTV TK gene. When this plasmid is used according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS, it will replace the DNA coding for amino acids 78 to 285 of the ILTV TK gene with DNA coding for the lacZ gene. The lacZ marker gene is under the control of the human cytomegalovirus (HCMV) immediate early (IE) gene promoter and also contains the pseudorabies virus (PRV) gX gene polyadenylation signal at the 3' end of the gene. A detailed description of the plasmid is given in Figures 5A-5D. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (42, 43). The plasmid vector is derived from an approximately 3002 base pair HindIII fragment of pSP64/65 (Promega). Fragment I is an approximately 1087 base pair HindlIl to BclI subfragment of the ILTV 2.4 kb HindIII fragment.

Fragment 2 is an approximately 5017 base pair Sall to Sall fragment containing the HCMV IE promoter. p-galactosidase (lacZ) marker gene, and PRV gX polyadenylation signal (see Figures 5A-5D). Fragment 3 is an approximately 675 base pair BclI to Hindlll subfragment of the ILTV 2.4 kb HindIII fragment.

HOMOLOGY VECTOR 544-55.12. The plasmid 544-55.12 was constructed for the purpose of deleting a portion of the US2 gene coding region from the ILT virus and inserting a foreign DNA. It incorporates a screenable marker, the E. coli uidA gene flanked by ILT virus DNA. The PRV gX promoter-E. coli uidA gene is inserted in the opposite transcriptional orientation to the ILTV WO 96/29396 PCT/US96/03916 US2 gene. Upstream of the uidA gene is an approximately 2300 base pair fragment of ILTV DNA which includes 41 amino acid codons at the 3' end of the US2 gene (SEQ ID NO 2: aa. 188-229). Downstream of the uidA gene is an approximately 809 base pair fragment of ILTV DNA which includes 22 amino acid codons at the 5' end of the US2 gene (SEQ ID NO 2: aa. 1-22).

When this plasmid is used according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS, it will replace the ILTV US2 DNA coding for amino acids 23 to 187 with DNA coding for the E. coli uidA gene. The uidA marker gene is under the control of the pseudorabies virus (PRV) gX promoter and also contains the herpes simplex virus type I thymidine kinase (HSV-1 TK) gene polyadenylation signal at the 3' end of the gene. A detailed description of the plasmid is given in Figures 6A-6D. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (42. 43). The plasmid vector is derived from an approximately 2958 base pair Asp7181 restriction fragment of a pSP 18/pSP 19 fusion such that the multiple cloning site is EcoRI/SacI/Asp718I/SacI/EcoRI. Fragment 1 is an approximately 2300 base pair Asp7181 to Dral subfragment (SEQ ID NO 1: Nucl. 1-405) of the ILTV 2.5 kb Asp718I fragment. Fragment 2 is an approximately 3039 base pair Xbal fragment containing the PRV gX promoter, the E. coli uidA gene. and the HSV-1 TK polyadenylation site (See Figures 6A- 6D). Fragment 3 is an approximately 809 base pair Xbal to Asp7181 subfragment of the ILTV 1097 bp Asp7181 fragment (SEQ ID NO 1: Nucl.

905-1714).

HOMOLOGY VECTOR 562-61.IF. The plasmid 562-61.1F was constructed for the purpose of deleting part of the gl gene from the ILT virus and inserting a foreign DNA. It incorporates a screenable marker, the E. coli uidA gene, flanked by ILT virus DNA. The PRV gX promoter-E. coli uidA gene is transcribed in the opposite direction to the ILTV gI gene promoter. The 983 base pair deletion begins 12 base pairs upstream of the translation initiation codon and deletes 324 of 363 amino acid codons at the 5' end of the ILTV gl gene (SEQ ID NO 11: aa. 325-363). When this plasmid is used according to WO 96/29396 PCT[US96/03916 -41the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS, it will replace the DNA coding for the ILTV gI gene with DNA coding for the E. coli uidA gene. A detailed description of the plasmid is given in Figures 7A-7D. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (42, 43). The plasmid vector is derived from an approximately 2647 base pair Asp7181 to HindIII fragment of pUC19.

Fragment 1 is an approximately 1619 base pair Asp718I to XbaI subfragment of the ILTV 8.0 kb Asp718I fragment (SEQ ID NO 1: Nucl. 7556-9175).

Fragment 2 is an approximately 691 base pair Xbal to Xhol fragment (SEQ ID NO 1: Nucl. 9175-9861) generated by the polymerase chain reaction (PCR).

The template was the ILTV 8.0 kb Asp7181 fragment. The upstream primer 92.09 (5'-CCTAGCACCCTTGTATCGCG-3'; SEQ ID NO. 55) sits down at a site 821 base pairs upstream of the ILTV gI gene and synthesizes DNA toward the 3' end of the gene. The downstream primer 92.11 CGCCTCGAGTCCCAATGAATAGGCATTGG-3'; SEQ ID NO. 56) sits down at a site 12 base pairs upstream of the translation start site of the ILTV gI gene and synthesizes DNA toward the 5' end of the gD gene. The product of the PCR reaction is 818 base pairs. This DNA fragment is digested with Xbal at the end (a restriction enzyme site present in the ILTV DNA) and XhoI at the 3' end (a restriction enzyme site created in the PCR primer-see underlined sequence) to create an approximately 691 base pair Xbal to Xhol fragment.

Fragment 3 is an approximately 3051 base pair Sall fragment containing the PRV gX promoter, the uidA gene. and the HSV-1 TK polyadenylation site (See Figures 6A-6D). Fragment 4 is an approximately 624 base pair Xhol to Hindlll fragment generated by PCR (SEQ ID NO 1: Nucl. 10,847-11,461). The template was the ILTV 8.0 kb Asp7181 fragment. The upstream primer 92.10 (5'-CGCCTCGAGGACCCATGGTTGCGTGCG-3'; SEQ ID NO. 57) sits down at a site 117 base pairs upstream from the translation termination codon within the ILTV gl gene. The downstream primer 92.08 CTCGTCCGAACGAGTTACAG-3'; SEQ ID NO. 58) sits down at a site 604 base pairs downstream of the translation termination site of the ILTV gl gene and within the ILTV gE gene. The PCR product (729 base pairs) is digested WO 96/29396 PCT/US96/03916 -42with XhoI which is a unique site generated by the upstream PCR primer (underlined) and with HindIII at a site within the ILTV gE gene. Restriction endonuclease digestion with Xhol and HindIII creates an approximately 624 base pair Fragment 4. Fragment 5 is an approximately 2700 base pair HindIII subfragment of the ILTV 8.0 kb Asp7181 fragment (SEQ ID NO 1: Nucl.

11,461-13,473 plus unsequenced DNA).

HOMOLOGY VECTOR 472-73.27. The plasmid 472-73.27 was constructed for the purpose of deleting a portion of the glycoprotein G (gG) gene coding region from the ILT virus and inserting a foreign DNA. It incorporates a screenable marker, the E. coli lacZ gene, flanked by ILT virus DNA. The HCMV IE promoter-E. coli lacZ gene is transcribed in the same direction to the ILTV gG gene promoter. The 874 base pair deletion of the ILTV gG gene extends from 60 nucleotides upstream of the translation initiation site to 814 nucleotides into the amino acid coding sequence, removing the coding capacity of 271 of 292 amino acids of the gG protein (SEQ ID NO When this plasmid is used according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS, it will replace the DNA coding for amino acids 1 to 271 of the ILTV gG gene with DNA coding for the E. coli lacZ gene. A detailed description of the plasmid is given in Figures 4A-4D. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (42. 43). The plasmid vector is derived from an approximately 2686 base pair Asp7181 restriction fragment of pUC 19 (Gibco, BRL).

Fragment I is an approximately 2830 base pair Asp7181 to Nhel subfragment of the ILTV 5164 bp Asp718I fragment (SEQ ID NO 1: Nucl. 1714-4544).

Fragment 2 is an approximately 5017 base pair Sall to Sall fragment containing the HCMV IE promoter, E. coli P3-galactosidase (lacZ) marker gene, and PRV gX polyadenylation signal (see Figures 4A-4D). Fragment 3 is an approximately 1709 base pair Sall to Asp7181 subfragment of the ILTV 5164 bp Asp7181 fragment (SEQ ID NO 1: Nucl. 5419-6878).

HOMOLOGY VECTOR 560-52.F1. The plasmid 560-52.FI was constructed WO 96/29396 PCTIUS96/03916 -43for the purpose of deleting part of the UL47-like gene, all of ORF4, and part of the ILTV gG gene from the ILT virus and inserting a foreign DNA. It incorporates a screenable marker, the E. coli uidA gene, flanked by ILT virus DNA. The PRV gX promoter-E. coli uidA gene is transcribed in the opposite direction to the ILTV UL47-like, ORF4, and gG gene promoters. The 2640 base pair deletion removes 442 of 511 amino acid codons at the 3' end of the UL47-like gene (SEQ ID NO the entire coding sequence of the ORF4 gene (SEQ ID NO 5) and 271 of 293 amino acid codons at the 5' end of the ILTV gG gene (SEQ ID NO When this plasmid is used according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS, it will replace the DNA coding for the ILTV UL47-like, ORF4 and gG genes with DNA coding for the PRV gX promoter-E. coli uidA gene. A detailed description of the plasmid is given in Figures 8A-8D. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (42, 43). The plasmid vector is derived from an approximately 2958 base pair Asp7181 restriction fragment of pSPl8/pSPI9 such that the multiple cloning site is EcoRI/SacI/Asp7181/SacI/EcoRI. Fragment 1 is an approximately 1066 base pair Asp718I to BssHII subfragment of the ILTV 5164 bp Asp7181 fragment (SEQ ID NO 1: Nucl. .1714-2777). Fragment 2 is an approximately 123 base pair Sail to Bcil subfragment of the ILTV 5164 bp Asp7181 fragment. Fragment 3 is an approximately 3027 base pair BamHI fragment containing the PRV gX promoter, the uidA gene, and the HSV-1 TK polyadenylation site (See Figures 8A-8D). Fragment 4 is an approximately 1334 base pair Bcl to Asp7181 subfragment of the ILTV 5164 bp Asp7181 fragment (SEQ ID NO 1: Nucl.

5544-6878).

HOMOLOGY VECTOR 579-14.G2. The plasmid 579-14.G2 was constructed for the purpose of deleting the entire gG gene and a portion of the g60 gene from the ILT virus and inserting a foreign DNA. It incorporates a PRV gX promoter and a screenable marker, the E. coli uidA gene, flanked by ILT virus DNA. The PRV gX promoter-E. coli uidA gene is transcribed in the same direction to the ILTV gG and g60 gene promoters. The 3351 base pair deletion WO 96/29396 PCTIUS96/03916 -44includes the entire coding sequence of the ILTV gG gene (SEQ ID NO 7) and 733 of 986 amino acid codons from the 5' end of the g60 gene (SEQ ID NO When this plasmid is used according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS, it will replace the DNA coding for the ILTV gG gene and amino acids 1 to 733 of the ILTV g60 gene with DNA coding for the E. coli uidA gene. A detailed description of the plasmid is given in Figures 9A-9D. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (42, 43). The plasmid vector pUC19 (Gibco, BRL) is derived from an approximately 2677 base pair Asp7181 to BamHI fragment. Fragment 1 is an approximately 2830 base pair Asp7181 to Nhel subfragment of the ILTV 5164 bp Asp7181 fragment (SEQ ID NO 1: Nucl. 1714-4544). Fragment 2 is an approximately 3051 base pair Sail fragment containing the PRV gX promoter, E. coli p-glucuronidase (uidA) marker gene, and an HSV-1 TK polyadenylation site (See Figures 9A-9D).

15 Fragment 3 is an approximately 1709 base pair Sall to BamHI subfragment of the ILTV 4545 base pair BamHI fragment (SEQ ID NO 1: Nucl. 7895-9604).

PLASMID 544-39.13. Plasmid 544-39.13 contains the p-glucuronidase expression cassette consisting of the PRV gX promoter, E. coli p-glucuronidase (uidA) marker gene, and an HSV-1 TK polyadenylation site. A detailed description of the marker gene is given in Figures 10A- 10D. It was constructed utilizing standard recombinant DNA techniques (42, 43) by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures 10A- 10D. The plasmid vector pSP71 (Promega) is derived from an approximately 3066 base pair Xmal to Smai fragment. Fragment 1 is an approximately 422 base pair Sall to EcoRI restriction subfragment of the PRV BamHI restriction fragment #10 Note that the EcoRI site was introduced at the location indicated in Figures 12A-12D by PCR cloning.

Fragment 2 is an approximately 1826 base pair EcoRI to SmaI fragment of the plasmid pRAJ260 (Clonetech). Note that the EcoRI and Xmal sites were introduced at the locations indicated in Figures 10A-10D by PCR cloning.

Fragment 3 is an approximately 784 base pair Xmal subfragment of the HSV- I1 WO 96/29396 PCT/US96/03916 BamHI restriction fragment Q Note that this fragment is oriented such that the polyadenylation sequence (AATAAA) is located closest to the junction with the E. coli uidA gene.

PLASMID 388-65.2. Plasmid 388-65.2 contains the P-galactosidase expression cassette consisting of the HCMV immediate early (IE) promoter, the E. coli lacZ marker gene, and the PRV gX gene polyadenylation site. A detailed description of the P-galactosidase expression cassette is given in Figures 11A- 11D. It was constructed utilizing standard recombinant DNA techniques (42.

43) by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures 11A-11 ID. The plasmid vector pSP72 (Promega) is derived from an approximately 3076 base pair PstI to PstI fragment. Fragment 1 is a 1154 base pair PstI to Avail fragment derived from a HCMV 2.1 kb Pstl fragment containing the HCMV IE promoter. Fragment 2 is a 3010 base pair BamHI to Pvull fragment derived from plasmid pJF751 (49) containing the E. coli lacZ gene. Fragment 3 is an approximately 750 base pair NdeI to Sall fragment derived from PRV BamHI #7 which contains the carboxy-terminal 19 amino acids and the polyadenylation signal of the PRV gX gene.

WO 96/29396 PCTfUS96/03916 -46-

EXAMPLES

Example 1 Complete sequence of the unique short region of Infectious Laryngotracheitis Virus (ILTV): The sequence of 13,473 base pairs of contiguous DNA from the short region of the ILT virus (SEQ. ID. NO. 1) was determined. This sequence contains the entire 13,098 base pair unique short region as well as 273 base pairs of repeat region at one end and 102 base pairs of repeat region at the other end. The unique short region contains 13 methionine initiated open reading frames (ORF) of greater than or equal to 110 amino acids (excluding smaller nested ORFs). All 13 ORFs were aligned to the Entrez release 6.0 virus division of the Genbank DNA database utilizing the IBI MacVector Protein to DNA alignment option (default settings). Eight of the ORFs exhibited significant homology to one or more other virus genes (see Table The nucleotide sequence numbers referred to below begin within the internal repeat sequence and end within the terminal repeat sequence. The unique short region begins at base pair 274 of SEQUENCE ID NO. 1.

WO 96/29396 PCTIUS96/03916 -47- Table I Sequence Homology between Infectious Laryngotracheitis Virus (ILTV) Open Reading Frames in the Unique Short Region and other Viral Proteins Open Reading Start(BP) End(BP) Length(aa) Genbank Fraine(ORF) _____Allignmenta 1 (RC)b 970 281 229 EI-I- US2 2 1059 2489 476 MDV PK 3 2575 4107 510 HSV-1 UL47 4 4113 4445 110 NSc 4 (RC) 4519 4139 126 NS 4609 5487 292 PRV gX 6 5697 8654 985 ILTV 6 (RC) 7826 6948 292 HSV-2 UL39 7 8462 9766 434 PRV 8 9874 10962 362 VZV gI 8 (RC) 11150 10617 177 NS 91 11159 12658 1499 VZV eE 10 112665 13447 1260 1NS aSequence allignrnent scored to the Entrez Release 6.0 of Genbank Virus Database.

b RC=Reverse Complement.

NS=No score above 120 was found.

Other Abbreviations: EHV= Equine herpesvirus; MvDV= Mareks disease virus; HSV-1= Herpes Simplex virus 1; PRV= Pseudorabies virus; ITLTV= Infectious laryngotracheitis virus; HSV-2= Herpes Simplex virus 2; VZV= Varicella-Zoster virus; BP= base pairs; aa= amino acids.

US2 gene The US2 gene consists of 690 base pairs and codes for a protein 229 amino acids in length and molecular weight approximately 25,272 daltons (SEQ. ID.

NO. 12, 13). The ILTV US2 is homologous to the Equine herpesvirus(EHV)-1I and EHV-4 US2 proteins. The US2 gene is transcribed from nucleotide 970 to WO 96/29396 PCT/US96/03916 -48- 281 on the reverse complement strand of the ILTV unique short region (SEQ.

ID. NO. The function of the US2 gene product is unknown.

Protein kinase gene The protein kinase gene consists of 1431 base pairs from nucleotide 1059 to 2489 and codes for a protein 476 amino acids in length and molecular weight approximately 54,316 daltons (SEQ. ID. NO. The ILTV protein kinase is homologous to the protein kinases from Mareks disease virus (MDV), Equine herpesvirus(EHV)-l and Pseudorabies virus (PRV), Varicella-Zoster virus (VZV), Simian varicella virus (SVV), and Herpes Simplex virus(HSV)-l and -2.

UL47-like gene The UL47-like gene is unique in its location within the unique short region of ILT virus. The UL47-like gene in all other known herpesviruses is located within the unique long sequence. The UL47-like gene consists of 1533 base pairs from nucleotide 2575 to 4107 and codes for a protein 510 amino acids in length and molecular weight approximately 57,615 daltons (SEQ. ID. NO. 3).

ORF4 ORF4 codes for a protein of unknown function. ORF4 consists of 333 base pairs from nucleotide 4113 to 4445 and codes for an open reading frame 110 amino acids in length and molecular weight approximately 12,015 daltons (SEQ. ID. NO. 4).

ORF4 Reverse Complement ORF4 Reverse Complement (RC) codes for a protein of unknown function.

ORF4 RC consists of 380 base pairs from nucleotide 4519 to 4139 and codes for an open reading frame 126 amino acids in length and molecular weight WO 96/29396 PCTIUS96/03916 -49approximately 13.860 daltons (SEQ. ID. NOS. 14, gG gene The gG gene consists of 879 base pairs from nucleotide 4609 to 5487 and codes for a glycoprotein 292 amino acids in length and molecular weight approximately 31,699 daltons (SEQ. ID. NO. ILTV gG glycoprotein is homologous to PRV gX, Bovine herpesvirus(BHV)-1.3 gG, EHV-1 gG and EHV-4 gG. Recombinant ILTV gG protein produced in a Swinepox virus vector or a Fowlpox virus vector can be purified (see Materials and Methods) and reacts to peptide antisera to ILTV gG. The peptide antisera reacts to ILTV gG from wild type virus, but not to viruses deleted for the ILTV gG gene.

Deletion of the gG gene results in an attenuated ILT virus that is useful as a vaccine against ILT disease in chickens (see table in Example 6) and also serves as a negative marker to distinguish vaccinated from infected animals.

gene The g60 gene has been identified as glycoprotein 60 (33, 53). The g60 gene consists of 2958 base pairs from nucleotide 5697 to 8654 and codes for a glycoprotein 985 amino acids in length and molecular weight approximately 106,505 daltons (SEQ. ID. NO. 6).

ORF6 Reverse Complement ORF6 RC consists of 878 base pairs from nucleotide 7826 to 6948 and codes for an open reading frame 292 amino acids in length and molecular weight approximately 32.120 daltons (SEQ. ID. NO. 16, 17). The ILTV ORF6 RC shares limited homology to portions of the HSV-1 and HSV-2 ribonucleotide reductase large subunit (UL39).

gD gene WO 96/29396 PCT/US96/03916 The expression of the gD glycoprotein in vectored fowlpox virus or herpesvirus of turkeys (33) is sufficient to raise a protective immune response in the chicken. The gD gene consists of 1305 base pairs from nucleotide 8462 to 9766 and codes for a glycoprotein 434 amino acids in length and molecular weight approximately 48.477 daltons (SEQ. ID. NO. 10, 11). The ILTV gD glycoprotein is homologous to the PRV g50, and the gD from HSV-1, MDV, IPV, and BHV-1.1. Monoclonal antibodies raised to ILT virus react specifically with gD protein from ILTV and also react to ILTV gD protein expressed in a Herpesvirus of Turkeys (HVT) virus vector. ILTV gD expressed in the HVT vector is useful as a subunit vaccine.

21 gene The gI gene consists of 1089 base pairs from nucleotide 9874 to 10,962 and codes for a glycoprotein 362 amino acids in length and molecular weight approximately 39,753 daltons (SEQ. ID. NO. The ILTV gI glycoprotein is homologous to the VZV gI. Recombinant ILTV gI protein expressed in a swinepox virus vector reacts to convalescent sera from ILTV-infected chickens.

Deletion of the gl gene results in an attenuated ILT virus that is useful as a vaccine against ILT disease in chickens. Recombinant viruses deleted for gl are safe in animal trials when vaccinated by a natural route directly into the respiratory tract. whereas parental virus causes lesions in 90% of the birds inoculated via the same route. Deletion of the gl gene serves as a negative marker to distinguish vaccinated from infected animals.

ORF8 Reverse Complement ORF8 Reverse Complement codes for a protein of unknown function. ORF8 RC consists of 533 base pairs from nucleotide 11,150 to 10,617 and codes for an open reading frame 177 amino acids in length and molecular weight approximately 19,470 daltons (SEQ. ID. NO. 18, 19).

WO 96/29396 PCT/US96/03916 -51gE gene The gE gene consists of 1500 base pairs from nucleotide 11,159 to 12.658 and codes for a glycoprotein 499 amino acids in length and molecular weight approximately 55,397 daltons (SEQ. ID. NO. The ILTV gE glycoprotein is homologous to the gE glycoproteins from VZV, Simian herpesvirus (SHV), EHV-1, HSV-1, and PRV. The ILTV gE is a neutralizing antigen useful as a subunit vaccine.

consists of 783 base pairs from nucleotide 12,665 to 13,447 and codes for a protein 261 amino acids in length and molecular weight approximately 27,898 daltons (SEQ. ID. NO. 9).

WO 96/29396 PCTfUS96/03916 -52- Example 2 S-ILT-004 S-ILT-004 is an infectious laryngotracheitis virus (ILTV) that has an approximately 620 base pair deletion of the thymidine kinase (TK) gene (28).

The gene for E. coli p-galactosidase (lacZ) was inserted in the place of the TK gene and is under the control of the HCMV immediate early (IE) promoter.

Transcription of the HCMV IE promoter-lac Z gene is in the opposite orientation to the TK promoter.

S-ILT-004 was constructed using homology vector 501-94 (see Materials and Methods) and S-ILT-001 (USDA ILTV Strain 83-2) in the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS. The transfection stock was screened by the BluogalTM SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The result of blue plaque purification was recombinant virus S-ILT- 004. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING OF DNA procedure. This analysis confirmed the presence of the p-galactosidase (lacZ) marker gene and the deletion of approximately 619 base pairs of the TK gene. The remaining TK gene sequence codes for protein including amino acids 1 to 77. and amino acids 286 to 363. The HCMV IE promoter-lacZ gene is in the opposite orientation to the TK gene transcription.

S-ILT-004 is attenuated by deletion of the ILTV TK gene, but retains other genes known to be involved in the immune response in chickens to ILT virus.

Therefore, S-ILT-004 may be useful as a killed vaccine to protect chickens from ILT disease.

WO 96/29396 PCT/US96/03916 -53- Example 3 S-ILT-009 S-ILT-009 is an infectious laryngotracheitis virus (ILTV) that has an approximately 498 base pair deletion of the ILTV US2 gene and an approximately 874 base pair deletion of the ILTV gG gene. The gene for E.

coli p-glucuronidase (uidA) was inserted in the place of the US2 gene and is under the control of the pseudorabies virus (PRV) gX promoter.

S-ILT-009 was constructed using homology vector 544-55.12 (see Materials and Methods) and S-ILT-002 in the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS. S-ILT-002 was constructed as described in Example 5 (S-ILT-014). The transfection stock was screened by the X-Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The resulting purification of a blue plaque was recombinant virus S-ILT-009. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING OF DNA procedure. This analysis confirmed the presence of the PRV gX promoter-p-glucuronidase (uidA) marker gene and the deletion of approximately 498 base pairs of the ILTV US2 gene and an approximately 874 base pair deletion of the ILTV gG gene.

However, during the Bluogal T M SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES, a deletion of the HCMV IE promoter-lacZ gene was detected within the existing ILTV gG deletion. The remaining insert into the ILTV gG deletion contains approximately 2000 base pairs of DNA of which all of the lacZ gene and part of the PRV gX polyadenylation site are missing. The deletion was characterized by detailed restriction mapping and determined to be slightly different from the S-ILT-014 deletion (See Example S-ILT-009 is attenuated by deletion of the ILTV US2 and gG genes, but retains other genes known to be involved in the immune response in chickens to ILT WO 96/29396 PCT/US96/03916 -54virus. Therefore, S-ILT-009 is useful as an attenuated live vaccine or as a killed vaccine to protect chickens from ILT disease as shown in the table. Since S- ILT-009 does not express the ILTV gG genes, it is utilized as a negative marker to distinguish vaccinated animals from infected animals as described previously.

WO 96/29396 WO 9629396PCTIUS96/039 16 Table II EFFICACY OF RECOMBINANT LIVE ILT VIRUS S-ILT-009 AGAINST VIRULENT INFECTIOUS LARYNGOTRACHEITIS VIRUS

CHALLENGE

Vaccine Gene(s) Dose Route Challenge' Protection b Deleted S-ILT-009 gG-. US2- 7.8x10 3 10' OS d S-ILT-009 gG-, US2- 1.56x10 3 10 OS 77% Controls OS 0% ASL 10 OS embryo____ 14 day old chicTsa: USDA Challenge virus =l.0X10 4 pfu b: Protection 4 healthy birds/total c: Intraocular d: Orbital Sinus WO 96/29396 PCT/US96/03916 -56- Example 4 S-ILT-011 S-ILT-011 is an infectious laryngotracheitis virus (ILTV) that has an approximately 983 base pair deletion of the ILTV gI gene. The gene for E. coli p-glucuronidase (uidA) was inserted in the place of the gI gene and is under the control of the pseudorabies virus (PRV) gX promoter. The PRV gX promoter-uidA gene is in the opposite orientation to the direction of transcription of the ILTV gI promoter.

S-ILT-011 was constructed using homology vector 562-61.1F (see Materials and Methods) and S-ILT-001 in the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS. The transfection stock was screened by the X- Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The result of blue plaque purification was recombinant virus S-ILT-011. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING OF DNA procedure. This analysis confirmed the presence of the P-glucuronidase (uidA) marker gene and the deletion of approximately 983 base pairs of the ILTV gI gene which deletes 325 of 363 amino acid codons from the 5' end of the gl gene.

S-ILT-011 is attenuated and is useful as a killed vaccine to protect chickens from ILT disease. S-ILT-011 shows a small plaque phenotype in tissue culture which is indicative of slow viral growth and attenuation. Since S-ILT-011 does not express the ILTV gl gene. it may be utilized as a negative marker to distinguish vaccinated animals from infected animals. As indicated in Example 1, ILTV-infected chickens make antibodies against ILTV gI protein.

WO 96/29396 PCT/US96/03916 -57- Example S-ILT-013 S-ILT-013 is an infectious laryngotracheitis virus (ILTV) that has an approximately 983 base pair deletion of the ILTV gI gene and an approximately 874 base pair deletion of the ILTV gG gene (and a deletion of the HCMV IE promoter lacZ marker gene making the lacZ gene nonfunctional). The gene for E. coli P3-glucuronidase (uidA) was inserted in the place of the gI gene and is under the control of the pseudorabies virus (PRV) gX promoter.

S-ILT-013 was constructed using homology vector 562-61.1 IF (see Materials and Methods) and S-ILT-014 in the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS. The transfection stock was screened by the X- Glue SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The result of blue plaque purification was recombinant virus S-ILT-013. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING OF DNA procedure. This analysis confirmed the presence of the p-glucuronidase (uidA) marker gene and the deletion of approximately 983 base pairs of the ILTV gI gene which removes 325 of 363 amino acid codons from the 5' end of the gl gene. This analysis also confirmed an approximately 874 base pair deletion of the ILTV gG gene and an approximately 1906 base pair insertion of a partial HCMV IE promoter-lacZ marker gene DNA. of which a portion of the HCMV IE promoter and almost none of the lacZ gene remains (see Example 6).

S-ILT-013 is attenuated and is useful as a killed vaccine to protect chickens from ILT disease. S-ILT-013 shows a small plaque phenotype in tissue culture which is indicative of slow viral growth and attenuation. Since S-ILT-013 does not express the ILTV gI or gG genes, ILTV gI and gG may be utilized as negative markers to distinguish vaccinated animals from infected animals.

WO 96/29396 PCT/US96/03916 -58- Example 6 S-ILT-014 S-ILT-014 is an infectious laryngotracheitis virus (ILTV) that has an approximately 874 base pair deletion of the ILTV gG gene and a deletion of the inserted HCMV IE promoter lacZ marker gene making the lacZ gene nonfunctional. S-ILT-014 was derived from a purified S-ILT-002 virus stock in which a deletion of the HCMV IE promoter lacZ marker gene occurred.

S-ILT-002 was constructed using homology vector 472-73.27 (See Materials and Methods) and S-ILT-001 in the DNA TRABSFECTION FOR GENERATING RECOMBINANT ILT VIRUS. The virus S-ILT-002 has a 874 base pair deletion within the ILTV gG gene and an insertion of the E. coli Pgalactosidase (lacZ) gene in place of the ILTV gG gene. However, during the BluogalTM SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES, a white plaque was picked which contained a deletion of the lacZ gene within the ILTV gG deletion.

This virus. S-ILT-014. was characterized by restriction mapping, DNA SEQUENCING and the SOUTHERN BLOTTING OF DNA procedure. This analysis confirmed the presence of an approximately 874 base pair deletion of the ILTV gG gene and approximately 1956 base pair insertion of a partial HCMV IE promoter lacZ marker gene DNA (2958 base pairs deleted). The remaining HCMV IE promoter lacZ marker gene DNA consists of an approximately 686 base pair DNA fragment of the approximately 1154 base pair HCMV IE promoter and an approximately 1270 base pair DNA fragment containing approximately 520 base pairs of the 3010 base pair P-galactosidase (lacZ) marker gene and all of the approximately 750 base pair PRV gX polyadenylation signal.

S-ILT-014 is useful as an attenuated live vaccine or as a killed vaccine to WO 96/29396 PCTIUS96/03916 -59protect chickens from ILT disease as indicated in the table below. Since S-ILT- 014 does not express the ILTV gG gene and ILTV-infected chickens make antibodies to gG as indicated in Example 1, ILTV gG is utilized as a negative marker to distinguish vaccinated animals from infected animals.

WO 96/29396 PCTfUS96/03916 Table III EFFICACY OF RECOMBINANT LIVE ILT VIRUS S-ILT-014 AGAINST VIRULENT INFECTIOUS LARYNGOTRACHEITIS VIRUS

CHALLENGE

Vaccine Gene(s) Dose Route Challenge' Protection' Deleted 0 S-ILT-014 gG- 1.08x10 10 C OS d 97% S-ILT-014 gG- 2.16xl0 3 10 OS 97% Controls OS 0% ASL embryo 05 14 day old chic a: USDA Challenge virus =l.0x10 4 5 phi b: Protection healthy birds/total c: Intraocular d: Orbitual Sinus WO 96/29396 PCTfUS96/03916 -61- Example 7 S-ILT-015 S-ILT-015 is an infectious laryngotracheitis virus (ILTV) that has an approximately 2640 base pair deletion of the UL47-like gene, the ORF4 gene, and ILTV gG gene. The gene for E. coli P1-glucuronidase (uidA) was inserted in the place of the UL47-like, ORF4, and gG genes and is under the control of the pseudorabies virus (PRV) gX promoter. The PRV gX promoter-uidA gene is in the opposite orientation to the direction of transcription of the ILTV UL47-like, ORF4. and gG promoters.

S-ILT-015 was constructed using homology vector 560-52.F1 (see Materials and Methods) and S-ILT-001 in the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS. The transfection stock was screened by the X- Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The result of blue plaque purification was recombinant virus S-ILT-015. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING OF DNA procedure. These results confirmed the presence of a 2640 base pair deletion which includes 442 of a total 511 amino acid codons at the 3' end of the UL47-like gene, all of the ORF4 gene and 271 of 293 amino acid codons of the 5' end of the gG gene.

S-ILT-015 is useful as an attenuated live vaccine or as a killed vaccine to protect chickens from ILT disease as indicated in the table below. Since S-ILT- 015 does not express the ILTV gG gene. ILTV gG is utilized as a negative marker to distinguish vaccinated animals from infected animals.

WO 96/29396 WO 9629396PCTIUS96/03916 -62- Table IV EFFICACY OF RECOMBINANT LIVE ILT VIRUS S-ILT-015 AGAINST VIRULENT INFECTIOUS LARYNGOTRACHEITIS VIRUS

CHALLENGE

Vaccine Gene(s) Dose Route Challenges Protection' Deleted S-ILT-015 gG-. 1.0X10 5 10 C OS d UL-47-like Controls 05 0% ASL 10 05 14 day old chicks a: USDA Challenge virus =l.0xl0 4 pfu b: Protection healthy birds/total c: Intraocular d: Orbital Sinus WO 96/29396 PCTIUS96/03916 -63- Example 8 S-ILT-017 S-ILT-017 is an infectious laryngotracheitis virus (ILTV) that has an approximately 3351 base pair deletion of the ILTV gG gene, ORF4 gene and the g60 gene. The gene for E. coli P-glucuronidase (uidA) was inserted in the place of the ILTV gG and g60 genes and is under the control of the pseudorabies virus (PRV) gX promoter.

S-ILT-017 was constructed using homology vector 579-14.G2 (see Materials and Methods) and S-ILT-001 in the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS. The transfection stock was screened by the X-Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The result of blue plaque purification was recombinant virus S-ILT-017.

S-ILT-017 is attenuated by deletion of the ILTV g60 and gG genes, but retains other genes known to be involved in the immune response in chickens to ILT virus. Therefore. S-ILT-017 may be used as a killed vaccine to protect chickens from ILT disease. Since S-ILT-017 does not express the ILTV gG or genes, it is used as a negative marker to distinguish vaccinated animals from infected animals.

WO 96/29396 PCTIUS96/03916 -64- Example 9 Recombinant infectious larvnaotracheitis viruses that express infectious bronchitis virus (IBV) spike and matrix protein genes: A homology vector is used to generate ILT viruses containing the IBV Arkansas spike protein gene. The recombinant ILT virus contains a deletion of one or more ILTV genes, including gG, US2, UL47-like, and ORF4, and the insertion of two foreign genes: the E. coli p-glucuronidase gene (uidA) and the IBV Arkansas spike protein gene. The uidA gene is under the control of the PRV gX promoter and the IBV Arkansas spike protein gene is under the control of the HCMV IE promoter.

To construct a homology vector containing the foreign genes inserted into the ILT virus, a DNA fragment containing the HCMV-IE promoter, the IBV Arkansas spike protein and the HSV-1 TK polyadenylation signal is inserted into a restriction enzyme site at the position of the deletion of the ILTV gG gene in the ILTV homology vector. A DNA fragment containing the PRV gX promoter and the E. coli p-glucuronidase (uidA) gene is inserted into a unique restriction enzyme site within the ILTV homology vector. A recombinant virus is constructed by combining the final homology vector containing the IBV Arkansas spike gene and the E coli p-glucuronidase (uidA) gene and S-ILT- 001 in the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS. The transfection stock is screened by the X-Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES to detect the presence of the uidA gene and by the BLACK PLAQUE ASSAY FOR FOREIGN GENE EXPRESSION to detect the presence of the IBV Arkansas spike protein.

A similar strategy is used to construct recombinant ILT viruses carrying the IBV Sl protein from Arkansas, Massachusetts,or Connecticut serotypes, IBV matrix protein from Arkansas. Massachusetts, or Connecticut serotypes, and WO 96/29396 PCTIUS96/03916 IBV nucleocapsid from Arkansas, Massachusetts, or Connecticut serotypes. The strategy is also used to construct recombinant ILT viruses carrying the Newcastle Disease virus (NDV) HN and F genes and the Infectious Bursal Disease virus (IBDV) polyprotein or portions thereof. The strategy is also used to construct recombinant ILT viruses carrying the Mareks Disease virus (MDV) gA, gD, and gB genes.

Recombinant ILT virus carrying these antigens are valuable as a multivalent vaccine to protect chickens from diseases caused by ILTV and one or more of the viruses IBV. NDV, IBDV, or MDV. Since the ILTV vaccines described here do not express ILTV gG, it is useful as a negative marker to distinguish vaccinated animals from infected animals.

Example Vaccines utilizing ILTV to express antigens from various disease causing microorganisms: Antigens from the following microorganisms are utilized to develop poultry vaccines: Chick anemia agent. Avian encephalomyelitis virus, Avian reovirus, Avian paramyxoviruses, Avian influenza virus ,Avian adenovirus, Fowl pox virus, Avian coronavirus. Avian rotavirus. Salmonella spp., E coli., Pasteurella spp., Haemophilus spp.. Chlamydia spp., Mycoplasma spp., Campylobacter spp., Bordetella spp.. Poultry nematodes, cestodes, trematodes, Poultry mites/lice, Poultry protozoa (Eimeria spp., Histomonas spp., Trichomonas spp.).

Example 11 A Genomic Map of Infectious Laryngotracheitis Virus and the Sequence and Organization of Genes Present in the Unique Short Region A cosmid library of the ILTV genome was created to facilitate restriction WO 96/29396 PCT[US96/03916 -66endonuclease mapping. Forty-three overlapping cosmids were analyzed by digestion with Asp7181 and Nod. Asp7181 was known to cut the genome relatively infrequently and it was found that Nod cut the genome less than ten times, which enabled cutting the vector away from the ILTV DNA insert.

Comparison of these cosmid digests allowed the order of the Asp7181 fragments covering 85% of the ILTV genome to be determined (Figure 12). On the long end of the genome, seven cosmids were identified which all contained a NotI site 0.9 kb from the end of the cloned insert; all other cosmid inserts had heterogeneous ends from shearing. This 0.9 kb fragment was used as a probe (P 1 in Figure 12) to genomic ILTV digested with Asp7l 81, NotI, or BamHI; the sizes of the genomic fragments that hybridized were identical to the size of the fragments excised from the cloned cosmid insert, indicating that the cloned insert extended all the way or very close to the end of the unique long. The 0.9 kb fragment did not hybridize to other bands in the ILTV digest, consistent with previous reports that this virus resembles PRV, and contains no long repeat Once the cosmid clones were ordered, the restriction sites for a more frequent cutting enzyme, BamHI, were mapped.

The resulting map indicated that the cosmid library did not include clones from the unique short portion of the genome. Cosmids spanning the unique short region of HVT (76) and PRV (83) have been found to be underrepresented in cosmid libraries. The Asp7181 fragments found in the cosmid clones with an Asp718I digest of wild type ILTV and identified fragments of 8.0, 5.1, and kb which were not represented in the cosmid library (Figure 13) were compared. These fragments were cloned into plasmids and hybridized to each other and to ILTV digested with BamHI. The Asp7181 2.5 and 8.0 kb fragments cross-hybridized, indicating that they contained sequence repeated in both clones. Fine mapping of the Asp718I 2.5 and 8.0 kb fragments showed them to contain 2.1 kb of identical sequence. Hybridization to ILTV digested with BamHI identified BamHI bands of 7.5, 6.5, and 4.5 kb which overlapped the Asp7181 fragments. These BamHI fragments were cloned and analyzed by restriction digestion and hybridization. This allowed the map of the entire WO 96/29396 PCTfUS96/03916 -67unique short region and some of the flanking short repeat to be elucidated (Figure 13). Subclones of this region were made, and the entire unique short region was sequenced.

To complete the genomic map, the map searched for an Asp7181 or BamHI fragment that spanned the region between the short repeat sequences of the or 2.5 kb Asp718I fragments mentioned above and the unique long region identified in the cosmid map. A 10 kb NotI fragment from the rightmost end of cosmid D5 (Figure 12) was hybridized to genomic ILTV digests on Southern blots. Interestingly, ladders of hybridizing bands were seen when the enzymes BamHI, Nod, and Asp718l were used. The bands corresponding to these ladders were not generally visible in ethidium bromide stained gels. Subsequent subcloning and mapping of the 10 kb D5 fragment indicated that it contained up to 5 repeats of an 856 bp segment, and that the cosmid insert ended within a repeat motif. HindIII, which cuts once within the repeat, was used to clone the 856 bp fragment. When this fragment (Figure 12, P2) was used to probe ILTV digested with Sfil, NotI, Asp7181, and BamHI, ladders of hybridization were again seen (Figure 14). These ladders arise from varying numbers of the 856 bp repeat in different viral molecules. Sfil cuts only once in this ILTV strain, and a ladder at very high molecular weight can be seen. Because the unique short is expected to invert, two overlapping Sfil ladders containing the unique short and terminal repeat should be present.; however, the bands are too large in this region to make this distinction. NotI and Asp7181 cut further away from the repeat, generating ladders beginning at 10.5 or 12 kb.

The Asp718I digest should generate two overlapping ladders, because one fragment is bounded by an Asp7181 site in the unique long, while the other is bounded by the end of the TR,. In contrast, only one ladder should be generated by the Notl digest. Comparison of Figure 14 lane c (NotI) with lane d (Asp7181) does suggest that in lane d a second ladder is superimposed on the first, starting somewhat higher. BamHI cuts close to the repeated region, and a ladder beginning at 3.4 kb is found. HindIIl cuts within the repeat and generates a strongly hybridizing 856 bp band, as well as the two flanking WO 96/29396 PCT/US96/03916 -68- HindIII fragments of about 1.1 and 2.5 kb, which each contain a portion of the repeated sequence. The presence of this 856 bp repeat accounted for the occasional observation of very fine submolar bands in ethidium bromide-stained Asp718I digests. It also accounted for the lack, in ethidium bromide-stained gels, of a molar or half-molar quantity Asp7181 or BamHI band greater than kb, which was expected to span this region based on analysis of the cosmid clones. Instead, because of the presence of the 856 bp repeat, this band exists as many submolar bands comprising the ladder. As can be seen in the BamHI digest, there can be thirteen or more repeats of the region. Comparison of the repeat sequence to the sequence submitted to GenBank by Johnson et al. (67) indicated that it corresponded (99% identity) to nucleotides 1140 to 1996 of their sequence, which is a region just upstream of the ILTV ICP4 gene. The relationship of the repeat to the surrounding sequence is depicted in Figure Restriction digests indicate that the region to the right of the repeat as shown is similar in the two strains; however, the position of the BamHI site indicated to the left of the repeat differs between them.

To identify the remainder of the short repeat from the 856 bp repetitive region to the BamHI fragments used for sequencing the unique short, the 8.0 kb Asp7l 81 fragment containing part of the short repeat was used as a probe to a second cosmid library of ILTV. One cosmid, clone 2F12, hybridized to the probe. Restriction endonuclease analysis of 2F12 and comparison to the cosmid map indicated that it was not a single contiguous cosmid, but was composed of two large non-contiguous fragments (see Figure 12). The break in the rightmost fragment was within a repeat of the 856 bp region. This fragment included at least two 856 bp repeats, and extended 4.6 kb through the remainder of the short repeat into the unique short.

To identify the end of the TR, the 6.6 kb Notl fragment spanning the unique long and the short internal repeat (IRJ) (P3 in Figure 2) was used as a probe.

It was noted that a 2.9 kb Nod fragment seen in gels stained with ethidium bromide was not represented in the restriction endonuclease map, and WO 96/29396 PCT/US96/03916 -69considered that it might represent the end of the TR,. Hybridization of a Notl digest of ILTV with P3 indicated that this was indeed the case (Figure 16).

The 2.9 kb NotI band hybridizes, as does the 6.6 kb band corresponding to the probe. In the BamHI digest, the predicted 13 kb fragment containing a portion of the IR, and a 3.5 kb fragment corresponding to the end of the TR, are evident. In the Asp7181 digest, an overlapping 2.7 kb fragment from the unique long hybridizes. and the high molecular weight ladder described previously was seen.

Sequencing of the ILTV unique short and flanking region identified nine open reading frames in the unique region and two (duplicated) in the repeat region as diagrammed in Figure 13 (SEQ UD NO:59). Comparison of the proteins encoded by these ORFs to the GenBank database (BLAST homology seacrh, National Center for Biological Information, NCBI) demonstrated identity for most of the potential proteins with other known herpesvirus gene products.

Table V summarizes the closest homologies found for each gene and gives the probability scores for those homologies as generated by the search program.

ORF2 (SEQ ID NO:63), the protein kinase (PK) gene (SEQ ID NO:63), is the most highly conserved of the ILTV ORFs to its herpes homologues. In contrast, the glycoprotein genes are less conserved. It should be noted that portions of the sequences of the ILTV protein kinase, gG, and ORF 5 genes have been published (69, 70 and 81); however, these genes were mapped to the unique long region. A description of each of the nine unique short genes and the two genes in the flanking short repeat follows.

The first open reading frame in the unique short encodes a 229 aa protein showing identity to other herpesvirus US2 proteins (SEQ ID NO:62). Like other US2 genes, it is in the opposite orientation to the remaining ORFs in the unique short. The coding sequence of the gene ends just within the unique short region, and a potential poly-A addition site is found 115 bases downstream in the short repeat. Two possible TATA promoters are found 37 and 70 bases upstream from the initiation codon.

WO 96/29396 PCTIUS96/03916 ORF2 encodes a protein kinase with strong identity to many other herpesvirus protein kinases and to cellular protein kinases The organization of the US2 and PK genes, with their 5' ends close together and their promoters possibly overlapping, is similar to that found in other herpesviruses. Two TATA sequences are present 14 and 49 bases upstream of the PK start codon, and two polyadenylation signals are found, one immediately after the stop codon. and one 50 bases downstream.

ORF3 encodes a 623 aa protein with similarity to the herpes simplex virus UL47 gene (SEQ ID NO:64). The program comparing this protein with other UL47 proteins projects a poor probability score for this homology. However, at least one of the regions of identity between ILTV and HSV UL47 corresponds to a region that is conserved among other herpesvirus UL47 homologues, suggesting that this identity is significant (Figure 17).

Additionally, it should be noted that equally poor probability scores for homology generated by comparisons of the gG or gl genes are also seen for certain homologue pairings, suggesting that these scores are not sufficient for determining homology. It is interesting that the ILTV UL47 gene, normally found in the unique long region of other herpesviruses, appears to have been transposed into the unique short in ILTV.

The fourth open reading frame encodes a 292 aa glycoprotein homologous to PRV gG (SEQ ID NO:65). Four N-linked glycosylation sites with the consensus sequence NXT or NXS are present. The protein has a signal sequence of 26 aa, which could be cleaved at G/AP, but lacks a transmembrane anchor. It is therefore likely that this protein is secreted, similar to other herpesvirus gG homologues. This gene has a consensus TATA sequence 83 bases upstream from the ATG start, and has two potential polyadenylation sites 73 and 166 bases downstream from the stop codon.

could encode a protein of 985 amino acids (SEQ ID NO:66). A WO 96/29396 PCT/US96/03916 -71hydrophobic signal sequence is found at the amino terminus, and a hydrophobic sequence is present at the carboxy terminus. Nine glycosylation sites are found, suggesting that this is a glycoprotein. ORF 5 contains an imperfect repeat, consisting of 30 to 36 bp repeated approximately 23 times from amino acid 431 to amino acid 677. The hydrophilic amino acid consensus sequence created by this repeat is FTQTPSTEPET/A. Comparison of ORF 5 with other herpesvirus sequences (Table V) shows similarity to the glycoprotein product from the equine herpesvirus 1 US5 gene (EUSS, 82). The low probability score for this identity arises primarily from the fact that both genes contain threonine-rich repeats. It is not clear whether this reflects homology in form, function, or both. Both the EUS5 and the ILTV ORF 5 genes are large, have similar positions among flanking genes in the unique short, have signal sequences, and encode glycoproteins. but other sequence similarities are not seen. It is interesting that the ORF 5 repeat region shows similarity to mucin genes, which also contain threonine rich repeats. The human mucin gene, for example, has the repeat GTOTPTTTPITTTTTVTPTPTPT, where 7 of the first 11 amino acids are identical to the ORF 5 repeat sequence. Again, whether this reflects a similarity in function of the encoded proteins is unclear. A TATA sequence is found 560 bases upstream of the start codon; the nearest consensus polyadenylation signal is at the end of the gl gene. This suggests that the ORF transcript may be coterminal with the gD transcript.

The open reading frame for the gD homologue (ORF 6) (SEQ ID NO:67) overlaps the end of ORF 5. Four in-frame methionines are found within the first 58 amino acids of the open reading frame, and it is not clear which is the actual translational start codon. Because a potential TATA promoter sequence is located only 6-9 bases upstream from the first possible ATG codon, this codon would probably not be within RNA transcribed from this promoter; however, there are several TATA sequences further upstream that may also be used to initiate transcription. The other three potential initiation codons are found at aa 23, 47, and 58 within this ORF. Comparison of the sequences surrounding the four ATGs with the eukaryotic translational initiation consensus WO 96/29396 PCT/US96/03916 -72sequence A/GCCATGG (71) suggests that the latter two ATG codons may be preferred translational start sites. The protein sequences derived from each of these starts were examined for the presence of eukaryotic signal sequences and signal cleavage sites. A start at aa 58 within the ORF would result in a signal peptide of 26 amino acids with a predicted cleavage site between two alanine residues. This same signal sequence would be positioned much further from the amino terminus and embedded in a more hydrophilic sequence if the other start sites were used. The start of ILTV gD was tentatively assigned to position 58, which would result in a protein 377 amino acids long. Of course, it is possible that more than one initiation codon is used in vivo. Experiments of Zelnik et al. (88) suggest that alternate in-frame ATG codons are used to initiate MDV and HVT gD transcription in vitro, though the in vivo situation was not addressed. Additional experiments on gD transcription and translation in ILTV are necessary to identify its translational start codon.

The ILTV gD homologue has a secretory signal sequence and a transmembrane helix (aa 352 372) at the carboxy terminus. Only one potential glycosylation site is found at position 250-252; this is of the form NPS, and may not be glycosylated due to the proline residue. There is some question, therefore, as to whether processed ILTV gD contains N-linked oligosaccharides. This would be similar to the gD homologue in pseudorabies virus, gp50, which also lacks N-linked glycosylation sites As in other herpesviruses, the gD coding sequence lacks a poly-A addition signal immediately following the gene, and the closest signal is at the end of the gl gene.

The seventh open reading frame encodes a protein of 362 aa and is most homologous to varicella zoster virus glycoprotein I (SEQ ID NO:68). The encoded protein shows all the characteristics of related gI glycoproteins, including a signal sequence with a potential cleavage site at positions 22 and 23 between a glycine and an isoleucine, a transmembrane helix at the carboxy terminus from 272 292, and four possible N-linked glycosylation sites. A TATA sequence is present 51 bases upstream from the methionine start codon.

WO 96/29396 PCT/US96/03916 -73- Two possible poly-A addition signals are found within the coding sequence for ILTV gI, and may be the signals used by the gD and ORF 5 transcription units upstream.

The gE gene (ORF 8) follows the gI. This gene is 499 aa long, and contains four N-linked glycosylation sites (SEQ ID NO:69). A signal sequence of 18 amino acids is present, and there are two and possibly three membraneassociated helices in the carboxy terminal portion of the protein. The gE gene has a TATA box 86 bases upstream of the start codon, and a potential poly-A addition signal just prior to the 3' end of the coding region. This may serve as the polyadenylation site for the gI gene.

The ninth open reading frame extends across the junction of the unique short and the short repeat, and could encode a protein of 260 amino acids (SEQ ID NO:70). This protein has no signal sequence or membrane anchor, but has one possible N-linked glycosylation site. In a search of GenBank, some similarity is found between this protein and BLRF2 of EBV, but the significance of this similarity is unknown. The poly-A addition signal in the short repeat may be utilized by this gene. A potential TATA sequence is found 178 bases upstream of the first ATG of this ORF.

The first open reading frame in the short repeat (SRORFI) (SEQ ID NOs: 61 and 71) encodes a 294 aa protein which displays homology to the gene product of MDV SORF3 (79 and 84) and HVT ORF3 In MDV and HVT, the corresponding gene is found as one copy in the unique short, and its function is unknown. No homology has been identified with mammalian herpesviruses; this gene appears to be specific to avian herpesviruses. MDV SORF3 has been deleted by Parcells et al. and does not appear to be absolutely required for infection in chickens.

SRORF2 encodes a protein of 278 amino acids with homology to other herpesvirus US10 genes (SEQ ID NOs:60 and 72). A zinc finger motif, found WO 96/29396 PCT/US96/03916 -74in the EHV-4 US 10, is highly conserved in the ILTV US10 (amino acids 201- 218); this suggests that the ILTV US10 gene is a DNA binding protein.

Regulatory sequences include a poly-A addition signal 163 bp after the stop codon; it is unclear where the promoter for this gene resides.

Discussion: The organization of the genes in the unique short region of ILTV is similar to that seen in other herpesviruses. Several genes encoding glycoproteins are present, and the order of these genes is similar to that seen in equine herpesvirus 1, particularly with respect to ORF 5. Similarities to avian herpesviruses are also evident in the presence of the avian-specific gene, SRORF1, and its position relative to US2 and PK, though it differs from HVT and MDV in that it is in the short repeat and is duplicated, also appearing downstream from the ORF 9 gene. The PK gene itself has the most identity to MDV and HVT PK genes; however, other genes are found to be more like their homologues in diverse herpesviruses such as EHV, PRV, and SHV SA8.

Unusual characteristics of the ILTV unique short are the inclusion of a gene normally found in the unique long, the UL47 homologue, and the presence of the unique gene. ORF 5, which contains a set of degenerate repeats.

This analysis of the structure of ILTV disagrees with previous reports.

Comparison of the sequences described here with those of the Australian ILTV isolate SA-2 indicates that a 32 kd protein described by Kongsuwan et al. is almost identical to the gG in this application, and the sequenced fragment of the g60 protein presented by Kongsuwan et al. (69) is part of the ORF 5 gene in this application. However, they identified the 5 kb Asp7181 fragment containing both of these genes as coming from the unique long region of SA-2 Recently, Guo et al. (62) reported the sequence of a region from the USDA challenge strain which they ascribed to the unique short on the basis of comparison to the map presented by Johnson et al. No identity was found between this sequence and the unique short sequence described here. Instead, WO 96/29396 PCT/US96/03916 the sequence described by Guo et al. (62) shows 98% identity to a sequence recently submitted to GenBank by Johnson et al. (67 and 68), which is reported to encode the ICP4 gene of ILTV. The BamHI sites within the ICP4 coding region generate two contiguous fragments of 1.2 and 1.7. kb (see Figure 15). In the map described here, two contiguous BamHI fragments of this size are found within the short repeats (Figure 12). In addition, the 856 bp repeat element, which is found just upstream of the ICP4 gene (Figure 15), was mapped in this application within the short repeats. This indicates that the ICP4 gene in the strain used in these studies is present in the IR and the TR. It is possible, but unlikely, that the Australian SA-2 vaccine strain underwent an unusual rearrangement which altered the relationship of the unique long, unique short, and short repeat. However, Guo et al. (62) used the same challenge strain as the one described in this application, and the sequence they reported is not in the unique short, but in the short repeats, similar to the ICP4 genes of other herpesviruses.

The gene encoded by ORF 5 contains threonine rich, degenerate repeats. These are similar in composition and in their repetitive nature to repeats found in mucin genes. This repeated region in mucin is modified by O-linked oligosaccharides and is highly hydrophilic. It is interesting to speculate on what the function of this somewhat similar region might be in infection, if it is expressed in toto in ILTV. At least a portion of this gene is known to be expressed, as Kongsuwan et al. (69) cloned and sequenced a fragment from it by probing a lambda gtl 1 library with a monoclonal antibody that was known to bind to a 60 kd ILTV protein (g60) on Western blots The relationship of such a 60 kd protein to the predicted 985 aa product from ORF 5 is unknown. Comparison of the application sequence with the complete sequence of the g60 coding region (81) shows a 98.5% homology between the SA-2 strain and the USDA strain. Interestingly, there is an insertion of a block of amino acids in g60 relative to the ORF 5 protein; this difference reflects one additional degenerate repeat sequence in the SA-2 strain.

WO 96/29396 PCT/US96/03916 -76- As mentioned above, Kongsuwan et al. (70) described an ILTV gene that encoded a 32 kd protein with similarity to PRV gG. A comparison of the ILTV gG protein sequence described in this application with their 32 kd protein found 10 amino acid differences in the first 273 residues of the protein. At amino acid 274, a deletion of one base pair in SA-2 relative to the USDA strain created a frame shift, such that 19 additional residues were found in the challenge strain as opposed to 26 in SA-2. A peptide was made from the carboxy terminal sequence elicited antisera in mice which reacted with ILTV gG; this indicates that the sequence described in this application reflects the actual carboxy terminus in the USDA strain. A similar situation was found when the ILTV gD protein described in this application was compared with the ILTV gD sequence submitted to GenBank by Johnson et al. Ten differences were found in the first 419 amino acids, after which a deletion of a base in the SA-2 strain relative to the the sequence described in this application caused the predicted carboxy termini to differ, with 15 more amino acids in the USDA strain and 9 in SA-2. These differences could arise from errors introduced during cloning and sequencing of these genes. It is also possible that the carboxy termini of the ILTV gG and gD genes are variable between these strains.

The 856 bp repeat unit identified within the short repeat is just upstream of the ICP4 gene described by Johnson el al. but, from the sequence alone, it does not appear to be repetitive in the SA-2 strain. The BamHI fragment containing this repetitive region is 2848 bp long in SA-2. The smallest repeat, seen faintly in the BamHl ladder of Figure 14, is 3.4 kb long. This is not quite large enough .to include two repeats. and suggests that other alterations between the two strains may exist in this region. A repeat of this sort has not been previously described for this or other ILTV strains, though the submolar nature of the bands may have obscured its presence. The appearance of the ladder is reminiscent of defective interfering particles, but it is not believed that this represents a case of defective interfering particles in the viral stock used here.

Several reasons for this follow. 1) Defective interfering particles are generally WO 96/29396 PCT/US96/03916 -77found when viruses are passaged at high multiplicity, and the ILTV viral stocks of this application were passaged at low multiplicity. In fact, viral stocks originating from a single picked plaque exhibited similar ladders when their DNA was subjected to Southern blot analysis, suggesting that a single viral particle containing a set number of repeats could regenerate the full range of the ladder after being grown for a short period of time. 2) If populations of defective interfering particles were present, one might expect to encounter digest fragments that would not be accommodated in the linear viral map (see.

for example, 77), yet all but one of the cosmids analyzed make a contiguous map, with Asp7181 bands identical to those present in genomic ILTV digests.

The exception. 2F12, was unusual in being the only one of several hundred cosmid clones screened which contained part of the unique short. This probably represented an aberrant cloning event, and not a widespread phenomenon related to defective viral particles. 3) Defective interfering particles often are present in larger molar amounts than standard viral particles, such that restriction fragments originating from the defective particles are overrepresented. In contrast, the bands of the 856 bp ladder are submolar, and are only rarely visible in ethidium bromide stained gels. 4) Defective interfering particles contain origins of replication. The 856 bp repeat itself does not contain a herpesvirus origin of replication as defined by the consensus sequence of Baumann et al. From these considerations it was concluded that varying numbers of 856 bp units are present in the short repeats of standard viral DNA from the USDA challenge strain of ILTV. Since fragments exist that contain thirteen or more repeats of the region, genomic DNA from ILTV could vary by over 11 kb in the short repeat regions. Repetitive regions have been identified in other herpesviruses; for example, Marek's disease virus contains a 132 bp repetitive sequence in the long repeat regions (61 and 73) and expansion of this repeat is associated with reduction of viral oncogenicity. The presence of the 856 bp tandem repeats in ILTV, in contrast, does not appear to affect viral pathogenicity, since this strain does cause severe clinical disease in chickens. It would be interesting to examine other ILTV strains for the presence of this repeat.

P OPER\MIRO\ 569) 96 ,1nd 1311A.-9 Mal 2. K 78 Table V indicates the ORFs of the ILTV unique short and the HSV nomenclature for these genes, in those cases where homology is found. The third column shows the best matches from the Blast homology search (NCBI), and the probability scores assigned by the program for the matches indicated. Smaller numbers indicate less likelihood that the match could occur randomly.

A genomic map of infectious laryngotracheitis virus (ILTV) and a 18.912 bp sequence containing the entire unique short region and a portion of the flanking short repeats is presented. In determining the genomic map, an 856 bp region repeated as many as 13 times was identified within the short repeats. The unique short sequence contains 9 potential open reading frames (ORFs). Six of these ORFs show homology to other known herpesvirus unique short genes. Using the herpes simplex virus nomenclature, these genes are the US2. protein kinase, and glycoproteins G, D, I, and E (SORFs 1, 2, 4, 6, 7, and 8, respectively). Interestingly, an open reading frame with homology to HSV-1 UL47 (SORF 15 3) is found in the unique short. One very large open reading frame (ORF 5) is present and contains a threonine rich, degenerate repeat sequence. This gene appears to be unique to ILTV among sequenced herpesviruses. Two ORFs were identified within the short repeat region. SRORF1 is homologous to a gene (SORF3) found in the unique short region in both MDV and HVT, and appears to be specific to avian herpesviruses. SRORF2 has 20 homology to HSV Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

WO 96/29396 WO 9629396PCTIUS96/03916 -79- Table V ORF HSV Best Matches Blast Score Homolog 1 US2 EHVlI EUS I 3.1x10' 3 EI-V4 EUSI 5.3 x10 2 HSV2 US2 6.7x 2 PK MDV PK 8.2x 10" 6 HVT PK 5.4x HSVI PK 4.1x10' 0 3 UL47 HSVl UL47 O.Oxl 1 EHV I UL47 9.9x MDV UL47 9.9x10' 4 gG PRV gG 5..3x1O- BI-IVI gG 1.7x10 2 EI-IigG 6.8xI0' ORF 5 EHVI EUS5 1.9X1O" Human mucin 1.lxlO*" WO 96/29396 WO 9629396PCTIUS96/03916 6 gD MDV gD 6.8xlO-' PRV g50 2.0x10 3 HVT gD 3.5xl10 3 7 gI VZV gI 4.2xI10 2 I-VT g1 7.9x 10- 2 SVV gI 4.3xl10' 8 gE SHV SA8 gE I.7x1O' HSV1I gE 1.lxtY 3 BH-V I gE I.5x10 2 9 ORE 9 EBV BLRF2 5.7xl0' SRI no HSV MDV t ORF3" 4.8xI101 homologue HVT "ORF3" 2.6x10-' SR2 US 10 EHV-4 USl10 1.2xl10 1 HSV-1 USIO 8.7x10'l EHV-1 USIO 8.7xl10l WO 96/29396 PCTIUS96/03916 -81-

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WO 96/29396 PCT/US96/03916 -87- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Wild, Martha A.

Cochran, Mark D.

(ii) TITLE OF INVENTION: RECOMBINANT INFECTIOUS LARYNGOTRACHEITIS VIRUS AND USES THEREOF (iii) NUMBER OF SEQUENCES: 72 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Cooper Dunham LLP STREET: 1185 Avenue of the Americas CITY: New York STATE: New York COUNTRY: U.S.A.

ZIP: 10036 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 23-MAR-1995

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/126,597 FILING DATE: 24-SEP-1993 (viii) ATTORNEY/AGENT INFORMATION: NAME: White, John P.

REGISTRATION NUMBER: 28,678 REFERENCE/DOCKET NUMBER: 39116-A (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (212) 278-0400 TELEFAX: (212) 391-0525 INFORMATION FOR SEQ ID NO:l: SEQUENCE CHARACTERISTICS: LENGTH: 13473 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1059..2489 (ix) FEATURE: NAME/KEY: CDS LOCATION: 2575..4107 WO 96/29396 WO 9629396PCTIUS96/03916 -88- (ix) FEATURE: NAME/KEY: CDS LOCATION: 4113. .4445 (ix) FEATURE: NAME/KEY: CDS LOCATION: 4609. .5487 (ix) FEATURE: NAME/KEY: CDS LOCATION: 5697. .8654 (ix) FEATURE: NAME/KEY: CDS LOCATION: 9874. .10962 (ix) FEATURE: NAME/KEY: CDS LOCATION: 11153. .12658 (ix) FEATURE: NAME/KEY: CDS LOCATION: 12665. .13447 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CCCGTGCCCC TAAAGGCCGC

ATGGTCGCCA.

TGGCGCGCGA

TGTGGTTATT

ACTATTCCGG

AGCGGGGTTT

CATCCGAGTC

TGCTGTACGG

GACTCAGAGA

AAT TTTTACC

CCATGCATAT

CAGATGACAA

GGTCGCCCCC

CCCCGGAAAC

CGTAACTCGC

CAAATCTAGA

CAGGCGCCAT

TCTAGCAGAG

ACCCTCCAAA

GACAATAACA

ATTACTGCTA

CAGCGGCCGC

CTTCGACTTA

GGAAACACAG

CCAGGGCGTT

CAGCGTTGTC

TCCGGTTTCG

GTCAGCGGTA

ATGGTAAAAC

CTCACCGAAC

TTCGTACAGA

CATAGTTTTC

ATTAGGTAGT

CGCCGCTCAG

TCAGGCCAAT

CGAGAAAGCT

TGCTACCCGC

CTTACTCATC

CCGCCCTTGT

CGACGCGGCG

AATAGACTCC

TATGCGGCCG

CTCAGGCTCG

TTGTCTCCGT

GTAATTAGGA

CCCAGCACCC

CCTGCGTCCA

CGAATCACGG

GATGCCGGGC

ACTGCGTGAA

TTGTAGTGCG

ACTCCACCCC

AAGTCCAAAT GTGACGTCGG CGGCCCACGC AA.CGCGGGCT

CGCGTACGCG

TTCTGCAAGG

AGCGTCGCCG

CGAGAAAAA

AGTTAGGTTT

AAGGGGCAAG

TTACCAAAAA

AAGTTTTTGG

ACAAATGGAA

AGCCGCTCCA

GTCTGCACAT

GCACGATTAC

CCAATTCTTT

AATCGACCGC

GCTATGATGT

TTATTATTG

CCCTCGC!CGC

CTAACGTCGG

TTTTGGCTGC

TACTTTAAA

AGTTGTCCAG

TATTTCCACT

CGCAGGGAGG

CTCTTTTGCG

CTCGGGACTT

CCTGGGAAGG

CGATAATGTA

CC-ATCCAGAA

AGGTCTCGAC

TTTATAAAGA

TCAATATTTG

GGCCCAGGCC

CGCCGCGGGG

CGTTCGCCAT

AACTTTACCG

ACTGATGG43T

CCTCTCTCAA

TTTAAAGCTG

GCATACGCGC

ACTCCAGGCG

GAAAACAGCT

CTCGGACGAT

TCCGAGAGCT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1073 1121 AGAAACTATA GTCACTTTTA CAGAAATATA ACGCTCTTAT ATATACAGCT TAGAGAAG ATG CGG TTT CGG CGC Met Arg Phe Arg Arg 1 ATC TGT TCA COC TCT AGG GCA GAA AAA CGA AGA AGA ACA ACC GAG AAT Ile Cys Ser Arg Ser Arg Ala Glu Lys Arg Arg Arg Thr Thr Glu Asn 15 WO 96/29396 PCTIUS96/03916 -89- CCG CTT ACC TCA AAA CGC GTT TGC GTA TTG GAT AGT TTC TCA CGG ACA 1169 Pro Leu Thr Ser Lys Arg Val Cys Val. Leu Asp Ser Phe Ser Arg Thr 30 ATG TCA TTG CGC CCC TAT GCA GAA ATT TTG CCG ACC GCG GAA GGC GTC 1217 Met Ser Leu Arg Pro Tyr Ala Giu Ile Leu Pro Thr Ala Giu Gly Val 45 GAG CGC CTC GCC GAA CTT GTT AGT GTG ACA ATG ACA GAA CGC GCG GAA 1265 Giu Arg Leu Ala Giu Leu Val Ser Val Thr Met Thr Giu Arg Ala Giu 60 CCT GTG ACA GAG AAT ACA GCT GTA AAC AGT ATC CC!C CCG GCT AAC GAG 1313 Pro Val Thr Giu Asn Thr Ala Val Asn Ser Ile Pro Pro Ala Asn Giu 75 80 AAC GGG CAG AAC TTC GCA TAT GCA GGC GAT GGG CCC TCG ACT ACT GAA 1361 Asn Gly Gin Aan Phe Ala Tyr Ala Gly Asp Gly Pro Ser Thr Thr Giu 95 100 AAA GTT GAC GGC TCG CAT ACA GAC TTC GAT GAA GCA TCG AGC GAC TAC 1409 Lys Val Asp Gly Ser His Thr Asp Phe Asp Glu Ala Ser Ser Asp Tyr 105 110 115 GCC GGC CCT GTC CCG CTC GCG CAA ACT AGA TTG AAG CAT TCG GAT GAA 1457 Ala Gly Pro Val Pro Leu Ala Gin Thr Arg Leu Lys His Ser Asp Giu 120 125 130 TTT CTT CAG CAC T TC CGA GTT TTA GAC GAT TTG GTG GAG GGG GCT TAC 1505 Phe Leu Gin His Phe Arg Val Leu Asp Asp Leu Val Glu Gly Ala Tyr 135 140 145 GGG TTT ATC TGC GGC CTC CGT CGC TAC ACC GAG GAA GAG CAA CGT CGA 1553 Gly Phe Ile Cys Gly Val Arg Arg Tyr Thr Glu Glu Clu Gin Arg Arg 150 155 160 165 AGA GGG GTT AAC AGT ACT AAC CAG GGG AAA TCA AAA TGT AAG CGC CTG 1601 Arg Gly Val Asn Ser Thr Asn Gin Gly Lys Ser Lys Cys Lys Arg Leu 170 175 180 ATA GCT AA TAT GTG AA AAT GGA ACA AGG GCG GCC TCT CAC CTG GAA 1649 Ile Ala Lys Tyr Val Lys Asn Gly Thr Arg Ala Ala Ser Gin Leu Giu 185 190 195 AAT GAA ATT TTG GTT CTC GGG CGC CTA AAT CAC GAG AAT GTT CTC AAG 1697 Asn Ciu Ile Leu Val Leu Gly Arg Leu Asn His Giu Asn Val Leu Lys 200 205 210 ATC CAG GAA ATC CTT CGG TAC CCG GAT AAT ACG TAC ATG TTA ACG CAG 1745 Ile Gin Clu Ile Leu Arg Tyr Pro Asp Asn Thr Tyr Met Leu Thr Gin 215 220 225 AGG TAT CAG TTC GAC TTC TAC AGC TAC ATG TAC CAT CAA GCC TTC CAC 1793 Arg Tyr Gin Phe Asp Leu Tyr Ser Tyr Met Tyr Asp Clu Ala Phe Asp 230 235 240 245 TGG AAA GAC AGT CCA ATG CTT AAA CAG ACT AGA CCC ATC ATG AAG CAG 1841 Trp Lys Asp Ser Pro Met Leu Lys Gin Thr Arg Arg Ile Met Lys Gin 250 255 260 CTC ATG TCA C CTC TCG TAT ATC CAT TCA AAG AAA CTC ATT CAC AGG 1889 Leu Met Ser Ala Val Ser Tyr Ile His Ser Lys Lys Leu Ile His Arg 265 270 275 GAC ATC AAA CTC GAA AAT AT? TTC TTA AAC TCC GAC GGC AAG ACA CTG 1937 Asp Ile Lys Leu Giu Asn Ile Phe Leu Asn Cys Asp Gly Lys Thr Val 280 285 290 WO 96/29396 PCT/US96/03916 CTG GGC GAC TTT GGA ACT GTC ACG CCT TTT GAA AAT GAG CGG GAG CCC 1985 Leu Gly Asp Phe Gly Thr Val Thr Pro Phe Glu Asn Glu Arg Glu Pro 295 300 305 TTC GAA TAT GGA TGG GTG GGG ACC GTG GCT ACT AAC TCT CCC GAG ATA 2033 Phe Glu Tyr Gly Trp Val Gly Thr Val Ala Thr Asn Ser Pro Glu Ile 310 315 320 325 CTC GCC AGG GAT TCG TAC TGT GAA ATT ACA GAC ATT TGG AGC TGC GGA 2081 Leu Ala Arg Asp Ser Tyr Cys Glu Ile Thr Asp Ile Trp Ser Cys Gly 330 335 340 GTA GTA TTG CTG GAA ATG GTA AGC CAT GAA TTT TGC CCG ATC GGC GAT 2129 Val Val Leu Leu Glu Met Val Ser His Glu Phe Cys Pro Ile Gly Asp 345 350 355 GGC GGG GGA AAT CCG CAC CAG CAA TTG CTG AAA GTT ATC GAC TCT CTC 2177 Gly Gly Gly Asn Pro His Gin Gin Leu Leu Lys Val Ile Asp Ser Leu 360 365 370 TCA GTT TGT GAT GAA GAG TTC CCA GAC CCC CCG TGT AAT CTG TAC AAT 2225 Ser Val Cys Asp Glu Glu Phe Pro Asp Pro Pro Cys Asn Leu Tyr Asn 375 380 385 TAT TTG CAT TAT GCG AGC ATC GAT CGC GCC GGA CAT ACG GTC CCG TCG 2273 Tyr Leu His Tyr Ala Ser Ile Asp Arg Ala Gly His Thr Val Pro Ser 390 395 400 405 CTC ATA CGG AAC CTC CAC CTT CCG GCG GAT GTG GAA TAC CCT CTA GTT 2321 Leu Ile Arg Asn Leu His Leu Pro Ala Asp Val Glu Tyr Pro Leu Val 410 415 420 AAA ATG CTT ACT TTT GAC TGG CGT TTG AGA CCC AGC GCG GCC GAA GTA 2369 Lys Met Leu Thr Phe Asp Trp Arg Leu Arg Pro Ser Ala Ala Glu Val 425 430 435 TTG GCA ATG CCA CTG TTT TCG GCT GAA GAG GAA CGG ACC ATA ACA ATT 2417 Leu Ala Met Pro Leu Phe Ser Ala Glu Glu Glu Arg Thr Ile Thr Ile 440 445 450 ATT CAT GGA AAA CAT AAA CCC ATC CGA CCC GAA ATC CGT GCG CGG GTG 2465 Ile His Gly Lys His Lys Pro Ile Arg Pro Glu Ile Arg Ala Arg Val 455 460 465 CCA CGG TCC ATG AGT GAA GGT TAATAATAAA GGACGGAGAT AGAGAACTGA 2516 Pro Arg Ser Met Ser Glu Gly 470 475 AGCGTCAGAT TTTTTTAAAA AAATAAATGA TCGAGAACTT ATGATTTGTC TTTCTTGA 2574 ATG ACC TTG CCC CAT CGA TTA ACG AAA AGA CCT TTC GCG CGT CGA TTC 2622 Met Thr Leu Pro His Arg Leu Thz Lys Arg Pro Phe Ala Arg Arg Phe 1 5 10 TGC TCG GTC TTT GTG ATA CAT TAT AGT GAG ACT AAA CTC GAC CGA TAT 2670 Cys Ser Val Phe Val Ile His Tyr Ser Glu Thr Lys Leu Asp Arg Tyr 25 AAC AAG ACA ATG TTA CTC TAT AGA CCG GAC TCA ACC ATG CGG CAT AGC 2718 Asn Lys Thr Met Leu Leu Tyr Arg Pro Asp Ser Thr Met Arg His Ser 40 GGA GGC GAC GCA AAT CAC AGA GGG ATA AGG CCG AGG CGG AAA TCT ATT 2766 Gly Gly Asp Ala Asn His Arg Gly Ile Arg Pro Arg Arg Lys Ser Ile 55 WO 96/29396 WO 9629396PCTIUS96103916 -91- GGA GCG TTT AGC GCG CGC GAA AAG ACT GGA AAA CGA AALT GCG CTG ACG Giy

GAA

Giu

GAA

Giu

GAG

Giu

GAT

Asp

CAG

Gin 145

TTT

Phe

GAG

Giu

CCG

Pro

TTT

Phe

TGC

Cys 225

ATT

Ile

CAC

His

GAG

Giu

GGG

Gly

CGC

Arg 305

GAC

Asp Ala Phe Ser Ala

AGC

Ser

TTT

Phe

GTC

Val

GCG

Aia 130

CCG

Pro

ACT

Thr

GCG

Aia

TTC

Phe

TTG

Leu 210

TTA

Leu

GTT

Val1

TGG

Trp

GAG

Giu

GCT

Al a 290

GAC

Asp

GCA

Ala AGC TCC Ser Ser GGC GGT Gly Gly 100 CTT TCT Leu Ser 115 GAG GAG Giu Giu TTC AAT Phe Asn CGA GCG Arg Ala TTG AAA Leu Lys 180 ACG CGA Thr Arg 195 GGG GAC Giy Asp CGA CAG Arg Gin AAC TGC Asn Cys AGA GGA Arg Giy 260 AAT ATT Asn Ile 275 GGT CTG Gly Leu AAG CGA Lys Arg GCC GTC Ala Val

TCC

Ser

AAG

Lys

CAG

Gin

GAG

Giu

GCC

Ala

CCA

Pro 165

GTA

Val

TCA

Ser

TCG

Ser

CAC

His

AAC

Asn 245

ATG

Met

CAA

Gin

TTC

Phe

CTG

Leu

CTC

Leu 325 Arg Giu Lys Thr Giy Lys Arg Asn Ala Leu Thr 70 75 TCC GAC ATG CTA GAT CCG TTT TCC ACG GAT AAG Ser Asp Met Leu Asp Pro Phe Ser Thr Asp Lys 90 TGG ACG GTA GAC GGA CCT GCC GAC ATT ACT GCC Trp Thr Val Asp Gly Pro Ala Asp Ile Thr Aia 105 110 GCA TGG GAC GTT CTC CAA TTA GTG AAG CAT GAA Ala Trp Asp Vai Leu Gin Let' Val Lys His Git' 120 1.25 AGA GTG ACT TAT GAG TCC AAA CCG, ACC CCG ATA Arg Vai Thr Tyr Giu Ser Lys Pro Thr Pro Ile 135 140 TGG CCG GAC GGG CCG AGT TGG AAC GCG CAG GAT Trp Pro Asp Giy Pro Ser Trp Asn Ala Gin Asp 150 155 160 ATA GTT TAT CCC TCT GCG GAG GTA TTG GAC GCA Ile Val Tyr Pro Ser Ala Git' Val Leu Asp Aia 170 175 GOG GCA TTC GTT AGC CGA GTT TTA CAA TGT GTA Gly Ala Phe Val Ser Arg Val Leu Gin Cys Val 185 190 AAG AAA AGC GTT ACG GTG CGG GAT GCG CAG TCG Lys Lys Ser Val Thr Vai Arg Asp Ala Gin Ser 200 205 TTC TGG AGA ATA ATG CAG AAC GTT TAC ACG GTT Phe Trp Arg Ile Met Gin Asn Val Tyr Thr Vai 215 220 ATA ACT CGA CTC AGG CAC CCT TCC AGC AAA AGC Ile Thr Arg Leu Arg His Pro Ser Ser Lys Ser 230 235 240 GAC CCT CTA TGG TAC GCC TAC GCG AAT CAA TTT Asp Pro Leu Trp, Tyr Ala Tyr Ala Asn Gin Phe 250 255 CGC GTG CCG TCG CTT AAA TTA GCC TCT CCC CCG Arg Val Pro Ser Leu Lys Let' Ala Ser Pro Pro 265 270 CAC GGC CCA ATG GCC GCC CTT TTT AGA AAC GCG His Cly Pro Met Ala Ala Val Phe Arg Asn Ala 280 285 CTG TGG CCT GCC ATG CGC GCA GCC TTT GAA GAG Leu Trp Pro Ala Met Arg Ala Ala Phe Giu Giu 295 300 TTA AGA GCA TGC CTG; TCT TC!A CTC GAT ATC ATG Leu Arg Ala Cys Leu Ser Ser Let' Asp Ile Met 310 315 320 CC TCG TTT CCA TTT TAC TGG CGC GGC GTC CAA Ala Ser Phe Pro Phe Tyr Trp Arg Cly Val Gin 330 335 2814 2862 2910 2958 3006 3054 3102 3150 3198 3246 3294 3342 3390 3438 3486 3534 3582 WO 96/29396 WO 9629396PCT1US96/03916

GAC

Asp

GCA

Ala

CAC

His

TAT

Tyr.

385

GGA

Gly.

CTG

Leu

TCC

Ser

AAA

Lys

GAG

Giu 465

AAT

Asn

CAT

His

ATG

Met 1

GCC

Al a

GCT

Al a

TCG

Ser

GTG

Val

A.CC

rhr

CTA

Leu

GCA

Ala 370

GAC

A.sp

GCC

A.la

GCG

Ala

GTG

Val

GCG

Ala 450

TTT

Phe

TTC

Phe

TTT

Phe

CTC

Leu

ATG

Met

ATT

Ile

GAC

Asp

ATA

Ile

TCG

Ser

GTG

Val 355

CTG

Leu

GAA

Giu

GAG

Giu GTG4 Val

OCA.

Ala 435

AGG

Arg TrC Phe

TGT

Cys

TGT

Cys

TGC

Cys

GGG

Giy

TCC

Ser

CAT

His

GAC

Asp

:GC

krg 340

;TG

VYal

;TA

Val k.CT Thr

GGT

Gly

GTT

Vai 420

A.CT

Thr

CGC

Arg

TTT

Phe

TTA

Leu

TCA

Ser 500

CCC

Pro

TCT

Ser

AGC

Ser

GCA

Ala

GAG

Giu

TTC

Phe

TTA

Leu

TTC

Phe

AGA

Arg

ACT

Thr 405

TGC

Cys

AGC

Ser

CCT

Pro

TAC

Tyr

AAC

Asn 485

GGG

Gly

CTT

Leu 5

GAG

Gi u

GAC

Asp

AGG

Arg

CCG

Pro

C

AG CCT GCG Giu Pro Ala :TG GCC GAG Leu Ala Giu -92- CTG GGC TGT TTG TCA GAG TAC TTT Leu Gly Cys Leu Ser Giu Tyr Phe 345 350 ACG GTC TTA GCG ACC ATG TTC GAC Thr Val Leu Ala Thr Met Phe Asp ATG AGO Met Arg 375 TAT ATA Tyr Ile 390 CTG TTA Leu Leu GCA AAC Ala Asix GCG TGC Ala Cys GGC ATG Gly Met 455 ATT GCG Ile Ala 470 ATT CTG Ile Leu 360 365 GCG CTG GCA GAC GGC AAT TTC GAT GAC Ala Leu Ala Asp Gly Asn Phe Asp Asp 380 GAC CCC OTT AAA AAC GAG TAC CTG AAC Asp Pro Val Lys Asn Giu Tyr Leu Asn 395 400 CGG GGC ATA GTG GCC TCC AAC ACC GCT Arg Gly Ile Val Ala Ser Asn Thr Ala 410. 415 ACC TAT TCG ACG ATA AGA AAA CTC CCG Thr Tyr Ser Thr Ile Arg Lys Leu Pro 425 430 AAT GTT GCC TAC AGG ACC GAA ACG CTG Asn Val Ala Tyr Arg Thr Giu Thr Leu 440 445 AGC GAC ATA TAC COG ATA TTA CAA AAA Ser Asp Ile Tyr Arg Ile Leu Gin Lys 460 TGG CTC CAG AGO GTT GCA ACA CAC GCA Trp Leu Gin Arg Val Ala Thr His Ala 475 480 AAG AGA AGC GTG GAT ACG GOC CCC CGC Lys Arg Ser Val Asp Thr Gly Pro Arg 490 495 3630 3678 3726 3774 3822 3870 3918 3966 4014 4062 4112 4160 4208 4256 4304 4352 4400

CCA

Pro

CTC

Leu

CTC

Leu

AGG

Arg

GAA

Giu

GTT

Val OCT COG AGA AGC GGC TGC AOC AGT TAAATAAA Ala Arg Arg Ser Gly Cys Ser Ser 505 510 OTO CCG ATT CAA TAT GAA GAC TTT TCG AAG Val Pro Ile Gin Tyr Giu Asp Phe Ser Lys 10 AAO AGG GAA AAG TTA GAG ACA TTC GTT AAA Lys Arg Glu Lys Leu Giu Thr Phe Val Lys 25 GAC CCO AGO GGG TCC TTA AGA TTT CTC ATT Asp Pro Arg Giy Ser Leu Arg Phe Leu Ile 40 ATT ATT GCA GAC GGA GTA COG TTT AAG CCG Ile Ile Ala Asp Giy Val Arg Phe Lys Pro 55 CGG GCT TCA GTT OCG CTG AGT ACC OCT GCC Arg Ala Ser Val Ala Leu Ser Thr Ala Ala 75 s0 70 OCT GGG AAA GTG AAA GCG CGA CGC TTA ACC Ala Gly Lys Val Lys Aia Arg Arg Leu Thr 90 TCA OTT COC GCG CCC GTA Ser Val Arg Ala Pro Val WO 96/29396 WO 9629396PCTfUS96/03916 -93- CCG CCC GOA GGC GCC GTT TCC GCG CGC CGG AAA TOG GAA ATA TGA TA Pro Pro Ala Gly Ala Val Ser Ala Arg Arg Lys Ser Giu Ile 100 105 110 AAAATGCTTG GCATTTGCGG GCGAAGAGGC GTGATCTGAA GGGCTCCAOA ATGACGTAAC TGAGCTACGC ATCCCTATAA AGTGTACSCG CTGACCGCTA GCCCATACAG TGTTACAGGA GGGGAGAGAG ACAACTTCAG CTCGAAGTCT GAAGAGACAT C ATG AGC GGC Met Ser Gly 1

TTC

Phe

TTG

Leu

AGC

Ser

ACC

Thr

GAA

Giu

GAA

Giu

GAC

Asp 100

TGC

Cys

AAA

Lys

TOT

Ser

ATC.

Ile

GTT

Val 180

TAC

Tyr

GAO

Asp

AGT

Ser

S

TGC

Cys

CCT

Pro

ACG

Thr

GGA

Gly

AGC

Ser

CTG

Leu

AGG

Arg

GTG

Val

GGC

Gly

TAC

Tyr 165

TAT

Tyr

ACC

Thr

GTA

Val AAC ATA Asn Ile GCA TOT Ala Ser TTC OCG Phe Pro ATT GAG Ile Glu GTG TCA Val Ser TTC GTG Phe Val TCT ACG Ser Thr GCG TO-T Ala 5cr CAG AGA Gin Arg 135 AAT GGA Asn Gly 150 GCC TAC Ala Tyr CTA GAO Leu Asp TAT TAC Tyr Tyr GCC TCG Ala Ser 215

GGA

Gly

GTA

Val1

TTC

Phe

GTG

Val

GTC

Val1

TTC

Phe

TGC

Cys

GAC

Asp 120

AGA

Arg

TC

Ser

CAG

Gin

GTC

Val

AGC

Ser 200 ccc Pro

TOG

Ser

TTA

Leu 25

GGG

Gly

ACG

Thr

GAG

Giu

GGG

Gly

TG

Trp 105

GCC

Al a

CTG

Leu

OTG

Leu

CTC

Leu

GGA

Gly 185

CTG

Leu

GAA

Glu ATT GCC ACC GTT TOC CTA GTA TGO TOG OTT Ile Ala Thr Val Ser Leu Val Cys Ser Leu 10 GGG GCG CCG GTA CTG GAO GGG OTC GAO TCG Gly Ala Pro Val Leu Asp Gly Leu Giu Ser 30 GGC AAA ATT ATA GCC CAG GCG TGC AAC CGC Gly Lys Ile Ile Ala Gin Ala Cys Asn Arg 45 GTC CCG TGG AGC GAC TAC TCT GGT OGC ACC Val Pro Trp, Ser Asp Tyr Ser Gly Arg Thr 60 GTG AAA TGG TTO TAO GGG AAT AGT AAT OC Val Lys Trp Phe Tyr Gly Asn Ser Asn Pro 75 s0 GTG GAT AGO GAA ACG GGO AGT GGA CAC GAG Val Asp Ser Glu Thr Gly Ser Gly His Giu 90 GOT OTA ATC OAT AAT CTG AAO GOG TOT GTG Ala Leu Ile His Asn Leu Asn Ala Ser Val 110 115 GGG ATA OOT GAT TTC GAO AAG CAG TGO GAA Gly Ile Pro Asp Phe Asp Lys Gin Cys Giu 125 130 000 TOO GGG GTG GAA OTT GGT AGT TAO GTG Arg Ser Gly Val Glu Leu Gly Ser Tyr Val 140 145 GTG OTG TAC OCA GGG ATG TAC GAT GOO GO Val Leu Tyr Pro Gly Met Tyr Asp Ala Gly 155 160 TOA GTG GGT GOG AAG GGA TAT ACC GGG TOT Ser Val Gly Gly Lys Gly Tyr Thr Giy Ser 170 175 COA AAO 000 GGA TGC CAC GAO CAG TAT GGG Pro Asn Pro Gly Cys His Asp Gin Tyr Gly 190 195 000 GAO GAG GOG TCA GAO TTA TOA TOT TAT Ala Asp Giu Ala Ser Asp Leu Ser Ser Tyr 205 210 OTO GAO GGT COT ATG GAG GAA GAT TAT TOO Leu Asp Giy Pro Met Giu Giu Asp Tyr Ser 220 225 4447 4507 4567 4617 46*65 4713 4761 4809 4857 4905 4953 5001 5049 5097 5145 5193 5241 5289 WO 96/29396 PTU9131 PCTIUS96/03916 -94- AAT TGT CTA GAC ATG CCC CCG CTA CGC CCA TGG ACA ACC GTT TGT TCG Asn Cys Leu Asp Met Pro Pro Leu Arg Pro Trp Thr Thr Val Cys Ser 230 235 240 CAT GAC GTC GAG GAG CAG GAA AAC GCC ACG GAC GAG CTT TAC CTA TGG His Asp Val Giu Giu Gin Giu Asn Ala Thr Asp Giu Leu Tyr Leu Trp 245 250 255 GAC GAG GAA TGC GCC GGT CCG CTG GAC GAG TAC GTC GAC GAA AGG TCA Asp Giu Giu Cys Ala Gly Pro Leu Asp Giu Tyr Val Asp Giu Arg Ser 260 265 270 275 GAG ACG ATG CCC AGG ATG GTT GTC TTT TCA CCG CCC TCT ACG CTC CAG Giu Thr Met Pro Arg Met Val Val Phe Ser Pro Pro Ser Thr Leu Gin 280 285 290 CAG TAGCCACCCG AGAGTGTTTT TTGTGAGCGC CCACGCAACA TACCTAACTG Gin CTTCATTTCT GATCAATTAT TGCGTATTGA ATAAATAAAC AGTACAAAAG CATCAGGTGT GGTTTGCGTG TCTGTGCTAA ACCATGGCGT GTGCGGGTGA AACCGTAAAT TACGTGATAA TAAATAGCAT AGGAGTTGGC GTGCAGCGTA TTTCGCCGAG AG ATG GGG ACA ATG Met Gly Thr Met TTA GTG Leu Val ACC GGC Thr Gly CAA GAG Gin Giu ACA TAT Thr Tyr ACC CCT Thr Pro CTG GAG Leu Giu CTG TCA Leu Ser GGG GTA Gly Val AGT CGG Ser Arg

TTG

Leu

AGA

Arg

AAC

Asn

CCG

Pro

ACT

Thr

CGC

Arg

TAT

TTC

Phe

GTC

Val 135

CGC

Arg

TTC

Phe

CTG

Leu

CCG

Pro

TTG

Leu

GGG

Gly

CC

Arg

GCT

Ala 120

ITA

Leu

CTT

Leu

TGC

Cys

GCG

Ala

CCT

Pro

CC

Arg

ACG

Thr

CCG

Pro 105

ACT

Thr

GTT

Val

TTC

Phe 10

CGA

Arg

GTG

Val

GAA

Giu

ATT

Ile

TCC

Ser 90

CGC

Arg

GCC

Gly

AAC

Asn

CTA

Leu

GTT

Val

CTC

Leu

GCC

Gly

CC

Arg 75

GCC

Ala

CCA

Pro

CAG

Gin

GCC

Ala CTT CCA GTA GCG GAC GCG C TTG CCG Leu Ala Val Ala Asp Ala Ala Leu Pro 15 TGG AAG GTC CCT CCG GGA GGA ACC ATC Trp Lys Val Pro Pro Cly Gly Thr Ile 30 C GAA TCG CCG GTC ACG GGA CAC GCG Ala Glu Ser Pro Val Thr Gly His Ala 45 GCC GTC AGC TTT CAG ATT TTT C GAC Ala Val Ser Phe Gin Ile Phe Ala Asp 60 TAC GOG CCT ACG GAG GAC GAA CTT GCA Tyr Gly Pro Thr Glu Asp Giu Leu Ala TCA GAC GCG GAC AAC GTG ACA TTT TCG Ser Asp Ala Asp Asn Val Thr Phe Ser 95 100 GAA ATT CAC GGA GCA TAC TTC ACC ATA Ciu Ile His Gly Ala Tyr Phe Thr Ile 110 115 ACC ACG GAA AGC ACC TAT TCG GTC ATC Ser Thr Giu Ser Ser Tyr Ser Val Ile 125 130 TCT CTG GAA CGG TCC CTG CCC CTG GAA Ser Leu Giu Arg Ser Val Arg Leu Glu 140 145 TTG GAG AAC GAG CCT ACA TGG GGC TCG Leu Gin Asn Giu Pro Thr Trp Gly Ser 5337 5385 5433 5481 5534 5594 5654 5708 5756 5804 5852 5900 5948 5996 6044 6092 6140 6188 ACG CCG TGC GAT GAA AAT TIT Thr Pro Cys Asp Giu Asn Phe 150 150 15 160 WO 96/29396 WO 9629396PCTUS96/03916

AAG

Lys 165

GCC

Ala

GCG

Ala

CCT

Pro

TCG

Ser

AGG

Arg 245

GGC

Gly

GGC

Gly

GCG

Ala

ACT

Thr

GCG

Ala 325

ACT

Thr

TCC

Ser

CCG

Pro

GGA

Gly

TCG

Ser 405

ACA

Thr CGT TGG TTA GGC CCC CCG TCG CC Arg Trp Leu Gly Pro Pro Ser Pr 170 r' TAT GTG CGA GAT AAC GAT GTC o Tyr Val Arg Asp Asn Asp Val 175 180

GTG

Val

GCC

Ala

AAA

Lys

CGC

Arg 230

AAC

Asn

CGG

Arg

AGC

Ser

ATT

Ile

GAA

Giu 310

CTC

Leu

CTG

Leu

CCG

Pro

CGC

Arg

TCA

Ser 390

ACA

Thr

CTG

Leu

TTG

Leu

CAG

Gin

AGA

Arg 215

ATA

Ile

GTT

Val

GCC

Ala

GTC

Val

CAA

Gin 295

ACG

Thr

GGG

Gly

CCT

Pro

ACC

Thr

AGC

Ser 375

GAA

Giu

ACT

Thr

TT

Phe

ACA

Thr

ACG

Thr 200

ATA

Ile

ACG

Thr

TCT

Ser

ATG

Met

CTC

Leu 280

CTG

Leu

ACC

Thr

AGC

Ser

CCG

Pro

CCC

Pro 360

ACT

Thr

ACT

Thr

CCT

Pro

CCT

Pro

AAA

Lys 185

GGG

Giy

GTA

Val

GTA

Val1

GAC

Asp

ATA

Ile 265

GCG

Al a

CAC

His

TTC

Phe

AAT

Asn

TTC

Phe 345

ACT

Thr

AGC

Ser

CTT

Leu

CTT

Leu

ACG

Thr 425 GCG CAG Ala Gin CTC ACG Leu Thr AAC GTC Asn Val TAC TCG Tyr Ser 235 GGG TTC Gly Phe 250 AAC GTT Asn Val TTT ACG Phe Thr CGG ATA Arg Ile GAC TGT Asp Cys 315 GTT CCC Val Pro 330 GAT ACC Asp Thr ACC GTG Thr Val GAT .ATG Asp Met TCG GTA Ser Val 395 ACC CTT Thr Leu 410 ACC GCG Thr Ala TAC ATT GGG GAG TGC TAC TCC AAC TCG Tyr Ile Gly Giu Cys Tyr Ser Asn Ser 190 195 TCT CTC AAC ATG ACC TTT TTC TAT TCG Ser Leu Asn Met Thr Phe Phe Tyr Ser 205 ,210 ACG TGG ACA ACC GGC GGC CCC TCC CCC Thr Trp Thr Thr Gly Gly Pro Ser Pro 220 225 TCG CGG GAG AAC GGG CAG CCC GTG TTG Ser Arg Giu Asn Gly Gin Pro Vai Leu 240 TTG GTT AAG TAC ACT CCC GAC ATT GAC Leu Val Lys Tyr Thr Pro Asp Ile Asp 255 260 ATT GCC AAT TAT TCG CCG GCG GAC TCC Ile Ala Asn Tyr Ser Pro Ala Asp Ser 270 275 GCC TTT AGG GAA GGA AAA CTC CCA TCC Ala Phe Arg Giu Gly Lys Leu Pro Ser 285 290 GAT ATG TCC GGG ACT GAG CCG CCG GGG Asp Met Ser Gly Thr Glu Pro Pro Gly 300 305 CAA AAA ATG ATA GA.A ACC CCG TAC CGA Gln Lys Met Ile Glu Thr Pro Tyr Arg 320 AGG GAC GAC TCT ATC CGT CCG GGG GCC Arg Asp Asp Ser Ile Arg Pro Gly Ala 335 340 OCA GCA CCT GAT TTC GAT ACA GGT ACT Ala Ala Pro Asp Phe Asp Thr Gly Thr 350 355 CCA GAG CCA GCC ATT ACT ACA CTC ATA Pro Giu Pro Ala Ile Thr Thr Leu Ile 365 370 GGA TTC 'rrC TCC ACG GCA CGT GCT ACC Gly Phe Phe Ser Thr Ala Arg Ala Thr 380 385 CCC GTC CAG GAA ACG GAT AGA ACT CTT Pro Val Gin Glu Thr Asp Arg Thr Leu 6236 6284 6332 6380 6428 6476 6524 6572 6620 6668 6716 6764 6812 6860 6908 6956 7004 400 CCA CTG ACT CCC GGT GAG TCA GAA AAT Pro Leu Thr Pro Gly Glu Ser Glu Asn 415 420 CCG GGG ATT TCT ACC GAG ACC CCG AGC Pro Gly Ile Ser Thr Glu Thr Pro Ser 430 435 WO 96/29396 WO 9629396PCTfUS96/03916 -96- ACC CAG AGT GCA GAA ACG GTG GTC TTT GCG Ala

ACT

Thr

GAA

Glu

ACT

Thr 485

AGT

Ser

GCA

Ala

CGT

Arg

GTT

Val

CCG

Pro 565

ATT

Ile

TCG

Ser

TTT

Phe

ACT

Ser

CCT

Pro 645

AGC

Ser

ACT

Thr

ACC

Thr

CCA

Ala

CAG

Gin

CCG

Pro 470

CAG

Gin

GCT

Ala

CCT

Pro

AC

Ser

TTT

Phe 550

AGC

Ser

TTT

Phe

ACT

Ser

TTC

Phe

ACA

Thr 630

TTC

Phe

GCC

Ala

GCA

Ala

GGG

Ciy CAT CAA His Ciu 440 ACT CCG Ser Pro 455 TCG TAT Trp, Tyr ACC CAC, Thr Gin GAA CAG Giu Gin GCC CAC Ala G1 n 520 ACG CCC Thr Pro 535 ACA CAG Thr Gin ACG GTA Thr Val ACT CGC Thr Arg CCC GAG Ala Ciu 600 ACT CAC Thr Gin 615 GAG CCC Ciu Pro ACC CCC Thr Arg GCA CCC Ala Pro CAG AC Gin Ser 680 ACC CAT Thr His 695 ACT ACA CAG Thr Thr Gin Thr Gin Ser Ala Giu Thr Val Val Phe

AGT

Ser

TTT

Phe

ATC

Ile

ATG

Met 505

ACC

Thr

CCC

Pro

ACT

Ser

CCC

Pro

ACT

Thr 585

CCC

Pro

CC

Ala

GAG

Ciu

ACT

Thr

GAG

Clu 665

ACA

Thr

AAT

Asn 445 450 ACC GAG TCC GAA ACC CC CGC TCC CAC ACT CAC Thr Ciu Ser Ciu Thr Ala Arg Ser Gin Ser Gin 460 465 ACT CAG ACT CCC AGT ACT GAA CAG CC CCT CTT Thr Gin Thr Pro Ser Thr Glu Gin Ala Ala Leu 475 480 CCA CAA ACC GAG C TTG TTT ACT CAC ACT CCC Ala Ciu Thr Ciu Ala Leu Phe Thr Gin Thr Pro 490 495 500 ACT TTT ACT CAC ACT CCC GGT GCA CAA ACC GAG Thr Phe Thr Gin Thr Pro Gly Ala Ciu Thr Clu 510 515 CCC ACC ACC ATA CCC GAG ATA TTT ACT CAC TCT Pro Ser Thr Ile Pro Ciu Ile Phe Thr Gin Ser 525 530 GAA ACC CCT CCC CCT CCC AGC C CC CCC GAG Ciu Thr Ala Arg Ala Pro Ser Ala Ala Pro Ciu 540 545 TCC ACT ACG GTA ACC GAG CTG TTT ACT CAG ACC Ser Ser Thr Val Thr Glu Val Phe Thr Gin Thr 555 560 AAA ACT ACT CTG ACT TCC ACT ACT GAA CCC CC Lys Thr Thr Leu Ser Ser Ser Thr Ciu Pro Ala 570 575 580 CAG ACC CC CCA ACT GAG CCC TTT ACT CAG ACT Gin Ser Ala Gly Thr Clu Ala Phe Thr Gin Thr 590 595 GAC ACT ATG CGA ACT CAG ACT ACT CAA ACA CAC Asp Thr Met Arg Thr Gin Ser Thr Giu Thr His 605 610 CCC ACT ACG GTA CCC AAA CCT ACT CAC ACT CCC Pro Ser Thr Val Pro Lys Ala Thr Gin Thr Pro 620 625 CTCG TTC ACT CAC ACT CCC ACT ACC CAA CCT CTC Val Leu Thr Gin Ser Pro Ser Thr Giu Pro Val 635 640 CTC CCC CCA GAG CCC CAA ATT ACT CAC ACC CCC Leu Cly Ala Giu Pro Ciu Ile Thr Gin Thr Pro 650 655 660 GTT TAT ACT CCC ACT TCG ACT ACC ATC CCA CAA Val Tyr Thr Arg Ser Ser Ser Thr Met Pro Giu 670 675 CCC CTG CCC TCC CAA AAC CCT ACC ACT TCC CGA Pro Leu Ala Ser Gin Asn Pro Thr Ser Ser Cly 685 690 ACT GAA CCC ACC ACT TAT CCA GTC CAA ACG ACA Thr Giu Pro Arg Thr Tyr Pro Val Gin Thr Thr 700 705 7052 7100 7148 7196 7244 7292 7340 7388 7436 7484 7532 7580 7628 7676 7724 7772 7820 WO 96/29396 PTU9/31 PCTIUS96/03916

CCA

Pro

ACT

Thr 725 ccc Pro

GAA

Giu

GAA

Giu

GGG

Gly

CAG

Gin 805

TGC

Cys

ACG

Thr

ACC

Thr

GTT

Val1

TAT

Tyr 885

GTG

Val

GGA

Gly

GTT

Val

GAA

Glu

CAT

His 710

GTT

Val

CTA

Leu

GTA

Val.

ACT

Thr

AAC

Asn 790

CTG

Leu

CTG

Leu

TCT

Ser.

TCT

Ser

GTT

Val 870

TTG

Leu

TG

Trp

GGC

Gly

ACT

Thr

AAC

Asn 950 ACC CAG AAA CTC Thr Gin Lys Leu GTT TCA GAA TTC Vai Ser Giu Phe 730 TTG GAC GTC AAA Leu Asp Val. Lys 745 ACG GCG ACT TGC Thr Ala Thr Cys 760 AAT TGG AAA GTA Asn Trp, Lys Val 775 AGT CCC GCC GGG Ser Pro Ala Gly TAC TAC AGA GTA Tyr Tyr Arg Vai 810 TCG TGC ACG AGC Ser Cys Thr Ser 825 CTT ATA GCG AGG Leu Ile Ala Arg 840 GAT CCG CAA ACG Asp Pro Gin Thr 855 CCG AGA TTC GAA Pro Arg Phe Glu ACG GCC ACG ACC Thr Ala Thr Thr 890 AAG AGC AGC GCG Lys Ser Ser Ala 905 AAT ATA TAC GAA Asn Ile Tyr Giu 920 ACG CGA AAG GAG Thr Arg Lys Glu 935 GOT GCT GCT CAG Gly Ala Ala Gin -97- TAC ACA GAA AAT AAG ACT TTA TCG TTT CCT Tyr Thr Giu Asn Lys Thr Leu Scr Phe Pro 715 720

CAT

His AT T Ile

OTT

Val

GAC

Asp

OTT

Val 795

ACC

Thr

CAT

His

GAA

Giu

GCA

Ala

TGG

TrD 875

OTT

Val

AGA

Arg

TGC

Cys GAG ATG TCG ACG GCA GAG TCG CAG ACG Giu Met Ser Thr Ala Giu Ser Gin Thr 735 740 GTA GAG GTG AAG TTT TCA AAC GAT GGC Val Giu Vai Lys Phe Ser Asn Asp Giy 750 755 TCC ACC OTC AAA TCT CCC TAT AGG GTA Ser Thr Val Lys Ser Pro Tyr Arg Val 765 770 CTC GTA GAT GTA ATO OAT GAA ATT TCT Leu Vai Asp Val Met Asp Oiu Ile Ser 780 785 TTT AAC AGT AAT GAG AAA TGG CAG AAA Phe Asn Scr Asn Glu Lys Trp Gin Lys 800 OAT GGA AGA ACA TC!G GTC CAG CTA ATG Asp Gly Arg Thr Ser Val Gin Leu Met 815 820 TCT CCG GAA CCT TAC TGT CTT TTC GAC Ser Pro Giu Pro Tyr Cys Leu Phe Asp 830 835 AAA GAT ATC GCG CCA GAG TTA TAC TTT Lys Asp Ile Ala Pro Giu Leu Tyr Phe 845 850 TAC TGC ACA ATA ACT CTG CCG TCC GGC Tyr Cys Thr Ile Thr Leu Pro Ser Giy 860 865 AGC CT AAT AAT GTT TCA CTO CCG GAA Ser Leu Asn Asn Va). Ser Leu Pro Giu 880 OTT TCG CAT ACC GCT GGC CAA AOT ACA Val 5cr His Thr Ala Gly Gin Scr Thr 895 900 GCA GOC GAG GCG TOG ATT TCT GGC CG Ala Gly Giu Ala Trp Ile Ser Gly Arg 910 915 ACC GTC CTC ATC TCA GAC GGC ACT CGC Thr Val Leu Ile Ser Asp Gly Thr Arg 925 930 7868 7916 7964 8 012 8060 8108 8156 8204 8252 8300 8348 8396 8444 8492 8540 8588 8636 AGO TGC TTA ACA AAC ACA TGG ATT GCG GTG Arg

GCG

Ala 955 Cys 940

CAG

Gin Lcu Thr CTG TAT Leu Tyr Asn Thr Trp Ile Ala Val 945 TCA CTC TTT TCT OGA CTT Ser Leu Phe Ser Gly Leu 960 GTG TCA GGA TTA TOC GGG AGC ATA Val 5cr Gly Leu Cys Gly Ser Ile 965 970 TCT OCT TTG TAC OCA ACG CTA TGG Ser Ala Leu Tyr Aia Thr Leu Trp, 975 980 WO 96/29396 WO 9629396PCTfUS96/03916 -98- ACC GCC ATT TAT TTT TGAGGAATGC TTTTTGGACT ATCGTACTGC TTTCTTCCTT Thr Ala Ile Tyr Phe 985

CGCTAGCCAG

CGCGCTAACC

CTGCGACGTT

AGAAAAAGAG

CGACGACGGC

CGTACAACTG

TCCTAGCACC

GCTCTCTGGC

AACTCTAGAA

GAAATGCTGG

CGCAGACACC

GCGCTGGAAT

CGTCCCGCAA

AAGCAGCGAA

TGGAGAAAAT

TGATCCAAAC

GACTACTAAA

GOTCGCGCTC

CCTAGAATAG

GAATAGCATA

AGCACCGCCG

ATACCGGCGG

GTCGAGCTCA

AAGGGGGGCC

GAGGACAAGT

CTATCTGAAT

CTTGTATCGC

CAATACTTGC

GTTAACGATC

ACAACAGAAC

AATACACGAC

AATTTGCTGA

GAAATTCCCG

AAGAAGGCCC

CCTGCCGCCC

TCGGATCCTG

AGTTCTAATG

GTGGGGCTAC

GTGGTTTCTT

CCGTCACGTA

TTGGCCCGTA

ACCCGATTTC

CTTTCGAGGC

ACTGTCCAAT

GCGCCGTTCA

GAAATGGCGC

TGACCCTGAA

GCTGTTTAAA

AGTATCAGAC

ACGCGGACGA

GGAAAAAGAA

CTGTAACCAA

CTCCAGAAGA

TCCCCGAAGA

ACTATTACAA

CCGTCTCCAT

TGGTTTGGAG

CCTACATGCC

CGACTACATT TTAGGCCGTC TAACAGATAC CTCACTAQGGG

TAACGTGGAC

CTCCGTCGTC

CTATAGAAAA

ATCTGCACAG

GGGACTGACT

AATCGGGAGA

GATCGGGTCG

TGGATTTCAA

CGTATATCGG

TCCTAGCGCG

GAAAGCGGAA

CTCGGAGGAC

CGACGAAGTC

TGACATGCCC

GCCCATATTZ

CATCGTAAAA

ACGCCTCACG

GACATGATAT

TGGTTCTACG

GAGTACAGGG

ATGTGGGCAG

ATATTCTCCC

TTTGCGCAAA

CAGCTTAACT

GGCGAACACC

GGATACGAAG

CCAGACCCTC

GGGCGCACCC

GACATGCAGG

CCCGAGGACA

GCCGTGATCC

GCGGCGTTCG

TGCGCGCGTA

G.CGCGCTCGA

TATCAAGAGG

CGGCGGCCAA

TGATTAAGGG

AATGTdGCGA

TGGACTATGT

CCACTGCTGC

CAGCTCTCGT

TTTTACCGTC

TTTATCCGAT

ATATTCTGCA

GTCCAGATAG

CGGACGCAGA

CAGAGGCTTC

CCGAGCACGA

CGGTGGAGGA

TAGCCTGCGC

GCTAATCGAG

8691 8751 8811 8871 8931 8991 9051 9111 9171 9231 9291 9351 9411 9471 9531 9591 9651 9711 9771 9831 9873 9894 9942 9990 10038 10086 10134 CTCATAATAT AAATCACATG CCAATGCCTA TTCATTGGGA CGTTCGAA.AA GC ATG GCA TCG CTA CTT GGA ACT Met 1

CTG

Leu Ala Ser Leu Leu Gly Thr GCT CTC Ala Leu CTT GCC GCG ACG Leu Ala Ala Thr GGA AAT CAC GTC Gly Asn His Val CTC GCA CCC TTC GGC GCG ATG GGA ATC Leu Ala Pro Phe Gly Ala Met Gly Ile 15 TCC GCC AGG ATT GAC GAC GAT CAC ATC Ser Ala Arg Ile Asp Asp Asp His Ile GTG ATC Val Ile GTG ATC Val Ile

ACT

Thr

TTC

Phe

ATG

Met GTC GCG CCT CGC Val Ala Pro Arg 45 CCT GGC CAG AGA Pro Gly Gin Arg TTT CGG TCT GAT Phe Arg Ser Asp CCC GAA GCT ACA ATT CA6A CTG CAG CTA TTT Prc Giu Ala Thr Ile Gin Leu Gin Leu Phe 50 CCC CAC AAA CCC TAC TCA GGA ACC GTC CGC Pro His Lys Pro Tyr Ser Gly Thr Val Arg 65 ATA ACA AAC CAG TGC TAC CAG GAA CTT AGC Ile Thr Asn Gin Cys Tyr Gin Giu Leu Ser 80 GTC GCG Val Ala WO 96/29396 PCTIUS96/03916 -99- GAG GAG CGC TTT GAA AAT TGC ACT CAT CGA TCG TCT TCT GTT TTT GTC 10182 Glu Glu Arg Phe Glu Asn Cys Thr His Arg Ser Ser Ser Val Phe Val 95 100 GGC TGT AAA GTG ACC GAG TAC ACG TTC TCC GCC TCG AAC AGA CTA ACC 10230 Gly Cys Lys Val Thr Glu Tyr Thr Phe Ser Ala Ser Asn Arg Leu Thr 105 110 115 GGA CCT CCA CAC CCG TTT AAG CTC ACT ATA CGA AAT CCT CGT CCG AAC 10278 Gly Pro Pro His Pro Phe Lys Leu Thr Ile Arg Asn Pro Arg Pro Asn 120 125 130 135 GAC AGC GGG ATG TTC TAC GTA ATT GTT CGG CTA GAC GAC ACC AAA GAA 10326 Asp Ser Gly Met Phe Tyr Val Ile Val Arg Leu Asp Asp Thr Lys Glu 140 145 150 CCC ATT GAC GTC TTC GCG ATC CAA CTA TCG GTG TAT CAA TTC GCG AAC 10374 Pro Ile Asp Val Phe Ala Ile Gin Leu Ser Val Tyr Gin Phe Ala Asn 155 160 165 ACC GCC GCG ACT CGC GGA CTC TAT TCC AAG GCT TCG TGT CGC ACC TTC 10422 Thr Ala Ala Thr Arg Gly Leu Tyr Ser Lys Ala Ser Cys Arg Thr Phe 170 175 180 GGA TTA CCT ACC GTC CAA CTT GAG GCC TAT CTC AGG ACC GAG GAA AGT 10470 Gly Leu Pro Thr Val Gin Leu Glu Ala Tyr Leu Arg Thr Glu Glu Ser 185 190 195 TGG CGC AAC TGG CAA GCG TAC GTT GCC ACG GAG GCC ACG ACG ACC AGC 10518 Trp Arg Asn Trp Gin Ala Tyr Val Ala Thr Glu Ala Thr Thr Thr Ser 200 205 210 215 GCC GAG GCG ACA ACC CCG ACG CCC GTC ACT GCA ACC AGC GCC TCC GAA 10566 Ala Glu Ala Thr Thr Pro Thr Pro Val Thr Ala Thr Ser Ala Ser Glu 220 225 230 CTT GAA GCG GAA CAC TTT ACC TTT CCC TGG CTA GAA AAT GGC GTG GAT 10614 Leu Glu Ala Giu His Phe Thr Phe Pro Trp Leu Glu Asn Gly Val Asp 235 240 245 CAT TAC GAA CCG ACA CCC GCA AAC GAA AAT TCA AAC GTT ACT GTC CGT 10662 His Tyr Glu Pro Thr Pro Ala Asn Glu Asn Ser Asn Val Thr Val Arg 250 255 260 CTC GGG ACA ATG AGC CZT ACG CTA ATT GGG GTA ACC GTG GCT GCC GTC 10710 Leu Gly Thr Met Ser Pro Thr Leu Ile Gly Val Thr Val Ala Ala Val 265 270 275 GTG AGC GCA ACG ATC GGC CTC GTC ATT GTA ATT TCC ATC GTC ACC AGA 10758 Val Ser Ala Thr Ile Gly Leu Val Ile Val Ile Ser Ile Val Thr Arg 280 285 290 295 AAC ATG TGC ACC CCG CAC CGA AAA TTA GAC ACG GTC TCG CAA GAC GAC 10806 Asn Met Cys Thr Pro His Arg Lys Leu Asp Thr Val Ser Gin Asp Asp 300 305 310 GAA GAA CGT TCC CAA ACT AGA AGG GAA TCG CGA AAA TTT GGA CCC ATG 10854 Glu Glu Arg Ser Gin Thr Arg Arg Glu Ser Arg Lys Phe Gly Pro Met 315 320 325 GTT GCG TGC GAA ATA AAC AAG GGC GCT GAC CAG GAT AGT GAA CTT GTG 10902 Val Ala Cys Glu Ile Asn Lys Gly Ala Asp Gln Asp Ser Glu Leu Val 330 335 340 GAA CTG GTT GCG ATT GTT AAC CCG TCT GCG CTA AGC TCG CCC GAC TCA 10950 Glu Leu Val Ala Ile Val Asn Pro Ser Ala Leu Ser Ser Pro Asp Ser 345 350 355 WO 96/29396 PTU9131 PCTIUS96/03916 -100- ATA AAA ATG TGATTAAGTC TGAATGTGGC TCTCCAATCA TTTCGATTCT Ile Lys Met 360 CTAATCTCCC AATCCTCTCA AAAGGGGCAG TATCGGACAC GGACTGGGAG GGGCGTACTA CACGATAGTT ATATGGTACA GCAGAGGCCT CTGAACACTT AGGAGGAGAA TTCAGCCGGG GAGAGCCCCT GTTGAGTAGG CTTGGGAGCA TATTGCAGG ATG AAC ATG TTA GTG Met Asn Met Leu Val

ATA

Ile

GTC

Val

AGA

Arg

AAC

Asn

TGC

Cys

GCG

Al a

AGC

Ser

ATC

Ile

TCG

Ser

ATG

Met 150

AAG

Lys

GAG

Glu

AAC

Asn

GTT

Val

CTC

Leu

AAC

Asn

GCG

Ala

TTT

Phe

GGA

Gly

TTC

Phe

ATG

Met

GAC

Asp 135

GAA

Glu

GCT

Ala

GCC

Ala

GAC

Asp

CTC

Leu

TTT

Phe

GTT

Val

TGC

Cys

CAT

His

CCC

Pro

GTC

Val

TGG

Trp 120

GAG

Glu

ACT

Thr

CCA

Pro

CAC

His

AAC

Asn 200

GCC

Ala

TTG

Leu

CCG

Pro

AAG

Lys

GAT

Asp

CTG

Leu

GTG

Val 105

ACA

Thr

GGA

Gly

ACA

Thr

GCA

Ala

GCG

Ala 185

AGC

Ser

TCT

Ser

GAA

Glu

GAA

Glu

ATG

Met

TTA

Leu

TCT

Ser

CTG

Leu

GAG

Glii

ATC

Ile

TCG

Ser

CCC

Pro 170

CAC

His

CCC

Pro TGT CTT CC Cys Leu Ala GGC ACT CAC Cly Thr Gln GGG ACT GTA Gly Thr Val 45 AAC CC GCC Lys Ala Ala 60 ATT TAC GAC Ile Tyr Asp 75 CCA AAC CTG Ala Asn Leu GCT TCT GGG CJly Ser Gly TAC GGA GGC Tyr Gly Gly 125 TAT TTT CGA Tyr Phe Arg 140 TAC AAC CTC Tyr Asn Val 155 CAC GAG GTG His Glu Val CTC GAA TTA Val Glu Leu ACC TAT GTG Thr Tyr Val 205

CGC

Arg

GCT

Ala 30

ATC

Ile

CAT

Asp

GGA

Gly

GTA

Val CTA ACT TTT GCG ACG CGA CAC Leu Thr Phe Ala Thr Arg His 15 GTC CTC GGG GAA GAT GAT CCC Val Leu Gly Giu Asp Asp Pro AAA TGG ACA AAA GTC CTC CGG Lys Trp Thr Lys Val Leu Arg GTC TGC TCT TCG CCT AAC TAT Val Cys Ser Ser Pro Asn Tyr GGA AAG AAA GAC TCC CCG CCC Cly Lys Lys Asp Cys Pro Pro 80 ATT TTA CTA AAG CCC GGC GAA Ile Leu Leu Lys Arg Gly Glu 95 100 10999 11059 11119 11173 11221 11269 11317 11365 11413 11461 11509 11557 11605 11653 11701 11749 11797 11845 CTA CAC AAC AGC AAT ATA ACT AAT Leu His Asn Ser Asn Ile Thr Asn 110 115 CTG CTC TTT GAT CCT CTA ACT CGT Leu Leu Phe Asp Pro Val Thr Arg 130 CGG ATC TCT CAG CCA GAT CTG GCC Arg Ile Ser Gin Pro Asp Leu Ala 145 AGC CTT CTT TCG CAC CTA GAC GAG Ser Val Leu Ser His Val Asp Clu 160 165 GAG ATA GAC ACC ATC AAC CCG TCA Glu Ile Asp Thr Ile Lys Pro Ser 175 180 CAA ATG CTG CCC TTT CAT GAA CTC Gin Met Leu Pro Phe His Glu Leu 190 195 ACC CCT GTT CTT AGA CTC TTC CCA Thr Pro Val Leu Arg Val Phe Pro 210 CCG ACC GAG CAC CTA AAA TTT AAC GTT ACG TAT TCG TGG TAT GGG TTT Pro Thr 215 Glu His Val Lys Phe 220 Asn Val Thr Tyr Ser Trp Tyr Gly Phe 225 WO 96/29396 PTU9/31 PCTfUS96/03916

GAT

Asp 230

TAC

Tyr

TGC

Cys

TTG

Leu

GTG

Val

CTG

Leu 310

GTT

Val

CGT

Arg

CCG

Pro

GGT

Gly

GTA

Val 390

CAC

His ccc Pro

ATT

Ile

GAA

Glu

GAA

Giu 470

ATC

Ile

GTC

Val

CAT

His

CTC

Leu

CTG

Leu

CCG

Pro 295

GTG

Val

TCC

Ser

AAC

Asn

ATG

Met

AAC

Asn 375

ATT

Ile

CAA

Gin

AAA

Lys

TAT

Tyr

TTC

Phe 455

AGG

Arg

AAA

Lys

CCT

Pro

ATA

Ile

GAT

Asp 280

AAC

Asn

ATC

Ile

GAC

Asp

AGT

Ser

AAC

Asn 360

TAT

Tyr

CTT

Leu

ATG

Met

AAC

Asn

GAC

Asp 440

CGC

Arg

GCC

Al a

GAG

Glu

ACA

Thr

GGA

Giy 265

TGT

Cys

CTA

Leu

AGT

Ser

GGA

Gly

CCG

Pro 345

AAA

Lys

AAA

Lys

ACA

Thr

GGT

Gly

GAG

Glu 425

GAA

Giu

GAA

Giu

TTA

Leu

GAG

Glu

GAC

Asp 250

TCT

Ser

TCC

Ser

CGG

Arg

CCT

Pro

GAC

Asp 330

CGC

Arg

GTA

Val1

ATG

Met

TGC

Cys

ACT

Thr 410

TTT

Phe

GTA

Val1

GAT

Asp

AAC

Asn -101- TGC GAA GAA GTG AAA CTG TTC GAG CCG TGC GTA Cys Glu Glu Val Lys Leu Phe Giu Pro Cys Val 235 240 245 GGC AAA TGT CAG TTT CCC GCA ACC AAC CAG AGA Giy Lys Cys Gin Phe Pro Ala Thr Asn Gin Arg 255 260 GTC TTG ATG GCG GAA TTC TTG GGC GCG GCC TCT Val Leu Met Ala Giu Phe Leu Gly Ala Ala Ser 270 275 CGC GAT ACT CTA GAA GAC TGC CAC GAA AAT CGC Arg Asp Thr Leu Giu Asp Cys His Glu Asn Arg 285 290 TTC GAT TCG CGA CTC TCC GAG TCA CGC GCA GGC Phe Asp Ser Arg Leu Ser Glu Ser Arg Ala Gly 300 305 CTT ATA GCC ATC CCC AAA GTT TTG ATT ATA GTC Leu Ile Ala Ile Pro Lys Val Leu Ile Ile Val 315 320 325 ATT TTG GGA TGG AGC TAC ACG GTG CTC GGG AAA Ile Leu Gly Trp Ser Tyr Thr Val Leu Gly Lys 335 340 GTA GTA GTC GAA ACG CAC ATG CCC TCG AAG GTC Val Val Val Glu Thr His Met Pro Ser Lys Val 350 355 GTA ATT GGC AGT CCC GGA CCA ATG GAC GAA ACG Vai Ile Giy Ser Pro Gly Pro Met Asp Giu Thr 365 370 TAC TTC GTC GTC GCG GGG GTG GCC GCG ACG TGE Tyr Phe Val Vai Ala Gly Val Ala Ala Thr C .380 385 GCT CTG CTT GTG GGG AAA AAG AAG TGC CCC GCG Ala Leu Leu Val Gly Lys Lys Lys Cys Pro Ala 395 400 405 T.T TCC AAG ACC GAA CCA TTG TAC GCG CCG CTC Phe Ser Lys Thr Glu Pro Leu Tyr Ala Pro Leu 415 420 GAG GCC GGC GGG CTT ACG GAC GAT GAG GAA GTG Giu Ala Gly Gly Leu Thr Asp Asp Giu Giu Val 430 435 TAC.GAA CCC CTA TTT CGC GGC TAC TGT AAG CAG Tyr Giu Pro Leu Phe Arg Gly Tyr Cys Lys Gin 445 450 GTG AAT ACC =T TTC GGT GCG GTC GTG GAG GGA Val Asn Thr Phe Phe Gly Ala Val Val Glu Gly 460 465 TTT AAA TCC GCC ATC GCA TCA ATG GCA GAT CGC Phe Lys Ser Ala Ile Ala Ser Met Ala Asp Arg 475 480 485 11893 11941 11989 12037 12085 12133 12181 12229 12277 12325 12373 12421 12469 12517 12565 12613 12664 CTG GCA AAT AAA Leu Ala Asn Lys 490 AGC GGC Ser Gly AGA AGG AAT ATG GAT AGC TAT TAGTTGGTC Arg Arg Asn Met Asp Ser Tyr 495 500 WO 96/29396 WO 9629396PCTIUS96/03916

ATG

Met 1

GCG

Ala

TTA

Leu

AAT

Asn

GGG

Gly

TGC

Cys

ACA

Thr

AAA

Lys

TGT

Cys

GCG

Al a

GTC

Val1 155

ACG

Thr

CGC

Arg

CCC

Pro

GTC

Val

CTT

Leu 235

CCT

Pro

GCC

Ala

CCG

Pro

TAT

Tyr

GAA

Glu

TGC

Cys Ccc Pro

CTA

Leu

AAA

Lys

CAA

Gln 140

GAC

Asp

ACG

Thr

CTG

Leu

TCC

Ser

GGG

Gly 220

CCG

Pro

TTT

Phe

GCG

Al a

ACC

Thr

ACC

Thr

TCT

Ser

GCA

Ala

ATG

Met

TCA

Ser

CTT

Leu 125

AGA

Arg

GCT

Ala

GAA

Glu

AAG

Lys

GAA

Glu 205

GCA

Ala

CTC

Leu AAG ACC Lys Thr 5 GGC AAG Gly Lys AGA CTG Arg Leu CAG CCT Gin Pro GAC AAC Asp Asn TCG CGC Ser Arg CCT ACT Pro Thr TCT TCA Ser Ser 110 CCG GAT Pro Asp TAT ACT Tyr Thr GTT GCG Val Ala CAA ACG Gln Thr 175 CCG AAA Pro Lys 190 GGC ACG Gly Thr GCG ATA Ala Ile GCC GCG Ala Ala AGA GGG GCC Arg Gly Ala AAC AGG TTT Asn Arg Phe CGT GGC ACC Arg Gly Thr ATT CCC TGG Ile Pro Trp 50 CAC GCG TGT His Ala Cys 65 TCT ACC GTT Ser Thr Val 80 GAG TAT GGG Glu Tyr Gly GAC TSS GAG Asp Xaa Glu TCT CAA GCA Ser Gln Ala 130 GTT GAC GAG Val Asp Glu 145 GAC TTA GAA Asp Leu Glu 160 GAA AGT AAA Glu Ser Lys CCC ACA AAC Pro Thr Asn GTC GAG GCG Val Glu Ala 210 GCA AAC GAG Ala Asn Glu 225 TCG GCG GCC Ser Ala Ala 240 -102- GAA GAC Glu Asp AAG AAA TCG AGA AAT CGG GAA ATC Lys Lys Ser Arg Asn Arg Glu Ile 20 GGT AAG AAA ACT GCC GGA TTG TCC Gly Lys Lys Thr Ala Gly Leu Ser 35 AAC CCT AAA TTC TGC AGC C CC Asn Pro Lys Phe Cys Ser Ala Arg AAA GAC ACT TT TAT CGC AGG ACG Lys Asp Thr Phe Tyr Arg Arg Thr TCC ACT CAA CCC GAT TCC CCC CAC Ser Ser Gin Pro Asp Ser Pro His 85 CGC GTG CCC TCC GCA AAG CGC AAA Arg Val Pro Ser Ala Lys Arg Lys 100 105 CGC GCG Cly Ala 115 GCA CCG Ala Pro GTT TCG Val Ser ACG CC Thr Arg AAT AAG Asn Lys 180 GAG CAC Glu His 195 CCA TCG Pro Ser CTG GCT Leu Ala CCT GCC Ala Ala CAC CAA CCC CTA GTA TCC His Gin Pro Leu Val Ser 120 GCG CGA ACC TAT AGT TCT Ala Arg Thr Tyr Ser Ser 135 TCG CCA ACT CCG CCA GCC Ser Pro Thr Pro Pro Gly 150 GCG GAA CTT CCT GGC GCT Ala Glu Leu Pro Gly Ala 165 170 CTC CCC AAC CAA CAA TCG Leu Pro Asn Gln Gin Ser 185 GTC GGA GGG GAG CGG TGC Val Gly Cly Clu Arg Cys 200 CTC GCC ATC CTC TCG CGC Leu Gly Ile Leu Ser Arg 215 CGT ATG CGG AGO GCG TGT Arg Met Arg Arg Ala Cys 230 OGA ATA GTG GCC TGG GCC Cly Ile Val Ala Trp, Ala 245 250 12694 12742 12790 12838 12886 12934 12982 13030 13078 13126 13174 13222 13270 13318 13366 13414 13467 CCG GCG AGG GCC TTG Ala Ala Arg Ala Leu CAC AAA CAA COO CCC Gin Lys Gln Gly Arg 260 TAG CAGTAATAATA ACCACACAA WO 96/29396 PCT/US96/03916 -103-

ATATTG

INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 476 amino acids TYPE: amino acid TOPOLOGY: linear 13473 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEC Met 1 Arg Ser Thr Thr Pro Pro Ala Lys Val 145 Glu Lys Ala Glu Tyr 225 Asp Ara Arg Phe Arg Thr Phe Ala Glu Pro Ser Ser His 130 Glu Glu Cys Ser Asn 210 Met Glu Ile Thr Ser Glu Arg Ala Thr Ser 115 Ser Gly Gin Lys Gin 195 Val Leu Ala Met Glu Arg Gly Ala Asn Thr 100 Asp Asp Ala Arg Arg 180 Leu Leu Thr Phe Lys 260 Arg 5 Asn Thr Val Glu Glu Glu Tyr Glu Tyr Arg 165 Leu Glu Lys Gln Asp 245 Gin Ile Cys Ser Arg Pro Met Glu Pro 70 Asn Lys Ala Phe Gly 150 Arg Ile Asn Ile Arg 230 Trp Leu Leu Ser Arg 55 Val Gly Val Gly Leu 135 Phe Gly Ala Glu Gin 215 Tyr Lys Met Thr Leu 40 Leu Thr Gin Asp Pro 120 Gin Ile Val Lys Ile 200 Glu Gln Asp Ser Ser 25 Arg Ala Glu Asn Gly 105 Val His Cys Asn Tyr 185 Leu Ile Phe Ser Ala 265 ID NO:2: Ser Arg Ala 10 Lys Arg Val Pro Tyr Ala Glu Leu Val Asn Thr Ala 75 Phe Ala Tyr 90 Ser His Thr Pro Leu Ala Phe Arg Val 140 Gly Val Arg 155 Ser Thr Asn 170 Val Lys Asn Val Leu Gly Leu Arg Tyr 220 Asp Leu Tyr 235 Pro Met Leu 250 Val Ser Tyr Glu Cys Glu Ser Val Ala Asp Gin 125 Leu Arg Gin Gly Arg 205 Pro Ser Lys Ile Lys Val Ile Val Asn Gly Phe 110 Thr Asp Tyr Gly Thr 190 Leu Asp Tyr Gin His 270 Arg Arg Leu Asp Leu Pro Thr Met Ser Ile Asp Gly Asp Glu Arg Leu Asp Leu Thr Glu 160 Lys Ser 175 Arg Ala Asn His Asn Thr Met Tyr 240 Thr Arg 255 Ser Lys WO 96/29396 WO 9629396PCTIUS96/039 16 -104- Lys Asp Asn 305 Asn Ile Cys Val Cys 385 His Glu Ser Arg Ile 465 Leu Gly 290 Glu Ser Trp Pro Ile 370 Asn Thr Tyr Al a Thr 450 Arg Ile 275 Lys Arg Pro Ser Ile 355 Asp Leu Val Pro Ala 435 Ile Ala His Arg Asp Ile Thr Val Giu Pro Glu Ile 325 Cys Gly 340 Gly Asp Ser Leu Tyr Asn Pro Ser 405 Leu Val 420 Glu Val Thr Ile Arg Vai Leu Phe 310 Leu Val Giy Ser Tyr 390 Leu Lys Leu Ile Pro 470 Gly 295 Giu Ala Val Gly Val 375 Leu Ile Met Ala His 455 Arg Lys Leu 280 Asp Phe Tyr Gly Arg Asp Leu Leu 345 Giy Asn 360 Cys Asp His Tyr Arg Asn Leu Thr 425 Met Pro 440 Gly Lys Ser Met Glu Giy Trp Ser 330 Glu Pro Glu.

Al a Leu 410 Phe Leu His Asn Thr Vai 315 Tyr Met His Glu Ser 395 His Asp Phe Lys Ile Val 300 Gly Cys Val Gin Phe 380 Ile Leu Trp Ser Pro 460 Phe Leu 285 Thr Pro Thr Val Giu Ile Ser His 350 Gin Leu 365 Pro Asp Aisp Arg Pro Ala Arg Leu 430 Ala Glu 445 Ile Arg Asn Phe Ala Thr 335 Glu Leu Pro Al a Asp 415 Arg Glu Pro Cys Glu Thr 320 Asp Phe Lys Pro Gly 400 Val Pro Giu Glu Ser Giu Gly 475 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 510 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Thr Leu Pro His Arg Leu Thr Lys Arg Pro Phe Ala Arg Arg Phe 1 5 10 Cys Ser Val Phe Val Ile His Tyr Ser Giu Thr Lys Leu Asp Arg Tyr 25 Asn Lys Thr Met Leu Leu Tyr Arg Pro Asp Ser Thr Met Arg His Ser 40 Gly Gly Asp Ala Asn His Arg Gly Ile Arg Pro Arg Axg Lys Ser Ile 55 Gly Ala Phe Ser Ala Arg Glu Lys Thr Gly Lys Arg Asn Ala Leu Thr 70 75 WO 96/29396 PCTIUS96/03916 -105- Glu Glu Glu Asp Gin 145 Phe Glu Pro Phe Cys 225 Ile His Glu Gly Arg 305 Asp Asp Ala His Tyr 385 Gly Leu Ser Phe Val Ala 130 Pro Thr Ala Phe Leu 210 Leu Val Trp Glu Ala 290 Asp Ala Thr Leu Ala 370 Asp Ala Ala Ser Gly Leu 115 Glu Phe Arg Leu Thr 195 Gly Arg Asn Arg Asn 275 Gly Lys Ala Ser Val 355 Leu Glu Glu Val Ser Ser Ser Asp Met Leu Gly 100 Ser Glu Asn Ala Lys 180 Arg Asp Gin Cys Gly 260 Ile Leu Arg Val Arg 340 Val Val Thr Gly Val 420 Lys Gin Glu Ala Pro 165 Val Ser Ser His Asn 245 Met Gin Phe Leu Leu 325 Phe Leu Phe Arg Thr 405 Cys Trp Ala Arg Trp 150 Ile Gly Lys Phe Ile 230 Asp Arg His Leu Leu 310 Ala Glu Leu Met Tyr 390 Leu Ala Thr Trp Val 135 Pro Val Ala Lys Trp 215 Thr Pro Val Gly Trp 295 Arg Ser Pro Ala Arg 375 Ile Leu Asn Jal Asp 120 Thr Asp Tyr Phe Ser 200 Arg Arg Leu Pro Pro 280 Pro Ala Phe Ala Glu 360 Ala Asp Arg Thr Asp 105 Val Tyr Gly Pro Val 185 Val Ile Leu Trp Ser 265 Met Ala Cys Pro Leu 345 Thr Leu Pro Gly Tyr 425 Asp 90 Gly Leu Glu Pro Ser 170 Ser Thr Met Arg Tyr 250 Leu Ala Met Leu Phe 330 Gly Val Ala Val Ile 410 Ser Pro Gin Ser Ser 155 Ala Arg Val Gin His 235 Ala Lys Ala Arg Ser 315 Tyr Cys Leu Asp Lys 395 Val Thr Pro Phe Ser Thr Ala Leu Lys 140 Trp Glu Val Arg Asn 220 Pro Tyr Leu Val Ala 300 Ser Trp Leu Ala Gly 380 Asn Ala Ile Asp Val 125 Pro Asn Val Leu Asp 205 Val Ser Ala Ala Phe 285 Ala Leu Arg Ser Thr 365 Asn Glu Ser Arg Ile 110 Lys Thr Ala Leu Gin 190 Ala Tyr Ser Asn Ser 270 Arg Phe Asp Gly Glu 350 Met Phe Tyr Asn Lys 430 Asp Lys Thr Ala His Glu Pro Ile Gin Asp 160 Asp Ala 175 Cys Val Gin Ser Thr Val Lys Ser 240 Gin Phe 255 Pro Pro Asn Ala Glu Glu Ile Met 320 Val Gin 335 Tyr Phe Phe Asp Asp Asp Leu Asn 400 Thr Ala 415 Leu Pro WO 96/29396 PCTIUS96/03916 Ser Lys Glu 465 Asn His Ala 435 Arg Phe Cys Cys Thr Arg Phe Leu Ser 500 Ser Pro Tyr Asn 485 Gly Cys Met 455 Ala Leu Ala Asn 440 Ser Trp Lys Arg -106- Val Ala Tyr Arg Thr Glu Thr Leu 445 Asp Ile Tyr Arg Ile Leu Gln Lys 460 Leu Gln Arg Val Ala Thr His Ala 475 480 Arg Ser Val Asp Thr Gly Pro Arg 490 495 Arg Ser Gly Cys Ser Ser 505 510 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 110 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Leu Cys Pro Leu Leu Val Pro Ile Gln Tyr Glu Asp 1 5 10 Ala Met Gly Ser Glu Leu Lys Arg Glu Lys Leu Glu Thr 25 Ala Ile Ser Ser Asp Arg Asp Pro Arg Gly Ser Leu Arg 40 Ser Asp His Ala Arg Glu Ile Ile Ala Asp Gly Val Arg 55 Val Ile Asp Glu Pro Val Arg Ala Ser Val Ala Leu Ser 70 Ala Gly Lys Val Lys Ala Arg Arg Leu Thr Ser Val Arg 90 Pro Pro Ala Gly Ala Val Ser Ala Arg Arg Lys Ser Glu 100 105 Phe Phe Phe Phe Thr Ala Ile 110 Ser Val Leu Lys Ala Pro INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 292 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ser Gly Phe Ser Asn Ile Gly Ser Ile Ala Thr Val Ser Leu Val 1 5 10 Cys Ser Leu Leu Cys Ala Ser Val Leu Gly Ala Pro Val Leu Asp Gly 25 WO 96/29396 PCTIUS96/03916 -107- Leu Glu Ser Ser Pro Phe Pro Phe Gly Gly Lys Ile Ile Ala Gln Ala 40 Cys Asn Arg Thr Thr Ile Glu Val Thr Val Pro Trp Ser Asp Tyr Ser 55 Gly Arg Thr Glu Gly Val Ser Val Glu Val Lys Trp Phe Tyr Gly Asn 70 75 Ser Asn Pro Glu Ser Phe Val Phe Gly Val Asp Ser Glu Thr Gly Ser 90 Gly His Glu Asp Leu Ser Thr Cys Trp Ala Leu Ile His Asn Leu Asn 100 105 110 Ala Ser Val Cys Arg Ala Ser Asp Ala Gly Ile Pro Asp Phe Asp Lys 115 120 125 Gin Cys Glu Lys Val Gln Arg Arg Leu Arg Ser Gly Val Glu Leu Gly 130 135 140 Ser Tyr Val Ser Gly Asn Gly Ser Leu Val Leu Tyr Pro Gly Met Tyr 145 150 155 160 Asp Ala Gly Ile Tyr Ala Tyr Gln Leu Ser Val Gly Gly Lys Gly Tyr 165 170 175 Thr Gly Ser Val Tyr Leu Asp Val Gly Pro Asn Pro Gly Cys His Asp 180 185 190 Gin Tyr Gly Tyr Thr Tyr Tyr Ser Leu Ala Asp Glu Ala Ser Asp Leu 195 200 205 Ser Ser Tyr Asp Val Ala Ser Pro Glu Leu Asp Gly Pro Met Glu Glu 210 215 220 Asp Tyr Ser Asn Cys Leu Asp Met Pro Pro Leu Arg Pro Trp Thr Thr 225 230 235 240 Val Cys Ser His Asp Val Glu Glu Gln Glu Asn Ala Thr Asp Glu Leu 245 250 255 Tyr Leu Trp Asp Glu Glu Cys Ala Gly Pro Leu Asp .Glu Tyr Val Asp 260 265 270 Glu Arg Ser Glu Thr Met Pro Arg Met Val Val Phe Ser Pro Pro Ser 275 280 285 Thr Leu Gln Gin 290 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 985 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Gly Thr Met Leu Val Leu Arg Leu Phe Leu Leu Ala Val Ala Asp 1 5 10 WO 96/29396 PCTIUS96/03916 -108- Ala Ala Gly Gly Thr Gly Ile Phe Asp Glu Val Thr Tyr Phe Tyr Ser 130 Val Arg 145 Thr Trp Asp Asn Tyr Ser Phe Phe 210 Gly Pro 225 Gin Pro Pro Asp Pro Ala Lys Leu 290 Glu Pro 305 Thr Pro Arg Pro Leu Pro Thr Ile His Ala Ala Asp Leu Ala Phe Ser 100 Thr Ile 115 Val Ile Leu Glu Gly Ser Asp Val 180 Asn Ser 195 Tyr Ser Ser Pro Val Leu Ile Asp 260 Asp Ser 275 Pro Ser Pro Gly Tyr Arg Gly Ala 340 Thr Gly Arg Phe Gin Thr Thr Leu Leu Gly Ser Thr Lys 165 Ala Ala Pro Ser Arg 245 Gly Gly Ala Thr Ala 325 Thr Glu Tyr Pro 70 Glu Ser Val Arg Pro 150 Arg Val Ala Lys Arg 230 Asn Arg Ser Ile Glu 310 Leu Leu Asn Pro 55 Thr Arg Tyr Phe Val 135 Cys Trp Leu Gin Arg 215 Ile Val Ala Val Gin 295 Thr Gly Pro Leu 40 Pro Leu Gly Arg Ala 120 Leu Asp Leu Thr Thr 200 Ile Thr Ser Met Leu 290 Leu Thr Ser Prc Cys 25 Ala Pro Arg Thr Pro 105 Thr Val Glu Gly Lys 185 Gly Val Val Asp Ile 265 Ala His Phe Asn Phe 345 Arg Val Trp Lys Val Pro Pro Val Glu Ile Ser 90 Arg Gly Asn Asn Pro 170 Ala Leu Asn Tyr Gly 250 Asn Phe Arg Asp Val 330 Asp Leu Gly Arg 75 Ala Pro Gin Ala Phe 155 Pro Gin Thr Val Ser 235 Phe Val Thr Ile Cys 315 Pro Thr Ala Ala Tyr( Ser Glu Ser Ser 140 Leu Ser Tyr Ser Thr 220 Ser Leu Ile Ala Asp 300 Gin Arg Ala Glu Val Gly Asp Ile Thr 125 Leu Gln Pro Ile Leu 205 Trp Arg Val Ala Phe 285 Met Lys Asp Ala Ser Ser Pro Ala His 110 Glu Glu Asn Tyr Gly 190 Asn Thr Glu Lys Asn 270 Arg Ser Met Asp Pro 350 Pro Phe Thr Asp Gly.

Ser Arg Glu Val 175 Glu Met Thr Asn Tyr 255 Tyr Glu Gly Ile Ser 335 Asp Jal Gln Glu Asn Ala Ser Ser Pro 160 Arg Cys Thr Gly Gly 240 Thr Ser Gly Thr Glu 320 Ile Phe Asp Thr Gly 355 Thr Ser Pro Thr Pro Thr Thr Val Pro Glu Pro Ala Ile 360 365 WO 96/29396 PCTfUS96/03916 -109- Thr Thr Leu Ile Pro Arg Ser Thr Ser Asp Met Gly Phe Phe Ser Thr 370 375 380 Ala Arg Ala Thr Gly Ser Giu Thr Leu Ser Val Pro Val Gin Giu Thr 385 390 395 400 Asp Arg Thr Leu Ser Thr Thr Pro Leu Thr Leu Pro Leu Thr Pro Gly 405 410 415 Giu Ser Giu Asn Thr Leu Phe Pro Thr Thr Ala Pro Gly Ile Ser Thr 420 425 430 Giu Thr Pro Ser Ala Ala His Giu Thr Thr Gin Thr Gin Ser Ala Giu 435 440 445 Thr Val Val Phe Thr Gin Ser Pro Ser Thr Glu Ser Giu Thr Aia Arg 450 455 460 Ser Gin Ser Gin Glu Pro Trp Tyr Phe Thr Gin Thr Pro Ser Thr Giu 465 470 475 480 Gin Ala Ala Leu Thr Gin Thr Gin Ile Ala Glu Thr Giu Ala Leu Phe 485 490 495 Thr Gin Thr Pro Ser Ala Giu Gin Met Thr Phe Thr Gin Thr Pro Gly 500 505 510 Ala Giu Thr Giu Ala Pro Ala Gin Thr Pro Ser Thr Ile Pro Giu Ile 515 520 525 Phe Thr Gin Ser Arg Ser Thr Pro Pro Giu Thr Ala Arg Ala Pro Ser 530 535 540 Ala Ala.Pro Giu Val Phe Thr Gin Ser Ser Ser Thr Val Thr Glu Val 545 550 555 560 Phe Thr Gin Thr Pro Ser Thr Val Pro Lys Thr Thr Leu Ser Ser Ser 565 570 575 Thr Glu Pro Ala Ile Phe Thr Arg Thr Gin Ser Ala Gly Thr Glu Ala 580 585 590 Phe Thr Gin Thr Ser Ser Ala Glu Pro Asp Thr Met Arg Thr Gin Ser 595 603 605 Thr Giu Thr His Phe Phe Thr Gin Ala Pro Ser Thr Val Pro Lys Ala 610 615 620 Thr Gin Thr Pro Ser Thr Glu Pro Giu Vai Leu Thr Gin Ser Pro Ser 625 630 635 640 Thr Giu Pro Val Pro Phe Thr Arg Thr Leu Gly Ala Giu Pro Giu Ile 645 650 655 Thr Gin Thr Pro Ser Ala Ala Pro Giu Val Tyr Thr Arg Ser Ser Ser 660 665 670 Thr met Pro Glu Thr Ala Gin Ser Thr Pro Leu Ala Ser Gin Asn Pro 675 680 685 Thr Ser Ser Gly Thr Gly Thr His Asn Thr Glu Pro Arg Thr Tyr Pro 690 695 700 Val Gin Thr Thr Pro His Thr Gin Lys Leu Tyr Thr Glu Asn Lys Thr 710 715 720 WO 96/29396 WO 9629396PCT[US96/03916 -110- Leu Ser Phe Pro Thr Val Val Ser Glu Phe His Giu Met Ser Thr Ala 725 730 735 Glu Ser Gin Thr Pro Leu Leu Asp Val Lys Ile Val Glu Val Lys Phe 740 745 750 Ser Asn Asp Gly Giu Val Thr Ala Thr Cys Val Ser Thr Val Lys Ser 755 760 765 Pro Tyr Arg Val Giu Thr Asn Trp Lys Val Asp Leu Val Asp Val met 770 775 780 Asp Giu Ile Ser Gly Asn Ser Pro Ala Gly Val Phe Asn Ser Asn Giu 785 790 795 800 Lys Trp Gin Lys Gin Leu Tyr Tyr Arg Val Thr Asp Gly Arg Thr Ser 805 810 815 Val Gin Leu Met Cys Leu Ser Cys Thr Ser.His Ser Pro Giu Pro Tyr 820 825 830 Cys Leu Plie Asp Thr Ser Leu Ile Ala Arg Giu Lys Asp Ile Ala Pro 835 840 845 Giu Leu Tyr Phe Thr Ser Asp Pro Gin Thr Ala Tyr Cys Thr Ile Thr 850 855 860 Leu Pro Ser Gly Val Val Pro Arg Phe Giu Trp Ser Leu Asn Asn Val 865 870 875 880 Ser Leu Pro Giu Tyr Leu Thr Ala Thr Thr Vai Val Ser His Thr Ala 885 890 895 Gly Gin Ser Thr Vai Trp Lys Ser Ser Ala Arg Ala Gly Giu Ala Trp 900 905 910 Ile Ser Gly Arg Gly Gly Asn Ile Tyr Giu Cys Thr Val Leu Ile Ser 915 920 925 Asp Gly Thr Arg Val Thr Thr Arg Lys Giu Arg Cys Leu Thr Asn Thr 930 935 940 Trp Ile Ala Val Giu Asn Gly Ala Ala Gin Ala Gin Leu Tyr Ser Leu 945 950 955 960 Phe Ser Gly Leu Val. Ser Gly Leu Cys Gly Ser Ile Ser Ala Leu Tyr 965 970 975 Ala Thr Leu Trp, Thr Ala Ile Tyr Phe 980 985 INFORMATION FOR SEQ ID NO:7: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 362 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Met Ala Ser Leu Leu Gly Thr Leu Ala Leu Leu Ala Ala Thr Leu Ala 1 5 1.0 WO 96/29396 PCTfUS96/03916 Pro Phe Gly Ala Met Gly Ile Val Ile Thr Gly Asn His Val Ser Ala 25 *Arg Ile Asp Asp Asp His Ilie Val Ile Val Ala Pro Arg Pro Giu Ala 40 Thr Ile Gin Leu Gin Leu Phe Phe Met Pro Gly Gin Arg Pro His Lys 55 Pro Tyr Ser Gly Thr Val Arg Val Ala Phe Arg Ser Asp Ile Thr Asn 70 75 Gin Cys Tyr Gin Giu Leu Ser Giu Giu Arg Phe Giu Asn Cys Thr His 90 Arg Ser Ser Ser Val Phe Val Giy Cys Lys Val Thr Giu Tyr Thr Phe 100 105 110 Ser Ala Ser Asn Arg Leu Thr Gly Pro Pro His Pro Phe Lys Leu Thr 115 120 125 Ile Arg Asn Pro Arg Pro Asn Asp Ser Giy Met Phe Tyr Vai Ile Vai 130 135 140 Arg Leu Asp Asp Thr Lys Giu Pro Ile Asp Vai Phe Ala Ile Gin Leu 145 iSO 155 160 Ser Val Tyr Gin Phe Ala Asn Thr Ala Ala Thr Arg Giy Leu Tyr Ser 165 170 175 Lys Ala Ser Cys Arg Thr Phe Gly Leu Pro Thr Val Gin Leu Glu Ala 180 185 190 Tyr Leu Arg Thr Giu Giu Ser Trp Arg Asn Trp Gin Ala Tyr Val Ala 195 200 205 Thr Giu Ala Thr Thr Thr Ser Ala Giu Ala Thr Thr Pro Thr Pro Val 210 215 220 Thr Ala Thr Ser Ala Ser Giu Leu Giu Ala Giu His Phe Thr Phe Pro 225 230 235 240 Trp Leu Giu Asn Giy Val Asp His Tyr Glu Pro Thr Pro Ala Asn Giu 245 250 255 Asn Ser Asn Val Thr Val Arg Leu Gly Thr Met Ser Pro Thr Leu Ile 260 265 270 Gly Val Thr Val Ala Ala Val Val Ser Ala Thr Ilie Gly Leu Val Ile 275 280 285 Val Ile Ser Ile Val Thr Arg Asn Met Cys Thr Pro His Arg Lys Leu 290 295 300 Asp Thr Val Ser Gin Asp Asp Giu Giu Arg Ser Gin Thr Arg Arg Giu 305 310 315 320 Ser Arg Lys Phe Giy Pro Met Val Ala Cys Glu Ilie Asn Lys Gly Ala 325 330 335 Asp Gin Asp Ser Giu Leu Val Giu Leu Val Ala Ilie Val Asn Pro Ser 340 345 350 Ala Leu Ser Ser Pro Asp Ser Ile Lys Met 355 360 WO 96/29396 PCTIUS96/03916 -112- INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 499 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Asn Met Leu Val Ile Val Leu Ala Ser Cys Leu Ala Arg Phe Gly Thr Ser Lys Leu Ser Asp Gin 145 Ser Thr Pro Leu Ser 225 Phe Ala Leu Ala Glu Lys Ser Asp Lys Asn Pro 130 Pro His Ile Phe Arg 210 Trp Glu Thr Gly Thr Asp Val Pro Cys Arg Ile 115 Val Asp Val Lys His 195 Val Tyr Pro Asn Ala Arg Asp Leu Asn Pro Gly 100 Thr Thr Leu Asp Pro 180 Glu Phe Gly Cys Gin 260 Ala His Pro Arg Tyr Pro Glu Asn Arg Ala Glu 165 Ser Leu Pro Phe Val 245 Arg Ser Val Arg Asn Cvs 70 Ala Ser Ile Ser Met 150 Lys Glu Asn Pro Asp 230 Tyr Cys Leu Leu Asn Ala 55 Phe Gly Phe Met Asp 135 Glu Ala Ala Asp Thr 215 Val His Leu Let Phe Val 40 Cys His Pro Val Trp 120 Glu Thr Pro His Asn 200 Glu Lys Pro lie a Asp 280 Leu 25 Pro Lys Asp Leu Val 105 Thr Gly Thr Ala Ala 185 Ser His Glu Thr Gly 265 Cys Glu Glu Met Leu Ser 90 Leu Glu Ile Ser Pro 170 His Pro Val Glu Asp 250 Ser Ser Gly Gly Lys Ile 75 Ala Gly Tyr Tyr Tyr 155 His Val Thr Lys Cys 235 Gly Val Arg Thr Thr Ala Tyr Asn Ser Gly Phe 140 Asn Glu Glu Tyr Phe 220 Glu Lys Leu Asp Gin Val Ala Asp Leu Gly Gly 125 Arg Val Val Leu Val 205 Asn Glu Cys Met Thr 285 Ala lie Asp Gly Val Leu 110 Leu Arg Ser Glu Gin 190 Thr Val Val Gin Ala 270 Let Leu Val Lys Val Gly Ile His Leu Ile Val Ile 175 Met Pro Thr Lys Phe 255 Glu Glu Thr Leu Trp Cys Lys Leu Asn Phe Ser Leu 160 Asp Leu Val Tyr Leu 240 Pro Phe Asp 275 Cys His Glu Asn Arg Val Pro Asn Leu Arg Phe Asp Ser Arg Leu Ser 290 295 300 WO 96/29396 PTU9131 PCT[US96/03916 -11- Glu Ser Arg Ala Gly Leu Val Ile Ser Pro 305 Val Thr Met Pro Val 385 Lys Leu Asp Gly Ala 465 Ser Asp Leu Ile Ile Val 325 Val Leu Gly Lys 340 Pro Ser Lys Val 355 Met Asp Giu Thr 370 Ala Ala Thr Cys Lys Cys Pro Ala 405 Tyr Ala Pro Leu 420 Asp Giu Giu Val 435 Tyr Cys Lys Gin 450 Val Val Glu Gly Met Ala Asp Arg 485 Ser Tyr Vai Arg Pro Giy Val 390 His Pro Ile Glu Giu 470 Ser Asp Asn Ser Met Asn 360 Asn Tyr 375 Ile Leu Gin Met Lys Asn Tyr Asp 440 Phe Arg 455 Arg Aia Giy Pro 345 Lys Lys Thr Giy Giu 425 Giu Giu Leu Asp 330 Arg Val Met Cys Thr 410 Phe Vai Asp Asn Lys 490 Leu 315 Ile Val Val Tyr Aia 395 Phe Giu Tyr Val Phe 475 Ile Leu Val Ile Phe 380 Leu Ser Ala Glu Asn 460 Lys Al a Gly Vai Gly 365 Val Leu Lys Gly Pro 445 Thr Ser Ile Trp Giu 350 Ser Vai Val Thr Gly 430 Leu Phe Ala Pro Ser 335 Thr Pro Al a Gly Giu 415 Leu Phe Phe Ile Lys 320 Tyr His Giy Giy Lys 400 Pro Thr Arg Giy Al a 480 Ile Leu Ala Asn Ser Gly Arg Arg Asn Met 495 INFORMATION FOR SEQ ID NO:9: Wi SEQUENCE CHARACTERISTICS: LENGTH: 260 amino acids TYPE: amino acid TOPOLOSY; Linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ.ID NO:9: Met Pro Phe Lys Thr Arg Gly Ala Glu Asp Ala Ala Ala Gly Lys Asn 1 5 10 Arg Phe Lys Lys Ser Arg Asn Arg Giu Ile Leu Pro Thr Arg Leu Arg 25 Gly Thr Gly Lys Lys Thr Ala Gly Leu Ser Asn Tyr Thr Gin Pro Ile 40 Pro Trp Asn Pro Lys Phe Cys Ser Ala Arg Gly Giu Ser Asp Asn His 55 Ala Cys Lys Asp Thr Phe Tyr Arg Arg Thr Cys Cys Ala Ser Arg Ser 70 75 WO 96/29396 PCT/US96/03916 -114- Thr Val Ser Ser Gin Pro Asp Ser Pro His Thr Pro Met Pro Thr Glu Tyr Xaa Gin Asp 145 Leu Ser Thr Glu Asn 225 Ala Lys Gly Glu Ala 130 Glu Glu Lys Asn Ala 210 Glu Ala Gin Arg Gly 115 Ala Val Thr Asn Glu 195 Pro Leu Ala Gly Val Pro 100 Ala His Pro Ala Ser Ser Arg Ala 165 Lys Leu 180 His Val Ser Leu Ala Arg Ala Gly 245 Arg 260 90 Ser Gin Arg Pro 150 Glu Pro Gly Gly Met 230 Ala Lys Pro Leu 120 Thr Tyr 135 Thr Pro Leu Pro Asn Gin Gly Glu 200 Ile Leu 215 Arg Arg Arg 105 Val Ser Pro Gly Gin 185 Arg Ser Ala Lys Ser Ser Gly Ala 170 Ser Cys Arg Cys Lys Cys Ala Val 155 Thr Arg Pro Val Leu 235 Leu Lys Gin 140 Asp Thr Leu Ser Gly 220 Pro Ser Leu 125 Arg Ala Glu Lys Glu 205 Ala Ser 110 Pro Tyr Val Gin Pro 190 Gly Ala Ser Asp Thr Ala Thr 175 Lys Thr Ile Asp Ser Val Asp 160 Glu Pro Val Ala Ser 240 Leu Ala Ala Ile Val Ala Trp Ala Ala Ala Arg Ala Leu Gin INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1305 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1305 (xi) SEQUENCE DESCRIPTION: SEQ ID ATG CAC CGT CCT CAT CTC AGA CGG CAC TCG CGT TAC TAC GCG AAA GGA Met His Arg Pro His Leu Arg Arg His Ser Arg Tyr Tyr Ala Lys Gly 1 5 10 GAG GTG CTT AAC AAA CAC ATG GAT TGC GGT GGA AAA CGG TGC TGC TCA Glu Val Leu Asn Lys His Met Asp Cys Gly Gly Lys Arg Cys Cys Ser 25 WO 96/29396 PCTfUS96/03916 -115- GGC GCA GCT GTA TTC ACT CTT TTC TGG ACT TGT GTC AGG ATT ATG CGG 144 Gly Ala Ala Val Phe Thr Leu Phe Trp Thr Cys Val Arg Ile Met Arg 40 GAG CAT ATC TGC TTT GTA CGC AAC GCT ATG GAC CGC CAT TTA TTT TTG 192 Giu His Ile Cys Phe Val Arg Asn Ala Met Asp Arg His Leu Phe Leu 55 AGG AAT GCT TTT TGG ACT ATC GTA CTG CTT TCT TCC TTC GCT AGC CAG 240 Arg Asn Ala Phe Ti-p Thr Ile Val Leu Leu Ser Ser Phe Ala Ser Gin 70 75 s0 AGC ACC GCC GCC GTC ACG TAC GAC TAC ATT TTA GGC CGT CGC GCG CTC 288 Ser Thr Ala Ala Val Thr Tyr Asp Tyr Ile Leu Gly Arg Arg Ala Leu 90 GAC GCG CTA ACC ATA CCG GCG GTT GGC CCG TAT AAC AGA TAC CTC ACT 336 Asp Ala Leu Thr Ile Pro Ala Vai Gly Pro Tyr Asn Arg Tyr Leu Thr 100 105 110 AGG GTA TCA AGA GGC TGC GAC GTT GTC GAG CTC AAC CC!G ATT TCT AAC 384 Arg Val Ser Arg Gly Cys Asp Val Val Giu Leu Asn Pro Ile Ser Asn 115 120 125 GTG GAC GAC ATG ATA TCG GCG GCC AAA GAA AAA GAG AAG GGG GGC CCT 432 Val Asp Asp Met Ile Ser Ala Ala Lys Giu Lys Glu Lys Gly Gly Pro 130 135 140 TTC GAG GCC TCC GTC GTC TGG TTC TAC GTG ATT AAG GGC GAC GAC GGC 480 Phe Giu Ala Ser Val Val Ti-p Phe Tyr Val Ile Lys Gly Asp Asp Gly 145 150 155 160 GAG GAC AAG TAC TGT CCA ATC TAT AGA AAA GAG TAC AGG GAA TGT GGC 528 Giu Asp Lys Tyr Cys Pro Ile Tyr Arg Lys Giu Tyr Arg Giu Cys Gly 165 170 175 GAC GTA CAA CTG CTA TCT GAA TGC GCC GTT CAA TCT GCA CAG ATG TGG 576 Asp Val Gin Leu Leu Ser Giu Cys Ala Val Gin Ser Ala Gin Met Ti-p 180 185 190 GCA GTG GAC TAT Cr7T CCT AGC ACC CTT GTA TCG CGA A.AT GGC GCG GGA 624 Ala Val Asp Tyr Val Pro Ser Thr Leu Val Ser Arg Asn Gly Ala Gly 195 200 205 CTG ACT ATA TTC TCC CCC ACT GCT GCG CTC TCT GCC CAA TAC TTG CTG 672 Leu Thr Ile Phe Ser Pro Thr Ala Ala Leu Ser Gly Gin Tyr Leu Leu 210 215 220 ACC CTG AAA ATC GGG AGA TTT GCG CAA ACA GCT CTC GTA ACT CTA GAA 720 Thr Leu Lys Ile Gly Arg Phe Ala Gin Thr Ala Leu Val Thr Leu Giu 225 230 235 240 GTT AAC GAT CGC TGT TTA AAG ATC GGG TCG CAG CTT AAC TTT TTA CCG 768 Val Asn Asp Arg Cys Leu Lys Ile Gly Ser Gin Leu Asn Phe Leu Pro 245 250 255 TCG AAA TGC TGG ACA ACA GAA CAG TAT CAG ACT GGA TTT CAA GGC GAA 816 Ser Lys Cys Ti-p Thr Thr Giu Gin Tyr Gin Thr Gly Phe Gin Gly Giu 260 265 270 CAC CTT TAT CCG ATC GCA GAC ACC AAT ACA CGA CAC GCG GAC GAC GTA 864 His Leu Tyr Pro Ile Aia Asp Thr Asn Thr Arg His Ala Asp Asp Val 275 280 285 TAT CGG GGA TAC GAA GAT ATT CTG CAG CGC TGG AAT A.AT TTG CTG AGO 912 Tyr Arg Gly Tyr Giu Asp Ile Leu Gin Arg Trp Asn Asn Leu Leu Arg 290 295 300 WO 96/29396 WO 9629396PCTIUS96/03916

AAA

Lys 305

GAA

Glu

GAA

Glu

CAG

Gin

GAA

Giu

TAT

Tyr 385

AGT

Ser

GCG

AAG

Lys

ATT

Ile

AGC

Ser

GCA

Ala

GTC

Val 370

TAC

Tyr

TCT

Ser

GTC

AAT

Asn

CCC

Pro

AGC

Ser

GAG

Giu 355 ccc Pro

AAT

Asn

AAT

Asn

GCG

CCT

Pro

GCT

Ala

GAA

Glu 340

GCT

Ala

GAG

Giu

GAC

Asp

GCC

Ala

CTC

AGC

Ser

GTA

Val 325

AAG

Lys

TCT

Ser

GAC

Asp

ATG

Met

GTC

Val 405

GTG

Val -116- GCG CCA GAC CCT CGT CCA GAT AGC GTC CCG CAA Ala Pro Asp Pro Arg Pro Asp Ser Val Pro Gin 310 315 320 ACC AAG AAA GCG GAA GGG CGC ACC CCG GAC GCA Thr Lys Lys Ala Giu Gly Arg Thr Pro Asp Ala 330 335 AAG GCC CCT CCA GAA GAC TCG GAG GAC GAC ATG Lys Ala Pro Pro Glu Asp Ser Giu Asp Asp Met 345 350 GGA GAA AAT CCT GCC GCC CTC CCC GAA GAC GAC Gly Glu Asn Pro Ala Ala Leu Pro) Glu Asp Asp 360 365 ACC GAG CAC GAT GAT CCA AAC TCG GAT CCT GAC Thr Glu His Asp Asp Pro Asn Ser Asp Pro Asp 375 380 CCC GCC GTG ATC CCG GTG GAG GAG ACT ACT AAA Pro Ala Val Ile Pro Val Giu Giu Thr Thr Lys 390 395 400 TCC ATG CCC ATA TTC GCG GC TTC OTA GCC TGC Ser Met Pro Ile Phe Ala Ala Phe Val Ala Cys 410 415 GGG CTA CTG GTT TGG AGC ATC GTA AAA TGC GCG Gly Leu Leu Val Trp Ser Ile Val Lys Cys Ala 425 430 960 1008 1056 1104 1152 1200 1248 1296 1305 Ala Val Ala Leu 420 CGTA AGC TAA Arg Ser 435 INFORMATION FOR SEQ ID NO:il: SEQUENCE CHARACTERISTICS: LENGTH: 434 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Met His Arg Pro His Leu Arg Arg His Ser Arg Tyr 1 5 10 Glu Val Leu Asn Lys His Met Asp Cys Gly Gly Lys 25 Gly Ala Ala Val Phe Thr Leu Phe Trp Thr Cys Val 40 Glu His Ile Cys Phe Val Arg Asn Ala Met Asp Arg 55 Arg Asn Ala Phe Trp Thr Ile Val Leu Leu Ser Ser 70 75 Ser Thr Ala Ala Val Thr Tyr Asp Tyr Ile Leu Gly 90 Tyr Arg Arg His Phe Arg Ala Cys Ile Leu Ala Arg Lys Cys Met Phe Ser Ala Gly Ser Arg Leu Gin Leu WO 96/29396 PCTIUS96/03916 -117- Asp Arg Val Phe 145 Glu Asp Ala Leu Thr 225 Val Ser His Tyr Lys 305 Glu Glu Gin Glu Tyr 385 Ser Ala Val Asp 130 Glu Asp Val Val Thr 210 Leu Asn Lys Leu Arg 290 Lys Ilie Ser Ala Val 370 Tyr Ser Leu Thr 100 Ser Arg 115 Asp Met Ala Ser Lys Tyr Gin Leu 180 Asp Tyr 195 Ile Phe Lys Ile Asp Arg Cys Trp 260 Tyr Pro 275 Gly Tyr Asn Pro Pro Ala Ser Glu 340 Glu Ala 355 Pro Glu Asn Asp Asn Ala Ile Pro Ala Val Gly Ile Val Cys 165 Leu Val Ser Gly Cys 245 Thr Ile Glu Ser Val 325 Lys Ser Asp Met Val 405 Cys Ser Val 150 Pro Ser Pro Pro Arg 230 Leu Thr Ala Asp Ala 310 Thr Lys Gly Thr Pro 390 Ser Asp Ala 135 Trp Ile Glu Ser Thr 215 Phe Lys Glu Asp Ile 295 Pro Lys Ala Glu Glu 375 Ala Met Val 120 Ala Phe Tyr Cys Thr 200 Ala Ala Ile Gin Thr 280 Leu Asp Lys Pro Asn 360 His Val Pro Gly 105 Val Lys Tyr Arg Ala 185 Leu Ala Gin Gly Tyr 265 Asn Gin Pro Ala Pro 345 Pro Asp Ile Ile Pro Tyr Asn Arg Tyr Leu Thr 110 Glu Glu Val Lys 170 Val Val Leu Thr Ser 250 Gin Thr Arg Arg Glu 330 Glu Ala Asp Pro Phe 410 Leu Lys Ile 155 Glu Gin Ser Ser Ala 235 Gin Thr Arg Trp Pro 315 Gly Asp Ala Pro Val 395 Ala Asn Glu 140 Lys Tyr Ser Arg Gly 220 Leu Leu Gly His Asn 300 Asp Arg Ser Leu Asn 380 Glu Ala Pro 125 Lys Gly Arg Ala Asn 205 Gln Val Asn Phe Ala 285 Asn Ser Thr Glu Pro 365 Ser Glu Phe Ile Gly Asp Glu Gin 190 Gly Tyr Thr Phe Gin 270 Asp Leu Val Pro Asp 350 Glu Asp Thr Val Ser Asn Gly Pro Asp Gly 160 Cys Gly 175 Met Trp Ala Gly Leu Leu Leu Glu 240 Leu Pro 255 Gly Glu Asp Val Leu Arg Pro Gin 320 Asp Ala 335 Asp Met Asp Asp Pro Asp Thr Lys 400 Ala Cys 415 Ala Val Ala Leu Val Gly Leu Leu Val Trp Ser Ile Val Lys Cys Ala 430 Arg Ser 425 WO 96/29396 WO 9629396PCTIUS96/03916 -118- INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 690 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .689 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ATG GCG CCT GTA AAA GTG ACT ATA GTT TCT GCG GTC GAT TCG CAC TAC Met 1 Ala Pro Val Lys Val Thr Ile Val Ser Ala Val Asp Ser His Tyr AAA CTA CCT Lys Leu Pro

AAT

Asn TCT AGA TTT GAG Ser Arg Phe Glu CTC TCG GAT TCT GGA TGG AAA GAA Leu Ser Asp 5cr Gly Trp Lys Glu 25 TTG GTT CAC GCA GTG AAA ACT ATG Leu Val His Ala Val Lys Thr Met 40 GCG AGT TAC GAT CGT Ala Ser Tyr Asp Arg TCT CTG TAC GAA GTT Ser Leu Tyr Glu Val CCG AGT ACA Pro Ser Thr TCC GGG GAG Ser Gly Glu TTA TCG Leu Ser GTA ATC GTG CGC Val Ile Val Arg CCG GCA Pro Ala 55

CTG

Leu TTT TCC CTT CCC Phe Ser Leu Pro AGG ATG TGC Arg Met Cys 70 AGA CCC GTG ATT CGG TTC GGT GAG Arg Pro Val Ile Arg Phe Gly Glu 75 GGG GGC GAC CCG CCT GGA GTA AGT CCC GAG Gly Gly Asp Pro Pro Gly Val Ser Pro Glu TGG AGC GGC TTG GAC GCA Trp Ser Gly Leu Asp Ala 000 TTT TAC Gly Phe Tyr TTG TGG GTG Leu Trp Val 115 TTG TCA TCT Leu Ser Ser GGC GCG TAT GCC GCA AAA GAG TI'C CAT Gly Ala Tyr Ala Ala Lys Glu Phe His 105 110 CTG GOT ACC OCT Leu Oly Thr Ala GAC ATA Asp Ile 120 TGC ATG GCA GCT TTA AAC CTC Cys Met Ala Ala Leu Asn Leu 125 240 288 336 384 432 480 528 CCT GCG Pro Ala 130 CCA AAA ACT Pro Lys Thr TTC CTA ATT ACC GAA ACC GGA GGT AAA AAT TTT Phe Leu Ile Thr Glu Thr Gly Oly Lys Asn Phe 135 140 ATA TTT TTG GTA AAC OGA GAC AAG ACA ACG CTG Ile Phe Leu Val Asn Gly Asp Lys Thr Thr Leu 150 155 160

GAG

G1u 145 AGA GGA GTG GAA Arg Gly Val Glu TCT CTG AGT CAC Ser Leu Ser His CCA TCA GTC TGG ACA ACT CTT GCC CCT TCG AGC CTO Pro Ser Val Trp Thr Thr Leu Ala Pro Ser Ser Leu 165 170 175 WO 96/29396 PCTIUS96/03916 -119- AGA ACG CCC TGG CCG TAC AGC ACG GTA AAG TTT TTA AAA GTA AAA CCT 576 Arg Thr Pro Trp Pro Tyr Ser Thr Val Lys Phe Leu Lys Val Lys Pro 180 185 190 AAC TCG GCC GCA TAC TGT GTT TCC GAC TCG GAT GAT GGC GAA CGG CAG 624 Asn Ser Ala Ala Tyr Cys Val Ser Asp Ser Asp Asp Gly Glu Arg Gin 195 200 205 CCA AAA TTT TTT CTC GGG AGT CTA TTT AAG TCG AAG AAA CCC CGC TCC 672 Pro Lys Phe Phe Leu Gly Ser Leu Phe Lys Ser Lys Lys Pro Arg Ser 210 215 220 CCG CGG CGC CGA CGT TA G 690 Pro Arg Arg Arg Arg 225 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 229 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Ala Pro Val Lys Val Thr Ile Val Ser Ala Val Asp Ser His Tyr 1 5 10 Lys Leu Pro Asn Ser Arg Phe Glu Leu 5cr Asp Ser Gly Trp Lys Glu 25 Leu Val His Ala Val Lys Thr Met Ala 5cr Tyr Asp Arg Pro Ser Thr 40 Leu Ser Val Ile Val Arg Pro Ala Ser Leu Tyr Glu Val 5cr Gly Glu s0 55 Leu Phe Ser Leu Pro Arg Met Cys Arg Pro Val Ile Axg Phe Gly Glu 70 75 s0 Gly Gly Asp Pro Pro Gly Val Ser Pro Glu Trp Ser Gly Leu Asp Ala 90 Gly Phe Tyr His Leu 5cr 5cr Gly Ala Tyr Ala Ala Lys Glu Phe His 100 105 110 Leu Trp Val Leu Gly Thr Ala Asp Ile Cys Met Ala Ala Leu Asn Leu 115 120 125 Pro Ala Pro Lys Thr Phe.Leu Ile Thr Glu Thr Gly Gly Lys Asn Phe 130 135 140 Glu Arg Gly Val Glu ile Phe Leu Val Asn Gly Asp Lys Thr Thr Leu 145 150 155 160 Ser Leu Ser His Pro Ser Val Trp Thr Thr Leu Ala Pro Ser Ser Leu 165 170 175 Arg Thr Pro Trp Pro Tyr Ser Thr Val Lys Phe Leu Lys Val Lys Pro 180 185 190 Asn 5cr Ala Ala Tyr Cys Val Ser Asp Ser Asp Asp Gly Giu Arg Gin 195 200 205 WO 96/29396 WO 9629396PCTIUS96/03916 -120- Pro Lys Phe Phe Leu Gly Ser Leu Phe Lys Ser Lys Lys Pro Arg Ser 210 215 220 Pro Arg Arg Arg Arg 225 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 381 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .380 (xi) SEQUENCE DESCRIPTION: SEQ 1D NO: 14: ATO COT AGC TCA OTT ACG TCA TTG TOG AGC CCT TCA GAT CAC GCC TCT Met Arg Ser Ser Val Thr Ser Leu 1

TCG

Ser

GCG

Al a

COT

Arg

GCC

Al a

ATA

Ile 000 Gly

CTC

Leu

CCC

Pro

GAA

Glu

CGC

Arg 50

CGA

Arg

ATT

Ile

TCC

Ser

TTG

Leu

GCA

Ala

ACG

Thr 35

OCT

Ala

ACC

Thr

TCC

Ser

CTG

Leu

AGC

Ser 115

AAT

Asn 20

GCG

Ala

TTC

Phe

GCC

Gly

CTT

Leu

TCG

Ser 100

TCA

Ser 5 0CC Ala

CCT

Pro

ACT

Thr

TCO

Ser G CA Al a

CTG

Leu

GAC

Asp

AAG

Lys

GCG

Al a

TTC

Phe

TCT

Ser 70

TOO

Trp

OAA

Glu

CCC

Pro

CAT

His

GGC

Gly

CCA

Pro 55

ATC

Ile

TCC

Ser

ATA

Ile

ATO

Met Trp Ser Pro 5cr Asp His Ala Ser 20 TAT CAT ATT TCC GAT TTC COG CGC Tyr His Ile Ser Asp Phe Arg Arg 25 ACG GGC GCG CGA ACT GAG OTT AAG Thr Oly Ala Arg Thr Olu Val Lys GCA OCO OTA CTC AGC GCA ACT GAA Ala Ala Val Leu Ser Ala Thr Glu GGC TTA AAC CGT ACT CCG TCT OCA Oly Leu Asn Arg Thr Pro Ser Ala 75 ATO AGA AAT CTT AAG GAC CCC CTC Met Arg Asn Leu Lys Asp Pro Leu 90 TTA ACG AAT GTC TCT AAC TTT TCC Leu Thr Asn Val Ser Asn Phe Ser 105 110 TTC GAA AAG TCT TCA TAT TO Phe Olu Lys Ser Ser Tyr 125 48 96 144 192 240 288 336 380 381 INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: LENGTH: 126 amino acids WO 96/29396 PCTIUS96/03916 -121- TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met 1 Ser Ala Arg Ala Ile Gly Leu (xi) SEQUENCE Arg Ser Ser Val 5 Pro Ala Asn Ala Glu Thr Ala Pro Arg Ala Phe Thr Arg Thr Gly Ser Ile Ser Leu Ala Ser Leu Ser Leu 100 Leu Ser Ser Asp 115 DESCRIPTION: SEQ ID Thr Ser Leu Trp Ser 10 Lys His Phe Tyr His 25 Ala Gly Gly Thr Gly 40 Phe Pro Ala Ala Ala 55 Ser Ile Thr Gly Leu 70 Trp Ser Glu Met Arg 90 Glu Ile Ala Leu Thr 105 Pro Met Ala Phe Glu 120 Pro Ser Asp His Ile Ser Asp Phe Ala Arg Thr Glu Val Leu Ser Ala Asn Arg Thr Pro 75 Asn Leu Lys Asp Asn Val Ser Asn 110 Lys Ser Ser Tyr 125 Ala Arg Val Thr Ser Pro Phe Ser Arg Lys Glu Ala Leu Ser INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 879 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..878 (xi) SEQUENCE .DESCRIPTION: SEQ ID NO:16: ATG TGG TGT CGT TTG CAC TGG ATA ACT CCT CGG TTC AGT ATT ATG CGT Met Trp Cys Arg Leu His Trp Ile Ser Pro Arg Phe Ser Ile Met Arg 1 5 10 CCC GGT TCC CGA ACT GGT AGG GTT TTG CGA GGC CAG GGG TGT GCT CTG Pro Gly Ser Arg Thr Gly Arg Val Leu Arg Gly Gln Gly Cys Ala Leu 25 TGC AGT TTC TGG CAT CGT ACT CGA ACT CCG AGT ATA AAC CTC CGG TGC Cys Ser Phe Trp His Arg Thr Arg Thr Pro Ser Ile Asn Leu Arg Cys 40 WO 96/29396 PCTfUS96/03916 -122- CGC GCT CGG GGT CTG AGT AAT TTC CGG CTC TGC GCC CAG AGT CCG GGT Arg Ala Arg Gly Leu Ser Asn Phe Arg Leu Cys Ala Gin Ser Pro Gly 55 GAA AGG CAC AGG TTC GGT ACT CGG ACT CTG AGT CAA CAC CTC CGG CTC Glu Arg His Arg Phe Gly Thr Arg Thr Leu Ser Gin His Leu Arg Leu 70 75 TGT ACT CGG AGT CTG AGT AGC TTT CGG TAC CGT ACT CGG GGC CTG AGT Cys Thr Arg Ser Leu Ser Ser Phe Arg Tyr Arg Thr Arg Gly Leu Ser 90 GAA AAA GTG TGT TTC AGT ACT CTG AGT TCG CAT AGT GTC CGG CTC GGC- Giu Lys Val Cys Phe Ser Thr Leu Ser Ser His Ser Val Arg Leu Gly 100 105 110 ACT CGA AGT CTG AGT AAA GGC CTC AGT TCC CGC GCT CTG AGT CCG AGT Thr Arg Ser Leu Ser Lys Gly Leu Ser Ser Arg Ala Leu Ser Pro Ser 115 120 125 AAA AAT CGC CGG TTC AGT ACT CGA ACT CAG AGT AGT TTT CGG TAC CGT Lys Asn Arg Arg Phe Ser Thr Arg Thr Gin Ser Ser Phe Arg Tyr Arg 130 135 140 GCT CGG GGT CTG AGT AAA CAC CTC CGT TAC CGT ACT CGA ACT CTG TGT Ala Arg Gly Leu Ser Lys His Leu Arg Tyr Arg Thr Arg Thr Leu Cys 145 150 155 160 AAA AAC CTC CGG CGC CGC GCT CGG AGC GCG AGC GGT TTC GGG GGG CGT Lys Asn Leu Arg Arg Arg Ala Arg Ser Ala Ser Gly Phe Gly Gly Arg 165 170 175 GCT ACG AGA CTG AGT AAA TAT CTC GGG TAT CGT GCT CGG GGT CTG GGC Ala Thr Arg Leu Ser Lys Tyr Leu Gly Tyr Arg Ala Arg Gly Leu Gly 180 165 190 AGG TGC CTC GGT TTC TGC ACC CGG AGT CTG AGT AAA AGT CAT CTG TTC Arg Cys Leu Gly Phe Cys Thr Arg Ser Leu Ser Lys Ser His Leu Phe 195 .200 205 AGC ACT CGG AGT CTG AGT AAA CAA CGC CTC CGT TTC TGC GAT CTG CGT Ser Thr Arg Ser Leu Ser Lys Gin Arg Leu Arg Phe Cys Asp Leu Arg 210 215 220 CTG ACT AAG AGC CGC CTG TTC AGT ACT CGG AGT CTG AGT AAA ATA CCA Leu Ser Lys Ser Arg Leu Phe Ser Thr Arg Ser Leu Ser Lys Ile Pro 225 230 235 240 CGG TTC CTG ACT CTG GGA CCG CCC GGT TTC CGA CTC GCT ACT CGC ACT Arg Phe Leu Thr Leu Cly Pro Arg Gly Phe Arg Leu Gly Thr Arg Thr 245 250 255 CTG ACT AAA GAC CAC CCT TTC TGC ACT CTG GGT CTG TGT ACT TTC ATG Leu Ser Lys Asp His Arg Phe Cys Thr Leu Cly Leu Cys Ser Phe Met 260 265 270 TCC CGC GCT CGC GGT CTC GGT AGA AAT CCC CCC CGC GGT CGT AGG AAA Cys Arg Ala Arg Gly Leu Cly Arg Asn Pro Arg Arg Gly Arg Arg Lys 275 280 285 CAG TGT ATT TTC TG A Gin Cys Ile Phe 290 INFORMATION~ FOR SEQ ID NO:17: 192 240 288 336.

384 432 480 528 576 624 672 720 768 816 864 879 WO 96/29396 WO 9629396PCT[US96/03916 -123- SEQUENCE CHARACTERISTICS: LENGTH: 292 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 110:17: Met Pro Cys Arg Glu Cys Glu Thr Lys Al a 145 Lys Al a Arg Ser Leu 225 Arg Leu Trp Cys Arg Leu His Trp Ile Ser Pro Arg Phe Ser Ile Met Gly Ser Al a Arg Thr Lys Arg Asn 130 Arg Asn Thr Cys Thr 210 Ser Phe Ser Ser Phe Arg His Arg Val Ser 115 Arg Gly Leu Arg Leu 195 Arg Lys Leu Lys Arg Trp, Gly Arg Ser Cys 100 Leu Arg Leu Arg Leu 180 Gly Ser Ser Thr Asp rhr Hi.s Leu Phe Leu Phe Ser Phe Ser Arg 165 Ser Phe Leu Arg Leu 245 His Gly Arg Ser Gly 70 Ser Ser Lys Ser Lys 150 Arg Lys Cys Ser Leu 230 Gly Arg krg rhr n-sn 55 Thr Ser Thr Gly Thr 135 His Ala Tyr Thr Lys 215 Phe Pro Phe Val Arg 40 Phe Arg Phe Leu Leu 120 Arg Leu Arg Leu Arg 200 Gin Ser Arg Cys Leu 25 Thr Arg Thr Arg Ser 105 Ser Thr Arg Ser Gly 185 Ser Arg Thr Gly Thr Arg Gly Pro Ser Leu Cys Leu Ser 75 Tyr Arg 90 Ser His Ser Arg Gin Ser Tyr Arg 155 Ala Ser 170 Tyr Arg Leu Ser Leu Arg Arg Ser 235 Phe Arg 250 Leu Gly 1ln Ile A.la Gln rhr Ser Ala Ser 140 Thr Gly Al a Lys Phe 220 Leu Leu Leu Gly Asn Gin His Arg Val1 Leu 125 Phe Arg Phe Arg Ser 205 Cys Ser Gly Cys Cys Leu Ser Leu Gly Arg 110 Ser Arg Thr Gly Gly 190 His Asp Lys Thr Ser 270 Ala Arg Pro Arg Leu Leu Pro Tyr Leu Gly 175 Leu Leu Leu Ile Arg 255 Phe Arg Leu Cys Gly Leu Ser Gly Ser Arg Cys 160 Arg Gly Phe Arg Pro 240 Thr Met 260 Cys Arg Ala Arg 275 Gin Cys Ile Phe 290 265 Gly Leu Gly Arg 280 Asn Pro Arg Arg Gly Arg Arg Lys 285 INFORMATION FOR SEQ ID 110:18: WO 96/29396 PCT/US96/03916 -124- SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..533 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATG CTC CCA AGC CTA CTC AAC AGG GGC TCT CCC CGG CTG AAT TCT CCT 48 Met Leu Pro Ser Leu Leu Asn Arg Gly Ser Pro Arg Leu Asn Ser Pro 1 5 10 CCT AAG TGT TCA GAG GCC TCT GCT GTA CCA TAT AAC TAT CGT GTA GTA 96 Pro Lys Cys Ser Glu Ala Ser Ala Val Pro Tyr Asn Tyr Arg Val Val 25 CGC CCC TCC CAG TCC GTG TCC GAT ACT GCC CCT TTT GAG AGG ATT GGG 144 Arg Pro Ser Gln Ser Val Ser Asp Thr Ala Pro Phe Glu Arg Ile Gly 40 AGA TTA GAG AAT CGA AAT GAT TGG AGA GCC ACA TTC AGA CTT AAT CAC 192 Arg Leu Glu Asn Arg Asn Asp Trp Arg Ala Thr Phe Arg Leu Asn His 55 ATT TTT ATT GAG TCG GGC GAG CTT AGC GCA GAC GGG TTA ACA ATC GCA 240 lie Phe Ile Glu Ser Gly Glu Leu Ser Ala Asp Gly Leu Thr Ile Ala 70 75 ACC AGT TCC ACA AGT TCA CTA TCC TGG TCA GCG CCC TTG TTT ATT TCG 288 Thr Ser Ser Thr Ser Ser Leu Ser Trp Ser Ala Pro Leu Phe Ile Ser 90 CAC GCA ACC ATG GGT CCA AAT TTT CGC GAT TCC CTT CTA GTT TGG GAA 336 His Ala Thr Met Gly Pro Asn Phe Arg Asp Ser Leu Leu Val Trp Glu 100 105 110 CGT TCT TCG TCG TCT TGC GAG ACC GTG TCT AAT TTT CGG TGC GGG GTG 384 Arg Ser Ser Ser Ser Cys Glu Thr Val Ser Asn Phe Arg Cys Gly Val 115 120 125 CAC ATG TTT CTG GTG ACG ATG GAA ATT ACA ATG ACG AGG CCG ATC GTT 432 His Met Phe Leu Val Thr Met Glu Ile Thr Met Thr Arg Pro Ile Val 130 135 140 GCG CTC ACG ACG GCA GCC ACG GTT ACC CCA ATT AGC GTA GGG CTC ATT 480 Ala Leu Thr Thr Ala Ala Thr Val Thr Pro Ile Ser Val Gly Leu Ile 145 150 155 160 GTC CCG AGA CGG ACA GTA ACG TTT GAA TTT TCG TTT GCG GGT GTC GGT 528 Val Pro Arg Arg Thr Val Thr Phe Glu Phe Ser Phe Ala Gly Val Gly 165 170 175 TCG TA A 534 Ser WO 96/29396 WO 9629396PCTIUS96/039 16 -125- INFORMATION FOR SEQ ID NO:19: Wi SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Met Leu Pro Ser Leu Leu Asn Arg Gly Ser Pro Arg Leu Asn Pro Arg Arg Ile Thr His Arg His Ala 145 Lys Pro Leu Phe Ser Ala Ser Met 130 Leu Cys Ser Glu Ile Ser Thr Ser 115 Phe Thr Ser Gin Asn Glu Thr Met 100 Ser Leu Tkxr Glu Ser Arg Ser Ser Gly Se r Val1 Al a Ala Val Asn Gly 70 5cr Pro Cys Thr Al a 150 5cr Ser Asp 55 Glu Leu Asn Glu Met 135 Thr Al a Asp 40 Trp Leu 5cr Phe Thr 120 Glu Val Val 25 Thr Arg 5cr Trp Arg 105 Val Ile Thr Pro Ala Ala Ala 5cr 90 Asp Ser Thr Pro Tyr Pro Thr Asp 75 Ala 5cr Asn Met Ile 155 Asn Phe Phe Gly Pro Leu Pkie Thr 140 Ser Tyr Arg Glu Arg Arg Leu Leu Thr Lcu Phe Leu Val .110 Arg Cys 125 Arg Pro Val Gly Ser Pro Val Val Ile Gly Asn His Ile Ala Ile Ser Trp Glu Gly Val Ile Val Leu Ile 160 Val Pro Arg Arg Val Thr Phe Glu Ser Phe Ala Gly Val Gly 175 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic aci.d STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID GAATTCGAGC TCGGTACCCG GATAATACGT ACATGTTAAC GCAGAGGT WO 96/29396 PCTIUS96/03916 -126- INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GCTGACCGCT AGTCGACCTG CAGTGAATAA TAAAAT 36 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TGTCCGTCGA GATCCTCTAG AGTCGACGAA AGGTCAGAGA CGATGCCC 48 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CGGATCAGAA ACTCTTTCGG TACCCGGGAT CCTCTAGA 38 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 96/29396 PCT/US96/03916 -127- TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GAATACAAGC TTAGATGCAT ATTTACTCGA GCC 33 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID GGTTTGGCGG AGCGGATATG ATCTCGACCT GCAGTGAATA ATAAAATGTG T 51 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: TGTCCGTCGA GATCCTCTAG AGTCGAGATC AGCAAAATGT TCACGGGG 48 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 96/29396 PCTIUS96/03916 -128- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: AAGCTTGGCG TAATCATG 18 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GGAATTCGAG CTCGGTACCT CGTGGCGAGC GCAGGCGGC 39 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GGCCGAGTTA GGTTTTACTT TTCTAGAGGA TCCCCTCGAC GTCTGGGGCG C 51 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID TTGCTGCGTT CCCGGGGATC CTCTAGAATT AGGTAGTTTG TAGTGCGA 48 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: WO 96/29396 PCT/US96/03916 -129- LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TCAAGATCCA GGAAATCCTT CGGTACCGAG CTCGAATTCG TA 42 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: GAATTCGAGC TCGGTACCGA AAGCTACTCA GAC 33 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: CGCAAACAGC TCTCGTAACT CTAGAAGTTA ACGATCGCTG TT 42 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 96/29396 PCT/US96/03916 -130- (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GAATAGCATA CCAATGCCTA TTCATTGGGA CTCGACTCTA GAGGATCCCC GGGAACG 57 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID TCGAGGGGAT CCTCTAGAGT CGAGGGACCC ATGGTTGCGT GC 42 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: TTTACTAAAG CGCGGCGAAA GCTTCGTCGT GCTGGGTTCT GG 42 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 96/29396 PCTIUS96/03916 -131- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: AAGCTTGGCG TAATCATGGT C 21 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: GGAATTCGAG CTCGGTACCC GGATAATACG TACATGTTAA CGCAGAGG 48 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: ATCTATTGGA GCGTTTAGCG CGCGTCGACG AAAGGTCAGA GACGA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID CTGCTTCATT TCTGATCCCC GGGAACG 27 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: WO 96/29396 PCT/US96/03916 -132- LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: ACCACCCCCG CGCCCCAGAC GTCGAGGGGA TCAATTATTG CGTATTGAAT A 51 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: ATCAGAAACT CTTTCGGTAC CGAGCTCGAA TTC 33 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: GAATTCGAGC TCGGTACCCG GATAATACGT ACATGTTAAC GCAGAGGT 48 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 96/29396 PCT/US96/03916 -133- (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: GCTGACCGCT AGTCGACTCT AGAGGATCCC CTC 33 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID CGTTCCCGGG GATCCTCTAG AGTCGACGGC AGAGTCGCAG AC 42 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: TGATCCAAAC TCGGATCCTC TAGAGTCGAC INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: WO 96/29396 PCT/US96/03916 -134- AAGCTTGGGC TGCAGGTCGA CTCTAGAGGA TCCCCTCGAC GTCTGGGG 48 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: CACACCTTTG CGCATCTCCA CAGCTCAACA ATGAATTCCA TGTTACGTCC TGTAGAAACC INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: CAGGGAGGCA AACAATGAAT CAACAACTCT CCCGGGAGAT GGGGGAGGCT AACTGAAACA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID TGCTGCGTTC CCGGGGATCC TCTAGAGTCG ACCTGCAGCC CAAGC INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: WO 96/29396 PCT/US96/03916 -135- LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: TCTAGAGTCG ACCTGCAGTG AATAATAAAA TGTGTGTTTG TCCGAAAT 48 INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: CTCCATAGAA GACACCGGGA CCATGGATCC CGTCGTTTTA CAACG INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 105 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: TCGGCGGAAA TCCAGCTGAG CGCCGGTCGC TACCATTACC AGTTGGTCTG GTGTCAAAAA GATCTAGAAT AAGCTAGAGG ATCGATCCCC TATGGCGATC ATCAG 105 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 96/29396 PCT/US96/03916 -136- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: CCGTCGAGAT CCTCTAGAGT CGACCTGCAG GTCGAC 36 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID CCTAGCACCC TTGTATCGCG INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: CGCCTCGAGT CCCAATGAAT AGGCATTGG 29 INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N WO 96/29396 PCTIUS96/03916 -137- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: CGCCTCGAGG ACCCATGGTT GCGTGCG INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: CTCGTCCGAA CGAGTTACAG INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 18912 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: N ANTI-SENSE: N (ix) FEATURE: NAME/KEY: .CDS LOCATION: 697..1533 OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: complement OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: complement OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: 3694..5124 OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: 5210..7081 OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: 7245..8123 OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: 8333..11290 OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS (1900..2784) (2916. .3605) WO 96/29396 PCTfUS96/03916 -138- LOCATION: 11098. .12402 OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: 12510. .13598 OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: 13792. .15291 OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: 15298. .16080 OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: 16129. .17013 OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: complement (17380. .18216) OTHER INFORMATION: (xi) SEQUENTCE DESCRIPTION: SEQ ID NO:59: GGATCCCGAA GAGCTCTCCC AGAAGTTTTT CTTTTCGGAC GTATCGGAGG ACGAAGAACC

GGCACGCGGG,

AGTGGACTTG

TGACTTAGAC

CCGTGCGTTA

GCGGGTAGGC

TCCGAGGAGA

GAGGCCTCGA

CCTACTCTGG

AGCCTTCACG

CCCCCTTCAC

AGGAGCTGGA

GGCGAGGAGG

TTTATTGACG

TTGCGGTGCG

GCACTCTTAA

GAAGTAAAAA

CGAGCAGATA

ACATTTATTG

CAGAAGCGGA

GCCCGGCGCA

GCGACCCGGA

ACACGGGACA

ACAGCTCTCC

CGGCACCCGC

AAGACGGAGG

AGAAGAAGCG

GAGGAGACGC

CCCGCTCGAT

CGCGCGCCCC

ATCCGTAGCC

GTCGGAGGAA

CAGCTCCACC

GGCGCCGCCG

AAAGACCCAC

TTGTCATTTT

GCCTCGACCC

GGGGCAGAAC

CCATTCTCAT

TTTGCGACCG

GTCCAACTCG

GAGC-AGCCTG

GAGTCAGAGC

CCATTTGCTA

GGAAGGCTTC

CTTCTTCCTC

CTGCACGGCG,

CTCCCCCTCC

CCAGAACTTC

CCGGACATCC

GGTGCCGGGG

CCACGCAATC

TCCCCCGCGT

GGCCGCCAGG

TGACGAGGAA

ACGCAAGGCC

CTCCCACCCC

TTTCCCGCTC

CGCCGCCCCC

120 180 240 300 360 420 480 540 600 660 714 762 810 858 906 GCCCAGCACA ACCGCAGTAG ACCGCCCGGA CCGCTCTCCT CTAGACACAT CCCTAA ATG GAA AAC ATG CTC GAC Met Giu Asn Met Leu Asp 1 GGG TGC TAC CCG CTG GCG CTG ATG GAC AGC GAT CAC ATT ACT GCG CAC Gly Cys Tyr Pro Leu Ala Leu Met Asp Ser Asp His Ile Thr Ala His i5 GCG GTA CCT CGT GGC GAG CGC AGGO CGG CAA GGT GCC GCT GTC GCC TCG Ala Val Pro Arg Gly Giu Arg Arg Arg Gin Gly Ala Ala Val Ala Ser 30 TCG GAG TCG GCC GAC TCG GTA GAC CCG TGC ATT CGG ATC GCC TCG CGG Ser Giu Ser Ala Asp Ser Vai Asp Pro Cys Ile Arg Ile Ala Ser Arg 45 CTC TGG CGC GAG TTA GTC GAG ATA TCG TCC GAA CTC AAG GAC GGT TAC Leu Trp Arg Giu Leu Val Giu Ile Ser Ser Glu Leu Lys Asp Gly Tyr 60 65S WO 96/29396 PCTIUS96/03916 -139- GGA GAG TTC ACG TCA GCG AGA GAC CGC CGC AAC GCG CTG ATT GCT GCC 954 Gly Giu Phe Thr Ser Ala Arg Asp Arg Arg Asn Ala Leu Ile Ala Ala 80 AAC GAA CGG CTA CGT TCG GCT TTT CTG GGG GCC AGC CGG GCG ACG CGC 1002 Asn Glu Arg Leu Arg Ser Ala Phe Leu Gly Ala Ser Axg Ala Thr Arg 95 100 GGC CTA GGT TTG AGG CCG CGG TGG GCG TCG ACG GAG AGC GTC GCC AAC 1050 Gly Leu Gly Leu Arg Pro Arg Trp Ala Ser Thr Olu Ser Val Ala Asfl 105 1.10 1.15 TCC CCC ACT GAC CCG MAT AAC GGC AAC GGG TTG, GGA GAA TTA GAG GAG 1098 Ser Pro Thr Asp Pro Asn Asn Gly Asn Oly Leu Gly Glu Leu Giu Glu 120 125 130 GCA ATG GAA GGG ATC GAG GGC GAT TTC TOG CTC GAC TCT CTG GAC GOT 1146 Ala Met Oiu Gly Ile Giu Gly Asp Phe Trp Leu Asp Ser Leu Asp Gly 135 140 145 150 GAC COC TTC GAG GAC GAG AGC CGT ACC ATG CAG AGC GAG AAT ATO COT 1194 Asp Arg Phe Glu Asp Giu Ser Arg Thr Met Gin Ser Giu Asn Met Arg 155 160 165 TTC OTO ATC GAG AAA GAA CTG TTA TCC TGG CTG TCC CGA CAC CTG CCG 1242 Phe Val Ile Giu Lys Glu Leu Leu Ser Trp, Leu Ser Arg His Leu Pro 170 175 180 GCC GAC CTC GCG TCC GCC GAG CGA GAG ACC TCC CGG TCT CTC CTG GCG 1290 Ala Asp Leu Ala Ser Ala Glu Arg Giu Thr Ser Arg Ser Leu Leu Ala 190 195 GCC GGG CAC TGG TGC TGC TTG TOG CAC CCT COG CCG TGC CGC GAA GCO 1338 Ala Gly His Trp Cys Cys Leu Trp His Pro Arg Pro Cys Arg Giu Ala 200 205 210 TOT TTO TAC GAC TCG ATT TAC GTG CAG AGT CTT TTC TOC GTC GOG ACG 1386 Cys Leu Tyr Asp Ser le Tyr Val Gin Ser Leu Phe Cys Val Oly Thr 215 220 225 230 GGG AGA GTC CCG CAA TCG GAG ATG CGC COT CGC GMA TAC CTO 0CC GCC 1434 Gly Arg Val Pro Gin Ser Glu Met Arg Arg Arg Giu Tyr Leu Ala Ala 235 240 245 TTG COC 0CC GGC GCG GCT GCC 0CC MAC TCT CCC GMA GTG AOC GCC TCG 1482 Leu Arg Ala Oly Ala Ala Ala Ala Asn 5cr Pro Oiu Val Scr Ala Ser 250 255 260 ATC TTT OCG AGO GAC GCT GGA ATC GCG CTO OCO CTG OCO CGO CGC COT 1530 Ile Phe Ala Arg Asp Ala Gly Ile Ala Leu Ala Leu Ala Arg Arg Arg 265 270 275 TGA CGGGAGAATG ACGCCCTCTA GCGGCTTCCT TACCTCCOCG TCCCTGACAA 1583 CCTCGCGGGT TTTTACACTG TCCTCCGTCC ACTCTCCCCC CTCACCCACT CCGCGGCAGC 1643 GAAACACAAC CCCCCCCCCC CCCCAGAAAC GAGCGACACG CGAGCGCTGC GAAATAAATA 1703 AAGTAATATT ATTGTGTGTT TTTCACOTTG TTGCAATCGA GAGGCCGTTT GTCTGTCTGT 1763 GTCTGTGCGO AGCTAGGCTT TCCCOGOCGG CCCCGTTCCA CCGTTCGGTT AGGCCOOTGG 1823 COACGOACA TAGAOAAAGA TAOAGCGCGC GCCCTGGCGG CGAGAGGGTG TTGCGOGOT 188B3 AAATGGGACC CTGAGCTCAC CATTTTGGCO GGGGATTOCA CGGGTAMCAA AAAGCTCTCT 1943 WO 96/29396 PCTfUS96/03916 -140- CGCACATAAT GATTTCCCTT AAACAGTGGC TGTAAAAGCT TTCTTCGACT GGGACGCGCA CGTCCGGAGA CATGATCTTA TCGGTAGCTA

GGATCGAGTT

GCGCTATCAC

CGGCCGGCGC

CTCTGATATA

CAATGACTGG

TGCCCCAGAC

TCCACAGTCC

GCGCCGTGAT

CGCGGGCGGC

GCGACGCCCG

CTAAGTCCAA

GCCGGCCCAC

TCCGCGTACG

GTTTCTGCAA

CGAGCGTCGC

CCCGAGAAAA

CGAGTTAGGT

CGAAGGGGCA

GTTTACCAAA

GAAAGTTTTT

CCACAAATGG

CAAGCCGCTC

GGGTCTGCAC

GCGCACGATT

AACCAATTCT

CGAATCGACC

TTGGGAACCT

CAGGAGGCTC

GCTGCTCTGG

GAGCACCGGC

AACTTTGGGC

CTTGCTTTCC

TACCGCCGCT

ATCTTCGGCG

CTGTACGATC

CCAGAATCTC

ATGTGACGTC

GCAACGCGGG

CGTTTATTAT

GGCCCTCGCC

CGCTAACGTC

AATTTTGGCT

TTTACTTA

AGAGTTGTCC

AATATTTCCA

GGCGCAGGGA

AACTCTrrTG

CACTCGGGAC

ATCCTGGGAA

ACCGATAATG

TTCCATCCAG

GCAGAAACTA

TCGGGGAGGT

CGCACCATCT

TTATTCCAGT

AGCTCGACGG

ACGTCTCTTA

AACTCCCCGT

GCGACGGCGC

ACCTGCAGAC

ACCGCGGCCA

CCTCCGGGCA

GGAGGTCTCG

CTTTTATAAA.

TGTCAATATT

GCGGCCCAGG

GGCGCCGCGG

GCCGTTCGCC

AAAACTTAC

AGACTGATGG

CTCCTCTCTC

GG=TAAAGC

CGGCATACGC

TTACTCCAGG

GGGAPJAACAG

TACTCGGACG

AATCCGAGAG

TAGTCAC=

CAGAA.ATAT;

CACAGTTCAT

CTCCGGGAG

CCATGCCTAT

GCGCGGACCG

CGGCGGAGAA

TTTCCCACGC

TCGGCCCCCC

GCTTAGCCGC

TGCCCAGCCT

GATCGGGCCA

GGTCCGTGCC

ACATGGTCGC

GATGGCGCGC

TGTGTGGTTA

CCACTATTCC

GGAGCGGGGT

ATCATCCGAG

CGTGCTGTAC

GTGACTCAGA

AAAJATTTTTA

TGCCATGCAT

GCCAGATGAC

CGGGTCGCCC

CTCCCCGGAA

ATCGTAACTC

;CTCAAATCTA

TACAGGCGCC

ACGCTCTTA7

GAGGTGGGCC

GGTGAAGTIT

CCTTATCGCC

CGAGTGCGCC

AAAAGAAAGA

GGCGGCCCGG

AACTAACTCC

GGGCGCTATT

TTCCTTCCCT

AA1AGAAAATA

CCTAAAGGCC

CAACCCTCCA

GAGACA76TAA

TTIATTACTGC

GGCAGCGGCC

TTCTTCGACT

TCGGAAACAC

GGCCAGGGCG

GACAGCGTTG

CCTCCGGTTT

ATGTCAGCGG

AAATGGTAAA

CCCTCACCGA

,ACTTCGTACA

GCCATAGTTT

GAXTTAGGTA

ACGAACGCGC

GACAGAGGCA

GCGAGTCCGG

CCTCCCCGGG

ATGTCCGGCC

GGAATCTGCT

GACAGCGCGG

CGCGGGTCGT

TCAAAATACG

TCGCAACTCT

GCCGAGAAAG

AATGCTACCC

CACTTACTCA

TACCGCCCTT

GCCGACGCGG

TAAATAGACT

AGTATGCGGC

TTCTCAGGCT

TCTTGTCTCC

CGGTAATTAG

TACCCAGCAC

ACCCTGCGTC

ACCGAP.TCAC

GAGATGCCGG

TCACTGCGTG

GTTTGTAGTG

2003 2063 2123 2183 2243 2303 2363 2423 2483 2543 2603 2663 2723 2783 2843 2903 2963 3023 3083 3143 3203 3263 3323 3383 3443 3503 3563 3615 3675 3726 3774

ATCGCCGCTCAG

TCTAGCAGAG TCAGGCCAAT ACTCCACCCC GCTATGATGT ATATACAGCT TAGAGAAG ATG CGG TTT CGG CGC ATC TGT TCA CGC TCT AGG Met Arg Phe Arg Arg Ile Cys Ser Arg Ser Arg 1 5 GCA GAA A.AA CGA AGA AGA ACA ACC GAG AAT CCG CTT ACC TCA AAA CGC Ala Glu Lys Arg Arg Arg Thr Thr Glu Asn Pro Leu Thr Ser.Lys Arg 20 WO 96/29396 WO 9629396PCTIUS96/03916 -14 1- GTT TGC GTA Val Cys Val TTG GAT AGT TTC Leu Asp Ser Phe TCA CGG ACA ATG TCA TTG CGC CCC TAT Ser Arg Thr Met Ser Leu Arg Pro Tyr 35 GCA GAA Ala Glu ATT TTG CCG ACC Ile Leu Pro Thr GCG GAA GGC GTC GAG CGC CTC GCC GAA CTT Ala Giu Gly Val Giu Arg Leu Ala Glu Leu 50

GTT

Val AGT GTG ACA Ser Val Thr ATG ACA Met Thr 65 GAA CGC GCG Giu Arg Ala GAA CCT GTG ACA GAG AAT ACA Giu Pro Val Thr Glu Asn Thr 70 GCT GTA AAC AGT ATC CCC CCG GCT Ala Val Asn Ser Ile Pro Pro Ala AAC GAG AAC Asn Giu Asn TAT GCA GGC Tyr Ala Gly ACA CAC TTC Thr Asp Phe 110

GAT

Asp GGG CCC TCG ACT ACT GAA AAA Gly Pro Ser Thr Thr Giu Lys 100 CCC CAG AAC TTC CCA Cly Gin Asn Phe Ala GTT GAC GGC TCG CAT Val Asp Cly Ser His 105 GGC CCT GTC CCG CTC Gly Pro Val Pro Leu 120 CTT CAC CAC TTC CGA Leu Gin His Phe Arg 135 GAT GAA GCA Asp Ciu Ala TCG AGC Ser Ser 115 GAC TAC CC Asp Tyr Ala GCG CAA Ala Gin 125 ACT AGA TTG AAG Thr Arg Leu Lys

CAT

His 130 TCG GAT GAA TTT Ser Asp Gbu Phe

GTT

Val 140 TTA GAC CAT TTG Leu Asp Asp Leu GTG GAG Val Ciu 145 GGG GCT TAC GGG TTT ATC TGC GAC GTC Cly Ala Tyr Gly Phe Ile Cys Asp Val ISO 155 CCT CGC TAC ACC Arg Arg Tyr Thr GAA GAG CAA CGT Giu Clu Gin Arg CGA AGA GCG GTT AAC ACT ACT Arg Arg Cly Val Asn Ser Thr 165 170 CTG ATA GCT AAA TAT GTG AAA Leu Ile Ala Lys Tyr Val Lys 185 3822 3870 3918 3966 4014 4062 4110 4158 4206 4254 4302 4350 4398 4446 4494 4542 4590 AAC CAG GGG Asn Gin Gly AAT GGA ACA Asn Gly Thr 190

AAA

Lys 175 TCA AAA TGT AAG Ser Lys Cys Lys AGG GCG GCC TCT Arg Ala Ala Ser

CAG

Gin 195 CTG GAA AAT Leu Ciu Asn CTC AAG ATC Leu Lys Ilie CCC CGC Cly Arg 205 CTA AAT CAC GAG Leu Asn His Glu AAT G=T Asn Val 210 GAA ATT TTC CTT CTC Glu Ile Leu Vai Leu 200 CAG GAA ATC CTT CCC Gin Giu Ile Leu Arg 215 TAT CAG TTC GAC TTG Tyr Gin Phe Asp Leu 235

TAC

Tyr 220 CCC CAT AAT ACC Pro Asp Asn Thr ATG TTA ACG CAC AGC Met Leu Thr Gin Arg .230 TAC ACC TAC ATG Tyr Ser Tyr Met GAT GAA CC Asp Giu Ala TTC GAC TGC AAA CAC ACT CCA ATC Phe Asp Trp Lys Asp Ser Pro Met 245 250 CTT AAA CAG Leu Lys Gin TAT ATC CAT Tyr Ile His .270 ACT AGA Thr Arg 255 CGC ATC ATG AAC CAC CTC ATG TCA CCG GTC TCG Arg Ile Met Gin Leu Met Ser Ala Val Ser 265 TCA AAC AAA Ser Lys Lys CTC ATT Leu Ile 275 CAC ACG GAC ATC AAA CTC GAA AAT His Arg Asp Ile Lys Leu Giu Asn 280 ATT TTC Ile Phe 285 TTA AAC TGC GAC Leu Asn Cys Asp CCC AAG ACA Cly Lys Thr 290 CTC CTG CCC GAC TTT CCA ACT Val Leu Gly Asp Phe Cly Thr 295 WO 96/29396 WO 9629396PCTIUS96/03916 142- GAG CCC GTC ACG Val. Thr CCT TTT GAA AAT GAG CGG Pro Phe Giu Asn Giu Arg Giu Pro TTC GAA TAT GGA TGG GTG Phe Giu Tyr Gly Trp, Vai 310 315 4638 300 305

CG

Gly

TOT

Cys

GTA

Val

CAG

Gin

TTC

Phe 380

ATC

Ile

CTT

Leu

TGG

Trp

TCG

Ser

CCC

ACC

Thr

GAA

Glu

AGC

Ser

CAA

Gin 365

CCA

Pro

GAT

Asp

CCG

Pro

COT

Arg

GCT

Ala 445

ATC

GTG

Val

ATT

Ile

CAT

His 350

TTG

Leu

GAC

Asp

CGC

Arg

GCG

Ala

TTG

Leu 430

GAA

Giu

CGA

GCT

Ala

ACA

Thr 335

GAA

Giu

CTG

Leu

CCC

Pro

GCC

Ala

GAT

Asp 415

AGA

Arg

GAG

Giu

CCC

ACT

Thr 320

GAC

Asp

TTT

Phe

AAA

Lys

CCG

Pro

GGA

Gly 400

GTG

Val1

CCC

Pro

GAA

Ciu

GAA

AAC

Asn

ATT

Ile

TGC

Cys

OTT

Val

TGT

Cys 385

CAT

His

GAA

Giu

AC

Ser

CG

Arg

ATC

Ile 465 TCT CCC Ser Pro TOG AGC Trp Ser CCG ATC Pro Ile 355 ATC GAC Ile Asp 370 AAT CTG Asn Leu ACO GTC Thr Val TAC CCT Tyr Pro GC GCC Ala Ala 435 ACC ATA Thr Ile 450 COT CCG Arg Ala GAG ATA CTC GCC AGO GAT TCG TAC Ciu Ile Leu Ala Arg Asp Ser Tyr 325 330 TGC GGA GTA GTA TTG CTG GAA ATG Cys Gly Val Val Leu Leu Giu Met 340 345 GGC GAT CCC COG GGA AAT CCC CAC Ciy Asp Giy Gly Gly Asn Pro His 360 TCT CTC TCA OTT TGT GAT GAA GAG Ser Leu Ser Vai Cys Asp Giu Ciu 375 TAC AAT TAT TTG CAT TAT GCC AGC Tyr Asn Tyr Leu His Tyr Ala Ser 390 395 CCG TCC CTC ATA CCC AAC CTC CAC Pro Ser Leu Ile Arg Asn Leu His 405 410 CTA GTT AAA ATG CTT ACT TTT GAC Leu Val Lys Met Leu Thr Phe Asp 420 425 CAA OTA TTG OCA ATG CCA CTG TTT Giu Vai Leu Ala Met Pro Leu Phe 440 ACA ATT ATT CAT GCA AAA CAT AAA Thr Ile Ilie His Oly Lys His Lys 455 COG OTO CCA CCC TCC ATG ACT GAA *Arg Val Pro Arg Ser Met Ser Ciu 470 475 4686 4734 4782 4830 4878 4926 4974 5022 5070 5118 5174 5227 5275 5323 5371 5419 Pro Ile Arg Pro Giu GOT TAA TAATAAAGGA CGGAGATAGA GAACTGAAGC GTCAGATTTT TTTAAAAAAA Gly.

TAAATGATCG AGAACTTATG ATTTGTCTTT CTTGA ATO ACC TTG CCC CAT CCA Met Thr Leu Pro His Arg 1 TTA ACC AAA AGA CCT TTC C COT CGA TTC TGC TCG CTC TTT GTC ATA Leu Thr Lys Arg Pro Phe Ala Arg Arg Phe Cys Ser Val Phe Vai Ile i5 CAT TAT ACT GAG ACT AAA CTC GAC CGA TAT AAC AAG ACA ATG TTA CT-- His Tyr Ser Ciu Thr Lys Leu Asp Arg Tyr Asn Lys Thr Met Leu Leu 30 TAT AGA CCC CAC TCA ACC ATO COG CAT ACC OGA CCC GAC OCA AAT CAC Tyr Arg Pro Asp Ser Thr Met Arg His Ser Cly Gly Asp Ala Asn His 45 AGA CCC ATA AGO CCC ACG CGC AAA TCT ATT OGA CC TTT AGC CC CC Arg Ciy Ile Arg Pro Arg Arg Lys Ser Ile Oly Ala Phe Ser Ala Arg 60 65 WO 96/29396 PCTIUS96/03916 -143- GAA AAG ACT GGA AAA CGA AAT GCG CTG ACG GAA AGC AGC TCC TCC TCC Glu Lys Thr Gly Lys Arg Asn Ala Leu Thr Glu Ser Ser Ser Ser Ser 80 5467

GAC

Asp

ACGC

Thr

TGG

Trp

GTG

Val- 135

CCG

Pro

GTT

Val

GCA

Ala

AAA

Lys

TGG

Trp 215

ACT

Thr

CCT

Pro

GTG

Val

GGC

Gly

TGG

Trp 295

AGA

kTG 4et

;TA

lai

;AC

ksp 1.20 kCT rhr

GAC

A.sp

TAT

Tyr

TTC

Phe

AGC

Ser 200

AGA

Arg

CGA

Arg

CTA

Leu

CCG

Pro

CCA

Pro 280

CC,'

Prc

GC;

CTA GAT C Leu Asp 1 GAC GGA C Asp Gly I 105 OTT CTCC Val Leu TAT GAG Tyr Glu GGG CCG Gly Pro CCC TCT Pro Ser 170 OTT AGC Val Ser 185 GTT ACG Val Thr ATA ATG Ile Met CTC AGG Leu A±-g TGG TAC Trp Tyr 250 TCG CTT Ser Leu 265 ATG GCC Met Ala GCC ATG Ala Met TGC CTG CO TTT ~ro Phe TCC ACG OAT AAG Ser Thr Asp Lys 95r GAA TTT GGC GOT AAG TGG Giu Phe Gly Gly Lys Trp 100

CT

?ro 1ln Ser kGT Scr 155 3.CG

:GA

Arg

GTG

Val

CAG

Gin

CAC

His 235

GCC

Al a

AAA

Lys

GCC

Ala

CGC

Ar

TC~

GCC GAC Ala Asp TTA GTG Leu Val 125 AAA CCG Lys Pro 140 TGG AAC Trp Asn GAG GTA Giu Val GTT TTA Val Leu CGG GAT Arg Asp 205 AAC GTT Asn Val 220 CCT TCC Pro Sei TAC GCG LTyr Al TTA GCC Leu Al~ GTT TTI Val Phi 28 GCA GC( ;Ala Al.

300 r TCA CTI r Ser Le 5 ATT ACT GCC GAG GTC C= TCT CAG GCA Ile Thr Ala Giu Val Leu Ser Gin Ala 110 115 AAO CAT GAA GAT OCG GAG GAG GAG AGA Lys His Oiu Asp Ala Giu Oiu Giu Arg 130 ACC CCO ATA CAG CCG TTC AAT 0CC TGG Thr Pro Ile Gin Pro Phe Asn Ala Trp 145 150 GCG CAG GAT TTT ACT CGA GCO CCA ATA Ala Gin Asp Phe Thr Arg Ala Pro Ile 160 165 TTO GAC OCA GAG GCG TTG, AAA GTA GGG Leu Asp Ala Giu Ala Leu Lys Val Gly 175 180 CAA TOT OTA CCG TTC ACO CGA TCA AAG Gin Cys Val Pro Phe Thr Arg Scr Lys 190 195 GCG CAG TCG TTT TTG 000 GAC TCG TTC Ala Gin Ser Phe Leu Gly Asp Ser Phe 210 *TAC ACG GTT GTC TTA CGA CAG CAC ATA *Tyr Thr Val Val Leu Arg Gin His Ile 225 230 AGC AAA AGC ATT GTT AAC TGC AAC GAC Ser Lys Ser Ile Val Asn Cys Asn Asp 240 245 AAT CAA TTT CAC TOG AGA OGA ATG CGC SAsn Gin Phe His Trp Arg Giy Met Arg 255 260 TCT CCC CCG GAG GAG AAT ATT CAA CAC Ser Pro Pro Giu Glu Asn Ile Gin His 270 275 AGA AAC GCG 000, OCT GGT CTG TTC CTG Arg Asn Ala Gly Ala Gly Leu Phe Leu 5 290 7TTT GAA GAG COC GAC AAG CGA CTG TTA a Phe Giu Glu Arg Asp Lys Arg Leu Leu 305 310 C OAT ATC ATG GAC OCA 0CC GTC CTC OCO u Asp Ile Met Asp Ala Ala Val Leu Ala 320 325 5515 5563 5611 5659 5707 5755 5803 5851 5899 5947 5995 6043 6091 6139 6187 Arg Ala Cys Leu Se: 31! TCG TTT CCA TTT TAC TOG CGC GGC GTC Ser Phe Pro Phe Tyr Trp Arg Gly Val 330 335 CAA GAC ACC TCG CGC TTC GAG Gin Asp Thr Ser Arg Phe Glu 340 6235 WO 96/29396 WO 9629396PCTIUS96/03916

CCT

Pro

GCC

GCG

Al a

GAG

CTG

Leu 345

ACG

TGT TTG Cys Leu TTA GCG

TCA

Ser

ACC

GAG TA Giu T 350 ATG TT -144- ~C TTT GCA CTA GTG GTG TTA CTG ~r Phe Ala Leu Val Val Leu Leu 355 C GAC CAC GCA CTG GTA TTC ATG aie Asp His Ala Leu Val. Phe Met Ala Glu Thr Val Leu Ala Thr Met P) 360 365 370

AGG

Arg 375

ATA

Ile

TTA

Leu

AAC

Asn

TGC

Cys

ATG

Met 455

GCG

Ala

CTG

Leu

AGC

Ser

CTC

Leu

CTC

Leu 535

AGG

Arg

GAA

Giu

GTT

Val

;CG

Ala

GAC

Asp

CGG

Ttrg

ACC

Thr

AAT

Asn 440

AGC

Ser

TGG

Trp

AAG

Lys

TCG

Ser

GTG

Val 520

AAG

Lys

GAC

Asp

AT'I

I le

CGC

ArS

CTG

Leu ccc Pro

GGC

Gly

TAT

Tyr 425

GTT

Val

GAC

Asp

CTC

Leu

AGA

Arg

GAG

Giu 505

CCG

Pro

AGG

*Arg

*CCG

Pro

ATT

Sle

GCT

Ala 585 3CA Ala

GTT

Val PkTA Ile 410

TCG

Ser

GCC

Ala

ATA

Ile

CAG

Gin

AGC

Ser 490

AAG

Lys

ATT

Ile

GAA

Giu

AGG

Arg

GCA

Ala 57c

TC;

Se~ GAC C Asp

AAAJ

Lys 3 95

GTG

Val.

ACG

Thr

TAC

Tyr

TAC

Tyr

AGG

Arg 475

GTG

Vai

CGG

Arg

CAA

Gin

AAG

Lys

GGG

Gly 555

GAC

Asp

SGTT

Val GC AAT 3iY Asn 380 kAC Asn 3CC A.TA2 Ile A.GG2 Arg CGG2 Arg 460

GTT

Val1

GAT

Asp

CTG

Leu

TAT

Tyr

TTA

Leu 540

TCC

Ser

GGA

Giy

'AG

.lu cc Ser kGA Arg cC Thr 445 k.TA Ile

GCA

Al a

PLCG

Thr

CAG

Gin

GAA

Giu 525

GAG

Giiu

TTA

Lei.

GTA

Val TTC GAT GAC TAT GAC GAA ACT AGA TAT Phe Asp Asp Tyr Asp Giu Thr Arg Tyr 385 390 TAC CTG AAC GGA GCC GAG GGT ACT CTG Tyr Leu Asn Gly Ala Giu Gly Thr Leu 400 405 AAC ACC GCT CTG GCG GTG GTT TGC GCA Asn Thr Ala Leu Ala Vai Vai Cys Ala 415 420 AAA CTIC CCG TCC GTG GCA ACT AGO GCG Lays Leu Pro Ser Vai Ala Thr Ser Ala 430 435 GAA ACG CTG AAA GCG AGG CGC CCT GGC Giu Thr Leu Lys Ala Arg Arg Pro Gly 450 TTA CAA AAA GAG TTT TTC TTT TAC ATT Leu Gin Lys Giu Phe Phe Phe Tyr Ile 465 470 ACA CAC GCA AAT TTC TGT TTA AAC ATT Thr His Ala Asn Phe Cys Leu Asn Ile 480 485 GGG GCC CCG CCA TT TTG TTC AGG GCC Gly Ala Pro Pro Phe Leu Phe Arg Ala 495 500 CAG TTA AAT AAA ATG CTC TGC CCC CTT Gin Leu Asn Lys Met Leu Cys Pro Leu 510 515 GAC TTT TCG AAG GCC ATG GGG TCT GAG Asp Phe Ser Lys Ala Met Gly Ser Glu 530 ACA TTC GTT AAA GCT ATT TCC AGC GAC Thr Phe Val Lys Aia Ile Ser Ser Asp 545 550 AGA TT CTC ATT TCG GAC CAT GCA AGG Arg Phe Leu Ile Ser Asp His Ala Arg 560 565 CGG TT AAG CCG GTG ATA GAC GAG CCG Arg Phe Lys Pro Vai Ile Asp Giu Pro 575 580 6283 6331 6379 6427 6475 6523 6571 6619 6667 6715 6763 6811 6859 6907 6955 7003 7051 GCG CTG ACT Ala Leu Ser 590 ACC GCT Thr Ala CCC GCT GGG AAA GTG AAA Ala Ala Giy Lys Val Lys 595 C CGA CGC TTA Ala Arg Arg Leu 600 ACC TCA GTT CGC GC CCC GTA CCG GCC GCA GGC GCC Thr Ser Val. Arg Ala Pro Val Pro Giy Ala Cly Ala 605 610 WO 96/29396 PCTfUS96/03916 -145- GTT TCC GCG CGC CGG AAA TCG GAA ATA TGA TAAAAATGCT TGGCATTTGC Val Ser Ala Arg Arg Lys Ser Glu Ile 615 620 GGGCGAAGAG GCGTGATCTG AAGGGCTCCA CAATGACGTA ACTGAGCTAC GCATCCCTAT AAAGTGTACC CGCTGACCGC TAGCCCATAC AGTGTTACAG GAGGGGAGAG AGACAACTTC AGCTCGAAGT CTGAAGAGAC ATC ATG AGC GGC TTC ACT AAC ATA GGA TCG Met Ser Gly Phe Ser Asn Ile Gly Ser 1

ATT

Ile

GG

Gly

GGC

Gly

GTC

Val.

GTG

Val

GTG

Val.

GCT

Al a

GGG

Gly

CGC

Arg

GTG

Val

TCA

Ser 170

CCA

Pro

GCC

Ala ;cc kla

GCG

PAA

Lys

CCG

Pro

AAA

Lys

GAT

Asp

CTA

Leu

ATA

Ile

TCC

Ser

CTG

Leu 155

GTG

Val

AAC

Asn

GAC

Asp

ACC

Thr

CCG

Pro

ATT

Ile

TGG

Trp

TGG

Trp

AGC

Ser

ATC

Ile

CCT

Pro

GGG

Gly 140

TAC

Tyr

GGT

Cly ccc Pro

GAG

Glu

;TT

Val

;TA

Vl

WTA

Ile

A.GC

Ser

TTC

Phe

GAA

Giu

CAT

His

GAT

Asp 125

GTC

Val

CCA

Pro

CCC

Giy

GGA

Gly

C

Alr: 205 TCC CTA Ser Leu 15 CTG GAC Leu Asp GCC CAG Ala Gin GAC TAC Asp Tyr TAC GGG Tyr Cly ACG GGC.

Thr Gly 95 AAT CTG Asn Leu 11e TTC CAC Phe Asp CAA Ciu Leu GGG ATG Cly Met AAC CGA Lys Cly 175 TGC CAC Cys His 190 TCA GAC Ser Asp ,TA TGC TCG CTT TTG TGC GCA TCT GTA TTA Val Cys Ser Leu

;GG

Gly

;CG

Ala

TCT

Ser

A.AT

Asn 80

AGT

Ser

AAC

Asn

AAG

Lys

GT

Gly CTC GAG TCC, Leu Glu Ser 35 TGC AAC CC Cys Asn Arg 50 GGT CGC ACC Gly Arg Thr 65 ACT AAT CCC Ser Asn Pro GGA CAC GAG Cly His Ciu C TCT GTG Ala Ser Val 115 CAC TGC CAA Gin Cys Clu 130 ACT TAC CTG Ser Tyr Val Cys Ala Ser Val Leu CCT TTC CCC TTC GGG Pro Phe Pro Phe Cly ACG ATT GAG CTC ACC Thr Ile Glu Val. Thr GGA CTG TCA CTC GAG Giy Va. Ser Val Clu AGC TTC GTC TTC GGG Ser Phe Val. Phe Cly CTG TCT ACG TGC TGG Leu Ser Thr Cys Trp 105 ACC CC TCT CAC GCC Arg Ala Ser Asp Ala 120 GTG GAG AGA AGA CTC Val Gin Arg Arg Leu 135 GGC AAT GGA TCC CTG Gly Asn Gly Ser Leu iso TAC CCC TAC GAG CTC Tyr Ala Tyr Gin Leu 165 TAT CTA GAC GTC GGA Tyr Leu Asp Val. Cly 185 ACC TAT TAC AGC CTG Thr Tyr Tyr Ser Leu 200 GTA CCC TCG CCC GAA Val Ala Ser Pro Giu 215 7101 7161 7221 7271 7319 7367 7415 7463 7511 7559 7607 7655 7703 7751 7799 7847 7895 7943 TAC GAT GCC CCC ATC Tyr Asp 160 TAT ACC Tyr Thr CAC CAC Asp Gin TTA TGA Leu Ser Al1a Gly COG TCT Cly Ser TAT CCC Tyr Gly 195 TCT TAT Ser Tyr 210 CTC GAC GGT Leu Asp Cly 220 CCT ATC GAG CAA CAT Pro Met Ciu Clu Asp 225 TAT TCC AAT TCT CTA GAC ATC CCC Tyr Ser Asn Cys Leu Asp Met Pro 230 WO 96/29396 PCTIUS96/03916 -146- CCG CTA CGC CCA TGG ACA ACC GTT TGT TCG CAT GAC GTC GAG GAG CAG Pro Leu Arg Pro Trp Thr Thr Val. Cys Ser His Asp Val Giu Glu Gin 235 240 245 GAA AAC GCC ACG GAO GAG OTT TAC CTA TGG GAC GAG GAA TGC GCO GGT Glu Asn Ala Thr Asp Giu Leu Tyr Leu Trp Asp Giu Glu Cys Ala Gly 250 255 260 265 CCG CTG GAC GAG TAC GTC GAO GAA AGG TCA GAG ACG ATG CCC AGG ATG Pro Leu Asp Giu Tyr Val Asp Glu Arg Ser Giu Thr Met Pro Arg Met 270 275 280, GTT GTC TTT TCA COG CCC TOT ACG CTC CAG CAG TAG CCACCCGAGA Val Val Phe Ser Pro Pro Ser Thr Leu Gin Gin 285 290

GTGTTTTTTG

GTATTGAATA

ATGGCGTGTG

CAGCGTATTT

AGCGGACGCG

AACCATCCAA

TOOGOOGCCT

TCGOTACGGC

GGACAACGTG

CACCATAGGG

GGTCTTAGTT

TTTTTTGCAG

TGTGCGAGAT

CAACTCGGCG

AAGAATAGTA

CTCGTOGCGG

GTAOAOTCCC

GGACTCCGGC

TCAACTGCAC

CTGTCAAA

CTCTATCCGI

AGGTACTTOC

CAGCACTAGC

GGTACCCGTC

TCCCGGTGAC

CCCGAGCGOC

TGAGCGCCOA

AATAAACAGT

CGGGTGAAAC

CGCCGAGAGA

GCGTTGCCGA

GAGAACCTGG

GAAGGCGCCG

GCTACGGAGG

ACATTTCGC

GTATTCGCTA

AACGCCTCTC

AACGAGCCTA

AACGATGTCG

GCCCAGACGG

AACGTCACGT

GAGAACGGGC

GACATTGACG

AGCGTCCTCG

CGGATAGA TA

ATGATAGAAA

CCGGGGGCCA

COGACOOCCA

GATATGGGAT

CAGGAAACGG

TCAGAAAATA

;GCACATGAA-Z

CGCAACATAC CTAACTG OTT

ACAAAAGCAT

CGTAAATTAC

TGGGGACAAT

CCGGCAGATT

CGGTGCTCGC

TCAGCTTTCA

ACGAACTTGC

TGTCATATCG

CTGGCCAGAG

TGGAACGGTC

CATGGGG 070

CCGTGTITGAC

GGCTCACGTC

GGACAACCGG

AGCCCGTGTT

GOOGGGCCAT

CGTTTACGGC

TGTCCGGGALC

CCCCGTACCG

CTCTGCCTCC

CTACCGTGCC

TCTTCTCCAC

ATAGAACTCI

CACTGTTTCC

L TACACAGAC

CAGGTGTGGT

GTGATAATAA

GTTAGTGTTG

CTGCCGAGTT

GGAATCGCCG

GATTTTTGCG

ACTGGAGCGC

CCCGCGCCCA

CACGGAAAGC

CGTGCGCCTG

GAAGCGTTGG

AA.AAGCGCAG

TCTCAACATG

CGGCCTC

GAGGAACGTT

GATAAACGTT

CT7TAGGGAA

TGAGCCGCCG

AGOGCTCGOG

GTTCGATACC

AGAGCCAGC,

GGCACGTGCJ

TTCGACAACI

TACGACCGCC

CCAGAGTGCI

CATTTCTGAT

TTGOGTGTCT

ATAGCATAGG

CGCCTTTTCC

TGGAAGGTGC

GTCACGGGAC

GACACCCCTA

GGGACGTCCG

GAAATTCACG

AGC'TATTCGG

GAAACGCCGT

TTAGGCCCOC

TACATTGGGG

ACCTTTTTCT

CCCTCGOGCA

TCTGACGGGT

ATTGCCAATT

GGAAAACTCC

GGGACTGAAA

AGCAATGTTC

GCAGCACCTC

ATTACTACAL

7ACCGGATCAC

CAATTATTGC

GTGCTAAACC

AGTTGGCGTG

TACTTGCAGT

CTCCGGGAGG

ACGCGACATA

CTTTGCGCAT

CCTCAGACGC

GAGCATACTT

TCATCAGTCG

GCGATGAAAA

CGTCGCCTTA

AGTGCTACTO

ATTCGCCTAA

TAACGGTATA

TCTTGGTTAA

ATTCGOOGGO

CATOOGCGAT

OGACCTTCGA

CCAGGGACGA

ATTTCGATAC

TCATACCGCG

AAACTCTTTC

7991 8039 8087 8133 8193 8253 8313 8373 8433 8493 8553 8613 8673 8733 8793 8853 8913 8973 9033 9093 9153 9213 9273 9333 9393 9453 9513 9573 9633 9693 CCTCTTACCC TTCCACTGAC

CCGGGGATTT

GAAACGGTGG

OTACOGAGAC

TCTTACTCA

WO 96/29396 WO 9629396PCTIUS96/039 16 -147- GAGTCCGAGT ACCGAGTCGG TCAGACTCCG AGTACTGAAC GTTGTTTACT CAGACTCCGA AACCGAGGCA CCTGCCCAGA CACGCCCCCC GAAACCGCTC GAGTACGGTA ACGGAGGTGT TTCGAGTACT GAACCGGCGA TCAGACTTCG AGTGCCGAGC CACTCAGGCC CCGAGTACGG GTTGACTCAG AGTCCGAGTA GGAAATTACT CAGACCCCGA GCCAGAAACT GCACAGAGCA GACGCATAAT ACTGAACCGA CTACACAGAA AATAAGACTT GACGGCAGAG TCGCAGACGC CGATGGCGAA GTAACGGCGA TAATTGGAAA GTAGACCTCG GGTTTTTAAC AGTAATGAGA AACATCGGTC CAGCTAATGT TTTCGACACG TCTCTTATAG TGATCCGCAA ACGGCATACT KTGGAGCCTT AATAATGTTT TACCGCTGGC CAAAGTACAG TGGCCGGGGA GGCAATATAT TACGCGAAAG GAGAGGTGCT GGCGCAGCTG TATTCACTC7 TTTGTACGCA ACGCTATGGA GTACTGCTTT CTTCCTTCGCl GGCCGTCGCG CGCTCGACGC ACTAGGGTAT CAAGAGGCTC ATGATATCGG CGGCCAAAG; TTCTACGTGA TTAAGGGCG; TACAGGGAAT GTGGCGACGI TGGGCAGTGG ACTATGTTCC AAACCGCGCG G AGGCGGCTCT TI

GTGCTGAACA

CCCCGAGCAC

GCGCTCCGAG

TTACTCAGAC

TTTTTACTCG

CGGACACTAT

TACCGAAAGC

CCGAACCTGT

GCGCGGCACC

CACCCCTGGC

GGACTTATCC

TATCGTTTCC

CCCTATTGGA

CTTGCGTT1C

TAGATGTAAT

AATGGCAGAA

GCCTGTCGTG

CGAGGGAA;LA

GCACAATAAC

CACTGCCGGA

TGTGGAAGAC

ACGAATGCAC

TAACAAACAC

TTCTGGACI

*CCGCCATT1

*TAGCCAGAG(

GCTAACCATI

CGACGTTGT(

AAAAGAGAA(

LCGACGGCGAC

7ACAACTGCTA' 'TAGCACCC7

C

c c c ITCCCAGAGT CAGGAACCGT GGTATTTTAC ACTCAGACG CAGATCGCAG AAACGGAGGC ;ATGACTTTT ACTCAGACTC CGGGTGCAGA ;ATACCCGAG ATATTTACTC AGTCTCGTAG GCGGCGCCG GAGGTTTTTA CACAGAGTTC CCGAGCACG GTACCGAAAA CTACTCTGAG ;ACTCAGAGC GCGGGAACTG AGGCCTTTAC ;CGAACTCAG AGTACTGAA.A CACACTTTTT r'ACTCAGACT CCGAGTACAG AGCCGGAGGT 3CCTTTCACC CGGACTCTGG GCGCAGAGCC GGAGGTTTAT ACTCGGAGTT CGAGTACGAT CTCGCAAAAC CCTACCAGTT CGGGAACCGG PIGTGCAA.ACG ACACCACATA CCCAGAAACT TACTGTT-GTTI TCAGAATTCC ATGAGATGTC CGTCAAA.ATT GTAGAGGTGA AGTTTTCAAA CACCGTCAAA TCTCCCTATA GGGTAGAAAC GGATGAAATT TCTGGGAACA GTCCCGCCGG ACAGCTGTAC TACAGAGTAA CCGATGGAAG CACGAGCCAT TCTCCGGAALC CTTACTGTCT AGATATCGCG CCAGAGTTAT ACTTTACCTC ITCTGCCGTCC GGCGTTGTTC CGAGATTCGA ATATTTGACG GCCACGACCG TTGTTTCGCA CAGCGCGAGA GCAGGCGAGG CGTGGATTTC CGTCCTCATC TCAGACGGCA CTCGCGTTAC ATGGATTGCG GTGGAAAACG GTGCTGCTCA TGTGTCAGGA TTATGCGGGA GCATATCTGC TTTTTGA GGAATGCTTT TTGGACTATC ACCGCCGCCG TCACGTACGA CTACATTTTA CCGGCGGTTG GCCCGTATAA CAGATACCTC GAGCTCAACC CGATTTCTAA CGTGGACGAC GGGGGCCCTT TCGAGGCCTC CGTCGTCTGG GACAAGTACT GTCCAATCTA TAGAAAAGAG TCTGAATGCG CCGTTCAATC TGCACAGATG GTATCGCGAA ATGGCGCGGG ACTGACTATA 9753 9813 9873 9933 9993 10053 10113 10173 10233 10293 10353 10413 10473 10533 10593 10653 10713 10773 10833 10893 10953 11013 11073 11133 11193 11253 11310 11370 11430 11490 11550 11610 11670 11730 WO 96/29396 PCTfUS96/03916 -148- TTCTCCCCCA CTGCTGCGCT CTCTGGCCAA TACTTGCTGA CCCTGAAAAT CGGGAGATTT

GCGCAAACAG

CTTAACTTTT

GAACACCTTT

TACGAAGATA

GACCCTCGTC

CGCACCCCGG

ATGCAGGCAG

GAGGACACCG

GTGATCCCGG

GCGTTCGTAG

CTCTCGTAAC

TACCGTCGAA

ATCCGATCGC

TTCTGCAGCG

CAGATAGCGT

ACGCAGAAAG

AGGCTTCTGG

AGCACGATGA

TGGAGGAGAC

CCTGCGCGGT

TCTAGAAGTT

ATGCTGGACA

AGACACCAAT

CTGGAATAAT

CCCGCAAGAA

CAGCGAAAAG

AGAAAATCCT

TCCAAACTCG

TACTAAAAGT

CGCGCTCGTG

AACGATCGCT

ACAGAACAGT

ACACGACACG

TTGCTGAGGA

ATTCCCGC!TG

AAGGCCCCTC

GCCGCCCTCC

GATCCTGACT

TCTAATGCCG

GGGCTACTGG

GTTTAAAGAT

ATCAGACTGG

CGGACGACGT

AAAAGAATCC

TAACCAAGAA

CAGAAGACTC

CCGAAGACGA

ATTACAATGA

TCTCCATGCC

TTTGGAGCAT

CGGGTCGCAG

ATTTCAAGGC

ATATCGGGGA

TAGCGCGCCA

AGCGGAAGGG

GGAGGACGAC

CGAAGTCCCC

CATGCCCGCC

CATATTCGCG

CGTAAAATGC

GCGCGTAGCT

TCACGCTCAT

AA TCGAGCCTAG AATAGGTGGT TTCTTCCTAC ATGCCACGCC AATATAAATC ACATGGAATA.GCATACCAAT GCCTATTCAT TGGGACGTTC GAAAAJGC ATG GCA TCG CTC GCA Leu Ala T CC GCC Ser Ala GAA GCT Met Ala Ser

I

CCC TTC GGC Pro Phe Gly AGG ATT GAC Arg Ile Asp ACA ATT CAA Thr Ile Gin CTA CTT GGA ACT CTG GCT CTC CTT GCC GCG ACG Leu Leu Gly Thr Leu Ala Leu Leu Ala Ala Thr S GCG ATG GGA ATC GTG ATC ACT GGA AAT CAC GTC Ala Met Gly Ile Val Ile Thr Gly Asn His Val 20 25 GAC GAT CAC ATC GTG ATIC GTC GCG CCT CGC CCC Asp Asp His Ile Val Ile Val Ala Pro Arg Pro 40 CTG CAG CTA TTT TTC ATG CCT GGC CAG AGA CCC Leu Gin Leu Phe Phe Met Pro Gly Gin Arg Pro GGA ACC GTC CGC GTC GCG TTT CGG TCT GAT ATA Gly Thr Vai Arg Val Ala Phe Arg Ser Asp Ile 70 11790 11850 11910 11970 12030 12090 2.2150 12210 12270 12330 12390 12442 12502 12551 12599 12647 12695 12743 12791 12839 12887 12935 12983 CAC AAA CCC TAC His Lys Pro Tyr

TCA

Se r ACA AAC CAG Thr Asn Gin ACT CAT CGA Thr His Arg ACG TTC TCC Thr Phe Ser CTC ACT ATA Leu Thr Ile TGC TAC CAG Cys Tyr Gin TCG TCT TCT Ser Ser Ser 100 GCC TCG AAC Ala Ser Asn 115 CGA AAT CCT Arg Asn Pro 130 GAA CTT Giu Leu 85 AGC GAG GAG CGC TTT GAA AAT TGC Ser Giu Giu Arg Phe Glu Asn Cys GTr TTT GTC GGC TGT AAA GTG ACC GAG TAC Val Phe Val Gly Cys Lys Vai Thr Giu Tyr 105 110 AGA CTA ACC GGA CCT CCA CAC CCG TT AAG Arg Leu Thr Gly Pro Pro His Pro Phe Lys 120 125 CGT CCG AAC GAC AGC GGG ATG TTC TAC GTA Arg Pro Asn Asp Ser Gly Met Phe Tyr Val 135 140 ATT GTT CGG CTA GAC GAC ACC AAA GAA CCC ATT GAC GTC TTC GCG ATC Ile Val Arg Leu Asp Asp Thr Lys Glu Pro Ilie Asp Val Phe Ala Ile 145 150 155 WO 96/29396 PCTIUS96/03916 -149- CAA TTC GCG AAC ACC GCC GCG ACT CGC GGA CTC Gin Phe Ala Asn Thr Ala Ala Thr Arg Gly Leu 165 170 CAA CTA TCG GTG TAT Gin Leu Ser Val Tyr 160 TAT TCC AAG Tyr Ser Lys 175 GAG GCC TAT Glu Ala Tyr GTT GCC ACG Val Ala Thr CCC GTC ACT Pro Val Thr 225 TTT CCC TGG Phe Pro Trp 240 AAC GAA AAT Asn Glu Asn 255 CTA ATT GGG Leu Ile Gly GTC ATT GTA Val Ile Val AAA TTA GAC Lys Leu Asp 305 AGG GAA TCG Arg Glu Ser 320 GGG GCT GAC Gly Ala Asp 335 CCG TCT GCG Pro Ser Ala

GCT

Ala

CTC

Leu

GAG

Glu 210

GCA

Ala

CTA

Leu

TCA

Ser

GTA

Val

ATT

Ile 290

ACG

Thr

CGA

Arg TCG TGT CGC Ser Cys Arg 180 ACC TTC GGA TTA CCT ACC GTC CAA CTT Thr Phe Gly Leu Pro Thr Val Gin Leu 185 190

AGG

Arg 195

GCC

Ala

ACC

Thr

GAA

Glu

AAC

Asn

ACC

Thr 275

TCC

Ser

GTC

Val

AAA

Lys

ACC

Thr

ACG

Thr

AGC

Ser

AAT

Asn

GTT

Val 260

GTG

Val

ATC

Ile

GAG

Glu

ACG

Thr

GCC

Ala

GGC

Gly 245

ACT

Thr

GCT

Ala

GTC

Val

GAA

Glu

ACC

Thr

TCC

Ser 230

GTG

Val

GTC

Val

GCC

Ala

ACC

Thr AGT TGG Ser Trp 200 AGC GCC Ser Ala 215 GAA CTT Glu Leu GAT CAT Asp His CGT CTC Arg Leu GTC GTG Val Val 280 AGA AAC Arg Asn 295 AAC TGG CAA GCG TAC Asn Trp Gin Ala Tyr 205 GCG ACA ACC CCG ACG Ala Thr Thr Pro Thr 220 GCG GAA CAC TTT ACC Ala Glu His Phe Thr 235 GAA CCG ACA CCC GCA Glu Pro Thr Pro Ala 250 ACA ATG AGC CCT ACG Thr Met Ser Pro Thr 270 GCA ACG ATC GGC CTC Ala Thr Ile Gly Leu 285 TGC ACC CCG CAC CGA Cys Thr Pro His Arg 300 CGT TCC CAA ACT AGA Arg Ser Gin Thr Arg 315 TGC GAA ATA AAC AAG Cys Glu Ile Asn Lys 330 GTT GCG ATT GTT AAC Val Ala Ile Val Asn 350 ATG TGA TTAAGTCTGA 13031 13079 13127 13175 13223 13271 13319 13367 13415 13463 13511 13559 13608 13668 13728 13788 13836 13884 TCG CAA GAC GAC GAA GAA Ser

TTT

Phe Asp 310

CCC

Pro Asp

ATG

Met Glu

GTT

Val CAG GAT AGT GAA CTT GTG GAA Gin

CTA

Asp

AGC

Glu

CCC

Leu

GAC

Glu

ATA

Leu Ser Ser Pro Asp 355 Ser Ile Lys Met 360 ATGTGGCTCT CCAATCATTT CGATTCTCTA ATCTCCCAAT CCTCTCAAAA GGGGCAGTAT CGGACACGGA CTGGGAGGGG CGTACACGAT AGTTATATGG TACAGCAGAG GCCTCTGAAC ACTTAGGAGG AGAATTCAGC CGGGGAGAGC CCCTGTTGAG TAGGCTTGGG AGCATATTGC AGG ATG AAC ATG TTA GTG ATA GTT CTC GCC TCT TGT CTT GCG CGC CTA Met Asn Met Leu Val Ile Val Leu Ala Ser Cys Leu Ala Arg Leu 1 5 10 ACT TTT GCG ACG CGA CAC GTC CTC TTT TTG GAA GGC ACT CAG GCT GTC Thr Phe Ala Thr Arg His Val Leu Phe Leu Glu Gly Thr Gin Ala Val 25 WO 96/29396 PCT/US96/03916 -150- CTC GGG GAA GAT GAT CCC AGA AAC GTT CCG GAA GGG ACT GTA ATC AAA 13932 Leu Gly Glu Asp Asp Pro Arg Asn Val Pro Glu Gly Thr Val Ile Lys 40 TGG ACA AAA GTC CTG CGG AAC GCG TGC AAG ATG AAG GCG GCC GAT GTC 13980 Trp Thr Lys Val Leu Arg Asn Ala Cys Lys Met Lys Ala Ala Asp Val 55 TGC TCT TCG CCT AAC TAT TGC TTT CAT GAT TTA ATT TAC GAC GGA GGA 14028 Cys Ser Ser Pro Asn Tyr Cys Phe His Asp Leu Ile Tyr Asp Gly Gly 70 AAG AAA GAC TGC CCG CCC GCG GGA CCC CTG TCT GCA AAC CTG GTA ATT 1407.6 Lys Lys Asp Cys Pro Pro Ala Gly Pro Leu Ser Ala Asn Leu Val Ile 85 90 TTA CTA AAG CGC GGC GAA AGC TTC GTC GTG CTG GGT TCT GGG CTA CAC 14124 Leu Leu Lys Arg Gly Glu Ser Phe Val Val Leu Gly Ser Gly Leu His 100 105 110 AAC AGC AAT ATA ACT AAT ATC ATG TGG ACA GAG TAC GGA GGC CTG CTC 14172 Asn Ser Asn Ile Thr Asn Ile Met Trp Thr Glu Tyr Gly Gly Leu Leu 115 120 125 TTT GAT CCT GTA ACT CGT TCG GAC GAG GGA ATC TAT TTT CGA CGG ATC 14220 Phe Aso Pro Val Thr Arg Ser Asp Glu Gly Ile Tyr Phe Arg Arg Ile 130 135 140 TCT CAG CCA GAT CTG GCC ATG GAA ACT ACA TCG TAC AAC GTC AGC GTT 14268 Ser Gln Pro Asp Leu Ala Met Glu Thr Thr Ser Tyr Asn Val Ser Val 145 150 155 CTT TCG CAC GTA GAC GAG AAG GCT CCA GCA CCG CAC GAG GTG GAG ATA 14316 Leu Ser His Val Asp Glu Lys Ala Pro Ala Pro His Glu Val Glu Ile 160 165 170 175 GAC ACC ATC AAG CCG TCA GAG GCC CAC GCG CAC GTG GAA TTA CAA ATG 14364 Asp Thr Ile Lys Pro Ser Glu Ala His Ala His Val Glu Leu Gln Met 180 185 190 CTG CCG TTT CAT GAA CTC AAC GAC AAC AGC CCC ACC TAT GTG ACC CCT 14412 Leu Pro Phe His Glu Leu Asn Asp Asn Ser Pro Thr Tyr Val Thr Pro 195 200 205 GTT CTT AGA GTC TTC CCA CCG ACC GAG CAC GTA AAA TTT AAC GTT ACG 14460 Val Leu Arg Val Phe Pro Pro Thr Glu His Val Lys Phe Asn Val Thr 210 215 220 TAT TCG TGG TAT GGG TTT'GAT GTC AAA GAG GAG TGC GAA GAA GTG AAA 14508 Tyr Ser Trp Tyr Gly Phe Asp Val Lys Glu Glu Cys Glu Glu Val Lys 225 230 235 CTG TTC GAG CCG TGC GTA TAC CAT CCT ACA GAC GGC AAA TGT CAG TTT 14556 Leu Phe Glu Pro Cys Val Tyr His Pro Thr Asp Gly Lys Cys Gln Phe 240 245 250 255 CCC GCA ACC AAC CAG AGA TGC CTC ATA GGA TCT GTC TTG ATG GCG GAA 14604 Pro Ala Thr Asn Gln Arg Cys Leu Ile Gly Ser Val Leu Met Ala Glu 260 265 270 TTC TTG GGC GCG GCC TCT TTG CTG GAT TGT TCC CGC GAT ACT CTA GAA 14652 Phe Leu Gly Ala Ala Ser Leu Leu Asp Cys Ser Arg Asp Thr Leu Glu 275 280 285 GAC TGC CAC GAA AAT CGC GTG CCG AAC CTA CGG TTC GAT TCG CGA CTC 14700 Asp Cys His Glu Asn Arg Val Pro Asn Leu Arg Phe Asp Ser Arg Leu 290 295 300 WO 96/29396 PCTIUS96/03916

TCC

Ser C AAA C Lys 320

TAC

Tyr

CAC

His

GGA

Gly

GGG

Gly

AAA

Lys 400

CCA

Pro

ACG

Thr

CGC

Arg

GGT

Gly

GCA

Ala 480

ATG

Met

GAC

Asp

ATC

Ile

GAG

Glu 305

GTT

lal

ACG

rhr

ATG

Met

CCA

Pro

GTG

Val 385

AAG

Lys

TTG

Leu

GAC

Asp

GGC

Gly

GCG

Ala 465

TCA

Ser

GAT

As;

GCC

Ali

TT

Le

TCA

Ser

TTG

Leu

GTG

Val

CCC

Pro

ATG

Met 370

GCC

Ala

AAG

Lys

TAC

Tyr

GAT

Asp

TAC

Tyr 450

GTC

Val

ATG

Met

AGC

Se

GC(

Al k CC i Pr CGC G Arg A ATT A Ile I CTC G Leu C

TCG

Ser 355

GAC

Asp

GCG

Ala

TGC

Cys

GCG

Ala

GAG

Glu 435

TGT

Cys

GTG

Val

GCA

Ala

TAT

Tyr

GCG

a Ala 3 ACC o Thr

CA

la

TA

le

GG

Gly 340

LAG

Lys

GAA

Glu

ACG

Thr

CCC

Pro

CCG

Pro 420

GAA

Glu

AAG

Lys

GAG

Glu

GAT

As;

TAG

GGC

Gl

AG

Ar -151- GGC CTG GTG ATC AGT CCT CTT ATA GCC ATC CCC Gly Leu Val Ile Ser Pro Leu Ile Ala Ile Pro 310 315 GTC GTT TCC GAC GGA GAC ATT TTG GGA TGG AGC Val Val Ser Asp Gly Asp Ile Leu Gly Trp Ser 325 330 335 AAA CGT AAC AGT CCG CGC GTA GTA GTC GAA ACG Lys Arg Asn Ser Pro Arg Val Val Val Glu Thr 345 350 GTC CCG ATG AAC AAA GTA GTA ATT GGC AGT CCC Val Pro Met Asn Lys Val Val Ile Gly Ser Pro 360 365 ACG GGT AAC TAT AAA ATG TAC TTC GTC GTC GCG Thr Gly Asn Tyr Lys Met Tyr Phe Val Val Ala 375 380 TGC GTA ATT CTT ACA TGC GCT CTG CTT GTG GGG Cys Val Ile Leu Thr Cys Ala Leu Leu Val Gly 390 395 GCG CAC CAA ATG GGT ACT TTT TCC AAG ACC GAA Ala His Gin Met Gly Thr Phe Ser Lys Thr Glu 405 410 415 CTC CCC AAA AAC GAG TTT GAG GCC GGC GGG CTT Leu Pro Lys Asn Glu Phe Glu Ala Gly Gly Leu 425 430 GTG ATT TAT GAC GAA GTA TAC GAA CCC CTA TTT Val Ile Tyr Asp Glu Val Tyr Glu Pro Leu Phe 440 445 CAG GAA TTC CGC GAA GAT GTG AAT ACC TTT TTC GIn Glu Phe Arg Glu Asp Val Asn Thr Phe Phe 455 460 GGA GAA AGG GCC TTA AAC TTT AAA TCC GCC ATC Gly Glu Arg Ala Leu Asn Phe Lys Ser Ala Ile 470 475 CGC ATC CTG GCA AAT AAA AGC GGC AGA AGG AAT Arg Ile Leu Ala Asn Lys Ser Gly Arg Arg Asn 485 490 495 TTGGTC ATG CCT TT AAG ACC AGA GGG GCC GAA Met Pro Phe Lys Thr Arg Gly Ala Glu 0 1 AAG AAC AGG TTT AAG AAA TCG AGA AAT CGG GAA Lys Asn Arg Phe Lys Lys Ser Arg Asn Arg Glu 15 20 k CTG CGT GGC ACC GGT AAG AAA ACT GCC GGA TTG g Leu Arg Gly Thr Gly Lys Lys Thr Ala Gly Leu 0 35 14748 14796 14844 14892 14940 14988 15036 15084 15132 15180 15228 15276 15324 15372 15420 15468 15516 3

TCC

Ser

CGC

Arg

AAT

Asn

GGG

Gly TAT ACC CAG CCT ATT CCC TGG Tyr

GAA

Glu Thr

TCT

Ser Gin

GAC

Asp Pro Ile AAC CAC Asn His Pro Trp 50 GCG TGT Ala Cys 65 AAC CCT AAA TTC TGC AGC GCG Asn Pro Lys Phe Cys Ser Ala AAA GAC ACT TTT TAT CGC AGG Lys Asp Thr Phe Tyr Arg Arg WO 96/29396 PCTIUS96/03916 -152- ACG TGC TGC GCA TCG CGC TCT ACC GTT TCC AGT CAA CCC GAT TCC CCC 15564 Thr Cys Cys Ala Ser Arg Ser Thr Val Ser Ser Gin Pro Asp Ser Pro 80 CAC ACA CCC ATG CCT ACT GAG TAT GGG CGC GTG CCC TCC GCA AAG CGC 15612 His Thr Pro Met Pro Thr Glu Tyr Gly Arg Val Pro Ser Ala Lys Arg 95 100 105 AAA AAA CTA TCA TCT TCA GAC TGC GAG GGC GCG CAC CAA CCC CTA GTA 15660 Lys Lys Leu Ser Ser Ser Asp Cys Glu Gly Ala His Gin Pro Leu Val 110 115 120- TCC TGT AAA CTT CCG GAT TCT CAA GCA GCA CCG GCG CGA ACC TAT AGT 15708 Ser Cys Lys Leu Pro Asp Ser Gin Ala Ala Pro Ala Arg Thr Tyr Ser 125 130 135 TCT GCG CAA AGA TAT ACT GTT GAC GAG GTT TCG TCG CCA ACT CCG CCA 15756 Ser Ala Gin Arg Tyr Thr Val Asp Glu Val Ser Ser Pro Thr Pro Pro 140 145 150 GGC GTC GAC GCT GTT GCG GAC TTA GAA ACG CGC GCG GAA CTT CCT GGC 15804 Gly Val Asp Ala Val Ala Asp Leu Glu Thr Arg Ala Glu Leu Pro Gly 155 160 165 GCT ACG ACG GAA CAA ACG GAA AGT AAA AAT AAG CTC CCC AAC CAA CAA 15852 Ala Thr Thr Glu Gin Thr Glu Ser Lys Asn Lys Leu Pro Asn Gln Gin 170 175 180 185 TCG CGC CTG AAG CCG AAA CCC ACA AAC GAG CAC GTC GGA GGG GAG CGG 15900 Ser Arg Leu Lys Pro Lys Pro Thr Asn Glu His Val Gly Gly Glu Arg 190 195 200 TGC CCC TCC GAA GGC ACG GTC GAG GCG CCA TCG CTC GGC ATC CTC TCG 15948 Cys Pro Ser Glu Gly Thr Val Glu Ala Pro Ser Leu Gly Ile Leu Ser 205 210 215 CGC GTC GGG GCA GCG ATA GCA AAC GAG CTG GCT CGT ATG CGG AGG GCG 15996 Arg Val Gly Ala Ala Ile Ala Asn Glu Leu Ala Arg Met Arg Arg Ala 220 225 230 TGT CTT CCG CTC GCC GCG TCG GCG GCC GCT GCC GGA ATA GTG GCC TGG 16044 Cys Leu Pro Leu Ala Ala Ser Ala Ala Ala Ala Gly Ile Val Ala Trp 235 240 245 GCC GCG GCG AGG GCC TTG CAG AAA CAA GGG CGG TAG CAGTAATAAT 16090 Ala Ala Ala Arg Ala Leu Gin Lys Gln Gly Arg 250 255 260 AACCACACAA ATATTGACAA TAATAAACGC GTACGCGG ATG AGT AAG TGT TAT 16143 Met Ser Lys Cys Tyr 1 TGT CTC GCG CGC CAT CTT.TAT AAA AGC CCG CGT TGC GTG GGC CGG CGG 16191 Cys Leu Ala Arg His Leu Tyr Lys Ser Pro Arg Cys Val Gly Arg Arg 15 GTA GCA TTT GGA GGG TTG GCG ACC ATG TCG AGA CCT CCG ACG TCA CAT 16239 Val Ala Phe Gly Gly Leu Ala Thr Met Ser Arg Pro Pro Thr Ser His 30 TTG GAC TTA GCT TTC TCG GCG GCC TTT AGG GGC ACG GAC CTG CCC GGA 16287 Leu Asp Leu Ala Phe Ser Ala Ala Phe Arg Gly Thr Asp Leu Pro Gly 45 GGG AGA TTC TGG CGG GCG TCG CAG AGT TGC GAT ATT TTC TTT TGG CCC 16335 Gly Arg Phe Trp Arg Ala Ser Gin Ser Cys Asp Ile Phe Phe Trp Pro 60 WO 96/29396 PCTfUS96/03916 -153- GAT CTG GCC GGG GTG ATC GTA GAG GCC GCC CGC GCG TAT TTT GAA GGG Asp Leu Ala Ala Val Ile Val Gin Ala Ala Arg Ala Tyr Phe Giu Gly 75 80 AAG GAA AGG CTG GGG AGT GTG GAG GTC GGC GAA GAT ATC ACG GCG CAC Lys Giu Arg Leu Gly Ser Leu Gin Val Ala Giu Asp Ile Thr Ala His 95 100 GAC CCG CGA ATA GCG CCC GCG GCT AAG CGC GCC GTC GCA GCG GCG GTA Asp Pro Arg Ile Ala Pro Ala Ala Lys Arg Ala Val Aia Ala Ala Val 105 110 115 GGA CTG TGG ACC GCG CTG TCG GAG TTA GTT GGG GGG CCG AAC GGG GAG Gly Leu Trp Thr Ala Leu Ser Glu Leu Val Gly Giy Pro Asn Gly .Giu 120 125 130 TTG GAA AGC AAG GTC TGG GGC AAG GAG ATT CCC GGG GCC GCC GCG TGG Leu Glu Ser Lys Val Trp Gly Lys Gin Ile Pro Arg Ala Ala Ala Trp 135 140 145 GAA ATA AGA GAG GTG GGC AAA GTT GGA GTG ATT GGG CCG GAC ATT GTT Glu Ile Arg Asp Val Pro Lys Val Pro Val Ile Gly Pro Asp Ile Leu 150 155 160 165 TCT TTT TTC TCC GCC GCC GTC GAG CTG CGG GTG CTC TAT ATC AGA GCC Ser Phe Phe Ser Ala Ala Vai Glu Leu Pro Vai Leu Tyr Ile Arg Ala 170 175 180 CGG GGA GGG GCG GAG TCG CGG TCC GGG CAC TGG AAT AAC GAG AGC AGG Arg Gly Giy Ala His Ser Arg Ser Ala His Trp Asn Asn Gin Ser Ser 185 190 195 GCG CCG GCC GCG GGA CTC GCG GCG ATA AGG ATA GGC ATG GAG ATG GTG Aia Pro Ala Ala Gly Leu Ala Ala Ile Arg Ile Gly Met Giu Met Val 200 205 210 CGG AGC =T CTG GTG ATA GCG GTG CCT CTG TCA AAG TTC ACC CTG CG Arg Ser Leu Leu Val Ile Ala Leu Pro Leu Ser Asn Phe Thr Leu Pro 215 220 225 GAA GAG CTC CCC GAA GGT TCC CAA AAC TCG ATC CGC1 GCG TTC GTG GCC Giu Asp Leu Pro Giu Gly Ser Gin Asn Ser Ile Arg Ala Phe Val Ala 230 235 240 245 CAC CTC ATG AAC TGT GTA GCT ACC GAT AAG ATC ATG TCT GCG GAG GTG His Leu Met Asn Cys Val Ala Thr Asp Lys Ile Met Ser Pro Asp Val 250 255 260 CGC GTC CCA GTC GAA GAA AGC TrT TAG AGC CAC TGT TTA AGG GAA ATC Arg Val Pro Val Giu Giu Ser Phe Tyr Ser His Gys Leu Arg Glu Ile 265 270 275 ATT ATG TGC GAG AGA GICT TTT TGT TAG CCG TGC AAT CGC GGG GGA AAA Ile Met Gys Glu Arg Ala Phe Gys Tyr Pro Cys Asn Pro Pro Pro Lys 280 285 290 TGG TGA GGTGAGGGTG GGATTTAGGG CCGCAAGAGC CTGTGGG GAGGGGGCGC Trp.

295 GCTGTATCTT TCTGTATGTG GGGTGGGAC GGGCTAACG GAAGGGTGGA AGGGGGCG CCGGGAAAGG GTAGCTCGG AGAGACAGAG AGAGACAAAC GGCGTCTCGA TTGGAAGAAG GTGAAAAACA GAGAATAATA TTACTTATT TATTTGGCAG GGTCGGGTG TGGTGGTTT GTGGGGGGGG GGGGGGGTTG TGTTrGGTG CCGCGGAGTG GGTGAGGGGG GAGAGTGGAC 16383 16431 16479 16527 16575 16623 16671 16719 16767 16815 16863 16911 16959 17007 17063 17123 17183 17243 17303 WO 96/29396 PCT/US96/03916 -154.

GGAGGACAGT GTA-AAAACCC GCGAGGTTGT CAGGGACGCG GAGGTAAGGA

AGCCGCTAGA

GGGCGTCATT

CAAAGATCGA

CCAGGTATTC

GACTCTGCAC

AGCAGCACCA

GGTCGGCCGG

TATTCTCGCT

GCCAGAAATC

TATTCGGGTC

CTAGGCCGCG

CAATCAGCGC

CGGACGATAT

CCGAGTCGGC

GTACCGCGTG

GCATGTTTTC

GTGCTGGGCC

GATGTCCGGC

AAGTTCTGGP

GAGGGGGAGC

GCCGTGCAGC

AGGAAGAAG;

AAGCCTTCCC

AGCAAATGG(

CTCCCGTCAA

GGCGCTCACT

GCGACGGCGC

GTAAATCGAG

GTGCCCGGCC

CAGGTGTCGG

CTGCATGGTA

GCCCTCGATC

AGTGGGGGAG

CGTCGCCCGG

GTTGCGGCGG

CTCGACTAAC

CGACTCCGAC

CGCAGTAATG

CATTTAGGGA

GAGTTGGACG

GGTCGCAAACG

TGAGAATGGP

TTCTGCCCCC

GGTCGAGGCC

AAATGACAAC

TGGGTC71TT,

GGCGGCGCCC

CGGCGCCGCG CCAGCGCCAG

TCGGGAGAGT

ATCTCCGATT

TCGTACAAAC

GCCAGGAGAG

GACAGCCAGG

CGGCTCTCGT

CCTTCCATTG

TTGGCGACGC

TGGCGGCAGC

GCGGGACTCT

ACGCTTCGCG

ACCGGGAGGT

ATAACAGT.TC

CCTCGAAGCG

CCTCCTCTAA

CGCGATTCCA

CGCGCCGGCG

CCCCGTCCCG

GCGTCCCTCG

CGCAAGGCGG

ACGCAGAAAA

GCACGGCCGA GGGTGCCACA

CTCTCGCTCG

TTTCTCGATC

GTCACCGTCC

TTCTCCCAAC

TC!TCCGTCGA CGCCCACCGC CTGGCCCCCA GAAAAGCCGA

ACGTAGCCGT

TCTCTCGCTG

ACGTGAACTC

TCGCGCCAGA GCCGCGAGGC GAGGCGACAG CGGCACCTTG TGATCGCTGT CCATCAGCGC TGTGTCTAGA GGAGAGCGGT GCTACGGATT GCGCCGGGCG GGGCGCGCGT CCGCTTC!TGC TCGAGCGGGC A.ATAAATGTC CGTCTCCTCT ATCTGCTCGT GCTTCT T= TTTTACTTCT CTCCGTCTTT TAAGAGTGCG CGGGTGCCGC GCACCGCA-AT GAGAGCTGTC GTCAATAAAG GTCCCGTGTC C-TCCTCGCCC rCCGTAACCG

GATCCGAATG

CCGCCTGCGC

CAGCGGGTAG

CCGGGCGGTC

TGAAGGGGGG

GTGAAGGCTG

CAGAGTAGGG

CGAGGCCTCG

CTCCTCGGAT

CCTACCCGCC

AACGCACGGA

TCTAAGTCAG

SCGGACGCGA

A.CGAAACGCA

AGAGAGTCGA

CCGTTGCCGT

GGCCTCAAAC

TCGTTGGCAG

TCCTTGAGTT

CACGGGTCTA

TCGCCACGAG

CACCCGTCGA

TACTGCGGTT

GGGGCGGCGG

AGCGGGAAAG

GGGTGGGAGG

GCCTTGCGTC

TCCTCGTCAG

CTGGCGGCCG

CGCGGGGGAT

ATTGCGTGGG

17363 17423 17483 17543 17603 17663 17723 17783 17843 17903 17963 18023 18083 18143 18203 18263 18323 18383 18443 18503 18563 18623 18683 18743 18803 18863 18912 CTCTGACTCG GTGGAGCTGT AAGTCCACTC CCCGGCACCC CCGCGTGCCG GTTCTTCGTC AGGCTGCTCT TCCTCCGACT CCGGGTCGCT CCAGCTCCTC CTCCGATACG TCCGAAXAGA AAAACTTCTG GGAGAGCTCT TCGGGATCC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS-: LENGTH: 278 ami~no acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 140:60: WO 96/29396 WO 9629396PCTIUS96103916 -155- Met 1 Asp Gly Ile Glu Asn Ala Thr Leu Leu 145 Gin Leu Ser Arg Leu 225 Arg Pro Al a Glu His Ala Arg Leu Ala Ser Glu Gly 130 Asp Ser Ser Arg Pro 210 Phe Glu Giu Leu Asn Ile Ala Ile Lys Leu Arg Ser 115 Glu Ser Glu Arg Ser 195 Cys Cys Tyr Val Ala 275 4et rhri lal kla Asp Ile Ala 100 Val Leu Leu Asn His 180 Let Arc VaJ Lei Se~ 261 Arc Leu 5 Al a Ala Ser Gly Al a Thr Ala Glu *Asp Met 165 *Leu Leu G 1.

*Gly a Ala 245 ~AlE 0 g ArS Asp Gly Cys Tyr His Ala Val Pro 25 Ser Ser Glu Ser 40 Arg Leu Trp, Arg 55 Tyr Gly Glu Phe 70 Ala Asn Glu Arg Arg Gly Leu Gly 105 Asn Ser Pro Thr 120 Glu Ala Met Glu 135 Gly Asp Arg Phe 150 Arg Phe Val Ile Pro Ala Asp Leu 185 Ala Ala Gly His 200 Ala Cys Leu Tyr 215 Thr Gly Arg Val 230 Ala Leu Arg Ala Ser Ile Phe Ala 265 ;Arg Pro Leu Ala Leu Met Asp Ser Prg Gly Glu Arg Ala Glu Thr Leu 90 Leu Asp Gly Glu Glu 170 Al a Trp Asp Pro Gly 250 Asp Leu Ser 75 Arg Arg Pro Ile Asp 155 Lys Ser Cys Sex Gir 235 Al a Ser Val Ala Ser Pro Asn Glu 140 Glu Glu Al a Cys Ile 220 Ser Ala Val G1u Arg Ala Arg Asn 125 Gly Ser Leu Glu Leu 205 Tyr Glu Ala k.rg ksp Ile Asp Phe Trp 110 Gly Asp Arg Leu Arg 190 Trpa Met Al a Arg Pro Ser Arg Leu Ala Asn Phe Thr Ser 175 Glu His Gin Arg Asn 1ln Cys Ser Arg Gly Ser Giy Trp Met 160 Trp Thr Pro Ser Arg 240 Ser 255 Arg Asp Ala Gly Ile Ala Leu 270 INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 294 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: WO 96/29396 PCTIUS96/03916 Me Cy Pr Th

I]

A:

A

V,

G

A

1

G

L

C

-156t Ser Lys Cys Tyr Cys Leu Ala Arg His Leu T 1 5 10 -s Val Gly Arg Arg Val Ala Phe Gly Gly Leu A 25 o Pro Thr Ser His Leu Asp Leu Ala Phe Ser A 40 ir Asp Leu Pro Gly Gly Arg Phe Trp Arg Ala S 55 Le Phe Phe Trp Pro Asp Leu Ala Ala Val Ile V 70 75 la Tyr Phe Glu Gly Lys Glu Arg Leu Gly Ser I 90 sp Ile Thr Ala His Asp Pro Arg Ile Ala Pro 100 105 al Ala Ala Ala Val Gly Leu Trp Thr Ala Leu 115 120 ly Pro Asn Gly Glu Leu Glu Ser Lys Val Trp 130 135 rg Ala Ala Ala Trp Glu Ile Arg Asp Val Pro 45 150 155 ly Pro Asp Ile Leu Ser Phe Phe Ser Ala Ala 165 170 eu Tyr Ile Arg Ala Arg Gly Gly Ala His Ser 180 185 isn Asn Gln Ser Ser Ala Pro Ala Ala Gly Leu 195 200 ly Met Glu Met Val Arg Ser Leu Leu Val Ile 210 215 Asn Phe Thr Leu Pro Glu Asp Leu Pro Glu Gly 225 230 235 Arg Ala Phe Val Ala His Leu Met Asn Cys Val 245 250 Met Ser Pro Asp Val Arg Val Pro Val Glu Glu 260 265 Cys Leu Arg Glu Ile Ile Met Cys Glu Arg Ala 275 280 Asn Pro Pro Pro Lys Trp 290 INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 229 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein yr la la er ral Leu Ala Ser Gly 140 Lys Val Arg Ala Ala 22( Se3 Al Se Ph Lys Ser P: Thr Met S 30 Ala Phe A Gin Ser C Gin Ala A Gin Val Ala Lys 3 110 Glu Leu 125 Lys Gn Val Pro Glu Leu Ser Ala 190 Ala Ile 205 i Leu Pro 0 r Gln Asn a Thr Asp r Phe Tyr 270 e Cys Tyr 285 ro 15 er rg ys Lla Lla 95 Arg /al Ile Val Pro 175 His Arc Let Se: Ly 25 Se Pr Arg Arg Gly Asp Arg Glu Ala Gly Pro Ile 160 Val Trp Ile Ser r Ile 240 s Ile r His o Cys WO 96/29396 WO 9629396PCTIUS96/03916 7- DESCRIPTION: SEQ ID NO:62: (xi) SEQUENCE Met Ala Pro Val Lys Val Thr Ile Val *1 5 Ser Ala Val Asp Ser His Tyr 10 Lys Leu Leu Leu Gly Gly Leu Pro Leu Val Ser so Phe Gly Phe Trp Ala 130 Pro His Val Ser Asp Tyr Val 115 Pro Asn Ala Ile Leu Pro His 100 Leu Lys Ser Val Phe Thr Glu Leu 25 Met Ala 40 Ser Asp Ser Gly Ser Tyr Asp Arg Val Arg Pro Ala Ser Leu Tyr Pro Pro Leu Gly Thr 55 Arg Met 70 Gly Val Ser Ser Thr Ala Phe Leu 135 Ile Phe 150 Ser Val Tyr Ser Cys Ser Gly Asp 120 Leu Trp Thr Arg Pro Ala 105 Ile Thr Val Thr Val 185 Pro Glu 90 Tyr Cys Glu Asn Thr 170 Lys Val 75 Trp Ala Met Thr Gly 155 Leu Phe Glu Ile Ser Ala Ala Gly 140 Asp Ala Leu Val A.rg Gly Lys Ala 125 GI y Lys Pro Lys TrpI Pro Ser Phe Leu Glu 110 Leu Lys Thr Ser Val 190 .sys Ser Gly Gly Asp Phe Asn Asn Thr Ser 175 Lys Glu Thr Glu Glu Ala His Leu Phe Leu 160 Leu Pro Glu Arg Gly Val Glu Ser Arg Leu Thr Ser Pro His Trp, 180 Pro 165 Pro Asn Ser Ala Ala Tyr Cys Val Ser Asp Ser Asp Asp Gly Glu Arg Gin 195 200 ZUD Pro Pro 225 Lys 210 Arg Phe Phe Arg Arg 215 Arg Leu Phe Lys Ser Lys Pro Arg Ser INFORMATION FOR SEQID NO:63: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 476 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: Met Arg Phe Arg Arg Ile Cys Ser Arg Ser Arg Ala Glu Lys Arg Arg 1 5 10 Arg Thr Thr Glu Asn Pro Leu Thr Ser Lys Arg Val Cys Val Leu Asp 25 Ser Phe Ser Arg Thr Met Ser Leu Arg Pro Tyr Ala Glu Ile Leu Pro 40 WO 96/29396 PCT[US96/03916 Thr Thr G Pro P Pro S Ala I Lys Val 145 Giu Lys Ala Glu Tyr 225 Asp Arg Lys Asp Asn 305 Asn Ile Cys la lu ~ro er ~er iiS 130 Glu Cys Ser Asn 210 Glu Arg Ala Thr Ser 115 Ser Gly Gin Lys Gin 195 Val Gly Ala Asn Thr 100 Asp Asp Ala Arg Arg 180C Let Let Val Glu Glu Glu Tyr Giu Tyr Arg 165 Leu Glu Lys Giu Pro 70 Asn Lys Al a Phe Gly 150 Arg Ile Asn Ile Arg 55 Val Gly Val Gly Leu 135 Phe Gly Al a Glu Gin 8- Leu Ala Glu Thr Glu Asn Gin Asn Phe 90 Asp Gly Ser 105.

Pro Val Pro 120 Gin His Phe Ile Cys Asp Val Asf Scr 170 Lys Tyr Val 185 Ile Leu Val 200 Giu Ile Leu Leu Thr Ala His Leu Arg Val 155 Thr Lys Leu Ar Val Ala Tyr Thr Ala Val 140 Arg Asn Asn Gly Tyr 3er I Val Ala Asp Gin 125 Leu Arg Gin Gly Arg 205 Pro 1al ksn Gly Phe 110 Thr Asp Tyr Gly Thr 190 Leu Asp Thr Ser Asp

ASP

Arg Asp Thr Lys 175 Arg Asn Asn Met Ile Gly Glu Leu Leu Giu 160 Ser Ala His Thr 215 220 Met Leu Thr Gin Glu Ile Leu Gly 290 Giu Ser Trp Pro Ala Met Ile 275 Lys Arg Pro Ser Ile 355 Phe Lys 260 His Thr Glu Giu Cys 340 Gly Asp 245 Gin Arg Val1 Pro Ile 325 Gly Asp Arg 230 Trp Leu Asp Leu Phe 310 Leu Val Gly Lys MetI Ile Gly 295 Glu Ala Val1 Gly Asp Ser Lys 2 80L Asp Tyr Arg Leu Gly 360 Ser Al a 265 Leu Phe Gly Asp Leu 345 Asn Pro 250 Val1 Giu Gly Trp Ser 330 Glu Pro Tyr Gin Phe Asp Leu Tyr Ser Tyr Met 235 Met Ser Asn Thr Val 315 Tyr Met His Leu Tyr Ile Val 300 Gly Cys Val Gin Lys Ile Phe 285 Thr Thr Glu Ser Gin 365 Gin His 270 Leu Pro Vai Ile His 350 Leu Thr 255 5cr Asn Phe Ala Thr 335 Giu Leu Tyr 240 Arg Lys Cys Glu Thr 320 Asp Phe Lys Val Ile Asp Ser Leu Ser Val Cys Asp Glu Glu Phe Pro Asp Pro Pro 370 375 380 Cys Asn Leu Tyr Asn 385 Tyr 390 Leu His Tyr Ala Ser 395 Ilie Asp Arg Ala Gly 400 WO 96/29396 PCTIUS96/03916 -159- His Thr Val Pro Ser Leu Ile Arg Asn Leu His Leu Pro Ala Asp Val 405 410 415 Glu Tyr Pro Leu Val Lys Met Leu Thr Phe Asp Trp Arg Leu Arg Pro 420 425 430 Ser Ala Ala Glu Val Leu Ala Met Pro Leu Phe Ser Ala Glu Glu Glu 435 440 445 Arg Thr Ile Thr Ile Ile His Gly Lys His Lys Pro Ile Arg Pro Glu 450 455 460 Ile Arg Ala Arg Val Pro Arg Ser Met Ser Glu Gly 465 470 475 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 623 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: Met Thr Leu Pro His Arg Leu Thr Lys Arg Pro Phe Ala Arg Arg Phe 1 5 10 Cys Ser Val Phe Val Ile His Tyr Ser Glu Thr Lys Leu Asp Arg Tyr 25 Asn Lys Thr Met Leu Leu Tyr Arg Pro Asp Ser Thr Met Arg His Ser 40 Gly Gly Asp Ala Asn His Arg Gly Ile Arg Pro Arg Arg Lys Ser Ile 55 Gly Ala Phe Ser Ala Arg Glu Lys Thr Gly Lys Arg Asn Ala Leu Thr 70 75 Glu Ser Ser Ser Ser Ser Asp Met Leu Asp Pro Phe Ser Thr Asp Lys 90 Glu Phe Gly Gly Lys Trp Thr Val Asp Gly Pro Ala Asp Ile Thr Ala 100 105 110 Glu Val Leu Ser Gln Ala Trp Asp Val Leu Gln Leu Val Lys His Glu 115 120 125 Asp Ala Glu Glu Glu Arg Val Thr Tyr Glu Ser Lys Pro Thr Pro Ile 130 135 140 Gin Pro Phe Asn Ala Trp Pro Asp Gly Pro Ser Trp Asn Ala Gln Asp 145 150 155 160 Phe Thr Arg Ala Pro Ile Val Tyr Pro Ser Ala Glu Val Leu Asp Ala 165 170 175 Glu Ala Leu Lys Val Gly Ala Phe Val Ser Arg Val Leu Gln Cys Val 180 185 190 Pro Phe Thr Arg Ser Lys Lys Ser Val Thr Val Arg Asp Ala Gln Ser 195 200 205 WO 96/29396 PCTIUS96/03916 -160lie Met Phe Leu Gly Asp Ser Phe Trp Arg Gin Asn Val Tyr Thr Val 220 210 215 Val I 225 Ile I His Glu Gly Arg 305 Asp Asp Ala His Tyr 385 Gly Leu Ser Lys Glu 465 Asn Pro Lys Leu A Val Trp G1u Ala 290 Asp Ala Thr Leu Ala 370 Asp Ala Ala Val Ala 450 Phe Phe Phe Met rg sn Arg Asn Gly Lys Ala Ser Val 355 Leu Glu Glu Val Ala 435 Arg Ph( Cy Le Le 51 Gin H Cys A 2 Gly M 260 Ile C Leu Arg Val Arg 340 Val Val Thr Gly Val 420 Thr Arg Phe s Leu a Phe 500 u Cys 5 is Lsn 45 et 1n ?he Leu Leu 325 Phe Leu Phe Arg Thr 405 Cys Ser Prc Ty] As 48 Ar Pr Ile T 230 Asp P Arg V His C Leu Leu 310 Ala Glu Leu Met Tyr 390 Leu Ala Ala Gly Ile 470 n Ile 5 Ala o Leu hr Arg Leu A ro Leu Trp T 2 al Pro Ser L 265 ly Pro Met A 280 rp Pro Ala V 95 rg Ala Cys I er Phe Pro Pro Ala Leu 345 Qa Glu Thr 360 krg Ala Leu 375 Ile Asp Pro Leu Arg Gly Asn Thr Tyr 425 Cys Asn Val 440 Met Ser Asp 455 Ala Trp Leu Leu Lys Arg Ser Ser Glu 505 Leu Val Pro 520 Leu Lys Arg 535 rg His Pro Ser Ser Lys Ser yr 50 eu la et Leu ?he 330 ;ly Jal Ala Val Ile 410 Ser Ala Ile Gir Se 49 Ly Il Gl 235 Ala T Lys L Ala V Arg A 3 Ser S 315 Tyr I cys Leu Asp Lys 395 Val.

Thr Tyr Tyr Arg 475 Val 0 Arg Gln u Lys 240 yr eu al Lla 00 er Trp Leu kla Gly 380 Asn Ala Ile Arg Arg 46 Va As Le Ty Le 54 Ala A Ala S 2 Phe 285 Ala I Leu Arg Ser Thr 365 Asn Glu Ser Arg Thr 445 Ile L Ala p Thr u Gin r Glu 525 u Glu 0 sn er Arg Phe Asp Gly Glu 350 Met Phe Tyr Asn Lys 430 Glu Lei.

Th2 Gl G1 51 As Th Gin 255 Pro Asn Glu Ile Val 335 Tyr Phe Asp Leu Thr 415 Let Thr G1 Hi y Al 49 .i Le 0 p Ph r Ph Phe Pro Ala Glu Met 320 Gln Phe Asp Asp Asn 400 Ala Pro Leu i Lys s Ala 480 a Pro u Asn e Ser e Val Lys Ala Met Gly Ser Glu 530 Lys 545 Ala Ile Ser Ser Asp 550 Arg Asp Pro Arg Gly 555 Ser Leu Arg Phe Leu 560 WO 96/29396 WO 9629396PCTIUS96/039 16 Ile Pro Al a Val Ser Val Ala Pro 610 Asp His Ala Arg 565 Ile Asp Glu Pro 580 Gly Lys Val Lys 595 Gly Ala Gly Ala -161- Giu Ile Ile Ala Asp Gly Val Arg Phe Lys 570 575 Val Arg Ala Ser Val Ala Leu Ser Thr Ala 585 590 Ala Arg Arg Leu Thr Ser Val Arg Ala Pro 600 605 Val Ser Ala Arg Arg Lys Ser Glu Ile 615 620 INFORM~ATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 292 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met 1 Cys Leu Cys Gly Ser Gly Al a Gin Ser 145 Asp Thr (xi) SEQUENCE Ser Gly Phe Ser Ser.Leu Leu Cys Giu Ser Ser Pro Asn Arg Thr Thr Arg Thr Glu Gly Asn Pro Giu Ser His Glu Asp Leu 100 Ser Val Cys Arg 115 Cys Giu Lys Val 130 Tyr Val Ser Gly Ala Gly Ile Tyr 165 Gly Ser Val Tyr 180 Asn Ile Giv Ser Ile Ala Thr Val Ser Leu Val DESCRIPTION: SEQ ID Ala Ser Val Leu Gly Phe Ile Val 70 Phe Ser Ala Gln Asn 150 Al a Leu Pro Glu 55 Ser Val Thr Se r Arg 135 Gly Tyr Phe 40 Val Val Phe Cys Asp 120 Arg Ser Gln 25 Gly Thr Giu Gly Trp 105 Ala Leu Leu Leu Gly Val Val Val 90 Ala Gly Arg Val Ser 170 Ala Lys Pro Lys 75 Asp Leu Ile Ser Leu 155 Val Pro Ile Trp Trp Ser Ile Pro Gly 140 Tyr Gly Val Ile Ser Phe Giu His Asp 125 Val Pro Gly Leu Ala Asp Tyr Thr Asn 110 Phe Glu Gly Lys Asp Gin Tyr Gly Gly Leu Asp Leu Met Gly 175 Gly Al a Ser Asn Ser Asn Lys Gly Tyr 160 Tyr Asp Val Giy Pro Asn Pro Gly 185 Cys His Asp 190 Gin Tyr Gly 195 Tyr Thr Tyr Tyr Ser Leu Ala Asp Giu Ala Ser Asp Leu 200 Pro Met Glu Giu Ser Ser 210 Tyr Asp Val Ala Ser 215 Pro Glu Leu ASP Gly 220 WO 96/29396 PCTIUS96/03916 Asp 225 Val Tyr Glu Thr Tyr Cys Leu Arg Leu 290 Ser 5cr Trp Ser 275 Gin -162- Asn Cys Leu Asp Met Pro Pro Leu Arg Pro Trp Thr Thr 230 235 240 His Asp Val Glu Glu Gin Giu Asn Ala Thr Asp Giu Leu 245 250 255 Asp Glu Glu Cys Ala Gly Pro Leu Asp Glu Tyr Val Asp 260 265 270 Giu Thr Met Pro Arg Met Val Val Phe Ser Pro Pro Ser 280 285 Gin INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 985 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: Met Gly Thr Met Leu Val Leu Arg Leu Phe Leu Leu Ala Val Ala Asp I 10 Al a Gly Thr Ile Asp Val Ty'r Tyr Val 145 Thr Al a Gly Gly Phe Glu Thr Phe 5cr 130 Arg Trp, Leu Thr His Ala Leu Phe Thr Val Leu Gly Pro Ile Al a Asp Ala 5cr 100 Ile Ile Glu Ser Thr Gin Thr Thr Leu Leu Gly Ser Thr Lys 165 Gly Glu Tyr Pro 70 Glu Ser Val1 Arg Pro 150 Arg Val Arg Asn Pro 55 Thr Arg Tyr Phe Val 135 Cys Trp Leu Phe Leu 40 Pro Leu Gly Arg Al a 120 Leu Asp Leu Thr Cys 25 Al a Pro Arg Thr Pro 105 Thr Val Giu Gly Lys 185 Arg Val Glu Ile Ser 90 Arg Gly Asn Asn Pro 170 Ala V~al Leu Gly Arg 75 Ala Pro Gin Ala Phe 155 Trp Al a Al a Tyr Ser Giu Ser Ser 140 Leu Lys Glu Val Gly Asp Ile Thr 125 Leu Gin Val 3 C Ser Ser Ala Ala His 110 Glu Giu Asn I~ro Pro ?ro Val Phe Gin rhr Giu s0 Asp Asn Gly Ala Ser Ser Arg Ser Glu Pro 160 Vai Arg 175 Pro Ser Pro Tyr Asp Asn Asp Val Ala IS0 Gin TIyr Ile Gly Glu Cys 190 Tyr Scr Asn 195 Ser Ala Ala Gin Thr Gly Leu Thr Ser Leu Asn Met Thr 200 205 WO 96/29396 WO 9629396PCTJUS96/039 16 -163- Ile Val Asn Phe Phe Tyr Ser Pro Lys Arg 210 215 Val Thr Trp, Thr Thr Gly 220 Gly 225 Gin Pro Pro Pro Ser Pro Ser Arg 230 Ile Thr Val Tyr Ser Ser Arg Giu Asn Pro Asp Ala Val Ile Asp 275 Leu Asp 260 Ser Arg 245 Gly Gly Asn Val Ser Asp G Arg Ser b Lys Leu Pro Ser Ala Ilie Gin Glu 305 Thr Arg Asp Thr Al a 385 Asp Giu Glu Thr Ser 465 Gir Thi

AD~

290 Pro Pro Pro Thr Thr 370 Arg Arg Ser Thr Val 450 Gin Ala Gir Gli.

Pro Gly Tyr Arg Gly Al a 340 Gly Thr 355 Leu Ilie Ala Thr Thr Leu Giu Asn 420 Pro Ser 435 Val Phe Ser Gin Ala Leu Thr Pro 500 1Thr Giu 515 rhr Al~a 325 Thr Ser Pro Gly Ser 405 Thr Al a Thr Glu Thr 485 Glu 310 Leu Leu Pro Arg Ser 390 Thr Leu Al a Gin Prc 47( Gir 295 Thr Gly Pro Thr Ser 375 Giu Thr *Phe *His Ser 455 Trp i Thr ~et Z80 Leu rhr Ser Pro Pro 360 Thr Thr Pro Pro Git 44( Prc Ile 265 Ala His Phe Asfl Phe 345 Thr Ser Leu Leu Thr 425 Thr SSer r Phe liy 1 ~50 ?he krg Asp Vai 330 Asp Thr Asp Ser Thr 410 Thr Thr Thr Thr 'he lal rhr.

Ile Cys 315 Pro Thr Val Met Val 395 Leu Lieu Ilie Ala Asp 300 Gin Arg Al a Pro Giy 380 Pro Pro Val Ala Phe 285 Met Lys Asp Ala Giu 365 Phe Val Leu Lys Asn 270 Arg Ser Met Asp Pro 350 Pro Phe Gin Thr ryr.

255 Tyr Giu Gly Ile Ser 335 Asp Ala Ser Glu Pro 415 Gly 240 Thr Ser Giy Thr Giu 320 Ile Phe Ile Thr Thr 400 Gly Ala Pro Gly Ile Ser Thr 430 Gin Glu Gin 475 Thr Ser 460 Thr Ser Thr Ser Ala Ala Thr Giu Arg Giu 480 Gin Ile Ala Giu Thr Glu Ala 490 Leu Phe 495 Ser Ala Giu Gin Ala Pro Ala Gin 520 Arg Ser Thr Pro 535 Met 505 Thr Pro Thr Pro Glu Phe Ser Thr Thr Thr Al a 540 Gin Thr 510 Ile Pro 525 Arg Ala Pro Giu Pro Gly Ile Ser Phe Thr Gin Ser 530 Ala Ala Pro Giu Val 545 Phe 550 Thr Gin Ser Ser Thr Val Thr Giu Val 560 WO 96/29396 WO 9629396PCTfUS96/03916 -164- Phe Thr Gin Thr Pro Ser Thr Val. Pro 5

G

*ys Thr Thr Leu Ser ser ser 70 575 lin Ser Ala Gly Thr Glu Ala 590 Thr G Phe T3 Thr C Thr C 625 Thr Thr Thr Thr Val 705 Leu Giu Ser Pro Asp 785 Lys Val Cys Giu Leu 865 iu ~hr ;iu in ilu 'ln ~1et Ser 590 Gln Ser Ser Asn Tyr 770 Glu Trp Gir Let Let Prc Pro Ala I 580 Gin Thr S 595 Thr His I Thr Pro Pro Val Thr Pro 660 Pro Glu 675 Ser Gly Thr Thr Phe Pro Gin Thr 740 Asp Gly 7 5 5"" Arg Val Ile Ser Gin Lys 1Leu Met 820 Phe Asp 835 STyr Phe SSet.. Gly :le er ?he 2 er Pro 645 Ser Thr Thr Pro Thr 725 Pro Glu Glu Gl) Gir 801 cyl Tku Th: Va Phe TI Ser Phe *I 6 Thr C 630 Phe Ala Ala Gly His 710 Val.

Leu Val.

Thr Asn 790 Leu Leu r Ser r Ser 1. Val 870 hr Lia ~hr 15s ;iu rhr kla Gln Thr 695 Thr Val Let.

Th3 Asi 77! Se Ty: Se Le As 85 A-rg T 5 Giu P 60C Pro C Arg Pro Ser 680 His Gin Ser 1Asp Al a 760 *i Trp, Pro Tyr Cys Ile 840 p Pro 5 'hr 85 ro 1ia ;iu Lrhr 3iu 665 rhr n-sn Lays Glu Val.

745E Thr Lys Alz Ar~ Th~ 82' Al G I Asp Pro Val Leu 650 Val.

Pro Thr Leu Phe 730 Lys Cvs Val.

IGly ;Val r Ser a ArS n Th2 Thr Met Arg TI 605 Ser Thr Val F 620 Leu Thr Gin S 635 Gly Ala Giu Tyr Thr Arg Leu Ala Ser 685 Giu Pro Arg 700 Tyr Thr Giu 715 His G).u Met Ile Val Giu Val Ser Thr 765 Asp Leu Val 780 Phe Asn 795 Thr Asp Gly His Ser Pro Giu Lys Asp 845 *Ala Tyr Cys 860 a Trp Ser Leu 87 5 hr ro er ?ro er 670 31n rhr Asn Ser Val 750 Val.

Asp Ser Arg Gl.

830 Ile Th~ Asi Gin S Lys Pro E 6 Glu 655 Ser Asn Tyr Lys Thr 735 Lys Lys *Val.

*Asn Thr 815 1Pro Ala Sle ai Asn er lia ier Ile 3er Pro Pro Thr 720 Ala Phe Ser Met Glu 800 Ser Tyr Pro Thr Val 880 Pro Arg Phe Gli Ser Leu Pro Glu Gly Gin Ser Thr 900 Tyr 885 Leu Thr Ala Thr Thr Val Val Ser His Thr Ala R90 895 Val Trp Lys Ser Ser 905 Ala Arg Ala Gly Giu Ala Trp 910 WO 96/29396 PCTIUS96/03916 Ile Asp Trp 945 Phe Ala Ser Gly 930 Ile Ser Thr Gly Arg 915 Thr Arg Ala Val Gly Leu Leu Trp 980 Gly Val Glu Val 965 Thr Gly Asn Thr Thr 935 Asn Gly 950 Ser Gly Ala Ile -165- Ile Tyr Glu Cys Thr Val Leu Ile Ser 920 925 Arg Lys Glu Arg Cys Leu Thr Asn Thr 940 Ala Ala Gln Ala Gln Leu Tyr Ser Leu 955 960 Leu Cys Gly Ser Ile Ser Ala Leu Tyr 970 975 Tyr Phe 985 INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 434 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:67: Met His Arg Pro His Leu Arg Arg His Ser Arg Tyr Tyr Ala Lys Gly Glu Gly Glu Arg Ser Asp Arg Val Phe 145 Glu Asp Val Ala His Asn Thr Ala Val Asp 130 Glu Asp Val Leu Asn Ala Val Ile Cys Ala Phe Ala Ala Leu Thr 100 Ser Arg 115 Asp Met Ala Ser Lys Tyr Gin Leu 180 Lys Phe Phe Trp Val Ile Gly Ile Val Cys 165 His Met Asp Thr Val Thr 70 Thr Pro Cys Ser Val 150 Pro Leu Arg 55 Ile Tyr Ala Asp Ala 135 Trp Ile Phe 40 Asn Val Asp Val Val 120 Ala Phe Tyr Cys 25 Trp Ala Leu Tyr Gly 105 Val Lys Tyr Arg Gly Gly Lys Thr Met Leu Ile 90 Pro Glu Glu Val Lys 170 Val Cys Asp Ser 75 Leu Tyr Leu Lys Ile 155 Glu Gin Val Arg Ser Gly Asn Asn Glu 140 Lys Tyr Ser Arg Arg His Phe Arg Arg Pro 125 Lys Gly Arg Ala Asn 205 Cys Ile Leu Ala Arg Tyr 110 Ile Gly Asp Glu Gln Cys Met Phe Ser Ala Leu Ser Gly Asp Cys 175 Met Ser Arg Leu Gin Leu Thr Asn Pro Gly 160 Gly Trp Leu Ser Glu Cys Ala 185 190 Ala Val Asp 195 Tyr Val Pro Ser Thr Leu Val Ser Arg 200 Gly Ala Gly WO 96/29396 WO 9629396PCTIUS96/03916 Leu Thr 225 Val Ser Thr 210 Leu Asn Lys Ile Lys Asp Cys Ser Pro Gly Arg 230 Cys Leu 245 Thr Thr Thr 215 Phe Lys Giu -166- Ala Leu Gin Thr Giy Ser 250 Tyr Gin 265 Ser Ala 235 Gin Thr Gly 220 Leu Leu Gly His Leu Tyr 275 Tyr Arg Gly 290 Lys Lys Asn 305 Giu Ile Pro Giu Ser Ser Gin Ala Giu 355 Giu Val Pro 370 Tyr Tyr Asn 385 Ser Ser Asn Pro Ile Aia Asp Thr Asn Thr Arg His Tyr Pro Ala Giu 340 Aia Giu Asp Ala Giu Ser Val 325 L.ys Se r Asp Met Val.

405 Asp Aila 310 Thr Lys Gly Thr Pro 390 Ser Ile 295 Pro Lys Ala Glu Giu 375 Al a Met 280 Leu Asp

LYS

Pro Asn 360 His Val1 Pro Gin Pro Ala Pro 345 Pro Asp Ile Ile Arg Arg Glu 330 Glu Ala Asp Pro Phe Trp Pro 315 Gly Asp Al a Pro Val.

395 Ala Asn 300 Asp Arg Ser Leu Asn 380 Gliu Ala Gin Val Asn Phe Ala 285 Asn Ser Thr Giu Pro 365 Ser Giiu Tyr I Thr Phe Gin 2170 Asp Leu Val Pro Asp 350 Giu Asp Thr Val .eu .jeu .jeu 255 Giy Asp Leu Pro Asp 335 Asp Asp Pro Th r Ala Leu Glu 240 Pro Glu Val Arg Gin 320 Ala Met Asp Asp Lys 400 Cys 410 415 Ala Vai Ala Leu Val. Gly Leu Leu Val Trp Ser Ile Val Lys Cys Ala 430 Arg Ser 420 INFORMATION FOR SEQ ID NO:68; Wi SEQUENCE CHARACTERISTICS: LENGTH: 362 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: Met Ala Ser Leu Leu Gly Thr Leu Ala Leu Leu Ala Ala Thr Leu Ala 1 5 10 Pro Phe Giy Ala Met Giy Ile Val Ile Thr Gly Asn His Val Ser Ala 25 Arg Ilie Asp Asp Asp His Ile Val Ilie Val Ala Pro Arg Pro Glu Ala 40 WO 96/29396 PCTIUS96/03916 -167- Phe Met Pro Pro His Lys Thr Ile Gin Leu Gin Leu Phe 55 Gly Gin Arg S er Asp Ile Thr Asn Pro Gin Arg Ser Ile Arg 145 Ser Lys Tyr Thr Thr 225 Trp Asn Gly Val Asp 305 [yr Cys Ser Ala Arg 130 Leu Val Ala Leu Glu 210 Ala Leu Ser Val Ile 29 Th Ser Tyr( Ser Ser 115 Asn Asp Tyr Ser Arg 195 Ala Thr Glu Asn Thr 275 Ser r Val Gly Gln Ser 100 Asn Pro Asp Gin Cys 180 Thr Thr Ser Asn Val 260 Val Ile Ser Thr Val Arg Val Ala Phe A 70 Glu Leu Ser Glu Glu Arg 1 85 90 Val Phe Val Gly Cys Lys 1 105 Arg Leu Thr Gly Pro Pro 120 Arg Pro Asn Asp Ser Gly 135 Thr Lys Glu Pro Ile Asp 150 Phe Ala Asn Thr Ala Ala 165 170 Arg Thr Phe Gly Leu Pro 185 Glu Glu Ser Trp Arg Asn 200 Thr Thr Ser Ala Glu Ala 215 Ala Ser Glu Leu Glu Ala 230 Gly Val Asp His Tyr Glu 245 250 Thr Val Arg Leu Gly Thr 265 Ala Ala Val Val Ser Ala 280 Val Thr Arg Asn Met Cys 295 Gln Asp Asp Glu Glu Arg 310 Gly Pro Met Val Ala Cys 325 330 rg Phe Val His Met Val 155 Thr Thr Trp Thr Glu 235 Pro Met Thr Th Se 31 Gl Glu A Thr C Pro Phe 140 Phe Arg Val Gin Thr 220 His Thr Ser Ile Pro 300 r Gin 5 u Ile a Ile Lsn lu ?he Cyr Ala Gly Gln Ala 205 Pro Phe Pro Pro Gly 285 His Thr As Va Cys Thr H Tyr Thr I 110 Lys Leu I Val Ile l1e Gin Leu Tyr 175 Leu Glu 190 Tyr Val Thr Pro Thr Phe Ala Asn 255 Thr Leu 270 Leu Val Arg Lys Arg Arg n Lys Gly 335 l Asn Pro 350 is he Thr Val Leu 160 Ser Ala Ala Val Pro 240 Glu Ile Ile Leu Glu 320 Ala Ser Ser Arg Lys Phe Asp Ala Gin Leu Asp Ser 355 Ser 340 Ser Leu Val Glu Leu Val Al 345 Asp Ser Ile Lys Met 360 INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: LENGTH: 499 amino acids TYPE: amino acid TOPOLOGY: linear WO 9629396PCT[US96/03916 WO 96/29396 -168- (ii) MOLECULE (xi) SEQUENCE Met Asn Met Leu Val 1 5 Phe Ala Thr Arg His Gly Giu Asp Asp Pro Thr Lys Val Leu Arg Ser Ser Pro Asn Tyr TYPE: protein DESCRIPTION: SEQ ID Ile Val Leu Ala Ser 10 Val Leu Phe Leu Glu 25 Arg Asn Val Pro Giu 40 Asn Ala Cys Lys Met 55 Cys Phe His Asp Leu 70 NO :69: Cys Leu Ala Gly Thr Gin Gly Thr Val Lys Ala Ala Ile Tyr Asp 75 Arg Ala Ile Asp Gly Leu Val Lys Val Gly Thr Leu Trp Cys Lys Lys Asp Cys Pro Pro Ala Gly Pro Leu Ser Ala Asn Leu Val Leu Lys Ser Asn Asp Pro 130 Gin Pro 145 Ser His Arg Gly Giu Ser Phe Ile 115 Val Asp Val 100 Thr Thr Leu Asp Asn A.-g Al a Glu 165 Ile Ser Met 150 Lys Glu Asn Pro Met Asp 135 Glu Al a Ala Asp Thr 215 Val Trp, 120 Glu Thr Pro His Asn 200 Glu Val 105 Thr Gly Thr Ala Ala 185 Ser His Leu Glu Ile Ser Pro 170 His Pro Val1 Gly Tyr Tyr Tyr 155 His Val Thr Lys Cys 235 Ser Gly Phe 140 Asn Glu Giu Tyr Phe 220 Gly Gly 125 Arg Val Val Leu Val 205 Asn Leu 110 Leu Arg Ser Giu Gin 190 Thr Val Ile His Leu Ile Val Ile 175 Met Pro Thr Thr Ile Lys Pro 5cr Pro Leu Ser 225 Phe Ala Leu Cys Giu 305 Val Phe Arg 210 Trp Giu Thr Gly His 290 Ser Leu His 195 Val1 Tyr Pro Asn Ala 275 Giu Arg Ile 180 Glu Phe Gly Leu Pro Phe Leu Asn Phe Ser Leu 160 Asp Leu Val Tyr Leu 240 Pro Phe i Asp ai Ser Lys 320 Tyr Asp Val Lys Glu Glu Glu Glu Val Lys Cys Val Tyr His Pro Thr Asp Gly Lys Cys Gin 260 Al a Asn Ala Ile 245 Arg Se r Arg Gly Val1 325 Cys Leu Leu Leu Val Pro 295 Leu Val 310 Val Ser Ile Asp 280 Asn Ilie Asp Gly 265 Cys Leu Ser Gly Ser Val 5cr Arg Arg Phe Pro Leu 315 Asp Ile 330 Leu Asp Asp 300 Ile Leu Met Thr 285 Ser Ala Gly Gin Phe 255 Ala Gli 270 Leu G1~ Arg Le~ Ile Pr Trp Se 33 WO 96/29396 PCTIUS96/03916 -169- Thr Val Leu Gly Lys Arg Asn Ser Pro Arg Val Val Val Giu Thr His 340 345 350 Met Pro Ser Lys Val Pro met Asn Lys Val. Val Ile Gly Ser Pro Gly 355 360 365 Pro Met Asp Giu Thr Gly Asn Tyr Lys Met Tyr Phe Val Val. Ala.Gly 370 375 380 Val Ala Ala Thr Cys Val. Ile Leu Thr Cys Ala Leu Leu Val Gly Lys 385 390 395 400 Lys Lys Cys Pro Ala His Gin Met Gly Thr Phe Ser Lys Thr Giu Pro 405 410 415 Leu Tyr Ala Pro Leu Pro Lys Asn Giu Phe Giu Ala Gly Gly Leu Thr 420 425 430 Asp Asp Giu Glu Val Ile Tyr Asp Giu Val. Tyr Giu Pro Leu Phe Arg 435 440 445 Gly Tyr Cys Lys Gin Giu Phe Arg Giu Asp Val Asn Thr Phe Phe Gly 450 455 460 Ala Va]. Val Giu Gly Giu Arg Ala Leu Asn Phe Lys Ser Ala Ile Ala 465 470 475 480 Ser Met Ala Asp Arg Ilie Leu Ala Asn Lys Ser Gly Arg Arg Asn Met 485 490 495 Asp Ser Tyr.

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 260 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Pro Phe Lys Thr Arg Gly Ala Giu Asp Ala Ala Ala Gly Lys Asn 1 5 10 Arg Phe Lys Lys Ser Arg Asn Arg Giu Ile Leu Pro Thr Arg Leu Arg 25 Gly Thr Gly Lys Lys Thr Ala Gly Leu Ser Asn Tyr Thr Gin Pro Ile 40 Pro Trp Asn Pro Lys Phe Cys Ser Ala Arg Gly Giu Ser Asp Asn His 55 Ala Cys Lys Asp Thr Phe Tyr Arg Arg Thr Cys Cys Ala Ser Arg Ser 70 75 s0 Thr Val Ser Ser Gin Pro Asp Ser Pro His Thr Pro Met Pro Thr Giu 90 Tyr G].y Arg Vai Pro Ser Ala Lys Arg Lys Lys Leu Ser Ser Ser Asp 100 105 110 WO 96/29396 PCT/US96/03916 -170- Cys Glu Gly Ala His Gln Pro Leu Val Ser Cys Lys Leu Pro Asp Ser 115 120 125 Gin Ala Ala Pro Ala Arg Thr Tyr Ser Ser Ala Gln Arg Tyr Thr Val 130 135 140 Asp Glu Val Ser Ser Pro Thr Pro Pro Gly Val Asp Ala Val Ala Asp 145 150 155 160 Leu Glu Thr Arg Ala Glu Leu Pro Gly Ala Thr Thr Glu Gln Thr Glu 165 170 175 Ser Lys Asn Lys Leu Pro Asn Gln Gln Ser Arg Leu Lys Pro Lys Pro 180 185 190 Thr Asn Glu His Val Gly Gly Glu Arg Cys Pro Ser Glu Gly Thr Val 195 200 205 Glu Ala Pro Ser Leu Gly Ile Leu Ser Arg Val Gly Ala Ala Ile Ala 210 215 220 Asn Glu Leu Ala Arg Met Arg Arg Ala Cys Leu Pro Leu Ala Ala Ser 225 230 235 240 Ala Ala Ala Ala Gly lie Val Ala Trp Ala Ala Ala Arg Ala Leu Gin 245 250 255 Lys Gln Gly Arg 260 INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: LENGTH: 294 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: Met Ser Lys Cys Tyr Cys Leu Ala Arg His Leu Tyr Lys Ser Pro Arg 1 5 10 Cys Val Gly Arg Arg Val Ala Phe Gly Gly Leu Ala Thr Met Ser Arg 25 Pro Pro Thr Ser His Leu Asp Leu Ala Phe Ser Ala Ala Phe Arg Gly 40 Thr Asp Leu Pro Gly Gly Arg Phe Trp Arg Ala Ser Gln Ser Cys Asp 55 Ile Phe Phe Trp Pro Asp Leu Ala Ala Val Ile Val Gln Ala Ala Arg 70 75 Ala Tyr Phe Glu Gly Lys Glu Arg Leu Gly Ser Leu Gln Val Ala Glu 90 Asp Ile Thr Ala His Asp Pro Arg Ile Ala Pro Ala Ala Lys Arg Ala 100 105 110 Val Ala Ala Ala Val Gly Leu Trp Thr Ala Leu Ser Glu Leu Val Gly 115 120 125 WO 96/29396 PCTfUS96/03916 -171- Gly Pro Asn Gly Glu Leu Giu Ser Lys Val Trp Gly Lys Gin Ile Pro 130 135 140 Arg Ala Ala Ala Trp Glu Ile Arg Asp Val Pro Lys Val Pro Val Ile 145 150 155 160 Gly Pro Asp Ile Leu Ser Phe Phe Ser Ala Ala Val Glu Leu Pro Val 165 170 175 Leu Tyr Ile Arg Ala Arg Gly Gly Ala His Ser Arg Ser Ala His Trp 180 185 190 Asn Asn Gin Ser Ser Ala Pro Ala Ala Gly Leu Ala Ala Ile Arg Ile 195 200 205 Gly Met Glu Met Val Arg Ser Leu Leu Val Ile Ala Leu Pro Leu Ser 210 215 220 Asn Phe Thr Leu Pro Giu Asp Leu Pro Glu Gly Ser Gin Asn Ser Ile 225 230 235 240 Arg Ala Phe Val Ala His Leu Met Asn Cys Val Ala Thr Asp Lys Ile 245 250 255 Met Ser Pro Asp Val Arg Val Pro Val Giu Glu Ser Phe Tyr Ser His 260 265 270 Cys Leu Arg Glu Ile Ile Met Cys Glu Arg Ala Phe Cys Tyr Pro Cys 275 280 285 Asn Pro Pro Pro Lys Trp 290 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 278 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NC:72: Met Giu Asn. Met Leu Asp Gly Cys 7yr Pro Leu Ala Leu Met Asp Ser i 5 10 Asp His Ile Thr Ala His Ala Val Pro Arg Giy Glu Arg Arg Arg Gin 25 Gly Ala Ala Val Ala Ser Ser Glu Ser Ala Asp Ser Val Asp Pro Cys 40 Ile Arg Ile Ala Ser Arg Leu Trp Arg Giu Leu Vai Giu Ile Ser Ser 55 Glu Leu Lys Asp Gly Tyr Gly Glu Phe Thr Ser Ala Arg Asp Arg Arg 70 75 Asn Ala Leu Ile Ala Ala Asn Glu Arg Leu Arg Ser Ala Phe Leu Gly 90 Ala 5cr Arg Ala Thr Axg Gly Leu Gly Leu Arg Pro Arg Trp Ala Ser 100 105 110 WO 96/29396 PTU9/31 PCTIUS96/03916 Thr Leu Leu 145 Gin Leu Ser Arg Leu 225 Arg Pro Al a Glu Gly 130 Asp Ser Ser Arg Pro 210 Phe Glu Glu Leu Ser G1u Ser Glu Arg Ser 195 Cys Cys Tyr Val Al a 275 Val Leu Leu Asn His 180 Leu Arg Val Leu Ser 260 Arg Ala Glu Asp Met 165 Leu Leu Glu Gly Al a 245 Al a Arg Asn Glu Gly 150 Arg Pro Al a Al a Thr 230 Ala Ser Arg Ser Ala 135 Asp Phe Ala Ala Cys 215 Gly Leu Ile Pro 120 Met Arg Val Asp Gly 200 Leu Arg Arg Phe -172- Thr ASP Glu Gly Phe Glu Ile Glu 170 Leu Ala 185 His Trp, Tyr Asp Val Pro *Ala Gly 250 *Ala Arg 265 Pro Asn Ile Glu 140 Asp Glu 155 Lys Glu Ser Ala Cys Cys Ser Ile 220 Gin Ser 235 Ala Ala Asp Ala Asfl 125 Gly Ser Leu Glu Leu 205 Tyr Glu Ala Gly Gly Asp Arg Leu Arg 190 Trp Val Met Ala Ile 270 Asfl Phe Thr Ser 175 Glu His Gln Arg Asn 255 Ala Gly TrP Met 160 Trp Thr Pro Ser Arg 240 Ser Leu

Claims

1. A recombinant infectious laryngotracheitis virus comprising an infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the infectious laryngotracheitis viral genome. wherein the deletion is in the glycoprotein G (gG) gene.

2. The recombinant infectious laryngotracheitis virus of claim 1. further characterized by a deletion in the US2 gene.

3. The recombinant infectious laryngotracheitis virus of claim 1, further characterized by a deletion in the ORF4 gene and a deletion in the UL47-like gene.

4. The recombinant infectious laryngotracheitis virus of claim 1, further characterized by a deletion in the glycoprotein 60 (g60) gene. The recombinant infectious laryngotracheitis virus of claim 1, further characterized by a deletion in the glycoprotein I (gI) gene.

6. The recombinant infectious laryngotracheitis virus of claim 1, further characterized by a deletion in the thymidine kinase (TK) gene.

7. The recombinant infectious laryngotracheitis virus of claim 1, which further comprises a foreign gene inserted within a non-essential site of the infectious laryngotracheitis viral genome, wherein the foreign gene is capable of being expressed in a recombinant infectious laryngotracheitis infected host cell.

8. The recombinant infectious laryngotracheitis virus of claim 7, wherein the foreign gene is inserted into a gene selected from a group consisting WO 96/29396 PCT[US96/03916 -174- of the US2 gene, UL47-like gene, ORF4 gene, glycoprotein G (gG) gene, glycoprotein 60 (g60) gene, and glycoprotein I (gl) gene.

9. The recombinant infectious laryngotracheitis virus of claim 7. wherein the foreign gene encodes a screenable marker. The recombinant infectious laryngotracheitis virus of claim 9, wherein the screenable marker is E. coli B-galactosidase.

11. The recombinant infectious laryngotracheitis virus of claim 9. wherein the screenable marker is E. coli B-glucuronidase.

12. The recombinant infectious laryngotracheitis virus of claim 7, wherein the foreign gene encodes an antigenic polypeptide.

13. The recombinant infectious laryngotracheitis virus of claim 12, wherein the antigenic polypeptide, when introduced into the host cell, induces production of protective antibodies against an avian disease causing agent from which the antigen is derived or derivable.

14. The recombinant infectious laryngotracheitis virus of claim 13, wherein the antigenic polypeptide is derived or derivable from a group consisting of infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus. The recombinant infectious laryngotracheitis virus of claim 13, wherein the antigenic polypeptide is derived or derivable from a group consisting of avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian influenza virus, avian adenovirus, fowl pox virus, avian coronavirus. avian rotavirus, chick anemia agent, Salmonella spp. E. coli, Pasteurella spp., Bordetella spp., Eimeria spp., Histomonas spp., WO 96/29396 PCTfUS96/03916 -175- Trichomonas spp., Poultry nematodes, cestodes, trematodes, poultry mites/lice, poultry protozoa.

16. The recombinant infectious laryngotracheitis virus of claim 7. wherein the foreign gene is under control of an endogenous upstream promoter.

17. The recombinant infectious laryngotracheitis virus of claim 7, wherein the foreign gene is under control of a heterologous upstream promoter.

18. The recombinant infectious laryngotracheitis virus of claim 17. wherein the promoter is selected from a group consisting of the HCMV IE promoter. PRV gX promoter, and BHV-1.1 VP8 promoter.

19. A recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the glycoprotein gG gene, so that upon replication the recombinant infectious laryngotracheitis virus produces no glyccprotein gG.

20. A recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the glycoprotein gl gene, so that upon replication, the recombinant infectious virus produces no glycoprotein gl.

21. A recombinant infectious laryngotracheitis virus of claim 20. which further comprises a deletion in the glycoprotein gG gene so that upon replication, the recombinant virus produces no glycoprotein gG.

22. The recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in WO 96/29396 PCTIUS96/03916 -176- a gene selected from a group consisting of the US2 gene. the UL47-like gene, and the glycoprotein g60 gene.

23. A recombinant infectious laryngotracheitis virus of claim 22, wherein the foreign gene is inserted in the gene selected from a group consisting of the US2 gene, UL-47 like gene, ORF4 gene and glycoprotein gene.

24. The recombinant infectious laryngotracheitis virus of claim 23. wherein the foreign gene encodes a screenable marker. The recombinant infectious laryngotracheitis virus of claim 24, wherein the screenable marker is E. coli B-galactosidase.

26. The recombinant infectious laryngotracheitis virus of claim 24, wherein the screenable marker is E. coli B-glucuronidase.

27. The recombinant infectious laryngotracheitis virus of claim 23, wherein the foreign gene encodes an antigenic polypeptide.

28. The recombinant infectious laryngotracheitis virus of claim 27, wherein the antigenic polypeptide, when introduced into the host cell, induces production of protective antibodies against an avian disease causing agent from which the antigen is derived or derivable.

29. The recombinant infectious laryngotracheitis virus of claim 28, wherein the antigenic polypeptide is derived from or derivable from a group consisting of infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus. The recombinant infectious laryngotracheitis virus of claim 28, wherein the antigenic polypeptide is derived from or derivable from a group WO 96/29396 PCTIUS96/03916 -177- consisting of avian encephalomyelitis virus, avian reovirus, avian paramyxovirus. avian influenza virus. avian adenovirus, fowl pox virus. avian coronavirus, avian rotavirus, chick anemia agent. Salmonella spp.. E. coli., Pasteurella spp., Bordetella spp., Eimeria spp., Histomonas spp., Trichomonas spp., Poultry nematodes, cestodes, trematodes. poultry mites/lice, poultry protozoa.

31. The recombinant infectious laryngotracheitis virus of claim 23, wherein the foreign gene is under control of an endogenous upstream infectious laryngotracheitis virus promoter.

32. The recombinant infectious laryngotracheitis virus of claim 23, wherein the foreign gene is under control of a heterologous upstream promoter.

33. The recombinant infectious laryngotracheitis virus of claim 32, wherein the promoter is selected from a group consisting of HCMV IE promoter, PRV gX promoter, and BHV-1.1 VP8 promoter.

34. A vaccine for infectious laryngotracheitis virus comprising an effective immunizing amount of the recombinant infectious laryngotracheitis virus of claim I and a suitable carrier. A multivalent vaccine for infectious laryngotracheitis and for one or more of other avian diseases comprising an effective immunizing amount of the recombinant virus of claim 13 and a suitable carrier.

36. A method of immunizing chickens or other poultry against infectious laryngotracheitis which comprises administering to said chickens or other poultry an effective immunizing amount of the vaccine of claim 34. P \OPER\MRO\ 53690 9(%,nd I3.doc-9 May. 2(Mx) 178

37. A method of distinguishing chickens or other poultry which are vaccinated with the vaccine of claim 19 from those which are infected with a naturally-occurring infectious laryngotracheitis virus which comprises analysing samples of body fluids from chickens or other poultry for the presence of glycoprotein gG and at least one other antigen normally expressed in chicken or other poultry infected by a naturally-occurring infectious laryngotracheitis virus, the present of those antigens normally expressed in infected chickens but the absence of glycoprotein gG being indicative of vaccination with the vaccine of claim 19 and not infection with an naturally-occurring infectious laryngotracheitis virus.

38. A homology vector for producing a recombinant infectious laryngotracheitis virus by deleting DNA which encodes a screenable marker, which has been inserted into the infectious laryngotracheitis virus genomic DNA, which comprises a double stranded DNA molecule consisting essentially of a double-stranded DNA to be deleted, which is flanked on each side by a double stranded DNA homologous to the infectious laryngotracheitis virus glycoprotein gG gene, glycoprotein gI gene, US2 gene, or UL-47 like gene.

39. The recombinant infectious laryngotracheitis virus according to any one of claims 1 to 33 as hereinbefore described with reference to the figures and/or Examples.

40. The vaccine of claim 34 as hereinbefore described with reference to the figures o:o: and/or examples.

41. The multivalent vaccine of claim 35 as hereinbefore described with reference to the figures and/or examples.

42. The method of claim 36 as hereinbefore described with reference to the figures and/or examples.

43. The method of claim 37 as hereinbefore described with reference to the figures WV0 and/or examples. P OPEPLWRO\ 536j90 96a nld 1311do.-J Ma 2)XH)

179- 44. The homology vector of claim 38 as hereinbefore described with reference to the figures and/or examples. DATED this NIINTH day of MAY, 2000 SYNTRO CORPORATION by DAVIES COLLISON CAVE Patent Attorneys for the applicant(s) **j