AU6250500A - Recombinant infectious laryngotracheitis virus and uses thereof - Google Patents

Description

Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION DIVISIONAL APPLICATION

(ORIGINAL)

Name of Applicant: Actual Inventor(s): Address for Service: Invention Title: Syntro Corporation Martha A. WILD Mark D. COCHRAN DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, Victoria 3000.

"Recombinant infectious laryngotracheitis virus and uses thereof' The following statement is a full description of this invention, including the best method of performing it known to us: oP Australia Documents received on: OCT 2000 Batch No: Q:\OPER\JMS\2345585 279 DIVPROV.DOC 5/10/00 RECOMBINANT INFECTIOUS LARYNGOTRACHEITIS VIRUS AND USES

T-IEREOF

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.

10 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 birdsearly in an outbreak. In order to design a more efficacious, attenuated vaccine, the i 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 Herperoviridae, 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 *10 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 15 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 PCT/US96/03916 -3 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 b 5 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.

20 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 6 5 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 non- 10 essential 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.

15 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).

20 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 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 gl 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 S: 10 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 20 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 15 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 20 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 PCT/US96/03916 -7- SUMMARY OF THE INVENTION The present invention provides a recombinant. attenuated infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome 4 5 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 10 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.

*oooo 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 10 nucleotide sequences of Figures IA-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 15 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 20 and end within the terminal repeat sequence. The unique short region begins at base pair 2910.

Figure 2: Asp718 1 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 O 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 1 10 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 20 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, gl, 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 o-igin 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 PCT/US96/03916 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).

10 Figures .0 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 15 junctions between fragments are also shown (SEQ ID NO's: 24.

25, 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 20 is also given. Restriction sites in brackets indicate the oo. 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 PCT/US96/03916 -Il- -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 10 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, 20 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 PCT/US96/03916 -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 10 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), -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 20 579-14.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), -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 10 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 15 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-11C: 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 PCT/US96/03916 WO 96/29396 -14- Figure 12: remnants 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, Asp7181, Noll, 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 2F12 contains two non-contiguous sections. Three probes used to characterize the ILTV genome are indicated as PI, P2, and P3. P1 is a 0.9 kb Notl fragment found at the terminus of the unique long region, P2 is the 856 bp HindIII fragment found in multiple copies within the short repeat, and P3 Is a 6.6 kb Nodl fragment used to identify the fragments at the end of the terminal repeat.

The region sequenced. and the positions of the Asp7181, BamHl.

NolI. 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 Hindlll Nodl Asp7181 or BamHI was probed with an 856 bp HindlII fragment from the short repeat.

Positions of molecular weight markers are indicated.

Figure 13: Figure 14: WO 96/29396 PCT/US96/03916 Fieure 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 Notl Asp7181 and BamHl was probed with the 6.6 kb Notl 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: 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. VR 2427). The S-ILT-014 virus has been deposited pursuant to the Budapest Treaty on the International 10 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. VR 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 :20 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 PCT/US96/03916 -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 20 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 15 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 20 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.

15 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 Sprotease 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 PCTIUS96/03916 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 S: such antigenic polypeptide are: equine influenza virus type A/Alaska 91 neuraminidase and hemagglutinin, equine influenza virus type A/Prague 56 S1 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 20 virus, equine encephalitis virus, equine rhinovirus and equine rotavirus.

*o 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 5 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 gl gene, so that upon replication, the recombinant virus produces no glycoprotein gl.

20 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 PCT/IUS96/03916 -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 ORFIO 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 15 infectious laryngotracheitis virus of the present invention.

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

20 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 15 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 5 rotavirus, chick anemia agent. Salmonella spp., E. coli, Pasteurella spp..

15 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 20 larvneotracheitis 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 PCTUS96/03916 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 20 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 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 WO 96/29396 PCT/US96/03916 -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..

Bordeiella 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., 20 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 chickensor 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 are vaccinated with an effective immunizing amount of a recombinant infectious laryngotracheitis virus which produces no giycoprotein 15 gl 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 gl of the infectious laryngotracheitis virus and at least one other antigen normally expressed in chickens or other poultry infected by a naturally-occurring 20 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 gl 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 gl 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 gi 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 gl 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 gl.

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 20 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 gl gene. US2 gene.

or UL-47 like gene. Preferred embodiments of this invention are the homology WO 96129396 PCT1US96/03916 -29vectors designated Homology Vector 544-55.12. Homology Vector 562-61.1 F, Homology Vector 472-73.27. Homology Vector 560-52.171 and Homology Vector 579-14.G2.

This invention provides an isolated nucleic acid molecule encoding a US 1 0 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). ORES gene (SEQ ID NO: 66). gD gene (SEQ ID NO:67), gI 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 USIO gene see, (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), gi 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 :10 ml of viral stock containing 10'-106 pfu in 1X 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 1 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-HCI pH 7.5, 1 mM EDTA, and 1.5 mM MgCI 2 and were incubated for 15 minutes at 4*C. Twenty five pls 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 pl of 0.5 M EDTA. 50 l of SDS, and 25 gl 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 PCTIUS96/03916 -31the cell media supematants (see above). The mixture was then treated at for 1-16 hours, followed by two extractions with phenol saturated with 100 mM Tris-HC1, 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 pl TE/flask and could be used at this point for further 10 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 Sresuspended 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 4l of 10 mg/ml RNase A was added, and the preparation was centrifuged 5 minutes at 1000 x g. Twentyfive 4l of 20 SDS and 25 pil 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 0 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 41 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 MgC,, 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"S-dATP (New England Nuclear). Reactions using both the dGTP mixes and the dlTP 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 Ml 3 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.

S 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 Svariation.

SOUTHERN BLOTTING OF DNA. The general procedure for Southern blotting was taken from Maniatis et al. (1982) and Sambrook, et.al.(1989) (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 Genius T non-radioactive labeling kit from Boehringer-Mannheim. Hybridization was overnight at 65C. Filters were washed twice with 40 mM Na,HPO,, pH 7.2, 1 mM EDTA, 5% SDS and then twice with 40 mM Na 2 HPO,, 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 PCT/US96/03916 -33- Mannheim GeniusTM non-radioactive detection kit.

DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS. The method is based upon the CaCl, 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 10 equal volume of a buffer containing 50 mM MOPS (pH 6.95), 280 mM NaCI, and 1.5 mM NaHPO, was added to the DNA/CaCI, 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 ug of ILTV DNA (obtained from S-ILT-001) in 0.5 ml of WO 96/29396 PCTIUS96/03916 -34mM Tris-HCI 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-HCI 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 0 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 Hindll and Aval to remove the tetracycline gene, and the ends were filled in with Klenow polymerase (FILL IN REACTION). The polylinker from pWEIS (Stratagene) was ligated into this vector. The polylinker was isolated by digestion with EcoRl, 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, pSY 1005, was modified at the EcoRI site to create pSY1626 by blunt-ended insertion of a 1.5 kb HindlII-BamHl 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 3-galactosidase or 0-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 10 substrate X-Glucuro Chx (5-bromo-4-chloro-3-indolyl-p-D-glucuronic acid Cyclohexylanunonium salt, Biosynth AG) was used. Plaques that expressed active marker enzyme turned blue. The blue plaques were then picked onto S.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, pH 7 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 10 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 YM30 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-HCI, pH 7.0. Three ml fractions were collected and assayed by Western blot. A peptide antibody against ILTV g. G 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 39C. 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'-10' 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 MgCI, and incubated 15', 4'C.

To this was added 25 pl/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 15 supematant was added (per original flask) 10 pl of 0.5 M EDTA, 50 1l of S SDS, and 25 l 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 supematant. 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 pl 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-HC1, 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 NaCI 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°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 HindIll and AvaI 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 EcoRl site to create pSY1626 by blunt-ended insertion of a 1.5 kb HindII-BamHI fragment from pNEO (P-L Biochemicals) containing the kanamycin resistance gene. PSY1626 was cut and made blunt at the BamHl site for use as the cosmid vector. The ligation mixture was packaged using Gigapack XL (Stratagene) according to the o manufacturer's directions. Colonies were selected on LB plates containing kanamycin.

SEQUENCING. Manual sequencing was performed using "S-dATP (NEN) with the BRL Sequenase Kit which uses the dideoxyribonucleotide chain termination method described by Sanger el 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 10 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 HindIll fragment of pSP64/65 (Promega). Fragment 1 is an approximately 1087 base pair HindIll to Bcfl subfragment of the ILTV 2.4 kb HindIll 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 Bcll to HindlHI subfragment of the ILTV 2.4 kb Hindll 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 1 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 8/pSP19 fusion such that the multiple cloning site is EcoRI/Sac/IAsp718IISacI/EcoRI. Fragment 1 is an approximately 2300 base pair Asp7181 to DraI subfragment (SEQ ID NO 1: Nucl. 1-405) of the ILTV 2.5 kb Asp7181 fragment. Fragment 2 is an approximately 3039 base pair Xbal fragment containing the PRV gX promoter, the E. coli uidA gene. and the HSV-I 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 562-61.1F. 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 gl 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 PCTIUS96/03916 -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 Asp7181 to XbaI subfragment of the ILTV 8.0 kb Asp7181 fragment (SEQ ID NO 1: Nucl. 7556-9175).

Fragment 2 is an approximately 691 base pair Xbal to XhoI fragment (SEQ ID 10 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 gl 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 XbaI at the 5' end (a restriction enzyme site present in the ILTV DNA) and XhoI at the 20 3' end (a restriction enzyme site created in the PCR primer--see underlined sequence) to create an approximately 691 base pair XbaI to XhoI 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 HindIlI 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 gI 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 HindiIl 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 Hindlll 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 10 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 15 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 20 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 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 5017 base pair Sall to Sall fragment containing the HCMV IE promoter, E. coli P-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.F1 was constructed WO 96/29396 PCT/US96/03916 -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 10 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 0* 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 pSP 18/pSP 19 such that the multiple cloning site is EcoRI/Sacl/Asp7181ISacl/EcoRI. Fragment 1 is an approximately 1066 base 0* pair Asp7181 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 Bcl 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 Bcll 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 PCTYUS96/03916 -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 pUC 19 (Gibco, BRL) is derived from an approximately 2677 base pair Asp7181 10 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 Sall fragment containing the PRV gX promoter, E. coli p-glucuronidase (uidA) marker gene, and an HSV-1 TK polyadenylation site (See Figures 9A-9D).

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 *i expression cassete 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 I0A-10D. The plasmid vector pSP71 (Promega) is derived from an approximately 3066 base pair Xmal to Smal 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 Smal fragment of the plasmid pRAJ260 (Clonetech). Note that the EcoRl and Xmal sites were introduced at the locations indicated in Figures 10A-10D by PCR cloning.

Fragment 3 is an approximately 784 base pair XmaI subfragment of the HSV-1 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 11 A- 11D. It was constructed utilizing standard recombinant DNA techniques (42.

10 43) by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in Figures 11A- ID. The plasmid vector pSP72 (Promega) is derived from an approximately 3076 base pair Pstl to PstI .i fragment. Fragment 1 is a 1154 base pair Pstl to AvaII fragment derived from o* a HCMV 2.1 kb PstI 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 Ndel 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.

S..o WO 96/29396 PCT/US96/03916 -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 10 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 S" 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 S2" unique short region begins at base pair 274 of SEQUENCE ID NO. 1.

S"

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 Frame(ORF) _____Allignmenta I (RC)b 970 281 229 EHV-I US2 2 1059 2489 476 MDV PK L0 3 2575 4107 510 HSV-l UL47 4 4113 4445 110 NSc 4 (RC) 4519 4139 126 NS 5 4609 5487 292 PRV gX 6 5697 8654 985 ILTV 6 (RC) 7826 6948 292 HSV-2 UL39 7 8 462 9766 434 PRV 8 9874 10962 362 VZV gI 8 (RC) 11150 10617 177 NS 9 11159 12658 499 VZV gE 10 12665 13447 260 ;NS a.

a

S

a S. a 0* *5 a.

a a 0 *5 a 6SO S *Oaa Sequence allignmcnt scored to the Entrez Release 6.0 of Genbank Virus Database.

bRC=Reverse Complement.

NS=No score above 120 was found.

Other Abbreviations: EHV= Equine herpesvirus; MDV= Mareks disease virus; H-S V-1= Herpes Simplex virus 1I PRV= Pseudorabies virus; ILTV= Infectious laryngotracheitis virus, HSV-2= Herpes Simplex virus 2; VZV= Varicella-Zoster virus; BP= base pairs; aa= amino acids.

US2 vene 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)- I 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 10 herpesvirus(EHV)-1 and Pseudorabies virus (PRV), Varicella-Zoster virus (VZV), Simian varicella virus (SVV), and Herpes Simplex virus(HSV)-I and

S.

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 PCT/US96/03916 -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 SEHV-4 gG. Recombinant ILTV gG protein produced in a Swinepox virus 10 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 15 as a negative marker to distinguish vaccinated from infected animals.

The g60 gene has been identified as glycoprotein 60 (33, 53). The g60 gene 20 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).

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.

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 gl 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 g gene results in an attenuated ILT virus that is useful as a vaccine against ILT disease in chickens. Recombinant viruses deleted for g 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 96129396 PCTIUS96IO39I 6 PaE eaene 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 (SHy.), EHV-1, HSV-1. and PRy. The ILTV gE is a neutralizing antigen usefu as a subunit vaccine.

ORFIO

*:ORFI10 consists of 783 base pairs from nucleotide 12,665 to 13,447 and codes a protein 261 amino acids in length and molecular weight approximately 27,898 daltons (SEQ. ID. NO. 9).

WO 96/29396 PCTIUS96/03916 -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 3-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 10 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 15 transfection stock was screened by the Bluogal M 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 20 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 I 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 15 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) 20 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/US96I0391 6 -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.

0000.

0 0 **0 0 0.

.o WO 96/29396 WO 9629396PCTIUS96/03916 Table I] 00..

.0 0a...

.00.

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, 0 S S-ILT-009 gG. US2- 1.56x10 3 10 OS 77% Controls 05 0% ASL T0 05 90/0 embryo 14 day old chicks a: USDA Challenge virus =1.0x10 4 pfu b: Protection healthy birds/total c: intraocular d: Orbital Sinus WO 96/29396 PCT/US96/03916 -56- Example 4 S-ILT-01 1 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 gl gene and is under the control of the pseudorabies virus (PRV) gX promoter. The PRV gX 10 promoter-uidA gene is in the opposite orientation to the direction of transcription of the ILTV gI promoter.

S. 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 15 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 20 confirmed the presence of the 1-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 gI 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 gl 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 gl 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 p-glucuronidase (uidA) was inserted in the place of the gl gene and is 10 under the control of the pseudorabies virus (PRV) gX promoter.

S-ILT-013 was constructed using homology vector 562-61.1F (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- 15 Glue SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING a 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 20 deletion of approximately 983 base pairs of the ILTV gl 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 gl or gG genes, ILTV gI and gG may be utilized as negative markers to distinguish vaccinated animals from infected animals.

F--

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 P- 15 galactosidase (lacZ) gene in place of the ILTV gG gene. However, during the o Bluogal

T

M 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.

20 This virus. S-ILT-014. was characterized by restriction mapping, DNA SEQUENCING and the SOUTHERN BLOTTNG 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 96129396 PCT1US96I03916 -59- Protect chickens from ILT disease as indicated in the table below. Since S-ILT- 014 does not express the ILTV gG gene and ILTV-inifected 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.

0* *0*6 o 0 0 *00 WO 96/29396 WO 9629396PCT11US96103916 Table III o o o EFFICACY OF RECOMBINANT LIVE ILT VIRUS S-ILT-014 AGAINST VIRULENT INFECTIOUS LARYNOOTRACHEITIS

VIRUS

CHALLENGE

Vaccine Gene(s) Dose Route Challenge' Protection' IDeleted 10X0 S-ILT-0 14 gG- 1.8x 0 1 C d97% S-ILT-0 14 gG- 2.16x10 3 10 OS 97% Controls OS 0% ASL embryo _10 105 l~14 y ldci a: USDA Challenge virus =L.OX10 4 .5 pfU b: Protection healthy birds/total c: Intraocular d: Orbitual Sinus WO 96/29396 PCT/US96/03916 -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 P-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 10 is in the opposite orientation to the direction of transcription of the ILTV UL47-like, ORF4. and gG promoters.

e.

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 15 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 20 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 96129396 WO 9629396PCT1US96/03916 -62- Table IV EFFICACY OF RECOMBINANT LIVE ILT VIRUS S-ILT-015 AGAINST VIRULENT INFECTIOUS LARYNGOTRACHEITIS VIRUS

CHALLENGE

Vaccine Gene(s) Dose Route Cballengea Protection' Deleted 10 S-ILT-015 gG-. l.0X10 3 10C OS d UL47-like Controls OS 0% ASL 10 OS embryo 14 day old 4ChiCFa: USDA Challenge virus =1.Ox 10" 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 PCT/US96/03916 -64- Example 9 Recombinant infectious larvneotracheitis 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.

0 To construct a homology vector containing the foreign genes inserted into the 15 ILT virus, a DNA fragment containing the HCMV-IE promoter, the IBV Arkansas spike protein and the HSV-l 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 96129396 PCTIUS96IO39 16 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 UI vaccines described ~:here do not express ITV gG, it is useful as a negative marker to distinguish vaccinated animals from infected animals.

Example Vaccines utilizing ILTV to exnress antigens from various disease causing MiCTOorganusms: Antigens from the following microorganisms are utilized to develop poultry 20 vaccines: Chick anemia agent. Avian encephalomyclitis virus, Avian reovirus, Avian pararnyxovi ruses, Avian influenza virus ,Avian adenovirus, Fowl pox virus, Avian coronavirus, Av'ian rotavirus. Salmonella spp., E col., Pasteurella spp., Haemophilus spp.. Chlamvydia spp., Mycoplasma spp., Cainpylobacter spp..

Bordetella spp.. Poultry nemnatodes, cestodes. tremnatodes, Poultry mites/lice, Poultry protozoa (Eimeria spp.. Histomonas spp., Trichomonas spp.).

Example 11 A Genomic MapR of Infectious LAryngotracheitis Virus and the Sequence !ad Organization of Genes Present in the Unique Short Region A cosmid library of the ILTV genome was created to facilitate restriction WO 96/29396 PCTIUS96/03916 -66endonuclease mapping. Forty-three overlapping cosmids were analyzed by digestion with Asp7181 and Notl. Asp7181 was known to cut the genome relatively infrequently and it was found that Notd 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 (PI in Figure 12) to genomic ILTV digested with Asp7181, Notl, 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 15 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 20 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 Asp718l 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 BamH1. The Asp7181 2.5 and 8.0 kb fragments cross-hybridized, indicating that they contained sequence repeated in both clones. Fine mapping of the Asp7181 2.5 and 8.0 kb fragments showed them to contain 2.1 kb of identical sequence. Hybridization to ILTV digested with BamHI identified BamHl 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 PCT/US9603916 -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 Asp7181 fragments mentioned above and the unique long region identified in the cosmid map. A 10 kb Notl 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, Notl, and Asp7181 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 15 a repeat motif. HindIll, 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, Notl, 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 20 strain, and a ladder at very high molecular weight can be seen. Because the unique short is expected to invert, two overlapping Sfi ladders containing the unique short and terminal repeat should be present.; however, the bands are too large in this region to make this distinction. Noll and Asp7181 cut further away from the repeat, generating ladders beginning at 10.5 or 12 kb.

The Asp7181 digest should generate two overlapping ladders, because one fragment is bounded by an Asp7l181 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 (Noll) 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 10 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 15 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 20 Asp7181 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 Nodl 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 Notl fragment seen in gels stained with ethidium bromide was not represented in the restriction endonuclease map, and WO 96/29396 PCTIUS96/03916 -69considered that it might represent the end of the TR,. Hybridization of a Nol digest of ILTV with P3 indicated that this was indeed the case (Figure 16).

The 2.9 kb Notl 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.

10 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 15 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 20 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 PCT/US96/03916 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 20 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 PCTIUS96/03916 -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 15 also contain threonine rich repeats. The human mucin gene, for example, has the repeat GTOTPTTTITTTVTPTPTPT, 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 20 polyadenylation signal is at the end of the gl gene. This suggests that the ORF 5 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 el 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 20 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 gl. 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 10 addition signal just prior to the 3' end of the coding region. This may serve as the polyadenylation site for the gl 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 15 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 el 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 US 10 genes (SEQ ID NOs:60 and 72). A zinc finger motif, found WO 96/29396 PCT/US96/03916 -74in the EHV-4 US10. 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, SRORFI, 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 15 downstream from the ORF 9 gene. The PK gene itself has the most identity 9 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 SAS.

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 20 the unique gene. ORF 5. which contains a set of degenerate repeats.

a 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 15 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 20 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 So.. 15 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 et 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 BamHI 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 96129396 PCTIUS96/03916 -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 Asp7 181 bands identical to those present in genomic ILTV digests.

The exception. 2FI2, 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 ethidiuni 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 Baumnann el 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, genornic DNA from ILTV could vary by over I I 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 30 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 'OPERM.RO\ ,W K. a-c I 'lIdoc-' M.l Ztt -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 Spresented. In determining the genomic map, an 856 bp region repeated as many as 13 10 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, 'oo' 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 9629396PCTIUS96/039 16 -79- Table V ORF HSV Best Matches Blast Score Hornolog I US2 EHV1 EUSI IW1xO EHV4 EUSI 5.3xlO-" HSV2 US2 6.7xI10 2 PK MDV PK 8.2xlO"* HVT PK 5.4x HSVI PK 4.1x1' 3 UL47 HSV1 UL47 6..0x 10-1 EHV I UL47 9.9x 107 MDV UL47 9.9)X10 1 4 gG PRV gG 5.3xIO W BHVI gG 1.7x EH-VI gG 6.8x10' ORF 5 EHVl EUSS 1.9X1O0" Human inucin 1.1x10 2 WO 96/29396 WO 9629396PCTIUS96/03916 6 gD MDV gD 6.8x]0-' PRV g50 2.0x10*' HVT gD 3.Sx10*' 7 gI VZV gi 4.2x 10* 2 HYT gI 7.9x 10- 2 SVV gI 4.3x 8 gE SHV SA8 gE I.7xIO' HSV I gE 1.1X10' BHV1I gE 1.5X 10, 2 9 ORF 9 EBV BLRF2 5.7x SRI no HSV MDV '0RF3 4.8x homnologue HVT "ORF3 2.6x10-' SR2 US 10 EHV-4 USIO 1.2x10' HSV-1 USIO 8.7x10'1 EHV-1 USIO 8.7xl10' WO 96/29396 PCT1US96/03916 -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 0 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk 0@ COMPUTER: IBM PC compatible C OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: o' FILING DATE: 23-MAR-1995

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: oo APPLICATION NUMBER: US 08/126,597 FILING DATE: 24-SEP-1993 090000 0 (viii) ATTORNEY/AGENT INFORMATION: NAME: White, John P.

REGISTRATION NUMBER: 28,678 REFERENCE/DOCKET NUMBER: 39116-A *ooS 0 TELECOMMUNICATION

INFORMATION:

TELEPHONE: (212) 278-0400 TELEFAX: (212) 391-0525 INFORMATION FOR SEQ ID NO:1: 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 9629396PCr/US96/039 16 -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

AATTTTTACC

CCATGCATAT

CAGATGACAA

GGTCGCCCCC

CCCCGGAAAC

CGTAACTCGC

CAAATCTAGA

CAGGCGCCAT

TCTAGCAGAG

ACCCTCCAAA

GACAATAACA

ATTACTGCTA

CAGCGGCCGC

CTTCGACTTA

GGAPJACACAG

CCAGGGCGTT

CAGCGTTGTC

TCCGGTTTCG

GTCAGCGGTA

ATGGTAAAAC

CTCACCGAAC

TTCGTACAGA

CATAGTTTTC

ATTAGGTAGT

CGCCGCTCAG

TCAGGCCAAT

CGAGAAAGCT

TGCTACCCGC

CTTACTCATC

CCGCCCTTGT

CGACGCGGCG

AATAGACTCC

TATGCGOCCO

CTCAGGCTCG

TTGTCTCCGT

GTAATTAGGA

CCCAGCACCC

CCTGCGTCCA

CGAATCACGG

GATGCCGGGC

ACTGCGTGAA

TTGTAGTGCG

AAGTCCAAAT GTGACGTCQO AGGTCTCGAC CGGCCCACGC AA.CGCGGGCT TrTATAAAGA CGCGTACGCG TTTATTATTG TCAATATTTG TTCTGCAAGG CCCTCGCCGC GGCCCAGGCC AGCGTCGCCG CTAACGTCGG CGCCGCGGGG CGAGAAAAAA TTTTGGCTGC CGTTCGCCAT AGT1TAGGTTT TACTrTTTAAA AACTTTACCG AAGGGGCAAG AGTrGTCCAG ACTGATGGGT TTACCAAAAA TATTCCACT CCTCTCTCAA AAGTrrGG CGCAGGGAGG TTTAA.AGCTG ACAAATGGAA CTC71TTGCG GCATACGCGC AGCCGCTCCA CTCGGGACTT ACTCCAGGCG GTCTGCACAT CCTGG3GAAGG GAAAACAGCT GCACGATTAC CGATAATGTA CTCGGACGAT CCAArCTTr CCATCCAGAA TCCGAGAGCT AATCGACCGC JIQAAACTATA GTCACTTTTA 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1 0'73 1121 ACTCCACCCC GCTATGATGT CAGAMATATA ACGCTCTTAT ATATACAGCT TAGAGAAG ATG7 CGG TTT CGG CGC Met Arg Phe Arg Arg 1 ATC TGT TCA CGC 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 96/29396PCIS6096 PCTIUS96/03916

CCG

Pro

ATG

Met

GAG

Giu

CCT

Pro

AAC

Msn

AAA

Lys

GCC

Ala Trr Phe

CG

Gly 150

AGA

Arg

ATA

Ile

AAT

Msn

ATC

Ile crT Leu

TCA

Ser

CGC

Arg

GTG

Val

GGG

Giy

GTT

Val

GGC

Gly

CTI

Leu 135 .ITlT Phe

GGG

Gly

GCT

Ala

GAA

Giu

CAG

Gin 215 -89- ACC TCA AAA CGC OTT TGC OTA TTG OAT ACT TTC TCA CGG ACA Thx Ser Lys Axg Val Cys Vai Leu Asp Ser Phe Ser Arg Thr 30 TTG CGC CCC TAT CCA GAA ATT TTG CCG ACC GCG GAA GGC GTC Leu Arg Pro Tyr Ala Giu Ile Leu Pro Thr Ala Glu Gly Val 45 s0 CTC GCC GAA CTT CTT AGT GTG ACA ATG ACA GAA CGC GCG GAA Leu Ala Giu Leu Val Ser Val Thr Met Thr Giu Arg Ala Giu 60 ACA GAG AAT ACA GCT GTA AAC AGT ATC CCC CCG GCT AAC GAG Thr Glu Msn Thr Ala Vai Msn Ser Ile Pro Pro Ala Msn Glu 75 so CAG MAC TTC GCA TAT GCA GCC GAT GGG CCC TCC ACT ACT GAA Gin Msn Phe Ala Tyr Ala Cly Asp Gly Pro Ser Thr Thr Clu 90 95 100 GAC GGC TCG CAT ACA GAC TTC GAT GAA GCA TCG AGC GAC TAC Asp Gly Ser Hi.s Thr Asp Phe Asp Giu Ala Ser 5cr Asp Tyr ID5 110 115 CCT GTC CCG CTC GCG CAA ACT AGA TTG AAG CAT TCG GAT GMA Pro Val Pro Leu Ala Gin Thr Arg Leu Lys His Ser Asp Glu 120 125 130 CAG CAC TTC CGA OTT TTA GAC GAT TTG GTC GAG GOG OCT TAC Gin His Phe Arg Val Leu Asp Asp Leu Val Giu Gly Ala Tyr 140 145 ATC TGC CCC GTC CGT CCC TAC ACC GAG QAA GAG CAA CGT CGA Ile Cys Oly Val Arg Arg Tyr Thr Giu Glu Ciu Gin Arg Arg 155 160 165 GTT AAC AGT ACT MAC CAG GGG MAA TCA AAA TGT MRG CGC CTG Val Msn Ser Thr Asn Gin Cly Lys Ser Lys Cys Lys Arg Leu 170 175 180 MAA TAT CT3 AAA MAT GGA ACA AGG GC GCC TCT CAG CTG GMA Lys Tyr Val Lys Asn Gly Thr Arg Ala Al1a Ser Gin Leu Glu 185 190 195 ATT TTC GTT CTC GCC CCC CTA MAT CAC GAG MAT OTT CTC MAG Ile Leu Val Leu Cly Arg Leu Asn His Glu Asn Val Leu Lys 200 205 210 GAA ATC CT COG TAC CCG GAT MAT ACG TAC ATG TTA ACG CAG Glu Ilie Leu Arg Tyr Pro Asp Msn Thr Tyr Met Leu Thr Gin 220 225 1169 12 17 1265 1313 1361 1409 14 57 1505 1553 1601 1649 AGO TAT CAG TTC GAC TTO TAC Arg Tyr Gin Phe Asp, Leu Tyr 230 235 TGG AAA GAC AGT CCA ATG CTT Trp Lys Asp Ser Pro Met Leu 250 CTC ATO TCA C GTC TCC TAT Leu Met Ser Ala Val Ser Tyr 265 GAC ATC AAA CTC GAA MAT ATT Asp Ile Lys Leu Glu Msn Ie 280 AGC TAC ATG TAC GAT GMA GCG TTC GAC 5cr Tyr met Tyr Asp Giu Ala Phe Asp 240 245 MAA CAC ACT AGA CCC ATC ATG MAG CAG Lys Gin Thr Arg Axg Ile Met Lys Gin 25S 260 ATC CAT TCA AAG AAA CTG ATT CAC AG Ile His Ser Lys Lys Leu Ile His Arg 270 275 TTC TTA AAC TGC GAC 0CC MAG ACA GTG Phe Leu Msn Cys Asp Gly Lys Tliz Val 285 290 1745 1793 1841 1989 1937 WO 96129396 PCTIUS96IO39 16 CTG GCC GAC TTT GGA ACT GTC ACG CCT 'TTT CM A.AT GAG CGG GAG CCC 1985 Leu Gly Asp Phe Gly Thr Val Thr Pro Phe Giu Asn Giu Arg Giu Pro 295 300 305 TTC GMA 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 Giu Ile 310 315 320 325 CTC GCC AGG GAT TCG TAC TGT GAA ATT ACA GAC ATT TGG ACC TGC GGA 2081 Leu A.1a Arg Asp Ser Tyr Cys Glu Ile Th~r Asp Ile Trp Ser Cys Cly 330 335 340 GTA GTA TTG CTC GAA ATC OTA ACC CAT GAA TTT TGC CCG ATC GOC GAT 2129 Val Val Leu Leu Glu Met Val Ser His Giu Phe Cys Pro Ile Giy Asp 345 350 355 GGC CCC GGA MAT CCG CAC CAG CMA TTG CTG MAA GTT ATC GAC TCT CTC 2177 ly Cly Gly Amn Pro His Gin Gin Leu Leu Lys Val Ile Asp Ser Leu 360 365 370 TCA GTT TGT CAT GMA GAG TTC CCA CAC CCC CCG TOT MAT CTG TAC MAT 2225 :.*Ser Val Cys Asp Clu Giu Phe Pro Asp Pro Pro Cys Asn Leu Tyr Asn 375 380 385 .TAT TTG CAT TAT C AC ATC GAT CCC CC GGA CAT ACG GTC CCC TCG 2273 Tyr Leu His Tyr Ala Ser Ile Asp Arg Ala Gly His Thz Vai Pro Ser 390 395 400 405 CTC ATA CGC MAC CTC CAC CTT CCG GCC GAT CTG-GAA TAC CCT CTA GTT 2321 Leu Ile Arg Asn Leu His Leu Pro Ala Asp Val Giu Tyr Pro Leu Val 410 415 420 AAA ATO CTT ACT TTT GAC TGG COT TTG AGA CCC AGC GCG GCC GAA GTA 2369 Lys Met Leu Thr Phe Asp Trp Arg Leu Arg Pro Set Ala Ala Clu Vai 425 430 435 TTC GCA ATG CCA CTG TTT TCG GCT GMA GAG GAA COG ACC ATA ACA ATT 241*7 Leu Ala met Pro Leu Phe Ser Ala Giu Giu Giu Arg Thr Ile Thr Ile 440 445 450 ATT CAT GGA AMA CAT AAA CCC ATC CGA CCC GAA ATC CCT C CCC CTG 2465 Ile His Cly Lys His Lys Pro Ile Arg Pro Glu Ile Arg Ala Arg Vai 455 460 465 CCA CCC TCC ATG ACT GMA GCT TMATMTAAA OGACGGAGAT AGAGAACTGA 2516 Pro Arg Ser Met Set Giu Gly 470 475 AGCGTCAGAT TTrMTAAAA AAATAAATGA TCCAGAACTT ATGATTTGTC TTTCTTGA 2574 ATG ACC TTC CCC CAT CGA TTA ACG AMA AGA CCT TTC GC CGT CGA TTC 2622 Met Thr Leu Pro His Arg L.eu Thx Lys Arg Pro Phe Ala Arg Arg Phe 1 5 10 TGC TCG CTC TTT GC ATA CAT TAT ACT GAG ACT AAA CTC CAC CGA TAT 2670 Cys Ser Val Phe Val Ile His Tyr Ser Glu Thi Lys Leu Asp Arg Tyr 25 MAC MAG ACA ATO TIA CTC TAT AGA CCC GAC TCA ACC ATG CCC CAT AGC 2718 Asn Lys Thr met Lou Leu Tyr Arg Pro Asp Ser Thr Met Arg His Set 40 GGA GOC GAC GCA M.T CAC AGA GGG ATA AGG CCC AGG CGG AAA TCT ATT 2766 Gly Gly Asp Ala Asn His Arg Gly Ile Arg Pro Arg Arg Ly's Set Ile so 55 WO 96129396 PCTfUS96103916 -91- GGA GCG TTT AGC GCG CGC GAA AAG ACT GGA AAA CGA MAT GCG CTG ACG 2814 Gly Ala Phe Ser Ala Arg Giu Lys Thr Gly Lys Arg Asn Ala Leu Thr 70 75 GAA AGC AGC TCC TCC TCC GAC ATG CTA GAT CCG TTT TCC ACG GAT AAG 2862 Glu Ser Ser Ser Ser Ser Asp Met Leu Asp Pro Phe Sex- Thr Asp Lys 90 GAA TTT GGC GGT AAG TGG ACG GTA GAC GGA CCT GCC GAC ATT ACT GCC 2910 Glu Phe Gly Gly Lys Trp Thr Val Asp Gly Pro Ala Asp Ile Thr Ala 100 105 110 GAG GTC CTT TCT CAG GCA TGG GAC GTT CTC CAA ?TA GTO MCG CAT GMA 2959- Glu Val Leu Ser Gin Ala Trp Asp Val Leu Gin Leu Val Lys His Giu 115 120 125 GAT GCG GAG GAG GAG AGA GTG ACT TAT GAG TCC AMA CCG ACC CCG ATA 3006 Asp Ala Giu Giu Giu Arg Val Thr Tyr Giu Ser Lys Pro Thr Pro Ile 130 135 140 CAG CCC TTC MAT CC TGG CCG GAC GGG CCC ACT TOG MC GCG CAG GAT 3054 Gin Pro Phe Asn Ala Trp Pro Asp Cly Pro Ser Trp Asn Ala Gin Asp :::145 150 155 160 TTT ACT CCA C CCA ATA CTT TAT CCC TCT GCG GAG GTA TrG GAC OCA 3102 **Phe Thr Arg Ala Pro Ile Val Tyr Pro Ser Ala Ciii Val Lou Asp Ala 16S 170 175 GAG C T70 AMA OTA CCC OCA TTC OTT AGC CGA GTT TTA CAA TG;T GTA 3150 G9. lii Ala Lou Lys Val Giy Ala Phe Val Ser Arg Val Leu Gin Cys Val 190 195 190 .9.CCG TTC ACG CGA TCA MAG MAA AGC OTT ACG GTG CGG GAT GCG CAC TCG 3198 Pro Phe Thr Arg Ser Lys Lys Ser Val Thr Val Arg Asp Ala Gin Ser 195 200 205 TTr 770 GGG GAC TCC TTC TGG AGA ATA ATG CAG AAC GTT TAC ACG CTT 3246 Phe Leu Gly Asp Ser Phe Trp, Arg Ile Met Gin Asn Val Tyr Thr Va 1 210 215 220 :*:TGC TTA CGA C-AG CAC ATA ACT CCA CTC AGC CAC CCT TCC ACC A.AA ACC 3294 Cys Leu Arg Gin His Ile Thr Arg Leu Arg His Pro Scr Ser Lys Ser *225 230 235 240 ATT GTT MAC TOC MAC CAC CCT CTA TGG TAC CCC TAC CC MAT CMA 777 3342 Ile Val Asn Cys As n Asp Pro Leu Trp Tyr Ala Tyr Ala Asn Gin Phe 245 250 255 CAC 7CC AGA GGA ATG COC CTG CCC 7CC CTT AAA TTA CC TCT CCC CCC 3390 His Tx-p Arg Gly Met Arg Vai Pro Ser Leu Lys Leu Ala Ser Pro Pro 260 265 270 GAG GAG MAT ATT CMA CAC CCC CCA ATG CCC GCC CTT TTT AGA MAC GCG 3430 Clii Cli Asn le Gin His Cly Pro Met Ala Ala Val Phe Arg Asn Ala 275 290 295 GGG CCT GOT CTG TTC CTC TCG CCT CCC ATC CGC OCA GCC TTT GMA GAG 3496 Cly Ala Cly Leu Phe Lou Trp Pro Ala Met Arg Ala Ala Phe Glu Ciu 290 295 300 CCC CAC MCG CGA CTG TTA AGA GCA TCC CTC TOT TCA CTC GAT ATC ATC 3534 Arg Asp Lys Arg Leu Lou Arg Ala Cys Lou Ser Ser Lou Asp Ile Met 305 310 315 320 CAC OCA CCC GTC CTC CC TCG TTT CCA TTT TAC TGG CCC CCC GTC CAA 3582 Asp Ala Ala Val Leu Ala Ser Phe Pro Phe Tyr Tx-p Arg Cly Val Gin 325 330 335 WO 96/29396 WO 9629396PCTfUS96/o3q16 GAC ACC Asp Thr GCA CTA Ala Leu CAC GCA His Ala 370 TAT GAC Tyr Asp 385 GGA GCC *.Gly Ala CTG GCG ::Leu Ala .*TCC GTO **.Ser Val SAA.A GCG Lys Ala 450 GAG TTT Giu Pkie 465 AAT TTC *Asn Phe CAT TTT His Phe

TCO

Ser

GTG

Val 355

CTG

Leu

GAA

Glu

GAG

Giu

GTG

Val

GCA

Ala 435

AGG

Arg

TTC

Phe

TGT

Cys

TGT

Cys -92- CGC TTC GAG CCT GCC CTG GGC TGT TTG TCA GAG TAC TTT Arg Phe Giu Pro Ala Leu Gly Cys Leu Ser Giu Tyr Phe 340 345 350 GTC TTA CTG GCC GAG ACG OTC TTA GCG ACC ATG TTC GAC Val. Leu Leu Ala Glu Thr Val Leu Ala Thr Met Phe Asp 360 365 GTA TTC ATG AGO GCG CTG GCA GAC GGC AAT TTC OAT GAC Val. Phe Met Arg Ala Leu Ala Asp Gly Msn Phe Asp Asp 375 380 ACT AGA TAT ATA GAC CCC OTT AAA AAC GAG TAC CTG AAC Thr Arg Tyr Ile Asp Pro Val Lys Asn Glu Tyr Leu Asn 390 395 400 GOT ACT CTG TTA CGG GGC ATA GTG GCC TCC AAC ACC GCT Gly Thz Leu Leu Arg Gly Ile Val Ala Ser Msn Thr Ala 405 410. 415 GTT TGC GCA AAC ACC TAT TCG ACG ATA ACGA AMA CTC CCG Val. Cys Ala Asn Tbx Tyr Ser Thr Ile Arg Lys Leu Pro 420 425 430 ACT AGC GCG TGC APT GTT GCC TAC AGG ACC GMA ACG CTG Thr Ser Ala Cys Msn Val Ala Tyr Arg Thr Giu Thr Leu 440 445 CC3C CCT GGC ATG AGC GAC ATA TAC CGG ATA TTA CMA A Arg Pro Gly*Met Ser Asp Ile Tyr Arg Ile Leu Gin LyB 455 460 TTT TAC AT1' GCC TGG CTC CAG AGG GTT GCA ACA CAC GCA Phe Tyr Ile Ala Trp Leu Gin Arg Val. Ala Thr His Ala 4'70 475 480 TTA MAC ATT CTG MAG AGA AGC GTG GAT ACG GOC CCC CGC Leu Msn Ile Leu Lys Arg Ser Val Asp Thr Gly Pro Arg 485 490 495 TCA GOO CCA OCT CGO AGA AGC GGC TGC AGC ACT TAAMTAAA Ser Gly Pro Ala Arg Arg Ser Gly Cys Ser Ser 500 505 510 3630 3678 3726 3774 3822 3870 3918 3966 4014 4062 4112 4160 4206 4256 4304 4352 4400 ATG CTC TOC Met 1

GCC

Al a

GCT

Ala

TCG

Ser

GTG

Val.

GCT

Ala Leu

ATG

Met

ATT

Ile

GAC

Asp so

ATA

Ile

GGG

Gly Cys

GG

Gly

TCC

Ser

CAT

His

GAC

Asp Lys CCC CTT CTC GTG CCC ATT CAA TAT GAA GAC TTT TCG MAG Pro Leu Leu Val Pro Ile Gin Tyr Giu Asp Phe Ser Lys 5 10 TCT GAG CTC MAG AGO GMA MO TTA GAG ACA TTC OTT AMA Ser Giu Leu Lys Arg Glu Lys Leu Glu Thr Phe Val Lys 25 AGC GAC AGO GAC CCC AGO GOG TCC TTA AGA TTT CTC ATT Ser Asp Arg Asp Pro Arg Gly ser Leu Arg Phe Leu Ile 40 GCA AGO GMA AT!' AT!' CCA GAC GGA GTA COG TTT MAG CCG Ala Arg iu Ile Ile Ala Asp Gly Val Arg Phe Lys Pro 55 GAG CCG OTT COO OCT TCA OTT GCG CTO ACT ACC GCT GCC Giu Pro Val Arg Ala Ser Val Ala Leu Ser Thr Ala Ala 70 75 s0 GTG AMA GCO CGA CGC TTA ACC TCA GTT CGC GCG CCC GTA Val Lys Ala Arg Arg Leu Thr Ser Vai Arg Ala Pro Val 90 WO 96129396 PC71US96/03916 -93- CCG CCC GCA GGC GCC GTT TCC GC CCC CGG AAA TCG GAA ATA TGA TA 4447 Pro Pro Ala Gly Ala Val Ser Ala Arg Arg Lys Ser Glu Ile 100 1.05 110 AAAATCCTTG GCATTTGCGG GCGAAGAGCC GTGATCTGAA GGGCTCCACA ATGACGTAAC 4507 TGAGCTACGC ATCCCTATAA AGTGTACSCG CTGACCGCTA GCCCATACAG TGTTACAGGA 4567 GGGGAGAGAG ACAACTTCAG CTCGAAGTCT GAAGAGACAT C ATG AGC GGC 4617 Met Ser Gly 1 TTC ACT AAC ATA CGA TCG ATT GCC ACC GTT TCC CTA GTA TGC TCCG CTT 4665 Phe Ser Asn Ile Cly Ser Ile Ala Thr Val Ser Leu Val Cys Ser Leu 10 TTG TCC GCA TCT GTA TTA GGG C CCG GTA CTG GAC GGG CTC GAG TCG 4713 Leu Cys Ala Ser Val Leu Gly Ala Pro Val Leu Asp Gly Leu Glu Ser **20 25s 30 AGC CCT TTC CCG TTC GGG GCC AAA ATT ATA CCC CAG GCC TGC AAC CCC 4761.

Ser Pro Phe Pro Phe Gly Gly Lys Ile Ile Ala Gin Ala CI's Asfl Arg 45 :ACC ACC ATT GAG GTG ACG GTC CCC TGG AGC GAC TAC TCT GCT CCC ACC 4809 Thr Thr Ile Glu Val Thr Val Pro Trp Ser Asp Tyr Scr Cly Arg Thr 6 GAA GCA GTO TCA GTC GAG GTG AAA TCC TTC TAC GGG AAT ACT AAT CCC 4857 Glu Cly Val Ser Val Glu Val Lys Trp Phe Tyr Gly Ass Ser Asn Pro 75 so CAA AGC TTC GTC TTC GGG CTC CAT ACC CAA ACC GGC AGT GGA CAC GAC 4905 Clu Scr Phe Val Phe Cly Val Asp 5cr Ciu Thr Gly 5cr Gly His Clu 90 GAC CTC TCT ACG TGC TGG G-T CTA ATC CAT AAT CTC AAC GCG TCT GTG 4953 Asp Lou ser Thr CI's Trp Ala Leu Ile Hi.s Asn Leu Asn Ala Ser Val 100 105 110 115 TGC AGG CC TC-T CAC GCC CCC ATA CCT CAT TTC GAC AAG CAC TGC GAA 5001 Cys Arg Ala Ser Asp Ala Cly Ile Pro Asp Phe Asp Lys Gin Cys Clu 120 1.25 130 AAA CTG CAG AGA AGA MT CGC 7CC CCC GTG GAA CTT GGT ACT TAC GTG 5049 Lys Val Gin Arg Arg Leu. Arg 5cr Giy Val Ciu Leu Cly Ser Tyr Val 135 140 145 TCT CCC AAT GGA TCC CTG GTG CTG TAC CCA CCC ATC TAC GAT CCC CCC 5097 Ser Cly Asn Cly Ser Leu Vai Leu Tyr Pro Gly Met TyrX Asp Ala Cly IS0 2.55 160 ATC.TAC GCC TAC CAG CTC TCA GTG CCT CCC AAG GGA TAT ACC CCC TCT 5145 Ile Tyr Ala Tyr Gin Leu Ser Val Cly Cly Lys Gly Tyr 7hz Gly Ser 165 170 175 CTT TAT CTA GAC GTC GGA CCA XAC CCC GGA TGC CAC GAC CAG TAT GGG 5193 Val Tyr Leu Asp Val Cly Pro Asn Pro Giy Cys His Asp Gin Tyr Gly 180 195 190 195 TAC ACC TAT TAC ACC CTC CCC GAC GAG C TCA GAC TTA TCA TCT TAT 5241.

Tyr Thr Tyr Tyr Ser Lou Ala Asp Glu Al cSr Asp Leu Ser SrTyr GAC GTA CCC TCC CCC GAA CTC GAC OCT CCT ATG GAG GAA GAT TAT TCC 5289 Asp Val Ala Ser Pro Giu Leu Asp Cly Pro Met Clu Clu Asp Tyr Ser 215 220 225 WO 96/29396 WO 9629396PCTIUS961O39 16 -94- AAT TGT CTA GAC ATG CCC CCG CTA CGC CCA TGG ACA ACC GTr 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 G2.u 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 Glu Cys Ala Gly Pro Leu Asp Giu Tyr Val Asp Glu 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 Gln 290 295 290 CAG TAGCCACCCG AGAGTGTTTT TTGTGAGCGC CCACGCAACA TACCTAACTG Gin CTTCATTiTCT GATCAATTAT TGCGTATTGA ATAAATAAAC AGTACAAAAG CATCAGGTGT GGTTTGCGTG TCTGTGCTAA ACCATGGCGT GTGCGGGTGA AACCGTAAAT TACGTGATAA TAAATAGCAT ACGAGTTGGC GTGCAGCGTA TTTCGCCGAG AG ATO GGG ACA ATG Met Gly Thr Met 00

S..

.*Io* 000.

Leu

ACC

Thr

CAA

Gin

ACA

Thr

ACC

Thr

CTG

Leu

CTG

Leu

GGG

Gly

ACT

Ser

GTG

Val

GCC

Gly

GAG

Glu

TAT

CCIT

Pro

GAG

Glu

TCA

Ser

GTA

Val

CG

Arg

TTG

Leu

AGA

Arg

AAC

Asn

CCC

Pro

ACT

Thr

CC

Arg

TAT

Tyr

TTC

Phe

GTC

Val 135 CGC CT? Arg Leu TTC TGC Phe Cys CTS C Leu Ala 40 CCC CCT Pro Pro TTC CC Lou Arg COG ACG Cly ?hr CGC CCG Arg Pro 105 GCT ACT Ala Thr 120 I""A CT? Lou Val TTC CTA CT? CCA OTA GCG GAC GCC C TTG CCG Phe Lou Leu Ala Val Ala Asp Ala Ala Leu Pro 10 15 CGA CT? TGG AAG G;TG CCT CCG OGA GQA ACC ATC Arg Val Tx-p Lys Val Pro Pro Gly Gly Thr le 30 GTG CTC GCG CAA TCG CCG GTC ACG GGA CAC C Val Leu Ala Giu Ser Pro Val Thr Gly His Ala 45 GAA CCC CCC GTC ACC =r CAG AT? TTT GCG GAC Giu Gly Ala Val Ser Phe Gin Ile Phe Ala Asp 60 AT? CGC TAC CCC CC? ACG GAG GAC GAA CTT OCA Ile Arg Tyr Gly Pro Thr Ciu Asp Giu Leu Ala 75 s0 T,-C CC TCA CAC GCG GAC AAC GTG ACA TTT TCG Scr Ala Ser Asp Al1a Asp Asn Val Thr Phe Ser 90 95 100 CGC CCA GAA ATT CAC GGA GCA TAC TTC ACC ATA Arg Pro Clu Ile His Gly Ala Tyr Phe Thr Ile 110 115 GCC CAG AGC AZ-G GAA AGC ACC TAT TCG GTC ATC Cly Gin Ser Tnr Giu Ser Ser Tyr Ser Val Ile 125 130 AAC GCC TCT CTG CAA CGG TCC GTG CCC CTG GA) Asn Ala Ser Lou Giu Arg Ser Val Arg Lou Giu 140 145 5337 5385 5433 5481 5534 5594 5654 5708 5756 5804 5852 5900 5948 5996 6044 6092 6140 6188 ACG CCC Thr Pro 150 TCC CAT GAA AAT TTT TTG CAG AAC GAG CCT ACA TCC CCC TCG Cys Asp Giu Asn Phe Leu Gin Asn Glu Pro Thr Trp Gly Ser 155 160 WO 96129396 WO 9629396PCTIUS96I03916 COT TOO TTA GGC CCC Arg Trp ILeu Gly Pro CCG TCG CCT TAT GTG CGA GAT AAC GAT GTC Pro Ser Pro Tyr Val Arg Asp Asn Asp Val 175 180 9 *9

GCC

Ala

GCG

Ala

CCT

Pro

TCG

Ser

AGO

Arg 245

GGC

Gly

GGC

Gly

GCO

Ala

ACT

Thr

GCG

Ala 325

ACT

Thr

TCC

Ser

CCG

Pro

GGA

Gly

TCG

Ser 405

GTG

Val

GCC

Ala

AAA

Lys

CGC

Arg, 230

AAC

Asn

CG

Arg

AGC

Ser Ile

GAA

Glu 310

CTC

Leu

CTG

Leu

CCG

Pro

CGC

Arg

TCA

Ser 390

ACA

Thr

TTG

I.eu

CAG

Gin

AGA

Arg 215

ATA

Ile

OTT

Val

GCC

Ala

OTC

Val

CAA

Gin 295

ACG

Thr

GGG

Giy

CCT

Pro

ACC

Thr

AGC

Ser 375

GAA

Giu

ACT

Thr ACA AAA Thr Lys 185 ACG GG Thr Gly 200 ATA GTA Ile Val ACG OTA Thr Val TCT GAC Ser Asp ATG ATA Met Ilie 265 CTC GCG Leu Ala 280 CTG CAC Leu His ACC TT-C Thr Phe AGC MAT Ser Asn CCG TTC Pro Phe 345 CCC ACT Pro Thr 360 ACT AGC Thr Ser ACT CTr Thr Leu~ CCT CTT Pro Leu GCG CAG TAC ATT GGG GAG TGC TAC TCC AAC TCG Ala Gin Tyr Ile Gly Giu Cys Tyr Ser Asn Ser 190 195 CTC ACO TCT CTC AAC ATG ACC TTT TTC TAT TCG Leu Thr Scr Leu Asn Met Thr Phe Phe Tyr Scr 205 '210O AAC OTC ACG TGG ACA ACC GOC GGC! CCC TCC CCC Asn Val Thr Trp Thr Thr Gly Gly Pro Ser Pro 220 225 TAC TCG TCG COG GAG AAC GGG CAG CCC GTG TTO Tyr Ser Ser Arg Glu Asn Gly Gin Pro Val Leu 235 240 GGG TTC TTG GTT MAG TAC ACT CCC GAC ATT GAC Gly Phe Leu Val Lys Tyr Thr Pro Asp Ile Asp 250 255 260 MAC OTT ATT GCC MAT TAT TCG CCOG CG GAC TCC Asn Val Ile Ala Asn Tyr Ser Pro Ala Asp Ser 270 275 TITT ACG GCC TTT AGO GAA GGA AAA CTC CCA TCC Phe Thr Ala Phe Arg Giu Giy Lys Lou Pro Ser 285 290 COO ATA CAT ATC TCC GGG ACT GAG CCG CCG GGG Arg Ile Asp Met Ser Gly Thr Giu Pro Pro Cly 300 305 GAC TGT CMA AMA ATG ATA GM6 ACC CCG TAC CGA Asp Cys Gin Lys Met Ile Glu Thr Pro Tyr Axg 315 320 GTT CCC AGG GAC GAC TCT ATC CGT CCG GGG CC Val Pro Arg Asp Asp 5cr Ile Arg Pro Giy Ala 330 335 340 GAT ACC GCA GCA CCT GAT TTC GAT ACA GOT ACT Asp Thr Ala Ala Pro Asp Phe Asp Thr Gly Thr 350 355 ACC GTG CCA GAG CCA GCC ATT ACT ACA CTC ATA Tkir Val Pro Giu Pro Ala Ile Thr Thr Leu Ile 365 370 GAT .ATG GGA CTC TTC TCC ACG GCA COT GCT ACC Asp Met Gly Phe Phe Ser Thr Ala Arg Ala Thr 390 385 TCG OTA CCC CTC CAG CMA ACG GAT AGA ACT CT Ser Val Pro Val Gin GIlu Thr Asp Arg Thr Lou 395 400 ACC CTT CCA CTG ACT CCC GGT GAG TCA GA AMT Thr Leu Pro Leu Thr Pro Giy Giu 5cr Giu Asn 410 415 420 6236 6284 6332 6380 6428 6476 6524 6572 6620 6668 6716 6764 6812 6860 6908 6956 7004 ACA CTG TTT Thr Leu Phe CCT ACG ACC C CCG COG ATT TCT ACC GAG ACC CCG AGC Pro Thr Thr Ala Pro Gly Ile Ser Thr Giu Thr Pro Ser 425 430 435 WO 96129396 WO 9629396PCTfIUS96/03916 00*0 0: 000.

GCG

Al a

ACT

Thr

GAA

Glu

ACT

Thr 485

AGT

Ser

GCA

Ala

CGT

Arg

CTT

Val

CCC

Pro 565

ATT

Ile

TCG

Ser Ph.

ACT

Ser CC'r Pro 645

AGC

Ser

GCA

Ala

CAG

Gin

CCG

Pro 470

CAG

Gin

GCT

Ala

CCT

Pro

AGC

Ser

TTT

Ph.

550

AGC

Ser Ph.

ACT

Se r TrC Ph.

ACA

Thr 630

TTC

Ph.

C

Ala

CAT

His

ACT

Ser 455

TOG

Trp

ACG

Thr

GAA

Clu

CC

Al a

ACG

Thr 535

ACA

Thr

ACG

Thr

ACT

Thr

CC

Ala

ACT

Thr 615

GAG

Glu

ACC

Thr

GCA

Al a

GAA

Ciu 440

CCC

Pro

TAT

Tyr

CAG

Gin

CAG

Gin

CAC

G1 n 520

CCC

Pro

CAG

Gin

OTA

Val

CGG

Arg

GAG

Clii 600

CAG

Gin

CCC

Pro

CCC

Arg

CCC

Pro ACT ACA Thr Thr ACT ACC Ser Thr TTT ACT CAG ACT Ph. Thr Gin 475 ATC GCA CAA Ile Ala Giu 490 ATG ACT TTT.

Met Thr Ph.

505 ACC CCC AC Thr Pro Ser CCC CAA ACC Pro Giu Thr ACT TCC ACT Ser Ser Ser 555 CCC AAA ACT Pro Lys Thr 570 ACT CAG AGC Thr Gin Ser 585 CCC GAC ACT Pro Asp Thr CCC CCC ACT Ala Pro Ser GAG CTC TTGr Ciu Val Leu 635 Thr

ACG

Thr

ACT

Thr ,hr

GCT

Ala 540

ACC

Thr

ACT

Thr

C

Ala

ATO

Met

ACC

Thr 620

ACT

Thr CCG AGT ACT GAA CAG C OCT CTT Pro Ser Thr Giu Gin Ala Ala Leu 480 GAG CC TTG TTT ACT CAG ACT CCC Glu Ala Leu Ph, Thr Gin Thr Pro 495 Soo CAC ACT CCC COT CCA GAA ACC GAG Gin Thr Pro Cly Ala Giu Thr Clu 510 515 ATA CCC GAG ATA TTT ACT CAG TCT Ile Pro Glu Ile Phe Thr Gin Ser 523 530 CCC GCT CCC AGC GCG CC CCC GAG Arg Ala Pro Ser Ala Ala Pro Giu 545 CTA ACG GAG GTG ACT CAG ACC Val Thr Ciu Val Ph. Thr Gin Thr 560 CTC AGT TCG AGT ACT GAA CCC C Lieu Scr Ser Ser Thr Giu Pro Ala 575 580 GGA ACT GAG CCC TTT ACT CAG ACT Cly Thr Clu Ala Ph. Thr Gin Thr 590 595 CCA ACT CAG ACT ACT OAA ACA CAC Arg Thr Gin Ser Thr Ciu Thr His 605 610 CTA CCC AAA CCT ACT GAG ACT CCC Val Pro Lys Ala Thr Gin Thr Pro 625 GAG ACT CCC ACT ACC GAA CCT GTC Gin Ser Pro Ser Tkir Giu Pro Val 640 -96- CAG ACC GAG ACT GCA GAA ACG GTC GTC TTT Gin Thr Gin Ser Ala Giu Thr Vai Val Phe 445 450 GAG TCG GAA ACC GC CGG TCC CAG ACT CAG Giu Ser Ciu Thr Ala Arg Ser Gin Ser Gin 460 465 7052 7100 7148 7196 7244 7292 7340 7388 7436 7484 7532 7580 7628 '7676 '7724 7772 '7820

ACT

Thr

GAG

Giu 665 CTG CCC GCA GAG CCC GAA ATT ACT CAG ACC CCC Leu Cly Ala Ciu Pro Ciu Ile Thr Gin Thr Pro 650 655 660 GT!' TAT ACT CCC ACT TCC ACT ACC ATC CCA GAA Val Tyr Thr Arg Ser Ser Ser Thr Met Pro Ciu 670 675 CCC CTG CCC TCC CAA AAC CCT ACC ACT TCG GGA Pro Leu Ala Ser Gin Asn Pro Thr Ser Ser Gly 695 690 ACT OAA CCC AGG ACT TAT CCA GTC CAA ACG ACA Thr Giu Pro Arg Thr Tyr Pro Vai Gin Thr Thr 700 705 ACT GCA CAG AGC ACA Thr Ala Cmn Ser Thr 680 ACC GGC ACO CAT AAT Thr Cly Thr His Asn 695 WO 96/29396 PTU9131 PCTIUS96JO3916 -97- CCA CAT Pro His 710 ACC CAG AAA CTC Thr Gin Lys Leu TAC ACA Tyr Thr 715 GAA AAT AAG ACT TTA TCC TTT CCT Giu Asn Lys Thr Leu Ser Phe Pro 720

ACT

Thr 725 GTT GTT TCA Val Val Ser GAA TTC Giu Phe 730 CCC CTA TTG GAC GTC AAA Pro Leu Leu Asp Val Lys 745 GAA GTA ACG GCG ACT TGC Glu Val Thr Ala Thr Cys CAT GAG ATG TCG ACG GCA GAG TCG CAG ACG His Giu Met Ser Thr Ala Glu Ser Gin Thr 735 740 AT? GTA GAG CTG AAG TTT TCA MAC GAT GGC Ile Val Giu Val Lys Phe Ser Asn Asp Gly 750 755 GTT TCC ACC GTC AAA TCT CCC TAT AGG GTA Val Ser Thr Val Lys Ser Pro Tyr Arg Val 765 770 a a a. a a a a GAA ACT MAT TGG AMA GTA CAC CTC GTA GAT GTA ATG GAT GMA ATT TCT Giu Thr Asn Trp Lys Val Asp Leu Val Asp Vai Met Asp Giu Ile Ser 775 780 785 GGG MAC Gly An 790 ACT CCC CCC GGG Ser Pro Ala Gly GTT TTT Val Phe 795 MAC AGT MAT GAG AMA TGG CAG AMA Asn Ser Asn Glu Lys Trp Gin Lys S00 CTG TAC TAC Leu Tyr Tyr AGA GTA Arg Val 810 ACG AC Thr Ser 825 ACC GAT OGA AGA ACA TCG GTC CAG CTA ATG Thr Asp Gly Arg Thr Ser Val Gin Leu met 815 820 TGC CTG TCC TGC Cys Leu Ser Cys CAT TC? CCG His Ser Pro ACG TCT CTT Thr Ser-Leu ACC TCT CAT Thr Ser Asp CC AGG GMA Ala Arg Giu AA CAT Lys Asp 845 TAC TGC Tyr Cys 860 GAM CCT TAC TGT CT? TTC CAC Glu Pro Tyr Cys Leu Phe Asp 830 835 ATC GCG CCA GAG TTA TAC TTT Ile Ala Pro Giu Leu Tyr Phe 850 ACA ATA ACT CTG CCG TCC GGC Thr Ile Thr Leu Pro Ser Gly 865 7869 7916 7964 8012 8060 8108 8156 8204 8252 8300 8348 8396 8444 8492 8540 8588 8636 CCG CAA ACG GCA Pro Gin Thr Ala a.

a a GTT GT? Val Val 970 CCC AGA T= GMA TGG Pro Arg Phe Clu Tro 9-75 AGC CTT MAT rSer Leu An MAT GTT TCA CTG CCG GMA Asn Val Set, Leu Pro Giu 890

TAT

Tyr ass TTG ACG GCC ACC Leu Thr Ala Thr ACC CTT Thr Val 890 CT? TCG CAT ACC OCT GGC CAA AC? ACA Val Ser His Thr Ala Gly Gin Ser Thr 895 900 CTG TGG MCG AGC Val Trp Lys Ser GCG AGA GCA GGC GAG GCG TGG ATT TC? CCC CGG Ala Arg Ala Gly Giu Ala Trp Ile Ser Gly Arg 910 915 GGA GGC MAT Gly Gly An GTT AC? ACG Val Thr Thr 93S

ATA

Ile 920 TAC GMA TGC Tyr Giu Cys CCA MAG GAG AGG Arg Lys Ciu Arg A.CC CTC CTC ATC TCA GAC GGC AC? CC Thr Val. Leu Ile Ser Asp Gly Thr Arg 925 930 TGC TTA ACA MAC ACA TGG ATT C GTG Cys Leu Thr Asn Thr Trp Ile Ala Val 940 945 CAG CTG TAT TCA CTC TTT TC? GGA CTT Gin Leu Tyr Ser Leu Phe Ser Giy Leu 960 GMA MC Giu An 950 GGT GC GC Gly Ala Ala CAG GCG Gin Ala 955 GTG TCA GGA TTA TGC GGG AGC ATA TCT GC TTG TAC GCA ACG CTA TGG Val Ser Cly Leu Cys Cly Scr Ile Ser Ala Leu Tyr Ala Thr Leu Trp 965 970 975 980 WO 96/29396 WO 9629396PCT1US96/03916 -98- ACC GCC ATT TAT TTT TGAOGAATGC TTTTTOGACT ATCGTACTGC TTTCTTCCTT Thr Ala Ile Tyr Phe 985 CGCTAOCCAG AOCACCGCCG CGCGCTAACC ATACCGGCGG CTGCGACGTT GTCGAGCTCA AGAAAAAGAG AAGGGGGGCC CGACGACGQC GAGOACAAGT CGTACAACTO CTATCTGAAT TCCTAGCACC CTTGTATCGC GCTCTCTGGC CAATACTTGC AACTCTAGAA GTTAACGATC GAAATGCTGG ACAACAGAAC CGCAGACACC AATACACGAC GCGCTOGAAT AALTTTGCTGA CGTCCCGCAk GAAATTCCCG AAGCAGCGAA AAGAAGG3CCC TGGAGAAAAT CCTOCCGCCC TGATCCAAAC TCGGATCCTO GACTACTAAA AGTTCTAATG G3GTCGCGCTC GTGGGGCTAC CCTAGAATAG GTGGTTTC-T

CCGTCACGTA

TTGGCCCGTA

ACCCGATTTC

CTTTCGAGGC CTCCGTCGTC ACTGTCCAAT CTATAGAAAA GCGCCGTTCA ATCTGCACAG GAAATGGCGC GGGACTGACT TGACCCTGAA AATCGGGAGA

TGGTTCTACG

GAGTACAGGG

ATGTGGGCAG

ATATTCTCCC

TTTGCGCAAA

CAG=TAACT

GGCGAACACC

GGATACGAAG

CCAGACCCTC

COACTACAT? TTAGGCCGTC TAACAGATAC CTCACTAGGG TAACGTGGAC GACATGATAT

GCTGTTAAA

AGTATCAGAC

ACGCGGACGA

GGAAAAAGAA

CTGTAACCAA

CTCCAGAAGA

TCCCCGA.AGA

ACTATTACAA

GATCGGGTCG

TGGA'ITTCAA

CGTATATCOG

TCCTAGCGCG

GCGCGCTCGA

TATCAAGAGG

CGOCGGCCAA

TGATTAAGGG

AATGTdGCGA

TGGACTATGT

CCACTGCTGC

CAGCTCTCGT

TTTTACCGTC

TTTATCCGAT

ATATTCTGCA

GTCCAGATAG

CGGACGCAGA

CAGAGGCTTC

CCGAGCACGA

CGCTGGAGGA

TAGCCTGCGC

GCTAATCGAG

GAAAGCGGAA GGGCGCACCC CTCOGAGGAC GACATGCAGG CGACGAAGTC CCCGAGGACA TGACATGCCC GCCGTGATCC 8691 8751 88l1 8871 8931 8591 9051 9111 9171 9231 9291 9351 9411 9471 9531 9591 9651 9711 9771 9831 9873 9894 9942 9990 10038 100866 10134 CCGTCTCCAT GCCCATATT- TGGTTTGGAG CATCGTAAAA

GCGGCGTTCG

TGCGCGCG;TA

CCTACATGCC ACGCCTCACG CTCATAATAT AAATCACATG GAATAGCATA CCAATGCCTA TTCATTGGGA CGTTCGAAAA GC ATG OCA TCG CTA C1-r OGA ACT Met Ala Ser Leu Leu Gly Thr 1 CTG OCT CTC CTT GCC GCG ACG CTC GCA CCC TTC GGC GCG ATG OGA ATC Leu Ala Leu Leu Ala Ala Thr Leu Ala Pro Phe Giy Ala Met Gly Ile 15 GTG ATC Val Ile ACT GGA AAT CAC GTC TCC GCC AGO ATT GAC GAC OAT CAC ATC Thr Gly Asn His Val Ser Ala Arg Ile Asp Asp Asp His Ile 30

GTG

Val

TTC

Phe ATC GTC GCG CCT CGC CCC GAA OCT ACA ATT CAA CTG CAG CTA TTT Ile Val Ala Pro Arg Prc Glu Ala Thir le Gin Leu Gin Leu Phe 45 5o ATG CCT GGC CAG AGA CCC CAC AAA CCC TAC TCA GGA ACC OTC CGC Met Pro Gly Gin Arg Pro His Lys Pro Tyr Ser Gly Thr Val Arg 65 GTC GCG TTT Val Ala Phe COG TCT GAT ATA ACA AAC CAG TGC TAC CAG GAA CTT AGC Arg Ser Asp Ile Thr Asn Gin Cys Tyr Gin Glu Leu Ser 80 PCTIUJS96IO39 16 WO 96129396 GAG GAG CC Glu Glu Arg TTT GAA AAT TGC Phe Glu Asn Cys

GGC

Gly

GGA

Gly 120

GAC

Asp

CCC

Pro

ACC

Thr

GGA

Gly

TGG

Trp 200

AAA

Lys

CCA

Pro

GGG

Gly

GAC

Asp

GCG

Ala 170

CCT

Pro

AAC

Asn

GTG

Val

CAC

His

ATG

Met

CTC

Val 155

ACT

Thr

ACC

Thr

TGG

Trp ACC GAG TAC Thr Glu Tyr 110 CCG TTr AAG Pro Phe Lys 125 TTC TAC GTA Phe Tyr Val 140 TTC GCG ATC Phe Ala Ile CGC GGA CTC Arg Gly Leu GTC CAA CTT Val Gin Leu 190 CA.A GCG TAC Gin. Ala Tyr -99- ACT CAT CGA TCG TCT TCT GTT TTT GJTC Thr His Arg Ser Ser Ser Val Phe Val 95 100 ACG TTC TCC GCC TCG AAC AGA CTA ACC Thr Phe Ser Ala Ser Asn Arg Lieu Thr 115 CTC ACT ATA CGA AAT CCT CGT CCG AAC Leu Thr Ile Arg Asn Pro Arg Pro Asn 130 135 ATT GTr CGG CTA GAC GAC ACC AAA GAA Ile Val Arg Leu Asp Asp Thr Lys Glv 145 150 CAA CTA TCG GTG TAT CAA TTC GCG AAC Gin Leu Ser Val Tyr Gin Phe Ala Asn 160 165 TAT TCC AAG GCT TCG TGT CGC ACC TTC Tyr Ser Lys Ala Ser Cys Arg Thr Phe 175 180 GAG GCC TAT CTC AGG ACC GAG GAA ACT Glu Ala Tyr Leu Arg Thr Glu Glu Ser 195 S S

S

*5 S S

S

*5 5 S *5

S.

S

*SS*

*5*t

S.

S

SSSS

S

*S*S

GTT CCC ACG Val Ala Thr GAG GCC ACG ACO ACC AGC Glu Ala Thr Thr Thr Ser 210 215 205 GCC GAG GCG ACA ACC CCC ACG CCC GTC ACT GCA ACC AGC GCC TCC GAA Ala Glu Ala Thr Thr Pro Thr Pro Val Thr Ala Thr Ser Ala Ser Glu 220 225 230 GAA GCG GAA CAC TT-, ACC TTT CCC TGG CTA GAA AAT GGC GTG GAT Leu Giu Ala Giu His Phe Thr Phe Pro Trp Leu Glu Asn Gly Vai Asp 235 240 245 CAT TAC GAA CCG ACA CCC GCA AAC GAA AAT TCA AAC GTT ACT GTC CGT His Tyr Glu Pro Thr Pro Ala Asn Giu Asn ser Asn Val Thr Val Arg 250 255 260 CTC COG ACA ATG AGC C=T ACG CrA ATT O G TA ACC GTG GCT GCC GTC Leu Cly Thr Met Ser Pro Thr Leu Ile Gly Val Thr Val Ala Ala Val 265 270 275 CTC AGC GCA ACG ATC GCC CTC GTC ATT CGrA ATT TCC ATC CTC ACC AGA 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 TCC CAA GAC GAC Asn Met Cys Thr Pro His Arg Lys Leu Asp Thr Val ser Gin Asp Asp 300 305 310 GAA GAA CG'r TCC CAA ACT AGA AGO GAA TCG CGA AAA TTT GGA CCC ATG Glu Glu Arg Sex Gin Thr Arg Arg Glu Ser Arg Lys Phe Cly Pro Met 315 320 325 G-TT GCG TGC GAA ATA AAC AAG GGC GCT GAC CAG GAT ACT GAA CTT GTG Val Ala Cys Giu Ile Asn Lys Gly Ala Asp Gin Asp Ser Giu Leu Val 330 335 340 GAA CTG GTT GCG ATT =T AAC CCC TCT GCC CTA AGC TCG CCC GAC TCA Glu Leu Val Ala Ile Val Asn Pro Ser Ala Leu Ser Ser Pro Asp Ser 345 350 355 10182 10230 10278 10326 10374 10422 10470 10518 10566 10614 10662 10710 10758 10806 10854 10902 10950 WO 96/29396 PCTfUS96/03916 -100- ATA AMA ATG TGATTAAGTC TGAATGTGGC TCTCCAATCA TTTCGATTCT Ile Lys Met 360 CTAATCTCCC AATCCTCTCA AAAGGGGCAG TATCGGACAC GGACTGGGAG GGGCGTACTA CACGATAGTT ATATGGTACA GCAGAGGCCT CTQAACACTT AG43AGGAGAA TTCAGCCGGG GAGAGCCCCT GTTGAGTAGG CTTGGGAGCA TATTCCAGG ATG MAC ATG TTA GTG Met Asri Met Leu Val 9e 0 000*

AGA

Arg

AAC

Asn

TGC

Cys 70

GCG

Ala

AC

Ser

ATC

Ile

TCG

Ser

ATG

Met

MAG

Lys

GTT

Val

CTC

Leu

AAC

Msn

GCG

Al a

TTT

Phe

GGA

Gly

TTC

Plie

ATG

Met

CAC

Asp 135

GMA

Glu

GCT

Al~a CTC GCC TCT Leu Ala Ser TTT TTG GMA Phe Leu Glu 25 OTT CCG GMA Val Pro Giu 40 TGC MAG ATG Cys Lys Met CAT GAT TTA AT? His Asp Leu Ile 75 CCC CTG TCT GCA Pro Leu Ser Ala 90 GTC GTG CTG GGT Val Val Leu Gl.y 105 TGG ACA GAG TAC Trp, Thr Glu Tyr 120 GAG GCA ATC TAT Clu Gly Ile Tyr ACT ACA TCC TAC Thr Thr Ser Tyr 155 CCA GCA CCG CAC Pro Ala Pro His 170 CT? GCG CCC CTA ACT TTT CCG ACG CGA CAC Leu Ala Arg Lou Tkir Phe Ala Thr Arg His 15 ACT CAG GC GTC CTC GOG GAA GAT GAT CCC Thr Gin Al a Val Leu Gly Glu Asp Asp Pro 30 ACT GTA ATC AAA TCG ACA AAA GTC CTC CG Thr Val Ile Lys Trp Tbhr Lys Val Leu Arg 45 C CCC CAT GTC TGC TCT TCG CCT MAC TAT Ala Ala Asp Val. Cys Ser Ser. Pro Asn Tyr 60 TAC GAC CGA GGA MAG AAA GAC TGC CCG CCC Tyr Asp Cly Cly Lys Lys Asp Cys Pro Pro 80 MAC CTC GTA AT? TTA CTA AAG CCC GGC GMA Msn Leu Val Ile Leu Leu Lys Arg Gly Glu 95 100 TCT GGG CTA CAC MC. AGC MAT ATA ACT MAT Ser Gly Leu His Asn Ser Asn Ile Thr Mgn 110 115 OGA CCC CTG CTC TTT GAT CCT OTA ACT CC? Cly Cly Lou Leu Phe Asp Pro Val Thr Arg 125 130 T1T CGA CGG ATC TCT CAC CCA CAT CTC CC Phe Axg Arg Ile Ser Gin Pro Asp Leu Ala 140 145 MAC CTC AGC CT? CTT TCG CAC GTA CAC GAG Msn Val Ser Val Leu Ser His Val Asp Clu 160 165 GAG GTG GAG ATA GAC ACC ATC AAG CCC TCA Glu Val Glu Ile Asp Thr Ile Lys Pro Ser 175 190 10999 11059 11119 11173 11221 11269 11317 11365 11413 11461 11509 11557 11605 11653 11'701 11"749 11797 11845 GAG CCC CAC CC CAC GTC CMA TTA CMA ATC CTG CCC TTT CAT GAA CTC Clu Ala His Ala His Val Glu Leu Gin Met Lou Pro Phe His Clu Leu l8S 190 195 MAC CAC MAC AGC CCC ACC TAT GTO ACC CCT CT? CT? AGA OTC TTC CCA Msn Asp Msn Ser Pro Thr Tyr Val Thr Pro Val Lou Arg Val Phe Pro 200 205 210 CCC ACC GAG CAC CTA AMA TTT MAC CT? ACG TAT TCG TGG TAT CCC ?TT Pro Thr Clu His Val Lys Phe Msn Val ?hr Tyr Ser Trp Tyr Gly Phe 215 220 225 WO 96/29396 PCTIIJS96/03916 -101- GAT GTC AAA GAG GAG TGC GAA GAA GTG AAA CTG TTC GAG CCG TGC GTA 1.1893 Asp Val. Lys Giu Glu Cys Glu Glu Val Lys Leu Phe Glu Pro Cys Val 230 235 240 245 TAC CAT CCT ACA GAC GGC AAA TGT CAG TTT CCC GCA ACC AAC CAG AGA 11941 Tyr His Pro Thr Asp Gly Lys Cys Gin Phe Pro Ala Thr Asri Gin Arg 250 255 260 TGC CTC ATA GGA TCT GTC TTG ATG GCG, GAA TTC TTG GCC GCC GCC TCT 11989 Cys Leu lie Gly Ser Val. Leu Met Ala Glu Phe Leu Gly Ala Ala Ser 265 270 275 TTG CTG GAT TCT TCC CCC GAT ACT CTA GAA GAC TOC CAC GAA AAT CGC 12037 Leu Leu Asp Cys Ser Arg Asp Thr Leu Glu Asp Cys His Giu Asn Arg 280 265 290 GTG CCG AAC CTA CGGG TTC GAT TCG CGA CTC TCC GAG TCA CGC GCA GGC 12065 :*Val Pro Asn Leu Arg Phe Asp Ser Arg Leu Ser Glu Ser Arg Ala Gly 00295 300 305 CTG GTG ATC AGT CCT CTT ATA GCC ATC CCC AAA OTT TTG ATT ATA GTC 12133 *Leu Val Ile Her Pro Leu Ile Ala Ile Pro Lys Val. Leu Ile Ile Val *60310 315 320 325 00 GTT TCC GAC GGA GAC ATT TTG GGA TGG AGC TAC ACG GTG CTC GGG AAA 12181 Val Ser Asp Gly Asp Ile Leu Gly Trp SrTyr TkirVa eGlLy 330 335 340 COT AAC AGT CCG CGC GTA GTA GTC GAA ACG CAC ATG CCC TCG AAG GTC 12229 Arg Asn Ser Pro Arg Vai Val. Val Giu Thr His Met Pro Ser Lys Vai i~.345 350 355 CCG ATG AAC AAA GTA GTA ATT GGC AGT CCC GGA CCA ATG GAC GAA ACG 12277 Pro Met Asn Lys Val Val Ile Gly Ser Pro Gly Pro Met Asp Giu Thr 360 365 370 0:.:GGT AAC TAT AAA~ ATG; TAC TTC GTC OTC GCG, GGG GTG GCC GCG ACG TOC 12325 Giy Asn Tyr Lys Met Tyr Phe Vai Vai Ala Giy Val Ala Ala Thr C% 375 390 385 TA ATT C1TT ACA TGC G17T CTG CTT GTC GGC AAA AAG AAG TGC CCC GCC 12373 e.Val Ile Leu Thr Cys Ala Leu Leu Val Gly Lys Lays Lys Cys Pro Ala 000390 395 400 405 CAC CAA ATG GOT AC-T -TT TCC AAG AC-- GAA CCA TTG TAC GCG CCG CTC 12421 His Gin met Gly Thr Phe Ser Lys Thr Oiu Pro Leu Tyr Ala Pro Leu 410 415 420 CCC AAA AAC GAG TTT GAG GCC GGC GGG CTT ACG GAC GAT GAG GAA GTG 12469 Pro Lys Asn Glu Phe Glu Ala Gly Gly Leu Thr Asp Asp Glia Glu Vai 425 430 435 AT? TAT GAC GAA GTA TAC.GAA CCC CTA TTT CGC GGC TAC TOT AAG CAG 12517 Ile Tyr Asp Glu'Va]. Tyr Giu Pro Leu Phe Arg Gly Tyr Cys Lys GIn 440 445 450 GAA TTC CGC GAA GAT OTG AAT ACC TTT TTC GGT OCO GTC GTG GAG GGA 12565 Glu Phe Arg Glu Asp Val. Asn Thr Phe Phe Gly Ala Val Val Giu GW 455 460 465 GAA AGG GCC TTA AAC Tr? AAA TCC GCC ATC GCA TCA ATG GCA GAT CGC 12613 Glu Arg Ala Leu Asn Phe Lys Ser Ala Ile Ala Her Met Ala Asp Arg 470 475 480 485 ATC CTG GCA AAT AAA AGC GGC AGA AGG AAT ATG GAT AGC TAT TAGTTGOTC 12664 Ile Leu Ala Asn Lys Ser Gly Azg Arg Asri Met Asp Ser TYyr 490 495 500 WO 96/29396 WO 9629396PCT[US96/03916 0 .00.

o o.

ATG

Met 1

GC

Al a

TTA

Leu

AAT

Asn

GGG

dly

TGC

Cys

ACA

Thr

AAA

Lys

TCT

Cys

C

Al a

GTC

Val1 155 CCT TTT Pro Phe GCC C Ala Ala CCC ACC Pro Thr TAT ACC Tyr Thr GAA TCT Glu Ser TGC GCA Cys Ala CCC ATG Pro Met

AAG

Lys

GGC

Gly

AGA

Arg

CAG

Gin

GAC

Asp

TCG

Ser

CCT

Pro

TCT

Ser 110

CCG

Pro

TAT

Tyr

GTT

Val

CAA

Gln

CCG

-102- ACC AGA GOG GCC GAA GAC Thr Arg Gly Ala Giu Asp 5 AAG AAC AGG TTT MAG MAA TCG AGA MAT CGG GMA ATC Lys Asn Arg Phe Lys Lys Ser Arg Asn Arg Giu Ile 20 CTG CGT GGC ACC GCT MG AAA ACT GCC OGA TTG TCC Leu Arg Gly Thr Gly Lys Lys Thr Ala Gly LIeu Ser 35 CCT ATT CCC TGG AAC CCT AAA TTC TGC AGC GCG CGC Pro Ile Pro Tx-p Asn Pro Lys Phe Cys Ser Ala Arg so0 551 AAC CAC C TGT AAA GAC ACT TTT TAT CGC AGG ACG Asri His Ala Cys Lys Asp Thr Phe Tyr Arg Arg Thr 65 CGC TCT ACC GTT TCC ACT CAA CCC CAT TCC CCC CAC Arg Ser Thr Val Ser Ser Gin Pro Asp Ser Pro His 80 85 ACT GAG TAT GCC CGC GTG CCC TCC GCA MCG CCC AAA Thr Glu Tyr Cly Arg Val Pro Sex- Ala Lys Arg Lys 100 105 TCA GAC TSS GAG GGC C CAC CAA CCC CTA GTA TCC Ser Asp Xaa Clu Gly Ala His Gin Pro Leu Val Ser 115 120 CAT TCT CMA GCA OCA CCC CC CGA ACC TAT ACT TCT Asp Ser Gin Ala Ala Pro Ala Arg Thr Tyr Ser Ser 130 135 ACT OTT GAC GAG GTT TCG TCG CCA ACT CCC CCA GGC Thr Val Asp Glu Val Ser Ser Pro Thr Pro Pro Gly 145 150 GCG GAC TTA GA.A ACG CGC GCG GMA CTT CCT GGC GCT Ala Asp Leu Glu Thr Arg Ala Glu Leu Pro Gly Ala 160 165 170 ACG GMA ACT AMA MT MGC CTC CCC MAC CMA CMA TCC Thr Glu Ser Lys Asn Lys Leu Pro Asn Gin Gin Ser 175 180 195 MAA CCC ACA MAC GAG CAC GTC CGA CCC GAG CGC TGC Lys Pro Thr Asri Clu His Val Gly Gly Glu Arg Cys

CTA

Leu

A

Lys

CMA

Gin 140

GAC

Asp

ACG

Thr

CTG

TCA

Ser

CTT

Leu 125

AGA

Arg

GCT

Ala

GMA

Glu

MCG

12694 12742 12790 12838 12886 12934 12982 13030 13078 13126 13174 13222 13270 13318 13366 13414 1346*7 Thr

CGC

Arg Lieu Lys Pro 190 CCC TCC GMA GCC ACG GTC GAG GCG Pro Ser Glii Gly Thr Val Glu Ala 205 210 GTC CCC GCA CC ATA GCA MAC GAG Val Cly Ala Ala Ile Ala Asn Ciii 220 225 CTT CCC CTC CCC C TCG GC CC Leu Pro Leu Ala Ala Ser Ala Ala 235 240 C CC AGG CCC TTG CAC MAA CAA Ala Ala Arg Ala Leu Gin Lys Cli 255 195 200 CCA TCG CTC CCC ATC CTC TCG CC Pro Ser Leu Gly Ile Leu Ser Arg 215 CTG OCT CCT ATG, CGG AGG C TOT Leu Ala Axg Met Arg Arg Ala Cys 230 CCT CCC GCA ATA GTC CCC TGG CC Ala Ala Cly Ile Val Ala Tx-p Ala 245 250 CCC CGC TAG CAGTAATMATA ACCACACAA Gly Arg 260 WO 96/29396 TIS6O96 PCTIUS96/03916 -103-

ATATTG

INFORMATION FOR SEQ ID NO:2: W1 SEQUENCE CHARACTERISTICS: LENGTH: 476 amino acids TYPE: amino acid TOPOLOGY: linear 13473 (ii) MOLECULE TYPE: protein 9 Met

I

Arg Ser Thr Thr Pro Pro Al a Lys Val1 145 Giu Lys Ala Glu Tyr 22S Asp (xi) SEQUENCE Arg Phe Arg Arg

S

Thr Thr Giu Asn Phe Ser Arg Thr 35 Ala Glu Gly Val so Giu Arg Ala Glu Pro Ala Asn Glu e5 Ser.Thr Thr Glu 100 5cr 8cr Asp Tyr 115 His Ser Asp Giu 130 Glu Gly Ala Tyr Giu Gin Arg Arg 165 Cys Lys Arg Leu 180 Ser Gln Leu Giu 195 Ann Val Lou Lys 210 Met Lou Thr Gin Glu Ala Phe Asp 245 DESCRIPTION: SEC Ile Cys Ser Arg Pro Met Glu Pro 70 An Lys Ala Phe Gly 150 Arg Ile Lou Ser Arg 55 Val Gly Val Gly Leu 135 Phe Gly Al a Thr Leu 40 Leu Thr Gin Asp Pro 120 Gin Ile Val1 Lys Scr 25 Arg Al a Giu An Gly 105 VIal His Cys An les ID NO: 2: Ser Arg Ala 10 Lys Arg Val Pro Tyr Ala Giu Leu Val Asn Thr Ala 175 Phe Ala Tyr 90 Ser His Thr Pro Lou Ala Phe Arg Val 140 Gly Val Arg 155 Ser Thx An 170 Glu Lys Cys Val Giu Ile Ser Val Val An Ala Giy Asp Phe 110 Gin Thr 125 Lou Asp Arg Tyr Gin Gly Arg 1s Leu Lou Thr 5cr Asp Asp Arg Asp Thr Arg Asp Pro Met Ile s0 Gly Giu Lou Lou Giu 160 Lys Scr 175 Gly Thr Ari Ala 190 Asn Giu Ile Leu Val Lou Giy 200 Arg Leu Ann His 205 215 Tyr Lou Asp Arg Leu 235 Tyr 220 Tyr Asp Tyr An Met Thr Tyr 240 Trp Lys Asp Ser Pro Met Lou Lys Gin Thr Arg 250 255 Ara Ile MetLeMtSe Lys Gln 260 Leu Met Ser Val 5cr Tyr Ile His Ser Lys 270 WO 96/29396 PCT/US96103916 -104- Lys Asp Asn 305 Asn Ile Cys Val Cys 385 His Glu Ser Arg Ile 465 Leu Ile 275 Gly Lys 290 Glu Arg Ser Pro Trp Ser Pro Ile 355 Ile Asp 370 Asn Leu Thr Val Tyr Pro Ala Ala 435 Thr Ile 450 Arg Ala Arg Val Pro Ile 325 Gly Asp Leu Asn Ser 405 Val Val Ile Val Asp Leu Phe 310 Leu Val Gly Ser Tyr 390 Leu Lys Leu Ile Pro 470 Ile Lys 280 Gly Asp 295 Glu Tyr Ala Arg Val Leu Gly Gly 360 Val Cys 375 Ieu His Ile Arg Met Leu Ala Met 440 His Gly 455 Arg Ser Leu Phe Gly Asp Leu 345 Asn Asp Tyr Asn Thr 425 Pro Lys Met Glu Gly Trp Ser 330 Glu Pro Glu.

Ala Leu 410 Phe Leu His Ser Asn Thr Vai 315 Tyr Met His Glu Ser 395 His Asp Phe Lys Glu 475 Ile Phe 285 Vai Thr 300 Gly Thr Cys Glu Val Ser Gin Gin 365 Phe Pro 380 Ile AIsp Leu Pro Trp Arg Ser Ala 445 Pro Ile 460 Gly Asn Phe Ala Thr 335 Glu Leu Pro Ala Asp 415 Arg Glu Pro Cys Glu Thr 320 Asp Phe Lys Pro Gly 400 Val Pro Glu Glu INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 510 amino acids TYPE: amino acid TOPOLOGY: iinear (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 Vai lie 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 Giy Asp Ala Asn His Arg Gly Ile Arg Pro Arg Arg Lys Ser Ile 55 Gly Ala Phe Ser Ala Arg Giu Lys Thr Giy Lys Arg Asn Ala Leu Thr 70 75 WO 96/29396 PCTIUS96/03916 -105- *..00 .00 0 0.

00 0 0 Glu Glu Glu Asp Gin 145 Phe Glu Pro Phe Cys 225 Ile His Glu Gly Arg 305 Asp Asp Ala Ser Phe Val Ala 130 Pro Thr Ala Phe Lou 210 Lau Val Trp Glu Ala 290 Asp Ala Thr Leu Ser Gly Leu 115 Glu Phe Arg Lau Thr 195 Gly Arg Asn Arg Asn 275 Gly Lys Ala Ser Val 355 Ser Gly 100 Ser Glu Asn Ala Lys 190 Arg Asp Gin Cys Gly 260 Ile Leu Arg Val Arg 340 Val Ser Lys Gin Glu Ala Pro 165 Val Ser Ser His Asn 245 Met G.n Phe Leu Lau 325 Phe Lou Ser Trp Ala Arg Trp 150 Ile Gly Lys Ph.

Ile 230 Asp Arg His Lau Leu 310 Ala Glu Leu Asp Thr Trp Val 135 Pro Val Ala Lys Trp 215 Thr Pro Val Gly Trp 295 Arg Ser Met Leu Asp 90 Val Asp Gly 105 Asp Val Leu 120 Thr Tyr Glu Asp Gly Pro Tyr Pro Ser 170 Phe Val Ser 185 Ser Val Thr 200 Arg Ile Met Arg Leu Arg Lou Trp Tyr 250 Pro Ser Lou 265 Pro Met Ala 280 Pro Ala Met Ala Cys Leu Phe Pro Ph.

330 Pro Pro Gin Ser Ser iss Ala Arg Val Gin His 235 Ala Lys Ala Arg Ser 315 Phe Ala Lou Lys 140 Trp Glu Val Arg Asn 220 Pro Tyr Leu Val Ala 300 Ser Asp Val 125 Pro Asn Val Lau Asp 205 Val Ser Ala Ala Phe 285 Ala Asp Thr His Pro Gin Asp 175 Cys Gin Thr Lys Gln 255 Pro Asn Glu Lys Ala Glu Ile Asp 160 Ala Val Se Val Ser 240 Phe Pro Ala Glu Ser Lou Asp lie Met Tyr T-p Axg Gly Val Gin 335 Pro Ala Lou Gly Cys Leu Ser Giu Tyr Phe 345 350 Ala Giu Thr Val Leu Ala Thr Met Phe Asp 360 365 His Ala Lou Val Phe Met 370 Ala Leu Ala Asp Gly 380 Asn Phe Asp Asp Tyr 385 Gly Leu Asp Ala Ala Glu Glu Val Thr Gly Val 420 Arg Thr 405 cys Tyr 390 Lau Ala Ile Leu Asn Asp Arg Thr Pro Gly Tyr 425 Val Ile 410 Ser Lys Asn Giu Tyr 395 Val Ala Ser Asn Thr lie Arg Lys 430 Lou Thr 415 Lau Asn 400 Ala Pro WO 96/29396 WO 9629396PCT(US96/03916 Ser Lys Glu 465 Asn His Al a Thr 435 Arg Arg Phe Phe Cys Leu Cys Ser 500 Cys Asn 440 Met Ser 455 Ala Trp Leu Lys Al a Arg -106- Val Ala Tyr Axg Thr Giu Thr Leu 445 Asp Ile Tyr Arg Ile LeL. Gln Lys 460 Leu Gin Azg Val Ala Thr His Ala 47 5 480 Arg Ser Val Asp Thr Gly Pro Arg 490 495 Arg Ser Gly Cys Ser Ser 505 510 a INFOR.MATION 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 Gin Tyr Glu Asp 1 5 10 Ala Met Gly Ser G1u 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 le Ile Ala Asp Gly Val Arg so 55 Val Ile Asp Glu Pro Val Arg Ala Ser Val Ala Leu Ser 65 70 Ala Gly Lys Val Lys A Ia Arg Arg Leu Thr Ser Val Arg 90 Pro Pro Ala Gly Ala Val Ser Ala Arg Arg LyE Ser Glu 100 105 Ph.

Phe Phe Phe Thr Ala Ile 110 Ser is Val Leu Lys Ala Pro INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: CA) 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 Vai Ser Leu Val 1 5 10 Cys Ser Leu Leu Cys Ala 5cr Val Leu Gly Ala' Pro Val Leu Asp Gly 25 WO 96129396 PCTIUS96/03916 -107- Leu Clu Ser Ser Pro Phe Pro Phe Gly Gly Lys Ile Ile Ala Gin Ala 40 Cys Asn Arg Thr Thr Ile Glii Val Thr Val Pro Trp Ser Asp Tyr Ser 5S Gly Arg Thr Giu Gly Val Ser Val Glu Val Lys Trp Phe Tyr Gly Asn 70 75 Ser Asn Pro Giu Ser Phe Val Phe Gly Val Asp Ser Giu 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 Giu Lys Val Gin Azrg Arg Leu Arg Ser Gly Val Glu Leu Cly 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 Ilie Tyr Ala Tyr Gin Leu Ser Val Gly Giy 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 Al1a Asp Giu Ala Ser Asp Leu 195 200 205 Ser Ser Tyr Asp Val Ala Ser Pro Glii Leu Asp Gly Pro Met Glu Glu 210 215 220 Asp Tyr Ser Asn Cys Leu Asp Met Pro Pro beu Arg Pro Trp, Thr Thr 225 230 235 240 Val Cys Ser His Asp Val Glu Glu Gin Glu Asn Ala Thr Asp Glu Leu 245 250 255 Tyr Leu Trp Asp Glu Glu Cys Ala Gly Pro Leu Asp.Giu Tyr Val Asp 260 265 270 Giu Arg Ser Glu Thr Met Pro Akrg Met Val Val Phe Ser Pro Pro Ser 275 280 285 Thr Leu Gin Gin 290 INFORMATION FOR SEQ ID NO: 6; Wi 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 S 10 WO 96129396 PCTfUS96/03916 -108- Ala Ala Leu Pro Thr Gly Arg Phe Cys Arg Val Trp Lys Val Pro Pro 25

S

S. 5555 Gly Thr Ile Asp Val yr Tyr Val 145 Thr Asp Tyr Phe Gly 225 Gin Pro Gly Gly Phe Glu Thr Phe Ser 130 Arg Trp Asn Ser Phe 210 Pro Pro Asp Thr His Ala Leu Phe Thr 115 Val Leu Gly Asp Ass 195 Tyr Ser Va1 Ile lie Ala Asp Ala Ser 100 Ile lie Glu Ser Val 180 Ser Ser Pro Leu Asp 260 Gin Thr Thr Leu Leu Gly Ser Thr Lys 165 Ala Ala Pro Ser Glu Tyr Pro 70 Glu Ser Val Arg Pro 150 Arg Val Ala Lys Arg 230 Asn Pro 55 Thr Arg Tyr Phe Val 135 ys Trp Leu Gin Arg 215 Ile Leu 40 Pro Leu Gly Arg Al a 120 Leu Asp Leu Thr Thr 200 Ile Thr Ala Pro Arg Thr Pro 105 Thr Val Glu Gly Lys 185 Gly Val Val Asp Val Glu Ile Ser 90 Arg Gly Asn Asn Pro 170 Ala Leu Asn Tyr Gly 25C Leu Gly Arg 75 Ala Pro Gin Ala Phe 155 Pro Gin Thr Val Ser 235 Phe Ala Ala Tyr' Ser Glu Ser Ser 140 Leu Ser Ser Thr 220 Ser Leu 3lu Val Gly Asp le Thr 125 Leu Gin Pro lie Leu 205 Trp Arg Val Ser Ser Pro Ala His 110 Glu Glu Asn Tyr Gly 190 Asn Thr Glu Lys Pro Phe Thr Asp Gly Ser Arg Glu Val 175 Glu Met Thr Asn Tyr 255 Val Gln 3iu Asn Ala Ser Ser Pro 160 Arg Cys Thr Gly Gly 240 Thr Arg Asn Val Ser 245 Gly Arg Ala Met Ile Asn Val lie Ala Ass Tyr Ser 270 Ala Phe Arg Giu Gly 285 Pro Ala Asp 275 5cr Gly Ser Val Leu Ala Phe Thr 280 Lys Leu Pro Ser Ala 290 Glu Pro Pro Gly Thr 305 Thr Pro Tyr Arg Ala 325 Arg Pro Gly Ala Thr 340 Asp Thr Gly Thr Ser 355 Ile Gin 295 Glu Thr 310 Leu Gly Leu Pro Pro Thr Leu Thr Ser Pro Pro 360 His Arg Phe Asp Asn Val 330 Phe Asp 345 Thr Thr lie Cys 315 Pro Thr Val Asp Met Ser 300 Gin Lys Met Arg Asp Asp Ala Ala Pro 350 Pro Glu Pro 365 Gly Ile Ser 335 Asp Ala Thr Glu 320 Ile Phe Ile PCTIUS96103916 WO 96/29396 -109- Thr Thr Leu Ile Pro Arg 370 Ser Thr Ser Asp Met Ala 385 Asp Giu Giu Thr Ser 465 Gin Thr Ala Phe Ala 545 Phe Arg Arg Ser Thr Val.

450 Gin Ala Gin Glu Thr 530 Ala Thr Ala Thr Thr Leu Giu Asn 420 Pro Ser 435 Val Phe Ser Gin Ala. Leu Thr Pro 500 Thr Glu s is Gin Ser Pro Giiu Gin Thr Gly Se r 405 Thr Al a Thr Giu Thr 485 Ser Ala Arg Val Pro 565 Ser 390 Thr Leu Al a Gin Pro 470 Gin Ala Pro Ser Phe 550 Ser Giu Thr Thr Pro Phe Pro His Glu 440 Ser Pro 4S5 Trp, Tyr Thr Gin Giu Gin Ala Gin 520 Thr Pro S35 Tkir Gin Thr Val Leu Leu Thr 425 Thr Ser Phe Ile Met S05 Thr Pro Ser Pro Ser Thr 410 Thr Thr Thr Thr Ala 490 Thr Pro Giu Ser Lys 570 Val 395 Leu Ala Gin Glu Gin 475 Glu Phe Ser Thr Ser 555 Thr Gly 380 Pro Pro Pro rhr Ser 460 T1hr Thr Thr Thr Ala 540 Thr Thr Val1 Leu Gly Gin 445 G lu Pro Giu Gin le 525 Arg Val Leu Gin Thr Ile 430 Ser Thr Ser Ala Thr 510 Pro Ala Thr Ser Glu Pro 415 Ser Al a Al a Thr Leu 495 Pro Giu Pro Glu Ser 575 Phe Phe Ser Thr Thr 400 Gly Thr Glu Arg Glu 480 Phe Gly Ile Ser Val 560 Ser Thr Glu Pro Ala Ilie 580 Phe Thr Arg Thr Gin Ser Ala Gly Thr Giu Ala 590 Thr Gin Ser Phe Thr Gin Thr Ser Ser Ala Giu Pro Asp Thr Met 59S 603 Thr Thr 625 Thr Thr Thr Thr Val 705 Glu Thr His Phe Phe 610 Gin Thr Pro Ser Thr 630 Glu Pro Vai Pro Phe 645 Gin Thr Pro Ser Ala 660 Met Pro Giu Thr Ala 675 Ser Ser Giy Thr Gly 690 Gin Thr Thr Pro His 710 Thr 615 Glu Thr Al a Gin Thr 695 Thr Gin Pro Arg Pro Ser 690 *His *Glr Al a Pro Giu Val Thr IAu 650 Glu Val 665 Thr Pro Asn Thr Lys Leu Ser Leu 635 Gly Tyr Giu Tyr 715 Thr 620 Thr Ala Thr Ala Pro Thr Val Pro Lys Gin Ser Pro Glu Pro Giu 655 Arg Ser Ser 670 Ser Gin Asn Arg Thr Tyr Giu Msn Lys U.a Ser 640 Ile Ser Pro Pro Thr 720 WO 96/29396 PCTUS96103916 -110- Leu Glu Ser Pro Asp 785 Lys Val Cys Glu Lau 865 Ser Gly Ile Asp Trp 945 Phe Ala Phe Pro Thr 725 Gin Thr Pro 740 Asp Gly Glu 755 Arg Val Glu Ile Ser Gly Gin Lys Gin 805 Leu Met Cys 820 Phe Asp Thr 835 Tyr Phe Thr Ser Gly Val Pro Glu Tyr 885 Ser Thr Vai 900 Gly Arg Gly 915 Thr Arg Val Ala Val Glu Gly Leu Val 965 Leu Trp Thr 980 Val Leu Val Thr Asn 790 Leu Leu Ser Ser Val 870 Leu Trp Gly Thr Asn 950 Ser Ala Val Leu Thr Asn 775 Ser Tyr Ser Leu Asp 855 Pro Thr Lys Asn Thr 935 Cly Gly Ile Ser Asp Ala 760 Trp Pro Tyr Cys Ile 840 Pro Arg Ala Ser Ile 920 Arg Ala Leu Tyr Glu Phe His 730 Val Lys Ile 745 Thr Cys Vai Lys Val Asp Ala Gly Val 795 Arg Val Thr 810 Thr Ser His 825 Ala Arg Glu Gin Thr Ala Phe Glu Trp 875 Thr Thr Vai 990 Ser Ala Arg 905 Tyr Glu Cys Lys Glu Arg Ala Gin Ala 955 Cys Gly Ser 970 Phe 985 Glu Val Ser Leu 780 Phe Asp Ser Lys Tyr 860 Ser Val Ala Thr Cys 940 Gin Met Ser Glu Val 750 Thr Val 765 Val Asp Asn Ser Gly Arg Pro Glu 830 Asp Ile 845 Cys Thr Leu Asn Ser His Gly Glu 910 Val Leu 925 Leu Thr Leu Tyr Thr 735 Lys Lys Val Asn Thr 815 Pro Ala Ile Asn Thr 895 Ala Ile Asn Ser Ile Ser Ala Leu Tyr 975 INFORMATION FOR SEQ ID NO:7: i) SEQUENCE CHARACTERISTICS: CA) LENGTH: 362 amino acids CB) TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Met Ala Ser Leu Leu Gly Thr Lou Ala Leu Leu Ala Ala Thr Lou Ala 1 5 10 PCTfUS96O39 16 WO 96/29396

-III-

Pro Phe GiY Ala Met Gay Ile Vai Ile Thr Gly Asn His Val Ser Ala 0 00.

0:0.

01% 000.

0.00 Arg Tlir Pro Gin Arg Ser Ile Arg 145 Ser Lys Tyr Thr Thr 225 Trp Ile Ile Tyr Cys Ser Ala Arg 130 Leu Val Ala Leu Giu 210 Al a Leu Asp Gin Ser Tyr Ser Ser 115 Asn Asp Tyr Ser Arg 195 Al a Thr Glu Asp Leu Gly Gin Ser 100 Asn Pro Asp Gin Cys 180 Thr Thr Ser Asp Gin Thr Giu Val Arg Arg Thr Phe Arg Giu Thr Al a His Leu Val Leu Phe Leu Pro Lys 150 Ala Thi- Giu Thr Se r 230 Ile Phe 55 Arg Ser Val Thr Asn 135 Giu Asn Phe Ser 5cr 215 Glu~ Val1 40 Phe Val Giu Gly Gly 120 Asp Pro Thr Gly Trp 200 25 Ile met Al a Giu Cys 105 Pro Ser Ile Ala Leu 185 Arg Val Pro Phe Arg 90 Lys Pro Gly Asp Al a 170 Pro Asn Ala Gly Arg 75 Pkie Val His Met Val 155 Thr Trp Pro Gin Ser Glu Thi- Pro Phe 140 Phe Arg Val Gin Thr 220 His Arg Arg Asp An Giu Phe 125 Tyr Ala Gly Gin Ala 205 Pro Phe Pro Pro Ile Cys Tyr 110 Lys Val1 Ile Lou Leu 190 Tyr Thr Th~r Glu Hiis Thr Thr Thr Leu Ile Gin 175 Glu Val Pro Phe Al a Lys Asn His Phe Thr Val Leu 160 Ser Ala Ala Val Pro 240 Ala Giu Ala Thr Leu Giu Ala Glu 235 Ann Gly Val Asp 245 His Tyr Giu Pro Thi- 250 Pro Ala Asn Giu 255 Asn Ser Ann Val Thr Val Arg 260 Gly Val Thr Val Ala Ala Val 275 Leu Gly Thr Met Ser Pro Thi- Leu Ile 265 270 5cr Ala Th- Ile Gly 285 Leu Val Ile Val Asp 305 Scr Asp Ala Ile Ser Ile Val Thr 290 Thi- Val Ser Gin Asp 310 Arg Lys Phe Gly Pro 325 Gin Asp Ser Giu Leu 340 Leu Ser Scr Pro Asp 355 Arg Asn 295 Asp Glu Met Vai Val Giu Ser Ile 360 met Glu Ala Leu 345 Lys Cys Axg Cys; 330 Val Met Thi- Pro 300 Ser Gin 315 Giu Ile Ala Ile His Thi- An Val Arg Lys Leu Arg Arg Glu 320 Lys Gly Ala 335 Ann Pro Ser 350 WO 96/29396 WO 9629396PCTILJS96I3916 -112- INFORMATION FOR SEQ ID NO:S: Wi SEQUENCE CHARACTERISTICS: LENGTH: 499 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 9*

S.

S

Met Phe Gly Thr Ser 65 Lys Ser Asp Gin 145 Se r (xi) Asn Met Ala Thz Glu As; Lys Val Ser Prc Asp Cy' Lys Ar5 Asn 11i Pro Va.

130 Pro As His Va

SEQUENCE

Leu Val Arg His Asp Pro -Leu Arg Ann Tyr Pro Pro 4 Gly Glu 100 e Thr Asn 1 Thr Arg p Leu Ala 1 Asp Glu 165 Val Leu Phe Leu Glu Gly Arg Asn Cvs 70 Ala Ser Ile Ser met 150 Lys Asn Ala 55 Phe Gly Phe Met Asp 135 Giu Al a Val1 40 cys His Pro Val Trp 120 Glu Thr Pro 25 Pro Lys Asp Leu Val 105 Thr Gly Thr Al1a Glu Met Leu Ser 90 Leu Glu Ile Ser Pro 1,70 Gly Lys Ile 75 Ala Gly Tyr Tyr His Thr Thr Ala Tyr.

A-n S er Gly Phe 140 Asn Glu G1n Val Al1a Asp Leu Gly Gly 125 Arg Val Val Ala Ile Asp Gly Val Leu 110 Leu Arg Ser Glu Val Lys Val Gly Ile His Leu Ile Val Ile 175 Leu Trp Cys Lys s0 Leu Asn Phe Ser Leu 160 Asp DESCRIPTION: SEQ ID NO:8: Ile Val Leu Ala Ser Cys Leu Ala Arg Leu Thr Thr Ilie Lys Pro Ser Giu Ala His Ala His Val Glu Leu Gin Met Leu 180 J. V w Pro Leu Ser 225 Phe Ala Leu Cys Phe Arg 210 Trp Giu Thr Gly His 290 His 195 Val Pro An Al a 275 Glu Giu Phe Gly Cys Gin 260 Ala Asn Leu Pro Phe Val 245 Arg Ser Arg An Pro Asp 230 Tyr Cys Leu Val Asp Thr 215 Vali His Leu Leu Pro 295 Asn Ser Pro 200 Giu His Val Lys Glu Giu Pro Thr Asp 250 Ile Gly Ser 265 Asp Cys Ser 280 Ann Leu Arg Thr Lys Cys 235 Gly Val Arg Pkie Tyr Phe 220 Glu Lys Leu Asp Asp 300 Val 205 Asn Giu Cys Met Thr 285 Ser rhr Val Val Gin Ala 270 Leu Arg Pro Val Thr Tyr Lys Leu 240 Phe Pro 255 Giu Phe Glu Asp Leu Ser PCTfUS96/03916 WO 96/29396 Glu 305 Val Thr Met Pro Val 385 Lys Leu .9 Asp Gly Ala 465 Ser *Asp IAsp Ser Leu Val Pro Met 370 Ala Lys Tyr Asp Tyr 450 Val Met Ser Arg Ala Gly Ile Ile Val 325 Leu Gly Lys 340 Ser Lys Val 355 Asp Glu Thr Ala Thr Cys Cys Pro Ala 405 Ala Pro Leu 420 Glu Glu Val 435 Cys Lys Gin Val Glu Gly Ala Asp Arg 485 Tyr Leu 310 Val Arg Pro Gly Val 390 His Pro Ile Glu Glu 470 Val Ser Asn Met Asn 375 Ile Gin Lys Tyr Phe 455 Arg Ile Asp Ser Asn 360 Tyr Leu Met Asn Asp 440 Arg Ala -113- Ser Pro Leu 315 Gly Asp Ile 330 Pro Arg Val 345 Lys Val Val Lys Met Tyr Thr Cys Ala 395 Gly Thr Phe 410 Glu Phe Glu 425 Ulu Val Tyr Glu Asp Val Leu Asn Phe 475 Ile Leu Val Ile Phe 380 Leu Ser Ala Glu Asn 460 Lys Ala Gly Val Gly 365 Val Leu Lys Gly Pro 445 Thr Ser Pro Ser 335 Thr Pro Al a Gly Glu 415 Leu Phe Phe Ile Lys 320 Tyr His Gly Gly Lys 400 Pro Thr Arg Gly Ala 480 Ile Leu Ala Asn Ser Gly Arg Arg Asn Met 495 9.

9 .99 9 9*99 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 260 amino acids TYPE: amino acid TOPOLOSY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ.ID Met Pro Phe Lys Thr Arg Gly Ala Glu Asp 1 5 10 Arg Phe Lys Lys Ser Arg Asn Axg Glu Ile 25 Gly Thr Gly Lys Lys Thr Ala Gly Leu 5er 40 Pro Trp Asn Pro Lys Phe Cys Ser Ala Arg 55 Ala Cys Lys Asp Thr Phe Tyr Arg Arg Thr 70 NO: 9: Ala Ala Ala Gly Lys Asn Leu Pro Thr Arg Leu Arg Asn Tyr Thr Gin Pro Ile Gly Glu Scr Asp Asn His Cys Cys Ala Ser Arg 75 WO 96/29396 WO 9629396PCTfUS96/03916 *5

S

Thr Tyr Xaa Gin Asp 145 Leu Ser Thr Glu Asn 225 Ala Lys Val Gi y Glu Ala 130 Glu Giu Lys Asn Ala 210 Glu Ala Gin Ser Ser Arg Val 100 Gly Ala 115 Al1a Pro Val Ser Thr Arg Asn Lys 180 Glu His 195 Pro Ser Leu Ala Ala Ala Gly Arg 260 Gin Pro Pro Ser His Gin Al a Arg Ser Pro 150 Ala Giu 165 Leu Pro Val Gly Leu Gly Arg Met 230 Gly Ile 245 Ser Lys Leu 120 Tyr pro Pro Gin Giu 200 Leu Arg Al a Pr Ar 10 Va Se Pr Gi Gi 18 Ax Se Al -114o His 90 g Lys 1 5cr r Ser o Gly y Ala 170 n Ser *g Cys *r Arg *a Cys .p Ala 250 Thr Lys Cys Ala Val 155 Thr Arg Pro Val Leu 235 Al a Pro Leu Lys Gin 140 Asp Thr Leu Ser Gly 220 Pro A.1 a Met Ser Leu 125 Arg Ala Glu Lys Giu 205 Ala Leu Arg Thr Ser Asp Thr Ala Thr 175 Lys Thr Ile Ala Leu 255 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) ANTrI-SENSE: NO (ix) FEATURE:- NAME/KEY: CDS LOCATION: 1. .1305 (xi) SEQUENCE DESCRIPTION: SEQ ID ATG CAC CGT CCT CAT CTC AGA COG CAC TCG COT 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 GOT GGA AAA COG TGC TGC TCA Giu Val Leu Asn Lys His Met Asp Cys Gly Gly Lys Arg Cys Cys Ser 25 WO 96/29396 WO 9629396PCTIUS96/03916 *5

S

S

S S *5

S.

S

*SSS

5555..

S.

S

S

*5*S

S

GGC GCA OCT Gly Ala Ala GAG CAT ATC Glu His Ile s0 AGG AAT GCT Arg Asn Ala AGC ACC GCC Ser Thr Ala GAC OCG CTA Asp Ala Leu AGO OTA TCA Arg Val Ser 115 GTG GAC GAC Vai Asp Asp 130 TTC GAG GCC Phe Oiu Ala 145 GAG GAC AAG Giu Asp-Lys GAC GTA CAA Asp Val Gin GCA OTG GAC Ala Val Asp 195 CTG ACT ATA Leu Thr Ile 210 ACC CTO AAA Thr Leu Lys 225 OTT AAC GAT Val Asn Asp TCG AAA TOC Ser Lys Cys CAC CTT TAT His Leu Tyr 275 OTA TTC Val Phe TOC TTT Cys Phe TTT TGG Phe Trp CCC GTC Ala Vai ACC ATA Thr Ile 100 AGA GOC Arg Oly ATG ATA Met Ile TCC OTC Ser Vai TAC TGT Tyr Cys 165 CTG CTA Leu Leu 180 TAT G7T Tyr Vai TTC TCC Phe Ser ATC 000 Ile Gly CGC TOT Arg Cys 245 TOO ACA Try, Thr 260 CCC ATC Pro Ile ACT CTT TTC Thr Leu Phe 40 GTA CGC AAC Vai Arg An 55 ACT ATC GTA Thr Ile Val 70 ACG TAC GAC Thr Tyr Asp CCG GCO OTT Pro Ala Val TOC GAC OTT Cys Asp Val 120 TCG 0CC GCC Ser Ala Ala 135 OTC TGG TTC Val Trp Phe 150 CCA ATC TAT Pro Ile Tyr TCT GAA TGC Ser Giu Cys CCT AGC ACC Pro Ser Thr 200 CCC ACT OCT Pro Thr Ala 215 AGA TTT 0CC Arg Phe Ala 230 -115- TOG ACT TGT GTC AGO ATT ATO COG Trp Thr Cys Val Arg Ile Met Arg OCT ATO GAC CGC CAT TTA TTT TTG Ala Met Asp Axg His Lieu Phe Leu CTG CTT TCT TCC TTC GCT AGC CAG Leu Leu Ser Ser Phe Ala Ser Gin 75 s0 TAC ATT TTA CGC COT COC GCG CTC Tyr Ile Leu Gly Arg Arg Ala Leu 90 GOC CCC TAT AAC AGA TAC CTC ACT Cly Pro Tyr Asn Arg Tyr Leu Thr 105 110 OTC GAO CTC AAC CCO ATT TCT AAC Val Olu Leu Asn Pro Ile Ser An 125 AAA GAA AAA GAG A.AG 000 CCC CCT Lys Glu Lys Glu Lys Gly Gly Pro 140 TAC OTG ATT AAC GGC GAC GAC GOC Tyr Val Ile Lys Gly Asp Asp Oly 155 160 AGA AAA GAG TAC AGO OAA TOT GOC Arg Lys Oiu Tyr Arg Giu Cys Gly 170 175 GCC OTT CAA TCT GCA CAG ATO TOG Ala Val Gin Ser Ala Gin Met Trp 295 190 CTT OTA TCO COA AAT GGC OCG GOA Leu Vai Ser Arg Ann Gly Ala Gly 205 0CC CTC TCT GGC CAA TAC TTC CTO Ala Leu Ser Oly Cmn Tyr Leu Leu 220 CAA ACA OCT CTC GTA ACT CTA GAA Gin Thr Ala Leu Val Thr Leu Giu 235 240 144 192 240 2988 336 384 432 480 529 576 624 672 720 TTA AAG ATC 000 TCO CAG CTT AAC TTT TTA CCG Leu

ACA

Thr

GCA

Ala Lys Ile Oly Ser 250 CAA CAG TAT CAG Giu Gin Tyr-Gin 265 GAC ACC AAT ACA Asp Thr Asn Thr Gin

ACT

Thr

CGA

Arg Leu Ann Phe Leu Pro 255 GGA TTT CAA GGC GAA Gly Phe Gin Gly Olu 270 CAC GCG GAC GAC OTA His Ala Asp Asp Val 295 768 816 864 280 TAT COG GGA TAC GAA OAT ATT CTO CAG 'COC TOG AAT AAT TTO CTG AGO Tyr Arg Giy Tyr Giu Asp Ile Leu Gin Arg Trp Ann Ann Leu Leu Arg 290 295 300 WO 96/29396 WO 9629396PCT1US96/03916 AA MG MhT CCT Lys Lys Asn Pro 305

GMA

Glu

GMA

Glu

CAG

Gin

GMA

Glu

TAT

Tyr 385

AGT

Ser

ATT

Ile

AGC

Ser

GCA

Ala

GTC

Val 370

TAC

Tyr

TCT

Ser CCC GCT Pro Al a AGC GAA Ser Glu 340 GAG GCT Glu Ala 355 CCC GAG Pro Glu MAT GAC Asn Asp MAT 0CC Asn Ala AGC GCG CCA Ser Ala Pro 310 GTA ACC MAG Val Thr Lys 325 MAG MG GCC Lys Lys Ala TCT GGA GMA Ser Gly Glu -116- GAC CCT COT CCA OAT AGC GTC CCG CMA Asp Pro Arg Pro Asp Ser Val. Pro Gin 315 320 AAA GCG GAA GG0 CGC ACC CCG OAC OCA Lys Ala Glu Gly Arg Thr Pro Asp Ala 330 335 CCT CCA GAA GAC TCG GAG GAC GAC ATG Pro Pro Glu Asp Ser Glu Asp Asp Met 345 350 MAT CCT GCC GCC CTC CCC GMA GAC GAC Asn Pro Ala Ala Leu Pro Giu Asp Asp 360 365 CAC OAT OAT CCA AAC TCG GAT CCT GAC His Asp Asp Pro Asn Ser Asp Pro Asp 380 GTG ATC CCG GTG GAG GAG ACT ACT AMA Val Ile Pro Val Glu Giu Thr Thr Lys 395 400 CCC ATA TTC GCG GCG TTC GTA 0CC TGC Pro Ile Phe Ala Ala Phe Val Ala Cys 410 415

S

S

S.

S

S.

S

S.

S

*5*S

S

S. S

S*

S S

GAC

Asp

ATC

Met

GTC

Val 405 ACC GAG Thr Oiu 375 CCC GCC Pro Ala 390 TCC ATG Scr Met 960 1008 1056 1104 1152 1200 1248 1296 1305 GCG GTC GCG CTC OTG 000 CTA CTG GTT TGG AGC ATC GTA AAA TGC GCG Ala Val Ala Leu Vai Oly Leu Leu Val TI-p Ser Ile Val Lys Cys Ala 420 425 430 COT AGC TMA Arg Ser 435 INF'ORM4ATION FOR SEQ ID NO:i1: 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 Ax-g Tyr Tyr Ala Lys Gly 1 5 10 is Glu Val Leu Asn Lys His Met Asp Cys Gly Gly Lys Arg Cys Cys Ser 25 Gly Ala Ala Val Phe Thr Leu Phe Trp Thr Cys Val Arg Ile Met Arg 40 G1u His Ile Cys Phe Val. Arg Asn Ala Met Asp Arg His Leu Phe Leu 55 Arg Ann Ala Phe Trp Thr Ile Val Leu Leu Ser Ser Pkxe Ala Ser Gin 70 s5o8 Ser Thr Ala Ala Val Thr Tyr Asp Tyr Ile Leu Gly Arg Arg Ala Leu 90 WO 96/29396 PCTIUS96/03916 a.

Asp Arg Val Phe 145 Glu Asp Ala Leu Thr 225 Val Ser His Tyr Lys 305 Glu Ala Val Asp 130 Glu Asp Val Val Thr 210 Leu Asn Lys Leu Arg 290 Lys lie Leu Ser 115 Asp Ala Lys Gin Asp 195 Ile Lys Asp ys Tyr 275 Gly Asn Pro Thr 100 Arg Met Ser Tyr Leu 180 Tyr Phe Ile Arg Trp 260 Pro Tyr Pro Ala Ile Gly Ile Val ys 165 Leu Val Ser Gly.

ys 245 Thr Ile Glu Ser Val 325 Pro Cys Ser Val 150 Pro Ser Pro Pro Arg 230 Leu Thr Ala Asp Ala 310 Thr Ala Asp Ala 135 Trp Ile Glu Ser Thr 215 Phe Lys Glu Asp Ile 295 Pro Lys Val Val 120 Ala Phe Tyr.

Cys Thr 200 Ala Ala Ile Gln Thr 280 Leu Asp Lys

I

vi

A]

Li 2

A

1

P

A]

G1

G:

75 2;

A:

G

P

A

-117- .y Pro

)S

Li Glu es Glu tr Val g Lys 170 La Val 35 iu Val La Leu ln Thr ly Ser 250 yr Gin 65 en Thr in Arg ro Arg .la Glu 330 Tyr Leu Lys Ile iss Glu Gin Ser Ser Ala 235 Gin Thr Arg Trp Pro 315 Gly Asn Asn Glu 140 Lys Tyr Ser Arg Gly 220 Leu Leu Gly His Asn 300 Asp Arg Arg Pro 125 Lye Gly Arg Ala Asn 205 Gin Va1 Asn Phe Ala 285 Asn Ser Th- Tyr 110 Ile Gly Asp Glu Gin 190 Gly Thr Phe Gin 270 Asp Leu Val Pro Leu Ser Gly Asp Cys 175 Met Ala Leu Leu Leu 255 Gly Asp Leu Pro Asp 335 Thr As Pro Gly 160 Gly Trp Gly Leu Glu 240 Pro Glu Val Arg Gin 320 Ala Glu Set Ser Giu Lys Lys Ala Pro Pro Glu Asp Ser Glu 340 345 Asp Asp Met 350 Gin Ala Giu Ala Ser Gly Glu Asn Pro Ala Ala Leu Pro Glu Asp Asp 355 360 365 Glu Val 370 Tyr Tyr 385 Set Ser Ala Val Arg Ser Pro Asn Asn Ala Glu Asp Ala Leu 420 Asp Met Val 405 Val Thr Pro 390 Ser Gly Glu His 375 Ala Val Met Pro Leu Leu Asp Ile le Val 425 Asp Pro Asn 380 Pro Val Glu 395 Phe Ala Ala 410 Trp Ser Ile Ser Asp Glu Thr Phe Val Val Lye 430 Pro Thr Ala 415 Cys Asp Lye 400 Cys Ala WO 96129396 PCTIUS96/03916 -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 DESCP.IPTION: SEQ ID NO:l2: ATG GCG CCT GTA AAA GTG ACT ATA GTT TCT GCG GTC GAT TCG CAC TAC 48 Met Ala Pro Val Lys Val Thr Ile Val Ser Ala Val Asp Ser His Tyr 1 5 10 AAA CTA CCT AAT TCT AGA TTT GAG CTC TCG GAT TCT GGA TGG AAA GAA 96 Lys Leu Pro Asn Ser Arg Phe Glu Leu Ser Asp Ser Gly Trp Lys Glu 25 TTG GTT CAC GCA GTG AAA ACT ATG GCG ACT TAC GAT COT CCC AGT ACA 144 Leu Val His Ala Val Lys Thr Met Ala Ser Tyr Asp Arg Pro Ser Thr 40 TTA TCC GTA ATC GTC CCC CCG GCA TCT CTG TAC GAA GTT TCC GGG GAG 192 Leu Ser Val Ile Val Arg Pro Ala Ser Leu Tyr Glu Val Ser Gly Glu 55 CTG TTT TCC CTT CCC AGG ATG TGC AGA CCC GTG ATT CGG TTC GCT GAG 240 Leu Phe Ser Leu Pro Arg Met Cys Arg Pro Val Ile Arg Phe Gly Glu 65 70 75 GGG GGC GAC CCG CCT GGA OTA ACT CCC GAG TGG AGC GGC TTG GAC GCA 288 Gly Gly Asp Pro Pro Gly Val Ser Pro Glu Trp Ser Cly Leu Asp Ala 90 CGG TTT TAC CAT TTC TCA TCT CCC GCC TAT CCC GCA AAA GAG TTC CAT 336 Gly Phe Tyr His Leu Ser Ser Gly Ala Tyr Ala Ala Lys Glu Phe His 100 105 110 TTC TGC GTG CTG GGT ACC OCT GAC ATA TCC ATG OCA GCT TTA AAC CTC 384 Leu Trp Val Leu Gly Thr Ala Asp Ile Cys Met Ala Ala Leu Asn Leu 115 120 125 CCT GCG CCA AAA ACT TTC CTA ATT ACC GAA ACC GGA GGT AAA ART TTT 432 Pro Ala Pro Lys Thr Phe Leu Ile Thr Clu Thr Gly Gly Lys Asn Phe 130 135 140 GAG AGA GGA GTG GAA ATA TTT TTG GTA ARC GGA GAC AAG ACA ACG CTG 480 Glu Arg Cly Val Glu Ile Phe Leu Val Asn Cly Asp Lys Thr Thr Leu 145 150 155 160 TCT CTG ACT CAC CCA TCA GTC TOG ACA ACT CTT GCC CCT TCG AGC CTG 526 Ser Lou Ser His Pro Ser Val Trp Thr Thr Lou Ala Pro Ser Ser Leu 165 170 175 WO 96/29396 PCT/US96/03916

AGA

Arg

AAC

Asn

CCA

Pro

CCG

Pro 225 (2) 0* S. S

S

S

S S -119- TAC AGC ACG GTA AAG TTT TTA AAA GTA AAA CCT Tyr Ser Thr Val Lys Phe Leu Lys Val Lys Pro 185 190 TGT GTT TCC GAC TCG GAT GAT GGC GAA CGG CAG Cys Val Ser Asp Ser Asp Asp Gly Giu Arg Gin 200 205 GGG AGT CTA TTT AAG TCG AAG AAA CCC CGC TCC Gly Ser Leu Phe Lys Ser Lys Lye Pro Arg Ser 215 220 TA G 576 624 672 690 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

S

S

*5

S

55** Lys Leu Leu Leu 65 Gly Gly Leu Pro Giu 145 Leu Val Ser 50 Phe Gly Phe Trp Ala 130 Arg Pro His Val Ser Asp Tyr Val 115 Pro Gly Asn Ala Ile Leu Pro His 100 Leu Lys Val Ser Arg Val Lye Val Arg Pro Arg 70 Pro Gly Leu Ser Gly Thr Thr Phe Glu lle 150 Phe Thr Pro 55 Met Val Ser Ala Leu 135 Glu Met 40 Ala Cys Ser Gly Asp 120 Ile Leu 25 Ala Ser Arg Pro Ala 105 Ile Thr Ser Asp Ser Gly Ser Leu Pro Glu 90 Tyr Cys Glu Tyr Val ~75 Trp Ala Met Thr Asp Glu Ile Ser Ala Ala Gly 140 Arg Val Arg Gly Lys Ala 125 Gly Trp Lye G1u Pro Ser Thr Ser Gly Glu Phe Gly Glu Leu Asp Ala Glu Phe His 110 Leu Asn Leu Lys Asn Phe Thr Thr Leu Phe Leu Val Asn Gly Asp Lys Ser Leu Ser His Pro Ser Val Trp Thr 165 Leu Ala Pro Ser Ser Leu 175 Arg Asn Thr Ser Pro Ala 195 Trp 180 Al a Pro Tyr Tyr Ser Thr Val Lys 185 Cys Val Ser Asp Ser 200 Phe Leu Lys Val 190 Asp Asp Giy Glu 205 Lys Pro Arg Gin WO 96129396 PCT1US96103916 -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) FEAUSE LOCATION: 1. .380 (xi) SEQUENCE DESCRIPTION: SEQ 1D NO:14: ATG CGT AGC TCA GTT ACG TCA TTG TGG AGC CCTL TCA GAT CAC GCC TCT 48 Met Arg 5cr Scr Val Thr Ser Leu Trp Ser Pro Ser Asp His Ala Ser 1 5 20 TCG CCC GCA AAT GCC AAG CAT TTT TAT CAT ATT TCC GAT TTC CGG CGC 96 Ser Pro Ala Asn Ala Lys His Phe Tyr His Ile Ser Asp Phe Arg Arg 25 GCG GAA ACG GCG CCT GCG GGC GGT ACG GGC GCG CGA ACT GAG GT1' AAG 144 *Ala Glu Thr Ala Pro Ala Gly Gly Thr Gly Ala Arg Thr Glu Val Lys 40 :CGT CGC GCT TTC ACT TTC CCAGCG GCAGCG GTA CTC AGC GCAACT GAA 192 Arg Arg Ala Phe Thr Phe Pro Ala Ala Ala Val Leu Ser Ala Thr Glu 55 GCC CGA ACC GGC TCG TCT ATC ACC GGC TTA AAC CGT ACT CCG TCT GCA 240 Ala Arg Thr (fly Ser 5cr Ile Thr Gly Leu Asri Arg Thr Prc Scr Ala 70 '75 so ATA ATT TCC CTT OCA TGG TCC GAA ATG AGA AAT CTT AAG GAC CCC CTC 298 Ile Ile 5cr Leu Ala Trp 5cr Glu Met Arg Asn Leu Lys Asp Pro Lecu 90 G TCC CTG TCG CTG GMA ATA GCT TTA ACG AAT GTC TCT AAC TTT TCC 336 Gly 5cr Leu Ser Leu Glu Ile Ala Leu Thr Asn Val 3cr Asn Phe Ser 100 105 110 CTC TTG AGC TCA GAC CCC ATG GCC rrC GAA AAG TCT TCA TAT TG 380 Leu Leu Ser Ser Asp Pro met Ala Phe Glu Lys Ser Ser Tyr 115 120 125 A 381 INF ORMA&TION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 126 amino acids WO 96/29396 WO 9629396PCTIUS96/039 16 -121- TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein .00.

see* 0 4. .00.

met Ser Ala Arg Ala Ile Gly Leu (xi) SEQUENCE Arg Ser Ser Val Pro Ala Asn Ala Glu Thr Ala Pro Arg Ala Phe Thz 50 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 Lys His Phe Tyr His Ala Gly Gly Thr Gly 40 Phe Pro Ala Ala Ala 5 Ser Ile Thr Gly Leu 70 Trp, 5cr Giu Met Ara 90 Glu Ile Al1a Leu Thr 105 Pro Met Ala Phe Glu 120 NO: Pro Ser Ile Ser Ala Ar-g Val Leu Asn Arg Asn Leu Asn Val Lys Ser Asp Asp Thr Ser Thr

LYS

Ser Ser 125 Ala Arg Val Thr Ser Pro Plie INFORMATION FOR SEQ ID NO:16: Wi 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._876 (xi) SEQUENCE.DESCRIPTION: SEQ ID NO:16: ATG TGO TGT CGT TTG CAC TGG ATA AGT 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 GOT AGO GTT TTG CGA GGC CAG GOG TGT GCT CTG Pro Gly Scr Arg Thr Gly Arg Val Leu Arg Gly Gln Gly Cys Ala Leu 25 TGC ACT TTC TGG CAT CGT ACT CGA ACT CCG ACT ATA AAC CTC CGG TGC Cys Ser Phe Trp His Arg Thr Arg Thr Pro Ser Ile Asn 1Leu Axg Cys 40 WO 96/29396 PCTIUS96O39 16 -122.

CGC GCT CGG OT CTG ACT AAT TTC CGG CTC TGC GCC CAG ACT CC OT 192 Arg Ala Arg Gly Lieu Ser Asn Phe Arg Lieu Cys Ala Gin Ser Pro Gly 55 GAA AGO CAC AO TTC OT ACT CGG ACT CTG AGT CAA CAC CTC COG CTC 240 Giu Arg His Arg Phe Gly Thr Arg Thr Leu Ser Gin His Leu Arg Leu 70 75 TOT ACT CGG ACT CTG ACT AGC TTT COO TAC COT ACT COG 0CC CTG AGT 288 Cys Thr Arg Ser Lieu Ser Ser Phe Arg Tyr Arg Thr Arg Gly Leu Ser 90 GAA AAA GTO TOT TTC ACT ACT CTG AGT TCG CAT AGT GTC COG CTC GGC. 336 Oiu Lys Val Cys Phe Ser Thr Lieu Ser Ser His Ser Val Arg Lieu Gly 100 105 110 0ACT CGA ACT CTG ACT AAA GGC CTC AGT TCC CGC OCT CTG ACT CCC AGT 384 *Thr Arg Ser Leu Ser Lys Gly Lieu Ser Ser Arg Ala Leu Ser Pro Ser 000115 120 125 *AAA AAT COC COG TTC ACT ACT COA ACT CAG ACT ACT TTT COG TAC COT 432 s.Lys Asn Arg Arg Phe Ser Thr Arg Thr Gin Ser Ser Phe Azg Tyr Arg S.*130 135 140 @0OCT COO GOT CTG ACT AAA CAC CTC COT TAC COT ACT CGA ACT CTG TOT 480 Ala Arg Oly Leu Ser Lys His Leu Arg Tyr Arg Tkxr Arg Thr Leu Cys 145 150 155 160 *AAA AAC CTC COO CCC COC OCT COG AOC OCO AGC GOT TTC 000 000 COT 528 *00Lys Asn Leu Arg Arg Arg Ala Arg Ser Ala Ser Gly Phe Gly Oly Arg 165 170 175 OCT ACG AGA CTO ACT AAA TAT CTC 000 TAT COT OCT COG GOT CTG GGC 576 Ala Thr Arg Lieu Ser Lys Tyr Leu Oly Tyr Arg Ala Arg Gly Lieu Gly @00180 185 190 AGO TOC CTC GOT TTC TOC ACC COG ACT CTC ACT AAA ACT CAT CTG TTC 624 Arg Cys Leu Gly Phe Cys Thr Arg Ser Lieu Se: Lys Ser His Lieu Phe *0195 200 205 *seAGC ACT COO ACT CTG ACT AAA CAA COC CTC COT TTC TOC OAT CTC CGT 672 **be Ser Thr Arg Ser Leu Ser Lys Gin Arg Leu Arg Phe Cys Asp Lieu Arg 210 215 220 CTG AGT AAO AOC CCC CTC TTC ROCT ACT CGG ACT CTG ACT AAA ATA CCA 720 Leu Ser Lys Ser Arg Leu Phe Se: Thr Azg ser Leu Ser Lys Ile Pro 225 230 235 240 COG TTC CTG ACT CTG OGA CCO CCC GOT TTC CGA CTC GOT ACT COG ACT 768 Arg Phe Leu Thr Leu Oly Pro Arg Cly Phe Arg Leu Gly Thr Arg Thr 245 250 255 CTC ACT AAA GAC CAC COT TTC TOC ACT CC OT CTG TOT ACT TTC ATG 816 Leu Se: Lys Asp His Arg Phe Cys Thr Leu Oly Leu Cys Ser Phe met 260 265 270 TOC CGC GCT COO GOT CTC GOT AGA AAT CCC COG COC! GOT COT AGO AAA 864 Cys Arg Ala Arg Cly Lieu Oly Arg Asn Pro Arg Arg Oly Arg Arg Lys 275 280 285 CAG TOT ATT TTC TO A 8'79 Gin Cys Ile Phe 290 IN~FORMATION FOR SEQ ID NO:17: WO 96/29396 PCTIUS96/03916 -123- SEQUENCE CHARACTERISTICS: LENGTH: 292 amino acids TYPE: amino acid TOPOLOGY; linear (ii) MOLECULE TYPE: protein 0 *5 met Pro Cys Arg Glu cys Glu Thr Lys Ala 145 Lys (xi) SEQUENCE Trp Cys, Arg Leu Gly Ser Arg Thr Ser Phe Trp His Ala Arg Gly Leu so Arg His Arg Phe Thr Arg Ser Leu Lys Val Cys Phe 100 Arg Ser Leu Ser 115 Asn Arg Arg Phe 130 Arg Gly Leu Ser Asn Leu Arg Arg 165 Oly Arg Ser Gly 70 Ser Ser Lys Ser Lys 150 Arg Arg Thr Asn 55 Thr Ser Thr Gly Thr 135 His Val Arg 40 Phe Arg Phe Leu Leu 120 Arg Leu Leu Tkir Arg Thr Arg Ser 105 Ser Tkxr Arg Arg Pro Leu Leu Tyr 90 Ser Ser Gln Tyr Gly Gin ser Ile Cys Ala Ser Gin 75 Arg Thx His Ser Arg Al a Ser Ser 140 Arg Thr 155 Gly Asn Gin His Arg Val1 Leu 125 Phe Axg DESCRIPTION: SEQ ID NO;.17: His Trp Ile Scr Pro Arg Pkie Ser Ile 10 Met Arg is Ala Leu Arg Cys Pro Gly Arg Leu Leu Ser Leu Gly Pro Ser Tyr Arg Leu Cys 16D Ala Arg Ser Ala Ser Gly Phe Gly Gly Arg 175 Ala Thir Arg Leu Ser Lys Tyr Leu 190 Gly Tyr Arg Ala Arg Gly Leu Gly 185 190 Arg Se r Leu 225 Arg Leu Cys Gin (2) Cys Leu Gly Phe Cys Thr Arg Se 195 200 Thr Arg Ser Leu 5cr Lys Gin Ar 210 215 Ser Lys Ser Arg Leu Phe Ser Tkh 230 Phe Leu Thr Leu Giy Pro Arg GI 245 Ser Lys Asp His Arg Phe Cys TI 2602 Arg Ala Arg Gly Leu Gly Arg A 275 290 Cys Ile Phe 290 INFORMATION FOR SEQ ID NO:18: r Leu ser g Leu Arg ~r Arg Ser 235 y~ Phe Arg 250 'ir Lou Gly an Pro Arg Lys Ser His Leu 205 Phe Cys Asp Leu.

220 Leu Ser Lys Ile Leu Gly Thr Arg 255 Let' CysSe5r Phe 270 Arg Gly Arg Arg 285 Phe Arg Pro 240 Thr Met Lys PCTIUS96/03916 WO 96/29396 -124- (j

ATG

Met 1

CCT

Pro

CGC

Arg

AGA

Arg

ATT

Ile

ACC

Thr

CAC

His

CGT

Arg

CAC

His

CC

Al1i 14!

GT(

Va.

TC

Se Wi SEQUENCE

CHARACTERISTICS:

LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY; linear ii) MOLECULE TYPE: DNA (genomic) .ii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: No (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 1. .533 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CTC CCA AGC CTA CTC AAC AGG GGC TC? CCC CGG CTG AAT TCT CCT Leu Pro Ser Lou Leu Asn Arg Gly Ser Pro Arg Leu Asfl Ser Pro 5 10 AAG TGT TCA GAG GCC TCT GC CTA CCA TAT AAC TAT CGT GTA GTA Lys cys Ser Glu Ala Scr Ala Val Pro Tyr Asn Tyr Arg Val Val 25 CCC TCC CAC TCC GTG TCC GA? ACT GCC CCT TTT GAG AGG AT? CCC Pro Ser Gln Ser Val Ser Asp Thr Ala Pro Phe Glu Arg Ile Gly 40 TTA GAG ART CGA AAT GAT TCG AGA CCC ACA TTC AGA CTT ART CAC Leu Glu Asn Az'g Ann Asp Trp, Arg Ala Thr Phe Arg Leu Asn His so 55 TTT AT? GAG TCG CCC GAG CTT ACC CCA GAC GGG TTA ACA ATC GCA Phe Ile Glu Ser Gly Glu Leu Ser Ala Asp Cly Lou Thr Ile Ala 70 75 s0 ACT TCC ACA AG? TCA CTA TCC TGG TCA C CCC TTC TTT AT? TCG Ser Ser Thr Ser Ser Lou Ser Trp Scr Ala Pro Lou Phe Ile Ser 90 GCA ACC ATC CC? CCA ART TT CCC GAT TCC CT? CTA CTT TOG CAR Ala Thr Met Cly Pro Asn Phe Arg Asp Ser Leu Leu Val Trp Glu 100 105 110 TCT TCG TCG TCT TG' GAG ACC GTG TCT RAT TTT CGG TGC GCC GTG Ser 5cr Ser Ser Cys Glu Thr Val Ser Ann Phe Arg Cys Gly Val 115 120 125 ATG TTr CTG GTG ACG ATG GAA AT? RCA ATO ACG AGO CCC ATC CT? Met Phe Leu Val Thr Met Glu Ie Thr Met Thr Arg Pro Ile Val 130 135 140 CTC ACO ACO CCA GCC ACG CT? ACC CCA AT? AGC GTA CCC CTC AT? Leu Thr Thr Ala Ala Thr Val Thr Pro Ile Ser Val Gly Lou Ile 5150 155 160 CCCG AGA COG ACA OTA RCC TTT GAA TT TCG TTT GC CC? GTC G 1 Pro Arg Arg Thr Val Thr Phe Glu Phe Ser Pkxe Ala Cly Val Gly 165 170 175 G TA A r 48 96 144 192 240 288 336 384 432 480 528 WO 96/29396 WO 9629396PCTfIUS96IO39 16 -125- INFORMATION FOR SEQ ID NO:19: Wi SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met Pro Arg Arg Ilie 65 Thr His Arg His Ala (xi) SEQUENCE Leu Pro Ser Leu Lys Cys Ser Glu Pro Ser Gin 5cr Leu Giu Msn Arg 50 Phe Ile Glu Ser Ser Ser Thr Ser Ala Thr Met Giy 100 Ser Ser Ser Ser 115 Met Phe Leu Val 130 Leu Thr Thr Ala Ala Vai Msn Giy 70 Ser Pro Cys Thr Al a 150 Ser Ala Ser Asp 40 Asp Trp, 55 Glu Leu Leu Ser Asn Phe Giu Thr 120 Met Glu 135 Thr Val Val 25 Thr Arg Ser Trp Arg 105 Val1 Ile Thr Pro Al a Al a Ala Ser 90 Asp 5cr Thr Pro Tyr Pro Thr Asp 75 Ala Ser Asn Met Ile 155 Msn Tyr Phe Glu Phe Arg Gly Leu Pro ILeu Leu Leu Pkxe Arg 125 Thr Arg 140 Ser Val Arg Arg Leu Thr Phe Val.

110 Cys Pro dly DESCRIPTION: SEQ ID NO:19:.

Leu Asn Arg Gly Ser Pro Arg Leu Asn Ser Pro Val Val Ile Gly Asn His Ile Ala so Ile Ser Trp Glu Gly Vai Ile Val *Leu Ile 160 *Val Gly 175 Val Pro Arg Arg Ser Val. Thr Phe Glu Phe ser Phe Ala Gly 170 INFORMATION FOR SEQ ID Wi 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 GAATTCGAGC TC!GGTACCCG GATAATACGT ACATGTTAAC GCAGAGGT WO 96/29396 C[S9/31 -126- INFORMATION FOR SEQ ID NO:.2i: Wi SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid CC) 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: Ci) SEQUENCE CHARACTERISTICS: WA LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genom~ic) (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: Wi SEQUENCE CHARACTERISTICS- LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 96/29396 PCTIUS96/03916 -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 Ci) SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) Ciii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID GGTTTGGCGG AGCGGATATG ATCTCGACCT GCAGTGAATA ATAAAATGTG T INFORMATION FOR SEQ ID NO:26: Wi SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single CD) 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: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs CB) TYPE: nucleic acid STRANDEDNESS: single CD) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) Ciii) HYPOTHETICAL: NO (iv) ANTI-SENSE: No WO 96129396 PCTIUS96/039 16 -128- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: AAGCTTGGCG TAATCATG 1 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 3 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 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 STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) Ciii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID 140:30: TTGCTGCGTT CCCGGGGATC CTCTAGAATT AGGTAGTTTG TAGTGCGA 48 INFORMATION FOR SEQ ID NO:31: Ci) SEQUENCE CHARACTERISTICS: WO 96/29396 PCTIUS96103916 -129- (A 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 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 FOPR SEQ ID NO:34: Wi SEQUENCE CHARACTERISTICS: LENGTH: S7 base pairs TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 96/29396 PCTIUS96/039 16 -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 Wi SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genornic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID TCGAGGGGAT CCTCTAGAGT CGAGGGACCC ATGGTTGCGT GC 42 INFORMATION FOR SEQ ID NO:36: Wi SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) M4OLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: TTTACTAAAG CGCGGCGAAA GCTTCGTCGT GCTGGGTTCT OG 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 PCTIUS96103916 -131- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: AAGCTTGGCG TA.ATCATGGT C 21 INFORMATION FOR SEQ ID NO: 38: Wi SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NC (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: GGAATTCGAG CTCGGTACCC GGATAATACG TACATGTTAA CGCAGAGG 48 INFORMATION FOR SEQ ID NO:39: Wi 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 SEQUENCE DESCRIPTION: SEQ ID NO:39: ATCTATTGGA GCGTTTAGCG CGCGTCGACG AAAGGTCAGA GACGA INFORMATION FOR SEQ ID Ci) 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 GOGAACG 27 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: WO 96129396 PCTIUS96/03916 -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 STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (geflomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: ATCAGAAACT CTTTCGGTAC CGAGCTCGAA TTC 33 INFORMATION FOR SEQ ID 140:43: 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 140:43: GAATTCGAGC TCGGTACCCG GATAATACGT ACATGTTAAC GCAGAGGT 46 C2) INFORMATION FOR SEQ ID 140:44: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 96/29396 PCTIUS96/039 16 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 1xi) 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 (genoniic) (iii) HYPOTHETICAL: NO ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID *CGTTCCCGGG GATCCTCTAG AGTCGACGGC AGAGTCGCAG AC 42 INFORMATION FOR SEQ ID 140:46: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) :(iii) HYPOTHETICAL: NO 'Goo.,(iv) ANTI-SENSE: NO0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: TGATCCAAAC TCGGATCCTC TAGAGTCGAC INFORMATION FOR SEQ ID NO:41: 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 PCTfUS96/03916 -134- AAGCTTGGGC TGCAGGTCGA CTCTAGAGGA TCCCCTCGAC GTCTGGGG 48 INFORMATION FOR SEQ ID NO:48: Wi SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRAI4DEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO SEQUENCE DESCRIPTION: SEQ ID NO:48; CACACCTTTG CGCATCTCCA CAGCTCAACA ATGAATTCCA TGTTACGTCC TGTAGAAACC INFORMATION FOR SEQ ID 140:49: Ci) SEQUENCE CHARACTERISTICS- LENGTH: 60 base pairs TYPE: nucleic acid goo.CC) STR.ANDEDNESS: single CD) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genoniic) Ciii) HYPOTHETICAL: NO 0 (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO0:49: CAGGGAGGCA AACAATGAAT CAACAACTCT CCCGGGAGAT GGGGGAGGCT AACTGAAACA INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: CA) 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 140:50: TGCTGCGTTC CCGGGGATCC TCTAGAGTCG ACCTGCAGCC CAAGC INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: WO 96129396 PCTIUS96O39 16 -135- LENGTH: 48 base pairs TYPE: nucleic acid STRAiNDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE. DNA (genomic) Ciii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: TCTAGAGTCG ACCTGCAGTG A)LTA)ATAAAA 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 (geflomic) (iii) HY1POTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: CTCCATAGAA GACACCGGGA CCATGGATCC CGTCGTTTTA CAACG INFORMATION FOR SEQ ID NO:53: i) SEQUENCE CHARACTERISTICS: LENGTH! 105 base pairs CB) TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA igenoamic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NIO (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 CHAR.ACTERISTICS: CA) LENGTH: 36 base pairs TYPE: nucleic acid CC) STRANDEDNESS: single CD). TOPOLOGY: linear WO 96/29396 PCTI[US96O39 16 -136- (ii) MOLECULE TYPE: DNA (genornic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: CCGTCGAGAT CCTCTAGAGT CGACCTGCAG GTCGAC 36 INFORMATION FOR SEQ ID NO:SS: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE:-nucleic acid STRANDEDNESS: single CD) 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: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genernic) *(iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: CGCCTCGAGT CCCAATGAAT AGGCATTGG 29 INFORM4ATION FOR SEQ ID NO:57: Ci) 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 96129396 WO 9629396PCfUS96103916 -137- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:.

CGCCTCGAGG ACCCATGGTT GCGTGCG INFORMATION FOR SEQ ID NO:S8: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomie) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: CTCGTCCGAA CGAGTTACAG INFORMATION FOR SEQ ID NO:59: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 19912 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE:.DNA (genomic) (iii) HYPOTHETICAL; N (iv) ANTI-SENSE: N (ix) FEATURE:

NAME/KEY:-CDS

LOCATION: 697. .1533 CD) OTHER INFORMATION: (ix) FEATURE: NAME/KEY:. CDS CB) LOCATION: complement (1900. .2784) CD) OTHER INFORMATION: (ix) FEATURE: CA) NAME/KEY: CDS LOCATION: complement (2916. .3605) CD) OTHER INFORMAATION: (ix) FEATURE.

NAME/KEY: CDS LOCATION- 3694. .5124 CD) OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: 5210. .7081 CD) OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: 7245. .8123 CD) OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: 8333. .11290 CD) OTHER INFORMATION: (ix) FEATURE.

NAME/KEY: CDS WO 96/29396 WO 9629396PCTIUS96/0391 6 -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: 15299. .16080 OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: 16129. .17013 OTHER INFORMATION: (ix) FEATURE: NAME/KEY: CDS LOCATION: complement (17380. .19216) OTHER INFORMATION: (xi) SEQUTENCE DESCRIPTION: SEQ ID NO:59:

C

0 0* GGATCCCGAA GAGCTCTCCC GGCACGCGGG AGGAGCTGGA AGTGGACTTG GGCGAGGAGG TGACTTAGAC TTTATGACG CCGTGCGTrA TTGCGGTGCG GCGGGTAGGC GCACTCTTAA TCCGAGGAGA GAAGTAAAAA GAGGCCTCGA CGAGCAGATA CCTACTCTGG ACATTTATTG AGCCTTCACG CAGAAGCGGA CCCCCTTCAC GCCCGGCGCA AGAA3TTTT

GCGACCCGGA

ACACGGGACA

ACAGCTCTCC

CGGCACCCGC

AAGACGGAGG

AGAAGAAGCG

GAGGAGACGC

CCCGCTCGAT

CGCGCGCCCC

ATCCGTAGCC

CTTTTCGGAC

GTCGGAGGAA

CAGCTCCACC

GGCGCCGCCG

AAAGACCCAC

TTGTCAT!-rT

GCCTCGACCC

GGGGCAGAAC

CCATTCTCAT

TY'TGCGACCG

GTCCAACTCG

GTATCGGAGG ACGAAGAACC

GAGCAGCCTG

GAGTCAGAGC

CCATTTGCTA

GGAAGGCTTC

CTrCTTCCTC

CTGCACGGCG

CTCCCCC!TCC

CCAGAACTTC

CCGGACATCC

GCCC.AGCACA

GGTGCCGGGG

CCACGCAATC

TCCCCCGCGT

GGCCGCCAGG

TGACGAGGAA

ACGCAAGGCC

CTCCCACCCC

T-ACCCGCTC

CGCCGCCCCC

ACCGCAGTAG

120 180 240 300 360 420 480 540 600 660 714 762 810 958 906 ACCGCCCGGA CCGCTCTCCT CTAGACACAT CCCTAA ATG GAA AAC ATG CTC GAC Met Glu Asn Met Leu Asp 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 Amp His Ile Thr Ala His i5 GCG GTA CCT CGT GGC GAG CGC AGG CGG CAA GGT GCC GCT GTC GCC TCG Ala Val Pro Arq Gly Glu 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 CGQ Ser Glu Set Ala Asp Set Val Asp Pro Cys Ile Arg Ile Ala Ser Arg 45 CTC TGG CGC GAG TTA GTc GAG ATA TCG TCC GAA CTC AAG QAC GOT TAC Leu Trp Arg Glu LeU Val Glu Ile Ser Set Giu Leu Lys Asp Gly Tyr 60 65 WO 9629396PCT1US96/03916 -139- GGA GAG TTC ACG TCA GCG AGA GAC CGC CGC AAC GCG CTG ATT GCT GCC 954 Gly Giu Phe Tkir Ser Ala Arg Asp Arg Arg Asn Ala Leu Ile Ala Ala 75 8085 AAC GAA CGG CTA CGT TCG GCT TT CTG GGG CCC AGC CGG GCG ACG CGC 1002 Ann Giu Arg Leu Arg Ser Ala Pkie Lou Gly Ala Ser Arg Ala Thr Arg 95 100 GGC C'TA GGT TTG AGG CCG CGG TGG OCG TCG ACG GAG AGC GTC GCC AAC 1050 Gly Lou Gly Leu Arg Pro Arg Trp Ala Ser Thr Glu Ser Val Ala Asn 105 110 TCC CCC ACT GAC CCG AAT AAC GGC AMC GGG TrG GGA GAA TTA GAG GAG 109B Ser Pro Thr Asp Pro Asn Asn Gly Ann Gly Lou Gly Glu Leu Giu Glu 120 125 130 GCA ATG GAA GGG ATC GAG GGC GAT TTC TGG CTC GAC TCT CTG GAC GGT 1146 :*:Ala Met Glu Gly Ile Glu Gly Asp Phe Trp Leu Asp Ser Lou Asp Gly 135 140 145 150 *GAC CGC TTC GAG GAC GAG ACC CG? ACC ATG CAG AGC GAG MAT ATG CGT 1194 ***Asp Arg Phe Glu Asp Giu Ser Arg Thr Met Gizi Ser Giu Ann Met Arg .**155 160 165 *TTC GTG ATC GAG AAA GMA CTG TTA TCC TO CTG TCC CGA CAC CTG CCG 1242 Phe Val Ile Glu Lys Giu Leu Lou Ser Trp Lou Ser Arg His Leu Pro 170 175 180 GCC CAC CTC GCG TCC GCC GAG CGA GAG ACC TCC CGG TCT CTC CTG GCG 1290 Ala Asp Lou Ala Ser Ala Giu Arg Giu Thr Ser Arg Ser Lou Leu Ala 185 190 195 GCC GGC CAC TOG TGC TGC TTG TGG CAC CCT CGG CCG TGC CCC GM. GCG 1338 Ala Gly His Trp Cys Cys Lou Trp His Pro Arg Pro Cys Arg Glu Ala 200 205 210 TGT TTG TAC CAC TCG ATT TAC GTG CAG ACT CT? TTC TGC GTC CCC ACG 1386 Cys Leisu Tyr Asp Ser le Tyr- Val Gin Ser Leu Phe Cys Val Gly Thr 215 220 225 230 GGCG AGA CTC CCG CAA TCG GAG ATG CGC COT COC GMA TAC CTG CC GCC 1434 Cly Arg Val Pro Gin ser Glu Met Arg Arg Arg Oiu Tyr Lou Ala Ala 235 240 245 TTG CCC CCC CC GCG GC GCC GCC AAC TCT CCC GMA CTG AGC GCC TCG 1482 Lou Arg Ala Gly Ala Ala Ala Ala Asn Ser Pro Glu Val Ser Ala ser 250 255 260 ATC TTT GCG AGO GAC GC GGA AT"' OCG CTG GCC; CTG GCG CCC CGC COT 1530 Ile Phe Ala Arg Asp Ala Gly Ile Ala Lou Al a Leu Ala Arg Arg Arg 265 270 275 TGA CGGGAGAATG ACGCCCCTA GCGGCrrCCT TACCTCCGCG TCCCTCACAA. 1563 CCTCGCGOGT Tz-rTACACTG TCCTCCGTCC ACTCTCCCCC CTCACCCACT CCGCGGCAOC 1643 GAAACACAAC CCCCCCCCCC CCCCAQAAAC GAGCGACACG CGAGCGCTGC GAAATAAATA 1703 AAGTAATATT ATITGTGT=T TTTCACGTTG TTGCAATCGA GAGGCCGTrT GTCTGTCTGT 1763 GTCTGTGCGG AGCTAGGCTT TCCCGGOCGG CCCCGTTCCA CCGTTCGGTT AGGCCGOTGG 1823 CGACGGGACA TAGAGAAAGA TAGAGCGCGC GCCCTGGCGG CGILGAGGTO TTGCGGGT 1883 AAATGGGACC CTGAGCTCAC CATTTTGGCG GGOATTICCA CGGGTAACAA AAAGCTCTCT 1943 WO 96/29396 WO 9629396PCTIUS96/0391 6 -140-

CGCACATAAT

CGTCCGGAGA

GGATCGAGTT

GCGCTATCAC

CGGCCGGCGC

CTCTGATATA

CAATGACTGG

TGCCCCAGAC

GATI-TCCCTT AAACAGTGGC CATGATCTTA TCGOTAGCTA TTGGGAACCT TCGGGGAGGT CAGGAGGCTC CGCACCATCT GCTGCTCTGG TTATTCCAGT GAGCACCQQC AGCTCGACGG AACTTTGGGC ACGTCTCTTA CrrGCTTTCC AACTCCCCGT 0* 0O S

S

@050

S

0000 0e 0 0*O 0*

S

S.

OSOS

S

0@S* TCCACAGTCC TACCGCCGCT( GCGCCGTGAT ATCTTCGGCG CGCGGGCGGC CTGTACGATC GCGACGCCCG CCAGAATCTCI CTAAGTCCAA ATGTQACGTCI GCCGGCCCAC GCAACGCGGGI TCCGCGTACG CGTTTATTAT' GTTTCTGCAA GGCCCTCGCC CGAGCGTCGC CGCTAACG;TC CCCGAGAAAA AATTTTGGCT CGAGTTAGGT TTTACTTTTA CGAAGGGGCA AGAGTTGTCC GTTTACCAAA AATATTTCCA GAAAGTTTT GGCGCAGGGA CCACAAATGG AACTCTTTG CAAGCCGCTC CACTCGGGAC GGGTCTGCAC ATCCTGGGAA% GCGCACGATT ACCGATAATG AACCAATTCT TTCCATCCAG

ICGACGGCGC

LCCTGCAGAC

XCCGCGG CCA

:CTCCGGGCA

;GAGGTCTCG

TTTTATAAA

rGTcAATATT

CGGCCCAGG

;vGCGCCGCGG

"CCG=CGCC

kAAACTTTAC

PGACTGATGG

CTCCTCTCTC

0G1-rAAAGC

CGGCATACGC

TTACTCCAGG

GGGAAAACAC

TACTCGGACC

AATCCGAGAC

TACTCACTr CAGAAATATj

TGTAAAAGCT

CACAGTTCAT

CTTCCQGGAG

CCATGCCTAT

GCGCGGACCG

CGGCGGAGAA

7TCCCACGC

TCGGCCCCCC

GCTTAGCCGC

TGCCCAGCCT

GATCGGGCCA

GGTCC=TCC

ACATGGTCGC

GATGGCGCGC

TGTGTGGTI'A

CCACTATI'CC

GGAGCGGGGT

ATCATCCGAG

CGTGCTGTAC

GTGACTCAGA

AAAAT-rrA

TGCCATGCAT

GCCAGATGAC

CGGGTCGCCC

CTCCCCGGAA

*ATCGTAACTC

CTCAAATCT;

r TACAGGCGC( k ACGCTCTTrA TTCTTCGACT GGCACGCGCA CAGGTGGGCC ACGAACGCGC

GGTGAAGTTT

CCTTATCOCC

CGAGTGCGCC

AAAAGAAAGA

GGCGGCCCGG

AACTAACTCC

GCGGCTATT

TTCCTTCCCT

AAAGAAAATA

CCTAAAGGCC

CAACCCTCCA

GAGACAATAA

TTATTACTGC

GGCAGCGGCC

TTCTTCGACT

TCGGAAACAC.

GGCCAGGGCG

GACAGCGTG

cCTccGGrT

ATGTCAGCGG

AAATGGTAAA

CCCTCACCGA

ACTTCGTACA

GCCATAGTTT

kGAkrAGGTA

;ACAGAGGCA

GCGAGTCCGG

CCTCCCCGGG

%TGTCCGGCC

;GAATCTGCT

;ACAGCGCGG

CGCGGGTCGT

rCAAAATACG

TCGCAACTCT

GCCGAGAA6AG

P.ATGCTACCC

CACTIACTCA

TACCGCCCTT

GCCGACGCGG

TAAATAG CT

PIGTATGCGGC

TTCTCAGGCr

TCTTGTCTCC

CGGTAATTAG

TACCCA6GCAC

ACCCTGCGTC

ACCGAATCAC

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 00 S. S 0 055S 5

OS''

CGAATCGACC

ACTCCACCCC

GCAGAAACTA

GCTATGATGT

ATCGCCGCTCAG

TCTAGCAGAG TCAGGCCAAT ATATACAGCT TAGAG AAG ATG CCC ?TT CGZC CGC ATC TGT TCA CCC TCT AGG Met Arg Pkie Arg Arg Ile Cys Ser Arg Ser Arg 1 5 GCA GAA AAA CGA AGA AGA ACA ACC GAG AMT CCG CT1' 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 9629396PCr/US96O3916 OTT TGC GTA TTG OAT ACT Val Cys Val Leu Asp Ser -14 1- TTC TCA COO ACA ATG TCA TTG CGC CCC TAT Phe Ser Arg Thr Met Ser Leu Arg Pro Tyr 35 3822

GCA

Ala

GTT

Val

OCT

Ala

TAT

Tyr

ACA

Thr

C

Ala

OTT

Val1

GAA

Glu

ACT

Ser

GTA

Val

GCA

Ala

GAC

Asp

CAA

Gin 125

TTA

Leu TTG CCC Leu Pro ACA ATG Thr Met ACT ATC Ser Ile so GAT CG Asp Oly GAT CAA Asp Giu AGA TTG Arg Leu OAT TTO Asp Leu I. I ACC GCG GAA GOC GTC GAG CGC CTC GCC GAA CTT Thr Ala Giu Giy Val Giu Arg Leu Ala Giu Leu 50 ACA GAA CGC GCG GAA CCT GTG ACA GAG AAT ACA Thr Ciu Arg Ala Giu Pro Val Thr Giu Asn Thr 65 70 CCC CCC OCT AAC GAG AAC G CAG AAC TTC OCA Pro Pro Ala Asn Ciu Asn Gly Cia Asn Phe Ala 85 CCC TCG ACT ACT GAA AAA OTT GAC COC TCG CAT Pro Ser Thr Thr Giu Lys Val Asp Oly Ser His 100 105 GCA TCG AGC GAC TAC GCC GOC CCT CTC CCC CTC Ala Ser Ser Asp Tyr Ala Gly Pro Val Pro Lou 115 120 AAC CAT TCC CAT GAA TTT CTT CAG CAC TTC CGA Lys His Ser Asp Clu Pkie Lou Gin His Phe Arg 130 13S OTO GAO GGG OCT TAC G Trr ATC TOC CAC OTC Val Oiu Oly Ala Tyr Gly Phe Ile Cys Asp Val 145 IS0 155 GAA GAG CAA CGT CGA AGA COO OTT AAC ACT ACT Giu Giu Gin Arg Arg Arg Oly Val Asn Ser Thr 165 170 AAA TOT AAC CCC CTC ATA GCT AAA TAT GTG AAA Lys Cys Lys Arg Lou Ile Ala Lys Tyr Val Lys IS0 195 CCC TAC ACC GAG Tyr

G

Gly Thr

AAA

Lys 175 Giu 160

TCA

Se r 3870 3918 3966 4014 4062 4110 4158 4206 4254 4302 4350 4398 4446 4494 4542 4590 AAT GGA ACA AGO 0CC GCC TCT CAC CTG GAA AAT OAA =TT TTGM OTT CTC Asn Cly Thr Arg Ala Ala Ser Gin Lou Ciu Ann Giu Ile Lou Val Lou 190 195 200 000 CGC CTA AAT CAC GAO AAT G= CTC AAG ATC CAG GAA ATC CTT CGG Gly Arg Leu Asn His Ciu Asn Val Leu Lys Ile Gin Glu Ile Leu Arg 205 210 215 TAC CCC OAT AAT ACG TAC ATG TTA ACG CAG AGG TAT CAG TTC GAC TTG Tyr Pro Asp Asn Thr Tyr Met Lou Thr Gin Arg Tyr Gin Phe Asp Lou 220 225 .230 235 TAC AGC TAC ATO TAC GAT GAA OCG TTC GAC TOG AAA GAC ACT CCA ATC Tyr Ser Tyr Met Tyr Asp Glu Ala Phe Asp Trp Lys Asp Ser Pro Met 240 245 250 CTT AAA CAC ACT AGA CGC ATC ATO AAO CAC CTC ATO TCA GCC OTC TCG Leu Lys Cmn Thr Arg Arg Ile Met Lys Gin Leu Met Ser Ala Val Ser 255 260 265 TAT ATC CAT TCA AAG AAA CTG ATT CAC AGG GAC ATC AAA CTC GAA AAT Tyr Ile His Ser Lys Lys Leu Ile His Arg Asp le Lys Lou Giu An 270 275 280 ATT TTC TTA AAC TGC GAC GCC AAG ACA GTC CTC GGC GAC TTr GGA ACT Ile Phe Lou Asn Cys Asp Gly Lys Thr Val Lou Cly Asp Phe Cly Thr 295 290 295 WO 96/29396 WO 9629396PCTIIJS96IO39 16 -142- GAA AAT GAG COG GAG CCC rrC GAA Giu Asn Giu Arg Giu Pro Phe Giu 305 310 GTC ACG CCT TrT Val Thr Pro Phe 300 TAT GGA TGG GTG Tyr Gly Trp Val 31.5 4638 0 0 OGG ACC Gly Thr TOT GAA Cys Glu GTA AOC Val Ser CAG CAA Gin Gin 365 TrC CCA Phe Pro 390 ATC OAT Ile Asp CTT CCG Leu Pro TOG CGT Trp Arg TCG OCT Ser Ala 445 CCC ATC Pro Ile

GTG

Val

ATT

Ile

CAT

His 350

TTG

Leu

GAC

Asp

CGC

Arg

GCG

Ala

TTG

Leu 430

GAA

Giu

CGA

Arg GCT ACT AAC TCT CCC GAG ATA CTC GCC AGG GAT TCG TAC Ala Thr Asn Ser Pro Glu Ile Leu Al a Arg Asp Ser Tyr 320 325 330 ACA GAC ATT TGG AGC TGC GGA GTA GTA TTG CTO GAA ATG Thr Asp Ile Trp Ser Cys Giy Val Val Leu Lou Giu Met 335 340 345 GAA TTT TGC CCO ATC GGC OAT GGC GGG GGA AAT CCG CAC Giu Phe Cys Pro Ile Gly Asp Oly Gly Giy Asn Pro His 355 360 CTG AAA 017r ATC GAC TCT CTC TCA GTr TOT GAT GAA GAG Leu Lys Vai Ile Asp Ser Leu Ser Val Cys Asp Glu Glu 370 375 CCC CCO TOT AAT CTG TAC AAT TAT '170 CAT TAT GCG AGC Pro Pro Cys Asn Lou Tyr Asn Tyr Leu His Tyr Ala Ser 38S 390 395 GCC GGA CAT ACG GTC CCG TCG CTC ATA CGG AAC CTC CAC Ala Oly His Thr Val Pro Ser Lou Ile Arg Asn Leu His 400 405 410 OAT OTG GAA TAC CCT CTA GTT AA.A ATG CTT ACT TTT GAC Asp Vai Giu Tyr Pro Lou Vai Lys Met Leu Thr Phe Asp 415 420 425 AGA CCC AOC GCG GCC GAA GTA TTO GCA ATG CCA CTO TT Arg Pro Ser Ala Ala Glu Val Leu Ala Met Pro Lou Phe 435 440 GAG GAA CGG ACC ATA ACA ATT ATT CAT GGA AAA CAT AAA Giu Giu Arg Thr Ile Thr Ile Ile His Gly Lys His Lys 450 455 CCC GAA ATC CGT GC CGG OTG CCA CGG TCC ATG AGT GAA Pro Giu Ile Arg Ala Arg Vai Pro Arg Ser Met Ser Glu 4686 4734 4782 4830 4878 4926 4974 5022 5070 5118 5174 5227 5275 5323 5371 5419 460 465 470 475 GGT TAA TAATAAAOOA CGGAGATAGA GAACTGAAGC GTCAGATTTT TTTAAAAAAA Gly.

TAAATGATCG AGAAC'17ATG ATTTGTCTTT C'17GA ATO ACC TTG CCC CAT CGA Met Thr Leu Pro His Arg 1 S TTA ACO AAA AGA CCT TrC GCG COT CGA TTC TGC TCG GTC TTT GTO ATA Leu Thr Lys Arg Pro Phe Ala Arg Arg Phe Cys Ser Val Phe Val Ile i5 CAT TAT AGT GAG ACT AAA CTC GAC CGA TAT AAC AAG ACA ATG TTA CT- His Tyr Sor Oiu Th r Lys Leu Asp Azg Tyr Asn Lys Thr Met Leu Lou 30 TAT AGA CCO GAC TCA ACC ATO COG CAT AGC GGA GOC GAC OCA AAT CAC Tyr Arg Pro Asp Ser Thr Met Arg His Ser Gly Gly Asp Ala Asn His 45 AGA G00 ATA AGG CCG AGO COG AAA TCT ATT GGA GCG TTT AOC GCG COC Arg Giy Ile Arg Pro Arg Arg Lys ser Ile Gly Ala Phe Ser Ala Arg 60 65 PCr[US96/03916 WO 96/29396 i. -143- GAA AAG ACT GGA AAA CGA AAT GCG CTG ACG GAA AGC AGC TCC TCC TCC Giu Lys Thr Gly Lys Arg Asn Ala Leu Thr Glu Ser Ser Ser Ser Ser 80 GAC ATG CTA GAT CCG TTT TCC ACG GAT AAG GAA TTT GGC GOT AAG TGG Asp Met Leu Asp Pro Ph. 5cr Thr Asp Lys Glu Phe Gly Gly Lys Trp 95 100 ACG GTA GAC GGA CCT GCC GAC ATT ACT 0CC GAG GTC CTT TCT CAG GCA Thr Val Asp Gly Pro Ala Asp Ile Thr Ala Glu Val Lou Ser Gin Ala 105 110 115 TGG GAC GTT CTC CAA TTA GTG AAG CAT OAA GAT OCG GAG GAG GAG AQA Trp Asp Val Leu Gin Leu Val Lys His Olu Asp Ala Glu Giu Giu Arg 120 125 130 GTG ACT TAT GAG TCC AAA CCG ACC CCG ATA CAG CCG TTC AAT GCC TG Val- Thr Tyr Giu Ser Lys Pro Thr Pro Ile Gil Pro Phe Asn Ala Trp 135 140 145 150 CCG OAC 000 CCC AGT TOG MAC OCO CAG OAT TTT ACT CGA GCG CCA ATA Pro Asp Gly Pro Ser Trp Asn Ala Gin Asp Ph. Thr Arg Ala Pro Ile 155 160 165 OTT TAT CCC TCT OCO GAG GTA TTG GAC GCA GAG 0CC TTG AMA OTA G Val Tyr Pro Ser Ala Giu Vai Leu Asp Ala Glu Ala Lou Lys Val Gly 170 175 180 OCA TTC GTT AGC CGA OTT TTA CAA TGT GTA CCG TTC ACG CGA TCA AAG Ala Ph. Val Ser Arg Val Leu Gin Cys Vai Pro Phe Thr Arg Ser Lys 195 190 195 AMA AGC OTT ACG OTO COG GAT CCAG TCC TTT TTYGGG0 GAC TCG TTC Lys Ser Val Thr Val Arg Asp Ala Gin Ser Phe Leu Gly Asp Ser Phe 200 205 210 TGG AGA ATA ATG CG A C GTI' TAC ACO OTT CTC TTA CGA CAG CAC ATA Trp Arg Ile Met Gin Msn Val Tyr Thr Val Val Lau Arg Gin His Ile 215 220 225 230 ACT CGA CTC AGO CAC CC? TCC AGC AMA AGC ATT OTT AAC TOC MAC GAC Thr Arg Leu Arg His Pro Ser Ser Lys Ser Ile Val Asn Cys Asn Asp 235 240 245 CCT CTA TOG TAC 0CC TAC 0CC MAT CMA TTT CAC TOG AGA GGA ATG COC Pro Leu Trp Tyr Ala Tyr Ala Msn Gin Phe His Trp Arg Gly Met Arg 250 255 260 GTG CCO TCO CTT AMA TTA-GCC TCT CCC CCC GAG GAG AAT AT? CAA CAC Vai Pro Ser Leu Lys Lou Ala Ser Pro Pro Ciu Giu Asn Ile Gin His 265 270 2"75 GGC CCA ATO 0CC 0CC OTT TTr AGA MAC GCG GOG OCT GOT CTO TTC CTG Gly Pro Met Ala Ala Val Ph. Arg Msn Ala Gly Ala Gly Lou Phe Lou 280 285 290 TOG CCT GCC ATO COC GCA 0CC TTT GMA GAG COC GAC AMG CGA CTG TTA Trp, Pro Ala Met Arg Ala Ala Phe Giu Giu Arg Asp Lys Arg Leu Leu 295 300 305 310 AGA OCA TGC CTG TCT TCA CTC GAT ATC ATG GACGOCA 0CC GTC CTC GCG Arg Ala Cys Lou Ser Ser Leu Asp Ile Met Asp Ala Ala Val Leu Ala 315 320 325 TCG TTT CCA TTT TAC TOO CCC GGC GTC CAA GAC ACC TCO COC TTC GAG Ser Phe Pro Phe Tyr Trp Arg Gly Val Gin Asp Thr Ser Arg Phe Oiu 330 335 340 5467 5515 5563 S6 i1 5659 5707 5755 5903 5851 5899 5947 5995 6043 6091 6139 6 187 6235 PCTIUS961039 16 WO 96/29396 CCT GCG CTG Pro Ala Leu 345 GGC TGT TTG Gly Cys Leu -144- TCA GAG TAC TTT GCA CTA GTG GTG TrA CTG Ser Giu Tyr Phe Ala Leu Val Val Leu Leu 350 355 GCC GAG ACG GTC Ala Giu Thr Val 360 AGG GCG CTG OCA Arg Ala Leu Ala 375 ATA GAC CCC T Ile Asp Pro Val TTA COG GGC ATA Leu Arg Gly Ile 410 AAC ACC TAT TCO Asn Thz Tyr Ser 425 TGC AAT GTT GCC Cys Asn Val Ala 440 ATO AGC GAC ATA Met Ser Asp Ile 455 GCG TOG CTC CAG Ala Trp Leu Gin CTG AAG AGA AGC Leu Lys Arg Ser 490 TTA GCG ACC ATG TTC GAC CAC GCA CTG GTA TTC ATG Leu Ala Thr Met Phe Asp His Ala Leu Val Phe Met 365 370 GAC GGC AAT TTC OAT GAC TAT GAC GAA ACT AGA TAT Asp Gly Asn Phe Asp Asp Tyr Asp Glu Thr Arg Tyr 380 385 390 AAA AAC GAG TAC CTG AAC GGA GCC GAG GGT ACT CTG Lys Asn Giu Tyr Leu Asn Oly Ala Glu Gly Thr Leu 395 400 405 GTG 0CC TCC AAC ACC OCT CTG GCG GTG GTT TGC GCA Val Ala Ser Ann Thr Ala Leu Ala Val Val Cys Ala 415 420 ACG ATA AGA AAA CTC CCG TCC 070 GCA ACT AGC GCG Thr Ile Arg Lys Leu Pro Ser Val Ala Thr Ser Ala 430 435 TAC AGG ACC GAA ACO CTO AAA OCO AOG CGC CCT GGC Tyr Arg Thr Olu Thr Leu Lys Ala Arg Arg Pro Gly 445 450 TAC CGG ATA TTA CAA AAA GAG TTI' TTC 777 TAC ATT Tyr Arg Ile Leu Gln L.ys Glu Phe Phe Phe Tyr Ile 460 465 470 AGO OTT GCA ACA CAC GCA AAT TTC TOT TTA AAC ATT Arg Val Ala Thr His Ala Asn Phe Cys Leu Ann Ile 490 485 6283 6331 6379 6 427 6475 6523 6571 6619 6667 070 GAT ACG GG Val Asp Thr Gly GCC CCG CCA 777 TTG 77C AGO 0CC Ala Pro Pro Phe Leu Phe Arg Al1a 495 500

AGC

Ser

CTC

Leu

CTC

Leu 535

AGO

Arg

GAA

Olu 077 Val 0CG Ala TCG GAG AAG CG Ser Glu Lys Arg

SOS

GTG CCO ATT CAA Val Pro Ilie 0Th 520 AAG AGO GAk AAG' Lys Arg Oiu Lys GAC CCG AGO GGG Asp Pro Arg Gly 555 ATT ATT GCA GAC Ile Ile Ala Asp 570 COO GCT TCA 077 Arg Ala Ser Val 585 CGA CGC TTA ACC Arg Arg Leu Thr 600

-TG

Leu

TAT

Tyr

TTA

Leu 540 7CC Ser Gly

OCG

Ala

TCA

Sei CAG CAG TTA AAT AAA ATG CT C TGC CCC C=T Gin Gin Leu Asn Lys Met Leu Cys Pro Leu 510 515 GAA GAC 777 TCG AAG GCC ATG 000 TCT GAG Giu Asp Phe Ser Lys Ala Met Gly Ser Giu 525 530 GAG ACA TTC OTT AAA OCT ATT 7CC AGC GAC Giu Thr Phe Val Lys Ala Ile Ser Ser Asp S45 550 TTA AGA TTT- CTC ATT TCO GAC CAT OCA AGO Leu Arg Phe Leu Ile Ser Asp His Ala Arg 560 565 07k CGG Ti-r AAG CCG 070 ATA GAC GAO CCG Val Arg Phe Lys Pro Val Ile Asp Giu Pro 575 580 CTG ACT ACC GCT GCC GCT G00 AAA 070 AAA *Leu Ser Thr Ala Ala Ala Gly Lys Val Lys 590 595 077 COC OCO CCC 07k CCG GOC OCA GGC 0CC Val Arg Ala Pro Val Pro Gly Ala Gly Ala 605 610 6763 6811 6859 6907 6955 7003 70S1 urujyclc I Ic WO 96/29396 rLAL -1~45- GTT TCC GCG cc CGG AAA TCG GAA ATA TGA TA.AAAATGCT TGGCATTTGC Val Ser Ala Arg Arg Lys Ser Giu Ile 615 620 GGCCGAAGAG GCGTGATCTG AAGGGCTCCA CAATGACGTA ACTGAGCTAC GCATCCCTAT AAAGTGTACC CGCTCACCGC TACCCCATAC AGTGTTAr-AG GAGGGGAGAG AGACAACTTC AOCTCGAAGT CTCAAGAGAC ATC ATG ACC GOC TTC ACT AAC ATA GGA TCG Met Ser Gly Phe Ser Asn Ile Gly Ser 1. U.77 7101 7161 7221 7271

ATT

Ile

GGG

Gly

GGC

Gly

GTC

Val

GTG

Val

GTG

Val

GCT

Ala

CC

Ala

GCG

Ala

AAA

Lys

CCG

Pro

AAA

Lys

GAT

Asp

CTA

Leu

ACC

Thr

CCG

Pro

ATT

Ile

TG

Trp 60

TGG

Trp

ACC

Ser

ATC

Ile OTT TCC CTA GTA TGC TCG CTT TTG TGC GCA TCT GTA TTA Val Ser Leu Val Cys Ser Leu Leu CyS Ala Ser Val Leu i5 20 GTA CTG GAC GGG CTC GAG TCG AGC CCT TTC CCG TTC GGG Val Leu Asp Giy Leu Giu Ser Ser Pro Phe Pro Phe Gly 35 ATA GCC CAG GCG TGC AAC CGC ACC ACG ATT GAG GTG ACO Ile Ala Gin Ala Cys Asn Arg Thr Thr Ile Giu Val Thr 50 AGC GAC TAC TCT OCT CCC ACC GAA GGA GTG TCA GTC GAG Ser Asp Tyr Ser Gly Arg Thr Glu Gly Val Ser Val Giu 65 TTC TAC COG AAT ACT AAT CCC GAA ACC TTC CTG TTC CCC Pkie Tyr Gly Asn Ser Asn Pro Glu Ser Phe Val Phe Cly so GMA ACG 0CC AGT GGA CAC GAG GAC CTG TCT ACG TOC TG Glu Thr Gly Ser Gly His Glu Asp Leu Ser Thr Cys Trp 95 100 105 CAT AAT CTC AAC C TCT CTG TCC AGG C TCT CAC GCC His Asn Leu Asn Ala Ser Val Cys Arg Ala Ser Asp Ala lic 115 120 7319 7367 7415 7463 7511 7559 17607 '7655 7703 7751 7799 7B47 7895 7943 GGG ATA Gly

CGC

Arg

CTG

Val

TCA

Ser 170

CCA

Pro Ile Tcc Ser

CTG

Leu 155

CTG

Val

AAC

Asn CCT CAT TTC CAC AAC CAG TCC CMA AAA CTC CAG AGA AGA CTG Pro Asp Phe Asp Lys Gin Cys Glu Lys Val Gin Arg Arg Leu 125 130 135 GGG GTG GMA C=T GOT ACT1 TAC 070 TCT GGC MAT OCA TCC CTC Gly Val Glu Leu Gly Ser Tyr Val Ser CJly Asn Oly Ser Leu 140 145 150 TAC CCA 000 ATG TAC GAT GCC GGC ATC TAC GCC TAC CAG CTC Tyr Pro Cly Met Tyr Asp Ala Gly Ile Tyr Ala Tyr Gin Leu 160 165 CCT CCC AAC GGA TAT ACC COG TCT GTT TAT CTA GAC GTC GGA Ciy Gly Lys Cly Tyr Thr Cly Ser Val Tyr Leu Asp Val Gly 175 i8O 185 CCC GGA TOC CAC CAC CAC TAT GGG TAC ACC TAT TAC ACC CTC Pro Cly Cys His Asp Gin Tyr Gly Tyr Thr Tyr Tyr Ser Leu 190 195 200 GAG C TCA GAC TTA TCA TC-r TAT GAC GTA CCC TCG CCC GAA Giu Ala Ser Asp Leu Ser Ser Tyr Asp Vai Ala Ser Pro Ciu 205 210 215 *GOT CCT ATC GAC GMA GAT TAT TCC AAT TGT CTA GAC ATC CCC Gly Pro Met Ciu Ciu Asp Tyr Ser Asn Cys Leu Asp Met Pro 220 225 230 0CC GAC Ala Asp CTC GAC Leu Asp WO 96/29396 PCTfUS96/03916 -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 Glu Giu Gin 235 240 245 GAA AAC GCC ACG GAC GAG CTT TAC CTA TGG GAC GAG GAA TGC GCC GGT Glu Asn Ala Thr Asp Glu Leu Tyr Leu Trp Asp Glu Giu Cys Ala Gly 250 255 260 265 CCG CTG GAC GAG TAC GTC GAC GAA AGG TCA GAG ACG ATG CCC AGG ATG Pro Leu Asp Glu Tyr Val Asp Glu Arg Ser Giu Thr Met Pro Arg Met 270 275 290, GTT GTC TTT TCA CCG CCC TCT ACG CTC CAG CAG TAG CCACCCGAGA Val Val Phe Ser Pro Pro Ser Thr Leu Gln Gin 285 290 a.

a a a a. a a.

a.

*a.a GTGTTTTTTG TGAGCGCCCA GTATTGAATA AATAAACAGT ATGGCGTGTG CGGGTGAAAC CAGCGTATTT CGCCGAGAGA AGCGGACGCG GCGTTGCCGA AACCATCCAA GAGAACCTGG TCCGCCGCCT GAAGGCGCCG TCGCTACGGC GCTACGGAGG GGACAACGTG ACATrTTCGC CACCATAGGG GTATTCGCTA GGTCTTAGTT AACGCCTCTC TTTTTTGCAG AACGAGCCTA TGTGCGAGAT AACGATGTCG CAACTCGGCG GCCCAGACGG AAGAATAGTA AACGTCACG7 CTCGTCGCGG GAGAACGGGC GTACACTCCC GACATTGACC GGACTCCGGC AGCGTCCTCC TCAACTGCAC COGATAGAtT) CTGTCAAAA ATGATAGAAJ CTCTATCCGT CCGGGGGCC

CGCAACATAC

ACAAAAG CAT

CGTAAATTAC

TGGGGACAA-

CCGGCAGATT

CGGTGCTCGC

TCAGC=TCA

ACGAACTTGC

TGTCATATCG

CTGGCCAGAG

TGGAACGGTC

CATGGGGCTC

CCGTGTITGAC

GGCTCACGTC

*GGACAACCGG

AGCCCGTGCTT

GCCGGGCCAT

CG!7ACGGC

.GTCCGGG.;C

SCCCCGTACCC

k CTCTGCCTCC

CTAACTGCTT

CAGGTGTGGT

GTGATAATAA

GTTAGTGTTG

CTGCCGAGTT

GGAATCGCCG

GATTTGCG

ACTGGAGCGC

CCCGCGCCCA

CACGGAAAGC

CCGTGCGCCTG

GAAGCGTTGG

AAAAGCGCAG

TCTCAACATG

CGGCCCCTCC

GAGGAACGTT

CATTTCTGAT

TTGCGTGTCT

ATAGCATAGG

CGCCZ"1rCC

TGGAAGGTGC

GTCACGGGAC

GACACCCCTA

GGGACGTCCG

GAAATTCACG

AGCTATTCGG

GAAACGCCGT

TTAGGCCCCC

TACATTGGGG

ACCTTTTTCT

CCCTCGCGCA

TCTGACGGGT

CAATTATTGC

3'TGCTAAACC

PLGTTGGCGTG

TACTTGCAGT

CTCCGGGAGG

ACGCGACATA

CTTTGCGCAT

CCTCAGACGC

GAGCATACTT

TCATCAGTCG

GCGATGAAAA

CGTCGCCTTA

AGTGCTACTC

ATTCGCCTAA

TAACGGTATA

TCTTGGTTAA

ATTCGCCGGC

CATCCGCGAT

CGACCTTCGA

CCAGGGACGA

ATTTCGATAC

7991 8039 8087 8133 8193 8253 8313 83'73 8433 8493 8553 8613 8673 8733 8793 8853 9913 8973 9033 9093 9153 9213 9273 9333 9393 9453 9513 9573 9633 9693 GATAAACGTT ATTGCCAATT C I IAGGGAA GGAAAACTCC TGAGCCGCCG GGGACTGAAA AGCGCTCGGG AGCMATGTTC GTCGATACC GCAGCACCTG

AGGTACTTCC

CAGCACTAGC

GGTACCCGTC

TCCCGGTGAG

CCCGAGCGCG

CCGACCCCCA CTACCGTGCC AGAGCCAGCC GATATGGGAT TCTT=CTCCAC GGCACGTGCT CAGGAAACGG ATAGAACTCT TTCGACAACT TCAGAAAATA CACTGTTTCC TACGACCGCG GCACATGAAA CTACACAGAC CCAGAGTGCA ArlACTACAC TCAkTACCGCG AccGGATC-AG AAACTCTTTC CCTCTTACCC TTCCACTGAC CCGGGGATTT C'rACCGAGAC GAAACGGTGG TCTTTACTCA WO 96/29396 WO 9629396PCTIUS96/039 16 -147- GAGTCCGAGT ACCGAGTCGG TCAGACTCCG AQTACTGAAC GTTGTTTACT CAGACTCCGA C AACCGAGGCA CCTGCCCAGA C CACGCCCCCC GAAACCGCTC G GAGTACGGTA ACGGAGGTGT TTCGAGTACT GAACCGGCGA TCAGACTTCG AGTGCCGAGC C CACTCAGGCC CCGAGTACGG GTTGACTCAG AGTCCGAGTA C GGAAATTACT CAGACCCCGA G GCCAGAAACT GCACAGAGCAC GACGCATAAT AC'TGAACCGAC CTACACAGAA AATAAGACTT I GACGGCAGAG TCGCAGACGC C CGATGGCGAA GTAACGGCGA TAATTQGAAA GTAGACCTCG GGTTTTTAAC AGTAATGAGA AACATCGGTC CAGCTAATGT TTTCGACACG TCTCTTATAG TGATCCGCAA ACGGCATACT ;.TGGAGCCT'r AATAATGTT TACCGC'rGGC CAAAGTACAG TGOCCGGQGA GGCAATATAT TACGCGAAAG GAGAGGTGCT GGCGCAGCTG TATrCACTCT TTTGTACGCA ACGCTATGGA GTACTGCII CCTTCG- GGCCGTCGCG CGCTCGACGC ACTAGGGTAT CAAGAGGCTG ATGATATCGG CGGCCAAAGA TCTACGTGA TTAAGGGCGA TACAGGGAAT GTGGCGACGT

AACCGCGCG

.GCGGCTCT

TGCTGAACA

CCCGAGCAC

CGCTCCGAG

TACTCAGAC

T-ACTCG

GOACACTAT

'ACCGAAAGC

CGAACCTGT

~CGCGGCACC

ACCCCTGGC

*,GACTTATCC

~ATCGTTTCC

CCTATTGGA

TrGCGTTTC ErAGATGTAAT

;ATGGCAGAA

3CCTGTC=T

CGAGGGAAAA

3CACAATAAC

CACTGCCGGA

TGTGGAAGAG

ACGAATGCAC

TAACAAACAC

TTTCTGGACT

CCGCCATTTA

TAGCCAGAGC

GCTAACCAT;

CGACGTTGTC

GTCCCAGAGT

TACTCAGACG

GATGACTTTT

GATACCCGAG

CGCGGtGCCG

CCCGAGCACG

GACTCAGAGC

GCGAACTCAG

TACTCAGACT

GCCTTTCACC CGGACTCTGG GCGCAGAGCC GGAGGTTTAT ACTCGGAGTT CGAGTACGAT CTCGCAAALAC CCTACCAGTT CGGGAACCGG AGTGCAkPACG ACACCACATA CCCAGAAACT TACTGTTGTT TCAGAAT'CC ATGAGATGTC CGTCAAAATT GTAGAGGTGA AGTTTTCAA6A CACCGTCA.AA TCTCCCTATA GGGTAGAAAC GQATGAAATT TCTGGGAACA GTCCCGCCGG ACAGCTGTAC TACAGAGTAA CCGATGGAAG CACGAGCCAT TCTCCGGAAC CTTACTGTCT AGATATCGCG CCAGAGTTAT ACTrTACCTC TCTGCCGTCC GGCGTTrG~rC CGAGATTCGA ATATTTGACG GCCACGACCG TTGTTTCGCA CAGCGCGAGA GCAGGCGAGG CGTGGATTTC COTCCTCATC TCAGACGGCA CTCGCGTTAC ATGGATTGCG GTGGAAAACG GTGCTGCTCA TGTGTCAGGA TrATGCGGGA GCATATCTGC TTTTTGA GGAATGCTT TTGGACTATC ACCGCCGCCG TCACGTACGA CTACATTTTA CCGGCGGTI'G GCCCGTATAA CAGATACCTC GAGCTCAACC CGATTTCTAA CGTGGACGAC GGGGGCCCrT TCGAGGCCTC CGTCGTCTGG CAGGAACCGT GGTATTTTAC CAGATCGCAG AAACGGAGGC ACTCAGACTC CGGGTGCAGA ATATTTACTC AGTCTCGTAG GALGGTTTTTA CACAGAGTTC GTACCGAAAA CTACTCTGAG GCGGGJLCTG AGGCCTTTAC AGTACTGAAA CACACTTTTT CCGAGTACAG AGCCGGAGGT 9753 9813 9873 9933 9993 10053 10113 10173 10233 10293 10353 10413 10473 10533 10593 10653 10713 10773 10833 10893 109S3 11013 11073 11133 11193 11253 11310 11370 11430 11490 11550 11610 11670 11730 AAAhGAGAAG

COACGGCGAG

ACAACTGCTA

GACAAGTACT

TCTGAATGCG

GTCCAATCTA TAQAAA.AGAG CC=TCAATC TGCACAGATC TGGGCAGTGG ACTATGTTCC TAGCACCCTI' GTATCGCGAA ATOOCGCOGG ACTGACTATA WO 96/29396 PCTIUS96/039 16 -148-

TTCTCCCCCA

GCGCAAACAG

CTTAACTTTT

GAACACCTTT

TACGAAGATA

GACCCTCGTC

CGCACCCCGG

ATGCAGGCAG

GAGGACACCG

GTGATCCCGG

GCGTTCGTAG

CTGCTGCGCT

CTCTCGTAAC

TACCGTCGAA

ATCCGATCGC

TTCTGCAGCG

CAGATAGCGT

ACGCAGAAAG

AGGCTTCTGG

AGCACGATGA

TGGAGGAGAC

CCTGCGCGGT

C'rCTGGCCAA

TCTAGAAGTT

ATGCTGGACA

AGACACCAAT

CTGGAATAAT

CCCGCAAG.AA

CAGCGAAAAG

AGAAAATCCT

TCCAAACTCG

TACTAAAAGT

CGCGCTCGTG

TACTTGCTGA CCCTGAAAAT CGGGAGATTT AACGATCGCT GTTTAAAGAT

CGGGTCGCAG

ACAGAACAGT ATCAGACTGG ATTTCAAGGC ACACGACACG CGGACGACGT ATATCGGGGA TTGCTGAGGA AAAAGAATCC TAGCGCGCCA ATTCCCGCTG TAACCAAGAA AGCGGAAGGG AAGGCCCCTC CAGAAGACTC GGAGGACGAC GCCGCCC!TCC CCGAAGACGA CGAAGTCCCC G.ATCCTGACT ATTACAATGA CATGCCCGCC TCTAATGCCG TCTCCATGCC

CATATTCGCG

GGGCTACTGG TTGGAGCAT CGTAA.AATGC 0 0* 0* 0 0 0 0 *00 C GCGCGTAGCT AA TCGAGCCTAG AATAGGTGGT TTCTTCCTAC ATGCCACGCC TCACGCTCAT AATATAAATC ACATGGAATA.GCATACCAAT GCCTATTCAT TGGGACGTTC GAAAAGC ATG GCA TCG CTA CT? GGA ACT CTG GCT CTC CTT GCC GCG ACG Met Ala Ser Leu Leu Gly Tkiz Leu Ala Leu Leu Ala Ala Thr 1 5 CTC GCA CCC TTC GGC GCG ATG GGA ATC GTG ATC ACT GGA AAT CAC GTC Leu Ala Pro Phe Gly Ala Met Gly Ile Val Ile Thr Gly Asn His Val 15 20 25 TCC GCC AGG AT? GAC GAC GAT CAC ATC GTG ATC GTC GCG CCT CGC CCC Ser Ala Axg ile Asp Asp Asp His Ile 'Val Ile Val Ala Pro Arg Pro 40 GAA GCT ACA AT? CAA CTG CAG CTA TTT ?TC ATG CCT GGC CAG AGA CCC Giu Ala Thr Ile Gin Leu Gin Leu ?hie Phe met Pro Gly Gin Arg Pro CAC AAA CCC TAC TCA GGA ACC GTC CGC GTC GCG TTT CGG TCT GAT ATA His Lys Pro Tyr Ser Gly Thr Val Arg Val Ala Phe Arg Ser Asp Ile 70 ACA AAC CAG TGC TAC CAG GAA CT? AGC GAG GAG CGC T GAA AAT TGC Thr Asn Gin Cys Tyr Gin Glu Leu Ser Giu Glu Arg Phe Glu Asn Cys so 85 ACT CAT CGA TCG TCT TCT GT? TTT GTC GGC TGT AAA GTG ACC GAG TAC Thr His Arg Ser Ser Ser Val Phe Val Gly Cys Lys Val Thr Giu Tyr 100 105 110 ACG TTC TCC GCC TCG AAC AGA CTA ACC OGA CC? CCA CAC CCC AAG Thr Phe Ser Ala Ser Asn Arg Leu Thr Gly Pro Pro His Pro Phe Lys 115 120 125 CTC ACT ATA CQA AAT CC? CGT CCG AAC GAC AGC CCC ATG TTC TAC GTA Leu ?hr Ile Arg Asn Pro Arg Pro Asn Asp Ser Giy Met Phe Tyr Val 130 135 140 AT? CT? CGG CTA GAC GAC ACC AAA GAA CCC AT? GAC GTC TC GCG ATC Ile Val Arg Leu Asp Asp Thr Lys Glu Pro Ile Asp Vai Phe Ala Ile 14S iSO 155 11790 11850 11910 11970 12030 12090 '2150 12210 12270 12330 12390 12442 12502 12551 12599 12647 12695 12743 12791 12839 12887 12935 12 983 WO 9629396PCTIUS96/039 16 -149- CAA CTA TCG GTG TAT CAA TTC GCG AAC ACC GCC GCG ACT CGC GGA CTC 13031 Gln Leu Ser Val Tyr Gin Phe Ala Asn Thr Ala Ala Thr Arg Gly Leu 160 165 1'70 TAT TCC AAG GCT TCG TOT CGC ACC TTC GGA TTA CCT ACC GTC CAA CTT 13079 Tyr Ser Lys Ala Ser Cys Arg Thr Phe Gly Leu Pro Thr Val. Gin Leu 175 180 185 190 GAG GCC TAT CTC AGG ACC GAG GAA AGT TGG CGC AAC TOG CAA GCG TAC 13127 Giu Ala Tyr Leu Arg Thr Glu Ciu Ser Trp Arg Asn Trp Gin Ala Tyr 195 200 205 OTT GCC ACG GAG GCC ACG ACG ACC AGC GCC GAG GCG ACA ACC CCG ACG 13175 Val Ala Thr Glu Ala Thr Thr Thr Ser Ala Glu Ala Thr Thr Pro Thr 210 215 220 CCC GTC ACT GCA ACC AGC GCC TCC GAA CTT GAA GCG GAA CAC TTT ACC 13223 Pro Val Thr Ala Thr Ser Ala Ser Oiu Leu Glu Ala Glu His Phe Thr 225 230 235 TTT CCC TGG CTA GAA AAT GGC GTG GAT CAT TAC GAA CCG ACA CCC GCA 13271 Phe Pro Trp Leu Glu Asn Gly Val Asp His Tyr Glu Pro Thr Pro Ala 240 245 250 :AAC GAA AAT TCA AAC GTT ACT GTC COT CTC 000, ACA ATG AGC CCT ACG 13319 *Asn Glu Asn Ser Asn Val Thr Val Arg Leu Gly Thr Met Ser Pro Thr *255 260 265 270 CTA ATT GGG GTA ACC GTG GCT GCC GTC GTG AGC GCA ACG ATC GGC CTC 13367 Leu Ile Gly Val Thr Val Ala Ala Val. Val Ser Ala Thr :le Gly Leu ***275 280 285 OTC ATT GTA ATT TCC ATC GTC ACC AGA AAC ATO TGC ACC CCG CAC CGA 13415 Val Ilie Vai Ile Ser Ile Val Thr Arg Asn Met Cys Thr Pro His Arg 290 295 300 .AAA TTA GAC ACG GTC TCG CAA GAC GAC GAA. GAA CGT TCC CAA ACT AGA 13463 *Lys Leu Asp Thr Val Ser Gin Asp Asp Glu Glu Arg Ser Gin Thr Arg 305 310 315 AGO GAM TCG CGA Akz TTT OGA CCC ATG GTT C TGC GAA ATA AAC AAO 13511 Arg Glu Ser Arg Lys Phe Gly Pro met Val Ala Cys Giu Ile Asn Lys 320 325 330 000 OCT GAC CAG OAT AGT GAA CTT GTG GAA CTG GTT C ATT GTT AAC 13559 Gly Ala Asp Gin Asp Ser Glu Leu Val Giu Leu Val Ala Ile Va). Asn 335 340 345 350 CCG TCT GC CTA AGC TCG CCC GAC TCA ATA AAA ATO TGA TTAAGTCTGA 13608 Pro Ser Ala Leu Ser Ser Pro Asp Ser Ile Lys Met 360 ATGTGCCTCT CCAATCATTT CGATTCTCTA ATCTCCCAAT CCTCTCAAAA GGGGCAGTAT 13668 CGGACACGGA CTGGGAGG CGTACACGAT AGTTATATGG TACAGCAGAG GCCTCTGAAC 13728 ACTTAGGAGG AGAATTCAGC CGGGGAGAGC CCCTGTTGAG TAGGCT TGGG AGCATATTGC 13789 AGO ATG AAC ATG TTA OTG ATA OTT CTC 0CC TCT TOT CTT 0CC CGC CTA 13836 Met Asn met Leu Val Ile Val Leu Ala Ser Cys Leu Ala Arg Leo 1 5 10 ACT TTT GCG ACG CGA CAC GTC CTC TTT Tl'O GM G GC ACT CAG OCT GTC 13884 Thr Phe Ala Thr Arg His Vai Leu Phe Leu Olu Oly Thr Gin Ala Val 20 25 PCTIUJS96/03916 WO 96/29396 CTC GGG Leu Gly GAl' GAT GAT CCC AGA AAC -150- GTT CCG GAl' Val Pro GlU 40 Glu Asp Asp

TGG

Trp

TGC

Cys

AAG

Lys

TTA

Leu

AAC

Asn

TTT

Phe

TCT

Ser

CTT

Leu 160

GAC

ACA

Thr

TCT

Ser

AAA

Lys

CTA

Leu

AGC

Ser

GAT

Asp

CAG

GIn 145

TCG

Ser

ACC

A

Lys

TCG

Ser

GAC

Asp

AAG

Lays

AAT

Asn

CCT

Pro 130

CCA

Pro

CAC

His

ATC

GTC

Val CC'r Pro

TGC

Cys

CGC

Arg

ATA

Ile 115

GTA

Val

GAT

Asp

GTA

Val

AAG

C'rG Leu

AAC

Asn

CCG

pro

GG-

dly 100

ACT

Thr

ACT

Thr

CTG

Leu

GAC

Asp

CCG

Pro Arg ASfl CGG AAC GCG Arg Asn Ala 55 TAT TGC TTT Tyr cys Phe 70 CCC GCG GGA Pro Ala Gly 85 GAl' AGC TrC Glu Ser Phe MAT ATC ATG Asn Ile Met CGT TCG GAC Arg Ser Asp 135 GCC ATG GMA Ala Met GL.

150 GAG AAG GCT Glu Lys Ali 165 TCA GAG GCC Ser Glu Al.

TGC AAG ATG AAG GCG GCC GAT GTC Cys Lys met Lys Ala Ala Asp Val CAT CAT TTA ATT TAC GAC GGA GGA His Asp Leu Ile Tyr Asp Gly Gly CCC CTG TCT GCA AAC CTG CTA ATT Pro iLeu Ser Ala Asn Leu Val Ile 90 GTC GTG CTG GGT TCT GOG CTA CAC Val Val Leu Gly Ser Gly Leu His 105 110 TGG ACA GAG TAC GGA CCC CTG CTC Trp Thr Glu Tyr Gly Gly Leu Leu 120 125 GAG GGA ATC TAT TTT CGA CGC ATC Glu Gly Ile Tyr Phe Arg Arg Ile 140 ACT ACA TCG TAC AAC GTC AGC GTT Thr Thr Ser Tyr Asn Val Ser Val 155 *CCA GCA CCG CAC GAG GTG GAG ATA Pro Ala Pro His Glu Val. Glu Ile 170 175 *CAC CCG CAC GTG GAA TTA CAA ATG 31 His Ala His Val Giu Leu Gin Met 185 190 13980 14028 1407.6 14124 14172 14220 14268 14316 14364 14412 14460 14508 14556 14604 14652 14700 GGG ACT GTA ATC AAA Gly Thr Val Ile Lys 13932 Asp Thr Ile Lys Pro CTC CCG TTT CAT GAA CTC AAC GAC MAC ACC CCC ACC TAT GTG ACC CCT Leu Pro Phe His Glu Leu Asn Asp Asn Ser Pro Thr Tyr Val Thr Pro 195 200 205 GTT CTTAGA GTC C CCA CCGACC GAG CAC GT A A A~C GTACG Val Leu Arg Val Phe Pro Pro Thr Glu His Val Lys Phe Msn Val Thr 210 215 220 TAT TCG TGC- TAT GGG =n -GAT GT AAA GAG GAG TGC GA;, GMA GTG AMA Tyr Ser Trp Tyr Cly Phe Asp Val Lys Clu Ciu Cys Clu Clii Val Lys 225 230 235 CTG TTC GAG CCG TGC CTA TAC CAT CC-- XCA GAC GGC AAA TCT CAG TT Leu Phe Glu Pro Cys VJal Tyr His Pro Tkir Asp (ily Lys Cys GIn Phe 240 245 250 255 CCC GCA ACC AAC CAG AGA TGC CTC ATA GGA TCT GTC TTG ATG CCG GAl' Pro Ala Thr Msn Gin Arg Cys Leu Ile Gly Ser Val Leu Met Ala Giu 260 265 270 TTC TTG GGC GCG GCC TCT TTG CTG GAT TGT TCC CGC CAT ACT CTA CAl' Phe Leu Giy Ala Ala Ser Leu Leu Asp Cys Ser Arg Asp Thr Leu Glu 275 280 285 GAC TGC CAC GAl' MT CCC GTG CCC MAC CTA CGG I-rC GAT TCG CGA CTC Asp Cys His Giu Asn Arg Val Pro Asn Leu Arg Phe Asp Ser Arg Leu 290 -295 300 WO 96/29396 PCTfUS96/03916 9* -151- TCC GAG TCA COC GCA GGC CTG GTG ATC AGT CCT CTT ATA GCC ATC CCC Ser Giu Ser Arg Ala Gly Leu Val Ile Ser Pro Leu Ile Ala Ile Pro 305 310 315 AAA OTT TTG ATT ATA GTC GTT TCC GAC GGA GAC ATT TTG GGA TGG AGC Lys Val Leu Ile Ile Val Val Ser A.sp Gly Asp Ile Leu Gly Trp Ser 320 325 330 335 TAC ACG GTG CTC GGG AAA COT AAC AGT CCG CGC GTA GTA GTC GAA ACG Tyr Thr Val Leu Gly Lys Arg Asn Ser Pro Arg Val Val Val Glu Thr 340 345 350 CAC ATG CCC TCG MAG GTC CCG ATG AMC AAA GTA GTA ATT GGC AGT CCC His met Pro Ser Lys Val Pro Met Asn Lys Val Val Ile Gly Ser Pro 355 360 365 GGA CCA ATG GAC GAA ACG GGT AAC TAT AAA ATG TAC TTC GTC GTC CCG Gly Pro Met Asp Giu Thr Gly Asn Tyr Lys Met Tyr Phe Val Vai Ala 370 375 380 GOO GTO GCC GCG ACG TGC GTA ATT CTT ACA TGC GCT CTG CTT OTG GGG Gly Val Ala Ala Thr Cys Val Ile Leu Thr Cys Ala Leu Leu Val Giy 385 390 395 AA AG AAG TGC CCC GCG CAC CAA ATO GOT ACT TTT TCC AAG ACC GAA Lys Lys Lys Cys Pro Aia His Gin Met Gly Thr Phe Ser Lys Thr Giu 400 405 410 415 CCA TTG TAC GCG CCG CTC CCC AAA AAC GAG TTT GAG GCC GGC GGG CTT Pro Leu Tyr Ala Pro Leu Pro Lys Asn Giu Phe Giu Ala Gly Oiy Leu 420 425 430 ACO GAC GAT GAG GAA =T ATT TAT GAC GMA GTA TAC GAA CCC CTA TTT Thr Asp Asp Oiu Giu Val Ile Tyr Asp Glu Val. Tyr Giu Pro Leu Phe 435 440 445 COC GGC TAC TOT AAG CAG GMA TTC CGC GAA GAT GTG MAT ACC TTT TTC Arg Gly Tyr Cys Lys GIn Giu Phe Arg Glu Asp Val Asfl Thr Phe Phe 450 455 460 GOT GCG GTC GTG GAG OA GMA AGO GCC TTA AMC TTT AMA TCC GCC ATC Gly Ala Val Val Giu Gly Oiu Arg Ala Leu Asn Phe Lys Scr Ala Ile 465 470 475 GCA TCA ATG OCA GAT CGC ATC CTG GCA MAT AMA AOC GGC AGA AGO MAT Ala 5cr Met Ala Asp Arg Ile Leu Ala Asn Lys Ser Gly Arg Arg Asn 480 485 490 495 ATG OAT AGC TAT TAG TTGGTC ATG CCT =1T MG ACC AGA GOO 0CC GMA Met Asp Ser Tyr .Met Pro Phe Lys Thr Arg Gly Ala Glu 500 1 GAC OCO 0CC GCG GGC MOG MC AGO TTT MAG MAA TCG AGA MAT COG GMA Asp Ala Ala Ala Gly Lys Asn Arg Phe Lys Lys Ser Arg Asn Arg Glu 15 20 ATC TTA CCG ACC AGA CTG COT 0CC ACC OCT MAG AMA ACT 0CC OA TTO Ile Leu Pro Thr Arg Leu Arg Gly Thr Gly Lys Lys Thr Ala Oly Leu 35 TCC MAT TAT ACC CAG CCT ATT CCC TOG MAC CC? AMA TTC TOC AGC OCG Ser Asn Tyr Thr Gin Pro Ile Pro Trp Asri Pro Lys Phe Cys Ser Ala so CGC COG GMA TCT GAC MAC CAC OCG TOT AAA GAC ACT TTT TAT COC AGO Arg Gly Oiu Ser Asp Asn His Ala Cys Lys Asp Thr Phe Tyr Arg Arg 65 14748 14796 14844 148 92 14940 14988 15036 15084 15132 15180 15228 15276 15324 15372 15420 15468 15516 WO 9629396PCTfUS96/03916 -152- ACG TGC TGC GCA TCG CGC TCT ACC OTT TCC AGT CAA CCC GAT TCC CCC 15564 Thr Cys Cys Ala Ser Arg Ser Tkir 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 IjCu 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 Al1a Ala Pro Ala Arg Thr Tyr Ser 125 130 135 TCT GCG CAA AGA TAT ACT OTT GAC GAG OTT TCG TCG CCA ACT CCG CCA 15756 Ser Ala Gin Arg Tyr Thr Val Asp Giu Val Ser Ser Pro Thr Pro Pro 140 145 150 0: GGC GTC GAC GCT GTT GCG CAC TTA GAA ACG CGC GCG GMA CTT CCT GGC 15804 Gly Val Asp Ala Val Ala Asp Leu Glu Thr Arg Ala Glu Leu Pro Gly 155 160 165 OCT ACG ACG GMA CMA ACG GMA ACT MAA MAT AAG CTC CCC AAC CAA CAA :LS852 A1a Thr Thr Glu Gin Thr Glu Ser Lys Asn Lys Leu Pro Asfl Gin Gin *170 175 180 185 *TCG CGC CTG MAG CCG AAA CCC ACA MAC GAG CAC OTC GGA GGG GAG CGG 15900 Ser Arg Leu Lys Pro Lys Pro Thr Asn Glu His Val Gly Gly Giu Arg 190 195 200 TGC CCC TCC GMA GGC ACG GTC GAG GCG CCA TCG CTC GGC ATC CTC TCG 15948 Cys Pro Ser Glu Gly Thr Val Giu Ala Pro Ser Leu Gly Ile Leu Ser 205 210 215 CCC GTC OGG GCA GCO ATA GCA MAC GAG CTG GCT CGT ATG COG AGG GCG 15996 Arg Val Gly Ala Ala Ile Ala Asn Giu Leu Ala Arg Met Arg Arg Ala 220 225 230 TGT CTT CCG CTC GCC GC TCG GCG CCC OCT GCC OGA ATA OTG GCC TOG 16044 Cys Leu Pro Leu Ala Ala Ser Ala Ala Ala Ala Gly Ile Val Ala Trp :**235 240 245 CCC CCG GCG AGG CCC TTG CAG AAA CAA GGG CGG TAG CAGTMATAAT 16090 Ala Ala Ala Arg Ala Leu Gln Lys G.1n Gly Arg 250 2.55 260 AACCACACA ATATTGACAA TMATAA6ACGC GTACCCGG ATG AGT MAG TOT TAT 16143 Met Ser Lys Cys Tyr 1 TGT CTC GC CGC CAT CTT. TAT AAM ACC CCG COT TGC GTG GGC COG CGG 16191 Cys Leu Ala Arg His Leu ?yr. Lys Ser Pro Arg Cys Val Gly Arg Arg 1s GTA GCA TTT GGA GGG TTG GC ACC ATG TCG AGA CCT CCC ACG TCA CAT 16239 Val Ala Phe Gly Gly Leu Ala Thr Met Ser Arg Pro Pro Thr Ser His 30 TTG GAC TTA OCT TTC TCG GCC GCC TTT AGG GGC ACG GAC CTG CCC GOA 16287 Leu Asp Leu Ala Phe Ser Ala Ala Phe Arg Gly Thr Asp Leu Pro Oly 45 GGG AGA -TTC TOG COG GC TCC CAG AGT TGC CAT-ATT TTC TTT TGG CCC 16335 Gly Arg Phe Trp Arg Ala Ser Gin Ser Cys Asp Ile Phe Phe Trp Pro 60 PCT1US96/03916 WO 96/29396 -153t"Cr eCC CGC GCG TAT TTT GAA GGG 16383 Asp Leu Ala Ala Val Ile Val Gin Ala Ala Arg Ala Tyr Phe Gilu Gly 75 s0 AAG GAA AGG CTG GGC AGT CTG CAG GTC GCC GAA GAT ATC ACG GCG CAC Lays Glu Arg Lau 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 Ala 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 Laeu Vai Gly Gly Pro Asfl Gly -Giu 120 125 130 TTG GAA AGC AAG GTC TGG GGC MAG CAG ATT CCC COG GCC GCC GCG TGG Leu Clu Ser Lys Val Trp Gly Lys Gin Ile Pro Arg Ala Ala Ala Trp 135 140 145 GMA ATA ACA GAC GTG CCC AAA GTT CCA GTC ATT COG CCG GAC ATTCT Glu Ile Arg Asp Val Pro Lys Val Pro Val Ile Giy Pro Asp Ile Laeu 150 155 160 165 TCT TTT TTC TCC GCC GCC GTC GAG CTG CCC GTG CTC TAT ATC AGA GCC Ser Phe Phe Ser Ala Ala Val Giu Lau Pro Val Leu Tyr Ile Arg Ala 170 1175 180 CGG GGA GO GCG CAC TCG CGG TCC CC CAC TGG MAT AAC CAG AGC AGC Arg Gly Giy Ala His Ser Arg Ser Ala Hi.s Trp ASfl An Gin SEr ser 185 190 195 GC CCG GCC CCC GCA CTC GC GCG ATA AGG ATA GCC ATG GAG ATG CTG Ala Pro Ala Ala Gly Leu Ala Ala Ile Arg Ile Gly Met Giu Met Vai 200 205 210 CGG AGC C= CTG CTG ATA GCG CTG CCT CTG TCA MAC TTC ACC CTC CCG Arg Ser Leu Leu Val Ile Ala Leu Pro Leu Ser Asn Phe Thr Leu Pro 215 220 225 GMA GAC CTC CCC GMA GG'T TCC CMA MC TCG ATC CGC 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 GC'T ACC GAT MAG ATC ATG TCT CCG GAC GTG His Leu Met Asn Cys Val Ala Thr Asp Lys Ile Met Ser Pro Asp Val 250 255 260 CGC CTC CCA GTC GMA CM AGC TTT TAC AGC CAC TGT TTA AGG GMA ATC Arg Val Pro Val Giu Ciu Ser Phe Tyr Ser His CyS Leu Arg Giu Ile 265 270 275 ATT ATG TGC GAG AGA CCT TTT TCT TAC CCC TGC MAT CCC CCG CCA MA Ile Met Cys Giu Arg Ala Phe Cys Tyr Pro Cys Asfl Pro Pro Pro Lys 280 295 290 TGG TGA GCTCAGGGTC CCATTTACCC CCGCAAC-ACC CTCTCGCCGC

CAGGGCGCGC

Trp.

295 GCTCTATCTI' TCTCTATGTC CCCTCGCCAC CGGcCTAACC GMCGGTGGA

ACGGGGCCGC

CCGGGAAAGC CTAGCTCCCC ACAGACACAG ACAGACAMAC GGCCTCTCGA

TTGCAACMC

GTGAAAAACA CACAATAATA T-rACTTTATr TATTTCGCAG CGCTCGCGTG TCGCTCCT1T CTGGGGGGGG GGGGGGGTTG TGTTTCGCTC CCCCGGACTG GGTGAGGGGG

GAGAGTGGAC

6431 6479 6527 .6575 16 623 16C71 16719 16767 16815 16863 16911 16959 17007 17063 17123 17183 17243 17303 PCT[US96/03916 WO 96/29396 -154- GGAGGACAGT GTAAAAACCC GCGAGGTTGT CAGGGACGCG GAGGTAAGGA

AGCCGCTAGA

GGGCGTCATT

CTCCCGTCAA

CAAAQATCGA GGCGCTCACT CCAGGTATTC GCGACGGCGC GACTCTGCAC GTAAATCGAG AGCAGCACCA GTGCCCGGCC GGTCGGCCGG CAGGTGTCGG TATTCTCGCT CTGCATGGTA GcCAGAAATC GCCCTCGATC TATTCGGGTC AGTGGGG-GAG CTAGGCCGCG CGTCGCCCGG CAATCAGCGC GTTGCGGCGG CGGACGATAT CTCGACTAAC CCGAGTCGGC CGACTCCGAC GTACCGCGTG CGCAGTAATG GCATGTTTTC CATTTAGGGA GTGCTGGGCC GAGTTGGACG GATGTCCGGC GGTCGCAAAC AAGTTCTGGA TGAGAATGG; GAGGGGGAGG TTCTGCCCC( GCCGTGCAGG GGTCGAGGCC AGGAAGAAGA AAATGACAA' AAGCCTTCCG TGGGTC TT AGCAAATGGC GGCGGCGCC CTC'rGACI'CG GTGGAGCTG CGGCGCCGCG C TCGGGAGAGT

T

ATCTCCGAT G

TCGTACAAAC

GCCAGGAGAG3

GACAGCCAGG

CGGCTCTCGT

CCTrCCATTG

TTGGCGACGC'

CTGGCC-CCCA

TCTCTCGCTG.

TCGCGCCAGA

GAGGCGACAG

TGATCGCTGT

,TGTGTCTAGA

GCTACGGATT

GGGCGCGCGT

kTCGAGCGGGC ScoTTCrCcT GCTCTcrrcT" CAGCGCCAG C 'GGCGGCAGC! C

;CGGGACTCT

LCGCTTCGCG

XCCGGGAGGT

k.TAACAGTTC

CCTCGAAGCG-

CCTCCTCTAA

TCTCCGTCGA

GAAAAGCCGA

ACGTGAACTC

GCCGcrGAGGC cGGCACCTTG

CCATCAGCGC

GGAGAGCGGT

GCGCCGGGCG

CCGCTTCTGC

AATAAATGTC

ATCTGCTCGT

GCGATTCCA C GCGCCGGCG C

:CCCGTCCCG

;CACGGCCGAC

CTCTCGCTCG

C

rTTCTCGATC

J

GTCACCGTCC

J

TTCTCCCAAC(

CGCCCACCGC

I

ACGTAGCCGT'

TCCGTAACCG

GATccGAATG

CCGCCTGCGC

CAGCGGGTAG

CCGGGCGGTC

TGAAGGGGGG

GTGAAGGCTG

CAGAGTAGGG

CGAGGCCTCG

CGTCCCTCG

GCAAGGCGG

,CGCAGAAAA

GGTGCCACA

;CGGACGCGA

ZCGAAACGCA

LGAGAGTCGA

!CGTTGCCGT

;GCCTCAAAC

rCGTTGGCAG rccTTGA=T

:ACGGGTCTA

rCGCCACGAG

:ACCCGTCGA

rACTGCGGTT

~GGCGGCGG

AGCGGGA4AG

GGGTGGGAGG

GCCTTGCGTC

TCCTCGTCAG

CTGGCGGCCG

CGCGGGGT

ATTGCGTGGG

CCCGGCACCC

17363 17423 17483 17543 17603 17663 17723 17783 17843 17903 17963 19023 18093 18143 18203 18263 19323 18393 19443 18503 18563 18623 18693 18743 19903 18863 18912 T1=TACTTCT CTCCTCGGAT CTCCGTC1TT TAAGAGTGCG CCTACCCGC-,

CGGGTGCCGC

GAGAGCTGTC

GTCCCGTGTC

CCGGGTCGCT

AAAACTTCTO

GCACCGCA-AT

GTCAATAAAG

-TCC7CGCCC

AACGCACGGA

TCTAAGTCAG

;,AGTCcACTC

AGGCTGCTCT

CTCCGATACG

TCCTCCGACT

TCCGAAAAGA

CCAGCTCCTC CCGCGTGCCG GTTCTTCGTC GGAGAGCTCT TCGGGATCC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 278 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 14:60: PCTIUS96/03916 WO 96/29396 -155- Met Giu Asfl met Leu Asp Gly CyS Tyr Pro 1 5 10 Leu Ala Leu Met Asp Ser 9* Asp Gly Ile Giu Asfl Ala Thr Leu Leu 145 Gin Leu Se r Arg His Ala Arg Leu Ala Ser Glu Gly 130 Asp Se r Se r Arg Pro 210 Ile Ala Ile Lys Leu Arg Ser 115 Glu Ser Glu Arg Ser 195 Cys Thr Val Ala Asp Ile Ala 100 Val Leu Leu Asn His 180 Ala Ala Ser Gly Ala Thr Ala Glu Asp Met 165 Leu His Ser Arg 70 Al a Arg Asn Giu Gly 150 Arg Pro Ser Leu 55 Gly Asn Gly Ser Ala 135 Asp Phe Al a 25 Giu Ser Ala 40 Trp Arg GlU Glu Phe Thr Glu Arg Leu 90 Leu Gly Leu 105 Pro Thr Asp 120 Met Glu Gly Arg Phe Giu Val Ile Giu 170 Asp Leu Ala 185 Gly His Trp 200 Leu Tyr Asp Asp S LeU Ser3 75 Arg Arg Pro Ile Asp 155 Lys Ser Cys Ser Gin 235 Al a Asp er tal Lla ;er Pro ksn Glu 14 0 Glu Glu Al a Cys IlE 22( Ala Val Pro Arg Gly Giu Arg Val Giu Arg Ala Arg Asfl 125 Gly Ser Leu Glu Leu 205 ~Tyr ksp Ile A.sp Phe Trp 110 Gly Asp Arg Leu Arg 190 Trp Val Arg Pro Se r Arg Leu Ala Asn Phe Thr Ser 175 Glu His Gln Gin Cys Arg Gly Ser Gly Trp Met 160 Trp Thr Pro Ser Arg 240 i Ser a Leu Leu Leu Ala Ala Arg Glu Ala Cys 215 Leu Phe Cys Val Gly Thr Gly Arg Val Pro Ser Glu Met Arc 225 Arg Pro Ala Giu Giu Leu Tyr Val Ala 275 Al a 245 Al a Arg 230 Al a Ser Arg Arg Phe A a A-1 a Ala Ala Gly Ile 270 INFORMATION FOR SEQ ID 140:61: SEQUENCE CHARACTERISTICS: LENGTH: 294 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO-61: PCTIUS96/03916 WO 96/29396 Met Cys Prc Thl 11 6' Al As Va G1 oAr 14 *o L

A

G

A

A

2

A

M

-156- Ser Lys Cys Tyr Cys Leu Ala Arg His Leu 5 Val Gly Arg Arg Val Ala Phe Gly Gly Leu o Pro Thr Ser His Leu Asp Leu Ala Phe Ser 40 r Asp Leu Pro Gly Gly Arg Phe Trp Arg Ala e Phe Phe Trp Pro Asp Leu Ala Ala Val Ile 5 7075 a Tyr Phe Glu Gly Lys Glu Arg Leu Gly Ser 85 90 p Ile Thr Ala His Asp Pro Arg Ile Ala Pro 100 105 1 Ala Ala Ala Val Gly Leu Trp Thr Ala Let 115 120 y Pro Asn Gly Glu Leu Glu Ser Lys Val Tr3 130 135 *g Ala Ala Ala Trp Glu Ile Arg Asp Val Pri 5 150 15 .y Pro Asp lie Leu Ser Phe Phe Ser Ala Al 165 170 eu Tyr Ile Arg Ala Arg Gly Gly Ala His Se 180 185 sn Asn Gln Ser Ser Ala Pro Ala Ala Gly Le 195 200 ly Met GIu Met Val Arg Ser Leu Leu Val Il 210 215 sn Phe Thr Leu Pro Glu Asp Leu Pro Glu G1 25 230 2: rg Ala Phe Val Ala His Leu Met Asn Cys Vi 245 250 let Ser Pro Asp Val Arg Val Pro Val Glu G 260 265 :ys Leu Arg Glu Ile Ile Met Cys Glu Arg A 275 280 sn 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 Tyr Ala Ala Ser Val Leu Ala u Ser p Gly 140 o Lys 5 a Val r Arg u Ali .e Al, 22 ly Se 35 al Al lu Se la P) Lys Ser Pro Arg 15 Thr Met Ser Arg 30 Ala Phe Arg Gly 45 Gin Ser Cys Asp Gin Ala Ala Arg Gin Val Ala Glu 95 Ala Lys Arg Ala 110 Glu Leu Val Gly 125 Lys Gln Ile Pro Val Pro Val Ile 160 Glu Leu Pro Val 175 Ser Ala His Trp 190 a Ala Ile Arg lie 205 a Leu Pro Leu Ser 0 r Gln Asn Ser Ile 240 a Thr Asp Lys Ile 255 .r Phe Tyr Ser His 270 ie Cys Tyr Pro Cys 285 WO 96/29396 WO 9629396PCT/US96/03916 -157- DESCRIPTION: SEQ ID NO:62: (xi) SEQUENCE Met Ala Pro Val Lys Val Thr Ile Val Ser Ala Val Asp Ser His TYr Lys Leu Leu Leu Gly Gly Leu Pro Glu 145 Ser Leu Val Ser Phe Gly Phe Trp Ala 130 Arg Leu Pro His Val Ser Asp Tyr Val 115 Pro Gly Ser An Ala Ile Leu Pro His 100 Leu Lys Val His Ser Val Val1 Pro Pro Leu Gly Thr Glu Pro 165 Arg Lys Arg Arg 70 Gly Ser Thr Phe Ile 150 Ser Phe Thr Pro 55 Met Val Ser Ala Leu 135 Phe Glu Met 40 Al a Cys.

Ser Gly Asp 120 Leu Leu 25 Al a Ser Arg Pro Ala 105 Ile Thr Val Ser Ser Leu Pro Glu 90 Tyr Cys Glu Asn Asp Tyr Tyr Val 75 Trp Ala Met Thr aly 155 Ser ASp Glu Ile Ser Ala Ala Gly 140 Asp Ala Gly Arg Val Arg Gly Lys Ala 125 GI y Lys Pro rrp Pro 5cr Phe Leu Glu 110 Leu Lys Thr Ser Lys Ser Gly Gly Asp Phe Asn An Thr Ser 175 Glu Thr Glu Glu Al a His Leu Phe Leu 160 Lieu 0* p I I C Val Trp, Thr Thr Leu 170 Arg Thr Pro Trp, 180 Pro Tyr Ser Thr Val 195 Lys Phe Leu Lys Val Lys Pro 190 Asn Ser Ala Ala Tyr Cys Val 5cr Asp 195 200 Ser Asp Asp 205 Lys Pro Arg ser

C.

0 C

C

C

Pro Pro 225 (2) 210 Arc Arg Leu Gly Arg Ser Leu Phe Lys Ser Lys 215 220 INFORMATION FOR SEQ ID NO:63: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 476 amino acids TYPE: amino acid CD) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO;63: Arg Phe Arg Arg Ile Cys Ser Arg 5cr Arg Ala Glu Lys Azg Arg 5 10 Thr Thr Glu Asn Pro Leu Thr 5cr Lys Axg Val Cys Val Leu Asp 25 Phe Ser Arg Thr Met Ser Leu Arg Pro Tyr Ala Giu Ile Leu Pro 40 Met 1 Arg Scr WO 96/29396 PCTIUS96/039 16 -158- Thr Ala Glu Gly Val Giu Arg Leu Ala Glu Leu Val Ser Val Thr Met so 55 Thr Giu Arg Ala Giu Pro Val Thr Glu As Thr Ala Val Asn Ser Ile 65 70 75 Pro Pro Ala Asn Glu Asn Gly Gin Asn Phe Ala Tyr Ala Gly Asp Gly 85 90 95 Pro ser Thr Thr Giu Lys Val Asp Gly Ser His Thr Asp Phe Asp Glu 100 105 110 Ala Ser Ser Asp Tyr Ala Gly Pro Val pro Leu Ala Gin Thr Arg Leu 115 120 125 Lys His Ser Asp Giu Phe Leu Gin His Phe Arg Val Leu Asp Asp Leu 130 135 140 Vai Giu Gly Ala Tyr Gly Phe Ile cys Asp Val Arg Arg Tyr Thr Giu 145 150 155 160 Giu Giu Gin Arg Arg Arg Giy Val Asn Ser Thr Asn Gin Gly Lys Ser "50*e165 170 175 Lys Cys Lys Arg Leu Ile Ala Lys Tyr Val Lys Asn Gly Thr Arg Ala 180 185 190 Ala Ser Gin Leu Giu Asn Giu Ile Leu Val Leu Gly Arg Leu Asn His 195 200 205 Glu Asn Val ieu Lys Ile Gin Giu Ile Leu Arg Tyr Pro Asp Asn Thr 210 215 220 09*e Tyr Met Leu Thr Gin Arg Tyr Gin Phe Asp Leu Tyr Ser Tyr Met Tyr 225 230 235 240 Asp Giu Ala Phe Asp Trp Lys Asp Ser Pro Met Leu Lys Gin Thr Arg 245 250 255 Arg Ile Met Lys Gir. Leu Met Ser Ala Vai Ser Tyr Ile His Ser Lys 260 265 270 Lys Leu Ile His Arg Asp Ile Lys Leu Giu Asn Ile Phe Leu Asn Cys s,275 280 285 Asp Gly Lys Thr Val Leu G1y Asp Phe Gly Thr Val Thr Pro Phe Giu 290 295 300 Asn Giu Arg Glu Pro Phe Giu Tyr Gly Trp Val Gly Thr Val Ala Thr 305 310 315 320 Asn Ser Pro Glu Ile Leu Ala Arg Asp Ser Tyr Cys Giu lie Thz Asp 325 330 335 Ile Trp ser Cys Giy Val Val Leu Leu Glu Met Val Ser His Giu Phe 340 345 350 Cys Pro Ile Gly Asp Gly Gly Gly Ann Pro His Gin Gin Leu Leu Lys 355 360 365 Val Ile Asp Ser Leu Ser Val Cys Asp Giu Giu Phe Pro Asp Pro Pro 370 375 380 Cys Asn Leu Tyr Asn Tyr Leu His Tyr Ala Ser Ile Asp Arg Ala Gly 385 390 395 400 WO 96/29396 PCTIfUS96/03916 -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 Psp Trp Arg Teu 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 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 Giu 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 Gin Ala Trp Asp Val Leu Gin 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 Gin 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 Gin Cys Val 180 185 190 Pro Phe Thr Arg Ser Lys Lys Ser Val Thr Val Arg Asp Ala Gin Ser 195 200 205 PCTUS96/03916 WO 96129396 Phe Leu Gly Asp Se 210 Val Leu Arg Gin Hi 225 lie Val Asn Cys As 24 His Trp Arg Gly Me 260 Glu Glu Asn lie G1 275 Gly Ala Gly Leu Pk 290 Arg Asp Lys Arg Lt 305 Asp Ala Ala Val LE 3: Asp Thr Ser Arg P1 340 Ala Leu Val Val L 355 His Ala Leu Val P 370 Tyr Asp Giu Thr A 385 Gly Ala Giu Gly T 4 Leu Ala Val Val C 420 Ser Val Ala Thr 1 435 Lys Ala Azg Arg 450 Glu Phe Phe Phe 465 Asn Phe Cys Leu Pro Phe Leu Phe 500 Lys Met Leu Cys 515 Lys Ala Met Gly 530 Lys Ala Ile Ser 545 r Phe s lie 230 .n Asp 5 t Arg .n His ie Leu !u Leu 310 eu Ala 25 ne Glu eu Leu he Met rg Tyz 39C hr Let ,05 'ys Al.

er Al Pro Gi Tyr I 47 Asn 11 485 Arg Al Pro L4 Ser G Ser A 5 -160- Trp Arg lie Met 215 Thr Arg Leu Arg Pro Leu Trp Tyr 250 Val Pro Ser Leu 265 Gly Pro Met Ala 280 Trp Pro Ala Met 295 Arg Ala Cys Leu Ser Phe Pro Phe 330 Pro Ala Leu Gly 345 Ala Glu Thr Val 360 Arg Ala Leu Ala 375 lie Asp Pro Vai i Leu Arg Gly Ile 41C Asn Thr Tyr Sei 425 a Cys Asn Val Ali 440 y Met Ser Asp I1 455 e Ala Trp Leu Gi 0 e Leu Lys Arg Se 49 a Ser Ser Giu L 505 eu Leu Val Pro 11 520 iu Leu Lys Arg G: 535 zp Arg Asp Pro A 50 Gin Asn Val Tyr Th 220 His Pro Ser Ser Ly 235 Ala Tyr Ala Asn G1 25 Lys Leu Ala Ser Pr 270 Ala Val Phe Arg As 285 Arg Ala Ala Phe GI 300 Ser Ser Leu Asp 11 315 Tyr Trp Arg Gly Vz 3: Cys Leu Ser Giu 350 Leu Ala Thr Met P1 365 Asp Giy Asn Phe A 380 Lys Asn Glu Tyr L 395 t Val Ala Ser Asn T 4 Thr lie Arg Lys L 430 i Tyr Arg Thr Giu 1 445 e Tyr krg lie Leu 460 n Arg Val Ala Thr I 475 r Val Asp Thr Gly 0 's Arg Leu Gin Gln 510 Le Gin Tyr Giu Asp 525 lu Lys Leu Giu Thr 540 rg Giy Ser Leu Arg 555 r Va s Se 24 n Ph 5 o Pr ,n Al *u Gl .e ME 32 1l GJ 'r P~ he A sp A eu A 4 hr A Ieu P :hr I 3n I His Ala 495 Leu Phe Phe Phe 1 r 0 e 0 la .u ti Ln lr pp sp nn 00 la ro eu ys Ala 480 Pro Asn Ser Val Leu 560 WO 96/29396 PCT/US96/03916 Ile Pro Ala Val Ser Asp His Ala Arg 565 Val Ile Asp Glu Pro 580 Ala Gly Lys Val Lys 595 Pro Gly Ala Gly Ala 610 -161- Giu Ile Ile Ala Asp Gly Val Arg Phe Lys 570 57 Val Arg Ala Ser Val Ala Leu Ser Thr Ala 585 590 Ala Arg ArS Leu Thr Scr Val Arg Ala Pro 600 605 Val Ser Ala Arg Arg Lys Ser Glu Ile 615 620 0 INFORMATION FOR SEQ ID W. SEQUENCE CHARACTERISTICS: LENGTH: 292 amino acids TYPE: amino acid TOPOLOGY; linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID) N Met Ser Gly Ph. Ser Asn Ile Gly 5cr Ile A 1 5 10 Cys Ser.Leu Leu Cys Ala Ser Val Leu Gly A 25 Leu Glu Ser Ser Pro Phe Pro Phe Gly GlyL 40 Cys Asn Arg Thr Thr Ile Glu Val Thr Val P 50 55 Gly Arg Thr Glu Gly Val Ser Val Glu Val I 70 Ser AXsn Pro Glu Ser Ph. Val Phe Gly Val as 90 Gly His Glu Asp Leu Ser Thr Cys Trp, Ala 100 105 Ala 5cr Val Cys Arg Ala Ser Asp Ala Gly 115 120 Gin Cys Giu Lys Val Gin Arg Arg Leu Arg 130 135 Ser Tyr Val Ser Gly Asn Gly 5Cr Leu Val 145 150 Asp Ala Gly Ile Tyr Ala Tyr Gin Leu Ser 165 170 Thr Gly Ser Val Tyr Leu Asp VJal Gly Pro 180 165 Gin Tyr Gly Tyr Thr Tyr Tyr Ser Leu Ala 195 200 Ser Ser Tyr Asp Val Ala Ser Pro Glu Leua 210 215 0:65: la Thr la, Pro .yz Ile 'ro Trp .ys Trp 75 ksp 5cr Leu Ile Ile Pro Ser Gl 14C Leu Tyl 155 Val G1, Asa Pri Asp G1 Asp GI 22 Val Ser L Val Leu Ile Ala G 4S Ser Asp Phe Tyr C Glu Thr His Asfl 110 Asp Phe 125 Val Giu Pro Gly rGly Lys o Gly Cys 190 u Ala Ser 205 v Pro Met eu Val .sp Gly in Ala 'yr Ser 1y Asn 31y Scr Leu Asn Asp Lys Leu Gly Met Tyr 160 Gly Tyr 175 His Asp Asp Leu Glu Giu WO 96/29396 PCTIUS96/03916 Asp 225 Va1 Tyr Glu Thr -162- Cys Leu Asp Met Pro Pro Leu Arg Pro Trp Thr Thr 230 235 240 Asp Val Glu Glu Gin Glu Asn Ala Thr Asp Glu Leu 245 250 255 Glu Glu Cys Ala Gly Pro Leu Asp Glu Tyr Val Asp 265 270 Thr Met Pro Arg Met Val Val Phe Ser Pro Pro Ser 280 285 0000 .0 0.0.

0.00 .0 0 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 A 1 5 10 Ala Ala Leu Pro Thr Gly Arg Phe Cys Arg Val Trp Lys Val P 25 3C Gly Gly Thr Ile Gin Giu Asn Leu Ala V'al Leu Ala Giu Ser F 35 40 Thr Gly His Ala Thr Tyr Pro Pro Pro Glu Gly Ala Val Ser I 55 Ile Phe Ala Asp Thr Pro Thr Lou Arg Ile Arg Tyr Gly Ala 70 75 Asp Giu Lou Ala Leu Glu Arg Gly Thr Ser Ala Ser Asp Ala 90 Val Thr Phe Ser Leu Ser Tyr Arg Pro Arg Pro Glu Ile His 100 105 110 Tyr Phe Thr le Gly Val Phe Ala Thr Gly Gln Ser Thr Glu 115 120 125 Tyr Ser Val Ile Ser Arg Val Leu Val Asn Ala Ser Lou Glu 130 135 140 Val Arg Leu Glu Thr Pro Cys Asp Glu Asn Phe Leu Gin An 145 150 155 Thr Trp Gly Ser Lys Arg Trp Lou Gly Pro Pro Ser Pro Tyr 165 170 Asp Ann Asp Val Ala Val Lou Thr Lys Ala Gin Tyr Ile Gly 180 195 190 Tyr Ser Ann Ser Ala Ala Gin Thr Gly Lou Thr Ser Leu Asn 195 200 205 la 'ro ,ro ?he rhr Asp Gly Ser Arg Glu Val 17E G1 Me Asp Pro Val Gin Glu Asn Ala Ser Ser Pro 160 Arg Cys t Thr PCT1US96/03916 WO 96/29396 0000 o 0 0000 0 a 0 0 0 1 Phe P1 21 Gly P~ 225 Gin P1 Pro Au Pro A.

Lys L 2 Glu P 305 Thr P Arg P Asp T Thr T 3 Ala 385 Asp I Glu Glu Thr Ser 465 Gin Thr Ala Phe Ala 545 ee

LO

ro ro ap La eu 90 ro ro ro hr *hr 70 Lg krg Ser rhi Val G1i Al.

G1 G1 Th 53 Al Tyr Ser Pro ser Pro Ser Val Leu Arg 245 Ile Asp Gly 260 Asp Ser Gly 275 Pro Ser Ala Pro Giy Thr Tyr Arg Ala 325 Gly Ala Thr 340 Gly Thr Sex 355 Leu Ile Prc Ala Thr GI1 Thr Leu Se: Glu Asn Th 420 Pro Ser Al 435 Val Phe Th i Ser Gin 31 a Ala ieu Th 48 n Thr Pro Si 500 u Thr Giu A 515 Lr Gin Ser A .a Pro Gu V -163- Lys Arg Ile Val Asn Va 215 Arg Ile Thr Val Tyr Se 230 23 Asn Vai Ser Asp Gly Ph 250 Arg Ala Met Ile Asr Va 265 Ser Vai Leu Ala Phe T1 280 Ile Gin Leu His Arg I] 295 Glu Thr Thr Phe Asp C) 310 3: Leu Gly Ser Asn Val PI 330 Leu Pro Pro Phe Asp TI 345 Pro Thr Pro Thr Thr V.

360 Arg Ser Thr Ser Asp M 375 Ser Glu Thr Leu Ser V 390 3 r Thr Thr Pro Leu Thr L 5 410 r Leu Phe Pro Thr Thr P 425 a Ala His Glu Thr Thr C 440 r Gin Ser Pro Ser Thr C 455 u Pro Trp Tyr Phe Thr 470 :r Gin Thr Gin lie Ala I5 490 r Ala Glu Gin Met Thr 505 la Pro Ala Gin Thr Pro 520 rg Ser Thr Pro Pro Glu 535 ai Phe Thr Gin Ser Ser 550 1 Thr Tr 220 r Ser Ar 5 ie Leu Va l Ile Al ir Ala P1 21 e Asp MI 300 ~s Gin L, 15 ro Arg A hr Ala A al Pro G 3 et Gly P 380 al Pro V 95 *eu Pro I la Pro C ;In Thr ;1u Ser 460 31n Thr 475 Glu Thr Phe Thr Ser Thr Thr Ala 540 Ser Thr 555 'p 11 ia iu e 'a sp a lu he 'al ;ly ;Ir I4 Pr

G!

GI

52 Ai

V;

Thr Thr Gi Glu Asn Gi 24 Lys Tr.Th 255 Asn Tyr Se 270 Arg Giu G1 Ser Gly T1 Met Ile G 3: Asp Ser I: 335 Pro Asp P 350 Pro Ala I: Phe Ser T Gin Giu T 4 Thr Pro G 415 Ile Ser 1 430 Ser Ala C a Thr Ala 2 o Ser Thr u Ala Leu 495 n Thr Pro 510 .e Pro Giu :g Ala Pro al Thr Glu y y 0 r Lu le he le hr hr 00 ly 'hr lu krg 31u 48o Phe Gly Ile Ser Val 560 WO 96/29396 -164- Phe Thr Gin Thr Pro Ser Thr Val Pro Lys Thr 565 570 PCTIUS96/03916 Thr Leu Ser Ser Ser 575 Thr Phe Thr Thr 625 Thr Thr Thr Thr Val 705 Leu Glu Thr Glu 610 Gin Glu Gin Met Ser 690 Gin Ser Pro Ala Ile Phe Gin 595 Thr Thr Pro Thr Pro 675 Ser Thr Phe 580 Thr Ser His Phe Pro Ser 'Val Pro 645 Pro Ser 660 Glu Thr Gly Thr Thr Pro Pro Thr 725 Ser Phe Thr 630 Phe Ala Ala Gly His 710 Thr Ala Thr 615 Glu Thr Ala Gin Thr 695 Thr Arg Glu 60C Gin Pro Arg Pro Ser 680 His Gin 585 Pro Ala Glu Thr Glu 665 Thr Asn Lys Asp Pro Va1 Leu 650 Val Pro Thr Leu Thr Ser Leu 635 Gly Tyr Leu Glu 715 Met Thr 620 Thr Ala Thr Ala Pro 700 Thr Thr Gin Ser Ala Gly Thr Glu Ala Arg 605 Val Gin Glu Arg ser 685 Arg Glu 590 Thr Pro Ser Pro Ser 670 Gin Thr Ann Gin Lys Pro Glu 655 Ser Asn Tyr Lys Ser Ala Ser 640 Ile Ser Pro Pro Thr 720 r Val Val Ser Giu Phe His Glu Met Ser 730 Thr Ala 735 Glu Set Gin Thr Pro Leu Leu Asp 740 Val Lys Ile Val Glu Val Lys Phe 745 750 Ser Ann Asp Gly ,7 5P" Glu Va Thr Ala Thr Cys Val 760 Pro Tyr Arg Val Glu 770 Asp Giu Ile Ser Gly 785 Lys Trp Gin Lys Gin 805 Val Gin Leu Met Cys 820 Cys Leu Phe Asp Thr 835 Glu Leu Tyr Phe Thr 850 Leu Pro Set Gly. Val 865 Ser Leu Pro Glu Tyr Gly Gin Ser Thr Va) 900 rhr Asn 1 775 Asn Set I 790 Leu Tyr Leu Ser Ser Leu Set Asp 855 Val Pro 870 Leu Thr Trp Lys rrp Pro Tyr Cys Ile 840 Pro Arc Al Se: Lys Ala Arg Thr 825 Ala Gin Phe i Thr r Sei 90' Val Asp Gly Val 795 Val Thr 810 Ser His Arg Clu Thr Ala Glu Tr; 875 Thr Va 890 Ala Ar Ser Thr Val Lys Ser 765 Leu Val Asp Val Met 780 Phe Ann Set Ann Glu 800 Asp Gly Arg Thr Ser 815 Ser Pro Giu Pro Tyr 830 Lys Asp Ile Ala Pro 845 Tyr Cys Thr Ile Thr 860 Set Leu Ann Ann Val 880 L Val Ser His Thr Ala 895 9 Ala Gly Gu Ala Trp 910 PCTIUS96/03916 WO 96129396 Ile Asp Trp 945 Phe Ala Gly Arg 915 Thr Arg Ala Val Gly Leu Leu Trp 980 Gly Asn Ile 920 Thr Thr Arg 935 Asn Gly Ala 950 Ser Gly Leu Ala Ile Tyr -165- Tyr Giu Cys Thr Val Leu Ile Ser 925 Lys Giu Arg Cys Leu Thr Asn Thr 940 Ala Gin Ala Gin Leu Tyr Ser Leu 955 960 Cys Gly ser Ile Ser Ala Leu Tyr 970 975 Phe 985

S

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 NO:67: Met His Arg Pro His Leu Arg Arg His Ser Arg Tyr Tyr A 1 5 10 Glu Val Leu Asn Lys His Met Asp Cys Gly Gly Lys Arg C 20 25 Gly Ala Ala Val Phe Thr Leu Phe Trp Thr Cys Val Arg I 35 40 Glu His Ile Cys Phe Val Arg Asn Ala Met Asp Arg His L 55 Arg Asn Ala Phe Trp Thr Ile Val Leu Leu Ser Ser Phe Z 70 75 Ser Thr Ala Ala Val Thr Tyr Asp Tyr Ile Leu Gly Arg 90 Asp Ala Leu Thr Ile Pro Ala Val Gly Pro Tyr Asn Arg 100 105 Arg Val Ser Arg Gly Cys Asp Val Val Giu Leu Asn Pro 115 110 125 Val Asp Asp Met Ile ser Ala Ala Lys Glu Lys Glu Lys 130 135 140 Phe Giu Ala Ser Val Val Trp Phe Tyr Val Ile Lys Gly 145 150 155 Glu Asp Lys Tyr Cys Pro Ile Tyr Arg Lys Glu Tyr Arg 165 170 Asp Val Gln Leu Leu Ser Giu Cys Ala Val Gin Ser Ala 180 185 Ala Val Asp Tyr Val Pro Ser Thr Leu Val Ser Arg Asn 195 200 205 la Lys Gly ys Cys Ser ie Met Arg ,eu Phe Leu la Ser Gin krg Ala Leu yr Leu Thr 110 Ile Ser Asn Gly Gly Pro Asp Asp Gly 160 Glu Cys Gly 175 Gin Met Trp 190 Gly Ala Gly WO 96/29396 -166- Leu Thr Ile Phe Ser Pro Thr Ala Ala Leu 210 215 PCTIUS96039 16 Ser Gly Gin Tlyr Leu Leu 220 Thr 225 Val Ser His Lys 305 Giu Glu Gin Glu Tyr 385 Ser Leu Asn Lys Leu Arg 290 Lys Ile Ser Al a Val 370 Tyr Ser Lys Asp Cys Tyr 275 Gly Asn Pro Ser Giu 355 Pro Asn Asn Ile Gly Arg CYS 245 Trp Thr 260 Pro Ile Tyr Glu Pro Ser Ala Val 325 Giu Lys 340 Ala Ser Giu Asp Asp Met Ala Val 405 Arg 230 Leu Tkxr Al a Asp Ala 310 Thr Lys Gly Thr P~ro 390 Phe Lys Glu Asp Ile 295 Pro Lys Ala Glu Giu 375 Al a A1a Ile Gin Thr 280 Leu Asp Lys Pro Asn.

360 His Val1 Gin Gly Tyr 265 Asn Gin Pro Ala Pro 345 Pro Asp Ile Thr Ser 2S0 Gin Thr Arg Arg Clu 330 Glu Ala Asp Pro Pho Ala 235 Gin Thr Arg Trp Pro 315 Gly Asp Al a Pro Val 395 Al a Leu Lou His Asfl 300

ASP

Arg Ser Leu Asn 380 Ala Val Asn Phe Ala 285 Asn Ser Thr Giu Pro 365 Ser Glu Phe Thr Phe Gin 270 Asp Leu Val Pro Asp 350 Glu Asp Thr Val Leu Glu 240 LeU Pro 255 Gly GiU Asp Val Lou Arg Pro Gin 320 Asp Ala 335 Asp Met Asp Asp Pro Asp Thr Lys 400 Ala Cys 415 Ser Met Pro Ile 410 Al a Arg Val Ala Lou 420 Ser Val Gly Leu Leu Trp Ser Ile Val Lys Cys Ala 430 INFORMATION FOR SEQ ID NO:68: 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 Lou Lou Gly Thr Lou Ala Lou Leu Ala Ala Thr Lou Ala 1 5 10 Pro Phe Gly Ala met Giy Ile Val Ile Thr Gly Asa His Val 5cr Ala 25 Arg Ilie Asp Asp Asp His Ile Val Ile Val Ala Pro Arg Pro Glu Ala 40 WO 96/29396 PCTIUS96/03916 Thr Ile Gin Leu Gin Leu Phe 55 -167- Phe Met Pro Val Ala Phe Gly Gin Arg Pro His Lys Ser Asp Ile Thr a a a.

a a a. a a a a a Pro Gin Arg Ser Ile Arg 145 Ser Lys Tyr Thr Thr 225 Tr-p T'yr Cys Ser Ala Arg 130 Leu Val Al a Leu Giu 210 Ala Leu Ser Ter Ser 115 Asn Asp Tyr Ser Arg 195 Ala Thr Glu Gly Gin Ser 100 Asn Pro Asp Gln cys 180 Thr Thr Ser Asn rhr Giu 85 Vai Arg Arg Thr Phe 165 Arg Glu Thr Ala Gly, 245~ Val Leu Phe Leu Pro Lys 150 Ala Thr Giu Thr Ser 230 Vai krg Ser Val Thr Asn 13 5 Giu ksn Phe Ser Ser 215 Giu Asp Giu Gly Gly 120 Asp Pro Thr Giy Trp 200 Al a Leu His Giu Cys 105 Pro Ser Ile Ala Leu 185 Arg Giu Giu Tyr Gly 265 rg ,ys Pro viy

A~SP

Ala 170 Pro Asn Al a Ala Giu 250 Val His Met Val 155 Thr Thr Trp Thr Giu 235 Pro Thr Pro Phe 140 Phe Arg Val Gin Thr 220 His Thr Giu Phe 125 Tyr Ala Gly Gin Ala 205 Pro Phe Pro Tyr 110 Lys Val Ile Leu Leu 190 Tyr Thr Thr Ala Thr Leu Ile Gin Tyr 175 Giu Val Pro Phe Asn 255 Phe Thr Val Leu 160 Ser Al a Al a Val Pro 240 Glu Phe Giu Asfl Cys Thr His Asn Ser Asn Val Thr Val Arg Leu 260 Thr Met Ser Pro Thr Leu Ile 270 Gly Vai Thr Val Ala Ala Val Val Ser Ala Thr 275 280 Val Ile Ser Ile Val Thr Arg AsrL Met CyS Thr 290 295 Asp Thr Val Ser Gin Asp Asp Giu Giu Arg Ser 305 310 315 Ser Arg Lys Phe G1y Pro Met Val Ala Cys Giu 325 330 Asp Gin Asp 5cr 'Ulu Leu Val Giu Leu Val Ala 340 345 Ala Leu Ser Ser Pro Asp Ser Ile Lys Met 355 360 INFORMATION FOR SEQ ID NO:'69: Wi SEQUENCE CHARACTERISTICS: LENGTH: 499 amino acids TYPE: amino acid TOPOLOGY; linear Ile Gly Leu 285 Pro His Arg 300 Gin Thr Arg Ile Asn Lys Ile Val Asn 350 Lys Arg Gly 3 Pro Leu Giu 320 Al a Ser Val Ile PCTIUS96/03916 WO 96129396 -168-

S

(ii) MOLECULE (xi) SEQUENCE Met As Met Leu Val 1 5 Phe Ala Thr Arg His Gly Glu Asp Asp Pro Thr Lys Val Leu Arg Ser Ser Pro Asn Tyr Lys Asp Cys Pro Pro Leu Lys Arg Gly Giu 100 Ser Asn Ile Thr Asn 115 Asp Pro Vai Thr Arg 130 Gin Pro Asp Leu Ala 145 Ser His Vai Asp Glu 165 Thr Ile Lys Pro Ser 180 Pro Phe His Glu Leu 195 Leu Arg Val Phe Prc 210 Trp Tyr Gly Phi 225 Phe Glu Pro Cys Va 24 Ala Thr Asn Gin Ax 260 Leu Gly Ala Ala Se 275 Cys His Giu Asn Ax 290 Glu Ser Arg Ala G] 305 Val Leu lie lie V 3; TYPE: protein DESCRIPTION: SEQ ID NO:69: Ile Val Leu Ala Ser cys Leu Al 10 Val Leu Phe Leu Glu Gly Thr G1 25 Arg Asn Val Pro Glu Gly Thr Va 40 4 Asn Ala Cys Lys Met Lys Ala Al 55 Cys Phe His Asp Leu Ile Tyr As 70 75 Ala Gly Pro Leu Ser Ala Asn Le 90 Ser Phe Val Val Leu Gly Ser GI 105 Ile Met Trp Thr Giu Tyr Gly G 120 1 Ser Asp Giu Giy Ile Tyr Phe X 135 140 Met Glu Thr Thr Ser Tyr Asn V 150 155 Lys Ala Pro Ala Pro His Glu V 170 Glu Ala His Ala His Val Glu L 185 Asn Asp Asn Ser Pro Thr Tyr V 200 2 Pro Thr Glu His Val Lys Phe 3 215 220 e Asp Val Lys Glu Giu Cys Glu 230 235 1 Tyr His Pro Thr Asp Gly Lys 5 250 g Cys Leu Ile Gly Ser Val Leu 265 r Leu Leu Asp Cys Ser Arg Asp 280 g Val Pro Asn Leu Arg Phe Asp 295 300 Ly Leu Val Ile Ser Pro Leu Iie 310 315 al Val Ser Asp Gly Asp lie Le.

25 330 a Arg Leu Thr n Ala Va Leu .1 Ile Lys Trp 5 .a Asp Val Cys ;p Gly Gly Lys

BC

!u Val Ile Leu Ly Leu His Asn 110 Ly Leu Leu Phe rg Arg lie Ser al Ser Val Leu 160 al Giu lie Asp 175 eu Gin Met Leu 190 ral Thr Pro Val sn Val Thr Tyr 31u Val Lys Leu 240 Cys Gin Phe Pro 255 Met Ala Glu Phe 270 Thr Leu Glu Asp 285 Ser Arg Leu Ser Ala Ile Pro Lys 320 Gly Trp Ser Tyr 335 PCTIUS961039 16 WO 96/29396 Thr Val Met Pro Pro Met 370 Val Ala 385 Lys Lys Leu Tyr Asp Asp Gly Tyr 450 Ala Val 465 Ser Met Asp Ser Leu Ser 355 Asp Ala Cys Ala Giu 435 Cys Val Al a Tyr Gly 340 Lys Glu Thr Pro Pro 420 Giu Lys Glu Asp

LYS

Val1 Thr Cys Ala 405 Leu Val Gin Gly Arg 485 Arg Asn Pro Met Gly Asn 375 Val Ile 390 His Gin Pro Lys Ile Tyr Glu Phe 455 Giu Arg 470 Ile Leu ser Asn 360 Tyr Leu Met Asn Asp 440 Arg Ala Ala Pr 34 Tk

GJ

G2 4:

G.

A

-169o Arg s~ Val rs Met ir Cys Ly Thr 410 Lu Phe lu Val lu Asp eu Asn sn Lys 490 Val Val Tyr Al a 395 Phe Giu Tyr Val Phe 475 Ser Val Ile Phe 380 Leu Ser Ala Glu Asn 460 Lys Gly V1al Gly 365 Val Leu Lys Gly Pro 445 Thr Ser Arg Glu 350 Ser Val Val Thr Giy 430 Leu Phe Ala Arg Thr Pro Ala Gly Giu 415 Leu Phe Phe Ile Asn 495 His Gly Gly Lys 400 Pro Thr Arg Gly Ala 480 Met 0 0 *00* 0 00*0 INFORMATION FOR SEQ ID 140:70: SEOUENCE 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 Giy Ala Glu Asp I S 10 Arg Phe Lys Lys Ser Arg Asn Arg Giu Ile 25 Gly Thr Gly Lys Lys Thr Ala Gly Leu Ser 40 Pro Trp Asn Pro LyS Phe Cys Ser Ala Arq 55 Ala Cys Lys Asp Thr Phe Tyr Arg Arg Thr 70 Thr Val Ser Ser Gin Pro Asp Ser Pro His 90 Tyr Gly Arg Val Pro Ser Ala Lys Arg Lys 100 105 0.

kla Ala Ala Gly Leu Pro Thr Arg Asn Tyr Thr Gin Gly Giu Ser Asp Cys Cys Ala Ser 75 Thr Pro Met Pro Lys Leu Ser Ser 110 Lys Leu Pro Arg Thr Ser Asn Arg Ile His Ser so Giu Asp WO 96/29396 PCTfUS96/03916 Cys Gin Asp 145 Leu Ser Thr Glu Asn 225 Ala Lys Glu Ala 130 Glu Glu Lys Asn Ala 210 Glu Ala Gln Gly Ala 115 Ala Pro Val Ser Thr Arg Asn Lys 180 Glu His 195 Pro Ser Leu Ala Ala Ala Gly Arg 260 His Ala Ser Ala 165 Leu Val Leu Arg Gly 245 Gin Arg Pro 150 Glu Pro Gly Gly Met 230 Ile Pro Leu 120 Thr Tyr 135 Thr Pro Leu Pro Asn Gln Gly Glu 200 Ile Leu 215 Arg Arg Val Ala -170- Val Ser Ser Ser Pro Gly Gly Ala 170 Gin Ser 185 Arg Cys Ser Arg Ala Cys Trp Ala 250 Cys Al a Val 155 Thr Arg Pro Val Leo 235 Ala Lys Gin 140 Asp Thr Leu Ser Gly 220 Pro Ala Leu 125 Arg Ala Glu Lys Glu 205 Ala Leo Arg Asp Thr Ala Thr 175 Lys Thr Ile Ala Leu 255 Ser Val Asp 160 Glu Pro Val Ala Ser 240 Gln 0O 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 1 S 10 Cys Val Gly Arg Arg Val Ala Phe Gly Gly Lou Ala Thr Met Ser 25 Pro Pro Thr Ser His Leu Asp Leu Ala Phe Ser Ala Ala Phe Arg 40 Thr Asp Lou Pro Gly Gly Arg Phe Trp Akrg Ala Ser Gin Ser Cys 55 Ile Phe Phe Trp Pro Asp Lou Ala Ala Val Ile Val Gin Ala Ala 70 75 Ala Tyr Phe Glu Gly Lys Glu Arg Leo Gly Ser Leu Gin Val Ala 90 Asp Ile Thr Ala His Asp Pro Arg Ile Ala Pro Ala Ala Lys Arg 100 105 110 Val Ala Ala Ala Val Gly Leu Trp Thr Ala Lou Ser Giu Lou Val 115 120 125 Arg Arg Gly Asp Arg Glu Ala Gly PCTUS96/03916 WO 96129396 Gly Arg 145 Gly Leu Asn Gly As 225 Arg Met Cys Asn Pro 130 Ala Pro Tyr As Met 210 Phe Ala Ser Leu Pro 290 Asn Ala Asp Ile Gin 195 Glu Thr Phe Pro Arg 275 Pro Gly Glu Ala Trp lie Leu 165 Arg Ala 180 Ser Ser Met Val Leu Pro Val Ala 245 Asp Val 260 Glu Ile Pro Lys Leu Glu 150 Ser Arg Ala Arg Glu 230 His Arg Ile Trp Glu 135 Ile Phe Gly Pro Ser 215 Asp Leu Val Met -171- Ser Lys Val Arg Asp Val Phe Ser Ala 170 Gly Ala His 285 Ala Ala Gly 200 Leu Leu Val Leu Pro Glu Met Asn Cys 250 Pro Val Glu 265 Cys Glu Arg 280 Trp Gly 140 Pro LYS 155 Ala Val Ser Arg Leu Ala Ile Ala 220 Gly Ser 235 Val Ala Glu Ser Ala Phe Lys Va1 Glu Ser Ala 205 Ieu Gin Thr Phe ys 285 Gin Pro Leu Ala 190 Ile Pro Asn Asp Tyr 270 Ile Val Pro 175 His Arg Leu Ser Lys 255 Ser Pro Pro Ile 160 Val Trp Ile Ser Ile 240 Ile His Cys 0090 0 00 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 278 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCZ DESCRIPTION: SEQ ID NC:72: Met Glu Asn Met Leu Asp Gly Cys lyr Pro Leu Ala Leu Met Asp Ser 1 5 10 Asp His Ile Thr Ala His Ala Val Pro Arg Gly 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 Glu Leu Val Glu Ile Ser Ser 55 Glu Leu Lys Asp Gly Tyr Gly Glu Phe Thr Ser Ala Arg Asp Arg Arg 70 75 so Asn Ala Leu Ile Ala Ala Asn Glu Arg Leu Arg Ser Ala Phe Leu Gly 90 Ala Ser Arg Ala Thr Arg Gly Leu Gly Leu Arg Pro Arg Trp Ala Ser 100 105 110 WO 96/29396 PCTIUS96iO39 16 -172- Thr Glu Ser Val Ala Asfl Ser Pro Thr Asp Pro Asn Asn Gly Asn Gly 115 120 125 Leu Gly Glu Leu Glu Glu Ala Met Glu Gly Ile Glu Gly Asp Phe Trp 130 135 140 Leu Asp 5cr Leu Asp Gly Asp Arg Phe Glu Asp Glu Ser Arg Thr Met 145 150 155 160 Gin Ser Glu Asn Met Arg Phe Val Ile Glu Lys Glu Leu Leu ser Trp 165 170 175 Leu Ser Arg His Leu Pro Ala Asp Leu Ala Ser Ala Glu Arg Glu Thx 180 185 190 Ser Arg Ser Leu Leu Ala Ala Gly His Trp Cys Cys Leu Trp His Pro 195 200 205 Arg Pro Cys Arg Glu Ala Cys Leu Tyr Asp Ser Ile Tyr Val Gin Ser *210 215 220 Leu Phe Cys Val Gly Thr Gly Arg Val Pro Gin Ser Glu Met Arg Arg :225 230 235 240 :Arg Glu Tyr Leu Ala Ala Leu Arg Ala Gly Ala Ala Ala Ala Asn Ser 245 250 255 Pro Glu Val Ser Ala Ser Ile Phe Ala Arg Asp Ala Gly Ile Ala Leu 260 265 270 Ala Leu Ala Arg Arg Arg 275

Claims (2)

173- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A nucleic acid encoding glycoprotein I of an infectious laryngotracheitis virus having the amino acid sequence shown in SEQ ID N0:7. 2. A nucleic acid encoding glycoprotein D of an infectious laryngotracheitis virus having an amino acid sequence shown in SEQ ID NO:11. 3. A recombinant DNA molecule comprising the nucleic acid molecule of claim 1 or 2 and DNA which is not derived from an infectious laryngotracheitis virus, wherein the recombinant molecule comprises no other gene from an infectious laryngotracheitis virus. S 4. The recombinant DNA molecule of claim 3, wherein the DNA encoding glycoprotein I or glycoprotein D is operatively .linked to a regulatory element derived from an infectious laryngotracheitis virus. 5. A recombinant DNA molecule comprising DNA encoding both glycoprotein I and glycoprotein D of an infectious laryngotracheitis virus and DNA which is not derived from an infectious laryngotracheitis virus, wherein the recombinant molecule comprises no other gene from an infectious laryngotracheitis virus. 6. A host cell comprising the nucleic acid of claim 1 or 2. 7. A host cell comprising the recombinant DNA molecule of any one P:\OPERUMS\2345585

174- of claims 3, 4 or 8. The nucleic acid of claim 1, wherein the nucleic acid consists of the nucleotide sequence from position 9874 to position 10,962 of the infectious laryngotracheitis virus nucleotide sequence (SEQ ID NO:1). 9. The nucleic acid of claim 2, wherein the nucleic acid consists of the nucleotide o sequence from position 8462 to position 9766 of the infectious laryngotracheitis virus nucleotide sequence (SEQ ID NO:1). 910 10. The nucleic acid of claim 1 or claim 2, substantially as hereinbefore described with reference to the figures and/or examples. 0g 11. The recombinant DNA molecule of any one of claims 3 to 5, substantially as hereinbefore described with reference to the figures and/or examples. 12. The host cell of claim 6 or claim 7, substantially as hereinbefore described with reference to the figures and/or examples. DATED this 5 th day of October 2000 Syntro Corporation DAVIES COLLISON CAVE Patent Attorneys for the Applicant Patent Attorneys for the Applicant