CA2216139A1

CA2216139A1 - Recombinant infectious laryngotracheitis virus and uses thereof

Info

Publication number: CA2216139A1
Application number: CA002216139A
Authority: CA
Inventors: Martha A. Wild; Mark D. Cochran
Original assignee: Individual
Current assignee: MSD International Holdings GmbH
Priority date: 1995-03-23
Filing date: 1996-03-21
Publication date: 1996-09-26
Anticipated expiration: 2016-03-21
Also published as: WO1996029396A1; AU5369096A; EP0822980A1; JPH11503009A; JP3964458B2; AU721451B2; CA2216139C; EP0822980A4

Abstract

The present invention provides a recombinant, attenuated infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene. This attenuated virus is useful as a vaccine against infectious laryngotracheitis virus. The present invention also provides a recombinant, attenuated infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the US2 gene, UL47-like gene, ORF4 gene or glycoprotein g60 gene. The present invention also provides a method for distinguishing chickens or other poultry vaccinated with a recombinant infectious laryngotracheitis virus which produces no glycoprotein gG from those infected with a naturally-occuring infectious laryngotracheitis virus.

Description

W 096/29396 PCTrUS96/03916 RECOMBINANT INFECTIOUS LARYNGOTRACHEITIS VIRUS AND
USES THERE;OF

This application is a con1inu~tion-in-part of U.S. Serial No. 08/126.597, filed September 24, l 993 which is hereby incorporated by reference into this application.

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

BACKGROUND OF THE INVENTION

Infectious laryngotr~eh.oitic virus is a herpesvirus that causes a le.~ dlcJly illness of varying virulence in chickens. Live ;~ A ILTV vaccines are available to protect against the ~lice~ce. but several reports have implicated vaccine viruses in the possible recurrence and spread of the disease (65 and 72), limiting vaccination to use in uninfected birds early in an outbreak. In order to design a more efficacious. ~tten1-~te-1 vaccine, the genomic org~ni7~ti-n of the ILTV virus has been studied.

'75 The ability to isolate viral DNA and clone this isolated DNA into bacterial p1~cmiclc 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 mo~ifiecl DNA is then reinserted into the viral genome to render the virus non-patho~enic. The resulting live virus may then be used in a vaccine to elicitan immune response in a host animal, and to protect the animal against a ~lice~ce.

CA 022l6l39 l997-09-22 W 096/29396 PCT~US96/03916 One group of animal viruses. the herpesviruses or Herpetoviridae, is an exarnpleof 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 i-lentifiecl that are non~ccenti~l for the replication of virus in ~itro in cell culture. Modifications in these regions ofthe DNA may lower the pathogenicity of the virus, i.e., ~ the virus. ~or example, inactivation of the thymidine kinase gene renders human herpes simplex virus non-pathogenic ( 1), and pseudorabies virus of swine non-pathogenic (2).
Removal of part of the repeat region renders human herpes simplex virus non-pathogenic (3, 4). A repeat region has been identified in Marek's disease virus that is associated with viral oncogenicity (5). A region in herpesvirus saimiri has similarlv been correlated with oncogenicity (6). Removal of part of the repeat region renders pseudorabies virus non-pathogenic (U.S. 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 (7, 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-ess~nti~l 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 (9), is an hll~ol~l~ patho~en of poultry in the USA, Europe, and Australia, responsible for egg production losses and death ( 10). It causes an acute disease of chickens which is characteriz.ed by respiratory depression, gpcrina and expectoration of bloody exudate. ~,'iral replication is limited to cells of the respiratory tract r wherein infection of the trachea gives rise to tissue erosion and hemorrhage.

W 096/29396 PCTrUS96/03916 In chickens, no drug has been effective in re~lllcing the degree of lesion formation or in decreasing clinical signs. Va~cin~tion of birds with various modified forms of the ILT virus derived by cell passage and/or tedious regimes of ~lmini~tration have been used to confer acceptable protection in susceptible chickens. Due to the limited degree of ~tteml~tion of current ILTV vaccines care must be taken to assure that the correct level of virus is m~intzlinPd enough to provide protection, but not enough to cause disease in the flock ( 1 1-21). Furtherrnore. these viruses may revert back to virulence, c~ ing 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, i.e.. thymidine kinase (27, 28), glycoprotein gB (27. 29. 30), ribonucleotide reAuct~ce ('7, 31). capsid p40 (31, 32).
1~
Furthermore~ Shepard. et al. (53) disclosed that several genes located in the unique long region of the infectious laryngotracheitis virus genomic DNA are non-t-c~enti~l for viral replication.

Applicants have une~cpecterll~ 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 wasfound that a deletion in the gl-coprotein G (gG) gene of the ILT virus results in an ~ nll~t~d ~ irus~ which is useful as a vaccine against subsequent attack by 2~ a virulent ILTV strains.

Applicants also found that a deletion in the glycoprotein I (gI) gene of the ~, unique short region also ~tten~tes the ILTV. Furthermore, it is contemplated that a deletion in the US2 gene, the UL47 like gene, and the glycoprotein g60 gene of the unique short region will also ~ttenll~te the ILTV.

ILTV can become latent in healthy ~nim~l~ which makes them potential carriers W 096129396 PCTfUSg6/03916 _4_ of the virus. For this reason, it is clearly advantageous to be able to distinguish z3nim~1c vaccinated with non-virulent virus from ~nim~lc infected with disease-causing wild-type or naturally-occurring virus. The development of differential vaccines and companion diagnostic tests has proven valuable in the management of pseudorabies disease (55). A similar dirf~cl~Lial marker vaccine would be of great value in the management of ILTV caused disease. The construction of dirre,~ ial ~ gnosticS has focused on the deletion of glyco~oteills.
Theoretically, the glycoprotein chosen to be the diagnostic marker should have the following characteristics: (1) the glycop-o~ - and its gene should be non-~ccf~nti~l 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 immnnity The ILT virus has been shown to specify at least four major glycoproteins as identified by monoclonal antibodies (M,- 205K, 115K, 90K and 60K). Three glycoproteins seem to be antigenically related (M,= 205K, 115K, and 90K) (3~36).

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

Of these, it is kno~n that the ILTV gB gene is an f~ccenti~l gene and would not be ~ iate as deletion marker genes. Furthermore, the gC gene of herpesviruses has been shown to make a significant contribution to protective immllnity as a target of neutralizing antibody (56) and as a target of cell-me~ t~d immunit (57). Therefore, the gC gene is not desirable as a deletion marker gene.

As to other glycoprotein ~nco~ling genes cited above, it is not kno~hn whether W O 96/29396 PCTrUS96/03916 _ ~ _ or not they would be suitable candidates for deletion in order to construct a recombinant ILT virus which can be used as a f~i~gnc~stic vaccine.

v Applicants have unexrecte~ly found that there are two glyco~lulcin 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 rli~gnostic vaccine. These are the glycoprotein gG gene and the glycoplotei" gI gene. By genetically engintoçring an ILT virus with a deletion in the glycoprotein G gene or the glycoprotein I gene. a ILT virus is produced which does not express any glycoplotein G or ~,lycoploteil 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 engin~ered, no exarnples of recombinant ILTV have been reported.
1~
The ability to ~ongin~er DNA viruses ~,vith large gennm~c, such as vaccinia virus and the herpesviruses, has led to the finding that these recombinant viruses canbe used as vectors to deliver vaccine antigens and therapeutic agents for ~nim~lc. The herpesviruses are attractive candidates for development as vectors because their host range is primarily limited to a single target species (37) and they have the capacity for establishing latent infection (38) that could providefor stable in VilV e~ es~ion of a foreign gene. Although several herpesvirus species have been engineered to express foreign gene products, recombinant infectious laryngotracheitis viruses e,.l"w~il,g foreign gene products have not 2~ been constructed. The infectious larvngotracheitis viruses described above may be used as vec~ors for the delivery of vaccine antigens from microorg~nicmc r~ncing important poultr,v ~iice~c~s Other viral antigens which may be included in a multivalent vaccine with an ILTV vector include infectious blonclliLis 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 tiic~ces Similarly the infectious laryngotracheitis viruses may be used as vectors for the delivery of th~ cuLic WO 96/29396 PCTrUS96/03916 agents. The th~ ulic 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 the a~:ulic agents from each of these classes of compounds in the form of anti-sense DNA, anti-sense RNA (39), - riboz~mes (40), suppressor tRNAs (41), hll~lr~lo~ nfl~ring double stranded RNA and numerous examples of protein th~la~ulics, from hormones. e.g., insulin, to lymphokines. e.g., illtel~l~ns and interleukins, to natural opiates.The discovery of these therapeutic agents and the elucidation of their structureand function does not neces~rily allow one to use them in a viral vector delivery system, however, because of the t;~ nt~tion necessary to clet~rrnine whether an ~,plo~-liate insertion site exists.

ILTV is classified as an alpha herpesvirus with a type D genome (78) composed of a unique long region and a unique short region flanked by inverted repeats.
A genomic restriction map of an Australian ILTV isolate (SA-2) was described by Johnson et al. (66). Using this map, Guo 2t 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 US~A challen,~e strain of ILTV, and reports characteristics of the putative genes present in the unique short region. The map disclosed herewith indicates that the sequence identified by Guo et al. (6~) is part of the short repeat sequence, and is not from the unique short. Other reports (69 and 70) describe the sequences of two ~enes, 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 o~p~ ion of the short region of ILTV is similar to other herpesviruses.

W 096/29396 PCT~US96/03916 SUMMARY OF THE INVENTION

The present invention provides a recombinant. ~ttPnll~tPr~ infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genomewhich contains a deletion in the glycoprotein gG gene. This ~ttPnll~tPd virus is useful as a vaccine against infectious laryngotracheitis virus.

The present invention also provides a recombinant, ~ ed infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genomewhich eontains a deletion in the US2 gene, UL47-like gene, ORF4 gene or glyeoprotein g60 gene.

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

W 096/29396 PCTrUS96/03916 BRIEF DESCRIPTION OF THE FIGURES

Fioures lA-lH: ' 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 107 base pairs of repeat region at the other end. The nucleotide sequences of Figures 1 A- 1 H begin with the intern~l repeat sequence and end within the terrnin~l repeat sequence. The unique short region begins at base pair 274 of this Figure. Sequence ID NO:S9 colll~ills the nucleotide sequence of 18,912 base paris of contiguous DNA from the unique short and repeat regions of the ILT virus. This sequence contains the entire 13,094 base pair unique short region as well as 2909 base paris of internal repeat region and 2909 base paris of short t~min~l repeat region. The nucleotide sequences begin with the internal repeat sequence and end within the terminal repeat sequence. The ~mique short region begins at base pair 2910.

Fi~ure 2: Asp718 I restriction enzyme map of the infectious laryngotracheitis virus (ILTV) USDA 83-2 genome. The upper diagram identifies the unique long (UL), internal repeat (IR)~ unique short (Us)~ and terrninal repeat (TR) sections found in the ILTV genome. A map of the Asp718 restriction en~ion~-clease sites in the ILTV genome is shown below. Letters A through O identify Asp718 I resh-iction endon~lcle~ce fr~gmPntc with "A" reprPS~nting the largest ~, fr~gm~nt Fragment "L" is the 2.5 kb Asp718 I fr~gmPnt fragment "H" is the 5164 bp Asp718 I fr~gment and fragment W 096/29396 PCTrUS96/03916 _9_ "G" is the 8.0 kb Asp718 I fr~gment The fr~gmentc marked 4 with asterisks contain a hypervariable region of approximately900 bp that is repeated from one to 12 times. Since no one ,. size predomin~t~c, these fr~gm~ntc 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.

Fi2~ure 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 co~ Lins 13 methionine initiated open reading frames (ORF) of greater than or equal to I 10 amino acids (excluding smaller nested ORFs). All 13 ORFs were aligned to the Entrez release 6.0 virus division of the GenbanL; DNA ~l~t~h~ce ~tili7ing the IBI MacVector Protein to DNA ~lignm~-nt option (default settings). Eight of the ORFs e.~hibited significant homology to one or more other virus genes:
unique short (US7)~ protein kinase (PK), unique long 47-like (I 'L 17-like). and glycoproteins gG, g60, gD, gI, and gE.

Fi~ures 4A-4B:
Detailed description of the DNA insertion in Homology Vector 472-73.27. Diagram showing the orientation of DNA fr~F~m-ntc 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 fr~gm~ntc are also shown (SEQ ID NO's: 20, 21. 77 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 W 096/29396 PCTrUS96/03916 location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets [] indicate the l c. ~ of sites which were destroyed during construction. The following abbreviations are used, infectious larvngotracheitis virus (ILTV), human cytomeg~lovirus immetli~t~ early (HCMV
IE), pseudorabies virus (PRV), lactose operon Z gene {lacZ), Escherichia coli (E. coli), polyadenvlation signal (poly A)~ and base pairs (BP).

Fi~ures 5A-~B:
Detailed description of the DNA insertion in Homology Vector 501-94. Diagram showing the orientation of DNA fr~gm~-nts assembled in plasmid 501-94. The origin of each fragment is indicated in the table. The sec~uences located at each of the junctions between fr~gm~ntc are also shown (SEQ ID NO's: 24, ''5. 26, and 27). The restriction sites used to generate each fragment as well as the synthetic linker se~uences 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 brackels [] indicate the remnants of sites which were destroyed during construction.

The following abbreviations are used, infectious laryngotracheitis virus (ILTV) . human cytomegalovirus immerii~te early (HCMV
IE). pseudorabies virus (PRV), lactose operon Z gene (lacZ), ~;scherichia coli (E. coli), polyadenylation signal (poly A), thvmidine kinase (TK). and base pairs (BP).

Fi~ures 6A-6B:
Detailed description of the DNA insertion in Homology Vector 544-55.12. Diagram showing the orientation of DNA fr~pm~ntc assembled in plasmid 544-55.12. The origin of each fragment W 096/29396 PCT~US96/03916 is indicated in the table. The sequences located at each of the junctions between fr~gment~ 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 fr~gment~ are described for each junction. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets [] indicate the 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.
coli), polvadenylation signal (poly A), and base pairs (BP).

Fi~ures 7A-7C:
Detailed description of the DNA insertion in Homology Vector 562-61.1 F. Diagram showing the orientation of DNA fr~gmt-nt~
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 fr~gmt-nt~ are also shown (SEQ ID NO's: 32, 33. 34 35. 36 and 37). The restriction sites used to gen~ldt~
each fragment as well dS 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 rernn~ntc of sites which were destroyed during construction.
The following abbreviations are used, infectious laryngotracheitis virus (ILTV)~ herpes simplex virustype 1 (HSV-1), pseudorabies virus (PRV), ~glucuronidase gene (uidA), ~scherichia coli (E.
coli), polyadenylation signal (poly A), and base pairs (BP).
FisJures 8A-8C:
Detailed description of the DNA insertion in Homology Vector W 096/29396 PCTrUS96/03916 560-52.F1. Diagram showing the orientation of DNA fr~gmentc assembled in plasmid 560-52.Fl. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fr~gm~nt~ 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 wereused to join the fr~gmentc are described for each junction. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets [] indicate the l ~l n ~ of sites which were destroyed during construction. The following abbreviations are used, infectious laryngotracheitis virus (ILTV) , herpes simplex virus type l (HSV- l ), pseudorabies virus (PRV), ~glucuronidase gene (uidA).
~scherichia coli (E. coli), polyadenylation signal (poly A), unique long 47 (UL47-like), open reading frame 4 (ORF4).
glycoprotein G (gG), and base pairs (BP).

Fi2~ures 9A-9B:
Detailed description of the DNA insertion in Homology Vector 579- 14.G2. Diagram showing the orientation of DNA fragm~ntc 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 fr~gmentc are also shown (SEQ ID NO's: 43, 44, 45, and 46). The restriction sites used to gc;~ dl~ each fragment as well as the synthetic linker sc.~u~llces which were used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elemf ntc is also given. Restriction sites in brackets [] in-lir~te the t~i of sites which were destroyed during construction. The following abbreviations are used, infectious laryngotracheitis virus (ILTV), herpes simplex virus type 1 (HSV-l), pseudorabies virus (PRV), ,B glucuronidase gene (uidA), Escherichia coli (E.

W 096/29396 PCTrUS96/03916 coli), polyadenylation signal (poly A), and base pairs (BP).

Fi~ures lOA-lOB:
Detailed description of the DNA insertion in Plasmid Vector ~i 544-39.13. Diagram showing the orientation of DNA frz~gmentcassembled in plasmid 544-39.13. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fr~gTn~-nt~ are also shown (SEQ ID NO's: 47, 48, 49, and 50). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fr~gm~nt~ are described for each junction. The synthetic linker sequences are nn~prlin~ by a heavy bar. The location of several gene coding regions and regulatory elements is also gi- en. Restriction sites in brackets [] indicate the 1~ remn~ntc of sites which were destroyed during construction. The following abbreviations are used, pseudorabies virus (PRV), ~glucuronidase gene (uidA), ~;scherichia coli (E. coli), herpes simplex virus type I (HSV-I), polyadenylation signal (poly A), and base pairs (BP).
Fi~ures 1 lA-l 1 C:
Detailed description of the DNA insertion in Plasmid Vector 388-65.~. Diagram showing the orientation of DNA fr:~nPn 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 fr~vm~nt~ are also shown (SEQ ID NO's: 51, 52, ~3. and 54). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fr~gm~nt~ are described for each junction. The ,~ 30 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 W 096/29396 PCTrUS96/03916 remn~nt~ of sites which were destroyed during construction. The following abbreviations are used, human cytomegalo~irus immediate early (HCMV IE)~ lactose operon Z gene (lacZ).
Escherichia coli (E. coli), pseudorabies. virus (PRV)~
polyadenylation signal (poly A), and base pairs (BP).

Figure 12: The genome of the ILTV virus, identifying the unique long (UL)~ unique short (US), internal repeat (IR), and terminal repeat (TR) is shown. The BamHI, Asp718I, NotI, and SfiI restriction maps of the virus are drawn lmtl~ nlh, with the highly repetitive region of the short repeats indicated by a set of wa~
lines. The position of the cocmi~c used to ~l~termine the map of ILTV are drawn beneath the restriction map. Note that cosmid 2F12 comains two non-contiguous sections. Th-ee probes used 1~ to characterize the ILTV genome are indicated as Pl, P2, and P3. Pl is a 0.9 kb NotI 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 Notl fragment used to identify the fr~gm~-ntc at the end of the terrninal repeat.

Fi~ure 13: The region sequenced. and the positions of the Asp71 8I, BamHI.
Norl~ and SfiI sites are shown. The and extent and orientation of the open reading frames found in the ILTV unique short and the fl~nking short repeat regions are indicated.

Fi~ure 14: Southern blot showing the repetition of an 856 bp element within the short repeat. Genomic ILTV DNA digested with SfiI
~ (a), HindIII (b), NotI (c), Asp718I (d), or BamHI (e) was probed with an 856 bp HindIII fragment from the short repeat Positions of molecular weight llldll~.:, are indicated.

Fi~ure 15: Depiction of the position of the 856 bp repeat region in the USDA strain, cc.~ ~cd to the same region from the SA-2 strain as described by Johnson et al. Three repeats are ~billcuily shown in the USDA strain. the region is not repeated in SA2.
B=BamHI~ H=HindlII, R-856 bp repeat.

FisJure 16: Southern blot identifying fr~gm~nt~ from the internal and terminal repeat that hybridized to a 6.6 kb NotI fragment cont~ining the junction of the unique long and the internal repeat. Genomic ILTV DNA digested with NotI (a), Asp718I
(b). and BamHI (c) was probed with the 6.6 kb NotI fragment.
Positions of molecular weight markers are indicated.

Fi~ure 17: 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 ~loteins 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 (60).

W 096/29396 PCT~US96/03916 DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a recombinant infectious laryngotracheitis virus c-lmpricing the infectious laryngotracheitis viral genome which contains a S deletion in the unique short region of the viral genom~ wherein the deletion is in the glyco~,oteill gG gene. Said deletion ~ s the virus. rendering it suitable for use as a vaccine against infectious laryngotracheitis virus. A
preferred embodiment of this invention is a recombinant infectious larvngotracheitis decign~terl S-ILT-014 (ATCC Accession No. 2427). The S-ILT-014 virus has been deposited ~ ua~lL to the Budapest Treaty on the Tnt~rn~tional Deposit of Microorg~ni.cmc for the Purposes of Patent Procedure with the Patent Culture Depository of the ~meric~n Type Culture Collection~
12301 Parklawn Drive, Rockville, Mar,vland 20852 U.S.A. on September 22.
1993 under ATCC Accession No. 2427). Another ~.eft"~d embodiment of this invention is a recombinant infectious laryngotracheitis virus tleciEn~tt~fl 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, e.g., the methods set forth in DNA TRANSFECTION FOR GENERATING
RECOMBINANT ILT VIRUS in Materials and Methods~ and the virus has not had genetic material eccenti~l for the replication of the infectious laryngotracheitis virus deleted.
The present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngoLIaclleilis viral genome which cc ~ ins a deletion in the glycoprotein gG gene and a deletion in the US2 gene. One l,leftl,~d embodiment of this invention is a recombinant infectious laryngotracheitis virus decign~t~d S-ILT-009. ?

The present invention further provides a recombinant laryngotracheitis virus W 096/29396 PCTrUS96/03916 comprising 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 S 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 infëctious laryngotracheitis virus which comprises the infectious laryngotracheitis viral genome which contains a deletion in the glycoploteill gG gene. a deletion in theORF4 gene, and a deletion in the UL47-like gene. A ~l~.led embodiment of this invention is a recombinant infectious laryngoL.~ch~il;c virus deeign~tt~l S-ILT-0 15 .
The present invention further provides a recombinant infectious laryngotracheitis virus which comprises the infectious laryngotr~ch~itic viral genome which contains a deletion in the glyco~loltill gG gene and a deletion in the glycoprotein g60 gene. A preferred embodiment of this invention is a recombinant infectious laryngotracheitis virus deciE~n~ted S-ILT-017.

The present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotr~chloitic 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 cont~ining a deletion in the glycoprotein gG gene and a deletion in the thymidine kinase (TK) gene.

The present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis virus genomewhich contains a deletion in the unique short region of the viral genome~
wherein the deletion in the glycu~loteh~ gG gene, and which also contains an insertion of a foreign gene. The foreign gene is inserted into a non-ecs~nti~l site of the infectious laryngotracheitis viral ~enome 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-ecc~nti~l 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-eccenti~l sites of the infectious laryngotracheitis viral genome are preferred sites for inserting a foreign gene into the virus: the thymidine kinase (TK) gene, the US2 gene, the UL47-like gene, the ORF4 gene, the glycoprotein gG gene. the glyco~lute~ll g60 gene, and the glycoproteingI gene.

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

The foreign gene which is inserted into a non-ecc~nti~l 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, ini~ectious laryngotracheitis virus (ILT) gB, infectious laryngotracheitis virus gI, in~ectious laryngotracheitis virus gD, infectious bursal disease virus (IBDV) VP2, infectious bursal disease virus VP3, infectious bursal disease virus VP4, CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 _19_ infectious 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., ~imeria spp., Histomonas spp Trichomonas spp.. Poultry nematodes, cesto~les~ 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 ~ ,.r~lOl~ (cIFN). Cytokines include, but are not limited to: transforming growth factor beta, epidermal growth factor family, fibroblast growth factors, hepatocyte growth factor. insulin-like growth factors, B-nerve growth factor.
platelet-derived growth factor, vascular endothelial gro~,vth factor, interleukin 1, IL- 1 receptor antagonist, interleukin 2, interleukin 3, interleukin 4, interleukin S. interleukin 6, IL-6 soluble rece~lol, interleukin 7, interleukin 8, interleukin 9. interleukin 10. interleukin 11, interleukin 12, interleukin 13.
angiogenin, chemokines. colony stimulating factors, granulocyte-macrophage colony stimulating factors. ervthropoietin, hll~l r~ , r~ g71mm~
le--kemi~ inhibitor- factor. oncostatin M~ pleiotrophin, secretory leukocyte protease inhibitor~ stem cell factor~ tumor necrosis factors, and soluble TNF
receptors. These c-tokines are from hllm~nc, bovine~ equine, feline, canine, porcine or avian. Recombinant ILT virus expressing cytokines is useful to .onh~n~ e the irnmune re~,onse when combined with vaccines c~ g allilgells of disease causing microorg~ni~m~

Recombinant infectious iaryngotracheitis virus ~A~vle~illg cytokines is used to enhance the immune response either alone or when combined with vaccines co.. ~ i.. p cytokines or antigen genes of disease causing microorg~ni~m~

Antigenic polypeptide of a human pathogen which are derived from human W O 96/29396 PCT~US96/03916 herpesvirus include, but are not limited to: hepatitis B virus and hepatitis C
virus hepatitis B virus surface and core antigens, hepatitis C virus, human immlmn~leficiency virus, herpes simplex virus- 1, herpes simplex virus-2. hurnancytom~g~lovirus, Epstein-Barr virus, Varicella-Zoster virus, human herpesvirus-6, human herpesvirus-7, human influenza, me~cle5 virus, h~nt~n virus~
pneumonia virus, rhinovirus, poliovirus, human respiratory syncytial virus.
retrovirus, human T-cell leukemia virus, rabies virus, mumps virus. malaria (Plasmodiumfalciparum), Bordetella pertussis, Diptheria. Rickettsia prowazekii, Borrelia berfdorferi, Tetanus toxoid, m~lign~nt tumor antigens.
The antigenic polvpeptide of an equine pathogen is derived from equine influen7~ virus, or equine herpesvirus. In one embodiment the antigenic polypeptide is equine influenza neuraminidase or hPm~g~lu~inhl. Examples of such antigenic polypeptide are: equine influen_a virus type A/Alaska 91 neu,dll.illidase and h~3m~glutinin~ equine influen_a virus type A/Prague 56 neur~minicl~ce and h~-m~gglutinin, equine influen_a virus type A/Miami 63 neur~mini~l~ce equine influenza virus type A/Kentucky 81 n~ dnli-lidase and hem~lutinin equine herpesvirus type 1 gly~,u~.u~ - B, and equine herpesvirus type 1 glycoprotein D, Streptococcus equi, equine infectious anemia virus, equine encephalitis virus. equine rhinovirus and equine rotavirus.

The antigenic polypeptide of an equine pathogen is derived from bovine respiratory syncytial virus or bovine parainfluenza virus, and is capable of being ..e~ed in a host infected by the recombinant infectious bovine rhinotracheitis virus. For example, the antigenic polypeptide is derived from bovine respiratory syncytial virus ~ hment protein (BRSV G), bovine rc~uild~oly syncytial virus fusion protein (BRSV F), bovine le:,~ildL~J.y syncytial virus nucleocapsid protein (BRSV N), bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza virus type 3 ht-m~gglutinin n~;u".. .,; l l i~l~ce The foreign gene may be put under control of an endogenous u~
infectious laryngotracheitis virus promoter, or it may be put under control of W 096/29396 PCTrUS96/03916 a heterologous U~ l promoter. The heterologous u~Lle~ll 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 genomewhich 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 glyco~ro~eil. gG.
The following recombinant viruses are preferred embo~1imentc of this invention:
A recombinant infectious laryngotr?chPitic virus ~lecign~tt-~l S-ILT-002~ S-ILT-014, S-ILT-009~ S-ILT-015, and S-ILT-017.

The present invention further provides a recombinant infectious 1~ laryngotracheitis v irus comprising the infectious laryngotracheitis viral genome which contains a deletion or other alteration in the unique short region of the viral genome. wherein the deletion or alteration is in the glycoprotein gI gene,so that upon replication~ the recombinant virus produces no glycoprotein gI.

The present invention further provides a recombinant infectious laryngotracheitis v irus 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 ~ lyco~rotein gG geneand in the glycoprotein gl gene. so that upon replication, the recombinant virus2~ produces no glycoprotein gG and no glycoprotein gI.

The present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genomewhich con~ins a deletion in the unique short region of the viral genome, wherein the deletion is in the US2 gene~ UL47-like gene, glycoploteiil g60 gene. It is contemplated that a deletion in any one of these genes will ~ le the virus, rendering it suitable to be used as a vaccine against infectious = =

W 096/29396 PCTIUS~6/03916 laryngotracheitis virus.

The present invention further provides a recombinant infectious laryngotracheitis virus which comprises a foreign gene inserted within the unique short region of the infectious laryngotracheitis viral genome, provided~
however, that the insertion is not in the protein kinase gene, the glycoprotein gD gene, the glycoprotein gE gene and the ORF10 gene. The foreign gene is inserted in such a way that it is capable of being t;~.essed 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 g60 gene.

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

The foreign gene thus inserted may encode a screenable marker, such as E. coli 13-galactosidase or E. coli 13-glucuronidase.

The foreign gene thus inserted may encode an antigenic polypeptide which.
when introduced into the host cell. induces production of plote.;Li~e antibodiesagainst an avian disease causin~ agent from which the antigen is derived or derivable. Such antigenic polvpeptide 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 infl~len7~ 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, Poultrv nPrn~todec cestodes, trematodes, poultry mites/lice, poultry protozoa.

W 096/29396 PCTrUS96/03916 The foreign gene thus inserted may be put under control of an endogenous u~usLIc~alll infectious laryngotracheitis virus promoter, or it may be put undercontrol of a heterologous upstream promoter. The heterologous u~ e~
promoter may be the HCMV IE promoter, the PRV gX promoter or BHV-1.1 S VP8 promoter.

The present invention further provides a vaccine for infectious laryngotracheitis virus which comprises a suitable carrier and an effective i.,....l--,i7inp amount of any of the recombinant infectious laryngotracheitis virus of the present invention. This vaccine may contain either inactivated or live recombinant virus.

Suitable carriers for the recombinant virus are well known in the art and include proteins, sugars. etc. One example of such a suitable carrier is a physiologically b~l~n~ed culture medium co"l;.;.. ;.. g one or more stabilizing agents such as hvdrolyzed 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 directlv after inactivation of the virus.

The present invention further provides a vaccine which comprises a suitable carrier and an eftective immunizing amour.t of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genomewhich contains a deletion in the unique short region of the viral genome, wherein the deletion is in the glycoprotein gG gene. A l,r~r~,.lc:d embodiment of this invention is a vaccine which comprises a suitable carrier and an effective immnni7ing amount of any one of the following viruses: recombinant infectious laryngotracheitis viruses ~IPcign~tç(l 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 ~lice~cçs which W 096/29396 PCTAUS~6/03916 -24-comprises an effective immunizing arnount 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-ecs~nti~l site of the viral genome s 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 en~eph~lomyelitis virus, avian reovirus, avian paramyxovirus. avianinfluen7~ virus. avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, chic~; anemia agent, Salmonella spp., ~. coli, Pasteurellc~ spp., Bordetella spp.. ~imeria spp., Histomonas spp., Trichomonas spp., poultry n~m~tocles cestodes, trematodes, poultry mites/lice, poultry protozoa The present invention further provides a vaccine which comprises a suitable carrier and an effective immllni7;ng amount of a recombinant infectious la~yngotrachei1is v irus comprising the infectious laryn~,oLldcheitis viral genome cont~ining a deletion or other alteration in the unique short region of the viral genome, wherein the deletion or alteration is in the glycu~loLeh~ 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 necign~t~o~l S-ILT-014, S-ILT-002, S-ILT-009, S-ILT-01~ and S-ILT-017 The present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing arnount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotr~t~h~itic viral genome which cont~inc a deletion or other alteration in the unique short region of the viral genome~ wherein the deletion or alteration is in the glycoprotein gI gene so that upon replication. the recombinant virus produces no glycu~lotehl gI.

The present invention further provides a vaccine which comprises a suitable carrier and an effective i.,.,.,u,.;,;.~g amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genomewhich 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 glyco~.oteill gI.

The present invention further provides a vaccine which comprises a suitable carrier and an effective ; 1 l l l l ~ ; l Ig amount of a recombinant infectiouslaryngotracheitis virus comprising the infectious laryngotr~-~h~-itic 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 g60 gene.

The present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genomewhich 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-eccenti~l sitein the viral genome.

The foreign gene encodes an antigenic polypeptide which inrluce5 host cell production of protective antibodies against an avian disease c~llcing agent fromwhich 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 CA 022l6l39 l997-09-22 W 096/29396 PCTrUS~6/03916 virus, avian encephalomyelitis virus, avian reovirus. avian ~..y~ovirus, avian influenza virus. avian adenovirus, fowl pox virus, avian coronavirus. avian rotavirus, chick anemia agent, Salmonella spp., ~. coli, Pasteurella spp., Bordetella spp., Eimeria spp., Histomonas spp., Trichomonas spp.. poul~
nematodes, cestodes, trematodes, poultry mites/lice, poultry protozoa.

The present invention further provides a vaccine which comprises a suitable carrier and an effective imml-ni7ing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genomewhich cont~inc an insertion of a foreign gene into a non-ec~enti~l site in the viral genome. The foreign gene encodes an antigenic polypeptide which induces host cell production of protective antibodies against an avian disease c:ausing 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, avianinfluenza virus. avian adenovirus, fowl pox virus, avian coronavirus~ avian rotavirus, chicl; anemia agent. Salmonella spp. ~. coli, Pasterurella spp., Bordetella spp. ~imeria 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 ~-imini~tering 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 ;~ g amount of a recombinant virus which produces no glycol"olein gG from those which are infected with a naturally-occurring infectious laryngotracheitis virus. This method comprises analyzing a sample of body fluid from the ~.hicL-~n~ or other W 096/29396 PCTrUS96/03916 poultry for the presence of glycoprotein gG of the infectious laryngotracheitis virus and at least one other antigen normally ~:x~.~s~ed in chickens or other poultry infected by a naturally-occurring infectious laryngotracheitis virus. The presence of antigen which is normally ~x~ ssed in chickens or other poultry S infected by a naturally-occurring infectious laryngotr~h~iti~ virus and theabsence 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 det~-nninl-d by ~i~tecting in the body fluid antibodies specific for the antigen and glycol,-oteill gG.

The present invention further provides a method for distinguishing chickens or other poultry which are vaccinated with an effective il..llll.~l;~;..g amount of a recombinant infectious laryngotracheitis virus which produces no giycoprotein gI from those which are infected with a naturally-occurring infectious lar,vngotracheitis virus. This method comprises analyzing a sample of body fluid from the chickens or other poultry for the presence of glycup~oteill gI ofthe infectious laryngotracheitis virus and at least one other antigen normally expressed in chickens or other poultry infected by a naturally-occurring infectious laryn~otracheitis virus. The presence of the antigen which is normally expressed in chickens or other poultry infected by a naturally-occurring infectious laryngotracheitis virus and the ~hsenr~e of glycoploteill gI
in the body fluid is indicative of being vaccinated with the recombinant vaccineand not infected with a naturally-occurring infectious laryngotracheitis virus.
The presence of the antigen and glycoprotein gI in the body fluid may be determined by detecting in the body fluid antibodies specific for the antigen and glyco,u.~ltelll gI.
-The present invention further provides a method for distinguishing chickens or other poultry which are vaccinated with an effective i--""~ ;"p amount of a recombinant virus which produces no glycoprotein gG and no glyco~rot~i.. gI
from those which are infected with a naturally-occurring infectious laryngotracheitis virus. This method comprises analyzing a sample of body fluid from the chickens or other poultry for the presence of glycoprotein gG andgI of the infectious laryngotracheitis virus and at least one other antigen normally ~ e~ed 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 ~sly~;o~lot~ gG and gI in the body fluid is indicative of being vaccinated with the vaccine and not infected with a naturally-occurring infectious laryngotracheitis virus. The presence of the antigen and glycop,o~ei~l gG and gI in the body fluid may be determined by detecting in the body fluid antibodies specific for the antigen and glycoproteingG and gI.

The present invention further provides a homology vector for producing a recombinant infectious laryngotracheitis virus by inserting a foreign DNA into the unique short region of the infectious laryngotracheitis genomic DNA, which comprises a double-stranded DNA molecule concicting ecs~nt~ y of a double-stranded foreign gene~ which is flanked on either side by the double-stranded DNA homologous to the DNA located in the unique short region of the genomic DNA. provided. houever, that the fl~nking sequences are not homologous to the glycoprotein gD ~2ene, the glycoprotein gE gene. the protein kinase gene, and the ORFIO gene. The foreign gene may encode a screenable marker. such as E coli B-g~l~e~Qsidase or E. coli B-glucuronidase.

The present invention further provides a homology vector for producing a recombinant infectious lar,vngotracheitis 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 concicting esserlti~lly 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, ~sly~ ot~ gI gene. US2 gene, or UL-47 like gene. Preferred embo~imPntc of this invention are the homology vectors ~ign~t~l Homology Vector 544-55.12, Homology Vector 562-61.1F, Homology Vector 472-73.27~ Homology Vector 560-52.F l and Homology Vector 579-14.G2.

This invention provides an isolated nucleic acid molecule encoding a US10 gene (SEQ ID NOs:60 and 70), AvSp gene (SEQ ID NOs: 61 and 71), US2 gene (SEQ ID NO:62)~ PK gene (SEQ ID NO:63), UL47 gene (SEQ ID
NO:64), gG gene (SEQ ID NO:65), ORF5 gene (SEQ ID NO: 66)~ gD gene (SEQ ID NO:67), gI gene (SEQ ID NO:68), gE gene (SEQ ID NO:69), or ORF9 gene (SEQ ID NO:70).

This invention provides an isolated polypeptide encoded by the US10 gene (SEQ ID NOs:60 and 70)~ AvSp gene (SEQ ID NOs: 61 and 71), US2 gene (SEQ ID NO:6 '). 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 NO:70).

CA 022l6l39 l997-09-22 W096129396 PCTrUS96/03916 EXPERIMENTAL DETAILS

Materials and Methods PREPARATION OF INFECTIOUS LARYNGOTRA(~ ;l l lS 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 h~tch.-~l from fertile eggs supplied by Hyvac) (50) in 225 cm' flasks with 0.5 ml of viral stock co~ g 105-106 pfu in lX Eagle's Basal Medium (modified) with Hank's salts (BME), 10% bromoethylamine(BEI)-treated fetal bovine serum (FBS), 1% ~ stock, 2% pennicillin/~LlGI~olllycin (P/S) stock, and 1% sodium bicarbonate stock (these co..l~ollents are obtained from Irvine Scientific or an equivalent supplier, and he.e~LGl the growth medium is referred to as complete BME medium). Viral stocks were then harvested 4-5 days later. Infected media and cells were resuspended in complete medium cont~ining 20% sterile whole milk and stored frozen at -70~C.

PREPARATION OF INFECTIOUS LARYNGOTRA~l~;lllS 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 5 minutes at 4~C. Because as much as 50% of the virus may be in the media, the supematants were saved and treated as will be described below. The cell pellets were le~ cllded in 1 ml PBS per tube, combined and centrifuged again at 1700 x g for 5 minutes The pellets were resllcr~ntl~d in 1 ml/flask of a buffer co.~t~ 10 mM Tris-HCI pH 7.5, 1 mM EDTA, and 1.5 mM MgCl2 and were in~nh~t.od for 15 minutes at 4~C. Twenty five ~ls of 20% NP40 per flask was added, and the mixture was then homogenized in a dounce homogenizer using an A pestle. The ~lcl,aldLion was centrifuged at 1700 x g for 10 minllt~s at 4~C and the supcllla~l~ was re~in~rl Ten ~1 of 0 5 M EDTA. 50 ~1 of 20%
SDS, and 25 ~1 of 10 mg/ml proteinase K was added to the ~ ..t (per original flask). In some cases, this was then combined with virus obtained from W 096/29396 PCTrUS96/03916 the cell media supernatants (see above). The mixture was then treated at 65~C
for 1-16 hours, followed by two extractions with phenol saturated with 100 mM
Tris-HCl, pH 8. DNA in the aqueous phase was then l,leci~i~a~ed with added 3 M sodium acetate (l/lOth volume) and 2.5 vols of 100% ethanol.
To obtain virus from the media~ the cell media ~u~ 1x were centrifuged at 23,500 x g for 30 min-ltec and drained well. The pellet was resuspended in the above proteinase K-cont~ining mixture as described. The DNA pellets were resl-~pton~led in 20 ~11 TE/flask and could be used at this point for further e~ -ents or treated further to remove RNA with pancreatic RNase A, followed by phenol extraction and ethanol p-eci~,i~Lion to obtain the DNA.

To prepare viral DNA ~.-i..i~)le~s, infected 10 cm. dishes were scraped into conical centrifuge tubes and centrifuged 5 ...i..~ s at 1000 x g. Cell media supernatants were kept and treated as above. The cell pellets were each resuspended in 0.5 ml of 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% NP40~
and in-~uh~t~d 10 minnt~s at room telll~e~d~ul~. Ten ~Ll of 10 mg/ml RNase A
was added, and the ~ ~dlion was centrifuged 5 Ill;ll~ 5 at 1000 x g. Twenty-five ~11 of 20 % SDS and 25 ~1 of 10 mg/ml proteinase K was added to the supernatant, and the entire ~lep~lion was added to the viral pellet from the cell media if it uas used. The mixture was incubated at 55-65~C for one hour, extracted with buffer-saturated phenol and ,~ ci~iL~led by the addition of 1 ml of ethanol. The Dl~.'A pellet was resuspended in 20 ~1 of TE and stored at 4~C.

POLYMERASE FILI~IN REACTION. DNA was resuspended in buffer CO~t~il.i,.g 50 mM Tris pH 7.4, 50 mM KCl, 5 mM MgCl2, 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 telllp,ldLIre. The DNA was phenol extracted and ethanol precipitated as above.

DNA SEQUENCING. Seq-~Pn-~inE~ was performed using the Sequenase Kit (US

~ -32-Biochemicals) and a35S-dATP (New Fn~ n~l Nuclear). Reactions using both the dGTP mixes and the dITP mixes were performed to clarify areas of compression. Alternatively, co~ ed areas were resolved on formamide gels.
Tt-mpl~ttqs were double-stranded plasmid subclones or single stranded M13 S subclones, and primers were either made to the vector just outside the insert to be sequenced, or to previously obtained sequence. Sequence obtained was assembled and compared using Dnastar software. Manipulation and comparison of sequences obtained was performed with IBI MacVector, Superclone and Supersee Align programs from Coral Software.
MOLECULAR BIOLOGICAL TECHNIQUES. Techniques for the manipulation of bacteria and DNA, including such procedures as digestion with restriction endonucleases, gel electrophoresis, extraction of DNA from gels.
Iigation, phosphorvlation with kinase, tre~fment with phosph~t~e, growth of bacterial cultures. transformation of bacteria with DNA, and other molecular biological methods are described ~42, 4~). The polymerase chain reaction (PCR) was used to introduce restriction sites convenient for the manipulation of various DNAs (44). In general amplified fr~gm~-nts were less than 500 base pairs in size and critical regions of amplified fr~pm~nt~ were confirmed by DNA sequencin~ Except as noted. these techniques were used with minor vanation.

SOUTHERN BLOTTING OF DNA. The general procedure for Southern blotting was taken from Maniatis et a/. (1982) and Sambrook, et.al.(l989) (42, 43). DNA was blotted to nylon membrane (Biorad Zetaprobe) in 0.4M NaOH
and prehybridized for 5 minut~s in a solution co,.~ 0.25 M Na2HPO4, pH
7.2, 1 mM EDTA. 7% SDS at 65~C. Labeled probe was added that had been labeled by random priming using a GeniusTM non-radioactive labeling kit from Boehringer-lvl~nnh.oim Hybridization was overnight at 65~C. Filters were washed twice with 40 mM Na.HPO4, pH 7.2, 1 mM EDTA, 5% SDS and then twice with 40 mM Na2HPO4, pH 7.2, 1 rnM EDTA, 1% SDS for 30 minlltec each at 65~C. Detection of bound probe was p~,~.,.,cd using the Boehringer W 096/29396 PCTrUS96/03916 ~nnhPim GeniusTM non-radioactive detection kit.

DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT
VIRUS. The method is based upon the CaC12 procedure of Chen and Okavama (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 col-t~;..i~.g the desired foreign DNA flanked by the ~lO~liate herpesvirus cloned sequences. Plasmid DNA ( 10-40 mg) was added to 250 ml of a solution having a final concentration of 0.25 M CaCl.. An equal volume of a buffer col-t~it,;~g 50 mM MOPS (pH 6.95), 280 mM NaCl, and 1.5 mM Na,HPO4 wasadded to the DNA/CaCl2 solution. After 10 minnt~c atroomt~lllpcldLIlre, the mixture was added drop~,vise to a 6 cm dish of CEK
cells on m~intt~n~nre rnedia. and placed at 39~C for 4 to 5 hours. The cells were rinsed once with PBS7 once with 20% glycerol in PBS for 2 ..,il,~eS, rinsed again with PBS and fed with m~ L~ re media. 1.5 ml of ILT viral stock was added to the media, and the ceiis were inrnh~teri overnight The next day~ fresh m~inten~n~e media was added, and the cells were inc~lb~te~l for two more days.
The transfection stock was harvested, aliquoted, and frozen at -70~C.

PROCEDURE FOR GENERATING ILTV SUBGENOMIC DNA
FRAGMENTS The abilit} to generate herpesviruses by cotransfection of cloned overlapping subgenomic fr~gmlontc has been demonstrated for pseudorabies virus (46). If deletions and/or insertions are ~ngin~ered directly into the cubgenomic fr~mentc prior to the cotransfection, this procedure resultsin a high frequency of viruses co.. ~i.. ;.. ~ the genomic alteration, greatly re-lucing 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 co. ~t~;--;--g ov~ g ILTV subgenomic fr~gmentc was gel,~.dlt:d as follows. USDA ILTV Strain 83-2 has been ~lecign~te~ S-ILT-001.
Appro~cim~tely 20 llg of ILTV DNA (obtained from S-ILT-OOl) in 0.5 ml of W 096/29396 PCTrUS~6/03916 10 mM Tris-HCI pH 8.0, 1 mM EDTA (TE) was sheared by passing it twice through a 25 guage needle as previously described (46). 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 co."~ ;.)g DNA of 35-50 kb were pooled, diluted twofold with TE. and precirit~t~(l with one tenth volurne of 3 M sodium acetate and 2.5 volumes of ethanol. The tubes were centrifuged for one hour at 109.000 x g at 10~C . Pellets were resllcpen~l~A~ transferred to microfuge tubes~ and precipitated with one tenth volume of 3 M sodium acetate and 2.5 volurnes 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, pleci~ t~;:dwith sodiurn 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 HindIII and AvaI to remove the tetracycline gene, and the ends were filled in with Klenow polymerase (FILL IN
REACTION) The polylinker from pWE15 (Stratagene) was ligated into this vector. The polvlinker was isolated bv digestion with EcoRI, the ends were filled in with Kleno~ polymerase (FILL IN REACTION), and the fragment was purified on a LMP-agarose gel. DNA ligation was p.,lr,l...ed in the presence of melted agarose The resulting cosmid, pSY1005, was modified at the: E;coRl site to create pSY l 626 b~ blunt-ended insertion of a 1.5 kb HindIII--BamHI
fragment from pNEO (P-L Biochemicals) Collt~ g the neomycin rçsict~nre gene. pSY1626 was cut and made blunt at the BamHI site~ and ligated with sheared ILTV fr~gmentc as described above. The ligation ~-.i~lu-e was packaged using Gig~r~rl' XL (Stratagene) according to the m~nnf~ rers instructions.
The p~ ging mixture was added to AGl cells (Stratagene) grown in the presence of maltose~ and colonies were selected on LB plates co"l;~;t~ g kanamycin. Cosmid subclones co,.~ ;.-g ILTV DNA were identified by COlllp~illg restriction enzyme maps of individual cosmid clones to each other and to ILVTV genomic DNA to obtain a contiguous sequence o-f ILTV

genomic DNA.

.
SCREEN FOR RECOMBINANT ILTV EXPRESSING ENZYMATIC
MARKER GENES. When the ~. coli ~-galactosidase or ~13-glucuronidase (uidA) marker gene was incorporated into a recombinant virus the plaques co~ g the recombinants were vi~n~li7~-~ by a simple assay. The enzymatic substrate was incorporated (300 ~lg/ml) into the agarose overlay during the plaque assay. For the lacZ marker gene the substrate Bluogal~M (halogenated indolyl-,B-D-galactosidase, Gibco BRL) was used. For the uidA marker gene the substrate X-Glucuro Chx (5-bromo-4-chloro-3-indolyl-,B-D-glucuronic acid Cyclohexylammonium salt, Biosynth AG) was used. Plaques that expressed active marker enzyme turned blue. The blue plaques were then picked onto fresh cells and purified by further blue plaque isolation. In recombinant virus strategies in which the enzymatic marker gene was removed, the assay involves plaque purifying white plaques from a background of parental blue plaques.
Viruses were typically purified with five to ten rounds of plaque purification.

SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT
ILTV USING BLACK PLAQUE ASSAYS. To analyze expression of foreign antigens ~ ed by recombinant ILT viruses, monolayers of CEK cells were infected with recombinant ILT virus~ overlaid with nutrient agarose media and inr~lb~t~d for 3-5 davs at 39~C. C)nce plaques have developed~ the agarose overlay was removed from the dish, the monolayer rinsed once with PBS, fixed with 100% methanol for 10 minnte~ at room t~ dLule and the cells air dried.
After re-hydrating the plate v~ith PBS, the primary antibody WdS diluted to the a~,oyl;ate dilution with PBS plus Blotto and incnh~ttocl with the cell monolayerfor 2 hours to overnight at room teln~eldlul~. Unbound antibody was removed from the cells by washing four times with PBS at room tenl~.dLulc. The ~lo~l.ate secondary antibody conjugate was diluted 1:500 with PBS and inrmhsltt~d with the cells for 2 hours at room ttlll~cld~ul~. Unbound secondary antibody was removed by washing the cells three times with PBS at room tt;lllp~.dL~c.The monolayer was rinsed in color development buffer (lOOmM

W 096/29396 PCTrUS96/03916 Tris pH 9.5/ 100mM NaCI/ SmM MgC12). and incubated 10 ~ nl~5 to overmght at room temperature with freshly ~-e~ d substrate solution (0.3 mglml nitro blue tetrazolium + 0.15 mg/ml 5-bromo-4-chloro-3-imdolyl pho~ph~t~e in color development buffer).The reaction was stopped by replacing the substrate solution with TE (lOmM Tris, pH7.51 1 mM EDTA). Plaques le~sillg the correct antigen stain black.

PURIFICATION OF ILTV gG FROM ILT VIRUS OR RECOMBINANT
VIRUSES EXPRESSING ILTV gG. ILTV gG was purified from the media of cells infected with either wild type ILTV or with FPV or SPV vectors e;,~illg 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 ct:l~Ll;ruge. The media was conc~..L.d~d in an Amicon concentrator using a YM30 ultrafiltration membrane at 15 psi. The co..c~ dle was dialyzed against 20 mM Tris-HCl, pH 7.0 and loaded onto a DEAE-Sephacel (Pharrnacia) column equilibrated with the same buffer. The m~tf~ l was eluted using a salt gradient from 0 to 1.5 M NaCI in 20 mM Tris-HCl, pH 7Ø Three ml fractions were collected and assayed by Western blot. A peptide antibody against ILTV
gG was used to identify fractions co~ -p 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, ~le~ign~t~d 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 S mg/ml trypsin and were then pelleted and le~ ~~ 1 at 1.3 x 106 cells/ml. Growth media (GM) was IX Eagle's Basal Mediurn (modified) with Hank's salts, with added 10% binary ethylPntoimin.q-treated fetal bovine serum (FBS), 2 mM glut~min~- 200 units/ml penicillin, 200 mg/ml streptomycin, and 8.9 rnM sodium bicarbonate (85). After ~ ,u~ ,ion, cells were plated and W 096/29396 PCTrUS96/03916 inf -lh~te-l at 39~C. Cells were rinsed and fed after 24 hours with m~ re media (MM), which is GM with 1% FBS. CKs were inocll1~t~?~1 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 105 106 pfu/ml.
PREPAlRATION OF VIRAL DNA. Cells and media from infected flasks were pelleted at 1700 g for 5' at 4~C. Sllp~ and cell pellet were initially treated separately. Virion particles were centrifuged out of the supçrn~t~nt at 23,500 g for 30 minllt~c The original cell pellet was rinsed with PBS and spun again. This pellet was resuspended in 1 ml/flask of a buffer co.. l~i.. ;.. g 10 mM
Tris-HCl pH 7.5, 1 mM EDTA, and 1.5 mM MgCl2 and incubated 15', 4 C.
To this was added 25 ~l/flask of 20% NP40, and the lllixlule was dounce homogenized using an A pestle. The ~ aldLion was c~;llL~iruged at 1700 g, 10', 4~C, and the SU~.lldti~lt was retained and the pellet discarded. To the SUpt:llldt~lL was added (per original flask) 10 ~l of 0.5 M EDTA, 50 ~11 of 20%
SDS, and 25 ~LI of 10 mg/ml ploteil.ase K This llliXIUle was used to resuspend the pellet of ~iral particles obtained by high speed c~ irugation of the first supernatant. The mixture was treated at 65 C for 1-16 hours, extracted twice with buffer-saturated phenol, and ~ i"i~L~d with added salt and ethanol. The resulting DNA pellet was resuspended in 100 lul 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., (83). Al~loxhllately 20 ~lg of ILTV DNA in 0.5 ml of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (TE) was sheared by passing it twice through a 25 gauge needle. The DNA was centrifuged through a 15-40% glycerol gradient in 50 rnM Tris-HCl, pH 8.0, 1 mM EDTA, 0.3 M NaCl for 5.5 h at 274,000 g. Fractions were analyzed on a 0.3% agarose gel, and those CO~ ;"g DNA of 35-50 kb were pooled, diluted twofold with TE, and ~le~ Ir~l with added salt and ethanol. The tubes were spun 1 h at 10~C and 109,000 g. Pellets were ~ e~l and W 096/29396 PCTrUS96/03916 lG~,leci~iLa~d with added salt and ethanol. The DNA was resuspended in TE
and the ends were made blunt by tre~tnn~nt with T4 DNA polymerase for 2 h at 15~C, in the presence of al,plo~liate buffer and 25 !lM dNTP~ followed by tre~tment with Klenow polymerase for 16 h at 15~C using 0.25 mM dNTP
The DNA was extracted with phenol and then ether, ~lc~ ed with added salt and ethanol, and resuspended in TE. This material was ligated overnight with 3 ~lg of cosmid vector pSY1626. Cosmid pSY1626 was made by digesting cosmid pHC79 (BRL) with HindIII and AvaI to remove the tetracycline gene.
The .c~ ;l,il.g fragment and the EcoRI digested polylinker from pWE15 (Stratagene) were filled in with Klenow polymerase and ligated together. The resllltinP cosmid vector, pSY1005, was modified at the EcoRI site to create pSY1626 by blunt-ended insertion of a 1.5 kb HindIII-BamHI fragment from pNEO (P-L Biochemicals) COIILit;,, i l~g the kanamycin r~?cict~nce gene. PSY1626was cut and made blunt at the BamHI site for use as the cosmid vector. The ligation mixture was packaged using Gi~p~ck XL (Stra~agene) according to the m~nllf~-~tnrer's directions. Colonies were selected on LB plates co..l~
kanamycin.

SEQUENCING. Manual sequencing was performed using 35S-dATP (NEN) with the BRL Sequenase Kit which uses the dideoxyribonucleotide chain tel~ Lion method described by Sanger et al. (80). Reactions using both - dGTP and dITP mixes were performed to clarify areas of co~ s~ion.
~ltern~tively~ colllplessed areas were resolved on 8% acrylamide gels that were 40% in formamide. Automatic fluorescence sequencing was performed using an Applied Biosystems (ABI) 373A DNA Sequencer. Subclones were made to facilitate seqllenrin~- Internal ~lilllCls were synth~ci7~1 on an ABI 392 DNA
synthPci7~r. 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 GenR~nk were l.c.rolllled at the NCBI using the BLAST network service (58).

W O 96/29396 PCTrUS96/03916 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 (28). 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 ~d"s,,l;~Lional orientation to the ILTV TK gene. U~L~C:a111 of the marker gene is an ~lo~illlately 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 a~.o~il..ately 675 base pair fragment of ILTV DNA which includes 80 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 cytom~g~lovirus (HCMV) imm~Ai~t~ 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-SD. It was constructed from the indicated DNA sources ~ltili~ing standard recombinant DNA techniques (42, 43). The plasmid vector is derived from an a~lo~inlately 3002 base pair HindlII fragment of pSP64/65 (Promega). Fragment 1 is an a~plo~ .ately 1087 base pair Hina~ll to Bcll subfragment of the ILTV 2.4 kb HindIII fr~gm~nt Fragment 2 is an approximately 5017 base pair Sall to SalI fragment cO"t~;"il-g the HCMV IE promoter~ ~-galactosidase (lacZ) marker gene, and PRV gX
polyadenylation signal (see Figures 5A-5D). Fragment 3 is an approximately 675 base pair Bcn to HinalII subfragment of the ILTV 2.4 kb HindlII
fr~ m~nt 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 illc~ olaLes a screenable marker. the . coli uidA gene flanked by ILT virus DNA. The PRV gX promoter--E. coli uidA gene is inserted in the OI)I)O'iL~ Ll~lsc,fl~Lional orientation to the ILTV

W 096/29396 PCTrUS~6/03916 US2 gene. Upstream of the uidA gene is an approximately 2300 base pair fragment of ILTV DNA which inclu(les 41 arnino acid codons at the 3' end of the US2 gene (SEQ ID NO 2: aa. 188-229). Downstream of the uidA gene is an ~p.o~ ately 809 base pair fragment of ILTV DNA which includes 2'2 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 ILT~ US2 DNA coding for arnino acids 73 to 187 with DNA coding for the ~. 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 thymidine kinase (HSV-l 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 lltiii7ing standard recombinant DNA techniques (4- . 43). The plasmid vector is derived from an approximately I 5 2958 base pair Asp7 1 8I restriction fragment of a pSP 18/pSP 19 fusion such that the multiple cloning site is ~coRI/SacI/Asp718I/SacI/EcoRI. Fragment 1 is an approximately 2300 base pair Asp718I to DraI subfragment (SEQ ID NO 1:
Nucl. 1-405) of the ILTV 2.5 kb Asp718I fr~gm~ont Fragment 2 is an approximately 3039 base pair ~aI fragment co~ g the PRV gX promoter, the E coli uidA gene. and the HSV-l TK polyadenylation site (See Figures 6A-6D). Fragment 3 is an approximately 809 base pair X~aI to Asp7 18I
subfragment of the ILTV 1097 bp Asp718I fragment (SEQ ID NO 1: Nucl.
905-1714).

HOMOLOGY VECTOR 56~-61.1F. The plasmid 562-61.1F was constructed for the purpose of deleting part of the gI gene from the ILT virus and insertinga foreign DNA. It incorporates a screenable marker, the E. coli uidA gene, flanked by ILT virus DNA. The PRV gX promoter-E. coli uidA gene is transcribed in the opposite direction to the ILTV gI gene promoter. The 983 base pair deletion begins 12 base pairs u~ of the translation initiation codon and deletes 324 of 363 amino acid codons at the 5' end of the ILTV gI
gene (SEQ ID NO 11: aa. 325-363). When this plasmid is used according to W 096/29396 PCTrUS96/03916 the 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 lltili7ing standard recombinant DNA techniques (42, 43). The plasmid vector is derived from an approximately 2647 base pair Asp71 8I to HindIII fragment of pUCI 9.
Fragment 1 is an apprnx;.,.~tely 1619 base pair Asp718I to X~aI subfragment of the ILTV 8.0 kb Asp718I fragment (SEQ ID NO 1: Nucl. 7556--9175).
Fragment 2 is an approximately 691 base pair X~aI to X71oI fragment (SEQ ID
NO 1: Nucl. 9175-9861) generated by the polymerase chain reaction (PCR).
The template was the ILTV 8.0 kb Asp718I fr~gm~nt The U~SIl~.alll primer 92.09 (5'-CCTAGCACCCTTGTATCGCG-3', SEQ ID NO. 55) sits down at a site 821 base pairs u~ e~ll of the ILTV gI gene and synshPCi7~s DNA
toward the 3' end of the gene. The downstream primer 92.11 (5'-I 5 CGCCTCGAGTCCCAATGAATAGGCATTGG-3 '; SEQ ID NO. 56) sits down at a site 12 base pairs u~ ~" of the translation start site of the ILTV gI gene and synthesizes DNA toward the 5' end of the gD gene. The product of the PCR reaction is 818 base pairs. This DNA fragment is digested with X~aI at the 5' end (a restriction en_yme site present in the ILTV DNA) and XhoI at the 3' end (a restriction enzyme site created in the PCR primer--see underlined sequence) to create an approximately 691 base pair X~aI to X7zoI fr~gm~-nt Fragment 3 is an approximatel~ 3051 base pair SalI fragment cont~ining the PRV gX promoter~ the uidA gene. and the HSV-I TK polyadenylation site (See Figures 6A-6D). Fragment 4 is an approximately 624 base pairX7zoI to HindJII
"5 fragment generated by PCR (SEQ ID NO 1: Nucl. 10,847--11,461). The template was the ILTV 8.0 kb Asp718I fr~gm~ont The up~ primer 92.10 (5'-CGCCTCGAGGACCCATGGTTGCGTGCG-3 '; SEQ ID NO. 57) sits down at a site 117 base pairs U~aLlc:alll from the translation termination codon within the ILTV gI gene. The downstream primer 92.08 (5'-CTCGTCCGAACGAGTTACAG-3'; SEQ ID NO. 58) sits down at a site 604 base pairs downstream of the translation ~ ;nn site of the ILTV gI gene and within the ILTV gE gene. The PCR product (729 base pairs) is digested with XhoI which is a unique site generated by the uy~L,e~hll PCR primer (underlined) and with HinalII at a site within the ILTV gE gene. Restriction endonuclease digestion with XhoI and HinalII creates an ~pl~xil~lately 624 base pair Fragment 4. Fragment 5 is an al~pl..x;,.,~t~ly 2700 base pair HindIII
subfragment of the ILTV 8.0 kb Asp718I fragment (SEQ ID NO 1: Nucl., 11,461--13,473 plus unsequenced DNA).

HOMOLOGY VECTOR 472-73.27. The plasmid 472-73.27 was constructed for the purpose of deleting a portion of the glycoprotein G (gG) gene coding region from the ILT virus and inserting a foreign DNA. It incorporates a screenable marker, the E. coli lacZ gene, flanked by ILT virus DNA. The HCMV IE promoter-E. coli lacZ gene is transcribed in the same direction to the ILTV gG gene promoter. The 874 base pair deletion of the ILTV gG gene extends from 60 nu~leotides U~ of the translation initiation site to 814 nucleotides into the amino acid coding sequence, removing the coding capacity of 271 of 292 amino acids of the gG protein (SEQ ID NO 7). When this plasmid is used according to the DNA TRANSFECTION FOR GENERATING
RECOMBINANT ILT VIRUS. it will replace the DNA coding for amirlo acids 1 to 271 of the ILTV gG gene with DNA coding for the E. coli lacZ gene. A
detailed description of the plasmid is given in Figures 4A-4D. It was constructed from the indicated DNA sources lltili7ing standard recornbinant DNA techniques (47. 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 Asp718I to NheI subfragment of the ILTV 5164 bp Asp718I fragment (SEQ ID NO 1: Nucl. 171~1 1544).
Fragment 2 is an approximately 5017 base pair SalI to SalI fragment col,l;~;ll;ll~
the HCMV IE promoter, E. coli ~-galactosidase (lacZ) marker gene, and PRV
gX polyadenylation signal (see Figures 4A-4D). Fragment 3 is an a~loxh.lately 1709 base pair Sall to Asp718I subfragment of the ILTV 5164 bp Asp718I fragment (SEQ ID NO 1: Nucl. 5419--6878).

HOMOLOGY VECTOR 560-52.Fl. The plasmid 560-52.Fl was constructed W O 96/29396 PCT~US96/03916 for 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 incu~o.dtes 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 4), the entire coding sequence of the ORF4 gene (SEQ ID NO S) and 271 of 293 amino acid codons at the 5' end of the ILTV
gG gene (SEQ ID NO 7). When this plasmid is used according to the DNA
TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS. it ~,vill replace the DNA coding for the ILTV UL47-like, ORF4 and gG genes ~,vith DNA coding for the PRV gX promoter--E. coli uidA gene. A detailed description of the plasmid is given in Figures 8A-8D. It was constructed from the indicated DNA sources ntili7ing standard recombinant DNA techniques (42, 43). The plasmid vector is derived from an ~ylu~ lately 2958 base pair Asp7 1 8I restriction fragment of pSP 18/pSP 19 such that the multiple cloning site is EcoRI/SacI/Asp718I/SacIlEcoRI. Fragment 1 is an apploxilllately 1066 base pair Asp718I to BssHI I subfragment of the ILTV 5164 bp Asp718I fragment (SEQ ID NO 1: Nucl. 1714-2777). Fragment 2 is an approximately 123 base pair SalI to Bcll subfragment of the ILTV 5164 bp Asp7 18I fr~tgment Fragment 3 is an approximately 3027 base pair BamHI fragment CO.-I;.;.~it~g the PRV gX
promoter, the uidA gene. and the HSV-l TK polyadenylation site (See Figures 8A-8D). Fragment 4 is an approximately 1334 base pair Bcll to Asp718I
subfragment of the ILTV 5164 bp Asp718I 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 includes 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
8). 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 ~ltili7ing standard recombinant DNA techniques (42, 43). The plasmid vector pUC19 (Gibco, BRL) is derived from an a~ hnately 2677 base pair Asp718I
to BamHI fr~gment Fragment 1 is an approximately 2830 base pair Asp718I
to MleI subfragment of the ILTV 5164 bp Asp718I fragment (SEQ ID NO 1:
Nucl. 171'1 1544) Fragment 2 is an ~ x;l~qtely 3051 base pair SalI
fr~grn-ont cont~inin~ the PRV gX promoter, E. coli ,B-glucuronidase (uidA) marker gene. and an HSV-l TK polyadenylation site (See Figures 9A-9D).
Fragment 3 is an ~ oxil.,ately 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 ~-glucuronidase expression cassette con~ictin~ of the PRV gX promoter, E. coli ~-glucuronidase (uidA) marker gene. and an HSV- I TK polyadenylation site. A clet~
description of the marker gene is given in Figures 1 OA-1 OD. It was consl:ructed lltili7in~ standard recombinant DNA techniques (42, 43) by joining restriction fr~m~ntc from the followin~ sources with the synthetic DNA sequences indicated in Figures I OA-I OD. The plasmid vector pSP71 (Promega) is derived from an approximately 3066 base pair XmaI to SmaI fr~gm~nt Fragment 1 is an appro~ ately 422 base pair SalI to EcoRI restriction subfragment of the PRV BamHI restriction fragment ~10 (47). Note that the EcoRI site was introduced at the location indicated in Figures 12A-12D by PCR cloning. .
Fragment 2 is an approximately 1826 base pair EcoRI to SmaI fragment of the plasmid pRAJ260 (Clonetech). Note that the EcoR~ and XmaI sites were introduced at the locations indicated in Figures lOA-lOD by PCR cloning.
Fragment 3 is an ~,o~hnately 784 base pair XmaI subfragment of the HSV-I

W O 96/29396 PCTrUS96103916 BamHI restriction fragment Q (48). Note that this fragment is oriented such thatthe polyadenylation se~lu~:.lce (AATAAA) is located closest to the junction withthe E. coli uidA gene.

S PLASMID 388-65.2. Plasmid 388-65.2 col~ s the ,B-galactosidase c~es~ion çzlc~ette con~i~ting of the HCMV imm~ te early (IE) promoter, the ~. coli lacZ mOEker gene, and the PRV gX gene polyadenyiation site. A ~let~iled description of the ~-g~ tos~ ce ~ ssion ç~ette is given in Figures 1 lA-1 lD. It was constructed lltili7ing standard recombinant DNA techniques (42.
43) by joining restriction fr~gm~nt~ from the following sources with the synthetic DNA sequences indicated in Figures 1 lA-l lD. The plasmid vector pSP72 (Promega) is derived from an approximately 3076 base pair PstI to PstI
fr~gm~nt Fragment 1 is a 1154 base pair PstI to ,4vaII fragment derived from a HCMV 2.1 kb PstI fragment co..~ ..;l.g the HCMV IE promoter. Fragment lS 2 is a 3010 base pair BamHI to PvuII fragment derived from plasmid pJF751 (4g) col-t~ the ~. coli lacZ gene. Fragment 3 is an ~lox;...~tely 750 base pair NdeI to SalI fragment derived from PRV BamHI #7 which contains the carboxy-terminal 19 amino acids and the polyadenylation signal of the PRV gX
gene.

CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 EXAMPLES

Example 1 Complete sequence of the unique short region of Infectious La~ tr..chc;li~ 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 fl.-t~rrnin~l This sequence contains the entire 13,098 base pair unique short region as well as 273 base pairs of repeat region at one end and 102 base pairs of repeat region at the other end. The unique short region conta.ins 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 Genh~nk DNA ~1~t~h~e ntili7ing the IBI
MacVector Protein to DNA alignment option (default settings). Eight of the ORFs exhibited si~nificant homology to one or m.ore other virus genes (see Table I). The nucleotide sequence numbers referred to below begin within the internal repeat sequence and end within the t~.rrnin~l repeat sequence. The unique short re~ion begins at base pair 274 of SEQUENCE ID NO. 1.

W 096/29396 PCTrUS96/03916 Table I
Sequence Homology between Infectious Laryngotracheitis Virus (ILTV) Open Reading Frames in the Unique Short Region and other Viral Proteins Open P~f- lin~ Start(BP) End(BP) Length(aa) G~n~
Frame(ORF) .~llignmenta 1 (Rc)b 970 281 229 EHV-l US2 io 3 2575 4107 510 HSV-l UL47 4 4113 4445 110 NSc 4 (RC) 4519 4139 126 NS
4609 5487 292 PRV gX
6 5697 8654 985 ILTV g60 6 (RC) 7826 6948 292 HSV-2 UL39 7 8462 9766 434 PRV g50 8 9874 10962 362 VZV gI
8 (RC) 11150 10617 177 NS
9 11159 12658 499 VZV gE

' Sequence ~llignrne~t scored to the Entrez Release 6.0 of Genbank Virus D~t~kRce, b RC=Reverse Complement.
' NS=No score above 120 was found.
Olher Abbreviations: EHV= Equine herpesvirus; MDV= Mareks disease virus;
HSV-l= Herpes Simplex virus l; PRY= Pseudorabies virus; Ir TV= Infectious laryngotr~rhPitic virus; HSV-2= Herpes Simplex virus 2; VZV=
Variceila-Zoster virus; BP= base pairs; aa= amino acids.

US2 gene The US2 gene consists of 690 base pairs and codes for a protein 229 amino acids in length and molecular weight ~ o~ t~ly 25,272 daltons (SEQ. ID.
NO. 12, 13). The ILTV US2 is homologous to the Equine herpesvirus(EHV)-l and EHV-4 US2 proteins. The US2 gene is transcribed from nucleotide 970 to W 096/29396 PCTrUSg6/03916 281 on the reverse complement strand of the ILTV unique short region (SEQ.
ID. NO. 1). The function of the US2 gene product is unknown.

Protein kinase ~ene s The protein kinase gene con~i~f~ of 1431 base pairs from nucleotide 1059 to 2489 and codes for a protein 476 arnino acids in length and molecular weight a~ ely 54,316 daltons (SEQ. ID. NO. 2). The ILTV protein kinase is homologous to the protein kinases from Mareks disease virus (MDV), Equine herpesvirus(EHV~l and -4, Pseudorabies virus (PRV), Varicella-Zoster virus (VZV), Simian varicella virus (SVV), and Herpes Simplex virus(HSV~-l and -2.

UL47--like ~ene 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 app.o~ ately 57,615 daltons (SEQ. ID. NO. 3).

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 frarne 110 amino acids in length and molecular weight a~ ~---ately 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 approximately 13.860 daltons (SEQ. ID. NOS. 14, 15).

~G ~ene 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 a~pro~i.,.~tely 31,699 daltons (SEQ. ID. NO. 5). ILTV gG glycoprotein is homoiogous to PRV gX, Bovine herpesvirus(BHV)-1.3 gG, EHV-l gG and EHV-4 gG. Recombinant ILTV gG protein produced in a Swinepox virus vector or a Fowlpox virus vector can be purified (see Materials and Methods) and reacts to peptide antisera to ILTV gG. The peptide antisera reacts to ILTV
gG from wild type virus, but not to viruses deleted for the ILTV gG gene.
Deletion of the gG gene results in an ~llr,.~ cl ILT virus that is useful as a vaccine against ILT disease in chir~n.~ (see table in Example 6) and also servesas a negative marker to distinguish vaccinated from infected z~nim~l~

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

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

~D ~ene W 096/29396 PCTrUS96/03916 -50-The ~.e~ion of the gD glycoprotein in vectored fowlpox virus or herpesvirus of turkeys (33) is sufficient to raise a protective immlme response in the chicLt~n 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, 1 1). The ILTV gD
glycoprotein is homologous to the PRV gSO, and the gD from HSV--1. MDV.
IPV, and BHV-l . l . 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.

ene 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 39,7j3 daltons (SEQ. ID. NO. 7). The ILTV gI glycoprotein is homologous to the VZV gI. Recombinant ILTV gI protein expressed in a swinepox virus vec~or reacts to convalescent sera from ILTV-infected chickens.
Deletion of the gl gene results in an ~ttenll~t~rl ILT virus that is useful as avaccine against ILT disease in chickens. Recombinant viruses deleted for gI are safe in animal trials when vaccinated by a natural route directly into the re:,~",atc.ly tract. whereas paren~al virus causes lesions in 90% of the birds inoc~ t~d via the same route. Deletion of the gI gene serves as a negative marker to distinguish vaccinated from infected ~nim~

ORF8 Reverse Complement ORF8 Reverse Complement codes for a protein of unknown function. 0RF8 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).

W 096/29396 PCTrUS96/03916 ~E ~ene The gE gene consists of 1500 base pairs from nucleotide 11.159 to 12.658 and codes for a glyco~lotein 499 amino acids in length and molecular weight approximately 55.397 daltons (SEQ. ID. NO. 8). The ILTV gE glycoprotein is honn.ol~gous to the gE g!ycoprot.eins from VZV, Sim.i~n. her~sviPus {~V), EHV-1, HSV-1 and PRV. The ILTV gE is a neutralizing antigen useful as a subunit vaccine.

ORF10 consists of 783 base pairs from nucleotide 12,665 to 13,447 and codes for a protein 261 amino acids in length and molecular weight app,oxi~-,ately 27,898 daltons (SEQ. ID. NO. 9).
,15 EYample 2 S-ILT-004 is an infectious laryngotracheitis virus (ILTV) that has an ~ o~illlately 620 base pair deletion of the thymidine kinase (TK) gene (28).
The gene for E. coli ,B-galactosidase (lacZ) was inserted in the place of the TKgene and is under the control of the HCMV immerli~te early (IE) promoter.
Transcription of the HCMV IE promoter--lac Z gene is in the opposite orientation to the TK promoter.

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

2~ S-ILT-004 is attenuated by deletion of the ILTV TK gene, but retains other genes k nown 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 ehirL-en~
from ILT disease.

W O 96/29396 PCTrUS96/03916 Example 3 S S-ILT-009 is an infectious laryngotracheitis virus (ILTV) that has an a~ x; "-~tely 498 base pair deletion of the ILTV US2 gene and an a~lo~illlately 874 base pair deletion of the ILTV gG gene. The gene for E
coli ~-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 Exarnple 5 (S-ILT-014). The transfection stock was screened by the X-Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING
ENZYMATIC MARKER GENES. The resulting purification of a blue plaque was recombinant virus S-ILT-009. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING OF DNA procedure. This analysis confirm~d the presence of the PRV gX promoter--~-glucuronidase (uidA) marker gene and the deletion of approximately 498 base pairs of the ILTV US2 gene and an ~ ro~ atelv 874 base pair deletion of the ILTV gG gene.
However, during the BluogalTM SCREEN FOR RECOMBINANT
HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES, a deletion of the HCMV IE promoter-lacZ gene was ~ietecte~l within the e~ ting ILTV gG
deletion. The rem~ining insert into the ILTV gG deletion contains applo"illlately 2000 base pairs of DNA of which all of the lacZ gene and part of the PRV gX polyadenylation site are mi~ing The deletion was characterized by detailed restriction mapping and determined to be slightly different from theS-ILT-014 deletion (See Example 5).
S-ILT-009 is ~tt~m~t~Cl by deletion of the ILTV US2 and gG genes, but retains other genes known to be involved in the immlme re;.~ullse in chickens to ILT

W 096129396 PCT~US96/03916 virus. Therefore, S-ILT-OO9 is useful as an ~llr~ cl live vaccine or as a killed vaccine to protect rhicl~n~ from ILT disease as shown in the table. Since S-ILT-OO9 does not express the ILTV gG genes, it is utilized as a negative marker to distinguish vaccinated ~nim~l~ from infected ~nim~l~ as described previously.

W 096/29396 PCTrUS96/03916 Table II
..

AGAINST VIRULENT INFECTIOUS LARYNGOTRACHEITIS VIRUS
CHALLENGE

Vaccine Gene(s) Dose Route Ch~ e- Protectionb Deleted S-ILT-009 gG-, US2- 7.8x103 IO ' OS d 70%
S-ILT-009 gG-. US2- 1.56x103 IO OS 77%
Controls OS 0%
ASL IO OS 90%
1 5 embryo 14 day old chicks a: USDA Challenge virus =1.0x104-~ pfu b: Protection = # healthy birds/total (%).
c: Intraocular d: Orbital Sinus W 096/29396 PCTrUS96/03916 Example 4 S-ILT-0 11 is an infectious laryngotracheitis virus (ILTV) that has an approximately 983 base pair deletion of the ILTV gI gene. The gene for E. coli ,B-glucuronidase (uidA! was inserted in the place of the gI gene and is under the control of the pseudorabies virus (PRV) gX promoter. The PRV gX
promoter--uidA gene is in the opposite orientation to the direction of s~ Lion of the ILTV gI promoter.

S-ILT-0 1 1 was constructed using homology vector 562-61.1 F (see Materials and Methods) and S-ILT-001 in the DNA TRANSFECTION FOR GENERATING
RECOMBINANT ILT VIRUS. The transfection stock was screened by the X-Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING
ENZYMATIC MARKER GENES. The result of blue plaque purification was recombinant virus S-ILT-011. This virus was characterized by reslriction mapping and the SOUTHERN BLOTTING OF DNA procedure. This analysis confirmed the presence of the ~-glucuronidase (uidA) marker gene and the deletion of appro~cimatel- 983 base pairs of the ILTV gI gene which deletes 325 of 363 arnino 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-0 1 1 shows a small plaque phenotype in tissue culture which is indicative of slow viral growth and i~llP~ Iion. Since S-ILT-011 does not express the ILTV gI gene~ it may be utilized as a negative marker to distinguish vaccinated ~nim~lc from infected ~nim~l~ As indicated in Example 1, ILTV--infected chickens make antibodies against ILTV gI protein.

.

Example .. .
S-ILT-013 is an infectious laryngotracheitis virus (ILTV) that has an approxim~tely 983 base pair deletion of the ILTV gI gene and an approximately 874 base pair deletion of the ILTV gG gene (and a deletion of the HCMV IE
promoter lacZ marker gene making the lacZ gene nonfunctional). The gene for ~. coli ~-glucuronidase (uidA) was inserted in the place of the gI gene and is under the control of the pseudorabies virus (PRV) gX promoter.

S-ILT-013 was constructed using homology vector 562-61.1 F (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-Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING
ENZYMATIC ~IARKER 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 conflrmed the presence of the ~;-glucuronidase (uidA) marker gene and the deletion of approximately 983 base pairs of the ILTV gI gene which removes 325 of 363 amino acid codons from the 5' end of the gI gene. This analysis also confirmed an a~ oxililately 874 base pair deletion of the ILTV gG gene and an approximatelv 1906 base pair insertion of a partial HCMV IE
promoter--lacZ n-arker 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 ~tt~nll~tion. Since S-ILT-013 does not express the ILTV gI or gG genes, ILTV gI and gG may be utilized as negative markers to distinguish v~l~cin~te~l ~nim~lc from h~fe.;lt:d ~nim~l~

W 096/29396 PCTrUS96/03916 E;xample 6 S-ILT-014 is an infectious laryngotracheitis virus (ILTV) that has an xilllately 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-007 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-00 1 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 ~-galactosidase (lacZ) gene in place of the ILTV gG gene. However, during the BluogalTM SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING
ENZYMATIC MARKER GENES, a white plaque was picked which contained a deletion of the lacZ gene ~ithin the ILTV gG deletion.

This virus~ S-ILT-01~ was characterized by restriction mapping, DNA
SEQUENCING and the SOUTHERN BLOTTING OF DNA procedure. This analysis confirrned the presence of an approxim~t~ly 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 rem~inin~ HCMV IE promoter lacZ marker gene DNA consists of an approxim~tely 686 base pair DNA fragment of the a~,oxilllately 1154 base pair HCMV IE promoter and an approximately 1270 base pair DNA fragment co.,L;~ approximately 520 base pairs of the 3010 base pair ~-galactosidase ~lacZ) marker gene and all of the approximately 750 base pair PRV gX
polyadenylation signal.

S-ILT-014 is useful as an ~ttenl~tto~l live vaccine or as a killed vaccine to -W 096/29396 PCTrUS96/03916 protect chickens from ILT disease as indicated in the table below. Since S-ILT-014 does not express the ILTV gG gene and ILTV--infected chickens make antibodies to gG as indicated in Example 1, ILTV gG is utilized as a negative marker to distinguish vaccinated ~nim~l.c from infected ~nim~lc -W096/29396 PCTrUS96/03916 Table III
e AGAlNST VIRULENT INFECTIOUS LARYNGOTRACHEITIS VIRUS
CHALLENGE
.

Vaccine Gene(s) Dose Route Ch~ nf~eY Protectionb Deleted S-ILT-014 ~Ci- 1.08x104 IO c OS d 97%
S-ILT-014 gG- 2.16x103 IO OS 97%
Controls OS 0%
ASL embryo IO OS 90%
14 day old chiccs a: USDA Challen~e virus =1.0x10~5 pfu b: Protection = # healthy birds/total (%).
c: Intraocular d: Orbitual Sinus W O 96/29396 PCT~US96/03916 Example 7 S-ILT-015 is an infectious lar,vngotracheitis virus (ILTV) that has an al)plo~ ately 2640 base pair deletion of the UL47--like gene, the ORF4 gene~
and ILTV gG gene. The gene for E. coli ~-glucuronidase (uidA) was inserted in the place of the UL47--like, ORF4, and gG genes and is under the control of the pseudorabies virus (PRV) gX promoter. The PRV gX promoter-uidA gene is in the opposite orientation to the direction of L~ s~ Lion of the ILTV
UL47--like, ORF4~ and gG promoters.

S-ILT-015 was constructed using homology vector 560-52.F 1 (see Materials and Methods) and S-ILT-001 in the DNA TRANSFECTION FOR GENERATING
RECOMBINANT ILT VIRUS. The transfection stock was screened by the X-Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING
ENZYMATIC MARKER GENES. The result of blue plaque purification was recombinant virus S-ILT-015. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING OF DNA procedure. These results confirmed the presence of a 2640 base pair deletion which includes 442 of a total 511 amino acid codons at the 3' end of the UL47--like gene, all of the ORF4 gene and 271 of 293 amino acid codons of the 5 end of the gG gene.

S-ILT-015 is useful as an ~tten--~t~l 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 ~nim~l~ from infected ~nim~l~

, W O 96/29396 PCTrUS96/03916 Table IV

AGAINST VIRULENT INFECTIOUS LARYNGOTRACHEITIS VIRUS
CHALLENGE

Vaccine Gene(s) Dose Route Ch~ n~e' Protectionb Deleted S-ILT-015 ~G-. 1.0x105 IO ' OS d 70%
UL47-like Controls OS 0%
ASL IO OS 90%.
embryo 14 day old chicks a: USDA Challen~e virus =1.0x10 5 pfu b: Protection = # healthy birds/total (%).
c: Intraocular d: Orbi~al Sinus W 096/29396 PCTrUS96103916 Example 8 t 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 ,B-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 kno~n 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 g60 genes, it is used as a negative marker to distinguish v~-çin~t~ nim~lc from infected ~nim~lc W 096/29396 PCTrUS96/03916 Example 9 Recombinant infectious lan~n~otracheitis viruses that express infectious bronchitis virus (IBV) sPike and matrix protein ~enes:
A homology vector is used to generate ILT viruses co. ,~ lg the IBV
~rk~n~ spike protein gene. The recombinant ILT virus CU~ S a deletion of one or more ILTV genes, including gG. US2, UL47--like, and ORF4, and the insertion of two foreign genes: the ~. coli ,B-glucuronidase gene (uidA) and theIBV ~rk~n~c spike protein gene. The uidA gene is under the control of the PRV gX promoter and the IBV Arkansas spike protein gene is under the control of the HCMV IE promoter.

To construct a homology vector col.li.;..i.,g the foreign genes inserted into the ILT virus, a DNA fragment cont~ininp 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 col~l;.;..i..~ the PRV gX
promoter and the E. coli ,B-~lucuronidase (uidA) gene is inserted into a unique restriction enzvme site within the ILTV homology vector. A recombinant virus is constructed b- combining the final homology vector c~..l;.;..;..g the IBV
Arkansas spike ~ene and the ~. c<7li ~-glucuronidase (uidA) gene and S-ILT-001 in the DNA TRANSFECTION FOR GENERATING RECOMBINANT
ILT VIRUS. The tr~nsfection 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 spi~;e protein.

A similar strategy is used to construct recombinant ILT viruses carrying the IBV S1 protein from Arkansas, M~c~-hll~ett~,or Connecticut s~i.oLyl,es, IBV
matrix protein from Arkansas~ c~ hl~ett~, or Connecticut serotypes, and IBV nucleocapsid from Arkansas, M~ss~rhl~setts 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 S to construct recombinant ILT viruses carrying the Mareks Disease virus (MDV) gA, gD, and gB genes.

Recombinant ILT virus carrying these antigens are valuabl~ as a multivalent vaccine to protect chickens from rlise~s~c caused by ILTV and one or more of the viruses IBV. N~)V, IBDV, or MDV. Since the ILTV vaccines described here do not express ILTV gG, it is useful as a negative marker to distin~uish v~cçin~t~ nim~ls from infected ~nim~lc Example 10 Vaccines l~tili7in~ ILTV to express anti~ens from various disease causin~
microor~nismS:

Antigens from the following microor~niSms are utilized to develop poultry vaccines: Chick anemia agent. Avian enreph~lomyelitis virus. Avian reovirus, Avian paramyxoviruses, Avian influenza virus ?Avian adenovirus, Fowl pox virus, Avian coronavirus~ Avian rotavirus. Salmonella spp., E coli., Pasteurellaspp., Haemophilus spp.. Chlamydia spp.. Mycoplasma spp., Campylobacter spp., Bordetella spp. Poultry nematodes, cestodes, trem~tocles~ Poultry mites/lice, Poultry protozoa (Eimeria spp.. Histomonas spp., Trichomonas spp.).

Example 11 -A Genomic Map of Infectious Larvn~oL,achcitis Virus and the Sequence and O,~ ion of Genes Present in the Unique Short Re~ion A cosmid library of the ILTV genome was created to f~ilit~t~ restriction CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 endonuclease mapping. Forty-three overlapping cocmi~1C were analyzed by digestion with Asp718I and NotI. Asp718I was known to cut the genome relatively infrequently (63), and it was found that NotI 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 Asp7 1 8I fr~gnnentccovering 85% of the ILTV ~enome to be d~l~ ",;"rd (Figure 12). On the long end of the genome, seven cocmiclc were i-lentifietl which all cont~in~d 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 (Pl in Figure 12) to genomic ILTV digested withAsp718I, NotI, or BamHI; the sizes of the genomic fr~gment~ that hybridized were identif ~1 to the size of the fr~ment~ excised from the cloned cosmid insert, indicating that the cloned insert extenrled 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, conc;ctçnt with previous reports that this virus resembles PRV, and colllaills no long repeat (66). Once the cosmid clones were ordered, the restriction sites for a more frequent cutting enzyme, BamHI, were mapped.

The resulting map indicated that the cosmid library did not include clones from the unique short portion of the genome. Cosmids cp~nning the unique short region of HVT (76) and PRV (83) have been found to be undc.l~ lcse~ d in cosmid libraries The Asp7181 fr~gm~ntc found in the cosmid clones with an Asp718I digest of wild type ILTV and identified fr~gmPnt~ of 8.0, 5.1, and 2.5 kb which were not rc~),esented in the cosmid library (Figure 13) were co,np~ed. These fragments were cloned into plasmids and hybridized to each other and to ILTV digested with BamHI. The Asp718I 2.5 and 8.0 kb fr~gmçntc cross-hybridized, indicating that they cc.l~ ed sequence ~ eait:d in both clones. Fine mapping of the Asp718I 2.5 and 8.0 kb fr~gmentc showed them to contain 2.1 kb of identical sequence. Hyhri-li7~tion to ILTV digested with BamHI identified BamHI bands of 7.5, 6.5, and 4.5 kb which ove~ cd the Asp71 8I fragments. These BamHI fr~m~ntc were cloned and analyzed by restriction digestion and hybri~li7~fi- n This allowed the map of the entire W 096/29396 PCTrUS96/03916 unique short region and some of the fl~nking short repeat to be elucidated (Figure 13). Subclones of this region were made, and the entire unique short region was sequenced.

S To complete the genomic map, the map searched for an Asp718I or BamHI
- fragment that sp~nn~-l the region between the short repeat sequences of the 8.0 or 2.5 kb Asp718I fr~gmPnt~ mentioned above and the unique long region icl.ontifi~l in the cosmid map. A 10 kb NotI fragment from the rightmost end of cosmid D5 (Figure 12) was hybridized to genomic ILTV digests on Southern blots. Il.l.,.. ,~Lillgly, ladders of hybridizing bands were seen when the enzymes BamHI, NotI, and Asp718I were used. The bands cGllG:~onding to these ladders were not generally visible in ethidium bromide stained gels. Subsequent subcloning and mapping of the 10 kb DS fragment in-lic~t~cl that it collldh~ed up to S repeats of an 856 bp segmPnt and that the cosmid insert ended within a repeat motif. HindIII, which cuts once within the repeat, was used to clone the 856 bp fr~gmPnt When this fragment (Figure 12, P2) was used to probe ILTV digested with SfiI, NotI, Asp718I, 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. SfiI cuts only once in this ILTV
strain~ and a ladder at very high molecular weight can be seen. Because the unique short is expected to invert, two ove~ pillg S.fiI ladders CO~ the unique short and terminal repeat (TR~) should be present.; however, the bands are too large in this region to make this f~i~tin~tion. NotI and Asp718I cut further away from the repeat, gen~.dling ladders beginning at 10.5 or 12 kb.
The Asp718I digest should generate two overlapping ladders, because one fragment is bour.ded by an Asp718I site in the unique long, while the other is bounded by the end of the TRs. In contrast, only one ladder should be generated by the NotI digest. Comparison of Figure 14 lane c (NotI) with lane d (Asp718I) does suggest that in lane d a second ladder is :~u~,.hll~osed on thefirst, stdrting somewhat higher. BamHI cuts close to the repe~t~l region, and a ladder be~;nn;ng at 3.4 kb is found. HindIII cuts within the repeat and g~ ,.ales a strongly hybridizing 856 bp band, as well as the two fl~nking W 096129396 PCTrUS96/03916 HindIII fr~gmt-ntc 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 Asp718I or BamHI band greater than 10 kb, which was expected to span this region based on analysis of the cosmid clones. Tnete~l because of the presence of the 856 bp repeat, this band exists as many submolar bands comprising the ladder. As can be seen in the BamHI
digest, there can be thirteen or more repeats of the region. Comparison of the repeat sequence to the sequence snbmitt~l to Gt-nR~nk by Johnson et al. (67) inrli~terl that it col.e:"~,onded (99% identity) to nucleotides 1140 to 1996 of their sequence, which is a region just u~Llc;~ll of the ILTV ICP4 gene. The relationship of the repeat to the surrounding sequence is depicted in Figure 15.Restriction digests indicate that the region to the right of the repeat as shown1.5 is similar in the two strains: however. the position of the BamHI site in-1ic~te-1 to the left of the repeat differs between them.

To identify the rpm~in~itor of the short repeat from the 856 bp lep~,liLi~e region to the Bam~I fr~gmentc used for sequencing the unique short, the 8.0 kb Asp718I fragment cons~ining part of the short repeat was used as a probe to a second cosmid librar~ of ILTV. One cosmid, clone 2F12, hybridized to the probe. Restriction endonuclease analvsis of 2F12 and colll~ison to the cosmid map indicated that it was not a single contiguous cosmid, but was composed of two large non-contiguous fr~gmentc (see Figure 12). The break in the rightmoet 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 rem~in~r of the short repeat into the unique short.

To identify the end of the TRs, the 6.6 kb NotI fragment s~ P the unique long and the short internal repeat (IRs) (P3 in Figure 2) was used as a probe.
It was noted that a 2.9 kb NotI fragment seen in gels stained with ethidium bromide was not re~l ~selll~d in the restriction endon~ ee map, and W 096/29396 PCTrUS96/03916 considered that it might lel~c;~G.ll the end of the TRs. Hybridization of a NotIdigest of ILTV with P3 in-1ic~t~d that this was indeed the case (Figure 16).
The 2.9 kb NotI band hybridizes, as does the 6.6 kb band colle~onding to the probe. In the BamHI digest, the predicted 13 kb fragment co.~ g a portion of the IRs and a 3.5 kb fragment col.G~onding to the end of the TRs are evident. In the Asp71 8I digest, an overlapping 2.7 kb fragment from the unique long hybridizes~ and the high molecular weight ladder described previously was seen.

Seql-~n~ing of the ILTV unique short and fl~nking region identified nine open reading frarnes 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 ri~t~b~ee (BLAST homology seacrh, National Center for Biological Information, NCBI) ri~m~nctrated identity for l$ most of the potential proteins with other known herpesvirus gene products.
Table V slln~m~rizes the closest homologies found for each gene and gives the probability scores for those homologies as generated by the search program.
ORF2 (SEQ ID NO:63), the protein kinase (PK) gene (SEQ ID NO:63), is the most highly conserved of the ILTV ORFs to its herpes homologues. In contrast, the glycoprotein genes are less conserved. It should be noted that portions of the sequences of the ILTV protein kinase, gG, and ORF S 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 fl~nking short repeat follows.
The first open reading frame in the unique short en~o-i~S 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 r~nn~ining ORFs in theunique 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 do~ L~G~ll in the short repeat. Two possible TATA promoters are found 37 and 70 bases u~LlGd--l from the initi~tion codon.

W 096/29396 PCTrUS96/03916 0RF2 encodes a protein kinase with strong identity to many other herpesvirus protein kinases and to cellular protein kinases . The org~ni7Rtion of the US2 and PK genes, with their 5' ends close together and their promoters possibly S ove,l~ lg, is similar to that found in other h~ e;,viluses. Two TATA
sequences are present 14 and 49 bases ulJSLlc,lll of the PK start codon, and twopolyadenylation signals are found, one immerliRt~ly after the stop codon. and one 50 bases downstrearn.

ORF3 encodes a 623 aa protein with similarity to the herpes simplex virus UL47 gene (SEQ ID NO:64). The program COlllpr~lllg this protein with other UL47 proteins projects a poor probability score for this homology. However, at least one of the regions of identity bc~ ,en ILTV and HSV UL47 corresponds to a region that is conserved among other herpesvirus UL47 homologues, suggesting that this identity is ~ignifiçRnt (Figure 17).
Additionally, it should be noted that equally poor probability scores for homology generated by comparisons of the gG or gI genes are also seen for certain homologue pairin~s, suggesting that these scores are not sufficient for detc,lllilling homology. It is interesting that the ILTV UL47 gene. norrnally found in the unique long region of other herpesviruses, appears to have been transposed into the unique short in ILl~l.

The fourth open reading frame encodes a 292 aa glyco~l,Jlcill homologous to PRV gG (SEQ ID NO:65). Four N-linked glycosylation sites with the con~en~C sequence NXT or NXS are present. The protein has a signal sequence of 26 aa. v~hich could be cleaved at G/AP, but lacks a transmembrane anchor. It is therefore likel~ that this protein is seclcLed, similar to other herpesvirus gG homologues. This gene has a conC~n~ TATA sequence 83 bases u~ ealll from the ATG start, and has t~,vo potential polyadenylation sites73 and 166 bases downstream from the stop codon.

ORFS could encode a protein of 985 amino acids (SEQ ID NO:66). A

W 096/29396 PCTrUS96/03916 hydrophobic signal sequence is found at the amino ~ IIIc, and a hydrophobic sequence is present at the carboxy t~ ",; ~ Nine glycosylation sites are found, s~l~gesting that this is a glycop~ cill. ORF S coll~illc an imperfect repeat.
~ ~ ct~ncictinf~ of 30 to 36 bp repeated approximately 23 times from arnino acid 431 to amino acid 677. The hydrophilic amino acid c~ c,lc sequence created by this repeat is FTQTPSTEPET/A. Comparison of ORF 5 with other herpesvirus sequences (Table V) shows similarity to the glyco~ ci.l product from the equine herpesvirus 1 US5 gene (EUS5, 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 fl~nking genes in the unique short, have signal sequences, and encode glycoproteins~ but other se4ucncc similarities are not seen. It is h.lelc~lhlg that the ORF 5 repeat region shows similarity to mucin genes, which also contain threonine rich repeats. The human mucin gene, for example, has the repeat GTO ~ 1 l 1 1 1 I V l ~ l, 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 Pnl~oded proteins is unclear. A TATA sequence is found 560 bases u~JsL~ I of the start codon; the nearest cnl-c~
polyadenylation signal is at the end of the gI gene. This suggests that the ORF

5 transcript mav be coterminal with the gD ~ s~ .L.

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 theactual translational start codon. Because a potential TATA promoter sequence is located only 6-9 bases u~ ~ll 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 U~ lC~Ill that may also be used to initiate Lla"SClil~liOn. The other three potential initiation codons arefound at aa 23, 47, and 58 within this ORF. ~omr~rison of the se.lu~,nces surrounding the four ATGs with the eukaryotic translational initiation co.~ .c~c W 096/29396 PCTrUS~6/03916 sequence A/GCCATGG (71) suggests that the latter two ATG codons may be pl~,r~:l.ed translational start sites. The protein sequences derived from each of these starts were .ox~min~-cl 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 recidues This same signal sequence would be positioned much further from the amino t~rminnc and ~mbe~(lP~l in a more hy~ul hilic sequence if the other start sites were used. The start of ILTV gD was tentatively ~ccign~orl 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 e~
al. (88) suggest that alternate in-frarne ATG codons are used to initiate l~IDV
and HVT gD transcription in vitro, though the in vivo situation was not addressed. Additional ~ hllcllL~ on gD transcription and translation in ILTV
are n~cess~ry to identify its translational start codon.

The ILTV gD homologue has a secretory signal sequence and a tr~ncm~-mhrane helix (aa 352-372) at the carboxy t~.---i--us. Only one potential glycosylation site is found at oosition 250-252; this is of the form NPS, and may not be glycosylated due to tne proline residue. There is some question, tnerefore, as to whether p.ocea:,ed ILTV gD contains N-linked oligos~cch~rides. This would be similar to the gD homologue in pseudorabies virus, gpS0, which also lacks N-linked glycosylation sites (75). As in other herpesviruses, the gD coding sequence lacks a polv-A addition signal imm~ tt-ly 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 glycc.~.ote". 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 30 23 between a glycine and an isoleucine, a Ll,.. c... hrane helix at the carboxy t~ lc from 272 - 2~2, and four possible N-linked glyco:jylation sites. A
TATA sequence is present 51 bases u~ u.. from the m~thionin~ start codon.

W 096/29396 PCTrUS96/03916 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.

S The gE gene (ORF 8) follows the gI. This gene is 499 aa long, and containsfour N-linked glycosylation sites (SEQ ID NO:69). A signal sequence of 18 amino acids is present, and there are two and possibly three mémbrane-associated helices in the carboxy t~rmin~l portion of the protein. The gE gene has a TATA box 86 bases upstream of the start codon, and a potential poly-A
addition signal just prior to the 3' end of the coding region. This may serve asthe polyadenylation site for the gI gene.

The ninth open reading frame extends across the junction of the unique short and the short repeat, and could encode a protein of 260 amino acids (SEQ ID
NO:70). This protein has no signal sequence or membrane anchor, but has one possible N-linked glycosylation site. In a search of GenR~nk, some similarity is found between this protein and BI,RF2 of E}3V, 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 U~ iLl~dlll of the first ATG of this ORF.
.

The first open reading frarne in the short repeat (SRORFl) (SEQ ID NOs: 61 and 71) encodes a ~94 aa protein which displays homology to the gene product of MDV SORF3 (79 and 84) and HVT ORF3 (87). In MDV and HVT, the co~ onding gene is found as one copy in the unique short, and its function is unknown. No homology has been iclentifiç~l with m~mm~ n herpesviruses;
this gene ap~e~s to be specific to avian herpesviruses. MDV SORF3 has been deleted by Parcells et al. (74). and does not appear to be absolutely required for infection in chickens.
SRORF2 encodes a protein of 278 arnino acids with homology to other herpesvirus US10 genes (SEQ ID NOs:60 and 72). A zinc finger motif, found W O 96/29396 PCTrUSg6103916 in 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 org~ni7~tion of the genes in the unique short region of ILTV is similar to that seen in other herpesviruses. Several genes encoding glycoplo~eills are present, and the order of these genes is similar to that seen in equine h~lye;tvil us 1, particularly with respect to ORF 5. Similarities to avian hc.~ vhuse~ are also evident in the ~resence of the avian-specific gene.
SRORF1, and its position relative to US2 and PK, though it differs from HVT
and MDV in that it is in the short repeat and is duplicated, also a~pe~i--g downstream from the ORF 9 gene. The PK gene itself has the most identity to MDV and HVT PK genes; however, other genes are found to be more like their homologues in diverse herpesviruses such as EHV, PRV, and SHV SA8.
Unusual characteristics of the ILTV unique short are the inclusion of a gene normally found in the unique long, the UL47 homologue, and the presence of the unique gene. ORF 5, which contains a set of degt~ .dlt: repeats.

This analysis of the s~ructure 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 af. (70) is almost iclerltic~l 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 Asp718I fragment CO..~ g both of these genes as coming from the unique long region of SA-2 (66). 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 CG~ al ison to the map presented by Johnson et af. (66). No identity was ~ound between this sequence and the unique short sequence described here. Tn~t~rl, W 096/29396 PCTrUS96/03916 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 fr~gm~ntc of 1.2 and 1.7 kb (see Figure 15). In the map described here~ t~vo contiguous BamHI fr~gm~ntc of this size are found within the short repeats (Figure 12). In addition. the 856 bp repeat - element, which is found just u~LIe~ll 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 IRs and the TRs. It is possible, but unlikel~, that the Australian SA-2 vaccine strain underwent an mllcll~l rearrangement which altered the relationship of the unique long, uniqueshort, and short repeat. However, Guo et al. (62) used the same ~h~llenge strain as the one described in this application, and the sequence they reported is not in the unique short, but in the short repeats, similar to the ICP4 genes of other herpesviruses.

The gene encoded by ORF S con~ins Lhleo~ e rich, degenerate repeats. These are similar in composition and in their ~ cLiLi~e nature to repeats found in mucin genes. This repeated region in mucin is ~nodified by O-linked oligo-c~rrll~rides and is highly hydrophilic It is hlL~,.e~Lil~g to speculate on what the function of this somewhat similar region might be in infection, if it is e:jsed 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 l library with a monoclonal antibody that was known to bind to a 60 kd ILTV protein (g60) on Western blots (86). 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 bcLw~cll thc SA-2 strain and the USDA strain. Interestingly, there is an insertion of a block of 10 amino acids in g60 relative to the ORF 5 ~,olcill~ this ~lirrc~cnce reflects oneadditional degenerate repeat sequence in the SA-2 strain.

W 096/29396 PCTrUS96/03916 As mentioned above, Kongsuwan et al. (70) described an ILTV gene that encoded a 32 kd protein with similarity to PRV gG. A c~ .A, ;~on of the ILTV gG protein sequence described in this application with their 3'7 kd proteinfound 10 amino acid dirr~l.,..ces 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 t~rninAl 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 i~ound when the ILTV gD protein described in this application was colllp~d with the ILTV gD sequence submitted to GenBank by Johnson et al. (68). Ten ~lirr~ lces were found in the first 419 amino acids, after which a deletion of a base in the SA-2 strain relative to the the sequence described in this application caused the predicted carboxy termini to differ, with 15 more amino acids in the USDA strain and 9 in SA-,. These differences could arise from errors introduced during cloning and seqllen~ing of these genes. It is also possible that the carboxv terrnini of the ILTV gG and gD genes are variable between these strains.
The 856 bp repeat unit identified within the short repeat is just u~sll~,~l, of the ICP4 gene described by Johnson e~ al. (67), but, from the sequence alone, it does not appear to be repetitive in the SA-2 strain. The BamHI fragment COI~tA;II;I~ this repetitive region is 2848 bp long in SA-2. The ~mAllest repeat, seen faintly in the BamHI ladder of Figure 14, is 3.4 kb long. This is not quitelarge enough to include two repeats. and suggests that other alterations betweenthe 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 a~pG~allce of the ladder is r~omini~cPnt of defective interfering particles, but it is not believed that this re~ ,s~nts a case of defective i,l-e,r~,l;"g particles in the viral stock used here.
Several reasons for this follow. 1) Defective i"lt;,r~lhlg particles are generally CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 found when viruses are passaged at high multiplicity, and the ILTV viral stocks of this application were passaged at low multiplicity. In fact~ viral stocks origin~tin~ from a single picked plaque exhibited similar ladders when their DNA was subjected to Southern blot analysis, sugge:,LiL.g that a single viral particle c~ g a set number of repeats could leg~t~ale the full range of the ladder after being grown for a short period of time. 2) If populations of defective i~ rt,.hlg particles were present, one might expect to encounter digest fr~gm~nt~ 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 Asp718I bands identical to those present in genomic ILTV digests.
The exception, 2F12, was unusual in being the only one of several hundred cosmid clones screened which cont~ined part of the unique short. This probably represented an aberrant cloning event, and not a widespread phPnnmPnon related to defective viral particles. 3) Defective interfering particles often are present in larger molar amounts than standard viral particles.
such -uhat restricLion fr~gmenTc ori~inz3Ting from Tne defeciive particies are 0~ ICS~ ef1 In contrast, the bands of the 856 bp ladder are submolar, and are only rarely visible in ethidium bromide stained gels. 4) Defective interfering particles contain origins of replication. The 856 bp repeat itself does not contain a herpesvirus origin of replication as defined by the consensus sequence of R~--rn~nn et al. (59). 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 fr~gmentc exist that contain thirteen or more repeats of the region, genomic DNA from ILTV
could vary by over l l kb in the short repeat regions. Repetitive regions have been identified in other herpesviruses; for exaTnple, Marek's disease virus contains a 132 bp .e~ e sequence in the long repeat regions (61 and 73) and expansion of this repeat is associated with re~ ction of viral oncogenicity. The ..
prese,lce of the 856 bp tandem repeats in ILTV, in contrast, does not appear to affect viral pathogenicity, since this strain does cause severe clinical disease in chickens. It would be intc.es~ing to ~, ....;..~ other ILTV strains for the presence of this repeat.

W 096/29396 PCTrUS96/03916 Table V indicates the ORFs of the ILTV unique short and the HSV
nom.-nt~l~tllre for these genes, in those cases where homology is found. The third column shows the best m~tch~c from the Blast homology search (NCBI)~
and the probability scores ~ccign~r1 by the program for the m~tc h--c indicated.Smaller nurnbers indicate less likelihood that the match could occur randomlv.

A genomic map of infectious laryngotracheitis virus (ILTV) and a 18.912 bp sequence cont~ining the entire unique short region and a portion of the fl~nkingshort repeats is presentec1 In ~ ...,i..;..g the genomic map, an 856 bp region repeated as many as 13 times was i-lentified within the short repeats. The unique short sequence contains 9 potential open reading frames (ORFs). Six of these ORFs show homology to other known hcl~e~vil us unique short genes.
Using the herpes simplex virus nnmton~ t lre these genes are the US2. protein kinase, and glycoproteins G, D, I, and E (SORFs 1, 2, 4, 6, 7, and 8, respectively). Interestingly, an open reading frame with homology to HSV-l UL47 (SORF 3) is found in the unique short. One very large open reading frame (ORF 5) is present and cc,..l;.;..c a Ihl.,~)lille rich, deg~ .ale repeat sequence. This gene appears to be unique to ILTV among sequenced herpesviruses. Two ORFs were identified ~vithin the short repeat region.
SRORFl is homologous to a ~ene (SORF3) found in the unique short region in both MDV and HVT. and appears to be specific to avian herpesviruses.
SRORF2 has homology to HSV US10.

W O 96/29396 PCTrUS96/03916 Table V

ORF HSV Best Matches Blast Score 5 . Homolog US2 EHVI EUSl 3.1x10-'3 EHV4 EUS 1 5.3xl 0-12 HSV2 US2 6.7x10-7 2 PK MDV PK 8.2x10-36 HVT PK 5.4x10-35 HSVl PK 4.1x10-3~
3 UL47 HSV 1 UL47 6~0x 10-' EHV l UL47 9.9xl O ' MDV UL47 9.9xlO-' 4 gG PRV gG 5.3x10-5 BHVl gG 1.7x10-2 EHVl gG 6.8xlO-I
ORF 5 EHVl EUS5 l.9x10~5 .
Human mucin l.lx1025 W 096/29396 PCTrUS96/03916 6 gD MDV gD 6.8xlO~

PRV g50 2.0x10-3 HVT gD 3.5xlO-3 7 gI VZV gI 4.2x10-' HVT gI 7.9xlO-~
SVV gI 4.3xlO ' 8 gE SHV SA8 gE 1.7xlO~

HSVl gE l.lx103 BHVl gE 1.5x10-2 9 ORF 9 EBV BLRF2 5.7xlO ' SRl no HSV MDV "ORF3" 4.8x104 homologue HVT"ORF3" 2.6xlO-' SR2 US 10 EHV-4 US10 1.2x10-' HSV-l US10 8.7xlO-' EHV-l US10 8.7xlO-' W 096/29396 PCT~US96/03916 REFERENCES:
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CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 SEQUENCE LISTING
(1) GENER~L lNrO~ATION:
(i) APPLICANT: Wild, Martha A.
Cochran, Mark D.
~ii) TITLE OF lNv~NllON: RECOMBINANT lNrr;~llOUS LARYNGOL~ACHEITIS VIRUS
AND USES THEREOF
(iii) NUMBER OF SEQUENCES: 72 (iv) COXkESPuN~N-~ ADDRESS:
A~ ~nD~ EE: Cooper & Dunham LLP
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(i) Sr;~ur.N~r; CHARACTERISTICS:
(A) LENGTH: 13473 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HY~Oln~llCAL: NO

(ix) FEATURE:
(A) NAME/REY: CDS
(B) LOCATION: 1059..2489 (ix) FEATURE:
(A) NAME/KEY: CDS
(B)- LOCATION: 2575..4107 CA 022l6l39 l997-09-22 096/29396 PCTrUS96/03916 (ix) FEATURE
(A) NAME/REY CDS
(B) LOCATION 4113 4445 (ix) FEATURE
(A) NAME/KEY CDS
(B) LOCATION 4609 5487 (ix) FEATURE
(A) NAME/KEY CDS
(B) LOCATION 5697 8654 (ix) FEA'lu~E
(A) NAME/~EY CDS
(B) LOCATION 9874 10962 (ix) FEATURE
(A) NAME/KEY CDS
(B) LOCATION 11153 12658 (ix) FEATURE
(A) NAME/KEY CDS
(B) LOCATION 12665 13447 (xi) ~u~ DESCRIPTION SEQ ID NO 1 CCC~l~CCCC TAAAGGCCGC CGAGAAAGCT AAGTCCAAAT GTGACGTCGG AG~l~lCGAC 60 AlG~LCGC~A ACC~l~AAA TGCTACCCGC CGGCCCACGC AACGCGGGCT TTTATAAAGA 120 l~l~lrATT ATTACTGCTA CCGCC~Ll~l TTCTGCAAGG CC~CGCCGC GGCCCAGGCC 240 AGCGGG~lll CTTCGACTTA AATAGACTCC CGAGAAAAAA TTTTGGCTGC C~ l-CGC~AT 360 CATCCGAGTC GGAAACACAG TATGCGGCCG iGTTAGGTTT TACTTTTAAA A~CTTTACCG 420 ~ ~lACGG CCAGGGCGTT CTCAGGCTCG AAGGGGCAAG A~l~ C AG ACTGATGGGT 480 GACTCAGAGA CAGC~ll~lC ~ CC~l TTACCAAAAA TATTTCCACT C~~ ~u~AA 540 AATTTTTACC TCCGGTTTCG GTAATTAGGA AA~llll~aG CGCAGGGAGG rlTAAAGCTG 600 CCATGCATAT GTCAGCGGTA CCCAGCACCC ACAAATGGAA ~l~-LlllGCG GCATACGCGC 660 GGTCGCCCCC CTCACCGAAC CGAATCACGG GTCTGCACAT C-~LGG~AAGG GAAAACAGCT 780 CGTAACTCGC CATAGTTTTC A~l~C~ L GAA CCAATTCTTT CCATCCAGAA TCCGAGAGCT 900 CAGGCGCCAT CGCCGCTCAG ACTCCACCCC GCTATGATGT CAGAAATATA ACG~- LlAT 10 20 Met Arg Phe Arg Arg Ile Cys Ser Arg Ser Arg Ala Glu Lys Arg Arg Arg Thr Thr Glu Asn W 096/29396 PCTrUS96/03916 Pro Leu Thr Ser Lys Arg Val Cys Val Leu Asp Ser Phe Ser Arg Thr ATG.TCA TTG CGC CCC TAT GCA GAA ATT TTG CCG ACC GCG GAA GGC GTC 1217 Met Ser Leu Arg Pro Tyr Ala Glu Ile Leu Pro Thr Ala Glu Gly Val Glu Arg Leu Ala Glu Leu Val Ser Val Thr Met Thr Glu Arg Ala Glu Pro Val Thr Glu Asn Thr Ala Val Asn Ser Ile Pro Pro Ala Asn Glu Asn Gly Gln Asn Phe Ala Tyr Ala Gly Asp Gly Pro Ser Thr Thr Glu Lys Val Asp Gly Ser His Thr Asp Phe Asp Glu Ala Ser Ser Asp Tyr Ala Gly Pro Val Pro Leu Ala Gln Thr Arg Leu Lys His Ser Asp Glu Phe Leu Gln His Phe Arg Val Leu Asp Asp Leu Val Glu Gly Ala Tyr Gly Phe Ile Cys Gly Val Arg Arg Tyr Thr Glu Glu Glu Gln Arg Arg Arg Gly Val Asn Ser Thr Asn Gln Gly Lys Ser Lys Cys Lys Arg Leu Ile Ala Lys Tyr Val Lys Asn Gly Thr Arg Ala Ala Ser Gln Leu Glu Asn Glu Ile Leu Val Leu Gly Arg Leu Asn His Glu Asn Val Leu Lys 200 ~05 210 Ile Gln Glu Ile Leu Arg Tyr Pro Asp Asn Thr Tyr Met Leu Thr Gln Arg Tyr Gln Phe Asp Leu Tyr Ser Tyr Met Tyr Asp Glu Ala Phe Asp Trp Lys Asp Ser Pro Met Leu Lys Gln Thr Arg Arg Ile Met Lys Gln ~ Leu Met Ser Ala Val Ser Tyr Ile His Ser Lys Lys Leu Ile His Arg 265 . 270 275 Asp Ile Lys Leu Glu Asn Ile Phe Leu Asn Cys Asp Gly Lys Thr Val CA 022l6l39 l997-09-22 096/29396 PCTrUS96/03916 Leu Gly Asp Phe Gly Thr Val Thr Pro Phe Glu Asn Glu Arg Glu Pro Phe Glu Tyr Gly Trp Val Gly Thr Val Ala Thr Asn Ser Pro Glu Ile Leu Ala Arg Asp Ser Tyr Cys Glu Ile Thr Asp Ile Trp Ser Cys Gly Val Val Leu Leu Glu Met Val Ser His Glu Phe Cys Pro Ile Gly Asp Gly Gly Gly Asn Pro His Gln Gln Leu heu Lys Val Ile Asp Ser Leu Ser Val Cys Asp Glu Glu Phe Pro Asp Pro Pro Cys Asn Leu Tyr Asn Tyr Leu His Tyr Ala Ser Ile Asp Arg Ala Gly His Thr Val Pro Ser Leu Ile Arg Asn Leu His Leu Pro Ala Asp Val Glu Tyr Pro Leu Val Lys Met Leu Thr Phe Asp Trp Arg Leu Arg Pro Ser Ala Ala Glu Val Leu Ala Met Pro Leu Phe Ser Ala Glu Glu Glu Arg Thr Ile Thr Ile Ile His Gly Lys His Lys Pro Ile Arg Pro Glu Ile Arg Ala Arg Val Pro Arg Ser Met Ser Glu Gly AGCGTCAGAT 'L 1 L L L-lAAAA AAATAAATGA TCGAGAACTT ATGATTTGTC 'L'L'l ~ L l-~A 2574 Met Thr Leu Pro His Arg Leu Thr Lys Arg Pro Phe Ala Arg Arg Phe Cys Ser Val Phe Val Tle His Tyr Ser Giu Thr Lys Leu Asp Arg Tyr Asn Lys Thr Met Leu Leu Tyr Arg Pro Asp Ser Thr Met Arg His Ser Gly Gly Asp Ala Asn His Arg Gly Ile Arg Pro Arg Arg Lys Ser Ile CA 022l6l39 l997-09-22 W 096/29396 PCT~US96/03916 GGA GCG TTT AGC GCG CGC GAA AAG ACT GGA A~A CGA AAT GCG CTG ACG 2814Gly Ala Phe Ser Ala Arg Glu Lys Thr Gly Lys Arg Asn Ala Leu Thr Glu Ser Ser Ser Ser Ser Asp Met Leu Asp Pro Phe Ser Thr Asp Lys Glu Phe Gly Gly Lys Trp Thr Val Asp Gly Pro Ala Asp Ile Thr Ala Glu Val Leu Ser Gln Ala Trp Asp Val Leu Gln Leu Val Lys His Glu Asp Ala Glu Glu Glu Arg Val Thr Tyr Glu Ser Lys Pro Thr Pro Ile Gln Pro Phe Asn Ala Trp Pro Asp Gly Pro Ser Trp Asn Ala Gln Asp Phe Thr Arg Ala Pro Ile Val Tyr Pro Ser Ala Glu Val Leu Asp Ala Glu Ala Leu Lys Val Gly Ala Phe Val Ser Arg Val Leu Gln Cys Val Pro Phe Thr Arg Ser Lys Lys Ser Val Thr Val Arg Asp Ala Gln Ser Phe Leu Gly Asp Ser Phe Trp Arg Ile Met Gln Asn Val Tyr Thr Val Cys Leu Arg Gln Hls Ile Thr Arg Leu Arg His Pro Ser Ser Lys Ser Ile Val Asn Cys Asn Asp Pro Leu Trp Tyr Ala Tyr Ala Asn Gln Phe CAC TGG AGA GGA ATG CGC GTG CCG TCG CTT A~A TTA GCC TCT CCC CCG 3390 His Trp Arg Gly Met Arg Val Pro Ser Leu Lys Leu Ala Ser Pro Pro Glu Glu Asn Ile Gln His Gly Pro Met Ala Ala Val Phe Arg Asn Ala Gly Ala Gly Leu Phe Leu Trp Pro Ala Met Arg Ala Ala Phe Glu Glu Arg Asp Lys Arg Leu Leu Arg Ala Cys Leu Ser Ser Leu Asp Ile Met Asp Ala Ala Val Leu Ala Ser Phe Pro Phe Tyr Trp Arg Gly Val Gln W 096/29396 PCTrUS96/03916 GAC ACC TCG CGC TTC GAG CCT GCG CTG GGC TGT TTG TCA GAG TAC TTT 3630Asp Thr Ser Arg Phe Glu Pro Ala Leu Gly Cys ~eu Ser Glu Tyr Phe Ala Leu Val Val Leu Leu Ala Glu Thr Val Leu Ala Thr Met Phe Asp His Ala Leu Val Phe Met Arg Ala Leu Ala Asp Gly Asn Phe Asp Asp TAT GAC GAA ACT AGA TAT ATA GAC CCC GTT AAA AAC GAG TAC CTG A~C 3774 Tyr Asp Glu Thr Arg Tyr Ile Asp Pro Val Lys Asn Glu Tyr Leu Asn Gly Ala Glu Gly Thr Leu Leu Arg Gly Ile Val Ala Ser Asn Thr Ala CTG GCG GTG GTT TGC GCA AAC ACC TAT TCG ACG ATA A~A AAA CTC CCG 3870 Leu Ala Val Val Cys Ala Asn Thr Tyr Ser Thr Ile Arg Lys Leu Pro Ser Val Ala Thr Ser Ala Cys Asn Val Ala Tyr Arg Thr Glu Thr Leu Lys Ala Arg Arg Pro Gly Met Ser Asp Ile Tyr Arg Ile Leu Gln Lys Glu Phe Phe Phe Tyr Ile Ala Trp Leu Gln Arg Val Ala Thr His Ala Asn Phe Cys Leu Asn Ile Leu Lys Arg Ser Val Asp Thr Gly Pro Arg 485 . 490 495 His Phe Cys Ser Gly Pro Ala Arg Arg Ser Gly Cys Ser Ser Met Leu Cys Pro Leu Leu Val Pro Ile Gln Tyr Glu Asp Phe Ser Lys Ala Met Gly Ser Glu Leu Lys Arg Glu Lys Leu Glu Thr Phe Val Lys Ala Ile Ser Ser Asp Arg Asp Pro Arg Gly Ser Leu Arg Phe Leu Ile Ser Asp ~is Ala Arg Glu Ile Ile Ala Asp Gly Val Arg Phe Lys Pro Val Ile Asp Glu Pro Val Arg Ala Ser Val Ala Leu Ser Thr Ala Ala GCT GGG AAA GTG A~A GCG CGA CGC TTA ACC TCA GTT CGC GCG CCC GTA 4400 Ala Gly Lys Val Lys Ala Arg Arg Leu Thr Ser Val Arg Ala Pro Val W 096129396 PCT~US96/03916 CCG CCC GCA GGC GCC GTT TCC GCG CGC CGG A~A TCG GAA ATA TGA TA 4447 Pro Pro Ala Gly Ala Val Ser Ala Arg Arg Lys Ser Glu Ile AAAATGCTTG GCATTTGCGG GCGAAGAGGC GTGATCTGA~ GGGCTCCACA ATGACGTAAC 4507 r Met Ser Gly Phe Ser Asn Ile Gly Ser Ile Ala Thr Val Ser Leu Val Cys Ser Leu Leu Cy8 Ala Ser Val Leu Gly Ala Pro Val Leu Asp Gly Leu Glu Ser AGC CCT TTC CCG TTC GGG GGC A~A ATT ATA GCC CAG GCG TGC AAC CGC 4761 Ser Pro Phe Pro Phe Gly Gly Lys Ile Ile Ala Glr. Ala Cys Asn Arg Thr Thr Ile Glu Val Thr Val Pro Trp Ser Asp Tyr Ser Gly Arg Thr Glu Gly Val Ser Val Glu Val Lys Trp Phe Tyr Gly Asn Ser Asn Pro Glu Ser Phe Val Phe Gly Val Asp Ser Glu Thr Gly Ser Gly His Glu GAC CTG TCT ACG TGC TGG G"T CTA ATC CAT AAT CTG AAC GCG TCT GTG 4953 Asp Leu Ser Thr Cys Trp Ala Leu Ile Hls Asn Leu Asn Ala Ser Val TGC AGG GCG TCT GAC GCC GGG ATA CCT GA. TTC GAC AAG CAG TGC GAA 5001 Cys Arg Ala Ser Asp Ala Gly Ile Pro Asp Phe Asp Lys Gln Cys Glu Lys Val Gln Arg Arg Leu Arg Ser Gly Val Glu Leu Gly Ser Tyr Val Ser Gly Asn Gly Ser Leu Val Leu Tyr Pro Gly Met Tyr Asp Ala Gly Ile Tyr Ala Tyr Gln Leu Ser Val Gly Gly Lys Gly Tyr Thr Gly Ser Val Tyr Leu Asp Val Gly Pro Asn Pro Gly Cys His Asp Gln Tyr Gly Tyr Thr Tyr Tyr Ser Leu Ala Asp Glu Ala Ser Asp Leu Ser Ser Tyr Asp Val Ala Ser Pro Glu Leu Asp Gly Pro Met Glu Glu Asp Tyr Ser W 096l29396 PCTrUS96/03916 AAT TGT CTA GAC ATG CCC CCG CTA CGC CCA TGG ACA ACC GTT TGT TCG 5337Asn Cys Leu Asp Met Pro Pro Leu Arg Pro Trp Thr Thr Val Cys Ser His Asp Val Glu Glu Gln Glu Asn Ala Thr Asp Glu Leu Tyr Leu Trp Asp Glu Glu Cys Ala Gly Pro Leu Asp Glu Tyr Val Asp Glu Arg Ser Glu Thr Met Pro Arg Met Val Val Phe Ser Pro Pro Ser Thr Leu Gln CAG TAGCCACCCG AGA~l~ l l L-l TTGTGAGCGC CCACGCAACA TACCTAACTG 5534 Gln Met Gly Thr Met Leu Val Leu Arg Leu Phe Leu Leu Ala Val Ala Asp Ala Ala Leu Pro Thr Gly Arg Phe Cys Arg Val Trp Lys Val Pro Pro Gly Gly Thr Ile Gln Glu Asn Leu Ala Val Leu Ala Glu Ser Pro Val Thr Gly His Ala Thr Tyr Pro Pro Pro Glu Gly Ala Val Ser Phe Gln Ile Phe Ala Asp Thr Pro Thr Leu Arg Ile Arg Tyr Gly Pro Thr Glu Asp Glu Leu Ala Leu Glu Arg Gly Thr Ser Ala Ser Asp Ala Asp Asn Val Thr Phe Ser Leu Ser Tyr Arg Pro Arg Pro Glu Ile His Gly Ala Tyr Phe Thr Ile Gly Val Phe Ala Thr Gly Gln Ser Thr Glu Ser Ser Tyr Ser Val Ile ~ Ser Arg Val Leu Val Asn Ala Ser Leu Glu Arg Ser Val Arg Leu Glu Thr Pro Cys Asp Glu Asn Phe Leu Gln Asn Glu Pro Thr Trp Gly Ser W 096/29396 PCTrUS96/03916 _95_ Lys Arg Trp Leu Gly Pro Pro Ser Pro Tyr Val Arg Asp Asn Asp Val Ala Val Leu Thr Lys Ala Gln Tyr Ile Gly Glu Cys Tyr Ser Asn Ser Ala Ala Gln Thr Gly Leu Thr Ser Leu Asn Met Thr Phe Phe Tyr Ser 200 205 ~ 210 Pro Lys Arg Ile Val Asn Val Thr Trp Thr Thr Gly Gly Pro Ser Pro Ser Arg Ile Thr Val Tyr Ser Ser Arg Glu Asn Gly Gln Pro Val Leu Arg Asn Val Ser Asp Gly Phe Leu Val Lys Tyr Thr Pro Asp Ile Asp Gly Arg Ala Met Ile Asn Val Ile Ala Asn Tyr Ser Pro Ala Asp Ser Gly Ser Val Leu Ala Phe Thr Ala Phe Arg Glu Gly Lys Leu Pro Ser Ala Ile Gln Leu His Arg Ile Asp Met Ser Gly Thr Glu Pro Pro Gly Thr Glu Thr Thr Phe Asp Cys Gln Lys Met Ile Glu Thr Pro Tyr Arg Ala Leu Gly Ser Asn Val Pro Arg Asp Asp Ser Ile Arg Pro Gly Ala Thr Leu Pro Pro Phe Asp Thr Ala Ala Pro Asp Phe Asp Thr Gly Thr Ser Pro Thr Pro Thr Thr Val Pro Glu Pro Ala Ile Thr Thr Leu Ile CCG CGC AGC ACT AGC GAT.ATG GGA ~C TTC TCC ACG GCA CGT GCT ACC 6860 Pro Arg Ser Thr Ser Asp Met Gly Phe Phe Ser Thr Ala Arg Ala Thr 37~ 380 385 ~ Gly Ser Glu Thr Leu Ser Val Pro Val Gln Glu Thr Asp Arg Thr Leu ~ 390 355 400 t Ser Thr Thr Pro Leu Thr Leu Pro Leu Thr Pro Gly Glu Ser Glu Asn Thr Leu Phe Pro Thr Thr Ala Pro Gly Ile Ser Thr Glu Thr Pro Ser 096/29396 PCTrUS96/03916 Ala Ala His Glu Thr Thr Gln Thr Gln Ser Ala Glu Thr Val Val Phe Thr Gln Ser Pro Ser Thr Glu Ser Glu Thr Ala Arg Ser Gln Ser Gln Glu Pro Trp Tyr Phe Thr Gln Thr Pro Ser Thr Glu Gln Ala Ala Leu Thr Gln Thr Gln Ile Ala Glu Thr Glu Ala Leu Phe Thr Gln Thr Pro Ser Ala Glu Gln Met Thr Phe Thr Gln Thr Pro Gly Ala Glu Thr Glu Ala Pro A'a G,n Thr Pro Ser Thr Ile Pro Glu Ile Phe Thr Gln Ser 520 52~ 530 Arg Ser Thr Pro Pro Glu Thr Ala Arg Ala Pro Ser Ala Ala Pro Glu Val Phe Thr Gln Ser Ser Ser Thr Val Thr Glu Val Phe Thr Gln Thr Pro Ser Thr Val Pro Lys Thr Thr Leu Ser Ser Ser Thr Glu Pro Ala Ile Phe Thr Arg Thr Gln Ser Ala Gly Thr Glu Ala Phe Thr Gln Thr Ser Ser Ala Glu Pro Asp Thr Met Arg Thr Gln Ser Thr Glu Thr His TTT TTC ACT CAG GCC CCG AGT ACG GTA CCG A~A GCT ACT CAG ACT CCG 7580 Phe Phe Thr Gln Ala Pro Ser Thr Val Pro Lys Ala Thr Gln Thr Pro Ser Thr Glu Pro Glu Val Leu Thr Gln Ser Pro Ser Thr Glu Pro Val Pro Phe Thr Arg Thr Leu Gly Ala Glu Pro Glu Ile Thr Gln Thr Pro Ser Ala Ala Pro Glu Val Tyr Thr Arg Ser Ser Ser Thr Met Pro Glu Thr Ala Gln Ser Thr Pro Leu Ala Ser Gln Asn Pro Thr Ser Ser Gly Thr Gly Thr His Asn Thr Glu Pro Arg Thr Tyr Pro Val Gln Thr Thr W 096/29396 PCTrUS96/03916 . -97-Pro His Thr Gln Lys Leu Tyr Thr Glu Asn Lys Thr Leu Ser Phe Pro Thr Val Val Ser Glu Phe His Glu Met Ser Thr Ala Glu Ser Gln Thr Pro Leu Leu Asp Val Lys Ile Val Glu Val Lys Phe Ser Asn Asp Gly Glu Val Thr Ala Thr Cys Val Ser Thr Val Lys Ser Pro Tyr Arg Val Glu Thr Asn Trp Lys Val Asp Leu Val Asp Val Met Asp Glu Ile Ser Gly Asn Ser Pro Ala Gly Val Phe Asn Ser Asn Glu Lys Trp Gln Lys Gln Leu Tyr Tyr Arg Val Thr Asp Gly Arg Thr Ser Val Gln Leu Met Cys Leu Ser Cys Thr Ser His Ser Pro Glu Pro Tyr Cys Leu Phe Asp Thr Ser.Leu Ile Ala Arg Glu Lys Asp Ile Ala Pro Glu Leu Tyr Phe Thr Ser Asp Pro Gln Thr Ala Tyr Cys Thr Ile Thr Leu Pro Ser Gly Val Val Pro Arg Phe Glu Trp Ser Le~ Asn Asn Val Se- Leu Pro Glu Tyr Leu Thr Ala Thr Thr Val Val Ser His Thr Ala Gly Gln Ser Thr Val Trp Lys Ser Ser Ala Arg Ala Gly Glu Ala Trp Ile Ser Gly Arg Gly Gly Asn Ile Tyr Glu Cys Thr Val Leu Ile Ser Asp Gly Thr Arg ~ Val Thr Thr Arg Lys Glu Arg Cys Leu Thr Asn Thr Trp Ile Ala Val ~ 935 940 945 Glu Asn Gly Ala Ala Gln Ala Gln Leu Tyr Ser Leu Phe Ser Gly Leu Val Ser Gly Leu Cys Gly Ser Ile Ser Ala Leu Tyr Ala Thr Leu Trp CA 022l6l39 l997-09-22 W 096/29396 PCTrUSg6/03916 ACC GCC ATT TAT TTT TGAGGAATGC TTTTTGGACT ATCGTACTGC ~ ~lLCCTT 8691 Thr Ala Ile Tyr Phe AGAAAAAGAG AAGGGGGGCC ~llLCGAGGC ~lCC~lC~LC TGGTTCTACG TGATTAAGGG 8931 CGACGACGGC GAGGACAAGT A~L~lC~AAT CTATAGAAAA GAGTACAGGG AATGTGGCGA 8991 TCCTAGCACC CTTGTATCG_ GAAATGGCGC GGGACTGACT ATAll~LCCC CCACTGCTGC 9111 G~L~l-l~GC CAATACTTGC TGACCCTGAA AATCGGGAGA TTTGCGCAAA CAG~L~LC~l 9171 AACTCTAGAA GTTAACGATC G~~ lAAA GATCGGGTCG CAGCTTAACT TTTTACCGTC 9231 C~LCCCGCAA GAAATTCCCG CTGTAACCAA GAAAGCGGAA GGGCGCACCC CGGACGCAGA 9471 GACTACTAAA AGTTCTAATG CC~l~lC~AT GCCCATATT_ GCGGCGTTCG TAGCCTGCGC 9711 GGTCGCGCTC GTGGGGCTAC L~lllGGAG CATCGTAAAA TGCGCGCGTA GCTAATCGAG 9771 CCTAGAATAG GTG~Lll~ 1 CCTACATGCC ACGCCTCACG CTCATAATAT AAATCACATG 9831 GAATAGCATA CCAATGCCTA TTCATTGGGA C~llC~AAAA GC 9873 Met Ala Ser Leu Leu Gly Thr Leu Ala Leu Leu Ala Ala Thr Leu Ala Pro Phe Gly Ala Met Gly Ile Val Ile Thr Gly Asn His Val Ser Ala Arg Ile Asp Asp Asp His Ile Val Ile Val Ala Pro Arg Prc Glu Ala Thr Ile Gln Leu Gln Leu Phe 40 45 5~ 55 Phe Met Pro Gly Gln Arg Pro His Lys Pro Tyr Ser Gly Thr Val Arg Val Ala Phe Arg Ser Asp Ile Thr Asn Gln Cys Tyr Gln Glu Leu Ser CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 _99 _ Glu Glu Arg Phe G1U Asn Cys Thr His Arg Ser Ser Ser Val Phe Val go 95 100 Gly Cys Lys Val Thr Glu Tyr Thr Phe Ser Ala Ser Asn Arg Leu Thr Gly Pro Pro His Pro Phe Lys Leu Thr Ile Arg Asn Pro Arg Pro Asn Asp Ser Gly Met Phe Tyr Val Ile Val Arg Leu Asp Asp Thr Lys Glu Pro Ile Asp Val Phe Ala Ile Gln Leu Ser Val Tyr Gln Phe Ala Asn Thr Ala Ala Thr Arg Gly Leu Tyr Ser Lys Ala Ser Cys Arg Thr Phe .GGA TTA CCT ACC GTC CAA CTT GAG GCC TAT CTC AGG ACC GAG GAA AGT 10470 Gly Leu Pro Thr Val Gln Leu Glu Ala Tyr Leu Arg Thr Glu Glu Ser 185 lgO 195 Trp Arg Asn Trp Gln Ala Tyr Val Ala Thr Glu Ala Th_ Thr Thr Ser Ala Glu Ala Thr Thr Pro Thr Pro Val Thr Ala Thr Ser Ala Ser Glu CTT GAA GCG GAA CAC TT. ACC TTT CCC TGG CTA GAA AAT GGC GTG GAT 10614 Leu Glu Ala Glu His Phe Thr Phe Pro Trp Leu Glu Asn Gly Val Asp His Tyr Glu Pro Thr Pro Ala Asn Glu Asn Ser Asn Val Thr Val Arg Leu Gly Thr Met Ser Pro Thr Leu Ile Gly Val Thr Val Ala Ala Val Val Ser Ala Thr Ile Gly Leu Val Ile Val Ile Ser Ile Val Thr Arg Asn Met Cys Thr Pro His Arg Lys Leu Asp Thr Val Ser Gln Asp Asp Glu Glu Arg Ser Gln Thr Arg Arg Glu Ser Arg Lys Phe Gly Pro Met Val Ala Cys Glu Ile Asn Lys Gly Ala Asp Gln Asp Ser Glu Leu Val Glu Leu Val Ala Ile Val Asn Pro Ser Ala Leu Ser Ser Pro Asp Ser 345 . 350 355 CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 ATA AAA ATG TGATTAAGTC TGAATGTGGC TCTCCAATCA TTTCGATTCT lO999 Ile Lys Met CTAATCTCCC AALC~l~u~A A~AGGGGCAG TATCGGACAC GGACTGGGAG GGGCGTACTA llG59 Met Asn Met Leu Val l 5 Ile Val Leu Ala Ser Cys Leu Ala Arg Leu Thr Phe Ala Thr Arg His Val Leu Phe Leu Glu Gly Thr Gln Ala Val Leu Gly Glu Asp Asp Pro Arg Asn Val Pro Glu Gly Thr Val Ile Lys Trp Thr Lys Val Leu Arg Asn Ala Cys Lys Met Lys Ala Ala Asp Val Cys Ser Ser Pro Asn Tyr Cys Phe His Asp Leu Ile Tyr Asp Gly Gly Lys Lys Asp Cys Pro Pro Ala Gly Pro Leu Ser Ala Asn Leu Val Ile Leu Leu Lys Arg Gly Glu Ser Phe Val Val Leu G y Ser Gly Leu His Asn Ser Asn Ile Thr Asn Ile Met Trp Thr Glu Tyr Gly Gly Leu Leu Phe Asp Pro Val Thr Arg Ser Asp Glu Gly Ile Tyr Phe Arg Arg Ile Ser Gln Pro Asp Leu Ala Met Glu Thr Thr Ser Tyr Asn Val Ser Val Leu Ser His Val Asp Glu Lys Ala Pro Ala Pro His Glu Val Glu Ile Asp Thr Ile Lys Pro Ser Glu Ala His Ala His Val Glu Leu Gln Met Leu Pro Phe His Glu Leu 185 l9G 195 Asn Asp Asn Ser Pro Thr Tyr Val Thr Pro Val Leu Arg Val Phe Pro t~

Pro Thr Glu His Val Lys Phe Asn Val Thr Tyr Ser Trp Tyr Gly Phe CA 022l6l39 l997-09-22 Asp Val Lys Glu Glu Cys Glu Glu Val Lys Leu Phe Glu Pro Cys Val Tyr His Pro Thr Asp Gly Lys Cys Gln Phe Pro Ala Thr Asn Gln Arg Cys Leu Ile Gly Ser Val Leu Met Ala Glu Phe Leu Gly Ala Ala Ser Leu Leu Asp Cys Ser Arg Asp Thr Leu Glu Asp Cys His Glu Asn Arg Val Pro Asn Leu Arg Phe Asp Ser Arg Leu Ser Glu Ser Arg Ala Gly Leu Val Ile Ser Pro Leu Ile Ala Ile Pro Lys Val Leu Ile Ile Val Val Ser Asp Gly Asp Ile Leu Gly Trp Ser Tyr Thr Val Leu Gly Lys Arg Asn Ser Pro Arg Val Val Val Glu Thr His Met Pro Ser Lys Val Pro Met Asn Lys Val Val Ile Gly Ser Pro Gly Pro Met Asp Glu Thr GGT AAC TAT AAA ATG TAC TTC GTC GTC GCG GGG GTG GCC GCG ACG TG~ 12325 Gly Asn Tyr Lys Met Tyr Phe Val Val Ala Gly Val Ala Ala Thr C~s GTA ATT CTT ACA TGC GrT CTG CTT GTG GGG AAA AAG AAG TGC CCC GCG 12373 Val Ile Leu Thr Cys Ala Leu Leu Val Gly Lys Lys Lys Cys Pro Ala CAC CAA ATG GGT A_T TTT TC_ AAG ACC GAA CCA TTG TAC GCG CCG CTC 12421 His Gln Met Gly Thr Phe Ser Lys Thr Glu Pro Leu Tyr Ala Pro Leu Pro Lys Asn Glu Phe Glu Ala Gly Gly Leu Thr Asp Asp Glu Glu Val ATT TAT GAC GAA GTA SAC.GAA CCC CTA TTT CGC GGC TAC TGT AAG CAG 12517 Ile Tyr Asp Glu Val Tyr Glu Pro Leu Phe Arg Gly Tyr Cys Lys Gln Glu Phe Arg Glu Asp Val Asn Thr Phe Phe Gly Ala Val Val Glu G~y ~ 455 460 465 Glu Arg Ala Leu Asn Phe Lys Ser Ala Ile Ala Ser Met Ala Asp Arg Ile Leu Ala Asn Lys Ser Gly Arg Arg Asn Met Asp Ser Tyr W 096/29396 PCTrUS96/03916 Met Pro Phe Lys Thr Arg Gly Ala Glu Asp Ala Ala Ala Gly Lys Asn Arg Phe Lys Lys Ser Arg Asn Arg Glu Ile Leu Pro Thr Arg Leu Arg Gly Thr Gly Lys Lys Thr Ala Gly Leu Ser Asn Tyr Thr Gln Pro Ile Pro Trp Asn Pro Lys Phe Cys Ser Ala Arg Gly Glu Ser Asp Asn His Ala Cys Lys Asp Thr Phe Tyr Arg Arg Thr Cys Cys Ala Ser Arg Ser Thr Val Ser Ser Gln Pro Asp Ser Pro His Thr Pro Met Pro Thr Glu Tyr Gly Arg Val Pro Ser Ala Lys Arg Lys Lys Leu Ser Ser Ser Asp Xaa Glu Gly Ala His Gln Pro Leu Val Ser Cys Lys Leu Pro Asp Ser Gln Ala Ala Pro Ala Arg Thr Tyr Ser Ser Ala Gln Arg Tyr Thr Val Asp Glu Val Ser Ser Pro Thr Pro Pro Gly Val Asp Ala Val Ala ~sp Leu Glu Thr Arg Ala Glu Leu Pro Gly Ala Thr Thr Glu Gln Thr Glu Ser Lys Asn Lys Leu Pro Asn Gln Gln Ser Arg Leu Lys Pro Lys Pro Thr Asn Glu ~is Val Gly Gly Glu Arg Cys Pro Ser Glu Gly Thr Val Glu Ala Pro Ser Leu Gly Ile Leu Ser Arg Val Gly Ala Ala Ile Ala Asn Glu Leu Ala Arg Met Arg Arg Ala Cys Leu Pro Leu Ala Ala Ser Ala Ala Ala Ala Gly Ile Val Ala Trp Ala 235 240 245 2S0 t Ala Ala Arg Ala Leu Gln Lys Gln Gly Arg A

CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 ~2) INFORMATION FOR SEQ ID NO:2:
( i ) ~ U~N~ CH~RACTERISTICS:
~A) LENGTH: 476 amino acids B) TYPE: amino acid D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:2:
Met Arg Phe Arg Arg Ile Cys Ser Arg Ser Arg Ala Glu Lys Arg Arg Arg Thr Thr Glu Asn Pro Leu Thr Ser Lys Arg Val Cys Val Leu Asp Ser Phe Ser Arg Thr Met Ser Leu Arg Pro Tyr Ala Glu Ile Leu Pro Thr Ala Glu Gly Val Glu Arg Leu Ala Glu Leu Val Ser Val Thr Met Thr Glu Arg Ala Glu Pro Val Thr Glu Asn Thr Ala Val Asn Ser Ile ~ro Pro Ala Asn Glu Asn Gly Gln Asn Phe Ala Tyr Ala Gly Asp Gly ~ro Ser.Thr Thr Glu Lys Val Asp Gly Ser His Thr Asp Phe Asp Glu Ala Ser Ser Asp Tyr Ala Gly Pro Val Pro Leu Ala Gln Thr Arg Leu Lys His Ser Asp Glu Phe Leu Gln His Phe Arg Val Leu Asp Asp Leu Val Glu Gly Ala Tyr Gly Phe Ile Cys Gly Val Arg Arg Tyr Thr Glu ~lu Glu Gln Arg Arg Arg Gly Val Asn Ser Thr Asn Gln Gly Lys Ser ~ys Cys Lys Arg Leu Ile Ala Lys Tyr Val Lys Asn Gly Thr Arg Ala Ala Ser Gln Leu Glu Asn Glu Ile Leu Val Leu Gly Arg Leu Asn His Glu Asn Val Leu Lys Ile Gln Glu Ile Leu Arg Tyr Pro Asp Asn Thr Tyr Met Leu Thr Gln Arg Tyr Gln Phe Asp Leu Tyr Ser Tyr Met Tyr ~sp Glu Ala Phe Asp Trp Lys Asp Ser Pro Met Leu Lys Gln Thr Arg ~r~ Ile Met Lys Gln Leu Met Ser Ala Val Ser Tyr Ile His Ser Lys W 096/29396 PCTrUS96/03916 Lys Leu Ile His Arg Asp Ile Lys Leu Glu Asn Ile Phe Leu Asn Cys Asp Gly Lys Thr Val Leu Gly Asp Phe Gly Thr Val Thr Pro Phe Glu Asn Glu Arg Glu Pro Phe Glu Tyr Gly Trp Val Gly Thr Val Ala Thr ~sn Ser Pro Glu Ile Leu Ala Arg Asp Ser Tyr Cys Glu Ile Thr Asp ~le Trp Ser Cys Gly Val Val Leu Leu Glu Met Val Ser His Glu Phe Cys Pro Ile Gly Asp Gly Gly Gly Asn Pro His Gln Gln Leu Leu Lys Val Ile Asp Ser Leu Ser Val Cys Asp Glu.Glu Phe Pro Asp Pro Pro Cys Asn Leu Tyr Asn Tyr Leu His Tyr Ala Ser Ile Asp Arg Ala Gly ~is Thr Val Pro Ser Leu Ile Arg Asn Leu His Leu Pro Ala Asp Val ~lu Tyr Pro Leu Val Lys Met Leu Thr Phe Asp Trp Arg Leu Arg Pro Ser Ala Ala Glu Val Leu Ala Met Pro Leu Phe Ser Ala Glu Glu Glu Arg Thr Ile Thr Ile Ile His Gly Lys His Lys Pro Ile Arg Pro Glu Ile Arg Ala Arg Val Pro Arg Ser Met Ser Glu Gly (2) INFORMATION FOR SEQ ID NO:3:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTP,: 510 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) ~yU~N~: DESCRIPTION: SEQ ID NO:3:
Met Thr Leu Pro His Arg Leu Thr Lys Arg Pro Phe Ala Arg Arg Phe ~ys Ser Val Phe Val Ile His Tyr Ser Glu Thr Lys Leu Asp Arg Tyr Asn Lys Thr Met Leu Leu Tyr Arg Pro Asp Ser Thr Met Arg His Ser Gly Gly Asp Ala Asn His Arg Gly Ile Arg Pro Arg Arg Lys Ser Ile Gly Ala Phe Ser Ala Arg Glu Lys Thr Gly Lys Arg Asn Ala Leu Thr W 096/29396 PCTrUS96/03916 Glu Ser Ser Ser Ser Ser Asp Met Leu Asp Pro Phe Ser Thr Asp Lys Glu Phe Gly Gly Lys Trp Thr Val Asp Gly Pro Ala Asp Ile Thr Ala Glu Val Leu Ser Gln Ala Trp Asp Val Leu Gln Leu Val Lys His Glu Asp Ala Glu Glu Glu Arg Val Thr Tyr Glu Ser Lys Pro Thr Pro Ile Gln Pro Phe Asn Ala Trp Pro Asp Gly Pro Ser Trp Asn Ala Gln Asp Phe Thr Arg Ala Pro Ile Val Tyr Pro Ser Ala Glu Val Leu Asp Ala Glu Ala Leu Lys Val Gly Ala Phe Val Ser Arg Val Leu Gln Cys Val Pro Phe Thr Arg Ser Lys Lys Ser Val Thr Val Arg Asp Ala Gln Ser Phe Leu Gly Asp Ser Phe Trp Arg Ile Met Gln Asn Val Tyr Thr Val Cys Leu Arg Gln ~is Ile Thr Arg Leu Arg His Pro Ser Ser Lys Ser Ile Val Asn Cys Asn Asp Pro Leu Trp Tyr Ala Tyr Ala Asn Gln Phe His Trp Arg Gly Met Arg Val Pro Ser Leu Lys Leu Ala Ser Pro Pro Glu Glu Asn Ile Gln His Gly Pro Met Ala Ala Val Phe Arg Asn Ala Gly Ala Gly Leu Phe Leu Trp Pro Ala Met ~rg Ala Ala Phe Glu Glu 290 25~ 300 Arg Asp Lys Arg Leu Leu Arg Aia Cys Leu Ser Ser Leu Asp Ile Met Asp Ala Ala Val Leu Ala Ser Phe Pro Phe Tyr Trp Arg Gly Val Gln Asp Thr Ser Arg Phe Glu Pro Ala Leu Gly Cys Leu Ser Glu Tyr Phe Ala Leu Val Val Leu Leu Ala Glu Thr Val Leu Ala Thr Met Phe Asp ~is Ala Leu Val Phe Met Arg Ala Leu Ala Asp Gly Asn Phe Asp Asp Tyr Asp Glu Thr Arg Tyr Ile Asp Pro Val Lys Asn Glu Tyr Leu Asn 385 390 ~95 400 Gly Ala Glu Gly Thr Leu Leu Arg Gly Ile Val Ala Ser Asn Thr Ala . 405 410 415 Leu Ala Val Val Cys Ala Asn Thr Tyr Ser Thr Ile Arg Lys Leu Pro CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 ~ -106-Ser Val Ala Thr Ser Ala Cys Asn Val Ala Tyr Arg Thr Glu Thr Leu Lys Ala Arg Arg Pro Gly Met Ser Asp Ile Tyr Arg Ile Leu Gln Lys Glu Phe Phe Phe Tyr Ile Ala Trp Leu Gln Arg Val Ala Thr His Ala Asn Phe Cys Leu Asn Ile Leu Lys Arg Ser Val Asp Thr Gly Pro Arg His Phe Cys Ser Gly Pro Ala Arg Arg Ser Gly Cys Ser Ser (2) INFORMATION FOR SEQ ID NO:4:
(i) ~U~N~ CHARACTERISTICS:
A) LENGTH: 110 amino acids B) TYPE: amino acid ,D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) S~yU~N~: DESCRIPTION: SEQ ID NO:4:
Met Leu Cys Pro Leu Leu Val Pro Ile Gln Tyr Glu Asp Phe Ser Lys ~la Met Gly Ser Glu Leu Lys Arg Glu Lys Leu Glu Thr Phe Val Lys Ala Ile Ser Ser Asp Arg Asp Pro Arg Gly Ser Leu Arg Phe Leu Ile Ser Asp His Ala Arg Glu Ile Ile Ala Asp Gly Val Arg Phe Lys Pro Val Ile Asp Glu Pro Val Arg Ala Ser Val.Ala Leu Ser Thr Ala Ala Ala Gly Lys Val Lys Ala Arg Arg Leu Thr Ser Val Arg Ala Pro Val Pro Pro Ala Gly Ala Val Ser Ala Arg Arg Lyc Ser Glu Ile (2) INFORMATION FOR SEQ ID NO:5:
(i) S~u~: CHARACTERISTICS:
A) LENGTH: 292 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~yu~N~-~ DESCRIPTION: SEQ ID NO:5:
Met Ser Gly Phe Ser Asn Ile Gly Ser Ile Ala Thr Val Ser Leu Val Cys Ser Leu Leu Cys Ala Ser Val Leu Gly Ala Pro Val Leu Asp Gly CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 Leu Glu Ser Ser Pro Phe Pro Phe Gly Gly Lys Ile Ile Ala Gln Ala Cys Asn Arg Thr Thr Ile Glu Val Thr Val Pro Trp Ser Asp Tyr Ser Gly Arg Thr Glu Gly Val Ser Val Glu Val Lys Trp Phe Tyr Gly Asn ~er Asn Pro Glu Ser Phe Val Phe Gly Val Asp Ser Glu Thr Gly Ser ~ly His Glu Asp Leu Ser Thr Cys Trp Ala Leu Ile His Asn Leu Asn Ala Ser Val Cys Arg Ala Ser Asp Ala Gly Ile Pro Asp Phe Asp Lys Gln Cys Glu Lys Val Gln Arg Arg Leu Arg Ser Gly Val Glu Leu Gly Ser Tyr Val Ser Gly Asn Gly Ser Leu Val Leu Tyr Pro Gly Met Tyr 14S 150 lS5 160 ~sp Ala Gly Ile Tyr A;a Tyr Gln Leu Ser Val Gly Gly Lys Gly Tyr ~hr Gly Ser Val Tyr Leu Asp Val Gly Pro Asn Pro Gly Cys His Asp Gln Tyr Gly Tyr Thr Tyr Tyr Ser Leu Ala Asp Glu Ala Ser Asp Leu Ser Ser Tyr Asp Val Ala Ser Pro Glu Leu Asp Gly Pro Met Glu Glu Asp Tyr Ser Asn Cys Leu Asp Met Pro Pro Leu Arg Pro Trp Thr Thr ~al Cys Ser His Asp Val Glu Glu Gln Glu Asn Ala Thr Asp Glu Leu ~yr Leu Trp Asp Glu Glu Cys Ala Gly Pro Leu Asp Glu Tyr Val Asp ~lu Arg Ser Glu Thr Me; Pro Arg Met Val Val Phe Ser Pro Pro Ser Thr Leu Gln Gln ~2) INFORMATION FOR SEQ ID NO:6:
i ) S~U~N~ CHARACTERISTICS:
~A) LENGTH: 955 amino acids ~B) TYPE: amino acid ,D) TOPOLOGY: linear ~ii) MOLECULE TYPE: protein (Xi) S~yU~N~ DESCRIPTION: SEQ ID NO:6:
Met Gly Thr Met Leu Val Leu Arg Leu Phe Leu Leu Ala Val Ala Asp 1 S 10 lS
.

CA 022l6l39 l997-09-22 Ala Ala Leu Pro Thr Gly Arg Phe Cys Arg Val Trp Lys Val Pro Pro ~ly Gly Thr Ile Gln Glu Asn Leu Ala Val Leu Ala Glu Ser Pro Val Thr Gly His Ala Thr Tyr Pro Pro Pro Glu Gly Ala Val Ser Phe Gln Ile Phe Ala Asp Thr Pro Thr Leu Arg Ile Arg Tyr Gly Pro Thr Glu ~sp Glu Leu Ala Leu Glu Arg Gly Thr Ser Ala Ser Asp Ala Asp Asn ~al Thr Phe Ser Leu Ser Tyr Arg Pro Arg Pro Glu Ile His Gly Ala Tyr Phe Thr Ile Gly Val Phe Ala Thr Gly Gln Ser Thr Glu Ser Ser Tyr Ser Val Ile Ser Arg Val Leu Val Asn Ala Ser Leu Glu Arg Ser Val Arg Leu Glu Thr Pro Cys Asp Glu Asn Phe Leu Gln Asn Glu Pro ~hr Trp Gly Ser Lys Arg ~rp Leu Gly Pro Pro Ser Pro Tyr Val Arg ~sp Asn Asp Val Ala Val Leu Thr Lys Ala Gln Tyr Ile Gly Glu Cys Tyr Ser Asn Ser Ala Ala Gln Thr Gly Leu Thr Ser Leu Asn Met Thr Phe Phe Tyr Ser Pro Lys Arg Ile Val Asn Val Thr Trp Thr Thr Gly Gly Pro Ser Pro Ser Arg Ile Thr Val Tyr Ser Ser Arg Glu Asn Gly ~ln Pro Val Leu Arg Asn Val Ser Asp Gly Phe Leu Val Lys Tyr Thr ~ro Asp Ile Asp Gly Arg Ala Met Ile Asn Val Ile Ala Asn Tyr Ser Pro Ala Asp Ser Gly Ser Val Leu Ala Phe Thr Ala Phe Arg Glu Gly Lys Leu Pro Ser Ala Ile Gln Leu His Arg Ile Asp Met Ser Gly Thr 290 2g5 300 Glu Pro Pro Gly Thr Glu Thr Thr Phe Asp Cys Gln Lys Met Ile Glu ~hr Pro Tyr Arg Ala Leu Gly Ser Asn Val Pro Arg Asp Asp Ser Ile ~rg Pro Gly Ala Thr Leu Pro Pro Phe Asp Thr Ala Ala Pro Asp Phe ~sp Thr Gly Thr Ser Pro Thr Pro Thr Thr Val Pro Glu Pro Ala Ile 355l 360 365 W 096/29396 PCTrUS96/039l6 Thr Thr Leu Ile Pro Arg Ser Thr Ser Asp Met Gly Phe Phe Ser Thr Ala Arg Ala Thr Gly Ser Glu Thr Leu Ser Val Pro Val Gln Glu Thr ~sp Arg Thr Leu Ser Thr Thr Pro Leu Thr Leu Pro Leu Thr Pro Gly ~lu Ser Glu Asn Thr Leu Phe Pro Thr Thr Ala Pro Gly Ile Ser Thr Glu Thr Pro Ser Ala Ala His Glu Thr Thr Gln Thr Gln Ser Ala Glu Thr Val Val Phe Thr Gln Ser Pro Ser Thr Glu Ser Glu Thr Ala Arg Ser Gln Ser Gln G1U Pro Trp Tyr Phe Thr Gln Thr Pro Ser Thr Glu ~ln Ala Ala Leu Thr Gln Thr Gln Ile Ala Glu Thr Glu Ala Leu Phe ~hr Gln Thr Pro Ser Ala Glu Gln Met Thr Phe Thr Gln Thr Pro Gly ~la Glu Thr Glu Ala Pro Ala Gln Thr Pro Ser Thr Ile Pro Glu Ile Phe Thr Gln Ser Arg Ser Thr Pro Pro Glu Thr Ala Arg Ala Pro Ser Ala Ala.Pro Glu Val Phe Thr Gln Ser Ser Ser Thr Val Thr Glu Val ~he Thr Gln Thr Pro Ser Thr Val Pro Lys Thr Thr Leu Ser Ser Ser ~hr Glu Pro Ala Ile Phe Thr Arg Thr Gln Ser Ala Gly Thr Glu Ala Phe Thr Gln Thr Ser Ser Ala Glu Pro Asp Thr Met Arg Thr Gln Ser 595 60~ 605 Thr Glu Thr His Phe Phe Thr Gln Ala Pro Ser Thr Val Pro Lys Ala Thr Gln Thr Pro Ser Thr Glu Pro Glu Val Leu Thr Gln Ser Pro Ser ~hr Glu Pro Val Pro Phe Thr Arg Thr Leu Gly Ala Glu Pro Glu Ile ~hr Gln Thr Pro Ser Ala Ala Pro Glu Val Tyr Thr Arg Ser Ser Ser ~hr Met Pro Glu Thr Ala Gln Ser Thr Pro Leu Ala Ser Gln Asn Pro Thr Ser Ser Gly Thr Gly Thr His Asn Thr Glu Pro Arg Thr Tyr Pro Val Gln Thr Thr Pro His Thr Gln Lys Leu Tyr Thr Glu Asn Lys Thr W 096/29396 PCTrUS96/03916 Leu Ser Phe Pro Thr Val Val Ser Glu Phe His Glu Met Ser Thr Ala ~lu Ser Gln Thr Pro Leu Leu Asp Val Lys Ile Val Glu Val Lys Phe Ser Asn Asp Gly Glu Val Thr Ala Thr Cys Val Ser Thr Val Lys Ser Pro Tyr Arg Val Glu Thr Asn Trp Lys Val Asp Leu Val Asp Val Met Asp Glu Ile Ser Gly Asn Ser Pro Ala Gly Val Phe Asn Ser Asn Glu ~ys Trp Gln Lys Gln Leu Tyr Tyr Arg Val Thr Asp Gly Arg Thr Ser ~al Gln Leu Met Cys Leu Ser Cys Thr Ser.His Ser Pro Glu Pro Tyr Cys Leu Phe Asp Thr Ser Leu Ile Ala Arg Glu Lys Asp Ile Ala Pro Glu Leu Tyr Phe Thr Ser Asp Pro Gln Thr Ala Tyr Cys Thr Ile Thr Leu Pro Ser Gly Val Val Pro Arg Phe Glu Trp Ser Leu Asn Asn Val ~er Leu Pro Glu Tyr Leu Thr Ala Thr Thr Val Val Ser His Thr Ala ~ly Gln Ser Thr Val Trp Lys Ser Ser Ala Arg Ala Gly Glu Ala Trp Ile Ser Gly Arg Gly Gly Asn Ile Tyr Glu Cys Thr Val Leu Ile Ser Asp Gly Thr Arg Val Thr Thr Arg Lys Glu Arg Cys Leu Thr Asn Thr Trp Ile Ala Val Glu Asn Gly Ala Ala Gln Ala Gln Leu Tyr Ser Leu Phe Ser Gly Leu Va~ Ser Gly Leu Cys Gly Ser Ile Ser Ala Leu Tyr ~la Thr Leu Trp Thr Ala Ile Tyr Phe (2) INFORMATION FOR SEQ ID NO:7:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 362 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~Uu~: DESCRIPTION: SEQ ID NO:7:
Met Ala Ser Leu Leu Gly Thr Leu Ala Leu Leu Ala Ala Thr Leu Ala CA 022l6l39 l997-09-22 W 096l29396 PCTrUS96/03916 Pro Phe Gly Ala Met Gly Ile Val Ile Thr Gly Asn His Val Ser Ala . Arg Ile Asp Asp Asp His Ile Val Ile Val Ala Pro Arg Pro Glu Ala Thr Ile Gln Leu Gln Leu Phe Phe Met Pro Gly Gln Arg Pro His Lys ' 50 55 60 Pro Tyr Ser Gly Thr Val Arg Val Ala Phe Arg Ser Asp Ile Thr Asn 7(~ 75 80 Gln Cys Tyr Gln Glu Leu Ser Glu Glu Arg Phe Glu Asn Cys Thr His Arg Ser Ser Ser Val Phe Val Gly Cys Lys Val Thr Glu Tyr Thr Phe Ser Ala Ser Asn Arg Leu Thr Gly Pro Pro His Pro Phe Lys Leu Thr Ile Arg Asn Pro Arg Pro Asn Asp Ser Gly Met Phe Tyr Val Ile Val Arg Leu Asp Asp Thr Lys Glu Pro Ile Asp Val Phe Ala Ile Gln Leu Ser Val Tyr Gln Phe Ala Asn Thr Ala Ala Thr Arg Gly Leu Tyr Ser Lys Ala Ser Cys Arg Thr Phe Gly Leu Pro Thr Val Gln Leu Glu Ala Tyr Leu Arg Thr Glu Glu Ser Trp Arg Asn Trp Gln Ala Tyr Val Ala Thr Glu Ala Thr Thr Thr Ser Ala Glu Ala Thr Thr Pro Thr Pro Val Thr Ala Thr Ser Ala Ser Glu Leu Glu Ala Glu His Phe Thr Phe Pro ~ Trp Leu Glu Asn Gly Val Asp His Tyr Glu Pro Thr Pro Ala Asn Glu Asn Ser Asn Val Thr Val Arg Leu Gly Thr Met Ser Pro Thr Leu Ile Gly Val Thr Val Ala Ala Val Val Ser Ala Thr Ile Gly Leu Val Ile Val Ile Ser Ile Val Thr Arg Asn Met Cys Thr Pro His Arg Lys Leu Asp Thr Val Ser Gln Asp Asp Glu Glu Arg Ser Gln Thr Arg Arg Glu Ser Arg Lys Phe Gly Pro Met Val Ala Cys Glu Ile Asn Lys Gly Ala Asp Gln Asp Ser Glu Leu Val Glu Leu Val Ala Ile Val Asn Pro Ser r . 340 345 350 Ala Leu Ser Ser Pro Asp Ser Ile Lys Met W 096/29396 PCTrUS96/03916 (2) lN~OR-I~TION FOR SEQ ID NO:8:
yU N~ ; CHARACTERISTICS:
(A) LENGTH: 499 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) MOLE~:u~E TYPE: protein -(xi) ~;yu~ ; DESCRIPTION: SEQ ID NO:8:
~et Asn Met Leu Val Ile Val Leu Ala Ser Cys Leu Ala Arg Leu Thr ~he Ala Thr Arg His Val Leu Phe Leu Glu Gly Thr Gln Ala Val Leu ~ly Glu Asp Asp Pro Arg Asn Val Pro Glu Gly Thr Val Ile Lys Trp Thr Lys Val Leu Arg Asn Ala Cys Lys Met Lys Ala Ala Asp Val Cys Ser Ser Pro Asn Tyr Cvs Phe His Asp Leu Ile Tyr Asp Gly Gly Lys ;0 75 80 ~ys Asp Cys Pro Pro Ala Gly Pro Leu Ser Ala Asn Leu Val Ile Leu ~eu Lys Arg Gly Glu Ser ~he Val Val Leu Gly Ser Gly Leu His Asn Ser Asn Ile Thr Asn Ile Met Trp Thr Glu Tyr Gly Gly Leu Leu Phe Asp Pro Val Thr Arg Ser Asp Glu Gly Ile Tyr Phe Arg Arg Ile Ser 130 . 135 140 Gln Pro Asp Leu Ala Met Glu Thr Thr Ser Tyr Asn Val Ser Val Leu lg5 150 ~ 155 160 ~er His Val Asp Glu Lys Ala Pro Ala Pro His Glu Val Glu Ile Asp ~hr Ile Lys Pro Ser Glu Ala His Ala His Val Glu Leu Gln Met Leu Pro Phe His Glu Leu Asn Asp Asn Ser Pro Thr Tyr Val Thr Pro Val Leu Arg Val Phe Pro Pro Thr Glu His Val Lys Phe Asn Val Thr Tyr Ser Trp Tyr Gly Phe Asp Val Lys Glu Glu Cys Glu Glu Val Lys Leu ~he Glu Pro Cys Val Tyr His Pro Thr Asp Gly Lys Cys Gln Phe Pro ~la Thr Asn Gln Arg Cys Leu Ile Gly Ser Val Leu Met Ala Glu Phe Leu Gly Ala Ala Ser Leu Leu Asp Cys Ser Arg Asp Thr Leu Glu Asp Cys His Glu Asn Arg Val Pro Asn Leu Arg Phe Asp Ser Arg Leu Ser CA 022l6l39 l997-09-22 W 096/29396 PCT~US96/03916 -l13-Glu Ser Arg Ala Gly Leu Val Ile Ser Pro Leu Ile Ala Ile Pro Lys Val Leu Ile Ile Val Val Ser Asp Gly Asp Ile Leu Gly Trp Ser Tyr Thr Val Leu Gly Lys Arg Asn Ser Pro Arg Val Val Val Glu Thr His Met Pro Ser Lys Val Pro Met Asn Lys Val Val Ile Gly Ser Pro Gly Pro Met Asp Glu Thr Gly Asn Tyr Lys Met Tyr Phe Val Val Ala Gly ~ 370 375 380 Val Ala Ala Thr Cys Val Ile Leu Thr Cys Ala Leu Leu Val Gly Lys Lys Lys Cys Pro Ala His Gln Met Gly Thr Phe Ser Lys Thr Glu Pro Leu Tyr Ala Pro Leu Pro Lys Asn Glu Phe Glu Ala Gly Gly Leu Thr Asp Asp Glu Glu Val Ile Tyr Asp ~lu Val Tyr Glu Pro Leu Phe Arg Gly Tyr Cys Lys Gln Glu Phe Arg Glu Asp Val Asn Thr Phe Phe Gly Ala Val Val Glu Gly Glu Arg Ala Leu Asn Phe Lys Ser Ala Ile Ala Ser Met Ala Asp Arg Ile Leu Ala Asn Lys Ser Gly Arg Arg Asn Met Asp Ser Tyr ~2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 amlno acids (B) ~YPE: amino acld (D) TOP3LOGY: line~r (ii) MOLECULE TYPE: proteln (xi) S~U~N~- DESCRIPTION: SEQ ID NO:9:
Met Pro Phe Lys Thr Arg Gly Ala Glu Asp Ala Ala Ala Gly Lys Asn 1 5 . 10 15 Arg Phe Lys Lys Ser Arg Asn Arg Glu Ile Leu Pro Thr Arg Leu Arg Gly Thr Gly Lys Lys Thr Ala Gly Leu Ser Asn Tyr Thr Gln Pro Ile Pro Trp Asn Pro Lys Phe Cys Ser Ala Arg Gly Glu Ser Asp Asn His . 60 Ala Cys Lys Asp Thr Phe Tyr Arg Arg Thr Cys Cys Ala Ser Arg Ser CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 Thr Val Ser Ser Gln Pro Asp Ser Pro His Thr Pro Met Pro Thr Glu ~yr Gly Arg Val Pro Ser Ala Lys Arg Lys Lys Leu Ser Ser Ser Asp Xaa Glu Gly Ala His Gln Pro Leu Val Ser Cys Lys Leu Pro Asp Ser Gln Ala Ala Pro Ala Arg Thr Tyr Ser Ser Ala Gln Arg Tyr Thr Val Asp Glu Val Ser Ser Pro Thr Pro Pro Gly Val Asp Ala Val Ala Asp ~eu Glu Thr Arg Ala Glu Leu Pro Gly Ala Thr Thr Glu Gln Thr Glu ~er Lys Asn Lys Leu Pro Asn Gln Gln Ser Arg Leu Lys Pro Lys Pro Thr Asn Glu His Val Gly Gly Glu Arg Cys Pro Ser Glu Gly Thr Val Glu Ala Pro Ser Leu Gly Ile Leu Ser Arg Val Gly Ala Ala Ile Ala Asn Glu Leu Ala Arg Met Arg Arg Ala Cys Leu Pro Leu Ala Ala Ser Ala Ala Ala Ala Gly Ile Val Ala Trp Ala Ala Ala Arg Ala Leu Gln ~ys Gln Gly Arg ~2) INFORMATION FOR SEQ ID NO:10:
(i) S~Qu~N~: CHARACTERISTICS:
(A) LENGTH: 1305 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
) ANTI-SENSE: NO

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1305 (xi) ~u~N~ DESCRIPTION: SEQ ID NO:10:

ATG CAC CGT CCT CAT CTC AGA CGG CAC TCG CGT TAC TAC GCG A~A GGA 48 Met His Arg Pro His Leu Arg Arg His Ser Arg Tyr Tyr Ala Lys Gly GAG GTG CTT AAC A~A CAC ATG GAT TGC GGT GGA AAA CGG TGC TGC TCA 96 Glu Val Leu Asn Lys His Met Asp Cys Gly Gly Lys Arg Cys Cys Ser W 096/29396 PCTrUS96/03916 GGC GCA GCT GTA TTC ACT CTT TTC TGG ACT TGT GTC AGG ATT ATG CGG 144Gly Ala Ala Val Phe Thr Leu Phe Trp Thr Cys Val Arg Ile Met Arg Glu His Ile Cys Phe Val Arg Asn Ala Met Asp Arg His Leu Phe Leu Arg Asn Ala Phe Trp Thr Ile Val Leu Leu Ser Ser Phe Ala Ser Gln Ser Thr Ala Ala Val Thr Tyr Asp Tyr Ile Leu Gly Arg Arg Ala Leu Asp Ala Leu Thr Ile Pro Ala Val Gly Pro Tyr Asn Arg Tyr Leu Thr Arg Val Ser Arg Gly Cys Asp Val Val Glu Leu Asn Pro Ile Ser Asn Val Asp Asp Met Ile Ser Ala Ala Lys Glu Lys Glu Lys Gly Gly Pro Phe Glu Ala Ser Val Val Trp Phe Tyr Val Ile Lys Gly Asp Asp Gly Glu Asp Lys Tyr Cys Pro Ile Tyr Arg Lys Glu Tyr Arg Glu Cys Gly Asp Val Gln Leu Leu Ser Glu Cys Ala Val Gln Ser Ala Gln Met Trp Ala Val Asp Tyr Val Pro Ser Tkr Leu Val Ser Arg Asn Gly Ala Gly Leu Thr Ile Phe Ser Pro Thr Ala Ala Leu Ser Gly Gln Tyr Leu Leu Thr Leu Lys Ile Gly Arg Phe Ala Gln Thr Ala Leu Val Thr Leu Glu Val Asn Asp Arg Cys Leu Lys Ile Gly Ser Gln Leu Asn Phe Leu Pro Ser Lys Cys Trp Thr Thr Glu Gln Tyr Gln Thr Gly Phe Gln Gly Glu His Leu Tyr Pro Ile Ala Asp Thr Asn Thr Arg His Ala Asp Asp Val Tyr Arg Gly Tyr Glu Asp Ile Leu Gln Arg Trp Asn Asn Leu Leu Arg CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 -ll6-Lys Lys Asn Pro Ser Ala Pro Asp Pro Arg Pro Asp Ser Val Pro Gln Glu Ile Pro Ala Val Thr Lys Lys Ala Glu Gly Arg Thr Pro Asp Ala Glu Ser Ser Glu Lys Lys Ala Pro Pro Glu Asp Ser Glu Asp Asp Met ' Gln Ala Glu Ala Ser Gly Glu Asn Pro Ala Ala Leu Pro Glu Asp Asp ~ 355 360 365 Glu Val Pro Glu Asp Thr Glu His Asp Asp Pro Asn Ser Asp Pro Asp Tyr Tyr Asn Asp Met Pro Ala Val Ile Pro Val Glu Glu Thr Thr Lys Ser Ser Asn Ala Val Ser Met Pro Ile Phe Ala Ala Phe Val Ala Cys Ala Val Ala Leu Val Gly Leu Leu Val Trp Ser Ile Val Lys Cys Ala Arg Ser (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 434 amino acids B) TYPE: a~ino acid ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) S~UU~N~ DESCRIPTION: SEQ ID NO:11:
Met His Arg Pro His Leu Arg Arg His Ser Arg Tyr Tyr Ala Lys Gly Glu Val Leu Asn Lys His Met Asp Cys Gly Gly Lys Arg Cys Cys Ser Gly Ala Ala Val Phe Thr Leu Phe Trp Thr Cys Val Arg Ile Met Arg Glu His Ile Cys Phe Val Arg Asn Ala Met Asp Arg His Leu Phe Leu Arg Asn Ala Phe Trp Thr Ile Val Leu Leu Ser Ser Phe Ala Ser Gln Ser Thr Ala Ala Val Thr Tyr Asp Tyr Ile Leu Gly Arg Arg Ala Leu W 096/29396 PCTrUS96/03916 ~ Asp Ala Leu Thr Ile Pro Ala Val Gly Pro Tyr Asn Arg Tyr Leu Thr - Arg Val Ser Arg Gly Cys Asp Val Val Glu Leu Asn Pro Ile Ser Asn Val Asp Asp Met Ile Ser Ala Ala Lys Glu Lys Glu Lys Gly Gly Pro Phe Glu Ala Ser Val Val Trp Phe Tyr Val Ile Lys Gly Asp Asp Gly Glu Asp Lys Tyr Cys Pro Ile Tyr Arg Lys Glu Tyr Arg Glu Cys Gly Asp Val Gln Leu Leu Ser Glu Cys Ala Val Gln Ser Ala Gln Met Trp Ala Val Asp Tyr Val Pro Ser Thr Leu Val Ser Arg Asn Gly Ala Gly Leu Thr Ile Phe Ser Pro Thr Ala Ala Leu Ser Gly Gln Tyr Leu Leu Thr Leu Lys Ile Gly Arg Phe Ala Gln Thr Ala Leu Val Thr Leu Glu Val Asn Asp Arg Cys Leu Lys Ile Gly Ser Gln Leu Asn Phe Leu Pro Ser Lys Cys Trp Thr Thr Glu Gln Tyr Gln Thr Gly Phe Gln Gly Glu His Leu Tyr Pro Ile Ala Asp Thr Asn Thr Arg His Ala Asp Asp Val Tyr Arg Gly Tyr Glu Asp Ile Leu Gln Arg Trp Asn Asn Leu Leu Arg Lys Lys Asn Pro Ser Ala Pro Asp Pro Arg Pro Asp Ser Val Pro Gln Glu Ile Pro Ala Val Thr Lys Lys Ala Glu Gly Arg Thr Pro Asp Ala Glu Ser Ser Glu Lys Lys Ala Pro Pro Glu Asp Ser Glu Asp Asp Met Gln Ala Glu Ala Ser Gly Glu Asn Pro Ala Ala Leu Pro Glu Asp Asp Glu Val Pro Glu Asp Thr Glu His Asp Asp Pro Asn Ser Asp Pro Asp Tyr Tyr Asn Asp Met Pro Ala Val Ile Pro Val Glu Glu Thr Thr Lys Ser Ser Asn Ala Val Ser Met Pro Ile Phe Ala Ala Phe Val Ala Cys Ala Val Ala Leu Val Gly Leu Leu Val Trp Ser Ile Val Lys Cys Ala ~,. 420 425 430 Arg Ser W 096/29396 PCTrUS96/03916 (2) INFORMATION FOR SEQ ID NO:12:
(i) ~yu~N~ CHARACTERISTICS:
(Al LENGTH: 690 base pairs (B~ TYPE: nucleic acid (C~ STRANDEDNESS: single (D,~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~OL~llCAL: NO
(iv) ANTI-SENSE: NO

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..689 (xi~ yu~ DESCPIPTION: SEQ ID NO:12:
ATG GCG CCT GTA A~A 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 AAA CTA CCT AAT TCT AGA TTT GAG CTC TCG GAT TCT GGA TGG A~A GAA 96 Lys Leu Pro Asn Ser Arg Phe Glu Leu Ser Asp Ser Gly Trp Lys Glu Leu Val His Ala Val Lys Thr Met Ala Ser Tyr Asp Arg Pro Ser Thr Leu Ser Val Ile Val Arg Pro Ala Ser Leu Tyr Glu Val Ser Gly Glu Leu Phe Ser Leu Pro Arg Met Cys Arg Pro Val Ile Arg Phe Gly Glu Gly Gly Asp Pro Pro Gly Val Ser Pro Glu Trp Ser Gly Leu Asp Ala GGG TTT TAC CAT TTG TCA TCT GGC GCG TAT GCC GCA A~A GAG TTC CAT 336 Gly Phe Tyr His Leu Ser Ser Gly Ala Tyr Ala Ala Lys Glu Phe His Leu Trp Val Leu Gly Thr Ala Asp Ile Cys Met Ala Ala Leu Asn Leu CCT GCG CCA AAA ACT TTC CTA ATT ACC GAA ACC GGA GGT A~A AAT TTT 432 Pro Ala Pro Lys Thr Phe Leu Ile Thr Glu Thr Gly Gly Lys Asn Phe Glu Arg Gly Val Glu Ile Phe Leu Val Asn Gly Asp Lys Thr Thr Leu Ser Leu Ser His Pro Ser Val Trp Thr Thr Leu Ala Pro Ser Ser Leu CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 Arg Thr Pro Trp Pro Tyr Ser Thr Val Lys Phe Leu Lys Val Lys Pro Asn Ser Ala Ala Tyr Cys Val Ser Asp Ser Asp Asp Gly Glu Arg Gln Pro Lys Phe Phe Leu Gly Ser Leu Phe Lys Ser Lys Lys Pro Arg Ser Pro Arg Arg Arg Arg ~2) INFORMATION FOR SEQ ID NO:13:
(i) ~u~N~: CHARACTERISTICS:
~A) LENGTH: 229 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:13:
Met Ala Pro Val Lys Val Thr Ile Val Ser Ala Val Asp Ser His Tyr Lys Leu Pro Asn Ser Arg Phe Glu Leu Ser Asp Ser Gly Trp Lys Glu Leu Val His Ala Val Lys Thr Met Ala Ser Tyr Asp Arg Pro Ser Thr Leu Ser Val Ile Val Arg Pro Ala Ser Leu Tyr Glu Val Ser Gly Glu Leu Phe Ser Leu Pro Arg Met Cys Arg Pro Val Ile Arg Phe Gly Glu Gly Gly Asp Pro Pro Gly Val Ser Pro Glu Trp Ser Gly Leu Asp Ala Gly Phe Tyr His Leu Ser Ser Gly Ala Tyr Ala Ala Lys Glu Phe His Leu Trp Val Leu Gly Thr Ala Asp Ile Cys Met Ala Ala Leu Asn Leu 115 120 ~25 Pro Ala Pro Lys Thr Phe Leu Ile Thr Glu Thr Gly Gly Lys Asn Phe Glu Arg Gly Val Glu Ile Phe Leu Val Asn Gly Asp Lys Thr Thr Leu Ser Leu Ser His Pro Ser Val Trp Thr Thr Leu Ala Pro Ser Ser Leu Arg Thr Pro Trp Pro Tyr Ser Thr Val Lys Phe Leu Lys Val Lys Pro ~ 180 185 190 Asn Ser Ala Ala Tyr Cys Val Ser Asp Ser Asp Asp Gly Glu Arg Gln W 096/29396 PCTrUS96/03916 Pro Lys Phe Phe Leu Gly Ser Leu Phe Lys Ser Lys Lys Pro Arg Ser Pro Arg Arg Arg Arg (2) INFORMATION FOR SEQ ID NO:14:
( i ) ~U~N~ CHARACTERISTICS:
~A) LENGTH: 381 base pairs ~B) TYPE: nucleic acid ~C) STRA~ c~: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..380 (xi) SEQU NCE DESCRIPTION: SEQ ID NO:14:

Met Arg Ser Ser Val Thr Ser Leu Trp Ser Pro Ser Asp His Ala Ser TCG CCC GCA AAT GCC .~AG CAT TTT TAT CAT ATT TCC GAT TTC CGG CGC 96 Ser Pro Ala Asn Ala Lys His Phe Tyr His Ile Ser ~sp Phe Arg Arg Ala Glu Thr Ala Pro Ala Gly Gly Thr Gly Ala Arg Thr Glu Val Lys Arg Arg Ala Phe Thr Phe Pro Ala Ala Ala Val Leu Ser Ala Thr Glu Ala Arg Thr Gly Ser Ser Ile Thr Gly Leu Asn Arg Thr Prc Ser Ala Ile Ile Ser Leu Ala Trp Ser Glu Met Arg Asn Leu Lys Asp Pro Leu GGG TCC CTG TCG CTG GAA ATA GCT TTA ACG A~T GTC TCT AAC TTT TCC 336 Gly Ser Leu Ser Leu Glu Ile Ala Leu Thr Asn Val Ser Asn Phe Ser Leu Leu Ser Ser Asp Pro Met Ala Phe Glu Lys Ser Ser Tyr ..
(2) INFORMATION FOR SEQ ID NO:15:
(i) ~:uu~ CHARACTERISTICS:
(A) LENGTH: 126 amino acids CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96103916 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) S~YU~N~ DESCRIPTION: SEQ ID NO:15:
Met Arg Ser Ser Val Thr Ser Leu Trp Ser Pro Ser Asp His Ala Ser ~er Pro Ala Asn Ala Lys His Phe Tyr His Ile Ser Asp Phe Arg Arg Ala Glu Thr Ala Pro Ala Gly Gly Thr Gly Ala Arg Thr Glu Val Lys Arg Arg Ala Phe Thr Phe Pro Ala Ala Ala Val Leu Ser Ala Thr Glu Ala Arg Thr Gly Ser Ser Ile Thr Gly Leu Asn Arg Thr Pro Ser Ala ~le Ile Ser Leu Ala Trp Ser Glu Met Arg Asn Leu Lys Asp Pro Leu ~ly Ser Leu Ser Leu Glu Ile Ala Leu Thr Asn Val Ser Asn Phe Ser ~eu Leu Ser Ser Asp Pro Met Ala Phe Glu Lys Ser Ser Tyr (2) INFORMATION FOR SEQ ID NO:16:
U~N~ CHARACTERISTICS:
~A~ LENGTH: 879 base pairs ~B~ TYPE: nucleic acid C~ STRANDEDNESS: single ,D,~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE- NO

(ix) FEATURE:
(A) NAME/~EY: CDS
(B) LOCATION: 1..878 (xi) S~yu~ DESCRIPTION: SEQ ID NO:16:

Met Trp Cys Arg Leu His Trp Ile Ser Pro Arg Phe Ser Ile Met Arg Pro Gly Ser Arg Thr Gly Arg Val Leu Arg Gly Gln Gly Cys Ala Leu Cys Ser Phe Trp His Arg Thr Arg Thr Pro Ser Ile Asn Leu Arg Cys CA 022l6l39 l997-09-22 Arg Ala Arg Gly Leu Ser Asn Phe Arg Leu Cys Ala Gln Ser Pro Gly Glu Arg His Arg Phe Gly Thr Arg Thr Leu Ser Gln ~is Leu Arg Leu 65 70 7s 80 Cys Thr Arg Ser Leu Ser Ser Phe Arg Tyr Arg Thr Arg Gly Leu Ser Glu Lys Val Cys Phe Ser Thr Leu Ser Ser His Ser Val Arg Leu Gly Thr Arg Ser Leu Ser Lys Gly Leu Ser Ser Arg Ala Leu Ser Pro Ser Lys Asn Arg Arg Phe Ser Thr Arg Thr Gln Ser Ser Phe Arg Tyr Arg Ala Arg Gly Leu Ser Lys His Leu Arg Tyr Arg Thr Arg Thr Leu Cys Lys Asn Leu Arg Arg Arg Ala Arg Ser Ala Ser Gly Phe Gly Gly Arg Ala Thr Arg Leu Ser Lys Tyr Leu Gly Tyr Arg Ala Arg Gly Leu Gly AGG TGC CTC GGT TTC TGC ACC CGG AGT CTG AGT A~A AGT CAT CTG TTC 624 Arg Cys Leu Gly Phe Cys Thr Arg Ser Leu Ser Lys Ser His Leu Phe 195 . 200 205 AGC ACT CGG AGT CTG AGT A~A CAA CGC CTC CGT TTC TGC GAT CTG CGT 672 Ser Thr Arg Ser Leu Ser Lys Gln Arg Leu Arg Phe Cys Asp Leu Arg Leu Ser Lys Ser Arg Leu Phe Ser Thr Arg Ser Leu Ser Lys Ile Pro Arg Phe Leu Thr Leu Gly Pro Arg Gly Phe Arg Leu Gly Thr Arg Thr Leu Ser Lys Asp His Arg Phe Cys Thr Leu Gly Leu Cys Ser Phe Met Cys Arg Ala Arg Gly Leu Gly Arg Asn Pro Arg Arg Gly Arg Arg Lys Gln Cys Ile Phe (2) INFORMATION FOR SEQ ID NO:17:

W 096/29396 PCTrUS96103916 1~
~ _ ( i ) S~;UU~;NC~!; CHARACTERISTICS:
(A~ LENGTH: 292 amino acids (8) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: protein (xi ) S~;YU~:N~; DESCRIPTION : SEQ ID NO: 17:
Met Trp Cys Arg Leu His Trp Ile Ser Pro Arg Phe Ser Ile Met Arg Pro Gly Ser Arg Thr Gly Arg Val Leu Arg Gly Gln Gly Cys Ala Leu Cys Ser Phe Trp H s Arg Thr Arg Thr Pro Ser Ile Asn Leu Arg Cys Arg Ala Arg Gly Leu Ser Asn Phe Arg Leu Cys Ala Gln Ser Pro Gly Glu Arg His Arg Phe Gly Thr Arg Thr Leu Ser Gln Eis Leu Arg Leu Cys Thr Arg Ser Leu Ser Ser Phe Arg Tyr Arg Thr Arg Gly Leu Ser Glu Lys Val Cys Phe Ser Thr Leu Ser Ser His Ser Val Arg Leu Gly Thr Arg Ser Leu Ser Lys Gly Leu Ser Ser Arg Ala Leu Ser Pro Ser Lys Asn Arg Arg Phe Ser Thr Arg Thr Gln Ser Ser Phe Arg Tyr Arg 130 ' 35 140 Ala Arg Gly Leu Ser Lys His Leu Arg Tyr Arg Thr Arg Thr Leu Cys Lys Asn Leu Arg Arg Arg Ala Arg Ser Ala Ser Gly Phe Gly Gly Arg Ala Thr Arg Leu Ser Lys Tyr Leu Gly Tyr Arg Ala Arg Gly Leu Gly Arg Cys Leu Gly Phe Cys Thr Arg Ser Leu Ser Lys Ser His Leu Phe Ser Thr Arg Ser Leu Ser Lys Gln Arg Leu Arg Phe Cys Asp Leu Arg Leu Ser Lys Ser Arg Leu Phe Ser Thr Arg Ser Leu Ser Lys Ile Pro Arg Phe Leu Thr Leu Gly Pro Arg Gly Phe Arg Leu Gly Thr Arg Thr ~ Leu Ser Lys Asp His Arg Phe Cys Thr Leu Gly Leu Cys Ser Phe Met Cys Arg Ala Arg Gly Leu Gly Arg Asn Pro Arg Arg Gly Arg Arg Lys , 275 280 285 Gln Cys Ile Phe (2) lN~O~ATION FOR SEQ ID NO:18:

W 096/29396 PCTrUS96/03916 (i) ~yu~N~ CE~RACTERISTICS:
(A) LENGTH: 534 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) EYPO~l~TICAL: NO
(iv) ANTI-SENSE: NO

(ix) FEATURE:
(A) NAME/XEY: CDS
(B) LOCATION: 1..533 (xi) ~yu~ DESCRIPTION: SEQ ID NO:18:

Met Leu Pro Ser Leu Leu Asn Arg Gly Ser Pro Arg Leu Asn Ser Pro Pro Lys Cys Ser Glu Ala Ser Ala Val Pro Tyr Asn Tyr Arg Val Val Arg Pro Ser Gln Ser Val Ser Asp Thr Ala Pro Phe Glu Arg Ile Gly Arg Leu Glu Asn Arg Asn Asp Trp Arg Ala Thr Phe Arg Leu Asn Eis Ile Phe Ile Glu Ser Gly Glu Leu Ser Ala Asp Gly Leu Thr Ile Ala Thr Ser Ser Thr Ser Ser Leu Ser Trp Ser Ala Pro Leu Phe Ile Ser His Ala Thr Met Gly Pro Asn Phe Arg Asp Ser Leu Leu Val Trp Glu Arg Ser Ser Ser Ser Cys Glu Thr Val Ser Asn Phe Arg Cys Gly Val Eis Met Phe Leu Val Thr Met Glu Ile Thr Met Thr Arg Pro Ile Val Ala Leu Thr Thr Ala Ala Thr Val Thr Pro Ile Ser Val Gly Leu Ile Val Pro Arg Arg Thr Val Thr Phe Glu Phe Ser Phe Ala Gly Val Gly Ser W 096/29396 PCTrUS96/03916 (2) INFORMATION FOR SEQ ID NO:l9:
yu~N~ CHARACTERISTICS:
(A) LENGTH: 177 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:l9:
Met Leu Pro Ser Leu Leu Asn Arg Gly Ser Pro Arg Leu Asn Ser Pro Pro Lys Cys Ser Glu Ala Ser Ala Val Pro Tyr Asn Tyr Arg Val Val Arg Pro Ser Gln Ser Val Ser Asp Thr Ala Pro Phe Glu Arg Ile Gly Arg Leu Glu Asn Arg Asn Asp Trp Arg Ala Thr Phe Arg Leu Asn His Ile Phe Ile Glu Ser Gly Glu Leu Ser Ala Asp Gly Leu Thr Ile Ala Thr Ser Ser Thr Ser Ser Leu Ser Trp Ser Ala Pro Leu Phe Ile Ser His Ala Thr Met Gly Pro Asn Phe Arg Asp Ser Leu Leu Val Trp Glu Arg Ser Ser Ser Ser Cys Glu Thr Val Ser Asn Phe Arg Cys Gly Val His Met Phe Leu Val Thr Met Glu Ile Thr Met Thr Arg Pro Ile Val Ala Leu Thr Thr Ala Ala Thr Val Thr Pro Ile Ser Val Gly Leu Ile Val Pro Arg Arg Thr Val Thr Phe Glu Phe Ser Phe Ala Gly Val Gly Ser (2) INFORMATION FOR SEQ ID NO:20:
(i) S~u~N~ CHARACTERISTICS:
(A) LENGTH: 48 base palrs (B) TYPE: nucleic acid (C) STR~n~nNF~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) H ~ lCAL: NO

(iv) ANTI-SENSE: NO
-(Xi) ~UU~N~: DESCRIPTION: SEQ ID NO:20:

W 096/29396 PCTrUS96/03916 (2) INFORMATION FOR SEQ ID NO:21:
(i) ~U~N~ CHARACTERISTICS:
'A) LENGTH: 36 base pairs B) TYPE: nucleic acid C) STR~Nn~n~F~s: single ,D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOl~TICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GCTGACCGCT AGTCGACCTG CAGTGAATAA TA~AAT 36 (2) INFORMATION FOR SEQ ID NO:22:
( i ) ~yu ~'N~ CHARACTERISTICS:
~A'l LENGTH: 48 base pairs ~B TYPE: nucleic acid C~ STRANDEDNESS: single ,D,~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~O~ CAL: NO
(iv) ANTI-SENSE: NO

(Xi) S~:~U~N~ DESCRIPTION: SEQ ID NO:22:
TGTCCGTCGA GAl~l~lAG AGTCGACGAA AGGTCAGAGA CGATGCCC 48 (2) INFORMATION FOR SEQ ID NO:23:
( i ) ~yU~N~ CHARACTERISTICS:
(A' LENGTH: 38 base pairs (B TYPE: nucleic acid (C~ STR~nFnNFCS single (D, TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~ul~lCAL: NO
(iv) ANTI-SENSE: NO
.

(Xi ) S~U~N~'~' DESCRIPTION: SEQ ID NO:23:
CGGATCAGAA A~lllCGG TACCCGGGAT CCTCTAGA 38 (2) INFORMATION FOR SEQ ID NO:24:
( i ) ~ ~:yU~N~' CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STR~N~ N~ S: single CA 022l6l39 l997-09-22 (D) TOPOLOGY: linear (ii) MOT~T~Cr~n~ TYPE: DNA (genomic) (iii) ~Y~u~ CAL: NO
(iv) ANTI-SENSE: NO

(Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:24:
GAATACAAGC TTAGATGCAT ATTTACTCGA GCC 33 .
(2) INFORMATION FOR SEQ ID NO:25:
( i ) ~yU~N~'~ CHARACTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~S single (D) TOPOLOGY: llnear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(Xi) ~-yU~N~ DESCRIPTION: SEQ ID NO:25:
GGrllGGCGG AGCGGATATG ATCTCGACCT GCAGTGAATA ATAAAATGTG T 51 (2) INFORMATION FOR SEQ ID NO:26:
( i ) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(Xi) S~UU~N~'~' DESCRIPTION: SEQ ID NO:26:
CC~lC~A GAlu~l~lAG AGTCGAGATC AGCAAAATGT TCACGGGG 48 (2) lN~ U~ ~ATION FOR SEQ ID NO:27:
( i ) ~yU~N~'~ C~ARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANn~n~: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~u~ lCAL: NO
(iv) ANTI-SENSE: NO

CA 022l6l39 l997-09-22 W O 96/29396 PCT~US96/03916 (Xi) ~:yU~N~ DESCRIPTION: SEQ ID NO:27:
AAGCTTGGCG TAATCATG . 18 (2) INFORMATION FOR SEQ ID NO:28:
( i ) ~yU~N~ CHARACTERISTICS:
A' LENGTH: 39 base pairs B~ TYPE: nucleic acid ~C~ STRPN~ )N~ S: single ~D~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~O~ CAL: NO
(iv) ANTI-SENSE: NO

(Xi) ~yU~'N~ DESCRIPTION: SEQ ID NO:28:
GGAATTCGAG ~lCG~LACCT CGTGGCGAGC GCAGGCGGC 39 (2) lN~OR L~TION FOR SEQ ID NO:29:
( i ) ~yU~N~ CHARACTERISTICS:
(A, LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iiij H~POTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
GGCCGAGTTA GGTTTTACTT TTCTAGAGGA lCCC~l-C~AC GTCTGGGGCG C 51 (2) INFORMATION FOR SEQ ID NO:30:
( i ) ~QU~N~- CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~Ol~llCAL: NO
(iv) ANTI-SENSE: NO

~xi) ~yu~N~ DESCRIPTION: SEQ ID NO:30:

'1 L~ L GC~ l 1' CCCGGGGATC CTCTAGAATT AGGTAGTTTG TAGTGCGA 48 (2) INFORMATION FOR SEQ ID NO:31:
( i ) ~yU~N~ CHARACTERISTICS:

CA 022l6l39 l997-09-22 W O 96/29396 PCTrUS96/03916 A) LENGTH: 42 base pairs B) TYPE: nucleic acid ~C) STRANDEDNESS: single tD) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(Xi) ~yu~N~ DESCRIPTION: SEQ ID NO:31:
TCAAGATCCA GGA~ATCCTT CGGTACCGAG CTCGA~TTCG TA 42 (2) INFORMATION FOR SEQ ID NO:32:
(i) ~yu~N~- CHARACTERISTICS:
A) LENGTH: 33 base pairs B) TYPE: nucleic acid ~C) STRAN~N~SS: slngle ,D) TOPOLOGY: linear (ii) MnT~CUT~ TYPE: DNA (genomic) (iii) ~Y~UL~.t~lCAL: NO
(iv) ANTI-SENSE: NO

(xi) ~uu~N-~ DESCRIPTION: SEQ ID NO:32:

(2) INFORMATION FOR SEQ ID NO:33:
(i) ~yU~N~: CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: slngle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~UL~L lCAL: NO
(iv) ANTI-SENSE: NO

(xi) S~YU~N-~ DESCRIPTION: SEQ ID NO:33:
CGCAAACAGC L~L~LAACT CTAGAAGTTA ACGATCGCTG TT 42 (2) INFORMATION FOR SEQ ID NO:34:
(i) S~yu~ CHARACTERISTICS:
(Al LENGTH: 57 base pairs (B~ TYPE: nucleic acid ~ (Cl STRANDEDNESS: single (D~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) W 096/29396 PCTrUS96/03916 (iii) ~r~uln~llCAL: NO
(iv) ANTI-SENSE: NO

(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:34:

(2) INFORMATION FOR SEQ ID NO:35:
( i ) ~yU~N~'~ CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~ul~llCAL: NO
(iv) ANTI-SENSE: NO

(xi) ~yu~ DESCRIPTION: SEQ ID NO:35:
TCGAGGGGAT CCTCTAGAGT CGAGGGACCC A~ lG~l- GC 42 (2) INFORMATION FOR SEQ ID NO:36:
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STR~Nul~:uNi~:SS: single (D) TOPOLOGY: linear ( ii) MoT~T~'cTTT~ TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:36:
TTTACTAAAG CGCGGCGAAA G~llC~lC~l G~lGGGLL~L GG 42 (2) INFORMATION FOR SEQ ID NO:37:
( i ) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STR~NnFnN~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~Ol~LlCAL: NO

(iv) ANTI-SENSE: NO

W 096/29396 PCTrUS96/03916 (Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:37:
AAG~LlGGCG TAATCATGGT C 2l ~2) INFORMATION FOR SEQ ID NO:38:
(i) ~Qu N~'~ CHARACTERISTICS:
A) LENGTH: 48 base pairs B) TYPE: nucleic acid C) STRANDEDNESS: sinsle ~,D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:38:
GGAATTCGAG ~lCG~lACCC GGATAATACG TACATGTTAA CGCAGAGG 48 (2) INFORMATION FOR SEQ ID NO:39:
(i) ~yU~:N~ CHARACTERISTICS:
(A~ LENGTH: 45 base pairs (B~ TYPE: nucleic acid (C STR~L)~ S: s~ngle (D~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~OL~LlCAL: NO
~ ) ANTI-SENSE: NO

(xi) ~yU~N~: DESCRIPTION: SEQ ID NO:39:

(2) INFORMATION FOR SEQ ID NO:40:
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTX: 27 base pairs (B) TYPE: nucleic acid (C) STR~N~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) S~UU~N~: DESCRIPTION: SEQ ID NO:40:

G~ ~ 1 ~ATT TCTGATCCCC GGGAACG 27 (2) INFORMATION FOR SEQ ID NO:4l:
(i) ~:yu~ CHARACTERISTICS:

CA 022l6l39 l997-09-22 W 096/29396 PCT~US96/03916 -l32-(A~ LENGTH: 51 base pairs ~B'~ TYPE: nucleic acid C~ STR~Nn~nN~S: single ,D, TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) ~yu~N~: DESCRIPTION: SEQ ID NO:41:

(2) INFORMATION FOR SEQ ID NO:42:
( i ) ~yU~N~ CHARACTERISTICS:
A LENGTH: 33 base pairs ~B TYPE: nucleic acid C STRANDEDNESS: single ~D, TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
tiv) ANTI-SENSE: NO

(Xi) ~yU~N~: DESCRIPTION: SEQ ID NO:42:
ATCAGAAACT ~--l''L''i CG~-AC CGAGCTCGAA TTC 33 (2) INFORMATION FOR SEQ ID NO:43:
( i ) ~yU~N~ CHARACTERISTICS:
(A'~ LENGTH: 48 base pairs (B TYPE: nucleic acid (Cl STRANDEDNESS: single (D,~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~Ol~llCAL: NO
(iv) ANTI-SENSE: NO

(Xi) S~yU~N~ DESCRIPTION: SEQ ID NO:43:

(2) INFORMATION FOR SEQ ID NO:44:
( i ) ~yU~N~ CHARACTERISTICS:
A'~ LENGTH: 33 base pairs ~B) TYPE: nucleic acid R~Nn~n~CS: single ~D,~ TOPOLOGY: linear (ii) M~T~ ~ TYPE: DNA (genomic) CA 022l6l39 l997-09-22 ~iii) ~Y~O~ CAL: NO
(ivJ ANTI-SENSE: NO

(Xi) ~yU~:N~ DESCRIPTION: SEQ ID NO:44:

(2) INFORMATION FOR SEQ ID NO:45:
( i ) ~UU~N~ CHARACTERISTICS:
A) LENGTH: 42 base pairs B) TYPE: nucleic acid ~C) STRANn~nN~S: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:45:
C~llCCCGGG GAlC~l-lAG AGTCGACGGC AGAGTCGCAG AC 42 (2) INFORMATION FOR SEQ ID NO:46:
( i ) ~yU~N~ CHARACTERISTICS:
~A~ LENGTH: 30 base pairs ~B TYPE: nucleic acid C~ STRANDEDNESS: single ~,D, TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) ~yu~NL~: DESCRIPTION: SEQ ID NO:46:
TGATCCA~AC TCGGATCCTC TAGAGTCGAC 30 (2) INFORMATION FOR SEQ ID NO:47:
(i) ~yU~N~ CHARACTERISTICS:
~A) LENGTH: 48 base pairs ~B) TYPE: nucleic acid C) STR~ S: single ,D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

WO 96/29396 PCT~US96/03916 AAGCTTGGGC TGCAGGTCGA CTCTAGAGGA ~lCCC~l-C~AC ~-l-~-l-GGGG 48 (2) INFORMATION FOR SEQ ID NO:48:
(i) ~uu~ CHARACTERISTICS:
(A LENGTH: 60 base pairs (B~ TYPE: nucleic acid (C STRANn~nN~ : single (D~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~Gln~llCAL: NO
(i~) ANTI-SENSE: NO

(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:48:
CACACCTTTG CGCATCTCCA CAGCTCAACA ATGAATTCCA TGTTACGTCC TGTAGA~ACC 60 (2) INFORMATION FOR SEQ ID NO:49:
(i) ~u~ CHARACTERISTICS:
~A'~ LENGTH: 60 base pairs B~ TYPE: nucleic acid C~ STRANDEDNESS: single ,D~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~ul~lICAL: NO
(iv) ANTI-SENSE: NO

(xi) S~yu~N~- DESCRIPTION: SEQ ID NO:49:

(2) INFORMATION FOR SEQ ID NO:50:
(i) S~Qu~N~ CHARACTERISTICS:
Al LENGTH: 45 base pairs B~ TYPE: nucleic acid Cl STRANDEDNESS: single - ~D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~O~ llCAL: NO
(i~) ANTI-SENSE: NO

(xi) ~Uu~N~ DESCRIPTION: SEQ ID NO:50:

(2) INFORMATION FOR SEQ ID NO:51:
(i) ~uu~: CHARACTERISTICS:

CA 022l6l39 l997-09-22 W O 96/29396 PCTrUS96/03916 ~A) LENGTH: 48 base pairs ~B) TYPE: nucleic acid ~C) STR~Nn~nN~.~s single ,~ ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ( iii ) ~Y~U-l~'l lCAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
TCTAGAGTCG ACCTGCAGTG AATAATA~AA ~L~-l~L~LLl-G TCCGA~AT 48 (2) INFORMATION FOR SEQ ID NO:52:
(i) ~yu~N~ CHARACTERISTICS:
~A) LENGTH: 45 base pairs B) TYPE: nucleic acid ~C) STRANDEDNESS: single - ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~POL~TICAL: NO
(iv) ANTI-SENSE: NO

(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:52:
CTCCATAGAA GACACCGGGA CCATGGATCC C~l~ l 'l-'L LA CAACG 45 (2) INFORMATION FOR SEQ ID NO:53:
( i ) ~ ~ yU~N~ CHARACTERISTICS:
(A) LENGTH: 105 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: slngle (D) TOPOLOGY: linear (ii) MOLECULE TY F : DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:53:
TCGGCGGAAA TCCAGCTGAG CGCCG~lCGC TACCATTACC A~. L~'l ~ LG GTGTCAAAAA 60 (2) INFORMATION FOR SEQ ID NO:54:

~i) ~yu~ CHARACTERISTICS:
~A) LENGTH: 36 base pairs B) TYPE: nucleic acid C) STR~Nn~nNFCS: single .D). TOPOLOGY: linear W 096/29396 PCTfUS96/03916 . -136-(ii) MOLECULE TYPE: DNA (genomic) (iii) nY~Oln~llCAL: NO
(iv) ANTI-SENSE: NO

(xi) ~yU~N~ DESCRIPTION: SEQ ID NO:54:
CC~G~AGAT CCTCTAGAGT CGACCTGCAG GTCGAC 36 (2) INFORMATION FOR SEQ ID NO:55:
(i) ~yu~N~: CHARACTERISTICS:
A~ LENGTH: 20 base pairs B~ TYPE: nucleic acid CI STRA~u~N~SS: single D,~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) nY~ln~llCAL: N
(iv) ANTI-SENSE: N

(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:55:

(2) lN~ORIL!TION FOR SEQ ID NO:56:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N

(xi) ~yu~-~- DESCRIPTION: SEQ ID NO:56:

(2) INFORMATION FOR SEQ ID NO:57:
(i) ~yu~N~ CHARACTERISTICS:
(A LENGTH: 27 base pairs (B~ TYPE: nucleic acid (C STR~NnFnN~S: single (DJ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) nY~vl~llCAL: N
(iv) ANTI-SENSE: N

W 096/29396 PCTrUS96/03916 (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:57:

(2) INFORMATION FOR SEQ ID NO:58:
(i) S~U~:NC~ CHARACTERISTICS:
'A) LENGTH: 20 base pairs B) TYPE: nucleic acid C) STR~N~ N~ : single ,D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(xi) ~Ub'N~'~ DESCRIPTION: SEQ ID NO:58:
c-lC~-lCC~AA CGAGTTACAG 20 (2) lN~-O~IATION FOR SEQ ID NO:59:
(i) ~U~NC~: CHARACTERISTICS:
(A) LENGTH: 18912 base pairs (B) TYPE: nucleic acid (C) STRPNn~nN~.~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHE.ICAL: N
(iv) ANTI-SENSE: N
(ix) FEATURE:
(A) NAME/KEY:.CDS
(B) LOCATION: 697..1533 (D) OTHER INFORMATION:
(ix) FEATURE:
(A) NAME/FEY: CDS
('3) LOCATION: complement ~1900..2784) tD~ OT~ER INFORMATION:
(ix) FEATURE:
~'A) NAME/KEY: CDS
B) LOCATION: complement (2916..3605) D) Ol~R INFORMATION:
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3694..5124 (D) OTHER INFORMATION:
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 5210..7081 (D) OTHER lN~O~ATION:
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 7245..8123 (D) OTHER INFORMATION:
~ (ix) 'FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 8333..11290 (D) OT~R INFORMATION:
(ix) FEATURE:
(A) NAME/REY: CDS

CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 tB) LOCATION: 11098..12402 (D) OTHER INFORMATION:
tix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 12510..13598 (D) Oi~R INFORMATION:
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 13792..15291 (D) OTHER INFORMATION:
(ix) FEATURE:
(A) NAME/ ~ Y: CDS
(B) LOCATION: 15298..16080 (D) OTHER INFORMATION:
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 16129..17013 (D) OTHER INFORMATION:
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: complement (17380..18216) (D) OTHER INFORMATION:

(xi) ~yu~ DESCRIPTION: SEQ ID NO:59:
GGATCCCGAA GAG~L~lCCC AGAAGTTTTT ~L~LlCGGAC GTATCGGAGG ACGAAGAACC 60 TGACTTAGAC TTTATTGACG ACAGCTCTCC GGCGCCGCCG CCALLLG-lA -LCCCCCGCGT 240 CCGTGCGTTA ~L 1 GCG~ 1 GCG CGGCACCCGC AAAGACCCAC GGAAGGCTTC GGCCGCCAGG 300 GC'GGGlAGGC GCACTCTTAA AAGACGGAGG TTGTCATTTT ~ L ~ ~ L 1 C~ LC TGACGAGGAA 360 GAGGCCTCGA CGAGCAGATA GAGGAGACGC GGGGCAGAAC ~LCCCC~-lCC CTCCCACCCC 480 CCTACTCTGG ACATTTATTG CCCGul~GAT CCATTCTCAT CCAGAACTTC LLlCCCGCTC 540 AGC~ll~ACG CAGAAGCGGA CGCGCGCCCC TTTGCGACCG CCGGACATCC CGC'CGCCCCC 600 CCCC~L1~AC GCCCGGCGCA ATCCGTAGCC GTCCAACTCG GCCCAGCACA ACCGCAGTAG 660 ACCGCCCGGA C~G~L~1C~1 CTAGACACAT CCCTAA ATG GAA AAC ATG CTC GAC 714 Met Glu Asn Met Leu Asp Gly Cys Tyr Pro Leu Ala Leu Met Asp Ser Asp His Ile Thr Ala His Ala Val Pro Arg Gly Glu Arg Arg Arg Gln Gly Ala Ala Val Ala Ser Ser Glu Ser Ala Asp ~er Val Asp Pro Cys Ile Arg Ile Ala Ser Arg Leu Trp Arg Glu Leu Val Glu Ile Ser Ser Glu Leu Lys Asp Gly Tyr CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 Gly Glu Phe Thr Ser Ala Arg Asp Arg Arq Asn Ala Leu Ile Ala Ala Asn Glu Arg Leu Arg Ser Ala Phe Leu Gly Ala Ser Arg Ala Thr Arg Gly Leu Gly Leu Arg Pro Arg Trp Ala Ser Thr Glu Ser Val Ala Asn Ser Pro Thr Asp Pro Asn Asn Gly Asn Gly Leu Gly Glu Leu Glu Glu Ala Met Glu Gly Ile Glu Gly Asp Phe Trp Leu Asp Ser Leu Asp Gly Asp Arg Phe Glu Asp Glu Ser Arg Thr Met Gln Ser Glu Asn Met Arg Phe Val Ile Glu Lys Glu Leu Leu Ser Trp Leu Ser Arg His Leu Pro Ala Asp Leu Ala Ser Ala Glu Arg Glu Thr Ser Arg Ser Leu Leu Ala Ala Gly His Trp Cys Cys Leu Trp His Pro Arg Pro Cys Arg Glu Ala TGT TTG TAC GAC TCG ~TT TAC GTG CAG AGT CTT TTC TGC GTC GGG ACG 1386 Cys Leu Tyr Asp Ser Ile Tyr Val Gln Ser Leu Phe Cys Val Gly Thr 51y Arg Val Pro Gln Ser Glu Met Arg Arg Arg Glu Tyr Leu Ala Ala Leu Arg Ala Gly Ala Ala Ala Ala Asn Ser Pro Glu Val Ser Ala Ser Ile Phe Ala Arg Asp Ala Gly Ile Ala Leu Ala Leu Ala Arg Arg Arg TGA CGGGAGAATG ACGCC'~ A GCGGC~lC~l TAC~LCCGCG LCC~l~ACAA 15&3 CCTCGCGGGT TTTTACACTG lC~lCC~CC A~l~lCCCCC CTCACCCACT CCGCGGCAGC 1643 AAGTAATATT A~ TTTCACGTTG TTGCAATCGA GAGGCC'~ ~l 1763 ~l~GCGG AGCTAGGCTT lCCCGGGCGG CCCC'~1C~A CC~LlCG~l L AGGCCG~.~G 1823 CGACGGGACA TAGAGAAAGA TAGAGCGCGC GCC~GGCGG CGAGAGGGTG TTGCGGGGGT 1883 AAATGGGACC CTGAGCTCAC CATTTTGGCG GGGGATTGCA CGGGTAACAA AAAG~l.L 1943 CA 022l6l39 l997-09-22 096/29396 PCTrUS96/03916 CGCACATAAT GA1.1 CC~ll AAACAGTGGC TGTAAAAGCT ~11~11~ACT GGGACGCGCA 2003 C~1CCGGAGA CATGATCTTA TCGGTAGCTA CACAGTTCAT GAGGTGGGCC ACGAACGCGC 2063 GGATCGAGTT TTGGGAACCT TCGGGGAGGT ~L lC~G~AG GGTGAAGTTT GACAGAGGCA 2123 CGGCCGGCGC GCTGCTCTGG TTATTCCAGT GCGCGGACCG CGA~1~CGCC C~LCCCCGGG 2243 CTCTGATATA GAGCACCGGC AGCTCGACGG CGGCGGAGAA AA~AGA~AGA A~l~LGCGGCC 2303 CAATGACTGG AA~111GGGC AC~1~1~11A 'l Ll CC~ACGC GGCGGCCCGG GGAATCTGCT 2363 GCGCCGTGAT A1~11CGGCG ACCTGCAGAC TGCCCAGCCT ~C~11CC~ TCAAAATACG 2543 CGCGGGCGGC~ CTGTACGATC ACCGCGGC_A GA1~GGGC~A A~AGAAAATA TCGCAACTCT 2603 GCGACGCCCG CCAGAATCTC CCTCCGGGCA G~'L~'~-1GCC CCTA~AGGCC GCCGAGA~AG 2663 CTAAGTCCAA ATGTGACGTC GGAG~1~1~ ACA1~CGC CAACC~C~A AATGCTACCC 2723 TCCGCGTACG CGTTTATTAT TGTCAATATT ~'1'~G-11A TTATTACTGC TACCGCCCTT 2843 L''l'-l ~-1 GCAA GGCC~CGCC GCGGCCCAGG CCACTATTCC GGCAGCGGCC GCCGACGCGG 2903 CGAGCGTCGC CGCTAACGTC GGCGCCGCGG GGAGCGGGGT 1~L1~ACT TAAATAGACT 2963 CCCGAGAAAA AATTTTGGCT G.-C-~11CGCC ATCATCCGAG TCGGA~ACAC AGTATGCGGC 3023 CGAGTTAGGT TTTACTTTTA AAAACTTTAC C~'1G~L~1'AC GGCCAGGGCG TTCTCAGGCT 3083 CGAAGGGGCA AGAGTTGTCC AGACTGATGG GTGACTCAGA GACAGCGTTG ~C1 1~L~-1CC 3143 GTTTACCAAA AATATTTCCA ~1C~ L ~ ~ ~1 C AAAATTTTTA C~1CCG~-1LL CGGTAATTAG 3203 CCACAAATGG AA~1~111G CGGCATACGC GCCAGATGAC AAATGGTAAA ACCCTGCGTC 3323 GGGTCTGCAC A1C~1GGGAA GGGAAAACAG ~CCCCGGAA A~L~C~LACA GAGATGCCGG 3443 AACCAATTCT TTCCATCCAG AATCCGAGAG CTCAAATCTA GAATTAGGTA ~L~LAGTG 3563 CGAATCGACC GCAGAAACTA TAGTCACTTT TACAGGCGCC A~CGCCG~l~AG 3615 ATATACAGCT TAGAGAAG ATG CGG TTT CG~ CGC ATC TGT TCA CGC TCT AGG 37.26 Met Arg Phe Arg Arg Ile Cys Ser Arg Ser Arg A1a G1U Lys Arg Arg Arg Thr Thr G1U ASn PrO Leu Thr Ser. Lys Arg W 096/29396 PCTrUS96/03916 GTT TGC GTA TTG GAT AGT TTC TCA CGG ACA ATG TCA TTG CGC CCC TAT 3822Val Cys Val Leu Asp Ser Phe Ser Arg Thr Met Ser Leu Arg Pro Tyr Ala Glu Ile Leu Pro Thr Ala Glu Gly Val Glu Arg Leu Ala Glu Leu Val Ser Val Thr Met Thr Glu Arg Ala Glu Pro Val Thr Glu Asn Thr Ala Val Asn Ser Ile Pro Pro Ala Asn Glu Asn Gly Gln Asn Phe Ala TAT GCA GGC GAT GGG CCC TCG ACT ACT GAA A~A GTT GAC GGC TCG CAT 4014 Tyr Ala Gly Asp Gly Pro Ser Thr Thr Glu Lys Val Asp Gly Ser His Thr Asp Phe Asp Glu Ala Ser Ser Asp Tyr Ala Gly Pro Val Pro Leu Ala Gln Thr Arg Leu Lys His Ser Asp Glu Phe Leu Gln His Phe Arg Val Leu Asp Asp Leu Val Glu Gly Ala Tyr Gly Phe Ile Cys Asp Val Arg Arg Tyr Thr Glu Glu Glu Gln Arg Arg Arg Gly Val Asn Ser Thr AAC CAG GGG A~A TCA AAA TGT AAG CGC CTG ATA GCT AAA TAT GTG AAA 4254 Asn Gln Gly Lys Se- Lys Cy5 Lys Arg Leu Ile Ala Lys Tyr Val Lys AAT GGA ACA AGG GCG GCC TCT CAG CTG GAA AAT GAA ~TT TTG GTT CTC 4302 Asn Gly Thr Arg Ala Ala Ser Gln Leu Glu Asn Glu Ile Leu Val Leu GGG CGC CTA AAT CAC GAG AAT G~ CTC AAG ATC CAG GAA ATC CTT CGG 4350 Gly Arg Leu Asn His Glu Asn Val Leu Lys Ile Gln Glu Ile Leu Arg Tyr Pro Asp Asn Thr Tyr Met Leu Thr Gln Arg Tyr Gln Phe Asp Leu Tyr Ser Tyr Met Tyr Asp Glu Ala Phe Asp Trp Lys Asp Ser Pro Met CTT A~A CAG ACT AGA CGC ATC ATG AAG CAG CTC ATG TCA GCG GTC TCG 4494 Leu Lys Gln Thr Arg Arg Ile Met Lys Gln Leu Met Ser Ala Val Ser Tyr Ile His Ser Lys Lys Leu Ile His Arg Asp Ile Lys Leu Glu Asn Ile Phe Leu Asn Cys Asp Gly Lys Thr Val Leu Gly Asp Phe Gly Thr W096/29396 PCTrUS96/03916 GTC ACG CCT TTT GAA AAT GAG CGG GAG CCC TTC GAA TAT GGA TGG GTG 4638Val Thr Pro Phe Glu Asn Glu Arg Glu Pro Phe Glu Tyr Gly Trp Val Gly Thr Vai Ala Thr Asn Ser Pro Glu Ile Leu Ala Arg Asp Ser Tyr Cys Glu Ile Thr Asp Ile Trp Ser Cys Gly Val Val Leu Leu Glu Met Val Ser His Glu Phe Cys Pro Ile Gly Asp Gly Gly Gly Asn Pro His CAG CAA TTG CTG A~A GTT ATC GAC TCT CTC TCA GTT TGT GAT GAA GAG 4830 Gln Gln Leu Leu Lys Val Ile Asp Ser Leu Ser Val Cys Asp Glu Glu Phe Pro Asp Pro Pro Cys Asn Leu Tyr Asn Tyr Leu His Tyr Ala Ser Ile Asp Arg Ala Gly His Thr Val Pro Ser Leu Ile Arg Asn Leu His Leu Pro Ala Asp Val Glu Tyr Pro Leu Val Lys Met Leu Thr Phe Asp TGG CGT TTG AGA C~C AGC GCG GCC GAA GTA TTG GCA ATG CCA CTG TTT 5022 Trp Arg Leu Arg Pro Ser Ala Ala Glu Val Leu Ala Met Pro Leu Phe Ser Ala Glu Glu Glu Arg Thr Ile Thr Ile Ile His Gly Lys His Lys Pro Ile Arg Pro Glu Ile Arg Ala Arg Val Pro Arg Ser Met Ser Glu 460 ~65 470 475 GGT TAA TAATAAAGGA CGGAGATAGA GAACTGAAGC GTCAGATTTT TTTA~AAAAA 5174 Gly TAAATGATCG AGAACTTATG A~ CTTGA ATG ACC TTG CCC CAT CGA 5227 Met Thr Leu Pro His Arg TTA ACG A~A AGA CCT TTC GCG C5T CGA TTC TGC TCG GTC TTT GTG ATA 5275 Leu Thr Lys Arg Pro Phe Ala Arg Arg Phe Cys Ser Val Phe Val Ile His Tyr Ser Glu Thr Lys Leu ASD Arg Tyr Asn Lys Thr Met Leu Leu 25 3~ 35 Tyr Arg Pro Asp Ser Thr Met Arg H.is Ser Gly Gly Asp Ala Asn His Arg Gly Ile Arg Pro Arg Arg Lys Ser Ile Gly Ala Phe Ser Ala Arg W 096/29396 PCTrUS96/03916 Glu Lys Thr Gly Lys Arg Asn Ala Leu Thr Glu Ser Ser Ser Ser Ser Asp Met Leu Asp Pro Phe Ser Thr Asp Lys Glu Phe Gly Gly Lys Trp Thr Val Asp Gly Pro Ala Asp Ile Thr Ala Glu Val Leu Ser Gln Ala Trp Asp Val Leu Gln Leu Val Lys His Glu Asp Ala Glu Glu Glu Arg GTG ACT TAT GAG TCC A~A CCG ACC CCG ATA CAG CCG TTC AAT GCC TGG 5659 Val~Thr Tyr Glu Ser Lys Pro Thr Pro Ile Gln Pro Phe Asn Ala Trp Pro Asp Gly Pro Ser Trp Asn Ala Gln Asp Phe Thr Arg Ala Pro Ile GTT TAT CCC TCT GCG GAG GTA TTG GAC GCA GAG GCG TTG A~A GTA GGG 5755 Val Tyr Pro Ser Ala Glu Val Leu Asp Ala Glu Ala Leu Lys Val Gly GCA TTC GTT AGC CGA GTT TTA CAA TGT GTA CCG TTC ACG CGA TCA A~G 5803 Ala Phe Val Ser Arg Val Leu Gln Cys Val Pro Phe Thr Arg Ser Lys Lys Ser Val Thr Val Arg Asp Ala Gln Ser Phe Leu Gly Asp Ser Phe Trp Arg Ile Met Gln Asn Val Tyr Thr Val Val Leu Arg Gln His Ile 215 220 . 225 230 Thr Arg Leu Arg ~ s Pro Ser Ser Lys Ser Ile Val Asn Cys Asn Asp Pro Leu Trp Tyr Ala Tyr Ala Asn Gln Phe ~is Trp Arg Gly Met Arg Val Pro Ser Leu Lys Leu Ala Ser Pro Pro Glu Glu Asn Ile Gln His Gly Pro Met Ala Ala Val Phe Arg Asn Ala Gly Ala Gly Leu Phe Leu Trp Pro Ala Met Arg Ala Ala Phe Glu Glu Arg Asp Lys Arg Leu Leu 1 Arg Ala Cys Leu Ser Ser Leu Asp Ile Met Asp Ala Ala Val Leu Ala Ser Phe Pro Phe Tyr Trp Arg Gly Val Gln Asp Thr Ser Arg Phe Glu CA 022l6l39 l997-09-22 CCT GCG CTG GGC TGT TTG TCA GAG TAC TTT GCA CTA GTG GTG TTA CTG 6283Pro Ala Leu Gly Cys Leu Ser Glu Tyr Phe Ala Leu Val Val Leu Leu Ala Glu Thr Val Leu Ala Thr Met Phe Asp His Ala Leu Val Phe Met Arg Ala Leu Ala Asp Gly Asn Phe Asp Asp Tyr Asp Glu Thr Arg Tyr Ile Asp Pro Val Lys Asn Glu Tyr Leu Asn Gly Ala Glu Gly Thr Leu 39" 400 405 Leu Arg Gly Ile Val Ala Ser Asn Thr Ala Leu Ala Val Val Cys Ala Asn Thr Tyr Ser Thr Ile Arg Lys Leu Pro Ser Val Ala Thr Ser Ala Cys Asn Val Ala Tyr Arg Thr Glu Thr Leu Lys Ala Arg Arg Pro Gly Met Ser Asp Ile Tyr Arg Ile Leu Gln Lys Glu Phe Phe Phe Tyr Ile Ala Trp Leu Gln Arg Val Ala Thr His Ala Asn Phe Cys Leu Asn Ile Leu Lys Arg Ser Val Asp Thr Gly Ala Pro Pro Phe Leu Phe Arg Ala Ser Ser Glu Lys Arg Leu Gln Gln Leu Asn Lys Met Leu Cys Pro Leu Leu Val Pro Ile Gln Tyr Glu Asp Phe Ser Lys Ala Met Gly Ser Glu 520 S~5 530 CTC AAG AGG GAA AAG TTA GAG ACA TTC GTT A~A GCT ATT TCC AGC GAC 6859 Leu Lys Arg Glu Lys Leu Glu Thr Phe Val Lys Ala Ile Ser Ser Asp Arg Asp Pro Arg Gly Ser Leu Arg Phe Leu Ile Ser Asp His Ala Arg Glu Ile Ile Ala Asp Gly Val Arg Phe Lys Pro Val Ile Asp Glu Pro Val Arg Ala Ser Val Ala Leu Ser Thr Ala Ala Ala Gly Lys Val Lys ~

Ala Arg Arg Leu Thr Ser Val Arg Ala Pro Val Pro Gly Ala Gly Ala GTT TCC GCG CGC CGG AAA TCG GAA ATA TGA TA~AAATGCT TGGCATTTGC 7101 Val Ser Ala Arg Arg Lys Ser Glu Ile A~AGTGTACC CGCTGACCGC TAGCCCATAC AGTGTTACAG GAGGGGAGAG AGA~AACTTC 7221 Met Ser Gly Phe Ser Asn Ile Gly Ser ATT GCC ACC GTT TCC CTA GTA TGC TCG CTT TTG TGC GCA TCT GTA TT~ 7319 Ile Ala Thr Val Ser Leu Val Cys Ser Leu Leu Cys Ala Ser Val Leu Gly Ala Pro Val Leu Asp Gly Leu Glu Ser Ser Pro Phe Pro Phe Gly ~ 30 35 40 GGC A~A ATT ATA GCC CAG GCG TGC AAC CGC ACC ACG ATT GAG GTG ACG 7415 Gly Lys Ile Ile Ala Gln Ala Cys Asn Arg Thr Thr Ile Glu Val Thr Val Pro Trp Ser Asp Tyr Ser Gly Arg Thr Glu Gly Val Ser Val Glu Val Lys Trp Phe Tyr Gly Asn Ser Asn Pro Glu Ser Phe Val Phe Gly Val Asp Ser Glu Thr Gly Ser Gly His Glu Asp Leu Ser Thr Cys Trp Ala Leu Ile His Asn Leu Asn Ala Ser Val Cys Arg Ala Ser Asp Ala llC 115 120 GGG ATA CCT GAT TTC GAC AAG CAG TGC GAA A~A GTG CAG AGA AGA CTG 7655 Gly Ile Pro Asp Phe Asp Lys Gln Cys Glu Lys Val Gln Arg Arg Leu Arg Ser Gly Val Glu Leu Gly Ser Tyr Val Ser Gly Asn Gly Ser Leu Val Leu Tyr Pro Gly Met Tyr Asp Ala Gly Ile Tyr Ala Tyr Gln Leu Ser Val Gly Gly Lys Gly Tyr Thr Gly Ser Val Tyr Leu Asp Val Gly Pro Asn Pro Gly Cys His Asp Gln Tyr Gly Tyr Thr Tyr Tyr Ser Leu lgO 195 200 Ala Asp Glu Ala Ser Asp Leu Ser Ser Tyr Asp Val Ala Ser Pro Glu Leu Asp Gly Pro Met Glu Glu Asp Tyr Ser Asn Cys Leu Asp Met Pro W096/29396 PCTrUS96/03916 Pro Leu Arg Pro Trp Thr Thr Val Cys Ser His Asp Val Glu Glu Gln Glu Asn Ala Thr Asp Glu Leu Tyr Leu Trp Asp Glu Glu Cys Ala Gly Pro Leu Asp Glu Tyr Val Asp Glu Arg Ser Glu Thr Met Pro Arg Met Val Val Phe Ser Pro Pro Ser Thr Leu Gln Gln ~-L~-l-l--l-l'l''LG TGAGCGCCCA CGCAACATAC CTAACTGCTT CAlll~L~AT CAATTATTGC 81S3 GTATTGAATA AATAAACAGT ACA~AAGCAT CAG~l~l~l TTGC~l~l~L GTGCTAAACC 8253 ATGGCGTGTG CGGGTGAAAC CGTA~ATTAC GTGATAATAA ATAGCATAGG AGTTGGCGTG 8313 CAGCGTATTT CGCCGAGAGA TGGGGACAAT GTTAGTGTTG C'GC~-1 -1''L'1-CC TACTTGCAGT 8373 AGCGGACGCG GCGTTGCCGA CCGGCAGATT CTGCCGAGTT TGGAAGGTGC ~LC~GGGAGG 8433 5GACAACGTG ACATTTTCGC TGTCATATCG CCCGCGCCCA GA~ATTCACG GAGCATACTT 8673 G~l~llAGTT AACGCCTCTC TGGAACGGTC CGTGCGCCTG GA~ACGCCGT GCGATGAAAA 8793 LlllllGCAG AACGAGCCTA CATGGGGCTC GAAGCGTTGG TTAGGCCCCC CGTCGCCTTA 8853 TGTGCGAGAT AACGATGTCG CC~l~,lGAC AAAAGCGCAG TACATTGGGG AGTGCTACTC 8913 CAACTCGGCG GC_CAGACGG GGCTCACGTC TCTCAACATG A~1L1L1~1 ATTCGCCTAA 8973 AAGAATAGTA AACGTCACGT GGACAACCGG CGGCCC~lCC CCCTCGCGCA TAACGGTATA 9033 ~lC~lCGCGG GAGAACGGGC AGCCC'~L~ll SAGGAACGTT TCTGACGGGT l~ll~llAA 9093 GGACTCCGGC AGC~LC~lCG CGTTTACGGC CTTTAGGGAA GGA~AACTCC CATCCGCGAT 9213 TCAACTGCAC CGCATAGATA ~lCCG~5AC TGAGCCGCCG GGGACTGA~A CGAC~llC~A 9273 CTGTCA~AAA ATGATAGAAA CCCCGTACCG AGCGCTCGGG A5CAATGTTC CCAGGGACGA 9333 CTCTATCCGT CCGGGGGCCA ~l~lGC~C GTTCGATACC GCAGCACCTG ATTTCGATA.C 9393 CAGCACTAGC GATATGGGAT l~ll~lC~AC GG Q CGTGCT ACCGGATCAG AAA~L~LLlC 9513 GGTACCCGTC CAGGAAACGG ATAGAACTCT TTCGACAACT C~l.LLACCC TTCCACTGAC 9573 lCCCG~L~AG TCAGAAAATA CA~l~lllCC TACGACCGCG CC~GG~ATTT CTACCGAGAC 9633 CCCGAGCGCG GCACATGAAA CTACACAGAC CCAGAGTGCA GAAACGGTGG TCTTTACTC'A 9693 CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 ~-11--1ACT CAGACTCCGA GTGCTGAACA GATGACTTTT ACTCAGACTC CGGGTGCAGA 9873 AACCGAGG Q CCTGCCCAGA CCCCGAG QC GATACCCGAG ATATTTACTC A~L~lC~lAG 9933 GAGTACGGTA ACGGAGGTGT TTACTCAGAC CCCGAGCACG GTACCGA~AA CTACTCTGAG 100 53 GTTGACTCAG AGTCCGAGTA CCGAACCTGT GC~111~ACC CGGACTCTGG GCGCAGAGCC 10293 CTACACAGAA AATAAGACTT TA1C~111-CC TA~1~11~-1--1 T QGAATTCC ATGAGATGTC 10 533 GACGG Q GAG TCGCAGACGC CCCTATTGGA CGT Q AAATT GTAGAGGTGA A~L11--1-~AAA 10593 CGATGGCGAA GTAACGGCGA ~11GC-~111C Q CCGTCAAA TCTCCCTATA GGGTAGAAAC 10653 G~11111AAC AGTAATGAGA AATGGCAGAA A QGCTGTAC TA QGAGTAA CCGATGGAAG 10773 AACATCGGTC CAGCTAATGT G~1~1~1G Q CGAGCCAT L ~1~CGGAAC CTTACTGTCT 10 833 TTTCGA Q CG ~1~1~11ATAG CGAGGGAAAA AGATATCGCG CCAGAGTTAT ACTTTACCTC 10893 TGATCCG QA ACGG Q TACT GCACAATAAC .~LGCC~lCC GGC~ll~L-LC CGAGATTCGA 10953 ATGGAGCCTT AATAATGTTT CACTGCCGGA ATATTTGACG GC Q CGACCG ~11~11-1-CGCA 11013 ~1GGCCGGGGA GGCAATATAT ACGAATGCAC C~1C~1~ATC T Q GACGGCA CTCGCGTTAC 11133 GGCG QGCTG TATTCACTCT TTTCTGGACT 1~1~1~AGGA TTATGCGGGA G QTATCTGC 11253 GTACTGCTTT ~11C~1 lCGC TAGCCAGAGC ACCGCCGCCG T QCGTACGA CTACATTTTA 11370 ACTAGGGTAT Q AGAGGCTG CGAC~11~1C GAGCTCAACC CGATTTCTAA CGTGGACGAC 11490 ATGATATCGG CGGC QAAGA AAAAGAGAAG GGGGGCCCTT TCGAGGCCTC C~-lC~-l~l GG 11550 TTCTCCCCCA CTGCTGCGCT CTCTGGCCAA TA~LLG~l~A CCCTGAAAAT CGGGAGATTT 11790 GCGCAAACAG ~L~lC~lAAC TCTAGAAGTT AACGATCGCT GTTTAAAGAT CGG~-LCGCAG 11850 CTTAACTTTT TA~C~lCGAA ATGCTGGACA ACAGAACAGT ATCAGACTGG ATTTCAAGGC 11910 GACC~LC~lC CAG~TAGCGT CCCGCAAGAA AllCCCG~l~ TAACCAAGAA AGCGGAAGGG 12090 CGCACCCCGG ACGCAGAAAG CAGCGAAAAG AAGGCCCCTC CAGAAGACTC GGAGGACGAC '2150 ATGCAGGCAG AGG~ll~rGG AGA~AATCCT GC~GCC~lCC CCGAAGACGA CGAAGTCCCC 12210 GAGGACACCG AGCACGATGA TCC~AACTCG GATCCTGACT ATTACAATGA CATGCCCGCC 12270 GTGATCCCGG TGGAGGAGAC TACTA~AAGT TCTAATGCCG TCTCCATGCC CATATTCGCG 12330 GC~llC~lAG CCTGCGCGGT CGCGCTCGTG GGGCTACTGG TTTGGAGCAT CGTAAAATGC 12390 GCGCGTAGCT AA TCGAGCCTAG AATAGGTGGT llCLL~lAC ATGCCACGCC 12442 Met Ala Ser Leu Leu Gly Thr Leu Ala Leu Leu Ala Ala Thr Leu Ala Pro Phe Gly Ala Met Gly Ile Val Ile Thr Gly Asn His Val TCC GCC ~GG ATT GAC GAC GAT CAC ATC GTG ATC GTC GCG CCT CGC CCC 12647 Ser Ala Arg Ile Asp Asp Asp His Ile Val Ile Val Ala Pro Arg Pro Glu Ala Thr Ile Gln Leu Gln Leu Phe Phe Met Pro Gly Gln Arg Pro 5~ 55 60 His Lys Pro Tyr Ser Gly Thr Val Arg Val Ala Phe Arg Ser Asp Ile Thr Asn Gln Cys Tyr Gln Glu Leu Ser Glu Glu Arg Phe Glu Asn Cys Thr His Arg Ser Ser Ser Val Phe Val Gly Cys Lys Val Thr Glu Tyr Thr Phe Ser Ala Ser Asn Arg Leu Thr Gly Pro Pro His Pro Phe Lys Leu Thr Ile Arg Asn Pro Arg Pro Asn Asp Ser Gly Met Phe Tyr Val Ile Val Arg Leu Asp Asp Thr Lys Glu Pro Ile Asp Val Phe Ala Ile CA 022l6l39 l997-09-22 Gln Leu Ser Val Tyr Gln Phe Ala Asn Thr Ala Ala Thr Arg Gly Leu Tyr Ser Lys Ala Ser Cys Arg Thr Phe Gly Leu Pro Thr Val Gln Leu Glu Ala Tyr Leu Arg Thr Glu Glu Ser Trp Arg Asn Trp Gln Ala Tyr Val Ala Thr Glu Ala Thr Thr Thr Ser Ala Glu Ala Thr Thr Pro Thr Pro Val Thr Ala Thr Ser Ala Ser Glu Leu Glu Ala Glu His Phe Thr Phe Pro Trp Leu Glu Asn Gly Val Asp His Tyr Glu Pro Thr Pro Ala Asn Glu Asn Ser Asn Val Thr Val Arg Leu Gly Thr Met Ser Pro Thr Leu Ile Gly Val Thr Val Ala Ala Val Val Ser Ala Thr Ile Gly Leu Val Ile Val Ile Ser Ile Val Thr Arg Asn Met Cys Thr Pro His Arg Lys Leu Asp Thr Val Ser Gln Asp Asp Glu Glu Arg Ser Gln Thr Arg 305 . 310 315 Arg Glu Ser Arg Lys Phe Gly Pro Met Val Ala Cys Glu Ile Asn Lys Gly Ala Asp Gln Asp Ser Glu Leu val Glu Leu Val Ala Ile Val Asn Pro Ser Ala Leu Ser Ser Pro Asp Ser Ile Lys Met ATGTGGCTCT CCAATCATTT CGAlL~l~l~A ATCTCCCAAT C~l~l~AAAA GGGGCAGTAT 13668 CGGACACGGA CTGGGAGGGG CGTACACGAT AGTTATATGG TACAGCAGAG GC~-l~l~AAC 13728 ACTTAGGAGG AGAATTCAGC CGGGGAGAGC CC~L~Ll~AG TAGGCTTGGG AGCATATTGC 13788 Met Asn Met Leu Val Ile Val Leu Ala Ser Cys Leu Ala Arg Leu Thr Phe Ala Thr Arg His Val Leu Phe Leu Glu Gly Thr Gln Ala Val = = --W 096/29396 PCT~US96103916 Leu Gly Glu Asp Asp Pro Arg Asn val Pro Glu Gly Thr Val Ile Lys TGG ACA A~A GTC CTG CGG AAC GCG TGC AAG ATG AAG GCG GCC GAT GTC 13980 Trp Thr Lys Val Leu Arg Asn Ala Cys Lys Met Lys Ala Ala Asp Val Cys Ser Ser Pro Asn Tyr Cys Phe His Asp Leu Ile Tyr Asp Gly Gly Lys Lys Asp Cys Pro Pro Ala Gly Pro Leu Ser Ala Asn Leu Val Ile Leu Leu Lys Arg Gly Glu Ser Phe Val Val Leu Gly Ser Gly Leu His Asn Ser Asn Ile Thr Asn Ile Met Trp Thr G1U Tyr Gly Gly Leu Leu TTT GAT CCT GTA ACT CGT TCG GAC GAG GGA ATC TAT TTT CGA C~G ATC 14220 Phe Asp Pro Val Thr Arg Ser Asp Glu Gly Ile Tyr Phe Arg Arg Ile Ser Gln Pro Asp Leu Ala Met Glu Thr Thr Ser Tyr Asn Val Ser Val Leu Ser His Val Asp Glu Lys Ala Pro Ala Pro His Glu Val Glu Ile Asp Thr Ile Lys Pro Ser Glu Ala His Ala His Val Glu Leu Gln Met Leu Pro Phe His Glu Leu Asn Asp Asn Ser Pro Thr Tyr Val Thr Pro Val Leu Arg Val Phe Pro Pro Thr Glu His Val Lys Phe Asn Val Thr TAT TCG TGC- TA~ GGG TTT GAT GT~ AAA GAG GAG TGC GAA GAA GTG AAA 14508 Tyr Ser Trp Tyr Gly Phe Asp Val Lys Glu Glu Cys Glu Glu Vai Lys CTG TTC GAG CCG TGC GTA TAC CA. CCT ACA GAC GGC AAA TGT AG TT. 14556 Leu Phe Glu Pro Cys Val Ty. His Pro Thr Asp ~1~ kys Cys Gln Phe Pro Ala Thr Asn Gln Arg Cys Leu Ile Gly Ser Val Leu Met Ala Glu Phe Leu Gly Ala Ala Ser Leu Leu Asp Cys Ser Arg Asp Thr Leu Glu Asp Cys His Glu Asn Arg Val Pro Asn Leu Arg Phe Asp Ser Arg Leu -W 096/29396 PCT~US96/03916 Ser Glu Ser Arg Ala Gly Leu Val Ile Ser Pro Leu Ile Ala Ile Pro AAA GTT TTG ATT ATA GTC GTT TCC GAC GGA GAC ATT TTG GG~ TGG AGC 14796 Lys Val Leu Ile Ile Val Val Ser Asp Gly Asp Ile Leu Gly TrF Ser TAC ACG GTG CTC GGG A~A CGT AAC AGT CCG CGC GTA GTA GTC GAA ACG 14844 Tyr Thr Val Leu Gly Lys Arg Asn Ser Pro Arg Val Val Val Glu Thr His Met Pro Ser Lys Val Pro Met Asn Lys Val Val Ile Gly Ser Pro Gly Pro Met Asp Glu Thr Gly Asn Tyr Lys Met Tyr Phe Val Val Ala Gly Val Ala Ala Thr Cys Val Ile Leu Thr Cys Ala Leu Leu Val Gly Lys Lys Lys Cys Pro Ala ~is Gln Met Gly Thr Phe Ser Lys Thr Glu Pro Leu Tyr Ala Pro Leu Pro Lys Asn Glu Phe Glu Ala Gly Gly Leu Thr Asp Asp Glu Glu Val Ile Tyr Asp Glu val Tyr Glu Pro Leu Phe Arg Gly Tyr Cys Lys Gln Glu Phe Arg Glu Asp Val Asn Thr Phe Phe Gly Ala Val Val Glu Gly Glu Arg Ala Leu Asn Phe Lys Ser Ala Ile Ala Ser Met Ala Asp Ars Ile Leu Ala Asn Lys Ser Gly Arg Arg Asn Met Asp Ser Tyr . Met Pro Phe Lys Thr Arg Gly Ala Glu Asp Ala Ala Ala.Gly Lys Asn Arg Phe Lys Lys Ser Arg Asn Arg Glu Ile Leu Pro Thr Arg Leu Arg Gly Thr Gly Lys Lys Thr Ala Gly Leu Ser Asn Tyr Thr Gln Pro Ile Pro Trp Asn Pro Lys Phe Cys Ser Ala Arg Gly Glu Ser Asp Asn ~i8 Ala Cys Lys Asp Thr Phe Tyr Arg Arg W 096/29396 PCT~US96/03916 -15~-Thr Cys Cys Ala Ser Arg Ser Thr Val Ser Ser Gln Pro Asp Ser Pro His Thr Pro Met Pro Thr Glu Tyr Gly Arg Val Pro Ser Ala Lys Arg Lys Lys Leu Ser Ser Ser Asp Cys Glu Gly Ala His Gln Pro Leu Val TCC TGT AAA CTT CCG GAT TCT CAA GCA GCA CCG GCG CGA ACC TA~ AGT 15708 Ser Cys Lys Leu Pro Asp Ser Gln Ala Ala Pro Ala Arg Thr Tyr Ser Ser Ala Gln Arg Tyr Thr Val Asp Glu Val Ser Ser Pro Thr Pro Pro Gly Val Asp Ala Val Ala Asp Leu Glu Thr Arg Ala Glu Leu Pro Gly GCT ACG ACG GAA CAA ACG GAA AGT AAA AAT AAG CTC CCC AAC CAA CAA. 15852 Ala Thr Thr Glu Gln Thr Glu Ser Lys Asn Lys Leu Pro Asn Gln Gln Ser Arg Leu Lys Pro Lys Pro Thr Asn Glu His Val Gly Gly Glu Arg Cys Pro Ser Glu Gly Thr Val Glu Ala Pro Ser Leu Gly Ile Leu Ser Arg Val Gly Ala Ala Ile Ala Asn Glu Leu Ala Arg Met Arg Arg Ala Cys Leu Pro Leu Ala Ala Ser Ala Ala Ala Ala Gly Ile Val Ala Trp Ala Ala Ala Arg Ala Leu Gln Lys Gln Gly Arg Met Ser Lys Cys Tyr TGT CTC GCG CGC CAT CTT.TAT AAA AGC CCG CGT TGC GTG GGC CGG CGG 16191 Cys Leu Ala Arg His Leu Tyr Lys Se_ Pro Arg Cys Val Gly Arg Arg Val Ala Phe Gly Gly Leu Ala Thr Met Ser Arg Pro Pro Thr Ser His Leu Asp Leu Ala Phe Ser Ala Ala Phe Arg Gly.Thr Asp Leu Pro Gly 40 . 45 50 Gly Arg Phe Trp Arg Ala Ser Gln Ser Cys Asp Ile Phe Phe Trp Pro W 096/29396 PCTrUS96/03916 Asp Leu Ala Ala Val Ile Val Gln Ala Ala Arg Ala Tyr Phe Glu Gly Lys Glu Arg Leu Gly Ser Leu Gln Val Ala Glu Asp Ile Thr Ala His Asp Pro Arg Ile Ala Pro Ala Ala Lys Arg Ala Val Ala Ala Ala Val Gly Leu Trp Thr Ala Leu Ser Glu Leu Val Gly Gly Pro Asn Gly Glu Leu Glu Ser Lys Val Trp Gly Lys Gln Ile Pro Arg Ala Ala Ala Trp GAA ATA AGA GAC GTG CCC A~A GTT CCA GTC ATT GGG CCG GAC ATT CTT 16623 Glu Ile Arg Asp Val Pro Lys Val Pro Val Ile Gly Pro Asp Ile Leu Ser Phe Phe Ser Ala Ala Val Glu Leu Pro Val Leu Tyr Ile Arg Ala Arg Gly Gly Ala His Ser Arg Ser Ala His Trp Asn Asn Gln e_ Ser Ala Pro Ala Ala Gly Leu Ala Ala Ile Arg Ile Gly Met Glu Met Val Arg Ser Leu Leu Val Ile Ala Leu Pro Leu Ser Asn Phe Thr Leu Pro Glu Asp Leu Pro Glu Gly Ser Gln Asn Ser Ile Arg Ala Phe Val Ala His Leu Met Asn Cys Val Ala Thr Asp Lys Ile Met Ser Pro Asp Val Arg Val Pro Val Glu Glu Ser Phe Tyr Ser His Cys Leu Arg Glu Ile Ile Met Cys Glu Arg Ala Phe Cys Tyr Pro Cys Asn Pro Pro Pro Lys TGG TGA GCTCAGGGTC CCATTTACCC CCGCAACACC ~l~LCGCCGC CAGGGCGCGC 17063 ~ Trp ~ 295 GCTCTATCTT TCTCTATGTC CCGTCGCCAC CGGC~LAACC GAACGG-LG~A ACGGGGCCGC 17123 CCGGGAAAGC CTAGCTCCGC ACAGACACAG ACAGACAAAC GGC~l ~A TTGCAACAAC 17183 GTGA~AAACA CACAATAATA TTACTTTATT TAlll~CAG CG~lCGC~lG LCG~LC~lll 17243 CTGGGGGGGG GGGGGG~llG ~1 ~'l''L'l CGCTG CCGCGGAGTG GGTGAGGGGG GAGAGTGGAC 17303 CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 GGGCGTCATT ~'-1~CC~'1'~AA CGGCGCCGCG CCAGCGCCAG CGCGATTCCA GC~1CC~1~G 17423 CA~AGATCGA GGCGCTCACT TCGGGAGAGT TGGCGGCAGC CGCGCCGGCG CGCAAGGCGG 17483 CCAGGTATTC GCGACGGCGC ATCTCCGATT GCGGGACTCT CCCC~'LCCCG ACGCAGAAAA 17543 GACTCTGCAC GTAAATCGAG TCGTACAAAC ACG~1-1~GCG GCACGGCCGA GGGTGCCACA 17603 GGTCGGCCGG CAG~-1~-1CGG GACAGCCAGG ATAACAGTTC ~1L1~L~ATC ACGAAACGCA 17723 TATTCTCGCT CTGCATGGTA CGGL'1'~'1C~-1' CCTCGAAGCG GTCACCGTCC AGAGAGTCGA 17783 GCCAGA~ATC GCC~1CGATC CCTTCCATTG C~'1C~-1'~'LAA ~1L~1CC~AAC CC~-L~1~GCCGT 17843 TAL'1'CGG~'LC AGTGGGGGAG TTGGCGACGC '1~'1CC~-LCGA CGCCCACCGC GGCCTCAAAC 17903 CAATCAGCGC GTTGCGGCGG 1'~'1'~''1CGCTG ACGTGAACTC TCCGTAACCG lC~-Ll~AGTT 18023 CGGACGATAT CTCGACTAAC TCGCGCCAGA GCCGCGAGGC GATCCGAATG CACGG~L~LA 18083 CCGAGTCGGC CGACTCCGAC GAGGCGACAG CGGCACCTTG CCGC~-1GCGC TCGCCACGAG 18143 GCA'1'~1L'1'1C CATTTAGGGA 1~1~1-1AGA GGAGAGCGGT CCGGGCGGTC TACTGCGGTT 18263 GAGGGGGAGG TTCTGCCCCG C~'1~1C~1~'1' ATCTGCTCGT CGAGGCCTCG GCCTTGCGTC 18503 GCCGTGCAGG GGTCGAGG-C G~11-1 1~A 1 TTTTACTTCT ~L~lCGGAT l~l~-~l~AG 18563 AAGCCTTCCG TGG~'1-~ L 1 lG CGGGTGCCGC GCACCGC~AT ~AACGCACGGA CGCGGGGGAT 18683 A~CAAATGGC GGCGGCGCCG GAGAGCTGTC GTCAATAAAG TCTAAGTCAG ATTGCGTGGG 18743 CTCTGACTCG GTGGAGCTGT GTCCCGTGTC ClC-l~GCCC ~AGTCCACTC CCCGGCACCC 18803 AGGCTGCTCT ~LC~-1CCGACT CCGGGTCGCT CCAGCTCCTC CCGC~'1'GCCG ~'1'1-~'L1C~-LC 18863 (2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 27B amino acids (B) TYPE amino acid (D) TOPOLOGY linear (ii) MOLECULE TYPE protein (Xi) SEQUENCE DESCRIPTION SEQ ID NO 60 CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 -15~-Met Glu Asn Met Leu Asp Gly Cys Tyr Pro Leu Ala Leu Met Asp Ser Asp His Ile Thr Ala His Ala Val Pro Arg Gly Glu Arg Arg Arg Gln Gly Ala Ala Val Ala Ser Ser Glu Ser Ala Asp Ser Val Asp Pro Cys Ile Arg Ile Ala Ser Arg Leu Trp Arg Glu Leu Val Glu Ile Ser Ser Glu Leu Lys Asp Gly Tyr Gly Glu Phe Thr Ser Ala Arg Asp Arg Arg ~65 70 75 80 Asn Ala Leu Ile Ala Ala Asn Glu Arg Leu Arg Ser Ala Phe Leu Gly Ala Ser Arg Ala Thr Arg Gly Leu Gly Leu Arg Pro Arg Trp Ala Ser Thr Glu Ser Val Ala Asn Ser Pro Thr Asp Pro Asn Asn Gly Asn Gly Leu Gly Glu Leu Glu Glu Ala Met Glu Gly Ile Glu Gly Asp Phe Trp Leu Asp Ser Leu Asp Gly Asp Arg Phe Glu Asp Glu Ser Arg Thr Met Gln Ser Glu Asn Met Arg Phe Val Ile Glu Lys Glu Leu Leu Ser Trp Leu Ser Arg His Leu Pro Ala Asp Leu Ala Ser Ala Glu Arg Glu Thr Ser Arg Ser Leu Leu Ala Ala Gly His Trp Cys Cys Leu Trp His Pro Arg Pro Cys Arg Glu Ala Cys Leu Tyr Asp Ser Ile Tyr Val Gln Ser Leu Phe Cys Val Gly Thr Gly Arg Val Pro Gln Ser Glu Met Arg Arg Arg Glu Tyr Leu Ala Ala Leu Arg Ala Gly Ala Ala Ala Ala Asn Ser Pro Glu Val Ser Ala Ser Ile Phe Ala Arg Asp Ala Gly Ile Ala Leu Ala Leu Ala Arg Arg Arg ~2) INFORMATION FOR SEQ ID NO:61.
U~N~ CHARACTERISTICS:
(A) LENGTH: 294 amino acids (B) TYPE: amino acid ~ (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~yu~N~: DESCRIPTION: SEQ ID NO:61:

CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 Met Ser Lys Cys Tyr Cys Leu Ala Arg His Leu Tyr Lys Ser Pro Arg Cys Val Gly Arg Arg Val Ala Phe Gly Gly Leu Ala Thr Met Ser Arg Pro Pro Thr Ser His Leu Asp Leu Ala Phe Ser Ala Ala Pne Arg Gly Thr Asp Leu Pro Gly Gly Arg Phe Trp Arg Ala Ser Gln Ser Cys Asp Ile Phe Phe Trp Pro Asp Leu Ala Ala Val Ile Val Gln Ala Ala Arg ~la Tyr Phe Glu Gly Lys Glu Arg Leu Gly Ser Leu Gln Val Ala Glu ~sp Ile Thr Ala His Asp Pro Arg Ile Ala Pro Ala Ala Lys Arg Ala Val Ala Ala Ala Val Gly Leu Trp Thr Ala Leu Ser Glu Leu Val Gly Gly Pro Asn Gly Glu Leu Glu Ser Lys Val Trp Gly Lys Gln Ile Pro Arg Ala Ala Ala Trp Glu Ile Arg Asp Val Pro Lys Val Pro Val Ile ~ly Pro Asp Ile Leu Ser Phe Phe Ser Ala Ala Val Glu Leu Pro Val ~eu Tyr Ile Arg Ala Arg Gly Gly Ala His Ser Arg Ser Ala His Trp Asn Asn Gln Ser Ser Ala Pro Ala Ala Gly Leu Ala Ala Ile Arg Ile Gly Met Glu Met Val Arg Ser Leu Leu Val Ile Ala Leu Pro Leu Ser Asn Phe Thr Leu Pro Glu Asp Leu Pro Glu Gly Ser Gln Asn Ser Ile ~rg Ala Phe Val Ala ~is Leu Met Asn Cys Val Ala Thr Asp Lys Ile ~et Ser Pro Asp Val Arg Val Pro Val Glu Glu Ser Phe Tyr Ser His ~ys Leu Arg Glu Ile Ile Met Cys Glu Arg Ala Phe Cys Tyr Pro Cys Asn Pro Pro Pro Lys Trp (2) INFORMATION FOR SEQ ID NO:62:

(i) S~u~N~ CHARACTERISTICS:
IA) LENGTH: 229 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein CA 022l6l39 l997-09-22 W 096/29396 PCT~US96/03916 (xi) S~yu~ DESCRIPTION: SEQ ID NO:62:
Met Ala Pro Val Lys Val Thr Ile Val Ser Ala Val Asp Ser His Tyr ~ys Leu Pro Asn Ser Arg Phe Glu Leu Ser Asp Ser Gly Trp Lys Glu Leu Val His Ala Val Lys Thr Met Ala Ser Tyr Asp Arg Pro Ser Thr Leu Ser Val Ile Val Arg Pro Ala Ser Leu Tyr Glu Val Ser Gly Glu Leu Phe Ser Leu Pro Arg Met Cys Arg Pro Val Ile Arg Phe Gly Glu ~ly Gly Asp Pro Pro Gly Val Ser Pro Glu Trp Ser Gly Leu Asp Ala ~ly Phe Tyr His Leu Ser Ser Gly Ala Tyr Ala Ala Lys Glu Phe His Leu Trp Val Leu Gly Thr Ala Asp Ile Cys Met Ala Ala Leu Asn Leu Pro Ala Pro Lys Thr Phe Leu ~ e Thr Glu Thr Gly Gly Lys Asn Phe Glu Arg Gly Val Glu Ile Phe Leu Val Asn Gly Asp Lys Thr Thr Leu ~er Leu Ser His Pro Ser Val Trp Thr Thr Leu Ala Pro Ser Ser Leu ~rg Thr Pro Trp Pro Tyr Ser Thr Val Lys Phe Leu Lys Val Lys Pro Asn Ser Ala Ala Tyr Cys Val Ser Asp Ser Asp Asp Gly Glu Arg Gln Pro Lys Phe Phe Leu Gly Ser Leu Phe Lys Ser Lys Lys Pro Arg Ser Pro Arg Arg Arg Arg t2) INFORMATION FOR SEQ ID NO:63:
(i) S~Qu~N~ CHARACTERISTICS:
tA) LENGTH: 476 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) S~yu~ DESCRIPTION: SEQ ID NO:63:

Met Arg Phe Arg Arg Ile Cys Ser Arg Ser Arg Ala Glu Lys Arg Arg ~rg Thr Thr Glu Asn Pro Leu Thr Ser Lys Arg Val Cys Val Leu Asp ~er Phe Ser Arg Thr Met Ser Leu Arg Pro Tyr Ala Glu Ile Leu Pro Thr Ala Glu Gly Val Glu Arg Leu Ala Glu Leu Val Ser Val Thr Met Thr Glu Arg Ala Glu Pro Val Thr Glu Asn Thr Ala Val Asn Ser Ile ~ro Pro Ala Asn Glu Asn Gly Gln Asn Phe Ala Tyr Ala Gly Asp Gly ~ro Ser Thr Thr Glu Lys Val Asp Gly Ser His Thr Asp Phe Asp Glu Ala Ser Ser Asp Tyr Ala Gly Pro Val Pro Leu Ala Gln Thr Arg Leu Lys His Ser Asp Glu Phe Leu Gln HiS Phe Arg Val Leu Asp Asp Leu Val Glu Gly Ala Tyr Gly Phe Ile Cys Asp Val Arg Arg Tyr Thr Glu ~lu Glu Gln Arg Arg Arg Gly Val Asn Ser Thr Asn Gln Gly Lys Ser ~ys Cys Lys Arg Leu Ile Ala Lys Tyr Val Lys Asn Gly Thr Arg Ala Ala Ser Gln Leu Glu Asn Glu Ile Leu Val Leu Gly Arg Leu Asn His Glu Asn Val Leu Lys Ile Gln Glu Ile Leu Arg Tyr Pro Asp Asn Thr Tyr Met Leu Thr Gln Arg Tyr Gln Phe Asp Leu Tyr Ser Tyr Met Tyr ~sp Glu Ala Phe Asp Trp Lys Asp Ser Pro Met Leu Lys Gln Thr Arg 2~5 250 255 ~rg Ile Met Lys Glr. Leu Mee Ser Ala Val Ser Tyr Ile H-s Ser LYR

Lys Leu Ile His Arg Asp Ile Lys Leu :;lu }:.sn Ile Phe Leu Asn Cys Asp Gly Lys Thr Val Leu Gly Asp Phe Gly Thr Val Thr Pro Phe Glu Asn Glu Arg Glu Pro Phe Glu Tyr Gly Trp Val Gly Thr Val Ala Thr ~sn Ser Pro Glu Ile Leu Ala Arg Asp Ser Tyr Cys Glu Ile Thr Asp ~le Trp Ser Cys Gly Val Val Leu Leu Glu Met Val Ser His Glu Phe Cys Pro Ile Gly Asp Gly Gly Gly Asn Pro His Gln Gln Leu Leu Lys Val Ile Asp Ser Leu Ser Val Cys Asp Glu Glu Phe Pro Asp Pro Pro Cys Asn Leu Tyr Asn Tyr Leu His Tyr Ala Ser Ile Asp Arg Ala Gly W 096/29396 PCTrUS96/03916 His Thr Val Pro Ser Leu Ile Arg Asn Leu His Leu Pro Ala Asp Val ~lu Tyr Pro Leu Val Lys Met Leu Thr Phe Asp Trp Arg Leu Arg Pro Ser Ala Ala Glu Val Leu Ala Met Pro Leu Phe Ser Ala Glu Glu Glu Arg Thr Ile Thr Ile Ile His Gly Lys His Lys Pro Ile Arg Pro Glu Ile Arg Ala Arg Val Pro Arg Ser Met Ser Glu Gly (2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 623 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) S~U~N~: DESCRIPTION: SEQ ID NO:64:
Met Thr Leu Pro His Arg Leu Thr Lys Arg Pro Phe Ala Arg Arg Phe ~ys Ser Val Phe Val Ile His Tyr Ser Glu Thr Lys Leu Asp Arg Tyr Asn Lys Thr Met Leu Leu Tyr Arg Pro Asp Ser Thr Met Arg His Ser Gly Gly Asp Ala Asn His Arg Gly Ile Arg Pro Arg Arg Lys Ser Ile Gly Ala Phe Ser Ala Arg Glu Lys Thr Gly Lys Arg Asn Ala Leu Thr ~lu Ser Ser Ser Ser Ser Asp Met Leu Asp Pro Phe Ser Thr Asp Lys ~lu Phe Gly Gly Lys Trp Thr Val Asp Gly Pro Ala Asp Ile Thr Ala Glu Val Leu Ser Gln Ala Trp Asp Val Leu Gln Leu Val Lys His Glu Asp Ala Glu Glu Glu Arg Val Thr Tyr Glu Ser Lys Pro Thr Pro Ile Gln Pro Phe Asn Ala Trp Pro Asp Gly Pro Ser Trp Asn Ala Gln Asp ~he Thr Arg Ala Pro Ile Val Tyr Pro Ser Ala Glu Val Leu Asp Ala ~lu Ala Leu Lys Val Gly Ala Phe Val Ser Arg Val Leu Gln Cys Val ~ro Phe Thr Arg Ser Lys Lys Ser Val Thr Val Arg Asp Ala Gln Ser Phe Leu Gly Asp Ser Phe Trp Arg Ile Met Gln Asn Val Tyr Thr Val Val Leu Arg Gln His Ile Thr Arg Leu Arg His Pro Ser Ser Lys Ser ~le Val Asn Cys Asn Asp Pro Leu Trp Tyr Ala Tyr Ala Asn Gln Phe ~is Trp Arg Gly Met Arg Val Pro Ser Leu Lys Leu Ala Ser Pro Pro Glu Glu Asn Ile Gln His Gly Pro Met Ala Ala Val Phe Arg Asn Ala Gly Ala Gly Leu Phe Leu Trp Pro Ala Met Arg Ala Ala Phe Glu Glu Arg Asp Lys Arg Leu Leu Arg Ala Cys Leu Ser Ser Leu Asp Ile Met ~sp Ala Ala Val Leu Ala Ser Phe Pro Phe Tyr Trp Arg Gly Val Gln ~sp Thr Ser Arg Phe Glu Pro Ala Leu Gly Cys Leu Ser Glu Tyr Phe Ala Leu Val Val Leu Leu Ala Glu Thr Val Leu Ala Thr Met Phe Asp His Ala Leu Val Phe Met Arg Ala Leu Ala Asp Gly A~n Phe Asp Asp Tyr Asp Glu Thr Arg Tyr Ile A~p Pro Val Lys A~n Glu Tyr Leu Asn ~ly Ala Glu Gly Th- Leu Leu Arg Gly Ile Val Ala Ser Asn Thr Ala ~eu Ala Val Val Cys Ala Asn Thr Tyr Ser Thr Ile Arg Lys Leu Pro Ser Val Ala Thr Ser Ala Cys Asn Val Ala Tyr Arg Thr Glu Thr Leu Lys Ala Arg Arg Pro Gly Met Ser Asp Ile Tyr Arg Ile Leu Gln Lys Glu Phe Phe Phe Tyr Ile Ala Trp Leu Gln Arg Val Ala Thr His Ala ~sn Phe Cys Leu Asn Ile Leu Lys Arg Ser Val Asp Thr Gly Ala Pro ~ro Phe Leu Phe Arg Ala Ser Ser Glu Lys Arg Leu Gln Gln Leu A~n Lys Met Leu Cys Pro Leu Leu Val Pro Ile Gln Tyr Glu Asp Phe Ser Ly~ Ala Met Gly Ser Glu Leu Lys Arg Glu Lys Leu Glu Thr Phe Val Lys Ala Ile Ser Ser Asp Arg Asp Pro Arg Gly Ser Leu Arg Phe Leu CA 022l6l39 l997-09-22 Ile Ser Asp His Ala Arg Glu Ile Ile Ala Asp Gly Val Arg Phe Lys Pro Val Ile Asp Glu Pro Val Arg Ala Ser Val Ala Leu Ser Thr Ala Ala Ala Gly Lys Val Lys Ala Arg Arg Leu Thr Ser Val Arg Ala Pro Val Pro Gly Ala Gly Ala Val Ser Ala Arg Arg Lys Ser Glu Ile (2) INFOR~.A~ION FOR S~Q ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 292 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) S~yu~ DESCRIPTION: SEQ ID NO:65:
Met Ser Gly Phe Ser Asn Ile Gly Ser Ile Ala Thr Val Ser Leu Val Cys Ser Leu Leu Cys Ala Ser Val Leu Gly Ala Pro Val Leu Asp Gly Leu Glu Ser Ser Pro Phe Pro Phe Gly Gly Lys Ile Ile Ala Gln Ala Cys Asn Arg Thr Thr Ile Glu Val Thr Val Dro Trp Ser Asp Tyr Ser Gly Arg Thr Glu Gly Val Ser Val Glu Val Lys Trp Phe Tyr Gly Asn 65. 70 75 80 Ser Asn Pro Glu Ser Phe Val Phe Gly Val Asp Ser Glu Thr Gly Ser Gly His Glu Asp Leu Ser Thr Cys Trp Ala Leu Ile His Asn Leu Asn Ala Ser Val Cys Arg Ala Ser Asp Ala Gly Ile Pro Asp Phe Asp Lys Gln Cys Glu Lys Val Gln Arg Arg Leu Arg Ser Gly Val Glu Leu Gly Ser Tyr Val Ser Gly Asn Gly Ser Leu Val Leu Tyr Pro Gly Met Tyr Asp Ala Gly Ile Tyr Ala Tyr Gln Leu Ser Val Gly Gly Lys Gly Tyr ~ Thr Gly Ser Val Tyr Leu Asp Val Gly Pro Asn Pro Gly Cys His Asp ; Gln Tyr Gly Tyr Thr Tyr Tyr Ser Leu Ala Asp Glu Ala Ser Asp Leu Ser Ser Tyr Asp Val Ala Ser Pro Glu Leu Asp Gly Pro Met Glu Glu W 096/29396 PCTrUS96103916 Asp Tyr Ser Asn Cys Leu Asp Met Pro Pro Leu Arg Pro Trp Thr Thr ~al Cys Ser His Asp Val Glu Glu Gln Glu Asn Ala Thr Asp Glu Leu ~yr Leu Trp Asp Glu Glu Cys Ala Gly Pro Leu Asp Glu Tyr Val Asp ~lu Arg Ser Glu Thr Met Pro Arg Met Val Val Phe Ser Pro Pro Ser Thr Leu Gln Gln (2) INFORMATION FOR SEQ ID NO:66:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 985 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~yu~N~: DESCRIPTION: SEQ ID NO:66:
Met Gly Thr Met Leu Val Leu Arg Leu Phe Leu Leu Ala Val Ala Asp ~la Ala Leu Pro Thr Gly Arg Phe Cys Arg val Trp Lys Val Pro Pro Gly Gly Thr Ile G~n Glu Asn Leu Ala Val Leu Ala Glu Ser Pro Val Thr Gly His Ala Thr Tyr Pro Pro Pro Glu Gly Ala Val Ser Phe Gln Ile Phe Ala Asp Thr Pro Thr Leu Arg Ile Arg Tyr Gly Ala Thr Glu ~sp Glu Leu Ala Leu Glu Arg Gly Thr Ser Ala Ser Asp Ala Asp Asn ~al Thr Phe Ser Leu Ser Tyr Arg Pro Arg Pro Glu Ile His Gly Ala Tyr Phe Thr Ile Gly Val Phe Ala Thr Gly Gln Ser Thr Glu Ser Ser Tyr Ser Val Ile Ser Arg Val Leu Val Asn Ala Ser Leu Glu Arg Ser Val Arg Leu Glu Thr Pro Cys Asp Glu Asn Phe Leu Gln Asn Glu Pro ~hr Trp Gly Ser Lys Arg Trp Leu Gly Pro Pro Ser Pro Tyr Val Arg ~sp Asn Asp Val Ala Val Leu Thr Lys Ala Gln Tyr Ile Gly Glu Cys ~yr Ser Asn Ser Ala Ala Gln Thr Gly Leu Thr Ser Leu Asn Met Thr CA 022l6l39 l997-09-22 W 096/29396 PCT~US96/03916 Phe Phe Tyr Ser Pro Lys Arg Ile Val Asn Val Thr Trp Thr Thr Gly Gly Pro Ser Pro Ser Arg Ile Thr Val Tyr Ser Ser Arg Glu Asn Gly ~ln Pro Val Leu Arg Asn Val Ser Asp Gly Phe Leu Val Lys Tyr Thr ~ro Asp Ile Asp Gly Arg Ala Met Ile Asn Val Ile Ala Asn Tyr Ser Pro Ala Asp Ser Gly Ser Val Leu Ala Phe Thr Ala Phe Arg Glu Gly Lys Leu Pro Ser Ala Ile Gln Leu His Arg Ile Asp Met Ser Gly Thr Glu Pro Pro Gly Thr Glu Thr Thr Phe Asp Cys Gln Lys Met Ile Glu ~hr Pro Tyr Arg Ala Leu Gly Ser Asn Val Pro Arg Asp Asp Ser Ile ~rg Pro Gly Ala Thr Leu Pro Pro Phe Asp Thr Ala Ala Pro Asp Phe Asp Thr Gly Thr Ser Pro Thr Pro Thr Thr Val Pro Glu Pro Ala Ile Thr Thr Leu Ile Pro Arg Ser Thr Ser Asp Met Gly Phe Phe Ser Thr Ala Arg Ala Thr Gly Ser Glu Thr Leu Ser Val Pro Val Gln Glu Thr ~sp Arg Thr Leu Ser Thr Thr Pro Leu Thr Leu Pro Leu Thr Pro Gly ~lu Ser Glu Asn Thr Leu Phe Pro Thr Thr Ala Pro Gly Ile Ser Thr Glu Thr Pro Ser Ala Ala ~is Glu Thr Thr Gln Thr Gln Ser Ala Glu 435 4~0 445 Thr Val Val Phe Thr Gln Ser Pro Ser Thr Glu Ser Glu Thr Ala Arg Ser Gln Ser Gln Glu Pro Trp Tyr Phe Thr Gln Thr Pro Ser Thr Glu ~ln Ala Ala Leu Thr Gln Thr Gln Ile Ala Glu Thr Glu Ala Leu Phe 48c 4~O 495 ~hr Gln Thr Pro Ser Ala Glu Gln Met Thr Phe Thr Gln Thr Pro Gly ~la Glu Thr Glu Ala Pro Ala Gln Thr Pro Ser Thr Ile Fro Glu Ile Phe Thr Gln Ser Arg Ser Thr Pro Pro Glu Thr Ala Arg Ala Pro Ser Ala Ala Pro Glu Val Phe Thr Gln Ser Ser Ser Thr Val Thr Glu Val CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 Phe Thr Gln Thr Pro Ser Thr Val Pro Lys Thr Thr Leu Ser Ser Ser Thr Glu Pro Ala Ile Phe Thr Arg Thr Gln Ser Ala Gly Thr Glu Ala Phe Thr Gln Thr Ser Ser Ala Glu Pro A~p Thr Met Arg Thr Gln Ser Thr Glu Thr His Phe Phe Thr Gln Ala Pro Ser Thr Val Pro Lys Ala Thr Gln Thr Pro Ser Thr Glu Pro Glu Val Leu Thr Gln Ser Pro Ser Thr Glu Pro Val Pro Phe Thr Arg Thr Leu Gly Ala Glu Pro Glu Ile Thr Gln Thr Pro Ser Ala Ala Pro Glu Val Tyr Thr Arg Ser Ser Ser Thr Met Pro Glu Thr Ala Gln Ser Thr Pro Leu Ala Ser Gln Asn Pro Thr Ser Ser Gly Thr Gly Thr His Asn Thr Glu Pro Arg Thr Tyr Pro Val Gln Thr Thr Pro His Thr Gln Lys Leu Tyr Thr Glu Asn Lys Thr Leu Ser Phe Pro Thr Val Val Ser Glu Phe His Glu Met Ser Thr Ala Glu Ser Gln Thr Pro Leu Leu Asp Val Lys Ile Val Glu Val Lys Phe Ser Asn As~ Gly Glu Val Thr Ala Thr Cvs Val Ser Thr Val Lys Ser 75~V 760 765 Pro Tyr Arg Val Glu Thr Asn Trp Lys Val Asp Leu Val Asp Val Met Asp Glu Ile Ser Gly Asn Ser Pro Ala Gly Val Phe Asn Ser Asn Glu Lys Trp Gln Lys Gln Leu Tyr Tyr Arg Val Thr Asp Gly Arg Thr Ser Val Gln Leu Met Cys Leu Ser Cys Thr Ser His Ser Pro Glu Pro Tyr Cys Leu Phe Asp Thr Ser Leu Ile Ala Arg Glu Lys Asp Ile Ala Pro Glu Leu Tyr Phe Thr Ser Asp Pro Glr. Thr Ala Tyr Cys Thr Ile Thr Leu Pro Se~ Gly Val Val Pro Arg Phe Glu Trp Ser Leu Asn Asn Val Ser Leu Pro Glu Tyr Leu Thr Ala Thr Thr Val Val Ser His Thr Ala Gly Gln Ser Thr Val Trp Lys Ser Ser Ala Arg Ala Gly Glu Ala Trp goo 905 910 Ile Ser Gly Arg Gl y Gly Asn Ile Tyr Glu Cys Thr Val Leu Ile Ser Asp Gly Thr Arg Val Thr Thr Arg Lys Glu Arg Cys Leu Thr Asn Thr Trp Ile Ala Val Glu Asn Gly Ala Ala Gln Ala Gln Leu Tyr Ser Leu ~, 945 950 955 960 Phe Ser Gly Leu Val Ser Gly Leu Cys Gly Ser Ile Ser Ala Leu Tyr Ala Thr Leu Trp Thr Ala Ile Tyr Phe (2) INFORMATION FOR SEQ ID NO: 67:
yU~!;N~ : CHARACTERISTICS:
(A) LENGTH: 434 amino acids ~B) TYPE: amino acid ( D ) TOPOLOGY: l inear ( ii ) MOLECULE TYPE: protein (xi) ~ ;Qu~;N~ DESCRIPTION: SEQ ID NO:67:
Met His Arg Pro His Leu Arg Arg His Ser Arg Tyr Tyr Ala Lys Gly Glu Val Leu Asn Lys His Met Asp Cys Gly Gly Lys Arg Cys Cys Ser Gly Ala Ala Val Phe Thr Leu Phe Trp Thr Cys Val Arg Ile Met Arg Glu His Ile Cys Phe Val Arg Asn Ala Met Asp Arg His Leu Phe Leu Arg Asn Ala Phe Trp Thr Ile Val Leu Leu Ser Ser Phe Ala Ser Gln Ser Thr Ala Ala val Thr Tyr Asp Tyr Ile Leu Gly Arg Arg Ala Leu Asp Ala Leu Thr Ile Pro Ala Val Gly Pro Tyr Asn Arg Tyr Leu Thr Arg Val Ser Arg Gly Cys Asp Val Val Glu Leu Asn Pro Ile Ser Asn 115 1~0 125 Val Asp Asp Met Ile Ser Ala Ala Lys Glu Lys Glu Lys Gly Gly Pro Phe Glu Ala Ser Val Val Trp Phe Tyr Val Ile Lys Gly Asp Asp Gly Glu Asp Lys Tyr Cys Pro Ile Iyr Arg Lys Glu Tyr Arg Glu Cys Gly Asp Val Gln Leu Leu Ser Glu Cys Ala Val Gln Ser Ala Gln Met Trp t 180 185 190 Ala Val Asp Tyr Val Pro Ser Thr Leu Val Ser Arg Asn Gly Ala Gly CA 022l6l39 l997-09-22 Leu Thr Ile Phe Ser Pro Thr Ala Ala Leu Ser Gly Gln Tyr Leu Leu Thr Leu Lys Ile Gly Arg Phe Ala Gln Thr Ala Leu Val Thr Leu Glu 225 230 235 240 b ~al Asn Asp Arg Cys Leu Lys Ile Gly Ser Gln Leu Asn Phe Leu Pro ~er Lys Cys Trp Thr Thr Glu Gln Tyr Gln Thr Gly Phe Gln Gly Glu His Leu Tyr Pro Ile Ala Asp Thr Asn Thr Arg His Ala Asp Asp Val Tyr Arg Gly Tyr Glu Asp Ile Leu Gln Arg Trp Asn Asn Leu Leu Arg Lys Lys Asn Pro Ser Ala Pro Asp Pro Arg Pro Asp Ser Val Pro Gln ~lu Ile Pro Ala Val Thr Lys Lys Ala Glu Gly Arg Thr Pro Asp Ala ~lu Ser Ser Glu Lys Lys Ala Pro Pro Glu Asp Ser Glu Asp Asp Met Gln Ala Glu Ala Ser Gly Glu Asr Pro Ala Ala Leu Pro Glu Asp Asp Glu Val Pro Glu Asp Thr Glu His Asp Asp Pro Asn Ser Asp Pro Asp Tyr Tyr Asn Asp Met ~ro Ala Val Ile Pro Val Glu Glu Thr Thr Ly~

~er Ser Asn Ala Val Ser Met Pro Ile Phe Ala Ala Phe Val Ala Cys ~la Val Ala Leu Val Gly Leu Leu Val Trp Ser Ile Val Lys Cys Ala ~rg Ser (2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS-(A) LENGTH: 36~ amino acids (B) TYPE: a~ino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~yu~ DESCRIPTION: SEQ ID NO:68:

Met Ala Ser Leu Leu Gly Thr Leu Ala Leu Leu Ala Ala Thr Leu Ala ~ro Phe Gly Ala Met Gly Ile Val Ile Thr Gly Asn His Val Ser Ala ~rg Ile Asp Asp Asp His Ile Val Ile Val Ala Pro Arg Pro Glu Ala CA 022l6l39 l997-09-22 Thr Ile Gln Leu Gln Leu Phe Phe Met Pro Gly Gln Arg Pro Xis Lys Pro Tyr Ser Gly Thr Val Arg Val Ala Phe Arg Ser Asp Ile Thr Asn ~ln Cys Tyr Gln Glu Leu Ser Glu Glu Arg Phe Glu Asn Cys Thr His g0 95 ~rg Ser Ser Ser Val Phe Val Gly Cys Lys Val Thr Glu Tyr Thr Phe Ser Ala Ser Asn Arg Leu Thr Gly Pro Pro His Pro Phe Lys Leu Thr Ile Arg Asn Pro Arg Pro Asn Asp Ser Gly Met Fhe Tyr Val Ile Val 130 ;35 140 Arg Leu Asp Asp Thr Lys Glu Pro Ile Asp Val Phe Ala Ile Gln Leu ~er Val Tyr Gln Phe Ala Asn Thr Ala Ala Thr Arg Gly Leu Tyr Ser ~ys Ala Ser Cys Arg Thr Phe Gly Leu Pro Thr Val Gln Leu Glu Ala Tyr Leu Arg Thr Glu Glu Ser Trp Arg Asn Trp Gln Ala Tyr Val Ala Thr Glu Ala Thr Thr Thr Ser Ala G1U Ala Thr Thr Pro Thr Pro Val Thr Ala Thr Ser Ala Ser Glu Leu Glu Ala Glu His Phe Thr Phe Pro ~~p Leu Glu Asn Gly Val Asp His Tyr Glu Pro Thr Pro Ala Asn Glu ~sn Ser Asn Val Thr Val Arg Leu Gly Thr Met Ser Pro Thr Leu Ile Gly Val Thr Val Ala Ala Val Val Ser Ala Thr Ile Gly Leu Val Ile Val Ile Ser Ile Val Thr Arg Asn Met Cys Thr Pro ~is Arg Lys Leu Asp Thr Val Ser Gln Asp Asp Glu Glu Arg Ser Gln Thr Arg Arg Glu ~er Arg Lys Phe Gly Pro Met Val Ala Cys Glu Ile Asn Lys Gly Ala ~sp Gln Asp Ser Glu Leu Val Glu Leu Val Ala Ile Val Asn Pro Ser Ala Leu Ser Ser Pro Asp Ser Ile Lys Met (2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 499 amino acids (B) TYFE: amino acid (D) TOPOLOGY: linear CA 022l6l39 l997-09-22 W 096/29396 PCT~US96/03916 (ii) MOLECULE TYPE: protein Ixi) S~YU~N~ DESCRIPTION: SEQ ID NO:69:
Met Asn Met Leu Val Ile Val Leu Ala Ser Cys Leu Ala Arg Leu Thr ,~

Phe Ala Thr Arg His Val Leu Phe Leu Glu Gly Thr Gln Ala Val Leu 20 25 30 ~
Gly Glu Asp Asp Pro Arg Asrl Val Pro Glu Gly Thr Val Ile Lys Trp Thr Lys Val Leu Arg Asn Ala Cys Lys Met Lys Ala Ala Asp Val Cys Ser Ser Pro Asn Tyr Cys Phe His Asp Leu Ile Tyr Asp Gly Gly Lys ~ys Asp Cys Pro Pro Ala Gly Pro Leu Ser Ala Asn Leu Val Ile Leu ~eu Lys Arg Gly Glu Ser Phe Val Val Leu Gly Ser Gly Leu His Asn Ser Asn Ile Thr Asn Ile Met Trp Thr Glu Tyr Gly Gly Leu Leu Phe Asp Pro Val Thr Arg Ser Asp Glu Gly Ile Tyr Phe Arg Arg Ile Ser Gln Pro Asp Leu Ala Met Glu Thr Thr Ser Tyr Asn Val Ser Val Leu ~er His Val Asp Glu Lys Ala Pro Ala Pro His Glu Val Glu Ile Asp ~hr Ile Lys Pro Ser Glu Ala His Ala His Val Glu Leu Gln Met Leu Pro Phe His Glu Leu Asn Asp Asn Ser Pro Thr Tyr Val Thr Pro Val Leu Arg Val Phe Pro Pro Thr Glu His Val Lys Phe Asn Val Thr Tyr Ser Trp Tyr Gly Phe Asp Val Lys Glu Glu Cys Glu Glu Val Lys Leu 225 ~30 235 240 ~he Glu Pro Cys Val Tyr His Pro Thr Asp Gly Lys Cys Gln Phe Pro ~la Thr Asn Gln Arg Cys Leu Ile Gly Ser Val Leu Met Ala Glu Phe Leu Gly Ala Ala Ser Leu Leu Asp Cys Ser Arg Asp Thr Leu Glu Asp Cys His Glu Asn Arg Val Pro Asn Leu Arg Phe Asp Ser Arg Leu Ser Glu Ser Arg Ala Gly Leu Val Ile Ser Pro Leu Ile Ala Ile Pro Lys Val Leu Ile Ile Val Val Ser Asp Gly Asp Ile Leu Gly Trp Ser Tyr Thr Val Leu Gly Lys Arg Asn Ser Pro Arg Val Val Val Glu Thr His Met Pro Ser Lys Val Pro Met Asn Lys Val Val Ile Gly Ser Pro Gly Pro Met Asp Glu Thr Gly Asn Tyr Lys Met Tyr Phe Val Val Ala Gly Val Ala Ala Thr Cys Val Ile Leu Thr Cys Ala Leu Leu Val Gly Lys Lys Lys Cys Pro Ala His Gln Met Gly Thr Phe Ser Lys Thr Glu Pro Leu Tyr Ala Pro Leu Pro Lys Asn Glu Phe Glu Ala Gly Gly Leu Thr Asp Asp Glu Glu Val Ile Tyr Asp Glu Val Tyr Glu Pro Leu Phe Arg Gly Tyr Cys Lys Gln Glu Phe Arg Glu Asp Val Asn Thr Phe Phe Gly Ala Val Val Glu Gly Glu Arg Ala Leu Asn Phe Lys Ser Ala Ile Ala Ser Met Ala Asp Arg Ile Leu Ala Asn Lys Ser Gly Arg Arg Asn Met Asp Ser Tyr (2) INFORMATION FOR SEQ ID NO:70:
( i ) ~h~UhN~ CHARACTERISTICS:
(A) LENGTH: 260 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) S~uuhN~h DESCRIPTION: SEQ ID NO:70:

Met Pro Phe Lys Thr Arg Gly Ala Glu Asp Ala Ala Ala Gly Lys Asn Arg Phe Lys Lys Ser Arg Asn Arg Glu Ile Leu Pro Thr Arg Leu Arg Gly Thr Gly Lys Lys Thr Ala Gly Leu Ser Asn Tyr Thr Gln Pro Ile Pro Trp Asn Pro Lys Phe Cys Ser Ala Arg Gly Glu Ser Asp Asn His ~ Ala Cys Lys Asp Thr Phe Tyr Arg Arg Thr Cys Cys Ala Ser Arg Ser Thr Val Ser Ser Gln Pro Asp Ser Pro His Thr Pro Met Pro Thr Glu Tyr Gly Arg Val Pro Ser Ala Lys Arg Lys Lys Leu Ser Ser Ser Asp CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 Cys Glu Gly Ala His Gln Pro Leu Val Ser Cys Lys Leu Pro Asp Ser Gln Ala Ala Pro Ala Arg Thr Tyr Ser Ser Ala Gln Arg Tyr Thr Val Asp Glu Val Ser Ser Pro Thr Pro Pro Gly Val Asp Ala Val Ala Asp ~eu Glu Thr Arg Ala Glu Leu Pro Gly Ala Thr Thr Glu Gln Thr Glu 165 1~0 175 ~er Lys Asn Lys Leu Pro Asn Gln Gln Ser Arg Leu Lys Pro Lys Pro Thr Asn Glu His Val Gly Gly Glu Arg Cys Pro Ser Glu Gly Thr Val Glu Ala Pro Ser Leu Gly Ile Leu Ser Arg Val Gly Ala Ala Ile Ala Asn Glu Leu Ala Arg Met Arg Arg Ala Cys Leu Pro Leu Ala Ala Ser Ala Ala Ala Ala Gly Ile Val Ala Trp Ala Ala Ala Arg Ala Leu Gln ~ys Gln Gly Arg (2) INFORMATION FOR SEQ ID NO:71:
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 294 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECU~E TYPE: protein (xi) s~uu~ DESCRIPTION: SEQ ID NO:71:
Met Ser Lys Cys Tyr Cys Leu Ala Arg His Leu Tyr Lys Ser Pro Arg ~ys Val Gly Arg Arg Val Ala Phe Gly Gly Leu Ala Thr Met Ser Arg Pro Pro Thr Ser His Leu Asp Leu Ala Phe Ser Ala Ala Phe Arg Gly Thr Asp Leu Pro Gly Gly Arg Phe Trp Arg Ala Ser Gln Ser Cys Asp Ile Phe Phe Trp Pro Asp Leu Ala Ala Val Ile Val Gln Ala Ala Arg ~la Tyr Phe Glu Gly Lys Glu Arg Leu Gly Ser Leu Gln Val Ala Glu ~sp Ile Thr Ala His Asp Pro Arg Ile Ala Pro Ala Ala Lys Arg Ala ~al Ala Ala Ala Val Gly Leu Trp Thr Ala Leu Ser Glu Leu Val Gly CA 022l6l39 l997-09-22 W 096/29396 PCTrUS96/03916 Gly Pro Asn Gly Glu Leu Glu Ser Lys Val Trp Gly Lys Gln Ile Pro Arg Ala Ala Ala Trp Glu Ile Arg Asp Val Pro Lys Val Pro Val Ile Gly Pro Asp Ile Leu Ser Phe Phe Ser Ala Ala Val Glu Leu Pro Val Leu Tyr Ile Arg Ala Arg Gly Gly Ala His Ser Arg Ser Ala His Trp Asn Asn Gln Ser Ser Ala Pro Ala Ala Gly Leu Ala Ala Ile Arg Ile Gly Met Glu Met Val Arg Ser Leu Leu Val Ile Ala Leu Pro Leu Ser Asn Phe Thr Leu Pro Glu Asp Leu Pro Glu Gly Ser Gln Asn Ser Ile Arg Ala Phe Val Ala His Leu Met Asn Cys Val Ala Thr Asp Lys Ile Met Ser Pro Asp Val Arg Val Pro Val Glu Glu Ser Phe Tyr Ser His Cys Leu Arg Glu Ile Ile Me~ Cys Glu Arg Ala Phe Cys Tyr Pro Cys Asn Pro Pro Pro Lys Trp (2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 278 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MCLECULE ~YPE: protein (xi) SEQUENG DESCRIPTION: SEQ ID NC:72:
Met Glu Asn Met Leu Asp Gly Cys ~yr Pro Leu Ala Leu Met Asp Ser Asp His Ile Thr Ala His Ala Val Pro Arg Gly Glu Arg Arg Arg Gln Gly Ala Ala Val Ala Ser Ser Glu Ser Ala Asp Ser Val Asp Pro Cys Ile Arg Ile Ala Ser Arg Leu Trp Arg Glu Leu Val Glu Ile Ser Ser Glu Leu Lys Asp Gly Tyr Gly Glu Phe Thr Ser Ala Arg Asp Arg Arg t Asn Ala Leu Ile Ala Ala Asn Glu Arg Leu Arg Ser Ala Phe Leu Gly Ala Ser Arg Ala Thr Arg Gly Leu Gly Leu Arg Pro Arg Trp Ala Ser Thr Glu Ser Val Ala Asn Ser Pro Thr Asp Pro Asn Asn Gly Asn Gly Leu Gly Glu Leu Glu Glu Ala Met Glu Gly Ile Glu Gly Asp Phe T~p Leu Asp Ser Leu Asp Gly Asp Ary Phe Glu Asp Glu Ser Arg Thr Met ~ln Ser Glu Asn Met Arg Phe Val Ile Glu Lys Glu Leu Leu Ser Trp ~eu Ser Arg His Leu Pro Ala Asp Leu Ala Ser Ala Glu Arg Glu Thr Ser Arg Ser Leu Leu Ala Ala Gly His Trp Cys Cys Leu Trp His Pro Arg Pro Cys Arg Glu Ala Cys Leu Tyr Asp Ser Ile Tyr Val Gln Ser Leu Phe Cys Val Gly Thr Gly Arg Val Pro Gln Ser Glu Met Arg Arg ~rg Glu Tyr Leu Ala Ala Leu Arg Ala Gly Ala Ala Ala Ala Asn Ser ~ro Glu Val Ser Ala Ser Ile Phe Ala Arg Asp Ala Gly Ile Ala Leu Al a Leu Al a Arg Arg Arg

Claims

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

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

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

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

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

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

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

8. The recombinant infectious laryngotracheitis virus of claim 7, wherein the foreign gene is inserted into a gene selected from a group consisting of the US2 gene, UL47-like gene, ORF4 gene, glycoprotein G (gG) gene, glycoprotein 60 (g60) gene, and glycoprotein I (gI) gene.

9. The recombinant infectious laryngotracheitis virus of claim 7, wherein the foreign gene encodes a screenable marker.

10. The recombinant infectious laryngotracheitis virus of claim 9, wherein the screenable marker is E. coli B-galactosidase.

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

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

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

14. The recombinant infectious laryngotracheitis virus of claim 13, wherein the antigenic polypeptide is derived or derivable from a group consisting of infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus. and Marek's disease virus.

15. The recombinant infectious laryngotracheitis virus of claim 13, wherein the antigenic polypeptide is derived or derivable from a group consisting of avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian influenza virus, avian adenovirus, fowl pox virus, avian coronavirus. avian rotavirus, chick anemia agent, Salmonella spp. E.
coli, Pasteurella spp., Bordetella spp., Eimeria spp., Histomonas spp., Trichomonas spp., Poultry nematodes, cestodes, trematodes, poultry mites/lice, poultry protozoa.

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

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

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

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

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

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

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

23. A recombinant infectious laryngotracheitis virus of claim 22, wherein the foreign gene is inserted in the gene selected from a group consisting of the US2 gene, UL-47 like gene, ORF4 gene and glycoprotein g60 gene.

24. The recombinant infectious laryngotracheitis virus of claim 23, wherein the foreign gene encodes a screenable marker.

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

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

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

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

29. The recombinant infectious laryngotracheitis virus of claim 28, wherein the antigenic polypeptide is derived from or derivable from a group consisting of infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus.

30. The recombinant infectious laryngotracheitis virus of claim 28, wherein the antigenic polypeptide is derived from or derivable from a group consisting of avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian influenza virus, avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, chick anemia agent, Salmonella spp., E. coli., Pasteurella spp., Bordetella spp., Eimeria spp., Histomonas spp., Trichomonas spp., Poultry nematodes, cestodes, trematodes, poultry mites/lice, poultry protozoa.

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

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

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

34. A vaccine for infectious laryngotracheitis virus comprising an effective immunizing amount of the recombinant infectious laryngotracheitis virus of claim 1 and a suitable carrier.

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

36. A method of immunizing chickens or other poultry against infectious laryngotracheitis which comprises administering to said chickens or other poultry an effective immunizing amount of the vaccine of claim 34.

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

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