Patent Publication Number: US-5632989-A

Title: Attenuated, live vaccine for Delaware strain IBDV

Description:
This application is a Continuation-in-Part of U.S. patent application Ser. No. 07/423,758, filed Oct. 18, 1989, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention pertains to a vaccine prepared from a live, but attenuated virus of the Delaware strain inducing infectious bursal disease in chickens. Specifically, a vaccine is prepared by screening potential attenuated virus candidates with a non-neutralizing specific monoclonal antibody. Further, a highly specific neutralizing monoclonal antibody is provided as an alternative, effective vaccine. 
     2. Background of the Prior Art 
     In the inventors&#39; related applications, including U.S. patent application Ser. No. 07/423,752, entitled MONOCLONAL ANTIBODIES FOR INFECTIOUS BURSAL DISEASE, VACCINES AND ASSAYS FOR USE THEREWITH, which in turn is a CIP application of U.S. application Ser. No. 07/061,083, now U.S. Pat. No. 4,956,452 and U.S. application Ser. No. 07/227,311, now U.S. Pat. No. 5,064,646 the development and study of a large number of monoclonal antibodies (Mab) specific for various strains of infectious bursal disease is discussed. As related in the above-cited U.S. application Ser. No. 07/423,752, the entire content of which is incorporated herein by reference, a panel of antibodies has been developed, each specific to a particular type of infectious bursal disease virus. That virus has been, by means of antibody assays, categorized into three separate classifications, classic, Delaware and GLS. At the time of filing of this application, the Delaware strain, of all pure strains, appears to be the dominant infectious bursal disease virus in the Eastern United States. Certainly, it is a substantial and continuing threat to the U.S. poultry industry. 
     Wild-type Delaware IBDV have been known for some time, and are commercially available. There are a number of vaccines for this Delaware-type IBDV commercially available, including both &#34;killed&#34; and &#34;live&#34; vaccines. A killed vaccine is prepared by culturing the virus itself, in e.g., chicken eggs and the like, and subsequently &#34;killing&#34; the virus by heat, chemical treatment and the like. In a live vaccine, the virus is present in a living form, but has been attenuated to reduce or eliminate its virulency, by serial passage through cell culturing and the like. Thus, the &#34;virus&#34; component of a live vaccine is attenuated. Methods for preparation thereof are known in the art, and do not constitute, per se, an aspect of this invention. No currently available vaccine for wild-type Delaware IBDV is based on or comprises a non-virus active element, such as a monoclonal antibody. 
     During the analysis and categorization of IBDV in the United States, all commercially available live, attenuated Delaware strain vaccines were assayed. One of the monoclonal antibodies used in that assay is designated BK9, and is expressed by the hybridomal cell line deposited at the ATCC under deposit number HB-10157. 
     It is apparent that in order to provide adequate coverage and protection against the Delaware strain infection, a live, attenuated vaccine must contain a virus that will produce antibodies recognizing the wild-type Delaware IBDV. That is, in the process of attenuation, the marker characteristics of the wild-type Delaware virus strain must not be lost. Surprisingly, in the course of testing commercially available vaccines, not one was demonstrated to bear the BK9 Mab marker, and thus not truly wild-type. Vaccines employing this type of virus will not, in fact, generate antibodies providing effective protection against wild-type Delaware IBDV infection. Thus, it remains a pressing need in the industry to provide a live, attenuated vaccine for Delaware IBDV. 
     Alternatively, it is known that for certain IBDV, vaccines can be prepared from monoclonal antibodies (Mab) which neutralize the virus. See, in particular, U.S. Pat. No. 4,956,452. It is a continuing goal to provide such Mab which neutralize the Delaware strain IBDV, and provide a vaccine based thereon. 
     SUMMARY OF THE INVENTION 
     The above goal, and others made clear by the invention disclosure set forth below, has been met by identification of the fact that the wild-type Delaware virus does not replicate well in serial cell culturing. Thus, live, attenuated viruses obtained by serial passaging of the wild-type Delaware strain IBDV frequently lose the IBDV characteristics desirable or necessary to induce the production of effective antibodies against the parental virus. A Delaware virus which lacks the BK9 Mab marker, i.e., to which that monoclonal antibody will not bind, will not or may not confer as effective protection, in a vaccine against the wild type. 
     When attenuating a wild-type Delaware IBDV, it is highly unlikely that any given adapt (the result of serial passaging) will retain the BK9 marker, and so provide effective coverage. The odds, in fact, on successful passaging are quite small. Therefore, adapts, as produced, must be screened for retention of the Delaware marker, by antigen capture enzyme linked immunosorbent assay (AC-ELISA) assay using the BK9 marker. As reflected in copending application, Attorney Docket Number 2284-018-0 CIP, the binding of the BK9 marker is a positive indication of the presence of the wild type Delaware strain. 
     A neutralizing Mab has been developed against the wild-type Delaware strain of IBDV. The antibody has been deposited and extensively mapped. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1-1e is a rendering of the complete nucleotide and deduced amino acid sequence (SEQ ID NOS: 1-2) of the Delaware IBDV structural protein(s) that is recognized by the Monoclonal Antibody 67. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION: 
     Six different cell culture adapted (i.e., chicken embryo fibroblast and the like) attenuated viruses were prepared from wild-type Delaware IBDV. Each was prepared by serial passaging through cell culture processes, conventional in the industry. Alternate methods of producing attenuated viruses, including cloning of the virus with deletion of nucleic acid sequences and site-directed mutagenesis, are also known, and are embraced in the invention. Of the six adapts prepared, five, when tested with the monoclonal antibody expressed by HB-10157 yielded negative results, that is, that had lost the BK9 antibody marker. Such viruses, although attenuated, will not or may not induce the production of antibodies effective in preventing Delaware IBDV infection. 
     The sixth adapt did indeed react positively with the BK9 monoclonal antibody, thus indicating that this attenuated virus still maintained an essential characteristics of a wild-type Delaware IBDV, and as prepared in a vaccine, should induce the production of antibody effective against wild-type Delaware IBDV infection. This sixth adapt is currently available from the University of Maryland or Intervet America and can be continuously monitored and maintained in chicken origin or vero cell lines located at Avrum Gudelsky Center, College of Veterinary Medicine, University of Maryland. However, it should be noted that access to this virus or these cell lines is not necessary to practice the invention. Specifically, the adapts retaining the BK9 marker, and thus providing effective vaccines, occur randomly, with a low probability for the given adapt, as discussed above. There appears to be a portion of the wild-type Delaware IBDV genomic population which does not replicate well during serial passage. However, the odds on successful reproduction of an attenuated virus, given appropriate screening measures is quite high, if a large enough family of adapts is made. Thus, one of ordinary skill in the art, practicing a plurality of serial passages, need only screen the results with the BK9 Mab, available from the deposit, made under Budapest Treaty conditions, in order to practice this invention. 
     Identification of the virus presence and confirmation of its virulence, is conducted in a fashion identical to that set forth in the copending application U.S. Application Ser. No. 07/423,752, incorporated herein by reference. Given the monoclonal antibody, these procedures are routine. 
     The vaccines themselves may be prepared by simple incorporation of the selected virus, confirmed by screening, and suspending or mixing it in a carrier. Appropriate dosage values can be determined through team trial and error techniques, sampling for the production of antibodies in the treated poultry individual. In general, dosage ranges will vary in a physiologically acceptable carrier, such as buffered saline, cell culture medium, Mareti&#39;s virus vaccine diluent, etc. The dosage is determined by the ability of the virus to replicate in the chicken and provide effective immunity with minimal pathologic damage. 
     Applicants have also developed a Mab, designated Mab 67, which neutralizes the wild-type Delaware strain IBDV. The murine monoclonal antibody is specific only for the Delaware-type virus, and does not neutralize or bind to other recognized IBDV viruses. Mab 67 does not bind to, or neutralize any of the currently available live or live-attenuated vaccine viruses which are licensed or currently available, intended to confer protection against Delaware-type IBDV. This further confirms the inadequacy of existing virus-based vaccines to confer protection against Delaware-type infection. 
     The monoclonal antibody was developed according to the method set forth in U.S. Pat. No. 4,956,452. Specifically, hybridoma cell lines were prepared according to standard procedure beginning with BALB/c mice, immunized with the Delaware virus strain, after purification. Hybridomas were prepared therefrom, and the resulting cell lines were assayed, through an enzyme-linked immunosorbent assay (ELISA) to identify those lines secreting an antibody that binds to at least one strain. The resulting cell line was cloned again, and injected into pristane primed mice, to produce ascitic fluid with higher titer values. Specific details as to the propagation of the IBDV strains used, production of the hybridomas, the ELISA employed, and the virus neutralization tests are set forth in U.S. Pat. No. 4,956,452, beginning at column 3, line 64 and continuing on to column 5, line 68. The disclosure of this patent is incorporated herein by reference. The hybridoma cell line expressing Mab 67 has been deposited under Accession No. ATCC-HB11122, deposited at the ATCC by David Snyder on Sep. 15, 1992. Further, the cell lines are continuously available from Avrum Gudelsky Center, College of Veterinary Medicine, University of Maryland. This Mab can be used not only as an assay to determine the presence of the Delaware-type IBDV, but in addition to the virus-based vaccine disclosed above, can be employed to prepare a vaccine conferring challenge protection against Delaware-type IBDV. To achieve temporary passive immunization at a uniform, standardized level, and to augment any maternally derived levels against Delaware-type IBDV field infection, one-day old chicks should be vaccinated with a vaccine comprising a pharmacologically acceptable carrier such as a phosphate buffered saline, cell culture medium, Mareti&#39;s virus vaccine diluent, etc., in which is present an amount of Mab 67 effective to provide enhanced protection for a period of time which allows the chicks to become more immunologically competent (about two-three weeks). The necessary level of protection can be conferred by a single dose of the vaccine administered to day-old chicks having a Mab concentration of between 1 microgram and 1 milligram, or repeated vaccinations having a smaller effective dose, but carried out over time. If repeated vaccinations are used, the dosage level should range within 10 micrograms to 1 milligram. The concentration level needed to vaccinate older chickens is expected to increase with the weight of the bird. Other administration protocols can be developed by those of skill in the art without the exercise of inventive skill. 
     It should be noted that in general, the pharmacologically acceptable carriers to be used with the virus-based vaccine discussed above can also be used in conjunction with the Mab-based vaccine addressed herein. 
     FIGS. 1-1E contains a full recitation of the nucleotide sequence (SEQ ID NO: 1) for the gene responsible for the expression of Delaware IBDV structural protein(s) recognized by Mab 67. Presented together with this information in FIGS. 1-1E is the amino acid sequence (SEQ ID NO: 2) for the Delaware IBDV protein(s) which will be recognized by Mab 67. As noted above, it is apparent that the virus quickly loses, in serial passage, its identifying characteristics, and accordingly, wholesale modification of the Mab, from a synthetic or site-specific mutagenesis aspect, may impair or destroy the antibody&#39;s ability to neutralize the virus. Nonetheless, based on the length of the amino acid required for the binding of the antibody, and studies applied to similar materials, it is expected that up to 10 percent of the amino acids of the structure can be modified or deleted, and up to 25 percent of the nucleotide sequence replaced or modified, particularly at the ends of the sequence, without loss of the binding and neutralizing ability of Mab 67. In particular, small modifications which do not affect conformal (quaternary) structure will not impede binding. Such modifications are clearly contemplated as one aspect of this invention. 
     Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be alternatively described or practiced otherwise than as specifically described herein. 
     
         __________________________________________________________________________
SEQUENCE LISTING                                                          
(1) GENERAL INFORMATION:                                                  
(iii) NUMBER OF SEQUENCES: 2                                              
(2) INFORMATION FOR SEQ ID NO:1:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 3180 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: unknown                                                 
(D) TOPOLOGY: unknown                                                     
(ii) MOLECULE TYPE: DNA (genomic)                                         
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 64..3099                                                    
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                   
GAATTCCTCCTTCTACAACGCTATCATTGATGGTTAGTAGAGATCAGACAAACGATCGCA60            
GCGATGACAAACCTGCAAGATCAAACCCACCAGATTGTTCCGTTCATA108                       
MetThrAsnLeuGlnAspGlnThrHisGlnIleValProPheIle                             
151015                                                                    
CGGAGCCTTCTGATGCCAACAACCGGACCGGCGTCCATTCCGGACGAC156                       
ArgSerLeuLeuMetProThrThrGlyProAlaSerIleProAspAsp                          
202530                                                                    
ACCCTGGAGAAGCACACTCTCAGGTCAGAGACCTCGACCTACAATTTG204                       
ThrLeuGluLysHisThrLeuArgSerGluThrSerThrTyrAsnLeu                          
354045                                                                    
ACTGTGGGGGACACAGGGTCAGGGCTAATTGTCTTTTTCCCTGGATTC252                       
ThrValGlyAspThrGlySerGlyLeuIleValPhePheProGlyPhe                          
505560                                                                    
CCTGGCTCAATTGTGGGTGCTCACTACACACTGCAGAGCAGTGGGAAC300                       
ProGlySerIleValGlyAlaHisTyrThrLeuGlnSerSerGlyAsn                          
657075                                                                    
TACAAGTTCGATCAGATGCTCCTGACTGCCCAGAACCTACCGGCCAGC348                       
TyrLysPheAspGlnMetLeuLeuThrAlaGlnAsnLeuProAlaSer                          
80859095                                                                  
TACAACTACTGCAGGCTAGTGAGTCGGAGTCTCACAGTAAGGTCAAGC396                       
TyrAsnTyrCysArgLeuValSerArgSerLeuThrValArgSerSer                          
100105110                                                                 
ACACTCCCTGGTGGCGTTTATGCACTAAACGGCACCATAAACGCCGTG444                       
ThrLeuProGlyGlyValTyrAlaLeuAsnGlyThrIleAsnAlaVal                          
115120125                                                                 
ACCTTCCAAGGAAGCCTGAGTGAACTGACAGATGTTAGCTACAACGGG492                       
ThrPheGlnGlySerLeuSerGluLeuThrAspValSerTyrAsnGly                          
130135140                                                                 
TTGATGTCTGCAACAGCCAACATCAACGACAAAATTGGGAACGTCCTA540                       
LeuMetSerAlaThrAlaAsnIleAsnAspLysIleGlyAsnValLeu                          
145150155                                                                 
GTAGGGGAAGGGGTAACCGTCCTCAGCTTACCCACATCATATGATCTT588                       
ValGlyGluGlyValThrValLeuSerLeuProThrSerTyrAspLeu                          
160165170175                                                              
GGGTATGTGAGGCTTGGTGACCCCATACCCGCTATAGGGCTTGACCCA636                       
GlyTyrValArgLeuGlyAspProIleProAlaIleGlyLeuAspPro                          
180185190                                                                 
AAAATGGTAGCAACATGTGACAGCAGTGACAGGCCCAGAGTCTACACC684                       
LysMetValAlaThrCysAspSerSerAspArgProArgValTyrThr                          
195200205                                                                 
ATAACTGCAGCCGATAATTACCAATTCTCATCACAGTACCAAACAGGT732                       
IleThrAlaAlaAspAsnTyrGlnPheSerSerGlnTyrGlnThrGly                          
210215220                                                                 
GGGGTAACAATCACACTGTTCTCAGCCAACATTGATGCCATCACAAGT780                       
GlyValThrIleThrLeuPheSerAlaAsnIleAspAlaIleThrSer                          
225230235                                                                 
CTCAGCGTTGGGGGAGAGCTCGTGTTCAAAACAAGCGTCCAAAGCCTT828                       
LeuSerValGlyGlyGluLeuValPheLysThrSerValGlnSerLeu                          
240245250255                                                              
GTACTGGGCGCCACCATCTACCTTATAGGCTTTGATGGGACTGCGGTA876                       
ValLeuGlyAlaThrIleTyrLeuIleGlyPheAspGlyThrAlaVal                          
260265270                                                                 
ATCACCAGAGCTGTGGCCGCAAACAATGGGCTGACGGCCGGCATCGAC924                       
IleThrArgAlaValAlaAlaAsnAsnGlyLeuThrAlaGlyIleAsp                          
275280285                                                                 
AATCTTATGCCATTCAATCTTGTGATTCCAACCAATGAGATAACCCAG972                       
AsnLeuMetProPheAsnLeuValIleProThrAsnGluIleThrGln                          
290295300                                                                 
CCAATCACATCCATCATACTGGAGATAGTGACCTCCAAAAGTGATGGT1020                      
ProIleThrSerIleIleLeuGluIleValThrSerLysSerAspGly                          
305310315                                                                 
CAGGCAGGGGAACAGATGTCATGGTCGGCAAGTGGGAGCCTAGCAGTG1068                      
GlnAlaGlyGluGlnMetSerTrpSerAlaSerGlySerLeuAlaVal                          
320325330335                                                              
ACGATCCATGGTGGCAACTATCCAGGAGCCCTCCGTCCCGTCACACTA1116                      
ThrIleHisGlyGlyAsnTyrProGlyAlaLeuArgProValThrLeu                          
340345350                                                                 
GTGGCCTACGAAAGAGTGGCAACAGGATCTGTCGTTACGGTCGCTGGG1164                      
ValAlaTyrGluArgValAlaThrGlySerValValThrValAlaGly                          
355360365                                                                 
GTGAGCAACTTCGAGCTGATCCCAAATCCTGAACTAGCAAAGAACCTG1212                      
ValSerAsnPheGluLeuIleProAsnProGluLeuAlaLysAsnLeu                          
370375380                                                                 
GTTACAGAATACGGCCGATTTGACCCAGGAGCCATGAACTACACGAAA1260                      
ValThrGluTyrGlyArgPheAspProGlyAlaMetAsnTyrThrLys                          
385390395                                                                 
TTGATACTGAGTGAGAGGGACCACCTTGGCATCAAGACCGTCTGGCCA1308                      
LeuIleLeuSerGluArgAspHisLeuGlyIleLysThrValTrpPro                          
400405410415                                                              
ACAAGGGAGTACACTGACTTTCGTGAGTACTTCATGGAGGTGGCCGAC1356                      
ThrArgGluTyrThrAspPheArgGluTyrPheMetGluValAlaAsp                          
420425430                                                                 
CTCAACTCTCCCCTGAAGATTGCAGGAGCATTTGGCTTCAAAGACATA1404                      
LeuAsnSerProLeuLysIleAlaGlyAlaPheGlyPheLysAspIle                          
435440445                                                                 
ATCCGGGCCATAAGGAGGATAGCTGTACCGGTGGTCTCTACATTGTTC1452                      
IleArgAlaIleArgArgIleAlaValProValValSerThrLeuPhe                          
450455460                                                                 
CCACCTGCCGCTCCTCTAGCCCATGCAATTGGGGAAGGTGTAGACTAC1500                      
ProProAlaAlaProLeuAlaHisAlaIleGlyGluGlyValAspTyr                          
465470475                                                                 
CTACTGGGCGATGAGGCACAGGCTGCTTCAGGAACCGCTCGAGCCGCG1548                      
LeuLeuGlyAspGluAlaGlnAlaAlaSerGlyThrAlaArgAlaAla                          
480485490495                                                              
TCAGGAAAAGCAAGGGCTGCCTCAGGCCGCATAAGGCAGCTGACTCTC1596                      
SerGlyLysAlaArgAlaAlaSerGlyArgIleArgGlnLeuThrLeu                          
500505510                                                                 
GCCGCCGACAAGGGGTACGAGGTAGTCGCGAATCTATTCCAGGTGCCC1644                      
AlaAlaAspLysGlyTyrGluValValAlaAsnLeuPheGlnValPro                          
515520525                                                                 
CAGAATCCCGTAGTCGACGGGATTCTTGCTTCACCCGGGATACTTCGC1692                      
GlnAsnProValValAspGlyIleLeuAlaSerProGlyIleLeuArg                          
530535540                                                                 
GGTGCACACAACCTCGACTGCGTGCTAAGAGAGGGTGCCACGCTATTC1740                      
GlyAlaHisAsnLeuAspCysValLeuArgGluGlyAlaThrLeuPhe                          
545550555                                                                 
CCTGTGGTCATTACGACAGTGGAAGACGCCATGACACCCAAAGCACTG1788                      
ProValValIleThrThrValGluAspAlaMetThrProLysAlaLeu                          
560565570575                                                              
AACAGCAAAATGTTTGCTGTCATTGAAGGCGTGCGAGAAGACCTCCAA1836                      
AsnSerLysMetPheAlaValIleGluGlyValArgGluAspLeuGln                          
580585590                                                                 
CCTCCATCTCAAAGAGGATCCTTCATACGAACTCTCTCCGGACACAGA1884                      
ProProSerGlnArgGlySerPheIleArgThrLeuSerGlyHisArg                          
595600605                                                                 
GTCTATGGATATGCTCCAGATGGGGTACTTCCACTGGAGACTGGGAGA1932                      
ValTyrGlyTyrAlaProAspGlyValLeuProLeuGluThrGlyArg                          
610615620                                                                 
GACTACACCGTTGTCCCAATAGATGATGTCTGGGACGACAGCATTATG1980                      
AspTyrThrValValProIleAspAspValTrpAspAspSerIleMet                          
625630635                                                                 
CTGTCCAAGGACCCCATACCTCCTATTGTGGGAAACAGTGGAAACCTA2028                      
LeuSerLysAspProIleProProIleValGlyAsnSerGlyAsnLeu                          
640645650655                                                              
GCCATAGCTTACATGGATGTGTTTCGACCCAAAGTCCCCATCCATGTG2076                      
AlaIleAlaTyrMetAspValPheArgProLysValProIleHisVal                          
660665670                                                                 
GCCATGACGGGAGCCCTCAACGCTTGTGGCGAGATTGAGAAAATAAGC2124                      
AlaMetThrGlyAlaLeuAsnAlaCysGlyGluIleGluLysIleSer                          
675680685                                                                 
TTCAGAAGCACCAAGCTCGCCACCGCACACCGGCTTGGCCTCAAGTTG2172                      
PheArgSerThrLysLeuAlaThrAlaHisArgLeuGlyLeuLysLeu                          
690695700                                                                 
GCTGGTCCCGGAGCATTCGATGTAAACACCGGGCCCAACTGGGCAACG2220                      
AlaGlyProGlyAlaPheAspValAsnThrGlyProAsnTrpAlaThr                          
705710715                                                                 
TTCATCAAACGTTTCCCTCACAATCCACGCGACTGGGACAGGCTCCCC2268                      
PheIleLysArgPheProHisAsnProArgAspTrpAspArgLeuPro                          
720725730735                                                              
TACCTTAACCTTCCATACCTTCCACCCAATGCAGGACGCCAGTACCAC2316                      
TyrLeuAsnLeuProTyrLeuProProAsnAlaGlyArgGlnTyrHis                          
740745750                                                                 
CTTGCCATGGCTGCATCAGAGTTCAAAGAGACCCCTGAACTCGAGAGC2364                      
LeuAlaMetAlaAlaSerGluPheLysGluThrProGluLeuGluSer                          
755760765                                                                 
GCCGTAAGAGCCATGGAAGCAGCTGCCAATGTGGACCCACTGTTCCAA2412                      
AlaValArgAlaMetGluAlaAlaAlaAsnValAspProLeuPheGln                          
770775780                                                                 
TCTGCACTCAGTGTGTTCATGTGGCTGGAAGAGAATGGGATTGTGACT2460                      
SerAlaLeuSerValPheMetTrpLeuGluGluAsnGlyIleValThr                          
785790795                                                                 
GACATGGCCAACTTCGCACTCAGCGACCCGAATGCCCATCGGATGCGA2508                      
AspMetAlaAsnPheAlaLeuSerAspProAsnAlaHisArgMetArg                          
800805810815                                                              
AATTTTCTTGCAAACGCACCACAAGCAGGCAGCAAGTCGCAAAGGGCC2556                      
AsnPheLeuAlaAsnAlaProGlnAlaGlySerLysSerGlnArgAla                          
820825830                                                                 
AAGTACGGGACAGCAGGCTACGGAGTGGAGGCCCGGGGCCCCACACCA2604                      
LysTyrGlyThrAlaGlyTyrGlyValGluAlaArgGlyProThrPro                          
835840845                                                                 
GAGGAAGCACAGAGGGAAAAAGACACACGGATCTCAAAGAAGATGGAG2652                      
GluGluAlaGlnArgGluLysAspThrArgIleSerLysLysMetGlu                          
850855860                                                                 
ACCATGGGCATCTACTTTGCAACACCAGAATGGGTAGCACTCAATGGG2700                      
ThrMetGlyIleTyrPheAlaThrProGluTrpValAlaLeuAsnGly                          
865870875                                                                 
CACCGCGGGCCAAGCCCCGGCCAGCTAAAGTACTGGCAGAACACACGA2748                      
HisArgGlyProSerProGlyGlnLeuLysTyrTrpGlnAsnThrArg                          
880885890895                                                              
GAAATACCGGACCCAAACGAGGACTATCTAGACTACGTGCATGCAGAG2796                      
GluIleProAspProAsnGluAspTyrLeuAspTyrValHisAlaGlu                          
900905910                                                                 
AAGAGCCGGTTGGCATCAGAAGAACAAATCCTAAGGGCAGCTACGTCG2844                      
LysSerArgLeuAlaSerGluGluGlnIleLeuArgAlaAlaThrSer                          
915920925                                                                 
ATCTACGGGGCTCCAGGACAGGCAGAGCCACCCCAAGCTTTCATAGAC2892                      
IleTyrGlyAlaProGlyGlnAlaGluProProGlnAlaPheIleAsp                          
930935940                                                                 
GAAGTTGCCAAAGTCTATGAAATCAACCATGGACGTGGCCCAAACCAA2940                      
GluValAlaLysValTyrGluIleAsnHisGlyArgGlyProAsnGln                          
945950955                                                                 
GGACAGATGAAAGATCTGCTCTTGACTGCGATGGAGATGAAGCATCGC2988                      
GlyGlnMetLysAspLeuLeuLeuThrAlaMetGluMetLysHisArg                          
960965970975                                                              
AATCCCAGGCGGGCTCCACCAAAGCCCAAGCCAAAACCCAATGCTCCA3036                      
AsnProArgArgAlaProProLysProLysProLysProAsnAlaPro                          
980985990                                                                 
ACACAGAGACCCCCTGGTCGGCTGGGCCGCTGGATCAGGACTGTCTCT3084                      
ThrGlnArgProProGlyArgLeuGlyArgTrpIleArgThrValSer                          
99510001005                                                               
GATGAGGACCTTGAGTGAGGCTCCTGGGAGTCTCCCGACACCACCCGCGCAGGCG3139               
AspGluAspLeuGlu                                                           
1010                                                                      
TGGACACCAATTCGGCCTTACAACATCCCAAATTGGATCCG3180                             
(2) INFORMATION FOR SEQ ID NO:2:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1012 amino acids                                              
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                   
MetThrAsnLeuGlnAspGlnThrHisGlnIleValProPheIleArg                          
151015                                                                    
SerLeuLeuMetProThrThrGlyProAlaSerIleProAspAspThr                          
202530                                                                    
LeuGluLysHisThrLeuArgSerGluThrSerThrTyrAsnLeuThr                          
354045                                                                    
ValGlyAspThrGlySerGlyLeuIleValPhePheProGlyPhePro                          
505560                                                                    
GlySerIleValGlyAlaHisTyrThrLeuGlnSerSerGlyAsnTyr                          
65707580                                                                  
LysPheAspGlnMetLeuLeuThrAlaGlnAsnLeuProAlaSerTyr                          
859095                                                                    
AsnTyrCysArgLeuValSerArgSerLeuThrValArgSerSerThr                          
100105110                                                                 
LeuProGlyGlyValTyrAlaLeuAsnGlyThrIleAsnAlaValThr                          
115120125                                                                 
PheGlnGlySerLeuSerGluLeuThrAspValSerTyrAsnGlyLeu                          
130135140                                                                 
MetSerAlaThrAlaAsnIleAsnAspLysIleGlyAsnValLeuVal                          
145150155160                                                              
GlyGluGlyValThrValLeuSerLeuProThrSerTyrAspLeuGly                          
165170175                                                                 
TyrValArgLeuGlyAspProIleProAlaIleGlyLeuAspProLys                          
180185190                                                                 
MetValAlaThrCysAspSerSerAspArgProArgValTyrThrIle                          
195200205                                                                 
ThrAlaAlaAspAsnTyrGlnPheSerSerGlnTyrGlnThrGlyGly                          
210215220                                                                 
ValThrIleThrLeuPheSerAlaAsnIleAspAlaIleThrSerLeu                          
225230235240                                                              
SerValGlyGlyGluLeuValPheLysThrSerValGlnSerLeuVal                          
245250255                                                                 
LeuGlyAlaThrIleTyrLeuIleGlyPheAspGlyThrAlaValIle                          
260265270                                                                 
ThrArgAlaValAlaAlaAsnAsnGlyLeuThrAlaGlyIleAspAsn                          
275280285                                                                 
LeuMetProPheAsnLeuValIleProThrAsnGluIleThrGlnPro                          
290295300                                                                 
IleThrSerIleIleLeuGluIleValThrSerLysSerAspGlyGln                          
305310315320                                                              
AlaGlyGluGlnMetSerTrpSerAlaSerGlySerLeuAlaValThr                          
325330335                                                                 
IleHisGlyGlyAsnTyrProGlyAlaLeuArgProValThrLeuVal                          
340345350                                                                 
AlaTyrGluArgValAlaThrGlySerValValThrValAlaGlyVal                          
355360365                                                                 
SerAsnPheGluLeuIleProAsnProGluLeuAlaLysAsnLeuVal                          
370375380                                                                 
ThrGluTyrGlyArgPheAspProGlyAlaMetAsnTyrThrLysLeu                          
385390395400                                                              
IleLeuSerGluArgAspHisLeuGlyIleLysThrValTrpProThr                          
405410415                                                                 
ArgGluTyrThrAspPheArgGluTyrPheMetGluValAlaAspLeu                          
420425430                                                                 
AsnSerProLeuLysIleAlaGlyAlaPheGlyPheLysAspIleIle                          
435440445                                                                 
ArgAlaIleArgArgIleAlaValProValValSerThrLeuPhePro                          
450455460                                                                 
ProAlaAlaProLeuAlaHisAlaIleGlyGluGlyValAspTyrLeu                          
465470475480                                                              
LeuGlyAspGluAlaGlnAlaAlaSerGlyThrAlaArgAlaAlaSer                          
485490495                                                                 
GlyLysAlaArgAlaAlaSerGlyArgIleArgGlnLeuThrLeuAla                          
500505510                                                                 
AlaAspLysGlyTyrGluValValAlaAsnLeuPheGlnValProGln                          
515520525                                                                 
AsnProValValAspGlyIleLeuAlaSerProGlyIleLeuArgGly                          
530535540                                                                 
AlaHisAsnLeuAspCysValLeuArgGluGlyAlaThrLeuPhePro                          
545550555560                                                              
ValValIleThrThrValGluAspAlaMetThrProLysAlaLeuAsn                          
565570575                                                                 
SerLysMetPheAlaValIleGluGlyValArgGluAspLeuGlnPro                          
580585590                                                                 
ProSerGlnArgGlySerPheIleArgThrLeuSerGlyHisArgVal                          
595600605                                                                 
TyrGlyTyrAlaProAspGlyValLeuProLeuGluThrGlyArgAsp                          
610615620                                                                 
TyrThrValValProIleAspAspValTrpAspAspSerIleMetLeu                          
625630635640                                                              
SerLysAspProIleProProIleValGlyAsnSerGlyAsnLeuAla                          
645650655                                                                 
IleAlaTyrMetAspValPheArgProLysValProIleHisValAla                          
660665670                                                                 
MetThrGlyAlaLeuAsnAlaCysGlyGluIleGluLysIleSerPhe                          
675680685                                                                 
ArgSerThrLysLeuAlaThrAlaHisArgLeuGlyLeuLysLeuAla                          
690695700                                                                 
GlyProGlyAlaPheAspValAsnThrGlyProAsnTrpAlaThrPhe                          
705710715720                                                              
IleLysArgPheProHisAsnProArgAspTrpAspArgLeuProTyr                          
725730735                                                                 
LeuAsnLeuProTyrLeuProProAsnAlaGlyArgGlnTyrHisLeu                          
740745750                                                                 
AlaMetAlaAlaSerGluPheLysGluThrProGluLeuGluSerAla                          
755760765                                                                 
ValArgAlaMetGluAlaAlaAlaAsnValAspProLeuPheGlnSer                          
770775780                                                                 
AlaLeuSerValPheMetTrpLeuGluGluAsnGlyIleValThrAsp                          
785790795800                                                              
MetAlaAsnPheAlaLeuSerAspProAsnAlaHisArgMetArgAsn                          
805810815                                                                 
PheLeuAlaAsnAlaProGlnAlaGlySerLysSerGlnArgAlaLys                          
820825830                                                                 
TyrGlyThrAlaGlyTyrGlyValGluAlaArgGlyProThrProGlu                          
835840845                                                                 
GluAlaGlnArgGluLysAspThrArgIleSerLysLysMetGluThr                          
850855860                                                                 
MetGlyIleTyrPheAlaThrProGluTrpValAlaLeuAsnGlyHis                          
865870875880                                                              
ArgGlyProSerProGlyGlnLeuLysTyrTrpGlnAsnThrArgGlu                          
885890895                                                                 
IleProAspProAsnGluAspTyrLeuAspTyrValHisAlaGluLys                          
900905910                                                                 
SerArgLeuAlaSerGluGluGlnIleLeuArgAlaAlaThrSerIle                          
915920925                                                                 
TyrGlyAlaProGlyGlnAlaGluProProGlnAlaPheIleAspGlu                          
930935940                                                                 
ValAlaLysValTyrGluIleAsnHisGlyArgGlyProAsnGlnGly                          
945950955960                                                              
GlnMetLysAspLeuLeuLeuThrAlaMetGluMetLysHisArgAsn                          
965970975                                                                 
ProArgArgAlaProProLysProLysProLysProAsnAlaProThr                          
980985990                                                                 
GlnArgProProGlyArgLeuGlyArgTrpIleArgThrValSerAsp                          
99510001005                                                               
GluAspLeuGlu                                                              
1010                                                                      
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