Protective malaria sporozoite surface protein immunogen and gene

A protein antigen (SSP2) on the surface of Plasmodium sporozoites is disclosed as a candidate immunogen for vaccination against malaria. This use of this protein, which is distinct from the extensively characterized circumsporozoite (CS) protein, will also facilitate research into host immunological responses to malaria. This antigen is detected by a monoclonal antibody (NYS4) which is specific for a 140 kilodalton (kD) protein on the sporozoite cell surface. Immunoreactive genomic clones are described which express this surface antigen gene and the primary nucleic acid sequence and the deduced amino acid sequence derived from this DNA sequence are disclosed. Unique repetitive sequences of amino acids are described which further demonstrate the distinction between SSP2 and the CS protein. A synthetic peptide containing repeating epitopes of SSP2 derived protein antigen and which are substantially shorter in length than the intact antigen are disclosed. The peptide when administered to a host elicits antibodies which bind to the SSP2 protein on the sporozoite surface. A recombinant plasmid bearing SSP2 DNA sequences expresses SSP2 epitopes in mammalian cells and the introduction of these transfected cells into mice elicits antibody and cytotoxic T-cell lymphocytic responses which confer partial protection to the recipient animals against challenge infection. In combination, the SSP2 protein and the CS protein elicit immunological responses in the mammalian host which confer 100% protection against challenge infection.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to an immunologically active protein, and the gene 
which encodes it, useful in vaccination against malaria. More 
particularly, this invention relates to a protein antigen on the surface 
of Plasmodium yoelii sporozoites detected by a monoclonal antibody NYS4, 
clones of genomic and complementary DNA sequences specific for this and 
closely related proteins from malarial parasites which, when administered 
as vaccine components either alone or in combination with other malarial 
antigens, can confer protective immunity. 
2. Description of the Prior Art 
Prevention of infection by human malarias would alleviate a major health 
problem in the tropical and sub-tropical areas of the world. The most 
promising method for the control of malaria appears to be the development 
and use of vaccines. 
Species of the genus Plasmodium are the etiological agents of malaria. 
These protozoan parasites have a complex life cycle involving reproduction 
in mammalian hosts and insect vectors. The infectious stage of the 
parasite is called the sporozoite. It is inoculated into a mammalian host 
by the bite of infected Anopheline mosquitos. 
One approach to producing a malaria vaccine is to attempt to induce an 
immune response against the infective sporozoites themselves. This has 
been done by immunization of human and animal models with radiation 
attenuated sporozoites. These attenuated sporozoites consistently protect 
against challenge with infectious sporozoites (Nussenzweig et al., Nature 
216:160, 1969, Clyde et al, Am. J. Med. Sci. 266:160, 1973, Rieckmann et 
al., Bull. W. H. O. 57, Suppl.1:261, 1979). 
A surface protein of Plasmodium sporozoites, designated the 
circumsporozoite protein (CSP) has been identified and well characterized 
(Nussenzweig and Nussenzweig, Cell 42:401, 1985). The CSPs of several 
Plasmodium species have been described. They all contain tandem repeats of 
short peptide sequences as well as regions of non-repetitive sequence. 
While the existence of tandem repeats is a conserved feature of all CSP 
genes, the particular amino acid sequences which are repeated vary both 
between and within species. On the other hand, the non-repetitive 
sequences are well conserved both within and between species. 
During the past decade, the primary strategy for malaria sporozoite vaccine 
development has been to produce vaccines that induce antibodies to the 
repeat region of the CSP. These antibodies are believed to prevent 
effective sporozoite invasion of hepatocytes (Mazier et al., Science 
231:156, 1986, Young et al., Science 228:958,1985, Ballou et al., Science 
228:996, 1985, Zavala, et al. Science 228: 1985, 1985). Thus far, 
protective immunity after immunization of humans (Ballou et al., Lancet 
1:1277, 1987, Herrington et al, Nature 328:257, 1987) and non human 
primates with CSP based vaccines (Collins, et al. Am. J. Trop. Med. Hyg. 
40:455, 1989) has been disappointing. Mice immunized with P. berghei 
subunit vaccines based on the CSP have been protected against moderate, 
but not against large challenge doses of sporozoites (Egan et al. Science 
236:453, 1987, Zavala et al. J. Ex. Med. 166:1591, 1987, Hoffman et al., 
J. Immunol. 142: 3581, 1989, Romero et al. Eur. J. Immunol. 18: 1951, 
1988). In the P. yoelii model system, mice have been immunized with a 
variety of synthetic peptides and recombinant proteins based on the CSP 
(Lal et al. Proc. Natl. Acad. Sci. USA 84:8647, 1987, Sedegah et al. in 
Technological Advances in Vaccine Development L. Lasky editor, New York, 
1988, Sedegah et al., Bull. W. H. O., in press, Charoenvit et al., Bull. 
W. H. O., in press). In the majority of experiments, mice developed high 
levels of antibodies to the CSP, but were not consistently protected 
against challenge with P. yoelii sporozoites. 
The failure of immunization with vaccines based on the CSP to provide the 
same solid immunity as immunization with radiation attenuated sporozoites 
suggests that although the CSP plays a role in immunity, there are other 
sporozoite antigens important in immunity to malaria. There is a need to 
identify, isolate and characterize these antigens so that they may be 
included in future malaria vaccines. 
SUMMARY OF THE INVENTION 
Accordingly, an object of this invention is a sporozoite surface antigen 
which is distinct from the CS protein. This antigen is designated 
Sporozoite Surface Protein 2 (SSP2). By SSP2 we refer both to the P. 
yoelii protein whose sequence is here disclosed and to the homologs of 
this protein which exist in other species of Plasmodium, including the 
human pathogens, P. falciparum, P. vivax, P. ovale, and P. malariae. These 
homologs may be recognized by their having both a predicted general 
structure similar to that of the P. yoelii SSP2 (FIG. 1), including a 
short hydrophobic leader sequence, a region containing perfect or 
degenerate tandem repeats of short peptide motifs, flanking regions 
containing non repetitive amino acid sequence, a membrane spanning domain 
and a cytoplasmic domain, and by their having an amino acid sequence 
which, at least in the non-repetitive regions of the sequence, bears 
substantial sequence similarity to the P. yoelii SSP2 amino acid sequence 
(Sequences 1 and 2). 
Another object of this invention is a vaccine containing a synthetic or 
recombinant fragment of the antigen SSP2 which may include all or part of 
the amino acid sequence shown as Sequence 2 and which causes the 
production of specific immune sera and immune cells and protects against 
malaria. 
A further object of the invention is a recombinant molecule containing all 
or part of the DNA and deduced amino acid sequence of SSP2. 
These and additional objects of the invention are accomplished by a protein 
antigen on the surface of Plasmodium sporozoites. The P. yoelii SSP2 
antigen is detected by a monoclonal antibody (NYS4) which is specific for 
a 140 kilodalton (kD) protein on the P. yoelii sporozoite cell surface. A 
synthetic peptide containing repeating epitopes of SSP2 derived protein 
antigen and which is substantially shorter in length than the intact 
antigen are also part of this invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
A protein antigen (SSP2) on the surface of Plasmodium sporozoites is a 
candidate immunogen for vaccination against malaria and facilitates 
research into host immunological responses to malaria. The P. yoelii SSP2 
antigen is detected by a monoclonal antibody (NYS4) which is specific for 
a 140 kilodalton (kD) protein on the sporozoite cell surface. 
Immunoreactive genomic clones express epitopes of this surface antigen 
gene and were used to clone the complete gene for P. yoelii SSP2. The DNA 
sequence of the gene encoding P. yoelii SSP2 and the predicted amino acid 
sequence (Sequences 1 and 2, respectively) show that it is distinct from 
the CSP. A synthetic peptide contains repeating epitopes of SSP2 derived 
protein antigen and is substantially shorter in length than the intact 
antigen. The peptide, when conjugated to an appropriate carrier molecule, 
for example keyhole limpet hemocyanin and administered to a host in an 
appropriate adjuvant, for example Freund's complete adjuvant, elicits 
antibodies which bind to the SSP2 protein on the sporozoite surface. A 
recombinant mammalian expression plasmid bearing SSP2 DNA sequences 
expresses SSP2 epitopes in mammalian cells and the introduction of these 
transfected cells into mice elicits antibody and cytotoxic T-cell 
lymphocytic responses which confer partial protection to the recipient 
animals against challenge infection. In combination, the SSP2 protein and 
the CS protein elicit immunological responses in the mammalian host which 
confer 100% protection against challenge infection. 
The only previously characterized sporozoite surface antigen, the CSP 
(Nussenzweig and Nussenzweig, Cell 42:401, 1985), is present on all 
malaria species studied to date. It is likely that homologs of SSP2 exist 
in the human malaria species, and that with the current art and the use of 
the immunological reagents and DNA and protein sequence information 
disclosed here, they will be identified and used both as vaccine 
candidates and as reagents for studies of the immune response to malaria 
and to putative malaria vaccines. 
Having described the invention, the following examples are given to 
illustrate specific applications of the invention including the best mode 
now known to perform the invention. These specific examples are not 
intended to limit the scope of the invention described in this 
application. 
EXAMPLE 1 
Clones from the Genomic Expression Library 
Parasites and DNA isolation. P. yoelii 17X(NL) parasites were obtained by 
blood passage in Balb/C mice. DNA isolation from parasite infected blood 
was as described (Wortman et al., Micro. Path., 6:227, 1989). P. yoelii 
genomic expression libraries were constructed using either 0.5-2.0 or 
2.0-7.0 kb fragments generated by partial DNase I digestion, addition of 
Eco RI linker oligomers, and ligation into the lambda cloning vector gtII 
by standard techniques. The recombinant phage were packaged in vitro and 
the resulting library was screened for antigen expressing clones with a 
1:20 dilution of a sporozoite surface specific antibody, NYS4, (Charoenvit 
et al.,Infect. Immun., 55:604, 1989). 
EXAMPLE 2 
Characterization of SSP2 Antigen 
Immuno-fluorescence and Western blotting. Immunofluorescence and Western 
blotting were carried out as previously described (Charoenvit et al., 
Infect. Immun., 55:604, 1987). Immunofluoresence showed that NYS4 reacts 
with a protein on the sporozoite surface. Western blotting showed that 
protein to have a molecular weight of 140 kD. 
Sequence analysis of the 140 kD antigen gene. A clone designated M4 reacted 
with NYS4. M4 was sequenced and used as a probe to screen the library 
containg 2.0-7.0 kb inserts. A clone, designated 10.1111, was found to 
contain a 4.7 kb insert which overlapped the complete sequence of M4. The 
DNA sequence of 10.1111 was determined by standard techniques It includes 
a 2481 bp open reading frame which encodes SSP2. The sequence of 10.1111 
and the deduced amino acid sequence of SSP2 is shown in FIG. 1. Like most 
other malaria antigens, SSP2 contains regions of repeating amino acids. 
One region consists of a repeating trimer, ProAsnAsn and the other is 
largely composed of the hexamer, AsnProAsnGluProSer. 
EXAMPLE 3 
Antibodies to AsnProAsnGluProSer recognize the surface of sporozoites and 
the 140 kD antigen 
The overall hydrophilicity of the repeating amino acid sequence 
AsnProAsnGluProSer suggested it as a potential antigenic determinant (Hopp 
and Woods, Proc. Natl. Acad. Sci. USA 78:3824, 1981). A synthetic peptide 
containing 3 copies of AsnProAsnGluProSer (18-mer) was strongly recognized 
by NYS4 in an ELISA and mice immunized with the peptide coupled to keyhole 
limpet hemocyanin produced antibodies that reacted with sporozoites in an 
IFAT and with the 140 kD antigen on western blots of sporozoite extracts. 
These results indicate that the deduced amino acid sequence of SSP2 
corresponds to the 140 kD antigen recognized by NYS4 and that the 
antigenic determinant of NYS4 is contained within the repeating hexamer 
AsnProAsnGluProSer. It is notable that this repetitive sequence bears no 
similarity to the major repeats of the P. yoelii CS protein, in which the 
consensus repeating elements are GlnGlyProGlyAlaPro and GlnGlnProPro (Lal 
et al., J. Biol. Chem., 262:2937, 1987). 
EXAMPLE 4 
Cytotoxic lymphocyte responses against SSP2 are protective 
We transfected a 1.5 kb fragment of the gene encoding SSP2 into P815 mouse 
mastocytoma cells. This fragment included amino acids 223 through 698 of 
Sequence 2 cloned into the mammalian expression vector, pcEXV3 (Miller and 
Germain, J. Exp. Med. 164:1578, 1986). Nine different clones were derived 
and each was used to stimulate in vitro spleen cells in culture from 
BALB/c mice immunized with irradiated P. yoelii sporozoites. SSP2 specific 
cytotoxic T lymphocytes (CTL) were only present in cultures of spleen 
cells from immunized mice. All cytotoxic activity was eliminated by in 
vitro depletion of CD8+T cells, but was unaffected by in vitro depletion 
of C04+T cells. In BALB/c mice, the protective immunity induced by 
immunization with irradiated P. yoelii sporozoites is eliminated by in 
vivo treatment of immune mice with antibodies to CD8+T cells, indicating 
that it is dependent on CTL. 
To determine if an immune response directed against SSP2 would provide 
protection, 8ALB/c mice were injected intraperitoneally 5 times at 2 week 
intervals with 2.times.10.sup.8 irradiated (10.sup.3 rads Cesium137) cells 
of a cloned line of P815 cells transfected with the SSP2 gene linked to 
pcEXV3. This cloned cell line is designated SSP2 3.9. Two weeks after the 
last dose, the mice were challenged with 200 P. yoelii sporozoites. After 
immunization with SSP2 3.9 cells, mice produced high levels of antibodies 
against SSP2 and sporozoites and CTL against SSP2. Four of the six mice 
were protected against challenge infection by immunization with 
mastocytoma cells expressing SSP2. Complete protection was demonstrated 
after immunization with a mixture of two transfected cell lines, SSP2 2.9, 
and an analogous line consisting of P815 cell transfected with the P. 
yoelii CSP gene and designated CSP 1.5. Furthermore, this regimen induced 
high levels of antibodies against sporozoites and antigen specific, C 
08+CTL To determine if this protective immunity, like that found after 
immunization of BALB/c mice with irradiated sporozoites, was dependent on 
CD8+T cells, we immunized mice with either SSP2 3.9 or CS 1.5 and then in 
vivo depleted with anti-CD8 antibody. The partial immunity induced by 
SSP2, or the CS protein, and the complete protection induced by the 
combination were completely reversed by depletion of CD8+T cells. 
EXAMPLE 5 
Identification of the homologs of SSP2 in human malaria species 
The protein characteristics, monoclonal antibody, and DNA sequence here 
disclosed can be used within the current art to detect proteins homologous 
to SSP2 in the human malaria species P. falciparum, P. malariae, P. vivax, 
and P. ovale. For example, oligonucleotide probes based on the DNA 
sequence or cloned fragments of the SSP2 gene may be used by standard 
techniques to screen genomic libraries constructed from human malaria 
species, the cloned gene encoding SSP2 may be expressed in a variety of 
prokaryotic and eukaryotic expression systems and crude or purified 
recombinant expressed SSP2 used to produce additional poly or monoclonal 
antisera or immune T lymphocytes which can be used to screen genomic 
expression or cDNA libraries in order to identify SSP2 homologs in human 
malaria species. The original monoclonal antibody NYS4 cannot be used to 
identify the SSP2 homologs in species other than P. yoelii. This is so 
because NYS4 recognizes an epitope contained within the repeated amino 
acid sequence AsnProAsnGluProSer. Since the repeated regions of malaria 
surface antigens are not conserved between species (Weber, Exp. Parasit. 
69:303, 1989) it is very unlikely that a monoclonal antibody directed at 
the repeat sequence of P. yoelii SSP2 will recognize SSP2 homologs in 
other Plasmodium species. 
EXAMPLE 6 
Use of human malaria homoloqs of SSP2 for vaccination of humans 
Given the DNA sequence of the human malaria homologs, readily obtainable 
with the present art, it is possible to design a large number of vaccine 
formulations including but not limited to 1) synthetic peptide vaccines 
based on the inferred amino acid sequence 2) recombinant proteins 
consisting of all or part of the SSP2 homolog protein expressed in any of 
a large number of protein expression systems including but not limited to 
phage lysogens, bacterial plasmids, yeast plasmids, mammalian cell 
expression plasmids and viruses, and insect viruses (e.g. baculovirus). 
Vaccines produced by these methods may be administered to humans in any 
pharmacologically active form and dosage with any pharmacologically 
appropriate adjuvant including but not limited to saline, aluminum 
hydroxide, and liposomes. In any of the above vaccines, components derived 
from the CSP may be included, particulary in light of our finding of the 
additive effect of immunizing mice with both SSP2 and the CSP. 
EXAMPLE 7 
Use of transfected cells expressinq SSP2 or its human malaria homologs to 
study the immune response of humans or animal models to malaria 
Human or murine cell lines transfected with expression vectors containing 
the SSP2 gene may be used to study the immune response of humans or 
experimental animals to naturally acquired or laboratory induced malaria, 
or to assess the effects of immunization against malaria with irradiated 
sporozoites or any other putative malaria vaccine. Transfected cells, for 
example, may serve as target cells in cytotoxicity assays to determine the 
presence of CTLs directed against SSP2 in humans or animals. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4673 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: N 
(iv) ANTI-SENSE: N 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Plasmodium yoelii 
(B) STRAIN: 17X(NL) 
(D) DEVELOPMENTAL STAGE: erythrocytic stage 
(F) TISSUE TYPE: Blood 
(G) CELL TYPE: erythrocytic stage 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Py-lambdagt11-2-7 kb genomic expression 
(B) CLONE: Py10.1111 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 718..3195 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CATTAAAACCATTTAAAAAAGTAAATTTTATAAATTTTGTTTAATTTTCTTTATATATAT60 
AATATATATATACATTTATATATACTCTTGTTCTTTTTATCGATTAAAAAAATATATAAT120 
ATCCATTATATTT ATTTTTTAACAATTAAAAATATATAAAATGTACCCCTTGTGCTTGAA180 
GCAACATTTTTTATATTTAACTGTTGTATCTTTTTTTACATATATTTGTTCACATTCTTT240 
GGGATGATATTAAATAATATAATTTTCGAAGAGAAATATTTTTAAATACTTTTTTTAGT G300 
CTTGCATTATTTTTATGATATATATTAACATTCATAAAATATATATTTGTTGAGTGTTGG360 
TTGCCAGTTTATTGAATTAGCTATATTTTTAAATACTAAATATATTTTTTTAAATTGGTT420 
ATGATCATATTCTAATCCGTATTATATTGCGTATGT GTATATATATAACGGAAAAAAAGG480 
AAAACATTTAATTTCCTCAGACGCTATTGAATTAAATTAACTATATATCAGTTTTATATA540 
AGAAAAGGTAACACACTCTCTCTCTATATATATATAATTGCAAACGTGTAGACATTTTTA600 
TATATGGCCAAAT AGTAAATACAAAATAATTCCTCACTTTTATTCTCTTACATATATTAT660 
AATACATACATAGACACATAATTTTACCCATTCCCCATTTCTCTTATAGACAGAAAC717 
ATGAAGCTCTTAGGAAATAGTAAATATATTTTTGTTGTGCTTTTATTA 765 
MetLysLeuLeuGlyAsnSerLysTyrIlePheValValLeuLeuLeu 
151015 
TGCATAAGCGTGTTCCTTAATGGTCAGGAAACTCTTGACGAAATAAAG 813 
CysIleSerValPheLeuAsnGlyGlnGluThrLeuAspGluIleLys 
202530 
TATAGTGAAGAAGTATGTACCGAACAAATCGACATTCATATATTACTA 861 
TyrSerGluGluValCysThrGluGlnIleAspIleHisIleLeuLeu 
354045 
GATGGTTCAGGAAGTATTGGTTATAGCAATTGGAAGGCTCATGTTATT909 
AspGlySerGlySerIleGlyTyrSerAsnTrpLysAlaHisValIle 
505560 
CCAATGCTTAATACTTTGGTTGATAACTTAAATATTTCAAATGATGAA957 
ProMetLe uAsnThrLeuValAspAsnLeuAsnIleSerAsnAspGlu 
65707580 
ATTAATGTATCTTTGACACTTTTTTCAACAAATTCACGTGAATTAATT1005 
IleA snValSerLeuThrLeuPheSerThrAsnSerArgGluLeuIle 
859095 
AAACTTAAAGGATATGGATCGACTAGTAAAGACTCGCTACGTTTTATA1053 
Lys LeuLysGlyTyrGlySerThrSerLysAspSerLeuArgPheIle 
100105110 
CTTGCACATCTCCAAAATAATTATTCACCAAATGGTAATACAAATTTA1101 
LeuAla HisLeuGlnAsnAsnTyrSerProAsnGlyAsnThrAsnLeu 
115120125 
ACGAGTGCATTATTGGTTGTTGATACTTTAATTAATGAAAGAATGTAT1149 
ThrSerAlaLe uLeuValValAspThrLeuIleAsnGluArgMetTyr 
130135140 
CGACCCGATGCAATACAATTAGCTATTATATTAACAGATGGTATCCCA1197 
ArgProAspAlaIleGlnL euAlaIleIleLeuThrAspGlyIlePro 
145150155160 
AATGATTTACCTAGATCTACTGCGGTTGTGCATCAATTAAAAAGAAAA1245 
AsnAspLeuProArg SerThrAlaValValHisGlnLeuLysArgLys 
165170175 
CATGTAAATGTAGCAATTATAGGTGTTGGTGCAGGTGTTAATAACGAA1293 
HisValAsnValAla IleIleGlyValGlyAlaGlyValAsnAsnGlu 
180185190 
TATAATAGAATTTTAGTTGGATGTGATAGATACGCACCATGCCCATAC1341 
TyrAsnArgIleLeuVa lGlyCysAspArgTyrAlaProCysProTyr 
195200205 
TACTCTTCTGGTAGTTGGAATGAAGCCCAAAATATGATAAAACCTTTT1389 
TyrSerSerGlySerTrpAsnG luAlaGlnAsnMetIleLysProPhe 
210215220 
CTTACTAAAGTTTGTCAGGAAGTAGAAAGAATTGCTCATTGTGGAAAA1437 
LeuThrLysValCysGlnGluValGluArg IleAlaHisCysGlyLys 
225230235240 
TGGGAAGAATGGAGTGAATGTTCTACTACTTGTGATGAAGGAAGAAAA1485 
TrpGluGluTrpSerGluCysSerThr ThrCysAspGluGlyArgLys 
245250255 
ATTAGAAGAAGACAAATATTACATCCTGGATGTGTTAGTGAGATGACT1533 
IleArgArgArgGlnIleLeuHisPr oGlyCysValSerGluMetThr 
260265270 
ACTCCATGTAAGGTTCGTGATTGCCCACAAATACCAATACCTCCTGTC1581 
ThrProCysLysValArgAspCysProG lnIleProIleProProVal 
275280285 
ATCCCTAATAAAATTCCAGAAAAGCCATCAAACCCAGAAGAACCAGTA1629 
IleProAsnLysIleProGluLysProSerAsn ProGluGluProVal 
290295300 
AATCCAAACGATCCAAACGATCCAAACAACCCAAACAACCCAAATAAC1677 
AsnProAsnAspProAsnAspProAsnAsnProAsnAsnPro AsnAsn 
305310315320 
CCAAACAACCCAAACAACCCAAATAACCCAAACAACCCAAACAACCCA1725 
ProAsnAsnProAsnAsnProAsnAsnProAsnAsnPr oAsnAsnPro 
325330335 
AACAACCCAAACAACCCAAACAATCCAAATAACCCAAATAACCCAAAC1773 
AsnAsnProAsnAsnProAsnAsnProAsnAsnProA snAsnProAsn 
340345350 
AACCCAAATAACCCAAATAACCCAAACAACCCAAATAACCCAAACAAC1821 
AsnProAsnAsnProAsnAsnProAsnAsnProAsnAsn ProAsnAsn 
355360365 
CCAAATAACCCAAATAACCCAAATAACCCAAATAACCCAAACGATCCA1869 
ProAsnAsnProAsnAsnProAsnAsnProAsnAsnProAsnAsp Pro 
370375380 
TCAAACCCAAACAACCACCCAAAAAGGCGAAACCCAAAAAGGCGAAAC1917 
SerAsnProAsnAsnHisProLysArgArgAsnProLysArgArgAsn 
385 390395400 
CCAAACAAGCCAAAACCAAACAAGCCAAACCCAAACAAGCCAAACCCA1965 
ProAsnLysProLysProAsnLysProAsnProAsnLysProAsnPro 
405410415 
AACGAACCATCAAACCCAAACAAGCCAAACCCAAACGAACCATCAAAC2013 
AsnGluProSerAsnProAsnLysProAsnProAsnGluProSerAsn 
420425430 
CCAAACAAGCCAAACCCAAACGAACCATCAAACCCAAACAAGCCAAAC2061 
ProAsnLysProAsnProAsnGluProSerAsnProAsnLysProAsn 
435440445 
CCAAATGAGCCATCAAACCCAAACAAGCCAAACCCAAATGAGCCATTA2109 
ProAsnGluProSerAsnProAsnLysProAsnProAsnGluProLeu 
450 455460 
AACCCAAACGAGCCATCAAATCCAAACGAGCCATCAAACCCAAATGCG2157 
AsnProAsnGluProSerAsnProAsnGluProSerAsnProAsnAla 
465 470475480 
CCATCAAACCCAAACGAACCATCAAACCCAAATGAACCATCAAACCCA2205 
ProSerAsnProAsnGluProSerAsnProAsnGluProSerAsnPro 
485490495 
AATGAGCCATCAAACCCAAACGAACCATCAAACCCAAATGAACCATCA2253 
AsnGluProSerAsnProAsnGluProSerAsnProAsnGluProSer 
50 0505510 
AACCCAAAAAAGCCATCAAACCCAAATGAGCCATCAAACCCAAATGAG2301 
AsnProLysLysProSerAsnProAsnGluProSerAsnProAsnGlu 
515 520525 
CCATTAAACCCAAATGAGCCATCAAACCCAAACGAACCATCAAACCCA2349 
ProLeuAsnProAsnGluProSerAsnProAsnGluProSerAsnPro 
530 535540 
AACGAACCATCAAACCCAGAAGAACCATCAAACCCTAAAGAGCCATCA2397 
AsnGluProSerAsnProGluGluProSerAsnProLysGluProSer 
545550 555560 
AACCCAAACGAACCATCAAACCCAGAAGAGCCAAACCCAGAAGAACCA2445 
AsnProAsnGluProSerAsnProGluGluProAsnProGluGluPro 
565 570575 
TCAAACCCTAAAGAGCCATCAAACCCAGAAGAGCCAATAAACCCAGAA2493 
SerAsnProLysGluProSerAsnProGluGluProIleAsnProGlu 
580 585590 
GAACTAAACCCAAAAGAGCCATCAAACCCAGAAGAATCGAACCCCAAA2541 
GluLeuAsnProLysGluProSerAsnProGluGluSerAsnProLys 
595600 605 
GAGCCAATAAACCCAGAAGAATCGAACCCCAAAGAGCCAATAAACCCA2589 
GluProIleAsnProGluGluSerAsnProLysGluProIleAsnPro 
610615 620 
GAAGATAATGAAAATCCATTGATAATACAAGATGAACCTATAGAACCC2637 
GluAspAsnGluAsnProLeuIleIleGlnAspGluProIleGluPro 
625630635 640 
AGAAATGATTCAAATGTAATACCAATTTTACCTATCATCCCACAAAAG2685 
ArgAsnAspSerAsnValIleProIleLeuProIleIleProGlnLys 
645650 655 
GGTAATAATATCCCAAGCAATCTACCAGAAAATCCATCTGACTCAGAA2733 
GlyAsnAsnIleProSerAsnLeuProGluAsnProSerAspSerGlu 
660665 670 
GTAGAATATCCAAGACCAAATGATAATGGTGAAAATTCAAATAATACT2781 
ValGluTyrProArgProAsnAspAsnGlyGluAsnSerAsnAsnThr 
675680 685 
ATGAAATCAAAAAAAAATATACCCAACGAGCCTATACCATCACCAGGT2829 
MetLysSerLysLysAsnIleProAsnGluProIleProSerProGly 
690695700 
GAT AACCCATATAAAGGTCACGAAGAAAGAATACCAAAACCTCATCGA2877 
AspAsnProTyrLysGlyHisGluGluArgIleProLysProHisArg 
705710715720 
TCAAATGATGACTATGTATATGATAATAATGTAAATAAAAATAATAAA2925 
SerAsnAspAspTyrValTyrAspAsnAsnValAsnLysAsnAsnLys 
725730735 
GATGAACCAGAAATTCCAAATAATGAGTATGAAGAGGATAAAAATAAA2973 
AspGluProGluIleProAsnAsnGluTyrGluGluAspLysAsnLys 
740745750 
A ACCAGTCTAAATCTAATAATGGATATAAAATTGCTGGTGGTATTATT3021 
AsnGlnSerLysSerAsnAsnGlyTyrLysIleAlaGlyGlyIleIle 
755760765 
GGAGGA TTAGCTATACTTGGATGTGCAGGTGTTGGTTATAATTTTATA3069 
GlyGlyLeuAlaIleLeuGlyCysAlaGlyValGlyTyrAsnPheIle 
770775780 
GCAGGAAGTAGCGCT GCAGGATTGGCTGGAGCAGAGCCTGCACCTTTT3117 
AlaGlySerSerAlaAlaGlyLeuAlaGlyAlaGluProAlaProPhe 
785790795800 
GAAGATGTAAT TCCAGATGATGACAAAGATATTGTTGAAAACGAACAG3165 
GluAspValIleProAspAspAspLysAspIleValGluAsnGluGln 
805810815 
TTTAAATTAC CTGAAGATAATGACTGGAACTAATTTTAATAAACGTATAT3215 
PheLysLeuProGluAspAsnAspTrpAsn 
820825 
ATCCACTTTATTATTCTTATATTACATACAAATCTGATATATGTTTGTCTTTTTT TTGCT3275 
TTTAAATATTATCTATGATTATATATATAATATACCTTTAAATAATAAATTCATAAATTC3335 
GCTTGTCTTTAAATTGTTTGTGTTTCTTTACACTTTATTCCTTTTTCCTGTTTTTGTTCC3395 
TTTTTTTTTGTATGATTAAGTTATTTTAAATT AACAGTTTGATAAATTGTCATCTTTTTA3455 
TGTTATTCATTCAATTATATATCCATTTATTTTCATATTTTTTTTTAACGATTTTTTTTT3515 
AACTATTTTTTTTTAACTAATTGTCTCGTTATAATATATATATTTATTTATACTCCAATA3575 
TTTAATGGTT ACAATTATTCTTAATATAAAAAAAAAAAAAAAAAAAAAAAACTTAAAAGT3635 
TAATAACATTTTTAGGTTTGTATATTTACACGGTATTTACTATTTTCAAAATAATTATGA3695 
ATAAAACAAAAAAGTGATAATACATAATAAAATGAATTCCTAAAAAAATAGACAA ATCCA3755 
CCAATATTATCGATAAAAAAGAAATAAACAAAATGTGATTATTTTAAAATTTACAAAACA3815 
TAAAAATAATGGTCTTAAGTTTTATGAACTAAAAAGTGTGATAAAAAAAAATGATGGAAT3875 
GTTAAAAAAGAGAATATCTAAAGTTGGCTCAT GATTTTTTGAAGTATTATCATCCTTATT3935 
ATACATATCTGAAATTTTTAATTTTTCATATAAACTTTTCGAAAATTCATAATTTTGTAT3995 
TTTCATATCTGTGTTATTATGTTTGGATTCATTTATTATATTATTTGTGTAAAAATTAAG4055 
ATGATATATT TTTAGCATATTTGACAAATTGTCAAATTCGTTGTATTCTGTTTTTGAAAA4115 
AATATGAGCATTTTTTTTAATATTATTATTCTCTTTCATTTTACAAAAAAATAAGAATCG4175 
ATTTTTTTTTTTTAAATCATTAAAATTAATTTTATTTTTAAGAGAAGCAATATCA TTTTC4235 
CAATTTATTTTCATTTTCTTCATTATTTGTAGTTGCATGTGTCCATTTTTTGTTTGGAGC4295 
ATATAAATTTATTAATTCTTTATTGCTTAATTCTTTATTTTGTAAATTTATTAATTTTGA4355 
GTATTGTATATATATTTCATCCATTTTTGTCT TGTTCATATTATTAGGAAAGGAAATAAT4415 
ATTCTTATTTGTATCAATACATTTATTTTTATTTTTCCTTTTATAAAAAATCGAATAAAA4475 
TTCTAATAACGCCATTACCTCTACCTTTTCATAATTTGAAGTATCATATAATGTGAATAA4535 
ATTTATAATG TTCCCTTCTAGTTCGTTTATTTTACATATTTCTTTTCCATATTGTATATT4595 
ACCAATCATGTTTTCGTTTGTTCTTTATTTATCTTATATCCCATTTTACTTATAGCTCGT4655 
GTTTCCTCCATTTTCTGG 4673 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 826 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetLysLeuLeuGlyAsnSerLysTyrIlePheValValLeuLeuL eu 
151015 
CysIleSerValPheLeuAsnGlyGlnGluThrLeuAspGluIleLys 
202530 
TyrSerGlu GluValCysThrGluGlnIleAspIleHisIleLeuLeu 
354045 
AspGlySerGlySerIleGlyTyrSerAsnTrpLysAlaHisValIle 
5055 60 
ProMetLeuAsnThrLeuValAspAsnLeuAsnIleSerAsnAspGlu 
65707580 
IleAsnValSerLeuThrLeuPheSerThrAsnSe rArgGluLeuIle 
859095 
LysLeuLysGlyTyrGlySerThrSerLysAspSerLeuArgPheIle 
10010511 0 
LeuAlaHisLeuGlnAsnAsnTyrSerProAsnGlyAsnThrAsnLeu 
115120125 
ThrSerAlaLeuLeuValValAspThrLeuIleAsnGluArgMetTyr 
130 135140 
ArgProAspAlaIleGlnLeuAlaIleIleLeuThrAspGlyIlePro 
145150155160 
AsnAspLeuProArgSerThrAla ValValHisGlnLeuLysArgLys 
165170175 
HisValAsnValAlaIleIleGlyValGlyAlaGlyValAsnAsnGlu 
180185 190 
TyrAsnArgIleLeuValGlyCysAspArgTyrAlaProCysProTyr 
195200205 
TyrSerSerGlySerTrpAsnGluAlaGlnAsnMetIleLysProPh e 
210215220 
LeuThrLysValCysGlnGluValGluArgIleAlaHisCysGlyLys 
225230235240 
TrpGluGluTrp SerGluCysSerThrThrCysAspGluGlyArgLys 
245250255 
IleArgArgArgGlnIleLeuHisProGlyCysValSerGluMetThr 
260 265270 
ThrProCysLysValArgAspCysProGlnIleProIleProProVal 
275280285 
IleProAsnLysIleProGluLysProSerAsnPro GluGluProVal 
290295300 
AsnProAsnAspProAsnAspProAsnAsnProAsnAsnProAsnAsn 
305310315320 
P roAsnAsnProAsnAsnProAsnAsnProAsnAsnProAsnAsnPro 
325330335 
AsnAsnProAsnAsnProAsnAsnProAsnAsnProAsnAsnProAsn 
340345350 
AsnProAsnAsnProAsnAsnProAsnAsnProAsnAsnProAsnAsn 
355360365 
ProAsnAsnProAsnAsnProAsn AsnProAsnAsnProAsnAspPro 
370375380 
SerAsnProAsnAsnHisProLysArgArgAsnProLysArgArgAsn 
385390395 400 
ProAsnLysProLysProAsnLysProAsnProAsnLysProAsnPro 
405410415 
AsnGluProSerAsnProAsnLysProAsnProAsnGluProSer Asn 
420425430 
ProAsnLysProAsnProAsnGluProSerAsnProAsnLysProAsn 
435440445 
ProAsnGluProS erAsnProAsnLysProAsnProAsnGluProLeu 
450455460 
AsnProAsnGluProSerAsnProAsnGluProSerAsnProAsnAla 
465470 475480 
ProSerAsnProAsnGluProSerAsnProAsnGluProSerAsnPro 
485490495 
AsnGluProSerAsnProAsnGluProSerAsn ProAsnGluProSer 
500505510 
AsnProLysLysProSerAsnProAsnGluProSerAsnProAsnGlu 
515520525 
Pr oLeuAsnProAsnGluProSerAsnProAsnGluProSerAsnPro 
530535540 
AsnGluProSerAsnProGluGluProSerAsnProLysGluProSer 
54555 0555560 
AsnProAsnGluProSerAsnProGluGluProAsnProGluGluPro 
565570575 
SerAsnProLysGluProSerA snProGluGluProIleAsnProGlu 
580585590 
GluLeuAsnProLysGluProSerAsnProGluGluSerAsnProLys 
595600 605 
GluProIleAsnProGluGluSerAsnProLysGluProIleAsnPro 
610615620 
GluAspAsnGluAsnProLeuIleIleGlnAspGluProIleGluPro 
625 630635640 
ArgAsnAspSerAsnValIleProIleLeuProIleIleProGlnLys 
645650655 
GlyAsnAsnIl eProSerAsnLeuProGluAsnProSerAspSerGlu 
660665670 
ValGluTyrProArgProAsnAspAsnGlyGluAsnSerAsnAsnThr 
675 680685 
MetLysSerLysLysAsnIleProAsnGluProIleProSerProGly 
690695700 
AspAsnProTyrLysGlyHisGluGluArgIleProLysProH isArg 
705710715720 
SerAsnAspAspTyrValTyrAspAsnAsnValAsnLysAsnAsnLys 
725730735 
AspGluProGluIleProAsnAsnGluTyrGluGluAspLysAsnLys 
740745750 
AsnGlnSerLysSerAsnAsnGlyTyrLysIleAlaGlyGlyIleIle 
755 760765 
GlyGlyLeuAlaIleLeuGlyCysAlaGlyValGlyTyrAsnPheIle 
770775780 
AlaGlySerSerAlaAlaGlyLeuAlaGlyAl aGluProAlaProPhe 
785790795800 
GluAspValIleProAspAspAspLysAspIleValGluAsnGluGln 
805810 815 
PheLysLeuProGluAspAsnAspTrpAsn 
820825 
Obviously, many 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 
practiced otherwise than as specifically described.